Ana Rolo

Neural tube closure: cellular, molecular and biomechanical mechanisms.

Neural tube closure has been studied for many decades, across a range of vertebrates, as a paradigm of embryonic morphogenesis. Neurulation is of particular interest in view of the severe congenital malformations – ‘neural tube defects’ – that result when closure fails. The process of neural tube closure is complex and involves cellular events such as convergent extension, apical constriction and interkinetic nuclear migration, as well as precise molecular control via the non-canonical Wnt/planar cell polarity pathway, Shh/BMP signalling, and the transcription factors Grhl2/3, Pax3, Cdx2 and Zic2. More recently, biomechanical inputs into neural tube morphogenesis have also been identified. Here, we review these cellular, molecular and biomechanical mechanisms involved in neural tube closure, based on studies of various vertebrate species, focusing on the most recent advances in the field. Summary: This Review discusses the cellular, molecular and biomechanical mechanisms involved in neural tube closure, focusing on the most recent advances in the field.

Epithelial Fusion during Neural Tube Morphogenesis

Adhesion and fusion of epithelial sheets marks the completion of many morphogenetic events during embryogenesis. Neural tube closure involves an epithelial fusion sequence in which the apposing neural folds adhere initially via cellular protrusions, proceed to a more stable union, and subsequently undergo remodeling of the epithelial structures to yield a separate neural tube roof plate and overlying nonneural ectoderm. Cellular protrusions comprise lamellipodia and filopodia, and studies in several different systems emphasize the critical role of RhoGTPases in their regulation. How epithelia establish initial adhesion is poorly understood but, in neurulation, may involve interactions between EphA receptors and their ephrinA ligands. Epithelial remodeling is spatially and temporally correlated with apoptosis in the dorsal neural tube midline, but experimental inhibition of this cell death does not prevent fusion and remodeling. A variety of molecular signaling systems have been implicated in the late events of morphogenesis, but genetic redundancy, for example among the integrins and laminins, makes identification of the critical players challenging. An improved understanding of epithelial fusion can provide insights into normal developmental processes and may also indicate the mode of origin of clinically important birth defects.

Unexpected activities of Smad7 in Xenopus mesodermal and neural induction

Neural induction is widely believed to be a direct consequence of inhibition of BMP pathways. Because of conflicting results and interpretations, we have re-examined this issue in Xenopus and chick embryos using the powerful and general TGFbeta inhibitor, Smad7, which inhibits both Smad1- (BMP) and Smad2- (Nodal/Activin) mediated pathways. We confirm that Smad7 efficiently inhibits phosphorylation of Smad1 and Smad2. Surprisingly, however, over-expression of Smad7 in Xenopus ventral epidermis induces expression of the dorsal mesodermal markers Chordin and Brachyury. Neural markers are induced, but in a non-cell-autonomous manner and only when Chordin and Brachyury are also induced. Simultaneous inhibition of Smad1 and Smad2 by different approaches does not account for all Smad7 effects, indicating that Smad7 has activities other than inhibition of the TGFbeta pathway. We provide evidence that these effects are independent of Wnt, FGF, Hedgehog and retinoid signalling. We also show that these effects are due to elements outside of the MH2 domain of Smad7. Together, these results indicate that BMP inhibition is not sufficient for neural induction even when Nodal/Activin is also blocked, and that Smad7 activity is considerably more complex than had previously been assumed. We suggest that experiments relying on Smad7 as an inhibitor of TGFbeta-pathways should be interpreted with considerable caution.

