Регенерация губок Halisarca dujardinii (класс Demospongiae) и Oscarella lobularis (класс Homoscleromorpha): клеточные механизмы и участие сигнального каскада Wnt тема диссертации и автореферата по ВАК РФ 00.00.00, кандидат наук Борисенко Илья Евгеньевич

• Борисенко Илья Евгеньевич
• кандидат наук
• 2022

Оглавление диссертации кандидат наук Борисенко Илья Евгеньевич

Введение

Глава 1. Обзор литературы

1Л.Клеточные и молекулярные механизмы регенерации у беспозвоночных

1.2.Роль сигнального пути Wnt в репаративных морфогенезах беспозвоночных

1.3.Клеточные и молекулярные механизмы регенерации у губок

Глава 2. Клеточные механизмы регенерации у НаШагеа ёщапИнИ и ОзеагвИа 1оЬы1ап$, представителей разных классов губок

2.1.Регенерация НаШагеа ёы]аг<Ипи

2.2.Регенерация ОзеагвНа 1оЬы1апз

Глава 3. Участие сигнального каскада Wnt в спецификации осей губок и регенерации

Заключение

Выводы

Благодарности

Материалы и методы

Список литературы

Введение

Введение диссертации (часть автореферата) на тему «Регенерация губок Halisarca dujardinii (класс Demospongiae) и Oscarella lobularis (класс Homoscleromorpha): клеточные механизмы и участие сигнального каскада Wnt»

Актуальность темы

Регенерация - процесс восстановления утраченной либо поврежденной части организма - широко распространена в животном царстве, при этом, регенеративные способности представителей разных типов животных неодинаковы [1]. Так, губки способны развиваться в полноценный организм из небольшой группы соматических клеток, а планарии - из небольшого фрагмента тела [2,3]. В то же время большинство повреждений у млекопитающих сопровождаются лишь формированием рубца. Насколько позволяют судить современные данные о репертуарах молекул, регулирующих морфогенезы (molecular toolkit: транскрипционные факторы, сигнальные молекулы, регуляторы клеточного цикла и цитоскелета), большинство многоклеточных находятся в равных условиях, т.к. репертуары таких молекул у них схожи.

Причины столь разительных отличий результатов восстановительных процессов при высокой степени сходства «инструментов», обеспечивающих их реализацию, остаются неясными. Изучение механизмов межклеточной коммуникации открыло для нас высокую консервативность ролей различных сигнальных путей в ходе развития различных животных, в частности - их участие в формировании осей полярности, пролиферации, дифференцировке, миграции клеток и апоптозе. Некоторые сигнальные механизмы, координирующие развитие, повторно активируются лишь в ходе регенерации, и предполагается, что различия в регенеративных способностях обеспечены отличиями в координации сигнальных путей в ходе регенерации, либо отличиями в составе сигнальных путей у разных животных [4-7].

На клеточном уровне процессы, происходящие при регенерации, различны среди Metazoa [1,8]. Так, с морфологической точки зрения традиционно принято выделять три регенерационных стратегии - эпиморфоз, морфаллаксис и трансдифференцировка [9-12]. Эпиморфоз характеризуется повышением пролиферативной активности клеток; пролиферация мезенхимных клеток, происходящая в специальном провизорном образовании - бластеме, предваряет формирование заново утраченной ткани, и обеспечивает накопление клеточного материала. Морфаллактическая регенерация происходит путем перестройки оставшегося клеточного материала, без активной пролиферации и роста [13]. Трансдифференцировка, или метаплазия, представляет собой необратимую трансформацию дифференцированных клеток одного типа в другой. Нередко она проходит через стадию, на которой клетки дедифференцируются и

приобретают мультипотентные свойства [11,14,15]. Однако возможно, что все три случая являются крайностями непрерывного континуума различных механизмов регенерации [4]. Общим показателем способности к регенерации является наличие в организме источника стволовых клеток или возможности дедифференцировки клеток для реализации того или иного механизма [16].

Клеточные механизмы регенерации у губок описаны преимущественно на светооптическом и электрономикроскопическом уровнях, а разнообразие объектов ограничено классами Demospongiae и Calcarea. Губки, при их ключевом филогенетическом положении и разнообразии эмбриональных морфогенезов, представляют собой ценный объект исследования для разработки таких фундаментальных вопросов, как переход к многоклеточности и ранняя эволюция формообразовательных процессов. Таким образом, мы считаем проведение сравнительного ультраструктурного исследования репаративных морфогенезов у губок из двух разных классов, Halisarca dujardinii (Demospongiae) и Oscarella lobularis (Homoscleromorpha), актуальным для всех сравнительных областей биологии, изучающих феномен регенерации - биологии развития, клеточной и эволюционной биологии, зоологии.

Сигнальный путь Wnt (Wnt-каскад) - один из ограниченного числа путей межклеточной сигнализации, управляющих развитием Metazoa. Секретируемые лиганды Wnt участвуют в регуляции формирования осей, органогенеза и морфогенетических движений - механизмах развития, приобретенных в ходе ранней эволюции многоклеточных животных. Сходные функции Wnt-сигналлинга показаны для представителей всех типов многоклеточных животных, от губок до млекопитающих. Так, у Cnidaria, наиболее просто организованных среди детально изученных животных, Wnt- каскад обеспечивает паттернирование вдоль орально-аборальной оси тела, контроль пролиферативного статуса эпителиальных клеток и поддержание популяции стволовых клеток в недифференцированном состоянии [17]. Именно Wnt-каскад обеспечивал спецификацию переднезадней оси вначале, до формирования кластеризованных и коллинеарно экспрессирующихся Hox генов [18]. В регенерации Wnt-каскад также обеспечивает регуляцию таких процессов, как становление осей формирующейся de novo части тела и пролиферация клеток [19-22].

Принимая во внимание доминирующую ныне точку зрения, что губки (Porifera) являются наиболее древним таксоном многоклеточных животных, и что сигнальный путь Wnt является новоприобретением многоклеточных животных, можно представить губок как первых многоклеточных животных, обладающих этим механизмом

межклеточной коммуникации [23-25]. Регенеративные процессы у губок хорошо изучены на морфологическом уровне, однако молекулярно-генетические механизмы их регуляции остаются малоизвестными [8,26-29]. Известно, что элементы Wnt-пути вовлечены в формирование осей тела и личинки губок, в эпителиальные морфогенезы. С точки зрения изучения такой глобальной проблемы, как эволюция механизмов молекулярно-генетической регуляции регенеративных процессов, нам представляется актуальным изучение роли Wnt-сигналлинга в регенерации губок. Их уникальное филогенетическое положение вкупе с широким спектром регенеративных ответов дает нам возможность максимально приблизиться к пониманию структуры и функции пути Wnt у последнего общего предка многоклеточных животных (last common ancestor).

Теоретическая и практическая значимость работы

Изучение механизмов регенерации животных имеет крайне важное значение для целого ряда вопросов фундаментальной биологии и биомедицины. Так, изучение клеточных механизмов репаративных морфогенезов имеет ключевое значение для биологии развития и эволюционной биологии, т.к. результаты сравнения поведения клеток в развитии и регенерации используются как дополнение данным молекулярной филогении, а также при анализе генных регуляторных сетей. Изучение молекулярных механизмов регенерации имеет значение также для фундаментальной молекулярной биологии и биомедицины. Данные о работе сигнальных каскадов, полученные на немодельных объектах, дополняют наши знания об их структуре и функциях, полученные на очень ограниченном числе видов животных и клеточных культурах. Накопление сведений о функционировании путей межклеточной коммуникации у большого вида моделей приближает нас к манипулированию регенеративным потенциалом, т.е. использованию в прикладных медицинских целях.

Цели и задачи

Цель данной работы: описать клеточные механизмы регенерации у представителей двух классов губок — Halisarca dujardinii (Demospongiae) и Oscarella lobularis (Homoscleromorpha), а также изучить участие в регенерации H. dujardinii сигнального каскада Wnt.

Задачи:

1. Описать события репаративной регенерации у губок H. dujardinii и O. lobularis на ультраструктурном уровне;

2. Оценить вклад пролиферации в процесс регенерации у данных видов;

3. Оценить вклад разных клеточных типов в процесс регенерации у данных видов;

4. Идентифицировать участников канонического (в-катенин-зависимого) сигнального пути Wnt у H. dujardinii;

5. Описать паттерны экспрессии лигандов Wnt-каскада у интактных губок H. dujardini;

6. Описать динамику и паттерны экспрессии лигандов Wnt-каскада у H. dujardinii в ходе регенерации.

Положения, выносимые на защиту

1. Репаративная регенерация у H. dujardinii осуществляется путем эпителио-мезенхимных и мезенхимо-эпителиальных преобразований. Из дедифференцированных клеток формируется регенерационная бластема, клеточными источниками которой служат хоаноциты, археоциты и эндопинакоциты. Однако активации пролиферации клеток в регенеративной бластеме не происходит.

2. Репаративная регенерация у O. lobularis осуществляется путем реорганизации неповрежденных тканей губки по типу эпителиальных морфогенезов, за счет трансдифференцировки хоаноцитов в экзопинакоциты. Клеточными источниками регенерации служат хоаноциты, экзо- и эндопинакоциты. Всплеска пролиферативной активности при регенерации не происходит.

3. У H. dujardinii обнаружены участники в-катенин-зависимого сигнального пути Wnt, а именно: лиганды Wnt (10 паралогов), рецепторы Frizzled (6 паралогов), секретируемые ингибиторы sFRP (4 паралога), корецептор LRP5/6, цитоплазматический мессенджер Dishevelled, транскрипционные факторы в-катенин и TCF, конститутивный ингибитор транскрипции Groucho, компоненты комплекса деструкции бета-катенина (APC, GSK3e и CK1).

4. Лиганды Wnt экспрессируются вдоль апико-базальной оси дефинитивной губки H. dujardinii, а также вдоль переднезадней оси личинки.

5. В ходе регенерации после удаления фрагмента тела H. dujardinii на границе раневой поверхности и интактной ткани наблюдается экспрессия одного из лигандов, HduWntK.

6. В ходе регенерации H. dujardinii путем развития из агрегатов клеток происходит активная экспрессия компонентов сигнального пути Wnt, а локализация транскриптов указывает на их участие в определении будущей оси тела и дифференцировке клеточных типов.

Научная новизна работы

Впервые на ультраструктурном уровне была описана регенерации губки Halisarca dujardinii после удаления фрагмента тела; у Oscarella lobularis впервые для класса Homoscleromorpha описана регенерация. Впервые проведен сравнительный анализ клеточных механизмов регенерации губок из разных классов с учетом новых данных по участию пролиферации в регенерации. Впервые определен состав участников Wnt-каскада у H. dujardinii, а также описана экспрессия Wnt у взрослых Demospongiae. Впервые показана локализация экспрессия Wnt в регенерации у губок. Впервые описана локализация транскриптов методом гибридизации нуклеиновых кислот in situ на примморфах. Проведенные комплексные исследования значительно расширяют данные по регенерации и ее регуляции у губок.

Публикации и апробация работы

По материалам диссертации опубликовано 17 работ: 7 научных статей в журналах, индексируемых системами WoS и/или Scopus, в том числе 3 в журналах первого квартиля SJR, и 12 публикаций в материалах международных и всероссийских конференций.

Основные положения и научные итоги диссертации были доложены в докладах на научных конференциях: 9th World Sponge Conference (04.11.2013 - 08.11.2013), 5th meeting of the European Society for Evolutionary Developmental Biology (22.07.2014 - 25.07.2014), Society for Molecular Biology and Evolution Conference (03.07.2016-07.07.2016), 4th International Congress on Invertebrate Morphology (ICIM4; 18.08.2017 - 23.08.2017); 10th World Sponge Conference NUI Galway (25.06.2017 - 30.06.2017), Всероссийская конференция «Морфогенез в индивидуальном и историческом развитии: онтогенез и формирование биологического разнообразия» (22.11.2017 - 24.11.2017), 2nd workshop of the COST Action 16203 MARISTEM: OMIC approaches to identify and characterize marine/aquatic invertebrate stem cells (08.04.2019 - 10.04.2019); Международная конференция «Marine biology, geology and oceanography — interdisciplinary studies based on the marine Stations and Labs. 80th anniversary of the Nikolai Pertsov White Sea Biological

Station» (19.11.2018 - 20.11.2018); «ЗООЛОГИЯ БЕСПОЗВОНОЧНЫХ - НОВЫЙ ВЕК» (19.12.2018 - 21.12.2018); XVIII Конференция-школа с международным участием «Актуальные проблемы биологии развития» (14.10.2019 - 19.10.2019).

