Copp, A. J. | bioRxiv Science

bioRxiv2023

Caudal Fgfr1 disruption produces localised spinal mis-patterning and a terminal myelocystocele-like phenotype in mice

Closed spinal dysraphisms are poorly understood neurodevelopmental malformations commonly classed as neural tube defects. Several, including terminal myelocystocele, selectively affect the distal lumbosacral spine. We previously identified a neural tube closure-initiating point, Closure 5, involved in forming the distal spine of mice. Here we document equivalent morphology of the caudal-most end of the closing posterior neuropore (PNP) in mice and humans, suggesting Closure 5 is conserved in humans. It forms in a region of active fibroblast growth factor (FGF) signalling and pharmacological blockade of FGF receptors (Fgfr) impairs Closure 5 formation in cultured mouse embryos. Conditional genetic deletion of Fgfr1 in caudal embryonic tissues with Cdx2Cre similarly impairs Closure 5 formation and leads to morphologically abnormal PNPs, which nonetheless achieve delayed closure although delayed. After PNP closure, a localised region of the distal neural tube of Fgfr1-disrupted embryos re-opens into a trumpet-like flared central canal between the presumptive hindlimbs, progressing to form a distal fluid-filled sac overlying ventrally flattened spinal cord. This phenotype resembles terminal myelocystocele. Histological analysis of spinal progenitor domains reveals regional and progressive loss of ventral spinal cord progenitor domains preceding cystic dilation of the central canal. Initially, the Shh and FoxA2-positive ventral domains are lost, resulting in Olig2-labelling of the ventral-most neural tube. The Olig2-domain is also subsequently lost, eventually producing a neural tube entirely positive for the dorsal marker Pax3. Thus, a terminal myelocystocele-like phenotype can arise after completion of neural tube closure due to localised spinal mis-patterning caused by disruption of Fgfr1 signalling.

developmental biology↗

bioRxiv2021

Severe neural tube defects due to failure of closure initiation can arise without abnormality of neuroepithelial convergent extension

Planar cell polarity (PCP) signalling is vital for initiation of neural tube closure in mice, with diminished convergent extension (CE) cell movements leading to a severe form of neural tube defect (NTD), termed craniorachischisis (CRN). Some human NTDs are also associated with PCP gene mutations, but affected individuals are generally heterozygous, whereas PCP homozygosity or compound heterozygosity is needed to produce CRN in mice. This suggests human NTDs may involve other genetic or environmental factors, that interact with partial loss of PCP function. We found that reduced sulfation OF glycosaminoglycans (GAGs) interacts with heterozygosity for the Lp allele of Vangl2 (a core PCP gene), to cause CRN in mice. Here, we hypothesised that this GAG-PCP interaction may regulate convergent extension movements, and hence lead to severe NTDs in the context of only partial loss of PCP function. Both Lp and null alleles of Vangl2 gave similar findings. Culture of E8.5 embryos in the presence of chlorate (a GAG sulfation inhibitor), or enzymatic cleavage of GAG chains, led to failure of NT closure initiation in the majority of Lp/+ embryos, whereas few +/+ littermates exhibited CRN. The chlorate effect was rescued by exogenous sulphate. Surprisingly, DiO labeling of the embryonic node demonstrated no abnormality of midline axial extension in chlorate-treated Lp/+ embryos that developed CRN. In contrast, positive control Lp/Lp embryos displayed severe convergent extension defects in this assay. Morphometric analysis of the closure initiation site revealed abnormalities in the size and shape of somites that flank the closing neural tube in chlorate-treated Lp/+ embryos. We conclude that severe NTDs involving failure of closure initiation can arise by a mechanism other than faulty neuroepithelial convergent extension. Matrix-mediated expansion of somites, flanking the closing neural tube, may be required for closure initiation.

developmental biology↗