Mutations in the Spliceosome Component CWC27 Cause Retinal Degeneration with or without Additional Developmental Anomalies.

Pre-mRNA splicing factors play a fundamental role in regulating transcript diversity both temporally and spatially. Genetic defects in several spliceosome components have been linked to a set of non-overlapping spliceosomopathy phenotypes in humans, among which skeletal developmental defects and non-syndromic retinitis pigmentosa (RP) are frequent findings. Here we report that defects in spliceosome-associated protein CWC27 are associated with a spectrum of disease phenotypes ranging from isolated RP to severe syndromic forms. By whole-exome sequencing, recessive protein-truncating mutations in CWC27 were found in seven unrelated families that show a range of clinical phenotypes, including retinal degeneration, brachydactyly, craniofacial abnormalities, short stature, and neurological defects. Remarkably, variable expressivity of the human phenotype can be recapitulated in Cwc27 mutant mouse models, with significant embryonic lethality and severe phenotypes in the complete knockout mice while mice with a partial loss-of-function allele mimic the isolated retinal degeneration phenotype. Our study describes a retinal dystrophy-related phenotype spectrum as well as its genetic etiology and highlights the complexity of the spliceosomal gene network.

Premature Vertebral Mineralization in hmx1-Mutant Zebrafish.

H6 family homeobox 1 (HMX1) regulates multiple aspects of craniofacial development, and mutations in HMX1 are linked to an ocular defect termed oculoauricular syndrome of Schorderet-Munier-Franceschetti (OAS) (MIM #612109). Recently, additional altered orofacial features have been reported, including short mandibular rami, asymmetry of the jaws, and altered premaxilla. We found that in two mutant zebrafish lines termed hmx1mut10 and hmx1mut150, precocious mineralization of the proximal vertebrae occurred. Zebrafish hmx1mut10 and hmx1mut150 report mutations in the SD1 and HD domains, which are essential for dimerization and activity of hmx1. In hmx1mut10, the bone morphogenetic protein (BMP) antagonists chordin and noggin1 were downregulated, while bmp2b and bmp4 were highly expressed and specifically localized to the dorsal region prior to the initiation of the osteogenic process. The osteogenic promoters runx2b and spp1 were also upregulated. Supplementation with DMH1-an inhibitor of the BMP signaling pathway-at the specific stage in which bmp2b and bmp4 are highly expressed resulted in reduced vertebral mineralization, resembling the wildtype mineralization progress of the axial skeleton. These results point to a possible role of hmx1 as part of a complex gene network that inhibits bmp2b and bmp4 in the dorsal region, thus regulating early axial skeleton development.

Incomplete Recovery of Zebrafish Retina Following Cryoinjury.

Zebrafish show an extraordinary potential for regeneration in several organs from fins to central nervous system. Most impressively, the outcome of an injury results in a near perfect regeneration and a full functional recovery. Indeed, among the various injury paradigms previously tested in the field of zebrafish retina regeneration, a perfect layered structure is observed after one month of recovery in most of the reported cases. In this study, we applied cryoinjury to the zebrafish eye. We show that retina exposed to this treatment for one second undergoes an acute damage affecting all retinal cell types, followed by a phase of limited tissue remodeling and regrowth. Surprisingly, zebrafish developed a persistent retinal dysplasia observable through 300 days post-injury. There is no indication of fibrosis during the regeneration period, contrary to the regeneration process after cryoinjury to the zebrafish cardiac ventricle. RNA sequencing analysis of injured retinas at different time points has uncovered enriched processes and a number of potential candidate genes. By means of this simple, time and cost-effective technique, we propose a zebrafish injury model that displays a unique inability to completely recover following focal retinal damage; an outcome that is unreported to our knowledge. Furthermore, RNA sequencing proved to be useful in identifying pathways, which may play a crucial role not only in the regeneration of the retina, but in the first initial step of regeneration, degeneration. We propose that this model may prove useful in comparative and translational studies to examine critical pathways for successful regeneration.

Hmx1 regulates urfh1 expression in the craniofacial region in zebrafish.

H6 family homeobox 1 (HMX1) regulates multiple aspects of craniofacial development as it is widely expressed in the eye, peripheral ganglia and branchial arches. Mutations in HMX1 are linked to an ocular defect termed Oculo-auricular syndrome of Schorderet-Munier-Franceschetti (MIM #612109). We identified UHRF1 as a target of HMX1 during development. UHRF1 and its partner proteins actively regulate chromatin modifications and cellular proliferation. Luciferase assays and in situ hybridization analyses showed that HMX1 exerts a transcriptional inhibitory effect on UHRF1 and a modification of its expression pattern. Overexpression of hmx1 in hsp70-hmx1 zebrafish increased uhrf1 expression in the cranial region, while mutations in the hmx1 dimerization domains reduced uhrf1 expression. Moreover, the expression level of uhrf1 and its partner dnmt1 was increased in the eye field in response to hmx1 overexpression. These results indicate that hmx1 regulates uhrf1 expression and, potentially through regulating the expression of factors involved in DNA methylation, contribute to the development of the craniofacial region of zebrafish.