Личный вклад автора

Сбор материала для всех исследований производился при личном участии автора. Большая часть исследований была проведена лично автором, в том числе микрохирургические операции, электронномикроскопические и светооптические исследования, адаптация протокола мечения и детекции синтеза ДНК с помощью этинилдезоксиуридина, выделение нуклеиновых кислот для секвенирования, клонирование генов и синтез меченных РНК-зондов, гибридизация in situ, филогенетический анализ, количественный анализ уровней экспрессии. Исключение составили: сбор животных O. lobularis, выполненный водолазным способом А. В. Ересковским; приготовление библиотек и NGS секвенирование, выполненное в Sars Centre for Marine Molecular Biology, и сборка транскриптома, проходившая при участи Marcin Adamski.

Анализ полученных данных проводился лично автором с участием коллег, представленных в коллективе авторов соответствующих публикаций.

Глава 1. Обзор литературы 1.1. Клеточные и молекулярные механизмы регенерации у беспозвоночных

Регенерационные способности распределены среди животных неравномерно [8]. Общие представления о потенциале восстановления после травмы имеются для большинства типов и классов животных. При этом исследования, позволяющие установить механизмы регенерации на клеточном и молекулярном уровне, проводятся на сравнительно небольшом репертуаре видов, в той или иной степени репрезентативных для типа или класса.

Тип Cnidaria. Классический модельный объект регенеративной биологии - Hydra -представляет собой гидроидный полип со слепо замкнутой кишкой, с хорошо выраженной орально-аборальной осью. На оральном конце тела гидры находится венчик щупалец, обрамляющий возвышающийся гипостом с ротовым отверстием в центре.

Механизмы регенерации тела Hydra изучались на модели рассечения тела животного поперек орально-аборальной оси. Наблюдаемые процессы отличались в зависимости от места рассечения: после декапитации происходило восстановление гипостома со щупальцами, после разрезания животного пополам каждая из половин регенерировала недостающую голову или ногу. При этом голова регенерирует по морфаллактическому пути после декапитации, и по эпиморфному - после разрезания животного поперек посредине [4]. Изначально описанную регенерацию головы относили к морфаллаксису потому, что инкубация оперированных животных в среде с колхицином не оказывала влияния на ход регенерации [30]. Помимо отсутствия активного деления клеток в пользу морфаллаксиса свидетельствуют отдельные описания трансдифференцировки: нейроны (конверсия фенотипа ганглионарных клеток в сенсорные эпидермальные) и секреторные клетки (железистые зимогенные клетки трансдифференцируются в слизистые) [31-34]. Более того, у гидромедузы Podocoryne трансдифференцировка является движущей силой регенерации [35,36].

Эпителизация раны после иссечения тела гидры пополам происходит за счет отростков, отходящих от основания эпителио-мышечных клеток. Образовавшаяся уплощенная регенеративная мембрана замещается обычным столбчатым эпителием в течение нескольких часов [31]. Фактором, изначально определяющим путь развития событий, является клеточный состав поврежденного участка: срединная часть полипа содержит большую часть стволовых клеток, тогда как в верхней части тела клетки коммитированы [37]. Доступность стволовых клеток в случае повреждения средней части

тела разрешает морфаллактическую регенерацию. Как было показано в экспериментах с мечеными предшественниками, пролиферации в области раны действительно не происходит. Стволовые клетки всегда компетентны к морфогенетическому сигналу, поэтому пролиферация не является обязательной стадией регенерации у гидры [38-40]. С другой стороны, блокировка клеточного цикла в S-фазе гидроксимочевиной приводит к замедлению регенерации. Таким образом, несмотря на отсутствие типичной бластемы, расположенной под раневой поверхностью, деления интерстициальных клеток, расположенных в средней части полипа и находящихся в G2-фазе, в норме имеют место и обеспечивают скорость регенерации, хотя и не являются совершенно необходимыми [41].

При изучении сигнального пути Wnt у гидры было показано, что после декапитации Wnt3 начинает экспрессироваться быстро и сильно в эпителиальных клетках, тогда как после разрезания животного поперек посредине первая волна активации обнаруживается в интерстициальных клетках, и лишь потом - в эпителии. Через 15 мин после разрезания животного пополам в половине, регенерирующей голову, наблюдается массивная, но краткосрочная, волна апоптоза в клетках-производных интерстициальной линии. Этот процесс специфичен для регенерации головы, т.к. в другой половине животного, регенерирующего ногу, уровень апоптоза остается прежним. В течение короткого промежутка времени подвергающиеся апоптозу клетки содержат Wnt3, который затем секретируется и вызывает транслокацию в-катенина в ядра интерстициальных клеток в течение последующих 1-1,5 часов. Активация сигнального пути Wnt вызывает волну пролиферации в интерстициальных клетках, что, по-видимому, является координирующим событием в дальнейшем ходе регенерации. Экспрессия Wnt3 является инструктивной и обеспечивает выполнение функции головного организатора, определяющего полярность регенерата. Временные паттерны активации экспрессии остальных шести из восьми имеющихся у Hydra Wnt различны в зависимости от места ампутации [4,42,43].

У другого Hydrozoa, Hydractinia echinata, декапитация сопровождается формированием типичной бластемы в течение 24 ч. [44]. Клетки в ней пролиферативно активны, а подавление делений с помощью рентгеновского излучения останавливает регенерацию. Кроме того, клетки бластемы экспрессируют маркеры интерстициальных клеток, такие как piwi1, vasa, pl10 и Ncol1. У интактного полипа интерстициальные клетки, как у гидры, сосредоточены в средней части тела. Нокдаун по маркерам i-клеток приводит к нарушению регенерации, но не формирования бластемы. Вкупе с наблюдениями EdU+-клеток in vivo, это свидетельствует о миграции стволовых клеток из средней части полипа в бластему под раневой поверхностью. Следует отметить, что при

ампутации ноги формирования бластемы не происходит, т.е. регенерация ноги и головы у H. echinata, как и у гидры, происходит разными путями.

Наличие типичных для Hydrozoa i-клеток не было показано для Anthozoa: пролиферативно активные клетки интактного кораллового полипа Nematostella vectensis равномерно распределены по его телу, в отличие от локализованных в середине тела i-клеток гидроидных полипов. Регенерация орального конца тела N. vectensis сопровождается активацией клеточных делений в районе раны с 18-24 ч после операции. Высокий уровень пролиферации поддерживается в формирующихся тканях, и блокировка ее гидроксимочевиной или нокодазолом предотвращает регенерацию. Подобно гидроидным полипам, регенерация аборальной части животного не сопровождается активацией делений клеток и происходит иным путем [45]. Несмотря на отсутствие i-клеток, с помощью экспериментов по пересадке тканей и pulse-chase мечению реплицирующейся ДНК у N. vectensis было показано наличие двух популяций стволовых клеток - быстро и медленно делящихся. Медленно делящиеся стволовые клетки локализованы в мезентериях полипа и мигрируют в эпителиальные ткани в ответ на повреждение. Мезентериальная популяция стволовых клеток необходима и достаточна для формирования бластемы и регенерации [46]. Вероятно, число субпопуляций стволовых клеток у Anthozoa не ограничено двумя, как следует из данных scRNA-seq [47].

Тип Ctenophora. Гребневики, за исключением представителей семейства Beroida, хорошо регенерируют удаленные органы, фрагменты тела и способны к регенерации целого тела из небольшого фрагмента (у отряда Platyctenida) [48]. Регенерация происходит быстро, за счет клеток, мигрирующих из мезоглеи и окружающих рану [49,50]. Хотя аборальный орган также способен регенерировать после полного удаления, он явно принимает участие в регуляции, в т.ч. в поддержании орально-аборальной оси тела взрослого животного [51]. Роль сигнального центра подтверждается экспрессией в аборальном органе комнонентов Wnt-каскада [52]. Вероятно, сохранение такого сигнального центра в регенерате позволяет избежать необходимости заново формировать орально-аборальную ось будущего нового животного.

Клеточные механизмы регенерации описан у гребневиков на примере модельного вида Mnemiopsis leidyi [50]. Пролиферирующие клетки у интактного животного в большинстве своем локализованы в основании щупалец, где за счет пролиферации и дифференцировки по типу конвеерной ленты происходит рост щупальца [53]. Единичные клетки обнаружены в аборальном органе и эпителии глотки. При удалении части тела гребневика происходит заживление раны без рубца, затем пролиферация в районе раны активируется, однако бластема не формируется. Пролиферирующие клетки имеют

локальное происхождение, и не мигрируют из известного места их скопления - корня щупальца, что показано в экспериментах с pulse-chase мечением синтезируемой ДНК. При этом блокировка пролиферации препятствует регенерации. Таким образом, разновидность эпиморфоза, лишенная бластемы, характерна для данного вида.

Развитие у гребневиков строго мозаично, и судьба отдельных бластомеров детерминируется и может быть прослежена уже со стадии 2-х клеточного зародыша [54,55]. При регенерации гребного ряда или щупальца вклад вносят только клетки той же линии, из которой удаленная структура формируется в нормальном развитии, т.е. при существовании популяции стволовых клеток, она должна была бы быть ограничена клеточной линией удаленного зачатка (lineage restricted) [56]. Однако, и щупальце, и гребной ряд, имеют свои пулы недифференцированных клеток, демонстрирующих экспрессию генов-маркеров (piwi и vasa) [52]; эти клетки не принимают участие в регенерации других структур и сами могут регенерировать при полном удалении щупальца или гребного ряда. Вероятно, генерализованная система мультипотентных клеток, вносящих вклад в регенерацию, отсутствует у гребневиков, и одним из возможных механизмов обеспечения источника клеточного материала в регенерации может быть дедифференцировка [51].

Геном M. leidyi был секвенирован и проанализированы сигнальные пути, участие которых в регенерации описано у других типов животных [57,58]. В геноме не были обнаружены многие компоненты в-катенин-независимого (неканонического) Wnt-каскада, FGF, Hedgehog, JAK/STAT сигналлингов, а также рецепторы и ферменты метаболизма ретиноевой кислоты. Кроме того, хотя компоненты канонического Wnt, TOR и MAP-киназного путей были обнаружены, их фармакологическое ингибирование при регенерации не привело к образованию какого-либо фенотипа. Принимая во внимание отсутствие явно выделенной системы стволовых клеток у взрослого животного, и отсутствие ряда функционально активных сигнальных механизмов, участвующих в регенерации у других животных, гребневики могут демонстрировать уникальную для типа программу репаративных морфогенезов [51].

Тип Platyhelminthes. Регенерация планарий изучалась на морфологическом уровне на протяжении более века [9]. После разрезания тела планарии (Dugesia gonocephala, D. lugubris и Dendrocoelum lacteum) происходит сильное мышечное сокращение в месте нанесения разреза, минимизирующее площадь раны [59]. Рабдиты, секреторные клетки, выбрасывают слизеподобное содержимое, покрывающее раневую поверхность [60]. Далее происходит эпителизация раны путем уплощения и миграции прилежащего эпидермиса [59,61]. В этот процесс вовлекается как дорзальный, так и вентральный эпидермис.

Источником клеточного материала при регенерации у планарий являются необласты - недифференцированные паренхимные клетки. Необласты являются единственными пролиферирующими клетками планарий. Небольшая популяция клеток в интактном животном пролиферирует постоянно; также имеется пул клеток, арестованных в G2 фазе, обеспечивающий пролиферативный ответ после кормления или при повреждении [62]. Элиминирование необластов с помощью рентгеновского излучения приводит к утрате регенерационных способностей в случае повреждения и дегенерации у интактных животных [63]. Следует упомянуть, что при истощении пула необластов в бластему могут включаться дедифференцированные клетки непаренхимного происхождения [64-66].

Интерпретация репаративного морфогенеза у планарии остается спорной. Факт участия необластов в регенерации глотки толковался разными авторами по-разному. Морган трактовал формирование новой глотки из оставшихся клеток тела (необластов) как перестройку, т.е. морфаллаксис, тогда как Кидо предположил, что необласты мигрируют из бластемы [67,68]. Однако позднее было показано, что ген тяжелой цепи миозина ((ЩМИС-Л), специфичный для мышц глотки, экспрессируется в необластах центральной части регенерата, но не в бластеме. Таким образом, глотка регенерирует из коммитированных необластов остатков тела [68]. Рабдиты также образуются из коммитированных необластов, расположенных в паренхимном пространстве, но не в бластеме [69].

