Variants in CLDN5 cause a syndrome characterized by seizures, microcephaly and brain calcifications.

The blood-brain barrier ensures CNS homeostasis and protection from injury. Claudin-5 (CLDN5), an important component of tight junctions, is critical for the integrity of the blood-brain barrier. We have identified de novo heterozygous missense variants in CLDN5 in 15 unrelated patients who presented with a shared constellation of features including developmental delay, seizures (primarily infantile onset focal epilepsy), microcephaly and a recognizable pattern of pontine atrophy and brain calcifications. All variants clustered in one subregion/domain of the CLDN5 gene and the recurrent variants demonstrate genotype-phenotype correlations. We modelled both patient variants and loss of function alleles in the zebrafish to show that the variants analogous to those in patients probably result in a novel aberrant function in CLDN5. In total, human patient and zebrafish data provide parallel evidence that pathogenic sequence variants in CLDN5 cause a novel neurodevelopmental disorder involving disruption of the blood-brain barrier and impaired neuronal function.

Loss of dlx5a/dlx6a Locus Alters Non-Canonical Wnt Signaling and Meckel's Cartilage Morphology.

The dlx genes encode transcription factors that establish a proximal-distal polarity within neural crest cells to bestow a regional identity during craniofacial development. The expression regions of dlx paralogs are overlapping yet distinct within the zebrafish pharyngeal arches and may also be involved in progressive morphologic changes and organization of chondrocytes of the face. However, how each dlx paralog of dlx1a, dlx2a, dlx5a and dlx6a affects craniofacial development is still largely unknown. We report here that the average lengths of the Meckel's, palatoquadrate and ceratohyal cartilages in different dlx mutants were altered. Mutants for dlx5a-/- and dlx5i6-/-, where the entire dlx5a/dlx6a locus was deleted, have the shortest lengths for all three structures at 5 days post fertilization (dpf). This phenotype was also observed in 14 dpf larvae. Loss of dlx5i6 also resulted in increased proliferation of neural crest cells and expression of chondrogenic markers. Additionally, altered expression and function of non-canonical Wnt signaling were observed in these mutants suggesting a novel interaction between dlx5i6 locus and non-canonical Wnt pathway regulating ventral cartilage morphogenesis.

Novel cross-regulation interactions between dlx genes in larval zebrafish.

The homeodomain-containing transcription factors dlx1a, dlx2a, dlx5a and dlx6a are expressed in the zebrafish brain in overlapping patterns and are important in vertebrate development. Previous work in mice have suggested the overlapping expression pattern is in part due to cross-regulatory interactions among the aforementioned dlx genes. However, the extent of these interactions and whether they are conserved among vertebrates remains to be determined. Through whole-mount in situ hybridization in zebrafish dlx mutants produced by CRISPR-Cas9 mutagenesis, cross-regulatory interactions between dlx1a, dlx2a, dlx5a and dlx6a were examined from 24 to 72 h post fertilization (hpf). Notably, and different from previous work done in mouse, zebrafish dlx2a-/- mutants continue to express dlx5a until 72hpf, whereas deletion of both enhancers within the dlx5a/dlx6a locus resulted in delayed dlx5a/dlx6a expression and relative increased dlx2a expression. These results suggest alternative regulatory elements and pathways exist to mediate dlx expression in zebrafish and may highlight evolutionary differences in gene interactions between vertebrates.