Ovol2 promoter mutations in mice and human illuminate species-specific phenotypic divergence.

Pathogenic variants in the highly conserved OVOL2 promoter region cause posterior polymorphous corneal dystrophy (PPCD) 1 by inducing an ectopic expression of the endothelial OVOL2 mRNA. Here we produced an allelic series of Ovol2 promoter mutations in the mouse model including the heterozygous c.-307T>C variant (RefSeq NM_021220.4) causing PPCD1 in humans. Despite the high evolutionary conservation of the Ovol2 promoter, only some alterations of its sequence had phenotypic consequences in mice. Four independent sequence variants in the distal part of the Ovol2 promoter had no significant effect on endothelial Ovol2 mRNA level or caused any ocular phenotype. In contrast, the mutation c.-307T>C resulted in increased Ovol2 expression in the corneal endothelium. However, only a small fraction of adult mice c.-307T>C heterozygotes developed ocular phenotypes such as irido-corneal adhesions, and corneal opacity. Interestingly, phenotypic penetrance was increased at embryonic stages. Notably, c.-307T>C mutation is located next to the Ovol1/Ovol2 transcription factor binding site. Mice carrying an allele with a deletion encompassing the Ovol2 binding site c.-307_-320del showed significant Ovol2 gene upregulation in the cornea endothelium and exhibited phenotypes similar to the c.-307T>C mutation. In conclusion, although the mutations c.-307T>C and -307_-320del lead to a comparably strong increase in endothelial Ovol2 expression as seen in PPCD1 patients, endothelial dystrophy was not observed in the mouse model, implicating species-specific differences in endothelial cell biology. Nonetheless, the emergence of dominant ocular phenotypes associated with Ovol2 promoter variants in mice implies a potential role of this gene in eye development and disease.

Validated WGS and WES protocols proved saliva-derived gDNA as an equivalent to blood-derived gDNA for clinical and population genomic analyses.

BACKGROUND: Whole exome sequencing (WES) and whole genome sequencing (WGS) have become standard methods in human clinical diagnostics as well as in population genomics (POPGEN). Blood-derived genomic DNA (gDNA) is routinely used in the clinical environment. Conversely, many POPGEN studies and commercial tests benefit from easy saliva sampling. Here, we evaluated the quality of variant call sets and the level of genotype concordance of single nucleotide variants (SNVs) and small insertions and deletions (indels) for WES and WGS using paired blood- and saliva-derived gDNA isolates employing genomic reference-based validated protocols. METHODS: The genomic reference standard Coriell NA12878 was repeatedly analyzed using optimized WES and WGS protocols, and data calls were compared with the truth dataset published by the Genome in a Bottle Consortium. gDNA was extracted from the paired blood and saliva samples of 10 participants and processed using the same protocols. A comparison of paired blood-saliva call sets was performed in the context of WGS and WES genomic reference-based technical validation results. RESULTS: The quality pattern of called variants obtained from genomic-reference-based technical replicates correlates with data calls of paired blood-saliva-derived samples in all levels of tested examinations despite a higher rate of non-human contamination found in the saliva samples. The F1 score of 10 blood-to-saliva-derived comparisons ranged between 0.8030-0.9998 for SNVs and between 0.8883-0.9991 for small-indels in the case of the WGS protocol, and between 0.8643-0.999 for SNVs and between 0.7781-1.000 for small-indels in the case of the WES protocol. CONCLUSION: Saliva may be considered an equivalent material to blood for genetic analysis for both WGS and WES under strict protocol conditions. The accuracy of sequencing metrics and variant-detection accuracy is not affected by choosing saliva as the gDNA source instead of blood but much more significantly by the genomic context, variant types, and the sequencing technology used.

MEIS-WNT5A axis regulates development of fourth ventricle choroid plexus.

