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Imágenes de embriones reales: ¿Cómo se conocen las diferentes zonas (organizador de velocidad, zonas marginales…)?

Imágenes de embriones reales: ¿Cómo se conocen las diferentes zonas (organizador de velocidad, zonas marginales…)?


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En muchos libros de texto, se dibujan figuras de embriones, pero en realidad, ¿cómo sabe el biólogo cuál es la zona de un embrión en estado de gastrulación? Excepto el labio dorsal aquí, no puedo localizar otra cosa.


Arkadia mejora la señalización relacionada con los nodos para inducir el mesendodermo

Los miembros relacionados con los nodos de la familia del factor de crecimiento transformante (TGF) -β regulan la inducción de mesodermo, endodermo y mesendodermo, un tejido específico del organizador de Spemann 1,2,3,4,5,6,7. No se comprende completamente cómo se forman estos diferentes tejidos en respuesta a las mismas moléculas de señalización. Se ha sugerido que los efectos dependientes de la concentración, mediados por cofactores y antagonistas extracelulares, son responsables de las diferencias 1,8,9,10. Aquí mostramos que la proteína nuclear Arkadia potencia específicamente la actividad inductora del mesendodermo de un subconjunto de miembros de la familia TGF-β. Las actividades combinadas de Arkadia y Xenopus nodal-related-1 son suficientes para inducir el mesendodermo y suprimir el mesodermo. Arkadia dorsaliza los tejidos ventrales, lo que da como resultado la inducción de la expresión génica específica del organizador. El bloqueo de la señalización nodal inhibe extracelularmente estos efectos. Arkadia influye en la actividad nodal cuando se coexpresa y puede funcionar en células adyacentes a las que producen la señal nodal. Nuestros hallazgos, junto con la observación de que los ratones mutantes Arkadia carecen de un nodo y un mesendodermo derivado de nodos, identifican a Arkadia como un modulador esencial de la cascada de señalización nodal que conduce a la inducción del organizador de Spemann.


Abstracto

Este año, 2019, marca el centenario del descubrimiento del embriólogo E. E. Just de lo que se conoce como el bloqueo rápido de la poliespermia. La observación de Just de los cambios sutiles que ocurren en la superficie del huevo durante la fertilización (y en la partenogénesis experimental) lo llevó a postular que el huevo, y de hecho cada célula, posee una propiedad que él llamó irritabilidad independiente, que representa la capacidad de la célula para responder de una manera fisiológicamente relevante a una variedad de señales o desencadenantes. En este artículo, sostengo que el concepto de irritabilidad independiente de Just informó a su contemporáneo Johannes Holtfreter cuando Holtfreter intentó explicar los fenómenos de inducción y competencia embrionarias y que Holtfreter, a su vez, influyó en Marc Kirschner y John Gerhart en su formulación de la teoría de la capacidad facilitada. variación. La influencia de Just es especialmente evidente en las presentaciones de Gerhart y Kirschner de lo que ellos llaman enlace débil—Una propiedad de los sistemas vivos que permite que los procesos y componentes centrales se mezclen y combinen de diferentes maneras para generar rasgos novedosos. Desafortunadamente, la conexión entre el trabajo de Holtfreter y el de Just ha permanecido oculta. Este artículo da ejemplos de fenómenos que exhiben un vínculo débil y expone el caso de que el concepto de irritabilidad independiente de Just, a través de Holtfreter, Gerhart y Kirschner, se ha infiltrado ampliamente en la biología celular y del desarrollo moderna.

“Sin embargo, los recientes grandes avances en [nuestra comprensión de la importancia de la superficie celular] requerirán que volvamos al libro de Just [La biología de la superficie celular] para reevaluar su relevancia contemporánea. Quizás, solo quizás, podría ser otro Mendel o Miescher, haciendo un trabajo fundamental que no fue apreciado durante décadas después de su muerte ”(Glass, 1984).


MATERIALES Y MÉTODOS

Ovocitos y embriones

Los ovocitos se desfolicularon y cultivaron manualmente como se describió anteriormente (Kofron et al., 1999). Los ovocitos fueron inyectados con oligos en medio de cultivo de ovocitos (OCM) usando dos inyecciones ecuatoriales por ovocito para oligos XTcf3, o una inyección vegetal para oligos VegT y Axin, cultivados a 18 ° C y fertilizados usando la técnica de transferencia de hospedador como se describió anteriormente (Zuck et al., 1998). Los experimentos de rescate se llevaron a cabo como se describe en el texto inyectando ARNm ecuatorialmente en ovocitos, 24 horas después del oligo (Fig. 2F), o inyectando en embriones en estadio de 4 células (Fig. 2E). Los huevos se separaron y fertilizaron utilizando una suspensión de esperma y los embriones se mantuvieron en 0,2 x MMR. Para las inyecciones de ARNm después de la fertilización (Fig. 2E), los embriones se desjelizaron y se transfirieron a Ficoll al 2% en 0,5 x MMR en la etapa de 1 célula. Los ARNm se diluyeron con agua destilada estéril y se inyectaron en blastómeros.

Para los ensayos de tapones de animales, los embriones de blástula media se colocaron en placas de agarosa al 2% en 1 × MMR y los tapones de animales se disecaron con unas pinzas afiladas. A continuación, las tapas se cultivaron en OCM hasta que los embriones hermanos alcanzaron la etapa de gástrula media o tardía. Para los explantes ecuatoriales, los embriones se colocaron en placas de agarosa al 2% en la etapa de blástula media y las regiones ecuatoriales se diseccionaron con agujas de tungsteno (Xanthos et al., 2001) y se cultivaron en OCM hasta que los embriones hermanos alcanzaron la etapa de neurula media. Para la separación en mitades dorsal y ventral en la etapa de gástrula, se marcó el lado dorsal de los embriones en la etapa de cuatro células utilizando cristales de azul Nilo. El eje dorsoventral fue reconocido en la etapa de cuatro células por las diferencias de pigmentación de los lados dorsal y ventral. Cuando los embriones de tipo salvaje alcanzaron la etapa 10, todos los lotes se colocaron en placas de agarosa al 2% en 1 × MMR pH 7,6 y se dividieron en mitades dorsal y ventral, y se congelaron en grupos de 4 semiembriones a intervalos de 2 horas a través de la gástrula. etapas.

Oligos y ARNm

Los oligodesoxinucleótidos antisentido utilizados fueron oligonucleótidos quiméricos de fosforotioato-fosfodiéster purificados por HPLC (Sigma / Genosys) con la composición de base:

XTcf3 T1: 5′-C * G * A * G * GGATCCCAGTC * T * T * G * G-3 ′.

XTcf3 T2: 5′-G * A * G * ATAACTCTGA * T * G * G-3 ′.

Los oligos de XTcf3 son completamente complementarios a las cuatro variantes de XTcf3. Los asteriscos (*) representan enlaces fosforotioato. Los oligonucleótidos se resuspendieron en agua filtrada esterilizada y se inyectaron en las dosis descritas en el texto. Longitud total XTcf3 en el vector pGlomyc se linealizó con XbaYo y coronado XTcf3 El ARNm se sintetizó usando el kit T7 mMessage mMachine (Ambion). Los ARN se extrajeron con fenol, se precipitaron con etanol y luego se resuspendieron en agua destilada estéril para inyección.

Análisis de la expresión génica mediante RT-PCR en tiempo real

Se preparó ARN total a partir de ovocitos, embriones y explantes usando proteinasa K y luego se trató con ADNasa libre de ARNasa como se describió previamente (Zhang et al., 1998). Se usó aproximadamente un sexto equivalente de embrión de ARN para la síntesis de ADNc con cebadores oligo (dT) seguido de RT-PCR en tiempo real y cuantificación usando el sistema LightCycler ™ (Roche) como lo describen Kofron et al. (Kofron et al., 2001). Los cebadores y las condiciones de ciclo usados ​​se enumeran en la Tabla 1. Los valores de expresión relativa se calcularon por comparación con una curva estándar generada por dilución en serie de ADNc de control no inyectado. Las muestras se normalizaron a niveles de ornitina descarboxilasa (ODC), que se utilizó como control de carga. Las muestras de agua sola o los controles que carecen de transcriptasa inversa en la reacción de síntesis de cDNA no dieron productos específicos en todos los casos.


