Study of the secundary neurulation in the chick embryo, a model to understand neural tube defects

Abstract

Body axis elongation is a hallmark of the vertebrate embryo, which also comprises the morphogenesis of the caudal neural tube (NT). The contribution of bipotential neuromesodermal progenitors (NMPs) to the cranio-caudal elongation of the embryo is beginning to be understood. However, the signalling pathways and tissue remodelling events required for shaping the caudal NT from NMPs remain largely unknown, even though the failure in this process generates caudal neural tube defects (NTDs). The caudal NT in amniote embryos forms by a process termed secondary neurulation (SN). SN shapes a secondary neural tube (SNT) through the lineage restriction and the mesenchymal-to-epithelial transition (MET) of NMPs into neural progenitor cells (NPCs), and the concomitant opening of a lumen de novo in the centre of the tissue. In human embryos, the development of the lumbar, sacral, coccygeal and equinal cord largely involves SN. The limited availability of human tissue to perform histological analyses at different developmental stages reinforces the need to use animal models to understand the events shaping the SNT, particularly since NTDs rank among the most common categories of birth defects, affecting 1 in every 1000 established pregnancies worldwide. Here, we combine the genetic manipulation of the chick embryo with an in vivo imaging technique to decipher the cellular events driving SNT formation and to demonstrate that TGF-b/SMAD3 signalling is required for proper SN, since its inhibition results in NTDs with multiple lumens. Our analysis demonstrates that the lineage restriction and the MET of Sox2+ T/Bra+ mesenchymal NMPs into Sox2+ T/Bra- epithelial neural progenitor cells (NPCs) are independent of SMAD3 activity. In the developing SNT, both the neural restriction and the MET tightly associate to the growing basement membrane (BM), which assembles in a dorso-ventral fashion. Hence, the first cells to adopt a neural identity and to undergo MET are those contacting the BM, located in the dorsal periphery of the medullary cord. On the contrary, centrally located cells remain mesenchymal, even to the very end of the process. It is between these two cell populations that small cavities of varied size and shape form, always at a one-cell distance from the BM, being SMAD3 also dispensable for lumen initiation. We found that the resolution of a single, centrally positioned continuous lumen in the SNT takes place through the intercalation of central cells, rather than through their programmed cell death. Indeed, results show an important novel activity for TGF-b/SMAD3 in the intercalation of central cells during lumen resolution. Notably, cell intercalation is always preceded by a cell division, either a symmetric II, which generates two intercalating daughter cells, or an asymmetric IC, which generates one intercalating and one central daughter cell. These two modes of division associate to different cranio-caudal levels, with II occurring cranially to IC. In addition, a third mode of division, the symmetric CC division, occurs in the caudal tail bud in order to generate two central mesenchymal NMPs and to expand the progenitor pool driving body axis elongation. Finally, we found lengthened primary cilia in sh-SMAD3 electroporated NPCs, a ciliopathy that might compromise the sensory functions of this organelle and ultimately contribute to the failure in central cell intercalation. Altogether, here we describe the cellular events driving SNT formation in the chick embryo and found a TGF-b/SMAD3-associated NTD. We anticipate our findings to be relevant to understand human SN and the embryonic origin of closed NTDs.