[en] Development of the posterior spinal cord involves secondary neurulation, a process poorly understood in which neural tube is formed by cavitation of the tail bud. Comprehension of secondary neurulation is required to understand the morphogenetic origin of high prevalence neural tube defects such as spina bifida. In zebrafish embryos neurulation goes through a stage of neural rod; a neural primordium that hollows a lumen in the middle. To form a single continuous lumen, cells converge to the tissue center, divide in stereotyped orientations perpendicular to it (the so-called C-Divisions), and form a tissue midline composed by apical polarity components that will later originate the central lumen. Polarizing events start with the centrosomes positioning in the midline under the control of unknown cues. Sonic Hedgehog has been widely described as a midline signaling protein in other systems, and its components are located in the apical cilium and the underlying basal body, formed by a centrosome. Shh expression onset occurs during gastrulation in embryonic midline structures —notochord and floor plate- and maintained along neurulation, suggesting a role of Shh in neural tube morphogenesis. Treatment with the Shh inhibitor cyclopamine disrupts the lumina! surface, while Shh pathway activation (Shh-GOF) produces partial to total lumen duplication. Shh-LOF/Shh-GOF display defects in neural lumen positioning and/or formation, confirming a role of Shh in lumen formation. Shh-LOF and Shh-GOF preserve their apicobasal polarity and tissue architecture. Analysis of the dynamics of tissue convergence in ShhGOF embryos shows that compared to the same WT stages the neural plate is wider and the neural plate borders stop converging later in development, suggesting delayed cell convergence movements. However comparison between Shh-GOF and WT cell trajectories to the midline reveals no differences in motion. We found that trajectories of cells committing C-Divisions had indistinguishable motion properties compared to their neighbouring cells, and that Shh-GOF cellular motion properties —velocity and persistency- are resistant to Shh activity. We next analyzed the behaviour of dividing cells and observed that mitosis progressively lengthened along neurulation. Detailed analysis of the mitotic phases shows that Shh-GOF cells show shortened mitoses from condensation to anaphase, but spend the same time –or slightly longer- in metaphase. C-Divisions need orient their metaphase plate so they divide perpendicularly to the midline, giving rise to daughter cells that lie at the sides of the prospective lumen. In C-Divisions, metaphase plate rotations are decreased and slower. We next assessed if division orientation was compromised along neural tube formation. Analysis of division orientation shows higher variability in ShhGOF embryos especially at early stages, a fact that would have morphogenetic consequences in the C-Divisions and lumen formation. With similar cell motion properties and similar metaphase duration between WT and ShhGOF mitotic cells, the latter have quicker mitoses, less metaphase rotations and the stereotyped orientation of polarizing C-Divisions is altered, thereby affecting lumen formation.

[es] Los vertebrados amniotas forman el tubo neural posterior por neurulación secundaria, un proceso apenas estudiado en el que el tubo abre un lumen en el centro. Proponemos el pez cebra (Danio rerio) como modelo para entender el origen embrionario de defectos derivados de fallos en la neurulación secundaria, como es la espina bífida. Durante la neurulación del pez cebra los progenitores neurales compaginan proliferación y convergencia hacia el centro del tejido. Tras la formación de un primordio neural condensado,las células orientan su eje de división perpendicular al eje central (C-Divisions), originando una linea media de polaridad apical que formará el lumen. La polarización empieza con la localización medial del centrosoma, que en interfase constituye la base del cilio. Los componentes de la vía Sonic Hedgehog (Shh) se hallan en el cilio, empezando shh a expresarse en estructuras mediales de la gástrula y néurula -futuras notocorda y placa del suelo-. La inhibición de Shh perturba la continuidad del lumen, y la activación de Shh (Shh-GOF)produce fenotipos de duplicación luminal, sugiriendo que Shh regula la localización/formación del lumen. La polaridad apicobasal de las células permanece intacta, pero el primordio neural es más ancho durante la neurulación. Sin embargo la motilidad de los núcleos y los cilios hacia la línea media es resistente a la actividad de Shh. La duración de las mitosis incrementa a lo largo de la neurulación, y las células Shh-GOF presentan mitosis más rápidas, sin acortamiento de la metafase. Lo mismo sucede en las C-Divisions, en las que la metafase presenta menor rotación y mayor variabilidad en el plano final de división, originando líneas medias ectópicas y duplicación del lumen. Los cambios en los niveles de activación de Shh conllevan consecuencias en la morfogénesis del lumen a través de la regulación de las divisiones durante la formación del tubo neural.