GRADIENTES PARA LOS EJES DEL CUERPO PRIMARIO EN VERTEBRADOS

Hay dos ejes corporales primarios, anteroposterior (AP) y dorsoventral (DV). Sin embargo, normalmente se supone que existe un solo organizador: el organizador de tipo Spemann. ¿Cómo puedo dos Los sistemas ortogonales de información posicional surgen bajo la influencia de un soltero ¿organizador? ¿Existe un segundo organizador que hasta ahora se haya pasado por alto? Hay buenos argumentos de que el organizador del patrón AP no es el organizador de Spemann sino toda la zona marginal (Meinhardt 2006). En la gástrula temprana, Wnt se produce en la zona marginal a excepción de la región organizadora (Christian y Moon 1993). Wnt proporciona información posicional para la separación en cerebro anterior y mesencéfalo (Kiecker y Niehrs 2001 Nordström et al. 2002 Dorsky et al. 2003). Este sistema de formación de patrones es evolutivamente muy antiguo. Una comparación de las expresiones génicas en la hidra y la gástrula de vertebrados temprana muestra una correspondencia sorprendente, lo que sugiere que el patrón del cerebro y el corazón de los vertebrados evolucionó a partir de un sistema que alguna vez fue responsable del patrón del cuerpo de un ancestro similar a una hidra (Fig. 2B). ) (Meinhardt 2002). En este punto de vista, el organizador de hidra y el blastoporo de vertebrado, es decir, la zona marginal, el anillo germinal, etc., son estructuras homólogas que son responsables del patrón AP.

En contraste con el organizador de hidra, el blastoporo vertebrado evolucionó a un anillo enorme con el organizador de Spemann formando un pequeño parche en este anillo. Se asume entonces que el organizador de Spemann modela el eje DV, pero lo hace indirectamente dando lugar a la línea media dorsal, la notocorda y la placa del piso, una "línea alta" y no un "punto alto" para el patrón DV. Ambas regiones organizadoras, el blastoporo para el AP y la línea media para el eje DV, forman un sistema de coordenadas casi cartesiano que permite un patrón combinatorio a lo largo de ambos ejes (Fig. 2C). La generación de una única “línea alta” larga y extendida para el patrón DV es un proceso sutil de formación de patrón. La solución para vertebrados no es la única. En los insectos, por ejemplo, un organizador dorsal ejerce un reprimiendo influencia, lo que hace que la línea media aparezca en el lado ventral opuesto (Meinhardt 2004, 2008), muy en contraste con los vertebrados en los que el organizador inicia y alarga la línea media dorsalmente. Este modelo proporciona una lógica para la ubicación dorsal o ventral del sistema nervioso central en vertebrados e insectos, respectivamente.


Modelo de escala dependiente de Chordin / Sizzled

Recientemente informamos sobre el mecanismo molecular de la escala en bisectados Xenopus embriones (Inomata et al. 2013). La mitad dorsal del embrión debe formar un gradiente de Chordin adecuado de acuerdo con el tamaño del embrión mediante la regulación de tres factores, síntesis-difusión-degradación. Sin embargo, si los tres factores cambian dinámicamente su valor, será difícil analizar el sistema de escala. Para eliminar esta complejidad, creamos artificialmente células fuente, que sintetizaban una cantidad constante de proteína Chordin sin verse afectadas por el tamaño del embrión o la actividad de las BMP (ensayo de reconstitución del eje D – V). Primero, los embriones fueron inyectados con β-catenina-MO y acordeón / noggin-MO (βCN-MO) para eliminar las células fuente (organizador) y la expresión de factores dorsalizantes endógenos, respectivamente (Fig.4) (Heasman et al. 2000 Oelgeschlager et al. 2003a). A continuación, utilizando estos embriones completamente ventralizados que carecían de gradiente, inyectamos localmente acorde ARNm para crear exógenamente un gradiente de Chordin en el embrión. Esto resultó en la formación de tres regiones distintas, las regiones dorsal (D), lateral (L) y ventral (V), como en el embrión de control. Cuando la tasa de producción de proteína Chordin se incrementó inyectando cuatro veces más acorde ARNm, los embriones demostraron un cambio moderado en la formación del eje D – V. Por lo tanto, la forma del gradiente de Chordin pareció estar regulada principalmente por degradación.

A partir de estos resultados, nos centramos en la asociación entre Chordin y Sizzled, que controla la estabilidad de la proteína Chordin (Fig. 2B). Para examinar la velocidad de difusión, realizamos la recuperación de la fluorescencia después del ensayo de fotoblanqueo (FRAP) o de espectroscopia de correlación de fluorescencia (FCS) y encontramos que Chordin y Sizzled, así como la forma secretora de mEGFP, se difundieron rápidamente. Por el contrario, la tasa de degradación de cada proteína fue claramente diferente. La proteína Sizzled se mantuvo estable en el embrión, mientras que la proteína Chordin (26,7 fmol por embrión) se degradó muy rápidamente con una vida media de aproximadamente 30 min. Esta inestabilidad de la proteína Chordin se bloqueó completamente por la cantidad en exceso de Sizzled, lo que indica que la mayor parte de la degradación de Chordin dependía de las metaloproteasas BMP1 y Xlr. Además, el gradiente de Chordin cambió dinámicamente su forma de empinado a poco profundo dependiendo de la concentración de Sizzled, incluso cuando la tasa de producción de Chordin se fijó mediante el ensayo de reconstitución del eje D – V. Estos resultados indicaron que la forma del gradiente de Chordin estaba regulada principalmente por la degradación, cuya velocidad dependía de la cantidad de proteína Sizzled.

Con base en la observación experimental, propusimos el modelo de escala dependiente de Chordin / Sizzled mencionado a continuación. Antes de la gastrulación, bmps y su gen objetivo chisporroteó se expresaron en todo el embrión (Fig. 5A). Sin embargo, cuando el organizador (células fuente) se formó localmente en el embrión en la etapa temprana de la gástrula, la Chordina sintetizada se difundió en el espacio extracelular y suprimió gradualmente la chisporroteó área de expresión inhibiendo la actividad de las BMP (Fig. 5A). Durante este proceso, la proteína Sizzled se acumuló gradualmente en el embrión debido a su baja tasa de degradación (Fig. 5B). Teniendo en cuenta la mitad dorsal del embrión, el chisporroteó El área de expresión fue rápidamente suprimida por la difusión de Chordin (Fig. 5A inferior). La acumulación de Sizzled se volvió más baja que en el embrión de control (Fig. 5B inferior). En este medio embrión dorsal de bajo chisporroteo, la degradación de Chordin se incrementó y formó un gradiente pronunciado adecuado para el embrión pequeño (Fig. 5C).

En este modelo de escala, propusimos que el tamaño del embrión regula la estabilidad de la proteína Chordin a través de la acumulación de la proteína Sizzled. Para abordar esta posibilidad, la producción de Chordin se fijó mediante el ensayo de reconstitución del eje D – V y el tamaño del embrión se modificó artificialmente mediante bisección. A pesar de la producción fija, se detectó la reducción de la proteína Chordin en los embriones bisectados. De acuerdo con el modelo de escala dependiente de Chordin / Sizzled, este cambio en la estabilidad de la proteína Chordin se eliminó cuando Sizzled fue agotado por morfolino. Además, construimos un modelo matemático basado en los resultados experimentales: (i) tasa de difusión idéntica de Chordin y Sizzled (ii) menor tasa de degradación de Sizzled que la de Chordin (iii) Regulación dependiente de Sizzled de la degradación de Chordin y (iv) supresión del área de expresión Sizzled por difusión Chordin. En este modelo matemático, confirmamos que tres regiones distintas, dorsal, lateral y ventral, podrían escalar al tamaño del embrión a través de la acumulación de Sizzled.


Discusión

los Xenopus El organizador de Spemann ha proporcionado un terreno de pesca fértil para el descubrimiento de proteínas secretadas que regulan el desarrollo. Se esperaba que pudieran aislarse nuevos factores de crecimiento, sin embargo, en cambio, se encontró que el organizador de Spemann media la inducción embrionaria a través de la secreción de una mezcla de antagonistas del factor de crecimiento (4, 5). En el presente estudio, utilizamos la secuenciación profunda para investigar la elección entre la epidermis y el tejido neural.

Un rico recurso transcriptómico.

El transcriptoma de las células del casquete animal que se habían disociado durante varias horas (provocando la neutralización), así como el de los explantes ectodérmicos microinyectados con una serie de ARNm que inducen tejido neural, como Acorde, Cerbero, y FGF8, se determinó mediante RNA-seq. También examinamos el efecto del inductor de endomesodermo. Xnr2, el inductor epidérmico BMP4, y el modificador de la competencia de inducción del mesodermo xWnt8 (32). Estos datos, que comprenden un mínimo de 45 × 109 nucleótidos secuenciados de ARN, se proporcionan en los conjuntos de datos S1-S3, que la comunidad de investigadores puede extraer fácilmente. Esto constituye un importante recurso abierto para los biólogos del desarrollo interesados ​​en la diferenciación de la capa germinal.

Aislamiento de un inhibidor de Wnt.

