Landmark discovery of eye defects caused by Pax6 gene mutations in humans, rodents, and even fruit flies combined with Pax6 gene expression studies in various phyla, led to the master control gene hypothesis postulating that the gene is required almost universally for animal visual system development. However, this assumption has not been broadly tested in genetically trackable organisms such as vertebrates. Here, to determine the functional role of the fish orthologue of mammalian Pax6 in eye development we analyzed mutants in medaka Pax6.1 gene generated by genome editing. We found that transcription factors implicated in vertebrate lens development (Prox1a, MafB, c-Maf, FoxE3) failed to initiate expression in the presumptive lens tissue of Pax6.1 mutant fish resulting in aphakia, a phenotype observed previously in Pax6 mutant mice. Surprisingly, the overall differentiation potential of Pax6.1-deficient retinal progenitor cells (RPCs) is not severely compromised, and the only cell types affected by the absence of Pax6.1 transcription factor are retinal ganglion cells. This is in stark contrast to the situation in mice where the Pax6 gene is required cell-autonomously for the expansion of RPCs, and the differentiation of all retina cell types. Our results provide novel insight into the conserved and divergent roles of Pax6 gene orthologues in vertebrate eye development indicating that the lens-specific role is more evolutionarily conserved than the role in retina differentiation.

• Keywords
• Pax6, eye evolution, gene expression, lens, retina, vision,
• Publication type
• Journal Article MeSH

The Pax6 gene is essential for eye and brain development across various animal species. Here, we investigate the function of Pax6 in the development of the anterior central nervous system (CNS) of the invertebrate chordate amphioxus using CRISPR/Cas9-induced genome editing. Specifically, we examined Pax6 mutants featuring a 6 bp deletion encompassing two invariant amino acids in the conserved paired domain, hypothesized to impair Pax6 DNA-binding capacity and gene regulatory functions. Although this mutation did not result in gross morphological changes in amphioxus larvae, it demonstrated a reduced ability to activate Pax6-responsive reporter gene, suggesting a hypomorphic effect. Expression analysis in mutant larvae revealed changes in gene expression within the anterior CNS, supporting the conserved role of Pax6 gene in brain regionalization across chordates. Additionally, our findings lend support to the hypothesis of a zona limitans intrathalamica (ZLI)-like region in amphioxus, suggesting evolutionary continuity in brain patterning mechanisms. ZLI region, found in both hemichordates and vertebrates, functions as a key signaling center and serves as a restrictive boundary between major thalamic regions.

• Keywords
• amphioxus, brain, chordates, evolution, eye, genome editing, pax6,
• Publication type
• Journal Article MeSH

Chromatin remodeling complexes are required for many distinct nuclear processes such as transcription, DNA replication, and DNA repair. However, the contribution of these complexes to the development of complex tissues within an organism is poorly characterized. Imitation switch (ISWI) proteins are among the most evolutionarily conserved ATP-dependent chromatin remodeling factors and are represented by yeast Isw1/Isw2, and their vertebrate counterparts Snf2h (Smarca5) and Snf2l (Smarca1). In this study, we focused on the role of the Snf2h gene during the development of the mammalian retina. We show that Snf2h is expressed in both retinal progenitors and post-mitotic retinal cells. Using Snf2h conditional knockout mice (Snf2h cKO), we found that when Snf2h is deleted, the laminar structure of the adult retina is not retained, the overall thickness of the retina is significantly reduced compared with controls, and the outer nuclear layer (ONL) is completely missing. The depletion of Snf2h did not influence the ability of retinal progenitors to generate all the differentiated retinal cell types. Instead, the Snf2h function is critical for the proliferation of retinal progenitor cells. Cells lacking Snf2h have a defective S-phase, leading to the entire cell division process impairments. Although all retinal cell types appear to be specified in the absence of the Snf2h function, cell-cycle defects and concomitantly increased apoptosis in Snf2h cKO result in abnormal retina lamination, complete destruction of the photoreceptor layer, and consequently, a physiologically non-functional retina.