The choroid plexus (ChP) produces cerebrospinal fluid and forms an essential brain barrier. ChP tissues form in each brain ventricle, each one adopting a distinct shape, but remarkably little is known about the mechanisms underlying ChP development. Here, we show that epithelial WNT5A is crucial for determining fourth ventricle (4V) ChP morphogenesis and size in mouse. Systemic Wnt5a knockout, or forced Wnt5a overexpression beginning at embryonic day 10.5, profoundly reduced ChP size and development. However, Wnt5a expression was enriched in Foxj1-positive epithelial cells of 4V ChP plexus, and its conditional deletion in these cells affected the branched, villous morphology of the 4V ChP. We found that WNT5A was enriched in epithelial cells localized to the distal tips of 4V ChP villi, where WNT5A acted locally to activate non-canonical WNT signaling via ROR1 and ROR2 receptors. During 4V ChP development, MEIS1 bound to the proximal Wnt5a promoter, and gain- and loss-of-function approaches demonstrated that MEIS1 regulated Wnt5a expression. Collectively, our findings demonstrate a dual function of WNT5A in ChP development and identify MEIS transcription factors as upstream regulators of Wnt5a in the 4V ChP epithelium.

Meis homeobox genes control progenitor competence in the retina.

The vertebrate eye is derived from the neuroepithelium, surface ectoderm, and extracellular mesenchyme. The neuroepithelium forms an optic cup in which the spatial separation of three domains is established, namely, the region of multipotent retinal progenitor cells (RPCs), the ciliary margin zone (CMZ)-which possesses both a neurogenic and nonneurogenic potential-and the optic disk (OD), the interface between the optic stalk and the neuroretina. Here, we show by genetic ablation in the developing optic cup that Meis1 and Meis2 homeobox genes function redundantly to maintain the retinal progenitor pool while they simultaneously suppress the expression of genes characteristic of CMZ and OD fates. Furthermore, we demonstrate that Meis transcription factors bind regulatory regions of RPC-, CMZ-, and OD-specific genes, thus providing a mechanistic insight into the Meis-dependent gene regulatory network. Our work uncovers the essential role of Meis1 and Meis2 as regulators of cell fate competence, which organize spatial territories in the vertebrate eye.

Multiple roles of Pax6 in postnatal cornea development.

Mammalian corneal development is a multistep process, including formation of the corneal epithelium (CE), endothelium and stroma during embryogenesis, followed by postnatal stratification of the epithelial layers and continuous renewal of the epithelium to replace the outermost corneal cells. Here, we employed the Cre-loxP system to conditionally deplete Pax6 proteins in two domains of ocular cells, i.e., the ocular surface epithelium (cornea, limbus and conjunctiva) (OSE) or postnatal CE via K14-cre or Aldh3-cre, respectively. Earlier and broader inactivation of Pax6 in the OSE resulted in thickened OSE with CE and limbal cells adopting the conjunctival keratin expression pattern. More restricted depletion of Pax6 in postnatal CE resulted in an abnormal cornea marked by reduced epithelial thickness despite increased epithelial cell proliferation. Immunofluorescence studies revealed loss of intermediate filament Cytokeratin 12 and diffused expression of adherens junction components, together with reduced tight junction protein, Zonula occludens-1. Furthermore, the expression of Cytokeratin 14, a basal cell marker in apical layers, indicates impaired differentiation of CE cells. Collectively, our data demonstrate that Pax6 is essential for maintaining proper differentiation and strong intercellular adhesion in postnatal CE cells, whereas limbal Pax6 is required to prevent the outgrowth of conjunctival cells to the cornea.

Identification of Nodal-dependent enhancer of amphioxus Chordin sufficient to drive gene expression into the chordate dorsal organizer.

The core molecular mechanisms of dorsal organizer formation during gastrulation are highly conserved within the chordate lineage. One of the key characteristics is that Nodal signaling is required for the organizer-specific gene expression. This feature appears to be ancestral, as evidenced by the presence in the most basally divergent chordate amphioxus. To provide a better understanding of the evolution of organizer-specific gene regulation in chordates, we analyzed the cis-regulatory sequence of amphioxus Chordin in the context of the vertebrate embryo. First, we generated stable zebrafish transgenic lines, and by using light-sheet fluorescent microscopy, characterized in detail the expression pattern of GFP driven by the cis-regulatory sequences of amphioxus Chordin. Next, we performed a 5'deletion analysis and identified an enhancer sufficient to drive the expression of the reporter gene into a chordate dorsal organizer. Finally, we found that the identified enhancer element strongly depends on Nodal signaling, which is consistent with the well-established role of this pathway in the regulation of the expression of dorsal organizer-specific genes across chordates. The enhancer identified in our study may represent a suitable simple system to study the interplay of the evolutionarily conserved regulatory mechanisms operating during early chordate development.

Minimal effect of conditional ferroportin KO in the neural retina implicates ferrous iron in retinal iron overload and degeneration.