Buscando genes de inducción neuronal activados por la disociación celular (que causa la activación de MAPK) (19) y buscando Cerbero, Acorde, y xWnt8 ARNm, identificamos una proteína que designamos como Bighead debido a su fenotipo de sobreexpresión. Inesperadamente, esta molécula no se expresó en el ectodermo de la última etapa de gástrula 12 cuando se prepararon las bibliotecas de RNA-seq. En esta etapa, el ARNm de cabeza grande se expresa en el endodermo, particularmente en el organizador de Spemann dorsal. La expresión del organizador se encuentra en el endodermo profundo, pero no se superpone con el endodermo anterior del borde de ataque (que da lugar al intestino anterior y al hígado), que expresa Cerberus y Dkk1 (24, 41). A la luz del requisito de Bighead para el desarrollo de la cabeza, parece que los antagonistas de Wnt deben emanar también de las regiones del endodermo más posteriores del organizador para potenciar completamente sus propiedades inductoras de la cabeza.

Es poco probable que la disociación de los casquetes animales induzca endodermo, ya que el marcador pan-endodérmico Sox17 no se expresa (Conjunto de datos S1). Parece probable que la disociación de los casquetes animales conduzca a la activación prematura de los dominios neurales de expresión de Bighead, que, en el embrión no perturbado, se observan en etapas posteriores de la neurula. La identificación de Bighead fue una suerte, porque resultó ser una proteína interesante.

Ya que X. laevis es alotetraploide, Bighead está codificado por dos genes de las formas S y L (20). Ambos codifican proteínas de aproximadamente 270 aa con un péptido señal y son secretadas. En experimentos de sobreexpresión, Bighead causó fenotipos muy similares al antagonista arquetípico de Wnt Dkk1 (41). Cabeza grande El ARNm expandió la expresión de varios marcadores de cabeza, bloqueó la expresión de la En2 Gen diana Wnt, previno la formación del eje secundario después de una sola inyección de xWnt8 ARNm y disminución de la inducción de los primeros objetivos de Wnt Siamois y Xnr3. Además, la adición de la proteína Bighead inhibió la señalización de Wnt canónica en los ensayos del gen indicador de luciferasa. Por lo tanto, Bighead se comporta como un antagonista canónico de señalización de Wnt, muchos de los cuales son conocidos por promover el desarrollo de la cabeza (48).

Las búsquedas exhaustivas de homólogos de Bighead en otros organismos mostraron que solo está presente en peces y anfibios. Por ejemplo, en el pez cebra, Bighead corresponde a LOC571755, una proteína de función desconocida. La proteína evolucionó rápidamente, pero sus seis cisteínas se conservaron en muchas especies. La predicción SWISS-MODEL sugiere que la región C-terminal de Bighead es compatible con la estructura cristalina del prodominio de TGF-βs como miostatina / GDF8 (38, 40) quizás parte de Bighead derivado de un dominio estructural en la prorregión de un TGF-β antiguo.

No se encontraron homólogos en reptiles, aves o mamíferos. La pérdida de genes es muy común durante la evolución. Por ejemplo, hemos descrito una antigua red autoorganizada de Chordin / BMP / Tolloid que regula el patrón D / V en vertebrados e invertebrados (49). Sin embargo, a pesar de esta profunda conservación, se perdieron algunos componentes de la red. La proteína morfogenética anti-dorsalizante (ADMP) es una BMP que se perdió en el ornitorrinco (Ornithorhynchus) (50). Las sFRPs Crescent y Sizzled están presentes en las aves y el ornitorrinco, pero no en los mamíferos superiores, que han perdido la yema de huevo. Además, los sFRP no están presentes en ningún invertebrado (51). Parece que el requisito embrionario para el nivel de regulación proporcionado por Bighead se perdió junto con la invención del amnios. A pesar de esto, nuestros estudios con el agotamiento de Bighead por OM demuestran un requisito notablemente fuerte para este gen en la formación de la cabeza durante el desarrollo de la rana.

¿Por qué tantos antagonistas de Wnt?

Bighead se suma a una gran lista de antagonistas de Wnt secretados. Estos incluyen las proteínas Dkk (48), sFRP, factor inhibidor de Wnt 1 (WIF-1) (52), SOST / Sclerostin (53), Notum (una hidrolasa que elimina el palmitoleoilato de Wnt en el espacio extracelular) (54), y Angptl4 (6). Además, proteínas transmembrana como Shisa (una proteína involucrada en el tráfico del receptor Frizzled a la superficie celular) (55), Tiki (una proteasa que escinde el término amino de Wnts) (56) y Znrf3 / RNF43 (una ubiquitina ligasa que se dirige a los receptores Frizzled y Lrp6 para la degradación lisosomal) (57, 58) regulan negativamente la señalización de Wnt.

Como se muestra en este estudio, Bighead se une a Lrp6, induciendo su rápida endocitosis en lisosomas. Como resultado, Lrp6 se elimina de la superficie de la célula y se degrada en endolisosomas. Este mecanismo molecular es muy similar al de los antagonistas de Wnt Dkk1 y Angptl4. Dkk1 se une a Lrp6 y Kremen1 / 2 y el complejo se internaliza. Angptl4 es una proteína secretada mejor conocida por su función como inhibidor de la lipoproteína lipasa (LPL), la enzima clave en la eliminación de triglicéridos del plasma sanguíneo (59). Estudios en Xenopus han demostrado que Angptl4 se une a los sindecanos de la superficie celular (que son proteoglicanos transmembrana) y que esta interacción desencadena la endocitosis de Lrp6 (6). En el caso de Bighead, no se sabe si se requiere un correceptor para la internalización de Lrp6. Sin embargo, lo que está claro es que estos tres antagonistas de Wnt conducen a la internalización de Lrp6 en una población endolisosómica que no participa en la generación de señales.

La existencia de tantos reguladores subraya la rica complejidad de la vía de señalización Wnt. Por lo general, pensamos en el Wnt canónico como una señal que simplemente aumenta los niveles de β-catenina nuclear para regular la transcripción por el factor de células T / factor de unión del potenciador linfoide (TCF / LEF). Sin embargo, Wnt tiene efectos adicionales. Por ejemplo, en la estabilización de proteínas dependiente de Wnt, cientos de proteínas celulares se estabilizan, lo que lleva a un aumento del tamaño de la célula (60, 61). Esto es causado por el secuestro de GSK3 dentro de endosomas tardíos / cuerpos multivesiculares (MVB) (62, 63), lo que disminuye la fosforilación de fosfodigrones en proteínas citosólicas que normalmente conducen a su degradación en proteasomas. Además de GSK3, otra enzima citosólica importante, la proteína arginina metiltransferasa 1 (PRMT1), se secuestra dentro de las MVB cuando los correceptores Wnt se endocitosan junto con su ligando Wnt (64). La reciente comprensión de que Wnt3a estimula en gran medida la endocitosis no mediada por receptores de BSA-DQ del medio extracelular (64) sugiere que Wnt es un importante regulador del tráfico de membranas. Proponemos que Lrp5 / 6 es un regulador importante no solo del tráfico de Wnts sino también de la absorción total de nutrientes y fluidos celulares. La endocitosis es una propiedad celular universal que podría ser regulada por Dkk1, Angptl4 y Bighead. Queda mucho por aprender sobre la fisiología de la notable vía de señalización Wnt (65, 66).


Agradecimientos

Agradecemos a F. Cong por proporcionar las construcciones ZNRF3 D. Koinuma por proporcionar las construcciones ALK C. Janda por proporcionar la construcción sustituta WNT R. Thomas para las células H1581. Agradecemos a G. Roth y Aska Pharmaceuticals Tokyo por proporcionar generosamente hCG. Agradecemos a NXR (RRID: SCR_013731), Xenbase (RRID: SCR_004337) y EXRC por Xenopus recursos. Agradecemos a Fabio da Silva por la lectura crítica del manuscrito. Se agradece el apoyo técnico experto de la instalación central de DKFZ para microscopía óptica y el laboratorio animal central de DKFZ. Este trabajo fue financiado por Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - SFB1324 - número de proyecto 331351713.


ENDOBLASTO EN HOZ (HIPOBLASTO SECUNDARIO)

El endoblasto falciforme forma una capa unicelular de gran extensión que prolifera desde el borde medial de la hoz de Rauber o endoblasto de unión en dirección centrípeta y craneal durante la incubación temprana (Callebaut y Van Nueten, 1994 Callebaut et al., 1999 Fig. 13). Principalmente contiene γ ooplasma.

El endoblasto falciforme pertenece al mismo linaje celular que la hoz de Rauber y tiene un comportamiento similar, pero está dominado por la hoz de Rauber

Si un fragmento de endoblasto de hoz de codorniz se coloca en la región anti-hoz de un blastodermo de pollo no incubado del que se ha raspado selectivamente la hoz de Rauber, entonces se desarrolla un embrión completo con un PS, endodermo definitivo, nódulo de Hensen y una placa neural en un dirección diametralmente opuesta, partiendo de la región antihoz (Callebaut et al., 2003a, b). Lo mismo ocurre cuando se coloca un fragmento de endoblasto falciforme de codorniz en la parte central aislada de un blastodermo de pollo no incubado (Callebaut et al., 2002c). El ooplasma subgerminal central colocado artificialmente en contacto con el material falciforme de Rauber o el endoblasto falciforme en cultivo, puede funcionar como sustrato para la proliferación celular con capacidades de inducción y / o regeneración nuevamente en la capa superior vecina (Callebaut et al., 2000c). Incluso la activación de la formación de embriones puede ocurrir por blastodiscos de codorniz no fertilizados (Callebaut et al., 2000d).