При разрезании тела планарии в месте повреждения формируется бластема. Ее образование происходит благодаря пролиферативному взрыву, происходящему рядом с местом повреждения. Образующиеся необласты скапливаются под раневым эпидермисом. Пролиферации в самой бластеме не обнаружено - ее рост происходит за счет постоянного притока необластов из остатков тела [62]. Предполагается, что бластема функционирует не только как место формирования некоторых органов (нервных ганглиев, фоторецепторов), но и как сигнальный центр, выполняющий обновление позиционной информации регенерата в соответствии с новой формой и размером тела [70]. Коммитированные необласты, распространенные по остатку тела планарии, подвергаются дифференцировке в соответствии с новой позиционной информацией.

Тип Echinodermata. Регенеративные способности иглокожих имеют существенные особенности от класса к классу. Общими чертами являются дедифференцировка или трансдифференцировка клеток, выступающие в качестве движущей силы регенерации Echinodermata [71-75]. Участие стволовых клеток в регенерации, и даже их присутствие у взрослых иглокожих не показано [76]. Кроме того, полная блокировка пролиферации не препятствует регенерации, что показывает ее независимость от деления клеток [77,78].

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SAINT-PETERSBURG STATE UNIVERSITY

Printed as manuscript

Borisenko Ilya Evgenievich

Regeneration of sponges Halisarca dujardinii (class Demospongiae) h Oscarella lobularis (class Homoscleromorpha): cellular mechanisms and participation of Wnt signaling pathway

1.5.23. Developmental biology, embryology Thesis for the degree of Candidate of Biological Sciences Translation from Russian

Supervisor: Doctor of Biological Sciences, Ereskovsky Alexander Vadimovich

Saint-Petersburg - 2021

Contents

Introduction...................................................................................................................................93

Chapter 1. Literature review..........................................................................................................99

1.1.Cellular and molecular mechanisms of regeneration in invertebrates.....................................99

1.2. The role of the Wnt signaling pathway in reparative morphogenesis in invertebrates.... 106

1.3.Cellular and molecular mechanisms of regeneration in sponges..........................................110

Chapter 2. Cellular mechanisms of regeneration in Halisarca dujardinii h Oscarella lobularis, representatives of different classes of sponges............................................................................118

2.1.Regeneration in Halisarca dujardinii....................................................................................118

2.2.Regeneration in Oscarella lobularis......................................................................................134

Chapter 3. Participation of the Wnt signaling cascade in the axis specification in sponges and

regeneration.................................................................................................................................148

Conclusion...................................................................................................................................159

Principal findings.........................................................................................................................162

Acknowledgements.....................................................................................................................163

Matherials and methods...............................................................................................................164

References...................................................................................................................................166

Introduction Relevance of work

Regeneration, the process of repairing the lost or damaged part of an organism, is widespread in the animal kingdom; however, regenerative abilities of representatives of different groups of animals are not the same [1]. For example, sponges are capable of developing into a complete organism from a small group of somatic cells, while planarians - from a small body fragment [2; 3]. At the same time, most injuries in mammals are accompanied only by scar formation. As far as current data on repertoires of molecules regulating morphogenesis (molecular toolkit: transcription factors, signaling molecules, cell cycle and cytoskeleton regulators) allow us to judge, most multicellular organisms are in equal conditions, since repertoires of such molecules are similar in them.

The reasons for such striking differences in the results of repair processes with a high degree of similarity of the "tools" providing their realization remain unclear. Studying the mechanisms of intercellular communication has revealed the highly conservative roles of various signaling pathways during development of various animals, in particular, their participation in the formation of polarity axes, proliferation, differentiation, cell migration and apoptosis. Some signaling mechanisms coordinating development are reactivated only during regeneration, and it is assumed that differences in regenerative abilities are provided by differences in the coordination of signaling pathways during regeneration, or by differences in the composition of signaling pathways in different animals [4-7].

At the cellular level, the processes occurring during regeneration are different among Metazoa [1; 8]. Thus, from the morphological point of view, it is traditionally accepted to distinguish three regenerative strategies - epimorphosis, morphallaxis and transdifferentiation [9-12]. Epimorphosis is characterized by increased proliferative activity of cells; proliferation of mesenchymal cells occurring in a special provisory formation - blastema, precedes the reforming of lost tissue, and provides accumulation of cellular material. Morphallactic regeneration occurs by restructuring the remaining cellular material, without active proliferation and growth [13]. Transdifferentiation, or metaplasia, is an irreversible transformation of differentiated cells of one type into another. Often, it passes through a stage in which the cells dedifferentiate and acquire multipotent properties [11; 14; 15]. However, it is possible that all three cases are extremes of a continuous continuum of different regeneration mechanisms [4]. A common indicator of the ability to regenerate is the presence of a source of

stem cells in the body or the possibility to dedifferentiate cells to implement one or another mechanism [16].

The cellular mechanisms of regeneration in sponges have been described mainly at the light and electron microscopy levels, and the diversity of studied objects is limited to the classes Demospongiae and Calcarea. Sponges, with their key phylogenetic position and diversity of embryonic morphogenesis, represent a valuable object of study for the development of such fundamental questions as the transition to multicellularity and the early evolution of shape-forming processes. Thus, we consider a comparative ultrastructural study of reparative morphogenesis in sponges from two different classes, Halisarca dujardinii (Demospongiae) and Oscarella lobularis (Homoscleromorpha), relevant to all comparative fields of biology studying the phenomenon of regeneration - developmental biology, cell and evolutionary biology, zoology.

The Wnt signaling pathway (Wnt cascade) is one of a limited number of intercellular signaling pathways controlling Metazoa development. Secreted Wnt ligands are involved in the regulation of axis formation, organogenesis, and morphogenetic movements - developmental mechanisms acquired during early evolution of multicellular animals. Similar functions of Wnt signaling have been shown for representatives of all types of multicellular animals, from sponges to mammals. Thus, in Cnidaria, the most easily organized among the detailed studied animals, Wnt cascade provides patterning along oral-aboral axis of the body, control of the proliferative status of epithelial cells and maintenance of the stem cell population in undifferentiated state [17]. It was the Wnt cascade that provided the specification of the anteroposterior axis in the beginning, before the emergence of clustered and collinearly expressed Hox genes [18]. In regeneration, Wnt cascade also provides the regulation of such processes as the formation of the axes of the forming de novo body part and cell proliferation [19-22].

Taking into account the currently dominant opinion that sponges (Porifera) are the most ancient taxon of multicellular animals and that the Wnt signaling pathway is a new acquisition of multicellular animals, we can present sponges as the first multicellular animals possessing this mechanism of intercellular communication [23-25]. Regenerative processes in sponges are well studied on morphological level, but molecular-genetic mechanisms of their regulation remain poorly known [8; 26-29]. It is known that Wnt-pathway elements are involved in the formation of sponge body and larval axes, in epithelial morphogenesis. From the point of view of studying such a global problem as the evolution of mechanisms of molecular-genetic regulation of regenerative processes, it seems relevant to us to study the role of Wnt-signaling in sponge regeneration. Their unique phylogenetic position coupled with a wide range of

regenerative responses gives us an opportunity to come as close as possible to understanding the structure and function of the Wnt pathway in the last common ancestor of multicellular animals (last common ancestor).

The theoretical and practical significance of the work

The study of the mechanisms of animal regeneration is extremely important for a number of fundamental biology and biomedicine questions. For example, the study of cellular mechanisms of reparative morphogenesis is of key importance for developmental and evolutionary biology, since the results comparing the behavior of cells in development and regeneration are used as a complement to the data of molecular phylogeny and in the analysis of gene regulatory networks. The study of molecular mechanisms of regeneration is also important for fundamental molecular biology and biomedicine. Data on the functioning of signaling cascades obtained on non-model organisms supplement our knowledge about their structure and functions obtained on a very limited number of animal species and cell cultures. The accumulation of data on the functioning of intercellular communication pathways in a large number of models brings us closer to the manipulation of regenerative potential, i.e. use for medical applications.

Goals and objectives

This work aim is to describe cellular mechanisms of regeneration in representatives of two classes of sponges - Halisarca dujardinii (Demospongiae) and Oscarella lobularis (Homoscleromorpha), and examine the involvement of the Wnt signaling cascade in the regeneration of H. dujardinii. Objectives:

1. Describe reparative regeneration events in the sponges H. dujardinii and O. lobularis at the ultrastructural level;

2. Evaluate the contribution of proliferation to regeneration in these species;

3. Evaluate the contribution of different cell types to the regeneration process in these species;

4. To identify participants of canonical (P-catenin-dependent) Wnt signaling pathway in H. dujardinii;

5. To describe the expression patterns of Wnt-cascade ligands in intact sponges of H. dujardini;

6. To describe the dynamics and expression patterns of Wnt-cascade ligands in H. dujardinii during regeneration.

Principal findings to be considered

1. Reparative regeneration in H. dujardinii is performed by epithelial-mesenchymal and mesenchymal-epithelial transformations. Regenerative blastema is formed from dedifferentiated cells, the cell sources of which are choanocytes, archaeocytes and endopinocytes. However, there is no activation of cell proliferation in the regenerative blastema.

2. Reparative regeneration in O. lobularis is carried out by reorganization of intact sponge tissues similar to epithelial morphogenesis, due to transdifferentiation of choanocytes into exopinacocytes. Choanocytes, exo- and endopinacocytes serve as cellular sources of regeneration. There is no increase in proliferative activity during regeneration.

3. In H. dujardinii, participants of ß-catenin-dependent Wnt signaling pathway were found, namely: Wnt ligands (10 paralogs), Frizzled receptors (6 paralogs), secreted sFRP inhibitors (4 paralogs), LRP5/6 coreceptor, cytoplasmic messenger Dishevelled, transcription factors ß-catenin and TCF, constitutive transcription inhibitor Groucho, components of beta-catenin destructor complex (APC, GSK3ß and CK1).

4. Wnt ligands are expressed along the apico-basal axis of the definitive H. dujardinii sponge, as well as along the anteroposterior axis of the larva.

5. During regeneration after removal of the H. dujardinii body fragment, expression of one of the ligands, HduWntK, is observed at the border of the wound surface and intact tissue.

6. During regeneration of H. dujardinii by development from cell aggregates, active expression of components of the Wnt signaling pathway occurs, and localization of transcripts indicates their involvement in determining the future body axis and differentiation of cell types.

The scientific novelty of the work

For the first time, regeneration of the sponge Halisarca dujardinii after removal of a body fragment has been described at the ultrastructural level; in Oscarella lobularis, regeneration has been described for the first time for the class Homoscleromorpha. For the first time, a comparative analysis of cellular regeneration mechanisms of sponges from different classes has been performed, taking into account new data on the participation of proliferation in regeneration. For the first time, the composition of Wnt-cascade participants in H. dujardinii has been determined and Wnt expression in adult Demospongiae has been described. The localization of Wnt expression in regeneration in sponges has been shown for the first time. The localization of transcripts by in situ nucleic acid hybridization on primorphs has been described

for the first time. This comprehensive study greatly extends the data on regeneration and its regulation in sponges.

Publication and approbation of work

On the materials of the dissertation 17 papers were published: 7 scientific articles in journals indexed by WoS and/or Scopus systems, including 3 in the journals of the first quartile of SJR, and 12 publications in the proceedings of international and all-Russian conferences.

The main provisions and scientific results of the thesis were presented in reports at scientific conferences: 9th World Sponge Conference (04.11.2013 - 08.11.2013), 5th meeting of the European Society for Evolutionary Developmental Biology (22. 07.07.2014 - 25.07.2014), Society for Molecular Biology and Evolution Conference (03.07.2016-07.07.2016), 4th International Congress on Invertebrate Morphology (ICIM4; 18.08.2017 - 23.08.2017); 10th World Sponge Conference NUI Galway (25.06.2017 - 30.06. 2017), All-Russian Conference "Morphogenesis in individual and historical development: ontogenesis and biodiversity formation" (22.11.2017 - 24.11.2017), 2nd workshop of the COST Action 16203 MARISTEM: OMIC approaches to identify and characterize marine/aquatic invertebrate stem cells (08.04.2019 - 10.04.2019); International Conference "Marine biology, geology and oceanography - interdisciplinary studies based on the marine Stations and Labs. 80th anniversary of the Nikolai Pertsov White Sea Biological Station" (19.11.2018 - 20.11.2018); "INVERTEBRATE ZOOLOGY - NEW AGE" (19.12.2018 - 21.12.2018); XVIII Conference-School with international participation "Actual problems of developmental biology" (14.10.2019 - 19.10.2019).