Iron-induced oxidative stress can cause or exacerbate retinal degenerative diseases. Retinal iron overload has been reported in several mouse disease models with systemic or neural retina-specific knockout (KO) of homologous ferroxidases ceruloplasmin (Cp) and hephaestin (Heph). Cp and Heph can potentiate ferroportin (Fpn) mediated cellular iron export. Here, we used retina-specific Fpn KO mice to test the hypothesis that retinal iron overload in Cp/Heph DKO mice is caused by impaired iron export from neurons and glia. Surprisingly, there was no indication of retinal iron overload in retina-specific Fpn KO mice: the mRNA levels of transferrin receptor in the retina were not altered at 7-10-months age. Consistent with this, levels and localization of ferritin light chain were unchanged. To "stress the system", we injected iron intraperitoneally into Fpn KO mice with or without Cp KO. Only mice with both retina-specific Fpn KO and Cp KO had modestly elevated retinal iron levels. These results suggest that impaired iron export through Fpn is not sufficient to explain the retinal iron overload in Cp/Heph DKO mice. An increase in the levels of retinal ferrous iron caused by the absence of these ferroxidases, followed by uptake into cells by ferrous iron importers, is most likely necessary.

Conditional knockout of hephaestin in the neural retina disrupts retinal iron homeostasis.

Iron accumulation has been implicated in degenerative retinal diseases. It can catalyze the production of damaging reactive oxygen species. Previous work has demonstrated iron accumulation in multiple retinal diseases, including age-related macular degeneration and diabetic retinopathy. In mice, systemic knockout of the ferroxidases ceruloplasmin (Cp) and hephaestin (Heph), which oxidize iron, results in retinal iron accumulation and iron-induced degeneration. To determine the role of Heph in the retina, we generated a neural retina-specific Heph knockout on a background of systemic Cp knockout. This resulted in elevated neural retina iron. Conversely, retinal ganglion cells had elevated transferrin receptor and decreased ferritin, suggesting diminished iron levels. The retinal degeneration observed in systemic Cp-/-, Heph-/- mice did not occur. These findings indicate that Heph has a local role in regulating neural retina iron homeostasis, but also suggest that preserved Heph function in either the RPE or systemically mitigates the degeneration phenotype observed in the systemic Cp-/-, Heph-/- mice.

Targeted deletion of Crb1/Crb2 in the optic vesicle models key features of leber congenital amaurosis 8.

The Crb1 and 2 (Crumbs homolog 1 & 2) genes encode large, single-pass transmembrane proteins essential for the apicobasal polarity and adhesion of epithelial cells. Crb1 mutations cause degenerative retinal diseases in humans, including Leber congenital amaurosis type 8 (LCA8) and retinitis pigmentosa type 12 (RP12). In LCA8, impaired photoreceptor development and/or survival is thought to cause blindness during early infancy, whereas, in RP12, progressive photoreceptor degeneration damages peripheral vision later in life. There are multiple animal models of RP12 pathology, but no experimental model of LCA8 recapitulates the full spectrum of its pathological features. To generate a mouse model of LCA8 and identify the functions of Crb1/2 in developing ocular tissues, we used an mRx-Cre driver to generate allelic combinations that enabled conditional gene ablation from the optic vesicle stage. In this series only Crb1/2 double knockout (dKO) mice exhibited characteristics of human LCA8 disease: locally thickened retina with spots devoid of cells, aberrant positioning of retinal cells, severely disrupted lamination, and depigmented retinal-pigmented epithelium. Retinal defects antedated E12.5, which is far earlier than the stage at which photoreceptor cells mainly differentiate. Most remarkably, Crb1/Crb2 dKO showed a severely attenuated electroretinogram at the eye opening stage. These results suggest that human LCA8 can be modeled in the mouse by simultaneously ablating Crb1/2 from the beginning of eye development. Importantly, they also indicate that LCA8 is caused by malfunction of retinal progenitor cells during early ocular development rather than by defective photoreceptor-Muller glial interaction, a mechanism proposed for RP12.

Liver-Specific, but Not Retina-Specific, Hepcidin Knockout Causes Retinal Iron Accumulation and Degeneration.

