The loop-tail mouse model displays open and closed caudal neural tube defects

ABSTRACT Neural tube defects (NTDs) are the second most common cause of congenital malformations and are often studied in animal models. Loop-tail (Lp) mice carry a mutation in the Vangl2 gene, a member of the Wnt-planar cell polarity pathway. In Vangl2+/Lp embryos, the mutation induces a failure in the completion of caudal neural tube closure, but only a small percentage of embryos develop open spina bifida. Here, we show that the majority of Vangl2+/Lp embryos developed caudal closed NTDs and presented cellular aggregates that may facilitate the sealing of these defects. The cellular aggregates expressed neural crest cell markers and, using these as a readout, we describe a systematic method to assess the severity of the neural tube dorsal fusion failure. We observed that this defect worsened in combination with other NTD mutants, Daam1 and Grhl3. Besides, we found that in Vangl2+/Lp embryos, these NTDs were resistant to maternal folic acid and inositol supplementation. Loop-tail mice provide a useful model for research on the molecular interactions involved in the development of open and closed NTDs and for the design of prevention strategies for these diseases.

Disease Models & Mechanisms: doi:10.1242/dmm.050175This table represents the different measurements carried out using the CAs to assess the severity of the phenotype observed in the different experimental groups of the study: Vangl2 +/Lp embryos; the double mutants Vangl2 +/Lp /Daam1 +/gt and Vangl2 +/Lp /Grhl3 +/Ct ; and Vangl2 +/Lp embryos under different supplementation conditions: NS, not supplemented; FA, supplemented with folic acid; MI, supplemented with myo-inositol; CI, supplemented with D-chiro-inositol; FA+CI, supplemented with the combination of folic acid and D-chiro-inositol.The incidence represents the percentage of embryos with CAs, including the total number of embryos analysed in brackets.The number of CAs per embryo is shown as mean ± SD with the total number of CAs in brackets.The significant increase in the number of CAs in Vangl2 +/Lp /Grhl3 +/Ct vs Vangl2 +/Lp embryos was determined using an unpaired t test (P<0.0001).The number of CAs that can be found in an embryo, from 1 to 7, is represented by the percentage of embryos that present a given number of CAs, with the total number of embryos shown in brackets.
The decrease in the percentage of Vangl2 +/Lp /Grhl3 +/Ct embryos that only had 1 or 2 CAs compared to Vangl2 +/Lp embryos was statistically significant as determined by Chi-square test (P<0.0001).The NT area affected by dorsal fusion failure is represented by the CA size in µm 3 shown as mean ± SD with the number of embryos analysed in brackets.The significant increase in the CA size in Vangl2 +/Lp /Daam1 +/gt and Vangl2 +/Lp /Grhl3 +/Ct vs Vangl2 +/Lp embryos was determined by unpaired t test (P=0.0002and P<0.0001, respectively).Late NT closure progression is represented by the percentage of CAs associated to a closed NT, including the total number of CAs studied in brackets.Tail curvature, in mm -1 , is represented by the mean ± SD of the inverse of the radius of the best-fit circumference plotted along the curvature of the tail, with the number of embryos analysed in brackets.Since the tail grows over time, this parameter was measured exclusively in E12.5 embryos.The significant changes in tail curvature observed in Vangl2 +/Lp /Grhl3  This tables summarises the characteristics of litters and pups of Vangl2 +/Lp dams under different supplementation conditions: NS, not supplemented; FA, supplemented with 10 ppm folic acid; MI, supplemented with 800 µg/g/day of myo-inositol; CI, supplemented with 800 µg/g/day D-chiro-inositol; FA+CI, supplemented with a combination of 10 ppm FA and 800 µg/g/day of CI.Dam weight gain (from E0.5 to E12.5), litter size, and crown-rump length are shown as mean ± SD, with the number of dams, litters, and embryos, respectively, shown in brackets.The percentage of live embryos was calculated by dividing the number of live embryos (subtracting the reabsorptions) by the total number of implants, shown in brackets.The Mendelian distribution and NTDs incidence (open spina bifida, exencephaly, and craniorachischisis) are shown as percentages with the number of embryos shown in brackets.The significant decrease in weight gain observed in Vangl2 +/Lp dams supplemented with CI vs NS dams was determined by one-way ANOVA followed by Dunnett's multiple comparison test (P=0.0285).This test was also used to study the litter size, percentage of live embryos, and crown-rump length.The Mendelian distribution was studied by Chi-square test.* P<0.05.‡ We observed the exencephaly phenotype -which had not been previously associated with the Wnt-PCP pathway-in all supplementation conditions except myo-inositol.This phenotype appeared after confinement for the Covid-19 pandemic, when the mouse line was maintained with a reduced number of animals.This strategy, as recently published (Moncaut and Hart-Johnson, 2021), may have been the cause of the inevitable acceleration of genetic drift and accumulation of mutations.This would also explain why this phenotype was not seen after myo-inositol supplementation, as these experiments were done before confinement.