The personal contribution of the author

Material for all studies was collected with the personal participation of the author. Most of the studies were performed personally by the author, including microsurgeries, electron microscopic and light-optical studies, adaptation of the labeling and DNA synthesis detection protocol using ethynyldeoxyuridine, isolation of nucleic acids for sequencing, gene cloning and synthesis of labeled RNA probes, in situ hybridization, phylogenetic analysis, quantitative analysis of expression levels. Exceptions were: collection of O. lobularis animals by diving by A. V. Ereskovsky; library preparation and NGS sequencing performed at the Sars Center for Marine Molecular Biology, and transcriptome assembly by Marcin Adamski.

The analysis of the data obtained was performed personally by the author with the participation of colleagues represented in the team of authors of the respective publications.

Chapter 1. Literature review 1.1. Cellular and molecular mechanisms of regeneration in invertebrates

Regenerative capabilities are distributed unevenly among animals [8]. General knowledge about the potential for recovery after injury is available for most phyla and classes of animals. At the same time, studies allowing to establish the mechanisms of regeneration at the cellular and molecular level are carried out on a relatively small repertoire of species representative to a greater or lesser extent of the phylum or class.

Phylum Cnidaria. The classical model object of regenerative biology, the Hydra, is a hydroid polyp with a blindly closed intestine, with a well-defined oral-aboral axis. At the oral end of the Hydra body is a crown of tentacles framing a towering hypostome with a mouth opening in the center.

The mechanisms of Hydra body regeneration were studied using the model of dissection of the animal body across the oral-aboral axis. The observed processes differed depending on the site of dissection: after decapitation, the hypostome with tentacles was regenerated; after cutting the animal in half, each of the halves regenerated the missing head or foot. In this case, the head regenerated by the morphallactic pathway after decapitation, and by the epimorphic pathway after cutting the animal across the middle [4]. Initially, the described head regeneration was attributed to morphallaxis because incubation of operated animals in medium with colchicine had no effect on the regeneration process [30]. In addition to the absence of active cell division, certain descriptions of transdifferentiation are in favor of morphallaxis: neurons (conversion of ganglion cell phenotype into sensory epidermal cells) and secretory cells (glandular zymogenic cells are transdifferentiated into mucous cells) [31-34]. Moreover, in the hydromedusa Podocoryne transdifferentiation is the driving force of regeneration [35; 36].

Epithelialization of the wound after excision of the hydra body in half occurs due to the processes that depart from the base of epithelio-muscular cells. The formed flattened regenerative membrane is replaced by normal columnar epithelium within several hours [31]. The factor initially determining the course of events is the cellular composition of the damaged area: the medial part of the polyp contains most of the stem cells, while the cells in the upper part of the body are committed [37]. The availability of stem cells in the case of damage of the middle part of the body allows morphallactic regeneration. As it was shown in experiments with labeled progenitors, proliferation in the wound area really does not occur. Stem cells are always competent to morphogenetic signal, so proliferation is not an obligatory stage of regeneration in hydra [38-40]. On the other hand, blocking of the cell cycle in the S-phase by hydroxyurea leads

to a delay in regeneration. Thus, despite the absence of typical blastema located under the wound surface, divisions of interstitial cells located in the middle part of the polyp and being in G2-phase take place normally and provide the regeneration rate, although they are not absolutely necessary [41].

When studying the Wnt signaling pathway in the hydra, it was shown that after decapitation Wnt3 starts to be expressed rapidly and strongly in epithelial cells, whereas after cutting the animal across the middle, the first wave of activation is found in interstitial cells, and only then - in epithelium. Fifteen minutes after cutting the animal in half, a massive, but shortlived, wave of apoptosis in interstitial derived cells is observed in the half regenerating the head. This process is specific to head regeneration, because in the other half of the animal regenerating the leg, the level of apoptosis remains the same. For a short period of time, apoptosis-prone cells contain Wnt3, which is then secreted and causes translocation of P-catenin into interstitial cell nuclei over the next 1-1.5 hours. Activation of Wnt signaling pathway induces a wave of proliferation in interstitial cells, which seems to be a coordinating event in the further course of regeneration. Wnt3 expression is instructive and provides the function of the head organizer determining the regenerate polarity. Temporal patterns of the activation of the expression of the other six out of eight available Hydra Wnt are different depending on the amputation site [4; 42; 43].

In another Hydrozoa, Hydractinia echinata, decapitation is accompanied by the formation of a typical blastema within 24 h. [44]. The cells in it are proliferatively active, and the suppression of divisions by X-rays stops regeneration. In addition, the blastema cells express interstitial cell markers such as piwi1, vasa, pl10 and Ncol1. In the intact polyp, interstitial cells, like in the hydra, are concentrated in the middle part of the body. Knockdown on i-cell markers results in impaired regeneration but not blastema formation. Together with the observations of EdU+ cells in vivo, this indicates the migration of stem cells from the middle part of the polyp to the blastema below the wound surface. It should be noted that blastema formation does not occur during leg amputation, i.e., regeneration of the leg and head in H. echinata, as well as in hydra, occurs by different pathways.

The presence of typical Hydrozoa i-cells has not been shown for Anthozoa: proliferatively active cells of the intact coral polyp Nematostella vectensis are evenly distributed throughout its body, in contrast to the localized in the middle of the body i-cells of hydroid polyps. Regeneration of the oral end of the body of N. vectensis is accompanied by activation of cell divisions in the wound area from 18-24 h after the operation. A high level of proliferation is maintained in the forming tissues, and blocking it with hydroxyurea or nocodazole prevents regeneration. Similar to hydroid polyps, aboral regeneration is not accompanied by activation of

cell division and occurs in a different way [45]. Despite the absence of i-cells, the presence of two populations of stem cells - rapidly and slowly dividing ones - was shown using experiments on tissue transplantation and pulse-chase labeling of replicating DNA in N. vectensis. Slowly dividing stem cells are localized in the mesenteric polyp and migrate to epithelial tissues in response to damage. The mesenteric population of stem cells is necessary and sufficient for blastema formation and regeneration [46]. Probably, the number of stem cell subpopulations in Anthozoa is not limited to two, as follows from scRNA-seq data [47].

Phylum Ctenophora. Ctenophores, with the exception of representatives of the family Beroida, regenerate well the removed organs, body fragments and are capable of regeneration of the whole body from a small fragment (in the order Platyctenida) [48]. Regeneration occurs rapidly, due to the cells migrating from the mesoglea and surrounding the wound [49; 50]. Although the aboral organ is also able to regenerate after complete removal, it clearly takes part in the regulation, including the maintenance of the oral-aboral axis of the adult animal body [51]. The role of signaling center is confirmed by the expression of Wnt cascade comnonents in aboral organ [52]. Probably, the preservation of such a signaling center in the regenerate avoids the necessity to rebuild the oral-aboral axis of the future new animal.

The cellular mechanisms of regeneration are described in ctenophores by the example of the model species Mnemiopsis leidyi [50]. The proliferating cells in the intact animal are mostly localized at the base of the tentacles, where the tentacle growth occurs due to proliferation and differentiation by the conveyor belt type [53]. Single cells are found in the aboral organ and pharyngeal epithelium. When a part of the crested ctenophore body is removed, the wound heals without a scar; then, proliferation in the wound area is activated, but the blastema is not formed. Proliferating cells have a local origin and do not migrate from the known place of their accumulation - the root of the tentacle, as shown in the experiments with pulse-chase labeling of synthesized DNA. At the same time, blocking of proliferation prevents regeneration. Thus, the variety of epimorphosis devoid of blastema is characteristic of this species.

Development in the comb jellies is strictly mosaic, and the fate of individual blastomeres is deterministic and can be traced back to the 2-cell embryo stage [54; 55]. In comb row or tentacle regeneration, only the cells of the same lineage from which the deleted structure is formed in normal development contribute, i.e., if a population of stem cells exists, it would have to be restricted to the cell line of the deleted primordium (lineage restricted) [56]. However, both the tentacle and the comb row have their own pools of undifferentiated cells, showing the expression of marker genes (piwi and vasa) [52]; these cells do not take part in the regeneration of other structures and can regenerate themselves when the tentacle or comb row is completely removed. Probably, the generalized system of multipotent cells contributing to the regeneration is absent in

the Ctenophora, and dedifferentiation may be one of the possible mechanisms for providing a source of cellular material in regeneration [51].

M. leidyi genome was sequenced and signaling pathways whose participation in regeneration has been described in other types of animals were analyzed [57; 58]. Many components of P-catenin-independent (noncanonical) Wnt cascade, FGF, Hedgehog, JAK/STAT signaling, and retinoic acid metabolism receptors and enzymes were not found in the genome. In addition, although components of the canonical Wnt, TOR, and MAP kinase pathways were detected, their pharmacological inhibition during regeneration did not result in any phenotype. Taking into account the absence of an explicitly isolated stem cell system in the adult animal, and the absence of a number of functionally active signaling mechanisms involved in regeneration in other animals, Ctenophora may demonstrate a reparative morphogenesis program unique to the phylum [51].

Phylum Platyhelminthes. Regeneration of planaria has been studied at the morphological level for more than a century [9]. After cutting the body of planaria (Dugesia gonocephala, D. lugubris and Dendrocoelum lacteum) there is a strong muscle contraction in the place of the cut, minimizing the wound area [59]. Rhabditis, secretory cells, eject a mucus-like content covering the wound surface [60]. Then, the wound epithelializes by flattening and migration of the adjacent epidermis [59; 61]. Both dorsal and ventral epidermis are involved in this process.

Neoblasts, undifferentiated parenchymal cells, are the source of cellular material during regeneration in planarians. Neoblasts are the only proliferating cells of planarians. A small population of cells in the intact animal proliferates continuously; there is also a pool of cells arrested in the G2 phase, providing a proliferative response after feeding or upon damage [62]. Elimination of neoblasts by X-rays leads to the loss of regenerative abilities in case of damage and degeneration in intact animals [63]. It is worth mentioning that when the pool of neoblasts is depleted, dedifferentiated cells of non-parenchymal origin can be included in the blastema [6466].

The interpretation of reparative morphogenesis in planaria remains controversial. The fact of participation of neoblasts in pharyngeal regeneration has been interpreted differently by different authors. Morgan interpreted the formation of a new pharynx from the remaining body cells (neoblasts) as rearrangement, i.e. morphallaxis, while Kido suggested that neoblasts migrate from the blastema [67; 68]. However, it was later shown that the myosin heavy chain gene (DjMHC-A) specific for pharyngeal muscles is expressed in the neoblasts in the central part of the regenerate, but not in the blastema. Thus, the pharynx regenerates from the commited neoblasts of the body residues [68]. Rhabditis also forms from commited neoblasts located in the parenchymal space, but not in the blastema [69].

When the body of a planarian is dissected, a blastema is formed at the site of the injury. Its formation occurs due to the proliferative explosion occurring near the injury site. The formed neoblasts accumulate under the wound epidermis. No proliferation in the blastema itself is detected - its growth occurs due to the constant inflow of neoblasts from the body remnants [62]. It is assumed that the blastema functions not only as a place of formation of some organs (nerve ganglia, photoreceptors), but also as a signaling center, which performs updating of positional information of the regenerate in accordance with the new shape and size of the body [70]. Committed neoblasts spread across the planarian body remnant are differentiated according to the new positional information.

Phylum Echinodermata. The regenerative abilities of echinoderms have significant features from class to class. The common features are the dedifferentiation or transdifferentiation of cells acting as the driving force of Echinodermata regeneration [71-75]. The participation of stem cells in regeneration, and even their presence in adult echinoderms has not been shown [76]. Moreover, complete blockage of proliferation does not prevent regeneration, which shows its independence from cell division [77; 78].