The liver secretes hepcidin (Hepc) into the bloodstream to reduce blood iron levels. Hepc accomplishes this by triggering degradation of the only known cellular iron exporter ferroportin in the gut, macrophages, and liver. We previously demonstrated that systemic Hepc knockout (HepcKO) mice, which have high serum iron, develop retinal iron overload and degeneration. However, it was unclear whether this is caused by high blood iron levels or, alternatively, retinal iron influx that would normally be regulated by retina-produced Hepc. To address this question, retinas of liver-specific and retina-specific HepcKO mice were studied. Liver-specific HepcKO mice had elevated blood and retinal pigment epithelium (RPE) iron levels and increased free (labile) iron levels in the retina, despite an intact blood-retinal barrier. This led to RPE hypertrophy associated with lipofuscin-laden lysosome accumulation. Photoreceptors also degenerated focally. In contrast, there was no change in retinal or RPE iron levels or degeneration in the retina-specific HepcKO mice. These data indicate that high blood iron levels can lead to retinal iron accumulation and degeneration. High blood iron levels can occur in patients with hereditary hemochromatosis or result from use of iron supplements or multiple blood transfusions. Our results suggest that high blood iron levels may cause or exacerbate retinal disease.

The Bmp signaling pathway regulates development of left-right asymmetry in amphioxus.

Establishment of asymmetry along the left-right (LR) body axis in vertebrates requires interplay between Nodal and Bmp signaling pathways. In the basal chordate amphioxus, the left-sided activity of the Nodal signaling has been attributed to the asymmetric morphogenesis of paraxial structures and pharyngeal organs, however the role of Bmp signaling in LR asymmetry establishment has not been addressed to date. Here, we show that Bmp signaling is necessary for the development of LR asymmetric morphogenesis of amphioxus larvae through regulation of Nodal signaling. Loss of Bmp signaling results in loss of the left-sided expression of Nodal, Gdf1/3, Lefty and Pitx and in gain of ectopic expression of Cerberus on the left side. As a consequence, the larvae display loss of the offset arrangement of axial structures, loss of the left-sided pharyngeal organs including the mouth, and ectopic development of the right-sided organs on the left side. Bmp inhibition thus phenocopies inhibition of Nodal signaling and results in the right isomerism. We conclude that Bmp and Nodal pathways act in concert to specify the left side and that Bmp signaling plays a fundamental role during LR development in amphioxus.

Polycomb repression complex 2 is required for the maintenance of retinal progenitor cells and balanced retinal differentiation.

Polycomb repressive complexes maintain transcriptional repression of genes encoding crucial developmental regulators through chromatin modification. Here we investigated the role of Polycomb repressive complex 2 (PRC2) in retinal development by inactivating its key components Eed and Ezh2. Conditional deletion of Ezh2 resulted in a partial loss of PRC2 function and accelerated differentiation of Müller glial cells. In contrast, inactivation of Eed led to the ablation of PRC2 function at early postnatal stage. Cell proliferation was reduced and retinal progenitor cells were significantly decreased in this mutant, which subsequently caused depletion of Müller glia, bipolar, and rod photoreceptor cells, primarily generated from postnatal retinal progenitor cells. Interestingly, the proportion of amacrine cells was dramatically increased at postnatal stages in the Eed-deficient retina. In accordance, multiple transcription factors controlling amacrine cell differentiation were upregulated. Furthermore, ChIP-seq analysis showed that these deregulated genes contained bivalent chromatin (H3K27me3+ H3K4me3+). Our results suggest that PRC2 is required for proliferation in order to maintain the retinal progenitor cells at postnatal stages and for retinal differentiation by controlling amacrine cell generation.

Autosomal-Dominant Corneal Endothelial Dystrophies CHED1 and PPCD1 Are Allelic Disorders Caused by Non-coding Mutations in the Promoter of OVOL2.