• Keywords
• Smarca5, Snf2h, apoptosis, cell cycle, photoreceptors, retina,
• MeSH
• Adenosine Triphosphatases * metabolism   MeSH
• Cell Nucleus metabolism   MeSH
• Chromatin * metabolism   MeSH
• Chromosomal Proteins, Non-Histone * metabolism   MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Cell Proliferation MeSH
• Chromatin Assembly and Disassembly * MeSH
• Retina MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Adenosine Triphosphatases * MeSH
• Chromatin * MeSH
• Chromosomal Proteins, Non-Histone * MeSH
• Smarca5 protein, mouse MeSH Browser

Members of the POU4F/Brn3 transcription factor family have an established role in the development of retinal ganglion cell (RGCs) types, the main transducers of visual information from the mammalian eye to the brain. Our previous work using sparse random recombination of a conditional knock-in reporter allele expressing alkaline phosphatase (AP) and intersectional genetics had identified three types of Brn3c positive (Brn3c+ ) RGCs. Here, we describe a novel Brn3cCre mouse allele generated by serial Dre to Cre recombination and use it to explore the expression overlap of Brn3c with Brn3a and Brn3b and the dendritic arbor morphologies and visual stimulus response properties of Brn3c+ RGC types. Furthermore, we explore brain nuclei that express Brn3c or receive input from Brn3c+ neurons. Our analysis reveals a much larger number of Brn3c+ RGCs and more diverse set of RGC types than previously reported. Most RGCs expressing Brn3c during development are still Brn3c positive in the adult, and all express Brn3a while only about half express Brn3b. Genetic Brn3c-Brn3b intersection reveals an area of increased RGC density, extending from dorsotemporal to ventrolateral across the retina and overlapping with the mouse binocular field of view. In addition, we report a Brn3c+ RGC projection to the thalamic reticular nucleus, a visual nucleus that was not previously shown to receive retinal input. Furthermore, Brn3c+ neurons highlight a previously unknown subdivision of the deep mesencephalic nucleus. Thus, our newly generated allele provides novel biological insights into RGC type classification, brain connectivity, and cytoarchitectonic.

• Keywords
• Brn3c, Cre recombinase, Pou4f3, deep mesencephalic nucleus, periaqueductal gray, retinal ganglion cells, superior colliculus, thalamic reticular nucleus, transcription factor,
• MeSH
• Alleles MeSH
• Gene Knock-In Techniques methods   MeSH
• Homeodomain Proteins genetics   metabolism   MeSH
• Integrases MeSH
• Brain cytology   metabolism   MeSH
• Mice MeSH
• Retinal Ganglion Cells cytology   metabolism   MeSH
• Transcription Factor Brn-3C genetics   metabolism   MeSH
• Visual Pathways cytology   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Cre recombinase MeSH Browser
• Homeodomain Proteins MeSH
• Integrases MeSH
• Pou4f3 protein, mouse MeSH Browser
• Transcription Factor Brn-3C MeSH

Genome duplication leads to an emergence of gene paralogs that are essentially free to undergo the process of neofunctionalization, subfunctionalization or degeneration (gene loss). Onecut1 (Oc1) and Onecut2 (Oc2) transcription factors, encoded by paralogous genes in mammals, are expressed in precursors of horizontal cells (HCs), retinal ganglion cells and cone photoreceptors. Previous studies have shown that ablation of either Oc1 or Oc2 gene in the mouse retina results in a decreased number of HCs, while simultaneous deletion of Oc1 and Oc2 leads to a complete loss of HCs. Here we study the genetic redundancy between Oc1 and Oc2 paralogs and focus on how the dose of Onecut transcription factors influences abundance of individual retinal cell types and overall retina physiology. Our data show that reducing the number of functional Oc alleles in the developing retina leads to a gradual decrease in the number of HCs, progressive thinning of the outer plexiform layer and diminished electrophysiology responses. Taken together, these observations indicate that in the context of HC population, the alleles of Oc1/Oc2 paralogous genes are mutually interchangeable, function additively to support proper retinal function and their molecular evolution does not follow one of the typical routes after gene duplication.

• MeSH
• Alleles MeSH
• Amacrine Cells metabolism   pathology   MeSH
• Retinal Bipolar Cells metabolism   pathology   MeSH
• Retinal Cone Photoreceptor Cells metabolism   pathology   MeSH
• Ependymoglial Cells metabolism   pathology   MeSH
• Genetic Loci MeSH
• Genotype MeSH
• Hepatocyte Nuclear Factor 6 genetics   metabolism   MeSH
• Homeodomain Proteins genetics   metabolism   MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Eye growth & development   pathology   MeSH
• Retina cytology   pathology   physiology   MeSH
• Retinal Ganglion Cells cytology   metabolism   MeSH
• Transcription Factors genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Hepatocyte Nuclear Factor 6 MeSH
• Homeodomain Proteins MeSH
• Onecut1 protein, mouse MeSH Browser
• ONECUT2 protein, mouse MeSH Browser
• Transcription Factors MeSH