Among the classes Crinoidea, Asteroidea and Ophiuroidea, the regeneration of the ophiura Amphiura filiformis is best described. It is characterized by autotomy - induced by external influence separation of the arm from the body. Distal to the amputation site, a blastema is formed, from which metameric segments of the arm are built up, within which the differentiation of new tissues takes place. At the morphological level, it has been shown that both migrating cells with the morphological features of multipotent cells and dedifferentiated cells contribute to the formation of the blastema [79]. The markers of muscle and skeletogenic lines of differentiation, troponin-1 and alpha-collagen-1, are not expressed in the blastema cells, but their expression begins in the segments of the forming arm long before they become functional. Expression of a set of transcription factors from the ETS, T-box and homeobox families is maintained in the distal part of the blastema, whereas with the isolation of metameric structures in the proximal part of the regenerate, expression of various factors is limited depending on the tissue that is being formed. Thus, the fate of blastema cells is regulated by combinations of transcription factors from at least these families [80]. The spicules of the newly formed arm are synthesized by mesenchymal cells; the population of these cells expressing markers of skeletogenic differentiation is not subject to proliferation, i.e. its replenishment comes from some other sources [81]. In addition, in Ophioderma brevispina, the dependence of regeneration on the activity of the Notch cascade has been demonstrated. When Notch activity is suppressed, regeneration is inhibited. Directly or indirectly, this signaling cascade regulates the expression of about 4.5 thousand genes during regeneration. Among them, extracellular matrix (ECM) and

intercellular adhesion proteins, components of intercellular signaling cascades, innate immune response and ubiquitinylation systems, as well as proteins responsible for apoptosis, survival, differentiation and cell proliferation are identified [82].

Members of the class Holothuroidea are notable not so much for their evisceration, i.e., their ability to eject their intestines in response to a stress, as for their ability to regenerate a functioning digestive system afterwards. In many holothurians, after evisceration, the tissues of the esophagus remain on the oral end of the animal's body, and the epithelium of the cloaca on the aboral end. From these two cell sources, two separate rudiments of the intestine, which grow towards each other due to the processes of dedifferentiation, proliferation, migration, and differentiation, are established [83-85]. In a number of species, such as Eupentacta fraudatrix, esteration occurs through the anterior part of the body, as a result of which the areas of the esophagus giving the anterior intestinal regenerate rudiment are not preserved. In this case, the foregut is formed by transdifferentiation and proliferation from the cells of the coelomic epithelium of mesodermal origin [73].

The cellular and molecular mechanisms of regeneration in Echinoidea, a sea urchin, remain virtually unstudied. It is known that pharmacological suppression of Notch signaling pathway significantly slows down regeneration, and Piwi- and Vasa-positive cells, presumably multipotent, are common in many adult animal tissues. The authors believe that their wide distribution is associated with potential participation in reparative regeneration [86].

Phylum Chordata. A large number of modern works on Ascidia is concentrated around the phenomenon of whole-body regeneration characteristic of colonial ascidians of the family Botryllidae [87]. The phenomenon consists in the restoration of a whole colony from a fragment of a blood vessel common to zooids. Many questions of this complex process remain unsolved, including which cells contribute to and how they form a new colony body. It is unambiguously known that undifferentiated piwi-positive cells found in blood and coelomic fluid proliferate after the operation and are a part of the forming epitheliums [88; 89]. Solitary ascidians of the genus Styela have much weaker regenerative abilities than colonial ones. Thus, an ascidia is capable of regenerating a syphon; moreover, only the proximal part of the animal (regenerant) has such an ability, and only if the branchial sac is preserved [90]. Using EdU labeling, it was shown that removal of the syphon causes proliferation of morphologically undifferentiated cells within the branchial sac. These cells expressing piwi and alkaline phosphatase, markers of stem cells, migrate to the wound surface and later form a blastema there. No proliferative response is observed in the wound area prior to blastema formation. Thus, in solitary ascidians, the removed syphon is restored by epimorphosis due to undifferentiated cells localized in the branchial sac.

The above studies demonstrate the role of stem cell systems in the regeneration of invertebrate animals belonging to different taxa.

1.2. The role of the Wnt signaling pathway in reparative morphogenesis in

invertebrates

In the course of regeneration, the organism has to solve for itself such tasks as (1) compensation of the lost tissues and (2) restoration of the disturbed spatial relations. The fulfillment of these tasks is not possible without intercellular communication systems. Molecular mechanisms, which ensure the cell's perception of information from the environment and neighboring cells, allow the cells to learn about the damage, trigger the proliferative response, provide directed migration and patterning of axes of both the developing and adult organism.

Currently, the number of models for the study of regeneration at the molecular genetic level among invertebrates is limited. Among them, regeneration of the hypostome in Hydra hydroid polyp, regeneration of the posterior part of the body in Schmidtea mediterranea, and regeneration of imaginal discs in Drosophila melanogaster. Wnt-signaling is shown to be involved in regeneration in all of the above models.

The activity of the Wnt pathway has been studied on the Hydra polyp the most extensively among invertebrates. The hydra hypostome is a classical organizer, a group of cells capable of specifying the fate of surrounding cells. The Hydra organizer is one of the key elements in the specification of the oral-aboral axis of the animal. Thus, transplantation of the hypostome to any other site of the hydra body leads to the induction of the secondary axis [93]. Inducing effect of hypostome is mediated by Wnt/P-catenin-dependent signaling: treatment of animals with alsterpaulone, an agent providing activation of Wnt/P-catenin-dependent pathway, results in formation of multiple organizers with increased expression of Wnt3, Wnt5 and Wnt8 in them [94; 95]. Moreover, 7 of 10 Wnt genes identified in Hydra are expressed in the hypostome of an adult animal [43].

The mechanism of regeneration when the oral end of the Hydra body is removed differs from that when the basal disc is removed [96]. After removal of the hypostome with tentacles, massive cell death occurs, and within 30-60 minutes about 50% of the cells undergo apoptosis. When the basal disc is removed, such extensive death does not occur, and the proportion of apoptotic cells is 7% with a baseline value of 1% for a non-regenerating animal [42]. In this case, only i-cells and their progeny (neurons and nematocytes) undergo apoptosis, while the epidermis and gastrodermis remain intact [97].

After removal of the upper half of the body, the dying cells are concentrated at the regenerating end of the body in the form of a thin layer. By labeling the synthesized DNA with bromodeoxyuridine (BrdU), it was shown that a zone of actively proliferating cells is located directly below the apoptotic cells [42]. By analogy with the regenerating imaginal discs of

Drosophila, it was suggested that the dying cells are a source of signal activating the proliferation of neighboring cells. Thus, apoptotic cells of the Drosophila imaginal disc secrete diffusible Wingless and Decapentaplegic proteins that induce proliferation near them [98; 99]. Indeed, during (but not before) the death of i-line cells in Hydra, within one hour after injury, the Wnt3 ligand is released from them [42; 100]. Probably, the protein is synthesized immediately at wounding or is present in the cells in the bound form. Apoptosis of i-cells occurs in caspase-dependent manner and is a necessary condition for regeneration; moreover, caspases are apparently involved in Wnt3 release from cells. Thus, when caspases are pharmacologically inhibited, apoptosis, Wnt3 release and head regeneration does not occur. At the same time, during normal basal disc regeneration, there is no mass apoptosis and Wnt3 release; when apoptosis is induced by local heating, there is release of this ligand and head regeneration instead of basal disc. Binding of Wnt3 to i-cells outside the apoptosis zone causes activation of Wnt/^-catenin-dependent pathway and nuclear translocation of [3-catenin. The experiments with inhibition of Wnt/p-catenin-dependent pathway show that this very event provides high proliferative activity of cells in the subterminal region of regenerating hydra [42; 96].

4 hours after the injury, the re-emergence of Wnt3 expression is observed, now together with Wnt9/10c and Wnt11. Wnt7, Wnt9/10a and Wnt16 start to be expressed after 12 hours; Wntl is expressed after 24 hours [4; 43]. Thus, the Wnt cascade regulates Hydra oral end regeneration by coordinated expression of various ligands, and Wnt3 plays the leading role in this regulation. Wnt participation in regeneration has also been shown in phylogenetically more basal Nematostella vectensis [101], but such mass apoptosis has not been described. Apparently, the role of Wnt is limited in N. vectensis by the patterning of oral-aboral axis: regeneration of aboral half of the body in the presence of Wnt exogenous activator leads to the formation of ectopic hypostome [102].

The planaria Schmidtea mediterranea is a free-living flatworm that has been a model organism of regenerative biology along with hydras and amphibians for more than a century. The genome of S. mediterranea contains 9 Wnt genes [19; 21; 103]. Six of them encode Wnt11 family orthologs; the remaining ones are Wnt1, Wnt2 and Wnt5 orthologs. The genes Wnt1, Wnt11-1, Wnt11-2, Wnt11-4, and Wnt11-5 show a shift in expression pattern towards the posterior end of the adult worm body, whereas Wnt11-3, Wnt11-6, and Wnt5 are expressed in a diffuse pattern throughout the body. In addition, there are three orthologs of secreted sFRP inhibitors: sFRP-1 and sFRP-2, which are expressed strictly at the anterior end of the body, whereas sFRP-3 is more widely distributed along the anteroposterior axis. The role of в-catenin in the specification of the anteroposterior axis of the planaria has been demonstrated using RNA interference: ectopic heads are formed in в-catenin RNA-interfered animals. The posterior

blastema that forms after tail amputation in P-catenin-deficient animals develops into heads. Together with the data on Wnt expression, this indicates the presence of a complex gradient of P-catenin activity (high in the posterior end of the body and low in the anterior end), although direct evidence of Wnt-cascade activation in S. mediterranea cells has not yet been obtained, and its mRNA is diffusely distributed along the anteroposterior axis [104; 105].

In the intact worm, the Wnt1 gene is expressed in a very small number of cells on the dorsal side of the posterior end of the body. In regeneration, however, its mRNA is abundantly present in all cells of the wound surface between 6 and 9 hours after tail or head amputation, as well as in the wound sites without amputation [105]. This means that wnt1 expression belongs to the early wound response regardless of the wound position [22]. In the posterior blastema of the head fragment regenerating the tail, the sequential appearance of Wnt gene mRNAs specific for the posterior end of the worm body is observed following the expression of Wnt1. Thus, Wnt11-5 appears one day after amputation, Wnt11-1 appears two days later, and Wnt11-2 appears four days later. In the anterior blastema of the posterior body fragment, single cells expressing sFRP appear. Later, the expression of inhibitors spreads along the anteroposterior axis, while the tail retains the expression of Wnt specific to the posterior end [105]. The described complex pattern of Wnt and sFRP expression indicates that Wnt-signaling can not only specify the major axis of the body, but also determine the so far undetermined boundaries of body parts (for example, by counter gradients of anterior sFRP-2 and posterior Wnt11-5). Thus, the Wnt/P-catenin-dependent pathway controls the specification of the anteroposterior blastema axis during regeneration in Schmidtea mediterranea and the adult animal homeostasis.

The imaginal discs of Drosophila represent the primordia of such adult insect structures as wings, legs, etc. [106].

The imaginal wing discs formed by the end of the third larval age of Drosophila contain about 75 thousand cells. At this stage, the cells composing the primordia of most of the definitive structures have molecular markers of belonging to one or another wing element. The notum (dorsal part of the thorax) develops from the most dorsal end of the disc. Wnt-signaling is involved in the regulation of wing development, since flies mutant for the Wnt homologue, the wingless gene, do not develop wings [106-108]. Induced expression of wingless at a certain time causes the formation of ectopic wings in the notum [109; 110]. During the second larval instar, wingless is expressed in the ventral part of the wing disc, specifying the wing field. Segregation of the imaginal disc into wing- and notum-forming areas occurs due to mutual antagonism between Wnt- and EGF-signaling, respectively.

When a small sector of the imaginal disc is excised, the wound is healed, after which the blastema is formed. The blastema grows and undergoes patterning under the influence of the

preserved structures of the imaginal disc. Fragmentation of the imaginal disc leads to the appearance of wingless expression at the wound edge [111]. The expression is controlled by an enhancer located in the 3'-region of wingless, which is activated upon damage [112]. Local overexpression of wingless in the intact disc induces the appearance of a zone of high proliferative activity and blastema formation [113; 114]. Together, these data suggest the importance of the role of Wnt in regeneration, however, apparently, the genetic programs triggered by Wnt in normal development of the imaginal disc and in its regeneration are different.

1.3. Cellular and molecular mechanisms of regeneration in sponges

Sponges (Porifera) are aquatic filter-feeding primitive animals. The body of sponges is bounded by two epithelial layers, the pinacoderm and the choanoderm. The pinacoderm is represented by flattened cells, pinacocytes, which form the outer covers of the sponge and line the canals of the aquiferous system. The exterior of the sponge is covered by exopinacoderm; at the point of contact with the substrate, exopinacocytes have a special structure and form the basopinacoderm. The choanoderm is formed by flagellate cells, choanocytes, lining the choanocytic chambers of the aquiferous system. The space between the outer layer of pinacocytes and the aquiferous system is filled with mesohyll consisting of txtracellular matrix (ECM) and various types of cells and skeletal elements. Water enters the choanocyte chambers through the inhalant canals of the aquiferous system lined by endopinacocytes. In the choanocyte chambers, the beating of choanocyte flagella ensures the movement of water as well as the trapping of food particles. From the choanocyte chambers, water enters the atrial cavity and leaves it through the osculum, an outlet tube [115].