Cuando se coloca endoblasto de hoz de codorniz en la región aislada antihoz de un blastodermo de pollo no incubado en cultivo, se desarrolla una placa neural temprana. Por el contrario, cuando se coloca un trozo de endoblasto de hoz de codorniz en la región antihoz de un pollo entero sin incubar en cultivo, no tiene ningún efecto inductor. Este hallazgo indica que la hoz de Rauber domina o inhibe el endoblasto falciforme colocado ectópicamente, que se deriva del mismo linaje celular. Este endoblasto falciforme, si se retira de la influencia de la hoz de Rauber, tiene potencias inductoras de gastrulación y / o neurulación en la capa superior del blastodermo no incubado, pero no influye en la formación de islas de sangre. El gen homeobox cHex se expresa en el endoblasto falciforme y falciforme de Rauber (Yatskievych et al., 1997). Las transcripciones de cHex también se detectaron en islas de sangre a partir de la etapa 4 (Hamburger y Hamilton, 1951) y en células endoteliales vasculares extraembrionarias e intraembrionarias. Debido a que hemos demostrado que la hoz de Rauber y el endoblasto de unión tienen un efecto inductor sobre la formación de islas de sangre, podemos postular una relación desconocida con el gen cHex.

Influencia del endoblasto falciforme en la neurulación y la gastrulación

La base molecular de la inducción neural se ha estudiado ampliamente en Xenopus laevis, y se encontró que estaba estrechamente acoplado al establecimiento del eje dorsoventral (De Robertis y Sasai, 1996 Hemmati-Brivanlou y Thomsen, 1995 Hemmati-Brivanlou y Melton, 1997). En las ranas, el ectodermo prospectivo es inducido por BMP. Por el contrario, un desarrollo neuronal requiere la inactivación de las BMP y se logra mediante la formación directa de complejos entre las BMP y los factores inductores de los nervios como la cordina, la noggin o la folistatina (Piccolo et al., 1996 Zimmermann et al., 1996). En el blastodermo de pollo en etapas tempranas, la epidermis prospectiva se caracteriza por la expresión del gen homeobox DLX5, que sigue siendo un marcador epidérmico durante la gastrulación y la neurulación y permite distinguirlo de la placa neural más central (Pera et al., 1999). ). Los últimos autores han demostrado que las señales verticales de la capa inferior son necesarias para el establecimiento de la placa neural mediante extirpaciones repetidas del endoblasto subyacente. En ausencia de las capas germinales inferiores, la epidermis se expandió hacia la región que normalmente forma la placa neural.

Knoetgen y col. (1999a, b) analizaron el GANF (Gallus pliegue neural anterior) - potencial inductor de varios tejidos en diferentes etapas durante el desarrollo del pollito mediante trasplante al margen exterior del área pelúcida, donde las células del epiblasto están destinadas a convertirse en epidermis (Spratt, 1952 Rosenquist, 1966 Schoenwolf y Sheard, 1990 Bortier y Vakaet, 1992 García-Martínez et al., 1993). Los trasplantes del nódulo de Hensen (HH3 + / HH4) en blastodermos completos llevaron a la inducción de una estructura neuroectodérmica con una fuerte expresión de GANF en su margen craneal. El injerto del proceso de cabeza joven (HH4) en el área craneal lateral pelúcida provocó un engrosamiento del epiblasto y una inducción de la expresión de GANF en las células yuxtapuestas.

Una molécula secretada llamada "Cerberus", que se expresa en el endodermo anterior, tiene la propiedad de inducir estructuras de cabeza ectópicas cuando se microinyecta en las regiones ventrales de Xenopus embriones (Bouwmeester et al., 1996 Bouwmeester, 1997). Pera y Kessel (1997) han descrito el patrón del ángulo del prosencéfalo del pollito por la placa precordal. Según estos autores también, la placa neural aviar es evidente antes de la entrada de las primeras células mesendodérmicas o mesodérmicas axiales, excluyendo la placa precordal y la notocorda como fuentes primarias de inducción neural. Durante la gastrulación temprana, las células invaginan a través de la punta de la línea de crecimiento y se diseminan radialmente para formar el endodermo definitivo (intestino) (Vakaet, 1970). Durante esta expansión radial, el último endodermo definitivo empuja el endoblasto falciforme también radialmente (Callebaut y Van Nueten, 1994 Fig. 13). El endoblasto falciforme hemicircular craneal se desliza por debajo de las células de la capa superior que se transforman en un anlage de placa neural hemicircular (Bortier y Vakaet, 1992). Las últimas células se localizan cerca de la antigua región anti-falciforme, exactamente en la concavidad de la media luna endófila desplazada cranealmente. El endoblasto falciforme restante más caudal se localiza debajo de la capa superior, lo que dará lugar a la región central del área de formación de PS. Esta evolución diferente en la región craneal (anti-hoz) frente a la región central (area centralis) probablemente puede explicarse por la diferente reactividad en estas dos regiones de la capa superior (Callebaut et al., 2002c).

La ausencia de inducción neural después de los experimentos de injerto con la capa profunda en blastodermos completos por Gallera y Nicolet (1969) y por Knoetgen et al. (1999a, b) probablemente se pueda explicar por la presencia total del material de hoz de Rauber. Este hallazgo indica también que las conclusiones anteriores de experimentos de injerto en blastodermos enteros no incubados (que contienen la hoz de Rauber, el organizador principal principal, o en blastodermos PS, que contienen el nodo de Hensen, un organizador principal secundario) deben reconsiderarse. Por tanto, no podemos estar de acuerdo con ninguna de las conclusiones de Knoetgen et al. (1999b) que el endoblasto por sí solo provoca cualquier cambio detectable en el ectoblasto del huésped adyacente después del trasplante o que el organizador aviar está confinado únicamente al nódulo de Hensen. Foley y col. (2000) estudiaron el papel eventual de la capa profunda temprana (endófilo y / o endoblasto falciforme) en la expresión de los marcadores moleculares Sox3 (Uwanogho et al., 1995) y Otx2 (Bally-Cuif et al., 1995) en el capa superior. Desde el estadio 6-7 de Hamburger y Hamilton en adelante, Sox3 se expresa específicamente en toda la placa neural del pollo y Otx2 se expresa en todo el prosencéfalo y el mesencéfalo. Foley y col. (2000) encontraron que la capa profunda temprana regula una fase transitoria temprana de la expresión de Otx2 y Sox3 en la capa superior adyacente. Por lo tanto, concluyeron que la capa profunda temprana no induce definitivamente el tejido neural o el prosencéfalo. Sin embargo, sus experimentos de trasplante no se realizaron en la hoz de Rauber, o en fragmentos de blastodermo sin endoblastos de unión, sino en blastodermos completos. Recientemente, Knezevic y Mackem (2001) encontraron evidencia de que dos genes, posteriormente asociados con el organizador de la gástrula (Gnot-1 y Gnot-2), son inducidos por las señales de la capa profunda en embriones pre-racha. Según estos últimos autores, estos genes quizás podrían regular la formación de ejes en el embrión temprano, lo que también podría explicar la inducción de una raya en la parte aislada del área central por el endoblasto falciforme (Callebaut et al., 2003b).


