Blindness associated with Usher Syndrome Type 1 (USH1) is typically characterized as rod photoreceptor degeneration, followed by secondary loss of cones. The mechanisms leading to blindness are unknown since most genetic mouse models only recapitulate auditory defects. We generated zebrafish mutants for one of the USH1 proteins, protocadherin-15b (pcdh15b), a putative cell adhesion molecule. Zebrafish pcdh15 is expressed exclusively in photoreceptors within calyceal processes (CPs), at the base of the outer segment (OS), and within the synapse. In our mutants, rod and cone photoreceptor integrity is compromised with early and progressively worsening abnormal OS disc growth and detachment, in part due to weakening CP contacts. These effects were attenuated or exacerbated by growth in dark and bright light conditions, respectively. We also describe novel evidence for structural defects in synapses of pcdh15 mutant photoreceptors. Cell death does not accompany these defects at early stages, suggesting that photoreceptor structural defects, rather than overt cell loss, may underlie vision deficits. Thus, we present the first genetic animal model of a pcdh15-associated retinopathy that can be used to understand the etiology of blindness in USH1.

SummaryProliferating progenitor cells undergo changes in competence to give rise to post-mitotic progeny of specialized function. These cell-fate transitions typically involve dynamic regulation of gene expression by histone methyltransferase (HMT) complexes. However, the composition, roles, and regulation of these assemblies in regulating cell-fate decisions in vivo are poorly understood. Using unbiased affinity purification and mass spectrometry, we identified the uncharacterized C2H2-like zinc finger protein ZNF644 as a G9a/GLP-interacting protein and co-regulator of histone methylation. In zebrafish, functional characterization of ZNF644 orthologs, znf644a and znf644b, revealed complementary roles in regulating G9a/H3K9me2-mediated gene silencing during neurogenesis. The non-overlapping requirements for znf644a and znf644b during retinal differentiation demarcate critical aspects of retinal differentiation programs regulated by differential G9a-ZNF644 associations, such as transitioning proliferating progenitor cells toward differentiation. Collectively, our data point to ZNF644 as a critical co-regulator of G9a/H3K9me2-mediated gene silencing during neuronal differentiation.

Highlights d MKRN2 physically interacts with GLE1 d MKRN2 co-depletion rescues the impact of GLE1 knockdown on zebrafish retinogenesis d MKRN2 selectively binds to the 3 0 UTR of a subset of mRNAs related to neurogenesis d MKRN2 knockdown results in enhanced export of its bound target mRNAs

Understanding the mechanisms that regulate the transition between the proliferative and a post-mitotic state of retinal progenitor cells (RPCs) is key to advancing our knowledge of retinal growth and maturation. In the present study we determined that during zebrafish embryonic retinal neurogenesis, two paired-type homeobox genes - vsx2 and dmbx1 - function in a mutually antagonistic manner. We demonstrate that vsx2 gene expression requires active Fgf signaling and that this in turn suppresses dmbx1 expression and maintains cells in an undifferentiated, proliferative RPC state. This vsx2-dependent RPC state can be prolonged cell-autonomously by knockdown of dmbx1, or it can be suppressed prematurely by the over-expression of dmbx1, which we show can inhibit vsx2 expression and lead to precocious neuronal differentiation. dmbx1 loss of function also results in altered expression of canonical cell cycle genes, and in particular up-regulation of ccnd1, which correlates with our previous finding of a prolonged RPC cell cycle. By knocking down ccnd1 and dmbx1 simultaneously, we show that RPCs can overcome this phenotype to exit the cell cycle on time and differentiate normally into retinal neurons. Collectively, our data provide novel insight into the mechanism that enables RPCs to exit the cell cycle through a previously unrecognized antagonistic interaction of two paired-type homeobox genes that are central regulators of an Fgf-vsx2-dmbx1-ccnd1 signaling axis.

The vertebrate retina develops from a specified group of precursor cells that adopt distinct identities and generate lineages of either the neural retina, retinal pigmented epithelium, or ciliary body. In some species, including teleost fish and amphibians, proliferative cells with stem-cell-like properties capable of continuously supplying new retinal cells post-embryonically have been characterized and extensively studied. This region, termed the ciliary or circumferential marginal zone (CMZ), possibly represents a conserved retinal stem cell niche. In this review, we highlight the research characterizing similar CMZ-like regions, or stem-like cells located at the peripheral margin, across multiple different species. We discuss the proliferative parameters, multipotency and growth mechanisms of these cells to understand how they behave in vivo and how different molecular factors and signalling networks converge at the CMZ niche to regulate their activity. The evidence suggests that the mature retina may have a conserved propensity for homeostatic growth and plasticity and that dysfunction in the regulation of CMZ activity may partially account for dystrophic eye growth diseases such as myopia and hyperopia. A better understanding of the properties of CMZ cells will enable important insight into how an endogenous generative tissue compartment can adapt to altered retinal physiology and potentially even restore vision loss caused by retinal degenerative conditions.

The proper development of the vertebrate retina relies heavily on producing the correct number and type of differentiated retinal cell types. To achieve this, proliferating retinal progenitor cells (RPCs) must exit the cell cycle at an appropriate time and correctly express a subset of differentiation markers that help specify retinal cell fate. Homeobox genes, which encode a family of transcription factors, have been accredited to both these processes, implicated in the transcriptional regulation of important cell cycle components, such as cyclins and cyclin-dependent kinases, and proneural genes. This dual regulation of homeobox genes allows these factors to help co-ordinate the transition from the proliferating RPC to postmitotic, differentiated cell. However, understanding the exact molecular targets of these factors remains a challenging task. This commentary highlights the current knowledge we have about how these factors regulate cell cycle progression and differentiation, with particular emphasis on a recent discovery from our lab demonstrating an antagonistic relationship between Vsx2 and Dmbx1 to control RPC proliferation. Future studies should aim to further understand the direct transcriptional targets of these genes, additional co-factors/interacting proteins and the possible recruitment of epigenetic machinery by these homeobox genes.

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