Four classes of sponges are currently distinguished: Calcarea (calcareous sponges), Demospongiae (demosponges), Homoscleromorpha, and Hexactinellida (glass sponges). Hexactinellida in the adult state have a syncytial structure and are the least studied.

Recovery processes in sponges have been studied mainly in representatives of the Demospongiae and Calcarea classes. The peculiarities of regenerative processes in sponges depend on the localization of injury, the degree of trauma, the level of organization of the species, and the stage of the life cycle, since in some sponges the features of organization change in the course of the life cycle [8]. For a long time, there have been ideas about the unlimited regenerative abilities of Porifera, which appeared due to experiments on the development of cell conglomerates after dissociation [3; 116]. Indeed, after dissociation of adult sponges of some species, their cells are able to aggregate and develop into primorphs - radially symmetric epithelialized aggregates inside which cells undergo dedifferentiation and provide development of a new animal [117; 118]. However, these processes are not successful in all species. Currently, this phenomenon is considered a case of whole-body regeneration [119].

Given the variety of forms of organization and the nature of injury, a unambiguous assessment of the regenerative abilities of sponges is difficult. Let us consider the classification based on the anatomical organization of the aquiferous system [8].

Among asconoid calcareous sponges, regeneration experiments were carried out on representatives of the species Leucosolenia complicata and L. variabilis. The sponge body is represented by a network of anastomosing basal tubes, from which blindly closed diverticulae

and oscular tubes depart. The body wall consists of pinacoderm, choanoderm, a thin layer of mesohyll with a large number of spicules, secretory cells, and is punctured by intracellular supply canals. When a small penetrating wound of the body wall was made, there was wound healing with restoration of tissues, pores and skeletal elements. When a large through-wound was inflicted, healing could also take place, but new diverticula or osseous section developed at the site of the injury. The extirpated fragments of osseous and basal tubes did not retain their former organization, but transformed into osseous and basal tubes of a new sponge, respectively [120-125].

Cellular mechanisms of regeneration turned out to be similar in all cases. The wound was closed by a bilayer regenerative membrane formed from transdifferentiating exopinacocytes and choanocytes. In case of a through wound, 12 hours after the formation of the membrane, dedifferentiated choanocytes started spreading along its inner side. No blastema formation was observed, but choanocytes adjacent to the wound lost their flagellum and collar for a while. The cells within the regenerative membrane did not divide, but all cells outside it retained proliferative activity at the level characteristic of an intact sponge.

The species specificity of reparative morphogenesis established for Syconoid sponges is explained by differences in the general organization of the definitive sponges. In the single-oscular, the more integrated Sycon raphanus, when the sponge body is cut along the apico-basal axis, fusion of the wound edges with preservation of the sponge polarity occurs. The halves and quarters of the body of multi-oscular S. lingua curve along the apico-basal axis and fuse in such a way that a blindly closed sponge is formed. The oscular region undergoes anarchization and ceases to function, while a new osculum develops elsewhere in the sponge. Also, development from small body areas is more successful in S. lingua [126]. Thus, the nature of the recovery process depends on the level of integration of the organism.

Reparative morphogenesis in Sycon has been described at the light-optical level [127-129] and by scanning electron microscopy [130]. Epithelialization of the wound occurs at the expenses of pinacocytes adjacent to the wound and choanocytes of the marginal zone - the peripheral part of the wound surface. The latter undergo transdifferentiation into exopinacocytes - they flatten and lose the microvillus collar; however, the flagellum is preserved [127-130]. Such transdifferentiated choanocytes infiltrate the intact exopinacoderm crawling over the wound surface. The formation of the regenerative membrane has been described on the process of regeneration from body fragments ("rings") obtained by cutting Sycon ciliatum into fragments across the apico-basal axis [130]. The wound epithelium, having covered the wound surface, continues to migrate in its plane and crawls into the lumen of the atrial cavity in the center of the "ring". On the inner side the exopinacoderm connects with a layer of endopinacocytes migrating

from the atrial cavity, resulting in formation of a two-layer regenerative membrane covering the central lumen of the "ring" (sponge atrium before injury). The regenerative membrane completely covers a slice of the sponge body, after which spicules begin to form in it, and later -osculum on one of the regenerate surfaces.

Among leuconoid sponges, regeneration experiments have been performed mainly on Demospongiae. As an example, let us consider the experiments carried out on the multi-oscular Halichondria panicea [122; 131; 132]. In the case of longitudinal incision of the body, healing of the wound, formation of deep epithelialized hollows in the sponge body or formation of small oscular tubercles (in the case of injuries of large outflow canals) took place. Small body fragments developed into a new sponge, and the morphological apico-basal axis developed de novo. The sides of the fragments did not contain exopinacoderm, i.e., they represented wound surfaces. Their epithelialization involved exo- and endopinacocytes, as well as nucleolated amebocytes. During the first day of the experiment amebocytes formed multilayered clusters on the wound surface, their upper layer flattened and differentiated into exopinacocytes. Further, during the rounding of the fragment, anarchyzation of the canals and chambers of the aquiferous system occurred, and the sponge lost its filtration ability. A part of choanocytes and endopinacocytes underwent phagocytosis, apparently making up for the lack of an inflow of nutrition from outside.

In freshwater demosponges Spongilla lacustris and Ephydatia fluviatilis, after removal of the apical section of the oscular tube, the exo- and endopinacoderm forming the tube is rapidly stretched and the original size of the functioning osculum is restored. Amebocytes are involved in the regeneration process, dividing and incorporating into the pinacoderm. During repeated surgeries the pool of amebocytes is depleted and choanocytes of the chambers adjacent to the oscular tube are included into the pinacoderm [133; 134].

In experiments on the cultivation of Geodia barretti choanosoma fragments, the possibility of regeneration of such fragments into a complete sponge was shown [135]. The authors note the formation of a regenerative membrane of "motile cells" as well as the accumulation of spherular cells at the periphery of the fragment. When studying the development of Chondrosia reniformis from body fragments, it was shown that the presence of an ectosome is essential for successful regeneration [136]. After rounding of the sponge fragment, reorganization of its aquiferous system took place. DNA synthesis was recorded during the first two weeks after the operation at a low level (less than 1% of the cells), but no incorporation of labeled nucleotides were recorded after 3 months of the experiment.

Regeneration of Halisarca dujardinii after cutting the animal body into several parts has been described at the light-optical and ultrastructural levels [137], and the manifestations of the

cellular response to the introduction of a foreign body have also been studied, which combines the participation of protective and regenerative processes [138]. The authors studied the development of sponges from small body fragments; this type of injury caused the emergence of radial symmetry in the regenerate and rearrangement of most of the aquiferous system. By labeling choanocytes with Indian ink particles, it was shown that after anarchyzation of the choanoderm, their fate could be different: a significant number of them remain in the mesohyll as dedifferentiated cells, some are incorporated into the exopinacoderm, some are transformed into endopinacocytes of the aquiferous canals. Epithelialization of the wound surfaces occurs due to stretching of the adjacent exopinacoderm as well as due to the evicting amebocytes.

A number of works are devoted to the analysis of cell proliferation at different stages of sponge life cycles. In particular, S.M. Efremova showed an increase in the proliferative activity of nucleus amebocytes during the development of somatic cell conglomerates of Ephydatia fluviatilis from 1.04 to 3.64% mitoses per hour [139]. The larval Baikalospongia bacillifera showed terminal differentiation of surface flagellate and eosinophilic cells as well as scleroblasts. DNA synthesis was observed in amebocytes, collenocytes, and choanocytes of the larva. The generation time for nucleated amebocytes was 5.0-5.6 h, for choanocytes 13.2-15.3 h. It is noteworthy that the proliferative pool of choanocytes was 100% [140]. The use of the colchicine method of mitosis blocking in parallel with thymidine autoradiography gave different values of the generation time of choanocytes of Hymeniacidon sinapium: 20 h for the colchicine method and 40 h for autoradiography [141]. The cell cycle of choanocytes was described in the tropical species Halisarca caerulea [142]. The duration of the entire cycle was 5.4 h and the duration of the S phase was 30 min. The volume of the growth fraction was 46.6% of choanocytes. The authors attribute such a high proliferation rate to the phenomenon of choanocyte shedding, which occurs in this species.

Changes in the proliferative activity of cells in the course of regeneration occupy a special place as a way to compensate for the lost cellular material. For this purpose, cell division activity was analyzed in the regeneration of Halisarca caerulea [143], Leucosolenia variabilis [144], Aplysina caverenicola [145] and Sycon ciliatum [130]. The kinetics of cell proliferation was assessed by the incorporation of exogenous nucleotide analogs into the DNA of dividing cells. The most proliferatively active population in all species was choanocytes. In H. caerulea, it was shown that the proportion of dividing cells in the first day sharply decreases in the area adjacent to the wound (7% of the total number of choanocytes in the studied area, compared to 46% in intact tissue), although their cell cycle duration remains the same [143]. The choanocytes in the chambers removed from the wound retain proliferative activity at the level of control values.

With the course of regeneration the proportion of proliferating cells near the wound increases and reaches the basic level. No proliferation activation was observed in mesohyl cells.

In the calcareous sponge L. variabilis, the regeneration of a through wound in the body wall is performed by the migration and transdifferentiation of choanocytes and exopinacocytes adjacent to the wound, which form a temporary formation - a regenerative membrane [144]. At the same time, neither in the tissues adjacent to the wound, nor in the remote parts of the sponge body there is an increase or suppression of proliferative activity: choanocytes, including those adjacent directly to the wound, retain the proliferative index at the level of about 15%. Thus, in L. variabilis, regeneration proceeds without changing the pool of proliferatively active cells. In another calcareous sponge, S. ciliatum, wound healing also occurs due to migration and transdifferentiation of adjacent cells [130], and no activation of proliferation is observed until the wound surface is closed, i.e. 12-24 hours. However, from this moment, reaching the maximum by 48 hours, there is an increase in the proportion of DNA-synthesizing cells. Their localization is not confined to the wound area - divisions are observed at a distance of 3-4 choanocyte chambers from the wound surface; moreover, dividing cells were not shown in the wound epithelium. Probably, in S. ciliatum, the only necessary mechanism of wound healing is transdifferentiation, and the generalized proliferative response is required at later stages to compensate for the cellular losses incurred during the closure of the wound surface.

Regeneration of the demosponge A. caverenicola occurs with the formation of blastema, which is formed from dedifferentiated cells under the wound surface [145]. At the stage of wound healing, cell division is suppressed in the 100-^m zone adjacent to the wound. Later, 2448 hours after the injury, dividing cells appear in the blastema. According to the shape and size of the nucleus, the authors suggest that these cells originate from the mesohyl. Their number increases with the course of regeneration, but the proportion of dividing cells remains lower in the tissues adjacent to the wound than in intact sponges. At the same time, the proportion of proliferatively active cells (choanocytes and archaeocytes) in the tissues remote from the wound does not change during regeneration and remains at the basic level.

Thus, we can see in the four studied species different types of proliferative response to damage: from complete absence of any changes, to active nonlocalized proliferation already after wound healing. This once again emphasizes the diversity of regenerative mechanisms even within the Porifera phyla.

Molecular biological studies of regeneration were performed on H. caerulea [28], regeneration from a body fragment of the sponge Chondrosia reniformis [29], regeneration from cell aggregates after dissociation [27] and from body fragments of S. ciliatum [130].