Biomechanical coupling facilitates spinal neural tube closure in mouse embryos

Significance Neurulation has been intensively studied in lower vertebrates, but marked species differences call into question the relevance of these models for human neural tube (NT) closure. Here, using mouse embryos, we demonstrate that mammalian neural fold apposition results from constriction of the open posterior NT, which is biomechanically coupled to the zippering point by an F-actin network. Using the Zic2 mutant model, we show that genetic predisposition to spina bifida, which likely underlies most human cases, directly affects the biomechanics of closure. We also identify a NT closure point at the caudal end of the embryo. Many spina bifida cases correspond to this anatomic portion of the NT, suggesting that this closure point may be important in humans as well. Neural tube (NT) formation in the spinal region of the mammalian embryo involves a wave of “zippering” that passes down the elongating spinal axis, uniting the neural fold tips in the dorsal midline. Failure of this closure process leads to open spina bifida, a common cause of severe neurologic disability in humans. Here, we combined a tissue-level strain-mapping workflow with laser ablation of live-imaged mouse embryos to investigate the biomechanics of mammalian spinal closure. Ablation of the zippering point at the embryonic dorsal midline causes far-reaching, rapid separation of the elevating neural folds. Strain analysis revealed tissue expansion around the zippering point after ablation, but predominant tissue constriction in the caudal and ventral neural plate zone. This zone is biomechanically coupled to the zippering point by a supracellular F-actin network, which includes an actin cable running along the neural fold tips. Pharmacologic inhibition of F-actin or laser ablation of the cable causes neural fold separation. At the most advanced somite stages, when completion of spinal closure is imminent, the cable forms a continuous ring around the neuropore, and simultaneously, a new caudal-to-rostral zippering point arises. Laser ablation of this new closure initiation point causes neural fold separation, demonstrating its biomechanical activity. Failure of spinal closure in pre-spina bifida Zic2Ku mutant embryos is associated with altered tissue biomechanics, as indicated by greater neuropore widening after ablation. Thus, this study identifies biomechanical coupling of the entire region of active spinal neurulation in the mouse embryo as a prerequisite for successful NT closure.

Rho GTPases in mammalian spinal neural tube closure

ABSTRACT Neural tube closure is an important morphogenetic event that involves dramatic reshaping of both neural and non-neural tissues. Rho GTPases are key cytoskeletal regulators involved in cell motility and in several developmental processes, and are thus expected to play pivotal roles in neurulation. Here, we discuss 2 recent studies that shed light on the roles of distinct Rho GTPases in different tissues during neurulation. RhoA plays an essential role in regulating actomyosin dynamics in the neural epithelium of the elevating neural folds, while Rac1 is required for the formation of cell protrusions in the non-neural surface ectoderm during neural fold fusion.

Overexpression of Grainyhead-like 3 causes spina bifida and interacts genetically with mutant alleles of Grhl2 and Vangl2 in mice

&NA; The genetic basis of human neural tube defects (NTDs), such as anencephaly and spina bifida (SB), is complex and heterogeneous. Grainyhead‐like genes represent candidates for involvement in NTDs based on the presence of SB and exencephaly in mice carrying loss‐of‐function alleles of Grhl2 or Grhl3. We found that reinstatement of Grhl3 expression, by bacterial artificial chromosome (BAC)‐mediated transgenesis, prevents SB in Grhl3‐null embryos, as in the Grhl3 hypomorphic curly tail strain. Notably, however, further increase in expression of Grhl3 causes highly penetrant SB. Grhl3 overexpression recapitulates the spinal NTD phenotype of loss‐of‐function embryos, although the underlying mechanism differs. However, it does not phenocopy other defects of Grhl3‐null embryos such as abnormal axial curvature, cranial NTDs (exencephaly) or skin barrier defects, the latter being rescued by the Grhl3‐transgene. Grhl2 and Grhl3 can form homodimers and heterodimers, suggesting a possible model in which defects arising from overexpression of Grhl3 result from sequestration of Grhl2 in heterodimers, mimicking Grhl2 loss of function. This hypothesis predicts that increased abundance of Grhl2 would have an ameliorating effect in Grhl3 overexpressing embryo. Instead, we observed a striking additive genetic interaction between Grhl2 and Grhl3 gain‐of‐function alleles. Severe SB arose in embryos in which both genes were expressed at moderately elevated levels that individually do not cause NTDs. Furthermore, moderate Grhl3 overexpression also interacted with the Vangl2Lp allele to cause SB, demonstrating genetic interaction with the planar cell polarity signalling pathway that is implicated in mouse and human NTDs.

04-P009 Notch activation is required for response to BMP inhibition in neural induction

if a simplified GRN (sGRN) is found in axolotl, an amphibian with primitive traits. We found that a simpler network does exist in the axolotl, with only one Mix type and two Nodal genes identified. Using morpholinos targeted to disrupt splicing, we demonstrate a single Nodal gene is required for the formation of the mesoderm and endoderm. The phenotype we observe recapitulates that seen in Xenopus when all Nodal activity is inhibited by treatment with a small molecule inhibitor (SB-431542). Unexpectedly, knockdown of the single Mix gene using a similar approach reveals a role for Mix in the specification of the mesoderm from the endoderm. We speculate this novel role has been previously obscured in Xenopus due to the presence of multiple Mix-like genes. We propose that GRN evolution is constrained in vertebrates in which germ cells are specified by epigenesis, a basal animal trait.