Neural tube closure defects result from fundamental failures of developmental processes during neurulation. Most common among these disorders is spina bifida, a failure of caudal tube closure, and anencephaly, a failure of rostral neural tube closure. A rare disorder, craniorachischisis, is a closure failure along the entire anterior-posterior axis from the midbrain-hindbrain boundary to the most caudal end of the neural tube. During neurulation in the mouse, the neural plate undergoes several morphological changes: neural folds are created, migrate toward the midline, and eventually fuse. As migratory cells intercalate with each other, the embryo is lengthened at the expense of the width within the medial-lateral plane in a process called convergent extension (CE). The first neural tube closure event (closure 1) begins at the boundary between the hindbrain and the cervical region and spreads both rostrally and caudally into the hindbrain and developing spinal cord, respectively. Thus, when closure 1 fails, the entire neural tube from the midbrain to the caudal neural tube remains open resulting in craniorachischisis1, 2
Currently, all known mutations that result in craniorachischisis in the mouse have been mapped to the vertebrate orthologs of components of the planar cell polarity (PCP) pathway, first identified for its role in tissue patterning of the fly1, 2
. Loss-of-function mutations in Vangl23, 4
double mutants7, 8
) as well as Ptk79
and Scribble10, 11
all cause craniorachischisis in the mouse, indicating that the non-canonical Wnt signaling pathway is critical for developmental events underlying the initiation of closure 1 during neurulation. Craniorachischisis in these mutants results from deficits in CE during the migratory movements required to initiate closure 112-14
Using a three-generation, forward-genetic screen for recessive mutations affecting neural development15
(Supplemental Figure 1
) we identified mouse line 811. Mutant 811 mice were easily distinguished from their littermates, as homozygous mutants within this allelic group developed craniorachischisis (). This mutant allele was found to segregate with Mendelian ratios with 48% (129/267) of parents carrying the allele and 12% (1/4 affected × 1/2 females are carriers) of embryos (232/1984) displaying the fully open neural tube (Supplemental Figure 2
). By embryonic day 18.5 (E18.5), approximately 33% of the mutants isolated were dead or dying but could still be scored and genotyped.
The mutation in mouse line 811 is Sec24b Y613
The genetic lesion underlying the craniorachischisis observed in line 811 was mapped to a 1.8 Mb region of chromosome 3 between rs13477397 and rs1347704 that contained 17 open reading frames. We sequenced the exons containing 5′UTR and coding sequence for the 14 loci without existing mouse models (Supplemental Methods; ). This sequence analysis revealed a single base pair substitution within all of the sequences analyzed. This point mutation was a T>A transversion in exon 9 of Sec24b. Two different transcript variants can be processed from the Sec24b locus of the mouse: ENMUST00000001079/NCBI (1079) and ENMUST00000098616 (98616) (, exon 9 highlighted in red). The T>A transversion is 2078T>A in 1079 and 1834T>A in 98616 (). The nucleotide substitution results in truncated proteins, Sec24b Y613 and Sec24b Y578 in 1079 and 98616, respectively (). Confirmation that the neural tube closure phenotype associated with line 811 is caused by the mutation in Sec24b (Sec24bY613) is provided by a second independently identified mouse mutant that harbors a distinct loss-of-function mutation in Sec24b (Sec24bS135X) and also exhibits chraniorachischisis (Frits Meijlink, Hubrecht Institute, Netherlands, personal communication). Sec24b is one of four mammalian orthologs of the yeast Sec24, and the first vertebrate Sec24 to be characterized in vivo.
Sec24b functions as a cargo-binding component of the COPII vesicle coat16-19
. These COPII vesicles are the primary pathway for active transport of secretory proteins from the ER to the Golgi. Thus, as an initial step in the forward secretion of nearly all non-ER-resident membrane and luminal proteins, COPII-mediated vesicle transport plays a key role in enabling the proper cellular localization of thousands of proteins. Sec23, the GTPase activating member of the complex, and Sec24 components form tight heterodimers in the cytosol, and this complex is then recruited to sites of active COPII budding – termed ER exit sites. Once recruited to the ER membrane, Sec24 proteins package cargo into the vesicle at sites of active budding. Based on co-crystal structural studies20
, it appears that Sec24b Y613 is truncated prior to the putative Sec23 binding site. Therefore, we predicted that Sec24b Y613 would lose its ability to associate with its Sec23 binding partners, rendering it functionally inactive. Indeed, we found that in contrast to wildtype Sec24b, mutant Sec24b Y613 failed to co-immunoprecipatate with either Sec23a or Sec23b in heterologous cells and was not concentrated at ER exit sites marked by another COPII component, Sec13 (Supplemental Figure 3
). Thus, Sec24bY613
functions as a loss-of-function allele encoding a protein that is incapable of associating with other components of the COPII complex and is not recruited to ER exit sites.
