Pigmentary glaucoma (PG) is a common glaucoma subtype that results from release of pigment from the iris, called pigment dispersion syndrome (PDS), and its deposition throughout the anterior chamber of the eye. Although PG has a substantial heritable component, no causative genes have yet been identified. We used whole exome sequencing of two independent pedigrees to identify two premelanosome protein (PMEL) variants associated with heritable PDS/PG. PMEL encodes a key component of the melanosome, the organelle essential for melanin synthesis, storage and transport. Targeted screening of PMEL in three independent cohorts (n = 394) identified seven additional PDS/PG-associated non-synonymous variants. Five of the nine variants exhibited defective processing of the PMEL protein. In addition, analysis of PDS/PG-associated PMEL variants expressed in HeLa cells revealed structural changes to pseudomelanosomes indicating altered amyloid fibril formation in five of the nine variants. Introduction of 11-base pair deletions to the homologous pmela in zebrafish by the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 method caused profound pigmentation defects and enlarged anterior segments in the eye, further supporting PMEL's role in ocular pigmentation and function. Taken together, these data support a model in which missense PMEL variants represent dominant negative mutations that impair the ability of PMEL to form functional amyloid fibrils. While PMEL mutations have previously been shown to cause pigmentation and ocular defects in animals, this research is the first report of mutations in PMEL causing human disease.

Normally folded prion protein (PrP) and its functions in healthy brains remain underappreciated compared with the intense study of its misfolded forms ("prions," PrP) during the pathobiology of prion diseases. This impedes the development of therapeutic strategies in Alzheimer's and prion diseases. Disrupting the zebrafish homologs of PrP has provided novel insights; however, mutagenesis of the zebrafish paralog did not recapitulate previous dramatic developmental phenotypes, suggesting redundancy with the paralog. Here, we generated zebrafish loss-of-function mutant alleles and dual mutants. Zebrafish and dual mutants resemble mammalian knockouts insofar as they lack overt phenotypes, which surprisingly contrasts with reports of severe developmental phenotypes when either or is knocked down acutely. Previous studies suggest that PrP participates in neural cell development/adhesion, including in zebrafish where loss of affects adhesion and deposition patterns of lateral line neuromasts. In contrast with the expectation that's functions would be redundant to , they appear to have opposing functions in lateral line neurodevelopment. Similarly, loss of blunted the seizure susceptibility phenotypes observed in mutants, contrasting the expected exacerbation of phenotypes if these prion gene paralogs were serving redundant roles. In summary, prion mutant fish lack the overt phenotypes previously predicted, and instead they have subtle phenotypes similar to mammals. No evidence was found for functional redundancy in the zebrafish prion gene paralogs, and the phenotypes observed when each gene is disrupted individually are consistent with ancient functions of prion proteins in neurodevelopment and modulation of neural activity.

Summary The transcription factor NRL (neural retina leucine zipper) has been canonized as the master regulator of photoreceptor cell fate in the retina. NRL is necessary and sufficient to specify rod cell fate and to preclude cone cell fate in mice. By engineering zebrafish, we tested if NRL function has conserved roles beyond mammals or beyond nocturnal species, i.e., in a vertebrate possessing a greater and more typical diversity of cone sub-types. Transgenic expression of Nrl from zebrafish or mouse was sufficient to induce rod photoreceptor cells. Zebrafish nrl −/− mutants lacked rods (and had excess UV-sensitive cones) as young larvae; thus, the conservation of Nrl function between mice and zebrafish appears sound. Strikingly, however, rods were abundant in adult nrl −/− null mutant zebrafish. Rods developed in adults despite Nrl protein being undetectable. Therefore, a yet-to-be-revealed non-canonical pathway independent of Nrl is able to specify the fate of some rod photoreceptors.

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Highlights-Nrl is conserved and sufficient to specify rod photoreceptors in zebrafish retina -Nrl is necessary for rod photoreceptors in early ontogeny of zebrafish larvae -Zebrafish Nrl is functionally conserved with mouse and human NRL -Remarkably, Nrl is dispensable for rod specification in adult zebrafish Abstract 1The transcription factor NRL (Neural Retinal Leucine-zipper) has been canonized, 2 appropriately enough, as the master regulator of photoreceptor cell fate in the retina. NRL 3 is necessary and sufficient to specify rod cell fate and to preclude cone cell fate in mice. 4By engineering zebrafish we tested if NRL function has conserved roles beyond mammals 5 or beyond nocturnal species, i.e. in a vertebrate possessing a greater and more typical 6 diversity of cone sub-types. Here, transgenic expression of a Nrl homolog from zebrafish 7 or mouse was sufficient to convert developing zebrafish cones into rod photoreceptors. 8Zebrafish nrl -/mutants lacked rods (and had excess UV-sensitive cones) as young larvae, 9thus the conservation of Nrl function between mice and zebrafish appears sound. These 10 data inform hypotheses of photoreceptor evolution through the Nocturnal Bottleneck, 11suggesting that a capacity to favor nocturnal vision is a property of NRL that predates the 12 emergence of early mammals. Strikingly, however, rods were abundant in adult nrl -/null 13 mutant zebrafish. Rods developed in adults despite Nrl protein being undetectable. 14 Therefore a yet-to-be-revealed non-canonical pathway independent of nrl is able to specify 15 the fate of some rod photoreceptors. 16 17 Keywords 18Nocturnal Bottleneck; Gene Regulatory Network; Visual system development; Evo-Devo; 20Rods and cones are the ciliary photoreceptors used by vertebrates to enable vision 21 across a broad range of circumstances. Rod photoreceptors enable vision in dim 22conditions, while cone photoreceptors convey wavelength-specific information, enable 23high acuity and can operate in brightly lit environments. Retinas with both rods and cones 24 are known as duplex retinas, and the basic features of the duplex retina are present even 25 among some of the earliest branching vertebrates, the lampreys [1-3]. 26The visual photoreceptors are among the best-studied neurons with respect to 27 developmental programs and gene regulatory networks. Photoreceptor precursor cells of 28 the developing mouse retina are thoroughly studied, and an elegantly simple gene 29 regulatory network determines all rod and cone cell fates. As the precursor cell exits its 30terminal mitosis, expression of the bZIP transcription factor NRL directs it to a rod fate 31(schematized in Fig. 1A); without NRL expression it develops as a cone [4][5][6][7]. With high 32 activity of the thyroid hormone receptor THRB, the presumptive cone will develop into the 33 medium (green) wavelength light-sensitive M-cone (the ancestral red cone, expressing 34 LWS opsin). Without THRB activity, it becomes a short wavelength (UV/blue) light-35 sensitive S-cone (the ancestral U...