We have isolated a novel Xenopus homeobox gene, Xrx1, belonging to the paired-like class of homeobox genes. Xrx1 is expressed in the anterior neural plate, and subsequently in the neural structures of the developing eye (neural retina and pigmented epithelium), and in other forebrain structures deriving from the anterior neural plate: in the pineal gland, throughout its development, in the diencephalon floor and in the hypophysis. Its rostral limit of expression corresponds to the chiasmatic ridge, which some authors consider as the anteriormost limit of the neural tube: thus, Xrx1 may represent one of the most anteriorly expressed homeobox genes reported to date. Moreover, its expression in organs implicated in the establishment of circadian rhythms, may suggest for Xrx1 a role in the genetic control of this function. Finally, analysis of Xrx1 expression in embryos subjected to various treatments, or microinjected with different dorsalizing agents (noggin, Xwnt-8), suggests that vertical inductive signals leading to head morphogenesis are required to activate Xrx1.

Highlights d Loss of SETD5 impairs brain development and neuronal functions d Setd5 mutant mice exhibit cognitive and sociobehavioral deficits d SETD5 loss leads to an unfavorable chromatin state for RNA elongation and splicing d SETD5 has intrinsic histone methyltransferase activity for H3K36

We have identified a transcription factor specifically expressed in the developing vertebrate eye. We named this gene vax2 because of the high degree of sequence similarity to the recently described vax1. Both in the human and mouse genomes, vax2 is localized in the vicinity of the emx1 gene. This mapping assignment, together with the previously reported colocalization of Vax1 and Emx2 in mouse, indicates that the vax and the emx genes may be organized in clusters. vax2 has a remarkable expression domain confined to the ventral portion of the prospective neural retina in mouse, human, and Xenopus. The overexpression of either the frog Xvax2 or the human VAX2 in Xenopus embryos leads to an aberrant eye phenotype and, in particular, determines a ventralizing effect on the developing eye. The expression domain of the transcription factor Xpax2, normally confined to the ventral developing retina, extends to the dorsal region of the retina after overexpression of vax2. On the other hand, the expression of Xvent2, a molecular marker of the dorsal retina, is strongly reduced. Furthermore, vax2 overexpression induces a striking expansion of the optic stalk, a structure deriving from the ventralmost region of the eye vesicle. Altogether, these data indicate that vax2 plays a crucial role in eye development and, in particular, in the specification of the ventral optic vesicle.

P23H is the most common mutation in the RHODOPSIN (RHO) gene leading to a dominant form of retinitis pigmentosa (RP), a rod photoreceptor degeneration that invariably causes vision loss. Specific disruption of the disease P23H RHO mutant while preserving the wild-type (WT) functional allele would be an invaluable therapy for this disease. However, various technologies tested in the past failed to achieve effective changes and consequently therapeutic benefits. We validated a CRISPR/Cas9 strategy to specifically inactivate the P23H RHO mutant, while preserving the WT allele in vitro. We, then, translated this approach in vivo by delivering the CRISPR/Cas9 components in murine Rho+/P23H mutant retinae. Targeted retinae presented a high rate of cleavage in the P23H but not WT Rho allele. This gene manipulation was sufficient to slow photoreceptor degeneration and improve retinal functions. To improve the translational potential of our approach, we tested intravitreal delivery of this system by means of adeno-associated viruses (AAVs). To this purpose, the employment of the AAV9-PHP.B resulted the most effective in disrupting the P23H Rho mutant. Finally, this approach was translated successfully in human cells engineered with the homozygous P23H RHO gene mutation. Overall, this is a significant proof-of-concept that gene allele specific targeting by CRISPR/Cas9 technology is specific and efficient and represents an unprecedented tool for treating RP and more broadly dominant genetic human disorders affecting the eye, as well as other tissues.