Given that other mouse mutants with craniorachischisis have deficits in CE12, 14, 21
, we analyzed the embryonic morphology of Sec24b
mutants during neurulation prior to neural tube closure. To do this, we examined Sec24bY613/Y613
mutants and control littermates at E8.5 for deficits in the length: width ratio. A decrease in this ratio reflects a developmental failure in the migratory movements required to lengthen the embryo and facilitate midline fusion14
. At the 6-, 7-, 8-, and 10-somite stages, Sec24bY613/Y613
mutants were shorter and wider than littermates, confirming a deficit in the morphogenic movements of CE (). These finding indicate that the developmental events underlying craniorachischisis in Sec24bY613
mutants are shared with other mouse mutants in the PCP-signaling pathway.
Sec24bY613/Y613 embryos have deficits in cochlear hair cell development and convergent extension
CE-deficient craniorachischisis is common to mouse mutants with deficits in the PCP-signaling pathway; thus, we sought to determine if other PCP-dependent phenotypes were also found in Sec24bY613/Y613 embryos. First, all mutants isolated at E18.5 were scored for omphalocele, a congenital birth defect in which the intestines, liver, and occasionally other organs develop outside of the abdomen because the tissues of the abdominal wall fail to fuse at the midline. We observed omphalocele in 45% (n=38) of the late gestation mutant embryos but in fewer than 1% (n=166) of littermate controls. Second, we examined the fusion of the eyelids in all of the E18.5 Sec24bY613/Y613 embryos. In nearly all of these mutants (99%, n=38), the upper and lower eyelids failed to fuse. In contrast, eyelid fusion failure was observed in only 2% (n=166) of littermate controls ().
Defects in the orientation of sensory hair cells of the cochlea and the vestibular system are also frequently observed in mice lacking core PCP components22
. Using immunological techniques to label the cilia of hair cells within these tissues, we examined the orientation of hair cells of the cochlea and vestibular system. In the cochlea, Sec24bY613/Y613
embryos exhibited deficits in the orientation of both the outer and inner hair cells. In addition, the alignment of both the outer and inner hair cells of the cochlea was abnormal in Sec24bY613/Y613
embryos with hair cells periodically falling out of phase from the row (). This defect was present across the entire organ of Corti. However, no differences between the Sec24bY613/Y613
mutants and littermate controls with respect to the orientation of hair cells in the vestibular system were observed. In addition, we examined the orientation of hair follicles of the back skin in Sec24bY613/Y613
embryos and their littermate controls. Hair follicle orientation of the mutants at this stage appeared relatively normal (data not shown). Therefore, in addition to craniorachischisis, Sec24bY613/Y613
mutants share most of the phenotypic characteristics of mice harboring mutations within components of the core PCP-signaling complex indicating that Sec24b might function by interacting with a component of the PCP pathway.
The role of Sec24b in the formation of COPII vesicles destined to transit between the ER and Golgi and the PCP-signaling dependent phenotypes found in Sec24bY613/Y613
mutants indicate that Sec24b Y613 might fail to properly sort and traffic a known member of the core PCP complex. Of the mouse mutants displaying craniorachischisis, five alleles code for proteins that are trafficked through the secretory pathway: Vangl2, Celsr1, Ptk7, Fzd6, and Fzd3. Of these genes, Vangl2
is the only dosage sensitive allele. Loss-of-function Vangl2
) heterozygotes have partial deficits with respect to neural tube closure that result in the looped-tail phenotype23
. Moreover, the Vangl2LP
allele genetically interacts with other members of the PCP signaling complex9, 10, 24
Given the functional centrality of Vangl2 in PCP signaling as well as the dosage sensitivity of Vangl2LP
, we tested for a genetic interaction between Sec24b
by crossing Vangl2+/LP
mice with Sec24b+/Y613
mice to create Vangl2+/LP; Sec24b+/Y613
embryos. In litters collected between E13.5 and E18.5, approximately 5% (n=40) of the Vangl2+/LP
embryos had a caudal neural tube closure defect. In striking contrast, 68% (n=18) of Vangl2+/LP; Sec24b+/Y613
embryos exhibited spina bifida. No littermates of other genotypes displayed this defect (). In addition, between late embryogenesis and four weeks of age, over 50% of the Vangl2+/LP; Sec24b+/Y613
mice died. The strong genetic interaction between Sec24b
suggests that Sec24b might directly regulate the trafficking and cell surface expression of Vangl2 during development.