In the first study of regeneration at the transcriptomic level, surface incisions on the body of H. caerulea sponges were used as an injury [28]. RNA isolated from intact sponges, after 2 hours and 12 hours after surgery, was sequenced. The sets of transcripts obtained from each library were compared and analyzed by two approaches - by the representation of transcripts classified by gene onthology (GO) and by differential expression evaluation in the RSEM package with a high level of variance fixed, because there were no biological or technical repetitions. With regard to GO classification, it was shown that 2 hours after injury there was an increase in signatures of processes such as response to biotic stimuli, apoptosis, carbohydrate metabolism, regulation of hormone levels, and embryonic development. The authors consider it logical to correlate the increase in the number of transcripts belonging to such processes with the early stages of regeneration - apoptosis, response to damage, activation or inhibition of metabolism. After 12 hours there is an increase in the number of transcripts attributed to genetic imprinting, response to TGF-P, polysaccharide metabolism, growth, maintenance of cell polarity, and adhesion. The authors associate some of these processes (imprinting and response to signaling molecules) with the events of de- and differentiation occurring during regeneration; another part (metabolism of polysaccharides, including extracellular matrix, cell polarity and adhesion) is directly related to morphogenetic events, cell migration and new tissue formation. Using differential expression assessment, a number of targets were found whose expression is significantly activated during regeneration: a1-chain collagen, a-actinin, vilin, EGF-containing Ca-binding protein, S-catenin, perlecan - elements of ECM and proteins responsible for mediating cytoskeleton binding to it. This pioneering work shows the potential of comparative-transcriptome studies of regeneration as applied to such a non-trivial model as sponges.

The role of TGF-P factors in C. reniformis regeneration was studied using a model of whole body development from its fragment [29]. The TGF-P signaling pathway was chosen as a participant in multiple processes in development (proliferation, differentiation, migration), as well as a widespread mechanism among Metazoa. Using scanning electron microscopy (SEM), immunocytochemistry, and labeling of choanocytes with ink particles, it was shown that the exopinacoderm on the wound surface is formed due to differentiation of archaeocytes, but not from dedifferentiated choanocytes. Six TGF-P ligand paralogs were found in C. reniformis, and 4 of them show differential expression at different stages of regeneration. One of the ligands, TGF6, is expressed early (up to 24 hours) in cells whose localization coincides with archaeocytes. This burst of expression coincides with the expression of a marker of archaeocytes (multipotent cells), Msi-1/Musashi, as well as with the timing of archaeocyte migration and differentiation on the wound surface. In addition, the pharmacological inhibitor of the TGF-P cascade, SB431542, suppresses exopinacoderm formation on the wound surface. Thus, by a

combination of molecular biological methods, the authors convincingly show the involvement of TGF-P in C. reniformis regeneration.

Another type of regeneration, development of the whole body from cell aggregates after sponge dissociation, was studied using a transcriptome approach in S. ciliatum [27]. Parallel analysis of the sets of transcripts at different stages of the process of cell aggregate development, and postlarval development (including metamorphosis), revealed the presence of a large number of common transcripts involved in both these processes. Thus, about 9 and 10 thousand genes are differentially expressed during regeneration and postlarval development, respectively; of these, about 50% of the transcripts are found in both processes. It was also shown that molecules participating in Wnt and TGF-P signaling cascades, transcription factors and regulators of apoptosis, ECM molecules and intercellular contacts are differentially expressed during development from the cell aggregate.

Also on S. ciliatum, for which the patterning of the definitive body axes by Wnt and TGF-P has been described, molecular events during development from the body fragment have been described [130]. In particular, for one of the TGF-P ligands, SciTGFbU, a sharp increase in the expression level (by 2 orders of magnitude) was shown as early as 3 hours after wounding, with a subsequent decrease in the number of transcripts up to 24 hours. The expression of another component of this signaling pathway, the secondary messenger SciSmadRa, is appreciably activated only 3 hours after wounding; before and after this period, the expression is observed at the basic level. Using in situ hybridization, it was shown that both genes (SciTGFbU and SciSmadRa) are expressed at the beginning of regeneration in an extensive zone adjacent to the wound surface, with its subsequent narrowing in the course of regenerative membrane formation. SciTGFbU expression is confined to exo- and endopinocytes, while SciSmadRa is expressed broadly and is not tissue-specific. Global evaluation of GO transcript sets by comparing RNA libraries at 3, 6, 12, and 24 hours of regeneration showed that early (3 and 6 hours) transcripts involved in activation of transcription, signal transduction, actin filaments, chromatin organization, but not in proliferation, apoptosis, and DNA repair are enriched. These data, together with the molecular mechanism of S. ciliatum body axis patterning [146], indicate that at least one of the mechanisms ensuring this in the adult sponge (TGF-P) is involved in the formation of the regenerate axis organization, and this involvement is evident long before the start of such characteristic cellular level events for regeneration as proliferation and apoptosis.

Previously, we provided data on the role of stem cells in the regeneration of cnidarians and flatworms. For sponges, this question is still far from being resolved, because even assuming that the main population of stem cells is archaeocytes, one of the molecular markers for them has been described only in the demospongia Ephydatia fluviatilis. Due to the lability of cell

differentiation in sponges, the participation of many cell types in regeneration was considered as transdifferentiation. However, in the light of the appearance of molecular data suggesting the presence of at least two types of polypotent cells in E. fluviatilis - archaeocytes and choanocytes [91; 92], we can consider such phenomena as participation of specialized cells (for example, choanocytes) in wound epithelization as a functioning stem cell system.

Despite a long history of extensive studies of regeneration in sponges, most of them have been performed at the light-optical and ultrastructural levels. Data on molecular mechanisms have been obtained in a few models and do not form a complete picture that allows us to describe the diversity of reparative morphogenesis in Porifera. Nevertheless, general morphological patterns of regenerative processes have been identified. We performed morphological descriptions of regeneration after comparable homotypic damage in two different models, evaluated the contribution of proliferation to recovery, and analyzed the involvement of a conserved molecular mechanism involved in regeneration in many animals, the Wnt signaling cascade, in regeneration in these species.

Chapter2. Cellular mechanisms of regeneration in Halisarca dujardinii h Oscarella lobularis, representatives of different classes of sponges

The cellular mechanisms underlying reparative morphogenesis vary from animal to animal. Such differences are widespread at the level of phyla and classes. For a comparative analysis of regeneration events at the cellular level, we described at the ultrastructural level the regeneration of representatives of two different sponge classes, Demospongiae (Halisarca dujardinii) and Homoscleromorpha (Oscarella lobularis), and evaluated the proliferative activity during regeneration by the incorporation of labeled DNA precursor.

2.1. Regeneration in Halisarca dujardinii

The Boreal-Arctic marine sponge Halisarca dujardinii lives mostly on sublittoral rocks and algae, and reaches 10 cm in diameter and 15 mm in thickness. The sponge has no organic or inorganic skeleton, is soft, smooth in shape. The body of the sponge consists of a peripheral ectosome and an internal choanosome. The ectosome reaches 27 p,m in thickness, and consists of three layers: (1) an outer layer of T-shaped exopinacocytes covered by a mucous cuticle, (2) a layer of collagen fibers with sparse spherular cells, and (3) an inner layer of dense collagen fibrils with exopinacocyte bodies embedded in it. The choanosome makes up the majority of the sponge body, and consists of choanocyte chambers, aquiferous canals, and the mesohyl. The mesohyl is a ECM with populations of resident cells: archaeocytes, lophocytes, spherular cells, granular, microgranular and vacuolar cells (Fig. 1, [147]).

Figure 1. The external view and morphology of the intact sponge Halisarca dujardinii. (A) H. dujardinii in nature. (B) Histological structure of the sponge. Different cell types on transmission electron microscopy (TEM): choanocyte chamber (C), exopinacocyte (D); endopinacocyte (E); archaeocyte (F); granular cell (G); spherular cell (H). Scales: A, 10 mm; B, 5 mm; C, 10 p.m; D, 2 p.m; E, 4 p.m; F, 1 p.m; G, H, 2 ^m. Notes: cc, choanocyte chamber; ch, choanocytes; ec, exhalant canal; enp, endopinacocyte; ep, exopinacoderm; exp, exopinacocyte; f, flagellum; g, granules; m, mesohyl; n, nucleus. From [147].

Microsurgical removal of the ectomosal region was chosen as a regeneration model (Fig. 2). The surgical site was chosen at a distance from the osculum or the border with the

basopinacoderm. The observed regeneration process in H. dujardinii was subdivided into three stages: I, formation of the "regenerative plug" (from the time of injury and up to 12 h), II, wound healing and formation of the "blastema" (from 12 to 36 h after injury ), and III, ectosome and choanosome recovery (36-72 h after injury ).

Figure 2. Schematic of the histological organization of H. dujardinii. The dotted line shows the area

removed during injury. From [147].

Immediately after surgery, the wound surface shrinks, causing the edges of the wound to protrude above it. During the operation, the peripheral part of the choanosome is inevitably damaged, but its underlying regions retain the structure and organization of the aquiferous system. The wound surface is covered with cellular debris, and numerous amoeboid-shaped cells can be found among the fibers of the ECM in the wound area. The choanocyte chambers and canals of the aquiferous system near the wound area lose organization: cells of these structures lose contact with their neighbors, and change their shape from trapezoidal (choanocytes) and flat (endopinocytes) to spherical or ameboid. These changes mark the beginning of dedifferentiation accompanying cell migration to the mesohyl. The fate of de- and transdifferentiated cells can be traced due to the presence of characteristic organelles in them. A natural marker of dedifferentiated choanocytes is the basal flagellar apparatus (basal corpuscle and accessory centriole) remaining in the cell. The collar of microvilli is reduced and disappears, and the

flagellum undergoes resorbtion, although some dedifferentiated choanocytes retain flagella up to the stages of differentiation into exopinacocytes. Endopinacocytes, flat in the intact state, take a spherical or amoeboid shape, and can be identified by a small nucleus devoid of a nucleus.

Stage I: formation of a "regenerative plug". During the first 6 hours after the injury, a "regenerative plug" forms in the wound area. It consists of mucus, bacteria, cell debris and individual mesohyl cells (Fig. 3, A-D). Vacuolar cells, phagocytes, and archaeocytes can also be found in the wound area. All cells except spherular ones have phagosomes, which indicates their participation in active phagocytosis of cellular debris (Fig. 3, E-G).

Figure 3. Halisarca dujardinii 6 hours after surgery. (A) Wound surface (marked with a dotted line) on SEM with debris on the surface, and the marginal zone surrounding the wound with ECM and

exopinacocytes. (B) Intact exopinacoderm, marginal zone, and wound on SEM. (C) Semi-thin section of the injured ectosome (1) and adjacent region of the choanosome (2). (D) Outer wound area with debris and dense ECM. (E) A dedifferentiated choanocyte in the wound area on the TEM. (F) Mesohyl amebocyte filled with phagosomes on TEM. (G) Endopinacocyte from the wound area filled with phagosomes on TEM. Scales: A, B, 30 p.m; C, 25 p.m; D, G, 2 p.m. de, debris; ECM, extracellular matrix; iex, intact exopinacoderm; mv, microvilli; mz, marginal zone; n, nucleus, pd, pseudopodia; ph,

phagosome. From [147].

Twelve hours after the injury, the wound surface appears completely covered with mucus and ECM, with minimal inclusion of debris on the surface (Fig. 4, A, B). The underlying region of the choanosome loses its organization (Fig. 4, C-F). The intact exopinacocytes surrounding the wound change the shape of their cytoplasmic outgrowths, which leads to separation of the outer layer of the exopinacoderm (Fig. 4, G). Reorganization of the ectosome begins with restoration of the middle layer of collagen fibers stacked in strands.

Figure 4. Halisarca dujardinii 12 hours after surgery. (A) Semi-thin section through the damaged section

of ectosome and adjacent region of choanosome. Insertion: middle layer of ectosome containing dense bundles of collagen fibers. (B) Wound surface covered with ECM on SEM. (C) Semi-thin section through the damaged choanosome. (D) Dedifferentiated cells of the damaged choanosome on TEM. (E)

Dedifferentiating endopinocytes in the damaged choanosome on TEM. (F) Dedifferentiated choanocytes with phagosomes retaining the basal body and accessory centriole on TEM (indicated by arrowhead). (G) Intact exopinacocytes surrounding the wound on SEM. ch, choanocytes; cho, choanosome; de, debris; ect, ectosome; ECM, extracellular matrix; enp, endopinocyte; exp, exopinacocyte; n, nucleus; ph, phagosomes. Scales: A-50 p,m, B-10 p,m; C-25 p,m; D-15 p,m; E-4 p,m; F-2 p,m; G-10 prn. From [147].

Stage II: wound healing and blastema formation. Twenty-four hours after the injury, the forming ectosome is a dense layer of collagen fibers (Fig. 5, A). Under the wound surface, dedifferentiated cells of different origin accumulate, including choanocytes and archaeocytes, multipotent cells of demosponges. This cluster of cells clearly resembles the blastema formed during regeneration in other animals (Fig. 5, B). The similarity is reinforced by the fact that the exopinacoderm and aquatic system are formed from these "blastema" cells.