Parte 3: Desarrollo temprano de la rana: cómo hacer un renacuajo o un gemelo

00: 00: 0708 Soy Richard Harland.
00: 00: 0808 Estoy en UC Berkeley.
00: 00: 0926 Y hoy les voy a contar sobre las actividades de señalización que dan lugar a
00:00:1322 the neural plate, the forerunner of the spinal cord and brain in vertebrate embryos.
00:00:1914 In previous talks, I've talked about the introduction to the Xenopus embryo, why it has some advantages
00:00:2509 for experiments.
00:00:2609 And I've talked about the cell shape changes that have happened in the. in the embryo
00:00:3015 that lead to the three-layered ectoderm/mesoderm/endoderm structure that is the case, the.
00:00:3625 the state at which neural tissue formation happens.
00:00:4120 Let's initially start by just reviewing the classic experiment, the organizer experiment
00:00:4520 done by Hilde Mangold and Hans Spemann.
00:00:4819 And this is the one that really set the stage for what I'm going to talk about.
00:00:5304 In this experiment, they used marked embryos.
00:00:5607 They used newts of different colors: a dark-colored, pigmented newt, and a light-colored newt.
00:01:0104 And what they were. asking the question, what happens if we move pieces of the embryo around?
00:01:0814 Do they differentiate according to how they normally were set up in the embryo?
00:01:1209 It's self-differentiating according to their original fate?
00:01:1502 Or do they adopt the fate of their surroundings?
00:01:1728 Are they told to become the fate of their surroundings?
00:01:2017 Well, there was one particular experiment that was quite spectacular,
00:01:2426 where not only did the cells self-differentiate, but they recruited cells from the rest of the embryo
00:01:3025 to make new structures, the phenomenon of embryonic induction.
00:01:3604 So here, what they did was to take this embryo, here. the dark embryo is the host, and there's
00:01:4212 this paler donor.
00:01:4328 And what they did was to cut out this dorsal lip region from the pale embryo, flip it around,
00:01:5013 and graft it in to the ventral side of the host.
00:01:5318 So, not only does this have its normal organizer side but it has a new piece of dorsal mesoderm
00:01:5904 stuck in the ventral side.
00:02:0028 And what they found was that this was enough to make the embryo twin.
00:02:0602 And that's shown down here in a representation, this particular one done by Andrea Wills,
00:02:1014 who was a student with me.
00:02:1204 So, you can see that there's a normal primary axis with a head with two eyes.
00:02:1619 Then down here, there's a secondary axis, which is fused down at the tail, but it's
00:02:2116 a complete, proper, organized secondary axis.
00:02:2524 Now, the important thing was, since they were using marked issues, they could tell
00:02:3006 what is the contribution of the graft and the host.
00:02:3228 And so here's a. a representation of the section that was made by Hilde Mangold.
00:02:3702 Here's the primary axis, with the tissues that should be familiar to us by now:
00:02:4121 the nervous system, the notochord, and the muscle.
00:02:4510 And here's the secondary axis.
00:02:4626 And there's the pale graft.
00:02:4801 The pale graft invariably contributes just to the midline tissues.
00:02:5215 Here, it's contributing to the notochord and a little bit of the somites.
00:02:5707 Sometimes it would contribute to a bit of the spinal cord.
00:02:5926 But the consistent observation is that it contributes to the midline tissues,
00:03:0420 whereas the bulk of these tissues are recruited from the host: most of the nervous system,
00:03:0810 the muscle, and so on.
00:03:1202 So this graft really must be instructing the surroundings to make this second axis and
00:03:1620 organize it properly.
00:03:1805 Here's a modern equivalent of the experiment, also done by Andrea Wills, where she's
00:03:2216 labeled the donor embryo with a red stain.
00:03:2515 And you can see in this twin, where the two axes have arranged themselves conveniently
00:03:3013 next to each other.
00:03:3117 Here is the red-labelled graft.
00:03:3308 And you can see above it the induced neural tissue.
00:03:3613 So, it's not a phenomenon just of the 1920s, but can be done in the current times.
00:03:4221 Now, I'm really going to emphasize that this induction mechanism was not obvious.
00:03:4714 And in fact, Warren Lewis, who is not as famous as Spemann and Mangold, tried a similar experiment
00:03:5313 earlier than they did.
00:03:5504 But what he concluded -- largely because the embryos were not marked --
00:03:5915 was that the result he was getting was exclusively the result of self-differentiation of the grafted tissue.
00:04:0521 And he couldn't see any induction, because the tissues were not marked.
00:04:0922 And so the bottom line is that he'd. because of this assumption of self-differentiation,
00:04:1506 he missed on the phenomenon of induction.
00:04:1800 And so we remember Spemann and Mangold, and less so Lewis.
00:04:2201 Okay, let's go back to the whole embryo and remind ourselves what we're looking at.
00:04:2713 So, we know from molecular mapping that the organizer is gonna be the top of this
00:04:3323 when it loops around again.
00:04:3428 We're going through gastrulation and neurulation.
00:04:3804 And when we loop around again. here's the organizer up here.
00:04:4013 It's going inside the embryo and is opposed to this neural plate, and is able to
00:04:4615 instruct it to make the neural tissue.
00:04:4907 So again, if we look at this MRI movie, we can see that dorsal mesoderm moving up
00:04:5604 against this overlying neural plate.
00:04:5811 And during this process, where it's opposed to the neural plate, it's in the right place
00:05:0207 to be inducing the neural tissue.
00:05:0407 So, that's the normal organizer that's going up there and is thought to be signaling
00:05:1005 to induce the neural plate.
00:05:1113 Okay, so we're going to discuss this more.
00:05:1501 But we first need to understand how we get to that position.
00:05:1724 And I'm not going to go through this in detail, but I'm going to give a brief summary of the
00:05:2126 initial events that happen to set up the organizer.
00:05:2517 And it comes down largely to the activity of two different signaling pathways: the Nodal/Smad2 pathway
00:05:3221 and the Wnt/beta-catenin pathway.
00:05:3512 We don't need to know about these pathways in detail, but what we do know is the way
00:05:4018 they're turned on in the embryo.
00:05:4224 So initially, when the egg is laid, it's got this axis from animal to vegetal,
00:05:4822 from the pigmented to the yolky side.
00:05:5122 And subsequently it was found that there are a number of pre-localized components in that
00:05:5515 polarized egg.
00:05:5717 And I'm going to talk about this red mRNA, messenger RNA, that's pre-localized,
00:06:0300 called vegt, first described by Mary Lou King's group.
00:06:0612 And there's also, slightly less well-characterized, activators of the Wnt/beta-catenin pathway
00:06:1119 down here.
00:06:1303 So initially, this is cylindrically symmetrical about the animal-vegetal axis.
00:06:1618 And an important process here, as is widespread in embryology, is the symmetry breaking.
00:06:2118 So, you have to go from a cylinder to a bilaterally symmetrical egg.
00:06:2523 And this is achieved during normal development because the sperm is going to enter on one side.
00:06:3213 That makes this giant aster.
00:06:3504 So, these astral microtubules extend throughout the egg cytoplasm during the first cell cycle.
00:06:4115 And not only do they serve to pull the maternal pronucleus towards it, but they also
00:06:4603 serve to bias the way that microtubules polymerize. polymerize in the outside cortex,
00:06:5101 the outer ten-micron layer.
00:06:5313 So as a result of this bias, there's an oriented array of microtubules that go around the embryo,
00:06:5928 here.
00:07:0028 And they act as tracks for carriage via kinesins of some of these purple components.
00:07:0622 There's a selectivity.
00:07:0822 The purple components that activate the Wnt/beta-catenin pathway get smeared out along the
00:07:1413 entire dorsal side of the embryo, whereas the red do not do this.
00:07:1826 They're sort of passively released from the vegetal cortex and spread in a graded way
00:07:2417 through the egg.
00:07:2517 So, we now have a broken symmetry, where we have the red going from vegetal to animal
00:07:2926 and the Wnt/beta-catenin concentrated from dorsal to ventral.
00:07:3504 Now, these two molecules get together and turn on the. the Nodal genes.
00:07:4000 The Nodal genes are signaling proteins in the TGF-beta superfamily.
00:07:4307 And they're turned on in the margin.
00:07:4515 And in normal development, there's a cooperativity between the purple Wnt signal and, now,
00:07:5026 this yellow protein produced from the vegt RNA.
00:07:5409 So, they get together and turn on these Nodal genes.
00:07:5802 And they turn them on at a higher level on the dorsal side and the ventral side.
00:08:0216 But they do turn them on everywhere.
00:08:0500 And these Nodal genes induce mesoderm.
00:08:0710 So, the cells that are initially naive get told, in this marginal zone, to become
00:08:1310 the prospective mesoderm.
00:08:1504 But where this interaction is the strongest -- the strongest interaction of beta-catenin
00:08:1922 and the Nodal gene expression -- that converges on the promoters of organizer gene and
00:08:2512 turns them on, especially in this dorsal region here.
00:08:2822 So, the marginal zone. the mesoderm goes all the way across in the equator,
00:08:3224 but the organizer is special in that it's only turned on at the convergence, the strongest convergence
00:08:3702 of these signals.
00:08:3804 Okay, so we've discussed that.
00:08:4024 And let's just contrast that with what happens if this cortical rotation doesn't occur.