The evolution of the vertebrate eye remains so far unresolved. Amphioxus frontal eye pigment cells and photoreceptors were proposed to be homologous to vertebrate photoreceptors and retinal pigmented epithelium, based on ultrastructural morphology and gene expression analysis in B. floridae. Here, we present comparative molecular data using two additional amphioxus species, a closely related B. lanceolatum, and the most divergent A. lucayanum. Taking advantage of a unique set of specific antibodies we characterized photoreceptors and putative interneurons of the frontal eye and investigated its neuronal circuitry. Our results corroborate generally conserved molecular fingerprint among cephalochordate species. Furthermore, we performed pharmacological perturbations and found that the Notch signaling pathway, a key regulator of retina development in vertebrates, is required for correct ratios among frontal eye cell types. In summary, our study provides a valuable insight into cell-type relationships in chordate visual organs and strengthens the previously proposed homology between amphioxus frontal eye and vertebrate eyes.

• Keywords
• Notch signaling, chordates, eye evolution, gene expression, interneurons, light detection, photoreceptors, vision,
• Publication type
• Journal Article MeSH

Paired box transcription factors play important role in development and tissue morphogenesis. The number of Pax homologs varies among species studied so far, due to genome and gene duplications that have affected PAX family to a great extent. Based on sequence similarity and functional domains, four Pax classes have been identified in chordates, namely Pax1/9, Pax2/5/8, Pax3/7, and Pax4/6. Numerous splicing events have been reported mainly for Pax2/5/8 and Pax6 genes. Of significant interest are those events that lead to Pax proteins with presumed novel properties, such as altered DNA-binding or transcriptional activity. In the current study, a thorough analysis of Pax2/5/8 splicing events from cephalochordates and vertebrates was performed. We focused more on Pax2/5/8 and Pax6 splicing events in which the paired domain is involved. Three new splicing events were identified in Oryzias latipes, one of which seems to be conserved in Acanthomorphata. Using representatives from deuterostome and protostome phyla, a comparative analysis of the Pax6 exon-intron structure of the paired domain was performed, during an attempt to estimate the time of appearance of the Pax6(5a) mRNA isoform. As shown in our analysis, this splicing event is characteristic of Gnathostomata and is absent in the other chordate subphyla. Moreover, expression pattern of alternative spliced variants was compared between cephalochordates and fish species. In summary, our data indicate expansion of alternative mRNA variants in paired box region of Pax2/5/8 and Pax6 genes during the course of vertebrate evolution.

• Keywords
• Pax258, Pax6, alternative splicing, paired domain, splice variants,
• Publication type
• Journal Article MeSH

Wnt/β-catenin signaling plays an essential role in the retinal pigment epithelium (RPE) determination. Since activity of Pax6 (together with Pax2) is also required for the RPE determination, we investigated a possible genetic interaction between Pax6 and Wnt/β-catenin signaling pathway by analyzing Pax6, β-catenin, and Pax6/β-catenin conditional knockout mice. Although Pax6 inactivation alone had no impact on initial specification determined by the expression of Mitf and Otx2, melanin pigmentation was reduced in the RPE. This suggests that along with Mitf and Otx2, Pax6 is required for the full differentiation of RPE. Reporter gene assays in vitro suggest that hypopigmentation is at least in part due to the direct regulation of genes encoding enzymes involved in melanin synthesis by Pax6, Mitf, and β-catenin. The RPE of a β-catenin/Pax6 double mutant was differentiated into the neural retina; however, the tissue was thinner than that of the conditional β-catenin mutant due to reduced proliferation. Together, our data demonstrate that Pax6 is required for the RPE differentiation by regulating pigmentation and accountable for hyperproliferation in the transdifferentiated RPE. In this context, Pax6 appears to function as a pleiotropic regulator, directing development of ocular tissues in concert with the signaling pathway and, at the same time, regulating expression of structural component of the eye, such as shielding pigment.

• MeSH
• beta Catenin genetics   metabolism   MeSH
• Homeodomain Proteins genetics   metabolism   MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Eye Proteins genetics   metabolism   MeSH
• Repressor Proteins genetics   metabolism   MeSH
• Retina cytology   metabolism   MeSH
• Retinal Pigment Epithelium metabolism   MeSH
• Wnt Signaling Pathway * MeSH
• Cell Transdifferentiation MeSH
• PAX6 Transcription Factor MeSH
• Paired Box Transcription Factors genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• beta Catenin MeSH
• Homeodomain Proteins MeSH
• Eye Proteins MeSH
• Pax6 protein, mouse MeSH Browser
• Repressor Proteins MeSH
• PAX6 Transcription Factor MeSH
• Paired Box Transcription Factors MeSH