Figure 5. Halisarca dujardinii 24 hours after surgery. (A) Regenerating ectosome with newly differentiating exopinacocytes on TEM. (B) Semi-thin section through the regenerating ectosome and the

adjacent region of the choanosome showing a cluster of differentiated cells (blastema). (C) Semi-thin section through the regenerating ectosome with newly degenerating (D) Wound surface with newly differentiating exopinacocytes and collagen fibers of the ectosome (co) on SEM. (E), (F) Different types

of differentiation of exopinacocytes from choanocytes (E) with characteristic nucleus shape, and archaeocytes (F), on TEM. (G) Semi-thin slice through the damaged choanosome with structures of the regenerating aquiferous system. (H) Dedifferentiated choanosome cells containing phagosomes on TEM. Scales: A, 5 pm; B, 50 pm; C, 15 pm; D, 15 pm; E, 10 pm; F, 2 pm; G, 100 pm; H, 5 p.m. bl, blastema; cc, choanocyte chamber; co, ectosome collagen layer; n, nucleus; nex, new exopinacocytes; ph,

phagosome. From [147].

Exopinacocytes differentiate individually, starting with cell migration from the blastema to the wound surface (Fig. 5, C, D). Upon reaching the wound surface, the cell flattens and occupies a position parallel to it. Ultrastructural markers show that the population of migrating cells includes archaeocytes, dedifferentiated choanocytes, endo- and exopinacocytes, and some mesohyl cells (Fig. 5, E, F). Then these cells begin to transform into T-shaped exopinacocytes characteristic of adult sponges: most of the cytoplasm together with the nucleus sinks into the ectosome as a cell outgrowth. Only a thin cytoplasmic bridge remains between the apical "hat" forming the sponge cover and the submerged part of the cell. The apical parts of cells take the form of a hexagonal plate.

The part of the choanosome underlying the wound remains anarchic at this time, showing clusters of individual cells instead of the characteristic epithelial structures of the aquatic system. These cells - mostly dedifferentiated choanocytes - contain numerous phagosomes (Fig. 5, G, H).

Stage III: recovery of the ectosome and choanosome. After 48 hours of regeneration, numerous newly differentiated exopinacocytes are present on the wound surface, but their continuous layer, the exopinacoderm, has not yet formed (Fig. 6, A). Numerous microvilli are formed on the apical surface of young exopinacocytes, indicating that these cells are mobile (Fig. 6, B). The bodies of these cells are lowered into the middle layer of the ectosome (Fig. 6, C, D).

During regeneration, choanosomes, aquiferous canals, and choanocyte chambers are formed from clusters of previously disaggregated endopinocytes and choanocytes, respectively (Fig. 6, D, E). The cells contact each other through interdigitations (Fig. 6, F). Individual choanocytes form groups of cells that resemble choanocyte chambers but are less compact (Fig. 6, G, H). Individual mesohyl cells still contain phagosomes.

Figure 6. Halisarca dujardinii 48 hours after surgery. (A) Intact and newly forming exopinacoderm on SEM. (B) Newly forming exopinacoderm, showing on SEM numerous small microvilli on the apical surface (shown with the arrowhead). (C) The body of the newly differentiating exopinacocyte on TEM. (D) A semi-thin section of the regenerating ectosome with the cortical layer and inhalant canals of the

aquiferous system. (E) Semi-thin section of regenerating ectosome and choanosome. (F) Contacts between choanocytes of the newly forming choanocyte chamber as interdigitations in the basal part of the

cells (shown by arrowheads); (G) The newly forming choanocyte chamber on TEM. (H) Intact choanocyte chamber of the same sponge on TEM. Scales: A, 10 ^m; B, C, 5 ^m; D, 20 ^m; E, 100 ^m; F, 5 p.m; G, H, 10 p.m. c, cortical layer; cc, choanocyte chamber; ch, choanocyte; exp, exopinacocyte; ic, bringing canal; iex, intact exopinacocyte; nex, new exopinacocyte. From [147].

Wound healing occurs 72 hours after surgery. By this time, the exopinacoderm of the wound surface is identical to the intact one (Fig. 7, A, B). The external hexagonal "hats" of exopinacocytes contact each other, forming a continuous exopinacoderm. Their surface is covered by a layer of glycocalyx. Mesohyl cells, choanocytes, and endopinacocytes lack phagosomes and have an ultrastructure typical of an intact sponge (Fig. 7, D).

Figure 7. Halisarca dujardinii 72 hours after surgery. (A) New exopinacoderm on SEM. (B)

Regenerating ectosome on TEM. (C) Semi-thin section through regenerating ectosome and choanosome. (D) New choanocyte chamber on the TEM. Scales: A, 20 ^m; B, 20 ^m; C, 50 p.m; D, 5 p.m. c, cortex; cc, choanocyte chamber; ch, choanocyte; cho, choanosome; ect, ectosome; exp, surface of the new

exopinacocyte. From [147].

Cell proliferation during regeneration. The cells of intact sponges actively incorporate 5-ethynyl-2'-deoxyuridine (EdU), an exogenous DNA precursor incorporated by DNA polymerase during replication. After incubation of sponges in seawater with the addition of EdU for 6 h, many cells, predominantly choanocytes, are labeled (Fig. 8, A-D). The surgery directly accompanied by labeling showed that many of the choanocytes migrating to the wound surface 6 hours after wounding demonstrated the presence of labeling in the nucleus (Fig. 8, C, E, F). However, 12 hours after surgery, no labeled cells were detected near the wound surface, although many choanocytes in the deep layers of the choanosome demonstrated active DNA synthesis (Fig. 8, G). This indicates that choanocytes migrating to the wound surface to differentiate into exopinacocytes stop DNA synthesis, whereas choanocytes in deep-lying choanocyte chambers (including disorganized ones) continue replication and - probably -division.

During reorganization of the aquiferus system, between 24 and 48 hours after surgery, labeled choanocytes and mesohyl cells were randomly distributed in the wound area (Fig. 8, H). Thus, no local activation of cell divisions in the wound area or in the deeper layers of the mesohyl was observed at this stage.

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Figure 8. DNA synthesis in intact H. dujardinii and during regeneration. (A) DNA synthesis in choanocytes of intact sponges after 6 hours of incubation in EdU. (B) Negative control for sample A incubated for 6 hours without EdU. (C) Inclusion of EdU in nuclear DNA and cell cytoplasm after 24

hours of labeling. (D) Negative control for sample A incubated for 24 hours without EdU. (E), (F) Wound surface after 24 hours of regeneration. (F) Fluorescence only in the EdU label spectrum. (G) Transverse section of the wound surface after 24 hours of regeneration. (H) Sagittal section of the wound surface at 24 hours of regeneration at the peripheral level. The wound boundary is shown as a dotted line. Blue is tubulin, red is DNA, green is EdU. cc, choanocyte chamber. Arrowheads show labeled nuclei, arrows show label in cytoplasmic granules of unknown nature. Scales: A-D, 10 pm; E, F, 20 pm; G, H, 30 pm.

From [147].

We have shown that regeneration in H. dujardinii proceeds through a series of stages, as previously described in other demosponges [131; 132; 148-150]: (1) wound closure and disintegration of structures adjacent to the wound; (2) formation of a mass of undifferentiated cells, the blastema; (3) epithelization of the wound surface; (4) reorganization of internal damaged structures.

The starting point of regeneration in multicellular animals is wound healing, which in one way or another involves the migration of cells to the site of damage [1; 151]. It was previously reported that partial wound epithelialization is observed in H. dujardinii after 24 hours of regeneration and is completely completed by 3 days [137; 138]. It was supposed that epithelialization of the wound surface in this species occurs due to gradual stretching of the intact exopinacoderm. However, we showed that epithelialization occurs due to inclusion of dedifferentiated mesohyl and choanocyte cells, while the peripheral exopinacoderm remains intact. The exopinacocytes covering the wound surface remain mobile up to 2 days of regeneration, as evidenced by microvilli on their surface.

Blastema in regenerative biology is a temporary specialized structure formed after an amputation or wound and consisting of undifferentiated cells capable of forming lost structures through subsequent cell differentiation [1]. During limb regeneration in vertebrates, the blastema is formed from the undifferentiated cells of the remaining tissues, which then proliferate and then differentiate into the original or other cell types [12; 151]. We call the blastema in H. dujardinii a mass of undifferentiated cells located under the wound surface. This temporary structure contains undifferentiated cells such as choanocytes, endo- and exopinacocytes, and multipotent cells - archaeocytes. All of these cell types migrate from the blastema to the wound surface to differentiate into a new epithelial structure, the exopinacoderm. Subsequently, the blastema cells also take part in the formation of the aquiferous system. Similar such blastema structures have also been described in other demosponges, such as Halichondria panicea [131], Hippospongia communis [148], Polymastia mamillaris [150] and Aplysina cavernicola [152].

We showed that the population of cells most actively synthesizing DNA in H. dujardinii are choanocytes. In addition to these, single EdU-positive cells, presumably archaeocytes, are observed in the mesohyl. These data coincide with the descriptions obtained earlier in other demosponges. For example, when studying the formation of choanocyte chambers in the freshwater sponge Ephydatia fluviatilis, it was shown that choanocytes rapidly divide [153]. In many tropical sponges, active proliferation and shedding of choanocytes maintains the cellular stability of the aquatic system [142; 143]. However, we did not observe an increase in blastema cell proliferation in H. dujardinii regeneration. Moreover, undifferentiated cells migrating to the wound surface in order to become exopinacocytes stop or slow down DNA synthesis, while choanocytes in the underlying tissues continue to actively divide.

Cell sources in regeneration remain one of the main issues of regenerative biology [14; 154; 155]. New cells can appear by different pathways, including (1) proliferation of resident stem cells [156], (2) division of terminally differentiated cells [154], (3) dedifferentiation of cells, which can act as precursors of other cell types, and transdifferentiation [15; 157]. Different pathways of cell source formation vary not only between different animal taxa, but can also occur in different tissues of the same organism [154].

Archaeocytes are considered to be toti- or multipotent cells within the Demospongiae class [158-160]. Archaeocytes have been shown to be among the most active participants of exopinacoderm regeneration mainly on the light-optical level [131; 132; 137; 148; 161]. However, the possibility of differentiation of archaeocytes into choanocytes during regeneration has been reported much less frequently [162; 163]. Nevertheless, based on the expression of EfPiwiA in archaeocytes and choanocytes, it was hypothesized that both archaeocytes and choanocytes are part of the demospongiosis stem cell system [92; 160]. Under certain conditions, choanocytes are capable of transforming into archaeocytes, i.e., even when differentiated, choanocytes retain the potencies characteristic of stem cells [92].

We have shown that in H. dujardinii there are three sources of new exopinacoderm in regeneration: choanocytes, archaeocytes, and endopinacocytes. Choanocytes are the main source of cellular material in regeneration. While archaeocytes undergo direct differentiation into a new cell type, choanocytes and pinacocytes go through a process of dedifferentiation in the blastema (Fig. 9, [147]).

Figure 9. Schematic of the regeneration process of H. dujardinii and cell sources of new choanocytes and exopinacocytes. (A) Intact sponge. (B) Stage I of regeneration: formation of a "regenerative plug". (C)

Stage II of regeneration: wound healing and formation of "blastema". (D) III stage of regeneration: restoration of ectosome and choanosome organization. Gray - exopinacocytes, blue - choanocytes, red -

archaeocytes. From [147].

Two main types, epithelial and mesenchymal, are distinguished among morphogenesis in multicellular organisms. In the first case, there is a movement of cell layers, in the second - of individual cells [164]. Transitions between epithelial and mesenchymal tissue states are called epithelial-mesenchymal (EMT) and mesenchymal-epithelial transitions (MET; [165; 166]). Both processes have been well studied in a number of model animals, and are involved in embryonic development, asexual reproduction and regeneration [165-169]. EMT and MET have a place in H. dujardinii regeneration as well as larval metamorphosis [170]. The epithelial-mesenchymal transition is the main mechanism that ensures blastema formation at the early stages of regeneration. Epithelial cells (choanocytes and endopinacocytes) from the damaged and nearby areas of the aquiferous system acquire a mesenchymal phenotype and migrate to the mesohyl. After the formation of the blastema, the reverse process, MET, occurs when the blastema cells re-form the epithelial structures of the ecto- and choanosoma.