00:08:4701 And so you can see here what. there are various tricks to. to cause this to happen.
00:08:5024 One is to irradiate the vegetal side of the embryo with ultraviolet light.
00:08:5413 And that prevents the polymerization of microtubules.
00:08:5726 The alternative is to eliminate beta-catenin production by using a reagent that
00:09:0209 blocks beta-catenin production.
00:09:0324 We'll come back to that.
00:09:0611 Either way, what happens is that we get the release of the vegt and we get the
00:09:1014 graded vegt protein, which turns on Nodal, but, in the case of the lack of cortical rotation,
00:09:1626 then this purple signal stays down here.
00:09:2001 And so as a result, there is no synergy on this side.
00:09:2225 There's no overlap between the signals.
00:09:2522 And so this whole marginal zone behaves like the ventral marginal zone.
00:09:3008 You get a. a ventralized type of embryo with no organizer.
00:09:3506 Okay, so that symmetry-breaking event back here was important.
00:09:3902 But we're gonna use this trick in the next experiment, that proves that we need organizer signal
00:09:4327 to get the neural plate to be formed.
00:09:4905 Before we go into that, we're just gonna discuss a little bit more about the graded Nodal signaling.
00:09:5327 Because there. a quite widespread view in the field is that this graded Nodal signaling
00:09:5823 is important in setting out the pattern of the marginal zone.
00:10:0217 It seems quite obvious that if there's going to be a graded signal with more on this side
00:10:0620 than that side, it should be used for something.
00:10:0922 So, we're going to get a graded response, and the phospho-Smad2 is the intracellular effector,
00:10:1515 which is known to be distributed like this.
00:10:1804 There really is graded expression of Smad2 going from dorsal to ventral.
00:10:2226 And then this proceeds as a wave across the embryo.
00:10:2505 So, it seems perfectly reasonable to think that in normal development what ought to be
00:10:3002 happening is that that signal will tell the embryo to make different kinds of mesoderm.
00:10:3618 And indeed, that whole idea is supported by this experiment, where we can take,
00:10:4127 from the blastula stage, naive ectoderm from this so-called animal cap and put it in culture.
00:10:4822 By itself, it will self-differentiate into epidermis.
00:10:5125 But if we add a signal, and we can use either Nodal or more conveniently, Activin,
00:10:5619 another member of the TGF-beta superfamily.
00:10:5908 If one adds increasing doses of that Activin signal, the mesoderm-inducing signal,
00:11:0416 one can get caps that develop in a more ventral way, making mesenchyme, whereas as one
00:11:0923 doses in the signal, more and more dorsal tissues, like muscle and ultimately a lot of notochord.
00:11:1519 So, those kinds of experiments, the description of the graded expression, as well as
00:11:2107 this result, where one's reconstructing what may be going on in the embryo, suggest that
00:11:2502 that graded signal may cause pattern.
00:11:2616 But I'm going to argue that's not true.
00:11:2913 So, just to sum up, this normal pattern in the marginal zone -- from notochord through
00:11:3502 muscle, kidney, and blood -- could in principle be set up by that graded Nodal signaling.
00:11:4204 But this was explicitly tested in a series of experiments by Ron Stewart and John Gerhard,
00:11:4724 and other very similar experiments by Jonathan Slack.
00:11:5102 And so what I want to review briefly is this experiment that shows that there's not enough
00:11:5617 information imparted by that early signal to give substantial pattern in the marginal zone.
00:12:0228 Now, what they did. they wanted to assess the effectiveness of organizer grafts.
00:12:0815 And they did this at the late blastula stage.
00:12:1021 So, this is a really early stage, before gastrulation goes on and before there's much to. allí.
00:12:1522 there certainly is pattern is the marginal zone later on.
00:12:1822 So, they took normal embryos and cut them in half, vertically, so they got two hemispheres.
00:12:2418 And they used that UV irradiation trick.
00:12:2607 So, they had a graft. they were able to graft these -- labeled grafts, of course --
00:12:3220 onto UV-irradiated hosts.
00:12:3320 So, they made this recombinant.
00:12:3611 In this case, the organizer is schematically illustrated in red.
00:12:4005 So, you've got a half organizer here.
00:12:4214 One of their first questions was, if you only put in a right organizer, do you only get
00:12:4612 a right embryo?
00:12:4714 And there are. the answer was no.
00:12:4822 You get a bilaterally symmetrical embryo.
00:12:5101 But also, in many cases this graft will give you a normal tadpole.
00:12:5517 So, you rescue development also, as shown by the lineage tracing experiment,
00:13:0009 from this ventralized half.
00:13:0215 But this is really the key one in my exper. in my view.
00:13:0615 So here, they've cut just 30 degrees off the dorsal midline axis, so they've cut the organizer
00:13:1228 into the right piece, and this piece has no organizer.
00:13:1607 Now, by the model I was discussing earlier, there should be some graded Activin signaling
00:13:2212 or graded. graded Nodal signaling in here that's inducing things like muscle.
00:13:2707 Well, we're gonna ask that question.
00:13:2819 We're gonna take this side and, again, fuse it to a naive ventralized piece,
00:13:3418 put them together, and ask what happens.
00:13:3806 And the result in most cases is there's absolutely no dorsal pattern in the embryo.
00:13:4411 And just to nail this home, I want to stress that.
00:13:4713 So, they're taking this ventralized piece and putting on this piece from a normal embryo
00:13:5305 that lacks just the organizer, but still has that dorsolateral prospective mesoderm
00:13:5714 that would make muscle if it were left alone.
00:14:0002 But in the context of this recombinant, you just get this completely ventralized embryo,
00:14:0518 as opposed to something that would make a little muscle and so on.
00:14:0819 So, this experiment shows I think quite well that the pattern that's induced by
00:14:1423 that graded Activin/Nodal signaling is not enough to have any permanent effect on this tissue.
00:14:2021 And that you actually need the organizer signaling.
00:14:2226 So, that sort of loss of organizer function proves that you need organizer signaling
00:14:2901 in normal development.
00:14:3219 Another is a sort of descriptive view, where we've looked at gene expression at different phases.
00:14:3715 And here's a case where we see two. expression of two different genes.
00:14:4016 This is noggin expressed in the organizer -- we'll come back to that -- and this
00:14:4422 prospective muscle gene, myod, that's expressed in a complementary way, in the non-organizer tissue.
00:14:5020 When it's first turned on, it's turned on fairly uniformly around the rest of the marginal zone.
00:14:5508 Later on, this expression turns off, and this gets enhanced by signaling from the organizer.
00:15:0022 But when it first turns on, it looks like the marginal zone is organized in a binary way.
00:15:0513 Now, this very rapidly changes.
00:15:0625 So, we see expression of genes such as this one, lhx1, that's high in the dorsal marginal zone
00:15:1127 and then graded off to the side.
00:15:1408 So, that's a later stage.
00:15:1524 We also see that with this split, where the blue and the brown genes are expressed
00:15:2010 in complementary domains at the end of the blastula stage, but very quickly become elaborated
00:15:2600 so that the brown gene, Wnt8, is restricted away from the organizer and just in the marginal zone.
00:15:3027 So, things are very dynamic.
00:15:3228 And one really has to look at this early stage to see this binary difference.
00:15:3624 By this stage, this tissue has already been instructed by the organizer to make muscle.
00:15:4125 But anyway, this descriptive experiment does support the idea that initially the marginal zone
00:15:4715 is split in a binary way and is not graded in this induction.
00:15:5017 So again, arguing that you need a signal from the organizer and it's not enough to have
00:15:5500 that graded Nodal signaling.
00:15:5824 This just reinforces that.
00:16:0106 And as that slides up, I'll say that we need. need now to figure out, what are these other signals?
00:16:0801 And at the time, there were a lot of experiments that were done using both cell biology,
00:16:1223 using secreted signals from cells and assaying them in embryos, and many of these signals do have
00:16:1716 important embryonic functions: fibroblast growth factors Nodals, Activins, and so on.
00:16:2318 But the dorsalizing molecules that are made from the organizer were not understood.
00:16:2816 So, how do we find those?
00:16:3027 And here I give much credit to Bill Smith, who's now a professor at UC Santa Barbara,
00:16:3418 who in the early '90s joined me and decided to use an expression cloning approach
00:16:4013 to try to find these molecules.
00:16:4122 And again, he used this trick of ventralizing embryos.
00:16:4513 But at the four-cell stage, he then took synthetic messenger RNAs made from a library.
00:16:5112 This library was a library of gastrula-specific RNAs in a plasmid that could be transcribed
00:16:5618 with this synthetic phage polymerase, SP6 polymerase, so that we could get a library of,
00:17:0204 in the first instance, 100,000 colonies, extract the DNA, and then transcribe
00:17:0820 that whole library of plasmids to make a complex mixture of synthetic RNA that we hoped mimicked
00:17:1315 what was in the embryo.
00:17:1524 Remarkably, that first injection of the synthetic RNA, when it was injected back at the four-cell stage,
00:17:2107 instead of these embryos looking like this -- complete belly pieces as Spemann would