Sec24b and Vangl2 genetically interact
Many transmembrane proteins are likely sorted into COPII vesicles via cargo binding sites common among all Sec24 proteins19
. However, the conservation of four distinct vertebrate paralogs suggests that each Sec24 may have evolved the ability to transport specific and essential cargos. To establish a direct measure of the ER export of Vangl2 and to assess the role of Sec24b in the trafficking of Vangl2, we used an in vitro
vesicle budding reaction25
. Using this assay, Vangl2-containing vesicles were formed in a COPII-dependent manner. More importantly, reactions supplemented with recombinant Sec24b, but not with the other Sec24 paralogs, substantially increased the amount of Vangl2 packaged into COPII vesicles. In contrast, another known COPII cargo protein Amyloid Precursor Protein (APP), which is packaged and transported in a COPII dependent manner, showed no specificity for any of the Sec24 paralogs (). Thus, Vangl2 is preferentially sorted by Sec24b during COPII vesicle formation from the ER.
Sec24b strongly enhances the ER export of Vangl2 but not Vangl2 looptail mutants
Two semi-dominant, loss-of-function alleles of Vangl2 have been mapped and isolated using classical genetics: Vangl2LP
. Heterozygotes of both alleles exhibit a looped-tail phenotype whereas homozygous mutants exhibit craniorachischisis. Both point mutations map to the cytosolic C-terminal domain of Vangl2 and could inhibit the ability of Vangl2 to interact with COPII or other cytosolic chaperones. Therefore, we assessed the ability of Vangl2 looptail mutant proteins to be packaged into COPII vesicles using the in vitro
vesicle budding assay. As before, Vangl2 was packaged into vesicles in a COPII dependent manner, but strikingly, neither Vangl2 S464N nor D255E was capable of entering COPII vesicles (). This effect was especially surprising given the conservative D255E substitution found in the Vangl2Lp-m1Jus
allele. Neither mutation depressed COPII budding, as other cargo proteins were still packaged normally (Supplemental Figure 4
). Correspondingly, Vangl2 D255E and S464N co-localize with Protein Disulfide Isomerase (PDI), an ER marker, and fail to reach the plasma membrane ().
Our findings suggest that the loss-of function phenotypes observed in Sec24bY613/Y613
mice result from trafficking defects in which the Vangl2 protein fails to package into COPII vesicles and exit the ER and are consistent with other studies showing a lack of Vangl2 plasma membrane localization in Vangl2LP
. To further test this idea, we used immunohistochemistry to analyze the subcellular localization of Vangl2 in the developing neural tube of both Sec24bY613/Y613
mutant mice. As a control, the subcellular localization of Beta-catenin, a membrane bound protein not associated with the PCP pathway, was assessed in the same tissue. Vangl2 protein was mislocalized to puncta within the cytoplasm of Sec24bY613/Y613
mutants while Vangl2 co-localized with Beta-catenin at the plasma membrane in wildtype embryos (). As previously reported26
, Vangl2 trafficking to the plasma membrane in Vangl2LP
mutants was aberrant (). In contrast, the pattern of subcellular distribution of Fzd3 in cells of the neural tube is comparable in wildtype, Sec24bY613/Y613
mice (Supplemental Figure 5
). These results are consistent with our in vitro
findings showing that Vangl2 is selectively sorted by Sec24b during COPII vesicle formation. Most fundamentally, these data indicate that Sec24b is essential for proper membrane localization of Vangl2 in vivo
Sec24bY613/Y613 embryos show aberrant subcellular localization of Vangl2 in the developing spinal cordin vivo
Here, we have identified Sec24b as a critical regulator of planar cell polarity signaling. In addition to the severe craniorachischisis phenotype, Sec24bY613 mutants have several other phenotypic abnormalities common to mouse mutants in components of the core PCP complex. Sec24b regulates cell surface expression of the dosage-dependent, core PCP protein Vangl2 by preferentially sorting this cargo into COPII vesicles. Additionally, Vangl2 looptail mutations prevent Vangl2 from being packaged into COPII vesicles and exiting the ER. Thus, it is likely that insufficient Vangl2 is brought to the cell surface during neurulation in Sec24b or Vangl2 mutants. This mislocalization of Vangl2 disrupts the entire PCP signaling cascade, resulting in a neural tube closure deficit similar to that observed in null alleles of other components of the core PCP complex. It is noteworthy that mice that are heterozygous for both Sec24bY613 and Vangl2LP have a propensity to develop spina bifida. Given that most human genetic diseases are not recessive but rather arise from the interactions of multiple loci, human mutations in Sec24b are likely to contribute to spina bifida.
Our analysis of Sec24b Y613 identifies a novel mechanism of protein regulation during development. Sec24b is one of four paralogs that function in the trafficking of secretory proteins between the ER and Golgi. Thus, loss of Sec24b function would be predicted to lead to deficits in the cell surface expression of some transmembrane proteins. Sec24b mutants, though clearly developmentally abnormal for neural tube closure, are relatively spared with respect to many other developmental processes. Vangl2 may be one of a small set of COPII cargo proteins whose expression is strictly dependent on Sec24b. Nonetheless, our findings indicate that cargo specificity has evolved between the four Sec24 paralogs with Sec24b being specifically required for trafficking of a core PCP component, Vangl2, and the establishment of planar cell polarity, convergent extension and neural tube closure.