Disease Models & Mechanisms • Supplementary information
We have used whole mount in situ hybridisation for Sox10 and corresponding sections to establish a protocol to assess the degree of severity of dorsal fusion impairment.The following parameters were measured.
1. Incidence.The parameter incidence refers to the percentage of embryos that present the phenotype, in this case the presence of cellular aggregates (CAs).This parameter can be measured from stereoscopic images of whole embryos after in situ hybridisation.Incidence can be considered as an indicator of the severity of dorsal neural tube (NT) closure failure.A high severity of the phenotype would correlate with a high incidence, as observed in Vangl2 +/Lp embryos that present a 100% incidence, whereas a milder phenotype would imply a lower incidence.

Number of regions affected by NT fusion failure.
Embryos can present one or more CAs, reflecting the number of regions affected by NT fusion failure.The number of CAs per embryo can be assessed from stereoscopic images of whole embryos after in situ hybridisation for Sox10.For this parameter, 0 is considered when an embryo does not present any CAs in a given zone.A worsening of the phenotype would imply an increase in the number of CAs per embryo, and an improvement a reduction therein.

Location of NT fusion failure.
The study of whole embryos revealed that most embryos developed more than one CA (Fig. 4B) and these were located within 3 specific zones: zone anterior to somite 29 (≤29s; Fig. 4D); intermediate zone, between somites 30 and 33 (30-33s; Fig. 4E,F); and the caudal zone, posterior to somite 34 (≥34s; Fig. 4G).The location of CAs with respect to these three defined anteroposterior regions can be assessed from stereoscopic images of whole embryos after in situ hybridisation for Sox10.As the presence of anterior CAs seems to be associated with an increase in the severity of the defect, worsening of the phenotype would lead to a greater presence of CAs in anterior positions, while a shift of the presence of CAs to more caudal areas would imply a milder phenotype.

NT affected by dorsal fusion failure.
Given that the CA appeared to fill the space left by a dorsal fusion failure, the size of the CAs can be measured as a readout of NT damage.To do so, CA area can be measured to from 40x micrographs of transverse sections (e.g., using ImageJ polygon tool or similar) noting that the area taken into consideration is the entire CA, as observed morphologically, and is not limited to the area marked with Sox10.The extent of damage in the dorsal NT is then calculated by summing up the areas of all the CAs found in an embryo, and multiplying this number by the thickness of the sections.For this parameter,

Supplementary Materials and Methods
0 is considered when an embryo does not present any CAs in a given zone.For this parameter, the severity of the phenotype correlated directly with the size of CAs, and thus, an improvement of the phenotype would result in a reduction of CA size, and a worsening of the phenotype would imply larger CAs.

Late NT closure progression.
In what we have termed "late NT closure progression" we found that some CAs were below/over a closed NT, indicating that although CAs formed where the NT had failed to fuse, the presence of the CA may have facilitated late closure, thereby attenuating dorsal damage.Thus, an increase in CAs associated with a closed NT would be related to an improvement of the phenotype, while an increase in CAs associated with an open NT would be expected to be found with more severe phenotypes.This parameter can be evaluated by visually determining whether each CA is associated with a closed or open NT using 40x micrographs of transverse sections.
6. Tail curvature.One of the most obvious characteristics of Vangl2 +/Lp mutants is the presence of a curled tail.The curvature of the caudal region or tail curvature (κ) can be measured from stereoscopic images of whole embryos by measuring the radius (r) of a circumference that is drawn (e.g., using ImageJ software) following the curvature of the tail embryos and applying the formula κ=1/r.Since this parameter is associated with caudal damage (De Castro et al., 2018), it would be expected that a greater curvature of the tail turn would be associated with more severe phenotypes than a lesser curvature.

Table S1 . General characteristics of the phenotype of the embryos analysed in the study using the CAs as a readout of dorsal NT closure failure.
The number of CAs in each zone the CA size (in µm 3 ) are shown as mean ± SD with the number of CAs and embryos, respectively, shown in brackets.The late NT closure progression is represented by the percentage of CAs associated to a closed NT, with the total number of CAs studied shown in brackets.The significant increase in the number and size of CAs in the ≤29s zone in Vangl2 +/Lp /Grhl3 +/Ct embryos and in the CA size in the 30-33s zone in Vangl2 +/Lp /Daam1 +/gt embryos compared to Vangl2 +/Lp was determined by two-way ANOVA followed by Šídák's multiple comparisons test.**** P<0.0001.

Protocol to assess the severity of the caudal neural tube fusion failure in Vangl2 +/Lp embryos using the cellular aggregates (CAs) as a readout.
DiseaseModels & Mechanisms: doi:10.1242/dmm.050175:Supplementary information Disease Models & Mechanisms • Supplementary information