00:17:2525 have called them -- they looked more like this.
00:17:2723 They had some tail structures, some muscle, and spinal cord.
00:17:3107 So, that RNA conferred a morphological rescue.
00:17:3421 At that point, we knew there was an active ingredient in the library, and it was
00:17:3909 just a question of sub-selection, where we would split the library into smaller and smaller pools,
00:17:4400 assaying those pools as we go along, and ask, is there a pool in there that
00:17:4908 confers this ability to dorsalize embryos?
00:17:5219 And sure enough, we. he did this a couple of times.
00:17:5502 The first time, he isolated a Wnt signal, which I won't discuss but is thought to mimic
00:17:5907 that early Wnt signal in dorsalizing the embryos.
00:18:0128 But for the purposes of this presentation, the second one he isolated was really exciting
00:18:0623 because it was completely new.
00:18:0824 And as a single RNA, as you can see in this picture, with increasing dose of the gene
00:18:1324 we called noggin RNA, you get this progressive rescue of structures to the normal state.
00:18:1917 And then when one overdoses the embryo, ultimately you end up with these little noggins,
00:18:2420 these little heads alone.
00:18:2520 So, that. that single RNA is able to transform, from the four-cell stage, a ventralized embryo.
00:18:3023 And if it's put in at enough dose, you get just a big head.
00:18:3604 This has been a very useful assay to isolate a number of other embryological activities,
00:18:4006 but that's the one I want to concentrate on, noggin.
00:18:4310 And so this is an in situ hybridization where we're looking at the messenger RNA
00:18:4810 that's expressed from the noggin gene in early development.
00:18:5100 And so let's look at this stage.
00:18:5319 This is a late blastula.
00:18:5504 Noggin has already turned on.
00:18:5613 And as you can see, it's turned on in just one side of the embryo.
00:18:5909 And we know this is the dorsal side.
00:19:0109 So, this gene is expressed. it not only has the right activity in these messenger
00:19:0614 RNA injections, but it's also expressed in the right place -- here's a vegetal pole view,
00:19:1025 remarkably it's a 60-degree sector, just the same as the sector that Ron Stewart identified
00:19:1622 in his activity assay -- and then in the gastrula stage it continues to be expressed
00:19:2408 in the dorsal marginal zone, in this involuting dorsal mesoderm that is lying just underneath
00:19:2915 the neural plate, and in the right place to induce the neural plate.
00:19:3218 Now, here's a neural. neurula stage where it continues to be expressed in the head,
00:19:3626 mesoderm, and notochord, again, in the right place to continue inducing the neural plate.
00:19:4304 So, the activity is promising, the extra expression is promising, but could we show that it
00:19:4817 had the right properties?
00:19:4924 So, we initially used a protein that we made in CHO cells that Teresa Lamb had transformed
00:19:5523 with noggin plasmids.
00:19:5624 But later in the collaboration with Regeneron Pharmaceuticals, they made a human noggin,
00:20:0210 particularly, Aris Economides, and gave us that recombinant human noggin for our experiments.
00:20:0706 So we were able to ask, now, can noggin protein mimic what we know are the embryological effects
00:20:1317 of grafting this tissue?
00:20:1705 So, again, this is the normal case, where noggin is expressed in this red dorsal mesoderm,
00:20:2218 and potentially instructing this overlying ectoderm to become neural plate.
00:20:2606 But how do we assay that activity?
00:20:2809 Well, again, we turned to our animal cap assay.
00:20:3021 And actually, we can turn to that assay in the gastrula stage, where the sensitivity
00:20:3616 of these cells up here has changed, and they're no longer responsive to the Activin/Nodal signal.
00:20:4402 But we know from recombination experiments that if we graft an organizer onto here
00:20:4903 neural induction will occur.
00:20:5111 So now we can replace the graft of organizer by soaking this tissue in noggin protein.
00:20:5720 And so this is a schematic, which we can do in the late blastula or the gastrula,
00:21:0124 where we take this prospective ectoderm off into culture.
00:21:0503 Normally, just makes a hollow ball of epidermis when left alone.
00:21:0825 As I mentioned before, with. with Activin or Nodal, you get a complex induction of
00:21:1504 dorsal mesodermal cell types.
00:21:1717 And those in turn can secondarily induce neural tissue.
00:21:1926 But the induction is not direct.
00:21:2120 The important thing for our purposes is that, when we treat this just with recombinant noggin,
00:21:2707 we get a clean neural induction.
00:21:2820 There's a little epidermis that's on the outside and an epithelial layer that's not so responsive,
00:21:3405 but all of the underlying cells, here, are trans. transformed into neural cells.
00:21:3809 So, we get a cleaner. clean neural tissue, and that can't be induced by any mesoderm
00:21:4300 because there's no mesoderm in this explant.
00:21:4611 We can also do this as at a time when the mesoderm can no longer be induced.
00:21:5004 So, if we do this at the gastrula stage instead of the late blastula stage, this experiment
00:21:5511 wouldn't work.
00:21:5611 Activin would have no effect.
00:21:5714 And yet noggin can still induce neural tissue.
00:22:0002 So this, then, really identified noggin as an authentic neural inducer, that it's expressed
00:22:0421 in the right place and time, and has the right activity to be doing the job in the normal embryo.
00:22:1117 Here are some pictures of the kind of results that we get.
00:22:1417 These are the works of. these are done by Anne Knecht in the lab.
00:22:1722 And so, here we have a molecular assay for neural tissue.
00:22:2022 This is a gene that's expressed throughout the nervous system.
00:22:2413 And here are some of these explants, the explants that lack noggin, and they don't stain
00:22:2922 for this gene.
00:22:3022 But the parallel explants that were soaked in noggin protein, as you can see, are robustly
00:22:3514 induced to make this marker gene, and so we can say they're neural.
00:22:3805 And that contrasts. again, I'll draw the contrast with the Activin mesoderm inducer.
00:22:4413 Here, we're looking down on the top of the embryo with the muscle and notochord in the middle.
00:22:4819 These are mesodermal structures that we've lit up with this collagen probe.
00:22:5226 If we take explants just like these ones, but treat them with Activin at the late blastula stage,
00:22:5714 we get lots of this mesoderm induced.
00:22:5909 But down here, we see that noggin induces no mesoderm.
00:23:0220 So, we do get neural tissue in the absence of mesoderm, and hence a clean neural induction.
00:23:0804 Just as an interesting side point -- I won't be discussing this much today -- that we
00:23:1315 also have to account for production of the entire neural plate from brain to spinal cord.
00:23:1811 So, what kind of tissue does noggin induce?
00:23:2111 Here we can use regional markers like this cement gland, this very anterior marker,
00:23:2612 or this forebrain-midbrain marker, otx2, and then this engrailed-2 is expressed just
00:23:3123 at the border of the hindbrain.
00:23:3326 And the noggin-treated explants will make these very anterior marker genes.
00:23:3904 they'll turn them on, but they don't turn on the more posterior ones.
00:23:4304 And so noggin, exclusively in this explant situation, induces anterior brain-like tissue.
00:23:5114 So, we have a molecule that works very well, but of course we then had to figure out how it works.
00:23:5820 And so, for this experiment, we for a long time labored under the delusion that
00:24:0220 we may have invented a new kind of signal transducer.
00:24:0600 At the time, it wasn't really appreciated that development gets by with a remarkably
00:24:0914 limited number of pathways.
00:24:1125 And so, here, what eventually turned out, with some very useful information from
00:24:1627 Chip Ferguson's lab, it was suggested that it may be impacting the BMP pathway.
00:24:2201 And Lyle Zimmerman was able to show that, because we had all these reagents in the lab
00:24:2523 at the. en el momento.
00:24:2725 And the way that noggin actually works is not by activating any new signal transduction pathway,
00:24:3304 but rather by interfering with the BMP signaling pathway.
00:24:3611 Normally, BMP binds to its two receptors, brings them together, and that has the consequence
00:24:4117 of ventralizing the embryo.
00:24:4325 In this case, when noggin is present, it binds tightly to BMP, prevents this interaction,
00:24:5009 and then by default, instead of being ventralized, the embryo is dorsalized.
00:24:5513 So I'll stress that, that it's the absence of this BMP signal that is instructive to the embryo
00:25:0104 and allows the embryonic structures to make dorsal structures rather than BMP-induced
00:25:0712 ventral structures.
00:25:0901 And satisfyingly, this crystal structure from Jay Groppe shows that noggin, as a dimer,
00:25:1423 sort of embraces the dimer of BMP.
00:25:1626 So, it's not surprising that it, as Lyle Zimmerman showed, prevents the BMP from binding their receptors.
00:25:2606 It's a very high-affinity reaction, as was shown here by this competition experiment.
00:25:2913 And this was done again by Lyle Zimmerman.
00:25:3126 Here took iodinated BMP4 and was able to bind it to a chimeric human noggin, which has an
00:25:3926 immunoglobulin tail which makes it easy to precipitate.
00:25:4303 And so if one simply mixes these together, you get a very active binding and precipitation.
00:25:4916 But by mixing in different doses of different kinds of other TGF-betas, we could work out
00:25:5510 the affinity of this interaction.
00:25:5708 And so, notably, if we take BMP4, the red one, and plot how that interferes