Congenital hereditary endothelial dystrophy 1 (CHED1) and posterior polymorphous corneal dystrophy 1 (PPCD1) are autosomal-dominant corneal endothelial dystrophies that have been genetically mapped to overlapping loci on the short arm of chromosome 20. We combined genetic and genomic approaches to identify the cause of disease in extensive pedigrees comprising over 100 affected individuals. After exclusion of pathogenic coding, splice-site, and copy-number variations, a parallel approach using targeted and whole-genome sequencing facilitated the identification of pathogenic variants in a conserved region of the OVOL2 proximal promoter sequence in the index families (c.-339_361dup for CHED1 and c.-370T>C for PPCD1). Direct sequencing of the OVOL2 promoter in other unrelated affected individuals identified two additional mutations within the conserved proximal promoter sequence (c.-274T>G and c.-307T>C). OVOL2 encodes ovo-like zinc finger 2, a C2H2 zinc-finger transcription factor that regulates mesenchymal-to-epithelial transition and acts as a direct transcriptional repressor of the established PPCD-associated gene ZEB1. Interestingly, we did not detect OVOL2 expression in the normal corneal endothelium. Our in vitro data demonstrate that all four mutated OVOL2 promoters exhibited more transcriptional activity than the corresponding wild-type promoter, and we postulate that the mutations identified create cryptic cis-acting regulatory sequence binding sites that drive aberrant OVOL2 expression during endothelial cell development. Our data establish CHED1 and PPCD1 as allelic conditions and show that CHED1 represents the extreme of what can be considered a disease spectrum. They also implicate transcriptional dysregulation of OVOL2 as a common cause of dominantly inherited corneal endothelial dystrophies.

Tcf7l1 protects the anterior neural fold from adopting the neural crest fate.

The neural crest (NC) is crucial for the evolutionary diversification of vertebrates. NC cells are induced at the neural plate border by the coordinated action of several signaling pathways, including Wnt/ß-catenin. NC cells are normally generated in the posterior neural plate border, whereas the anterior neural fold is devoid of NC cells. Using the mouse model, we show here that active repression of Wnt/ß-catenin signaling is required for maintenance of neuroepithelial identity in the anterior neural fold and for inhibition of NC induction. Conditional inactivation of Tcf7l1, a transcriptional repressor of Wnt target genes, leads to aberrant activation of Wnt/ß-catenin signaling in the anterior neuroectoderm and its conversion into NC. This reduces the developing prosencephalon without affecting the anterior-posterior neural character. Thus, Tcf7l1 defines the border between the NC and the prospective forebrain via restriction of the Wnt/ß-catenin signaling gradient.

The Gene Regulatory Network of Lens Induction Is Wired through Meis-Dependent Shadow Enhancers of Pax6.

Lens induction is a classical developmental model allowing investigation of cell specification, spatiotemporal control of gene expression, as well as how transcription factors are integrated into highly complex gene regulatory networks (GRNs). Pax6 represents a key node in the gene regulatory network governing mammalian lens induction. Meis1 and Meis2 homeoproteins are considered as essential upstream regulators of Pax6 during lens morphogenesis based on their interaction with the ectoderm enhancer (EE) located upstream of Pax6 transcription start site. Despite this generally accepted regulatory pathway, Meis1-, Meis2- and EE-deficient mice have surprisingly mild eye phenotypes at placodal stage of lens development. Here, we show that simultaneous deletion of Meis1 and Meis2 in presumptive lens ectoderm results in arrested lens development in the pre-placodal stage, and neither lens placode nor lens is formed. We found that in the presumptive lens ectoderm of Meis1/Meis2 deficient embryos Pax6 expression is absent. We demonstrate using chromatin immunoprecipitation (ChIP) that in addition to EE, Meis homeoproteins bind to a remote, ultraconserved SIMO enhancer of Pax6. We further show, using in vivo gene reporter analyses, that the lens-specific activity of SIMO enhancer is dependent on the presence of three Meis binding sites, phylogenetically conserved from man to zebrafish. Genetic ablation of EE and SIMO enhancers demostrates their requirement for lens induction and uncovers an apparent redundancy at early stages of lens development. These findings identify a genetic requirement for Meis1 and Meis2 during the early steps of mammalian eye development. Moreover, they reveal an apparent robustness in the gene regulatory mechanism whereby two independent "shadow enhancers" maintain critical levels of a dosage-sensitive gene, Pax6, during lens induction.

Asymmetric pitx2 expression in medaka epithalamus is regulated by nodal signaling through an intronic enhancer.

