Retinal Cone Mosaic in sws1-Mutant Medaka (Oryzias latipes), A Teleost.

Purpose: Ablation of short single cones (SSCs) expressing short-wavelength-sensitive opsin (SWS1) is well analyzed in the field of regenerative retinal cells. In contrast with ablation studies, the phenomena caused by the complete deletion of SWS1 are less well-understood. To assess the effects of SWS1 deficiency on retinal structure, we established and analyzed sws1-mutant medaka. Methods: To visualize SWS1, a monoclonal anti-SWS1 antibody and transgenic reporter fish (Tg(sws1:mem-egfp)) were generated. We also developed a CRISPR/Cas-driven sws1-mutant line. Retinal structure of sws1 mutant was visualized using anti-SWS1, 1D4, and ZPR1 antibodies and coumarin derivatives and compared with wild type, Tg(sws1:mem-egfp), and another opsin (lws) mutant. Results: Our rat monoclonal antibody specifically recognized medaka SWS1. Sws1 mutant retained regularly arranged cone mosaic as lws mutant and its SSCs had neither SWS1 nor long wavelength sensitive opsin. Depletion of sws1 did not affect the expression of long wavelength sensitive opsin, and vice versa. ZPR1 antibody recognized arrestin spread throughout double cones and long single cones in wild-type, transgenic, and sws1-mutant lines. Conclusions: Comparative observation of sws1-mutant and wild-type retinas revealed that ZPR1 negativity is not a marker for SSCs with SWS1, but SSCs themselves. Loss of functional sws1 did not cause retinal degeneration, indicating that sws1 is not essential for cone mosaic development in medaka. Our two fish lines, one with visualized SWS1 and the other lacking functional SWS1, offer an opportunity to study neural network synapsing with SSCs and to clarify the role of SWS1 in vision.

Transcriptional co-regulation of evolutionarily conserved microRNA/cone opsin gene pairs: implications for photoreceptor subtype specification.

The vertebrate retina contains two types of photoreceptor cells, rods and cones, which use distinct types of opsins and phototransduction proteins. Cones can be further divided into several subtypes with differing wavelength sensitivity and morphology. Although photoreceptor development has been extensively studied in a variety of vertebrate species, the mechanism by which photoreceptor subtypes are established is still largely unknown. Here we report two microRNAs (miRNAs), miR-726 and miR-729, which are potentially involved in photoreceptor subtype specification. In the medaka Oryzias latipes, the genes encoding miR-726 and miR-729 are located upstream of the red-sensitive opsin gene LWS-A and the UV-sensitive opsin gene SWS1, respectively, and are transcribed in the opposite direction from the respective opsin genes. The miR-726/LWS pair is conserved between teleosts and tetrapods, and the miR-729/SWS1 pair is conserved among teleosts. in situ hybridization analyses and fluorescence reporter assays suggest that these miRNAs are co-expressed with the respective opsins in specific cone subtypes. Potential targets of miR-726 and miR-729 predicted in silico include several transcription factors that regulate photoreceptor development. Functional analyses of cis-regulatory sequences in vivo suggest that transcription of the paired microRNA and opsin genes is co-regulated by common cis-regulatory modules. We propose an evolutionarily conserved mechanism that controls photoreceptor subtype identity through coupling between transcriptional and post-transcriptional regulations.

Cis-acting transcriptional repression establishes a sharp boundary in chordate embryos.

The function of bone morphogenetic protein (BMP) signaling in dorsoventral (DV) patterning of animal embryos is conserved among Bilateria. In vertebrates, the BMP ligand antidorsalizing morphogenetic protein (Admp) is expressed dorsally and moves to the opposite side to specify the ventral fate. Here, we show that Pinhead is an antagonist specific for Admp with a role in establishing the DV axis of the trunk epidermis in embryos of the ascidian Ciona intestinalis. Pinhead and Admp exist in tandem in the genomes of various animals from arthropods to vertebrates. This genomic configuration is important for mutually exclusive expression of these genes, because Pinhead transcription directly disturbs the action of the Admp enhancer. Our data suggest that this dual negative regulatory mechanism is widely conserved in animals.

A conserved non-reproductive GnRH system in chordates.

Gonadotropin-releasing hormone (GnRH) is a neuroendocrine peptide that plays a central role in the vertebrate hypothalamo-pituitary axis. The roles of GnRH in the control of vertebrate reproductive functions have been established, while its non-reproductive function has been suggested but less well understood. Here we show that the tunicate Ciona intestinalis has in its non-reproductive larval stage a prominent GnRH system spanning the entire length of the nervous system. Tunicate GnRH receptors are phylogenetically closest to vertebrate GnRH receptors, yet functional analysis of the receptors revealed that these simple chordates have evolved a unique GnRH system with multiple ligands and receptor heterodimerization enabling complex regulation. One of the gnrh genes is conspicuously expressed in the motor ganglion and nerve cord, which are homologous structures to the hindbrain and spinal cord of vertebrates. Correspondingly, GnRH receptor genes were found to be expressed in the tail muscle and notochord of embryos, both of which are phylotypic axial structures along the nerve cord. Our findings suggest a novel non-reproductive role of GnRH in tunicates. Furthermore, we present evidence that GnRH-producing cells are present in the hindbrain and spinal cord of the medaka, Oryzias latipes, thereby suggesting the deep evolutionary origin of a non-reproductive GnRH system in chordates.