00:26:0214 with binding of iodinated BMP4, we get this nice curve.
00:26:0714 And if we plot the half-maximal inhibition, it comes out that the interaction there is.
00:26:1202 it has a remarkably tight Kd of about 20 picomolar.
00:26:1626 Other BMPs, like BMP7, in blue here, have a lower affinity.
00:26:2305 And then yet other TGF-beta family members, like TGF-beta itself, have no measurable affinity.
00:26:2820 So, there is a variation in affinity, but very tight affinity for the BMP2 and -4 class.
00:26:3500 So, we come up with this general model that in normal development you set up the mesoderm
00:26:4026 with a special dorsal territory, this naive and fairly uniform ventral-lateral territory,
00:26:4626 and the rest of the patterning is mediated during gastrulation by dorsalizing signals
00:26:5128 like noggin that come from the dorsal-marginal zone, instruct the overlying epidermis
00:26:5713 to instead become nervous system, and instruct this ventral mesoderm to become things like muscle.
00:27:0222 So, is that it?
00:27:0420 Really, we've done this by add-back, but what about loss of function?
00:27:0900 And in the interim, a large number of other antagonists were discovered.
00:27:1213 And a lot of these were discovered by Eddy De Robertis' lab, so chordin and cerberus.
00:27:1916 Noggin, we discovered, Bill Smith discovered, as well as this Nodal-3 molecule.
00:27:2422 And follistatin had already been known about, but its activity as a dorsalizing molecule
00:27:2928 was worked out by Ali Brivanlou in Doug Melton's lab.
00:27:3302 So, looking at all these, they're all expressed in the organizer, and they all have some similar
00:27:3911 anti-BMP activities.
00:27:4113 You can see that they turn on at different times.
00:27:4316 This is the early stage, so some are turned on very early.
00:27:4718 And then some are turned on in slightly different territories.
00:27:5004 And perhaps that's important in how they work in detail, but so far, as far as we can tell,
00:27:5424 they all work essentially the same way.
00:27:5617 But there are a lot of them, and they probably have overlapping activity.
00:28:0108 And so if we try to knock down their activity, do we. can we knock down one and get a result,
00:28:0517 or do we have to knock down many?
00:28:0824 To do this experiment, we use morpholino oligonucleotides.
00:28:1114 These are synthetic, uncharged, and very stable oligonucleotides that can hybridize
00:28:1526 to the messenger RNA and interfere with translation, in this case.
00:28:2000 So, we can specifically use the information from base pairing to specifically knock down
00:28:2602 these individual RNAs.
00:28:2720 So, Mustafa Khokha, when he was in the lab, did this experiment using mixtures of morpholinos
00:28:3227 against follistatin, chordin, and noggin.
00:28:3517 And just as a side note, to make this simpler, we did it in the related species to Xenopus laevis,
00:28:4020 Xenopus tropicalis, which now had a sequenced genome, and so we could identify
00:28:4514 and design these oligonucleotides easily to target just single genes rather than two genes
00:28:5003 in the paleotetraploid, Xenopus laevis, genome.
00:28:5419 So, we can inject these morpholinos and then ask what happens.
00:28:5925 And we're going to use, for example, in the neurula stage, this neural marker, which at
00:29:0323 the mid-neurula stage has this nice ability to light up the neural plate.
00:29:1008 Because of worries about specificity of these reagents, which always become toxic if you
00:29:1404 put enough of them in, we do a specificity assay of rescuing.
00:29:1716 So, by cloning the pufferfish noggin, we could use that different sequence to put back BMP-antagonist activity,
00:29:2417 as we'll find out, and rescue the whole process.
00:29:2728 So, these are the kinds of results.
00:29:3009 So, we're going to do single, double, and triple knockdowns.
00:29:3410 And the results are pretty simple.
00:29:3527 When comparing the uninjected to the morpholino-injected, when we knocked down noggin we see no effect.
00:29:4203 Essentially no effect with follistatin.
00:29:4402 Some mild effect, as reported by De Robertis' group, for chordin.
00:29:4716 But then, when we knocked down two -- follistatin/noggin, chordin/noggin, chordin/follistatin --
00:29:5220 we see a more extreme effect.
00:29:5324 There's a smaller neural plate.
00:29:5511 Well, when we knocked down all three, there's a spectacular result, where now, instead of
00:29:5919 making the neural plate, the neural plate is eliminate. eliminated.
00:30:0328 Not only is the neural plate eliminated, but the underlying muscle is almost completely gone.
00:30:0908 And by this hedgehog control, the notochord and the floor plate are also gone.
00:30:1428 So, this is important to rescue.
00:30:1627 We can rescue it with this mixture of morpholinos, rescuing it with the pufferfish noggin.
00:30:2303 And as you see, we get back all of these tissues, demonstrating specificity.
00:30:2817 We can also rescue it by knocking down additional BMPs.
00:30:3206 So, instead of this horrendously high mixture of morpholinos, we're putting in
00:30:3614 even more morpholinos, but also knocking down BMPs.
00:30:3925 And again, you can see that rescue.
00:30:4102 So, we're pretty satisfied that this is a specific manipulation.
00:30:4421 So, knocking down the BMP antagonists eliminates the neural plate.
00:30:4921 You need the antagonists to make the neural plate.
00:30:5327 As we would expect, as you can see on the right of this picture, the loss of those
00:30:5814 dorsal structures is accompanied by a gain in ventrolateral structures.
00:31:0302 So here, for instance, let's. let's look at msx1.
00:31:0522 It's expressed just in the flank.
00:31:0802 But in this manipulated embryo, there's just a narrow stripe left of nonexpressing tissue.
00:31:1306 So, all these ventral tissues are expanded.
00:31:1708 We can also ask, when does this dorsal identity fail?
00:31:2010 When we knock down those antagonists, we clearly lose dorsal structures, but at what step does
00:31:2417 this happen?
00:31:2517 And of course, we would predict that it should fail at the time that normal genes are expressed,
00:31:2906 at the late blastula stage.
00:31:3128 We can start to look at that, and contrast the situation where we interfere with antagonist function
00:31:3802 in normal development, with what happens when we ventralize the embryo from
00:31:4212 the get-go, either with UV light or by depleting beta-catenin, actually also with a beta-catenin
00:31:4727 specific morpholino.
00:31:4902 So, in that case, we lose the beta-catenin purple signal, and we get just this ventralized embryo.
00:31:5611 Okay.
00:31:5711 So, in looking at those, we can see that in the. in the. in the morpholino-knockdown cases,
00:32:0515 where we've knocked down follistatin, chordin, and noggin, we have normal mesoderm,
00:32:0905 as marked by this brachyury gene, but in the case where we look for dorsal identity
00:32:1521 with the goosecoid marker -- here's the control, here's the follistatin/chordin/noggin knockdown --
00:32:2013 there's still dorsal identity.
00:32:2322 Whereas if we contrast that with the beta-catenin knockdown, where we've knocked down
00:32:2705 the early dorsal signal, then of course we never get a dorsal identity.
00:32:3023 So, there's a contrast here, showing that in the absence of the antagonists
00:32:3528 we still get a dorsal identity.
00:32:3713 But then that dorsal identity fails to execute. execute its function.
00:32:4404 And indeed, we can also look at these embryos to ask what happens to BMP signaling.
00:32:4818 And in particular, we can use this useful mark, vent2, because that's normally expressed
00:32:5313 everywhere except the organizer.
00:32:5515 And we also know it's a direct target of BMP signaling.
00:32:5921 So again, when we knock down the follistatin, chordin, and noggin, we see that that gap
00:33:0323 is filled in.
00:33:0508 So in other words, in the absence of the antagonists, there's a sign pretty early on of excess BMP signaling,
00:33:1100 which is going to mess up dorsal developments and lead to a ventralized embryo.
00:33:1519 And again, we get the similar effect, as we would expect, if we eliminate the organizer
00:33:2000 completely with beta-catenin.
00:33:2104 The same result is found with this early muscle marker, which.
00:33:2528 muscle development used to be thought to be development mostly on the graded signal
00:33:2911 from Nodal.
00:33:3011 But you can see here, clearly, that we need that BMP antagonist in order to amplify the
00:33:3506 expression of this muscle determinant in the early embryo.
00:33:3928 So overall, we conclude now that this pathway, of knockdown of BMP activity by these BMP antagonists,
00:33:4726 by a cocktail of BMP antagonists, is crucial to get dorsal development,
00:33:5224 and in order to get neural induction to occur.
00:33:5508 We can show that these molecules by themselves, as protein, induce neural tissue.
00:33:5826 We can show that the combination is essential.
00:34:0127 And they are expressed in the right time and the right place to be ex. executing this function.
00:34:0704 So all in all, it's a comprehensive statement that these are crucial for neural induction
00:34:1303 and dorsal development.
00:34:1513 We can add on the additional observation from the end, that even in the absence of.
00:34:2200 well, in the absence of these antagonists, the early events go by perfectly normal. normally,
00:34:2725 and we get dorsal specification in the marginal zone.
00:34:3108 But in the absence of the antagonists, that organizer can no longer execute its function.
00:34:3621 So all in all, we can conclude then that BMP antagonists are essential for
00:34:4202 the Spemann organizer phenomenon.
00:34:4327 Thank you.

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