The epithalamic region of fishes shows prominent left-right asymmetries that are executed by nodal signaling upstream of the asymmetry-determining transcription factor pitx2. Previous reports have identified that nodal controls the left-sided pitx2 expression in the lateral plate mesoderm through an enhancer present in the last intron of this gene. However, whether similar regulation occurs also in the case of epithalamic asymmetry is currently unresolved. Here, we address some of the cis-regulatory information that control asymmetric pitx2 expression in epithalamus by presenting a Tg(pitx2:EGFP) 116-17 transgenic medaka model, which expresses enhanced green fluorescent protein (EGFP) under control of an intronic enhancer. We show that this transgene recapitulates epithalamic expression of the endogenous pitx2 and that it responds to nodal signaling inhibition. Further, we identify that three foxh1-binding sites present in this enhancer modulate expression of the transgene and that the second site is absolutely necessary for the left-sided epithalamic expression while the other two sites may have subtler regulative roles. We provide evidence that left-sided epithalamic pitx2 expression is controlled through an enhancer present in the last intron of this gene and that the regulatory logic underlying asymmetric pitx2 expression is shared between epithalamic and lateral plate mesoderm regions.

Stage-dependent requirement of neuroretinal Pax6 for lens and retina development.

The physical contact of optic vesicle with head surface ectoderm is an initial event triggering eye morphogenesis. This interaction leads to lens specification followed by coordinated invagination of the lens placode and optic vesicle, resulting in formation of the lens, retina and retinal pigmented epithelium. Although the role of Pax6 in early lens development has been well documented, its role in optic vesicle neuroepithelium and early retinal progenitors is poorly understood. Here we show that conditional inactivation of Pax6 at distinct time points of mouse neuroretina development has a different impact on early eye morphogenesis. When Pax6 is eliminated in the retina at E10.5 using an mRx-Cre transgene, after a sufficient contact between the optic vesicle and surface ectoderm has occurred, the lens develops normally but the pool of retinal progenitor cells gradually fails to expand. Furthermore, a normal differentiation program is not initiated, leading to almost complete disappearance of the retina after birth. By contrast, when Pax6 was inactivated at the onset of contact between the optic vesicle and surface ectoderm in Pax6(Sey/flox) embryos, expression of lens-specific genes was not initiated and neither the lens nor the retina formed. Our data show that Pax6 in the optic vesicle is important not only for proper retina development, but also for lens formation in a non-cell-autonomous manner.

Onecut1 and Onecut2 transcription factors operate downstream of Pax6 to regulate horizontal cell development.

Genetic studies of the last decades strongly indicated that generation of particular retinal cell types is governed by gene regulatory networks of transcription factors and their target genes. The paired and homeodomain transcription factor Pax6 plays a pivotal role in retinal development as its inactivation in the retinal progenitor cell population leads to abolished differentiation of all retinal cell types. However, until now, only a few transcription factors operating downstream of Pax6 responsible for generation of individual retinal cell types have been identified. In this study, we identified two transcription factors of the Onecut family, Onecut1 and Onecut2, as Pax6 downstream-acting factors. Onecut1 and Onecut2 were previously shown to be expressed in developing horizontal cells, retinal ganglion cells and cone photoreceptors; however, their role in differentiation of these cell types is poorly understood. In this study, we show that the horizontal cell genesis is severely disturbed in Onecut-deficient retinae. In single Onecut1 and Onecut2 mutants, the number of horizontal cells is dramatically reduced while horizontal cells are completely missing in the Onecut1/Onecut2 compound mutant. Analysis of genes involved in the horizontal cell genesis such as Foxn4, Ptf1a, Prox1 and Lim1 showed that although horizontal cells are initially formed, they are not maintained in Onecut-deficient retinae. Taken together, this study suggests the model in which Pax6 regulates the maintenance of horizontal cells through the activation of Onecut1 and Onecut2 transcription factors.

vox homeobox gene: a novel regulator of midbrain-hindbrain boundary development in medaka fish?

The midbrain-hindbrain boundary (MHB) is one of the key organizing centers of the vertebrate central nervous system (CNS). Its patterning is governed by a well-described gene regulatory network (GRN) involving several transcription factors, namely, pax, gbx, en, and otx, together with signaling molecules of the Wnt and Fgf families. Here, we describe the onset of these markers in Oryzias latipes (medaka) early brain development in comparison to previously known zebrafish expression patterns. Moreover, we show for the first time that vox, a member of the vent gene family, is expressed in the developing neural tube similarly to CNS markers. Overexpression of vox leads to profound changes in the gene expression patterns of individual components of MHB-specific GRN, most notably of fgf8, a crucial organizer molecule of MHB. Our data suggest that genes from the vent family, in addition to their crucial role in body axis formation, may play a role in regionalization of vertebrate CNS.