Two alleles of an eyeless mutant, chokh (chk), were identified in ongoing zebrafish F(3) mutagenesis screens. Morphologically, chk mutants can be identified at 15 h post-fertilization by the failure of optic primordia to evaginate from the forebrain. The chk phenotype appears specific, as marker genes in the forebrain, midbrain, and pineal are expressed in normal temporal, spatial, and circadian patterns. Sequence analysis of the chk alleles revealed nonsense or missense mutations in the rx3 homeobox. Rx genes encode paired-type homeodomain transcription factors known to be key regulators of eye development in mouse, medaka, Xenopus, and zebrafish. To uncover novel Rx targets, we analyzed the expression of multiple eye development genes in chk. We find that expression of mab21l2, mab21l1 and rx2 are specifically absent in the eye field of chk embryos. Knockdown of Mab21l2 by antisense morpholino microinjections partially phenocopies the rx3 mutation, leading to microphthalmia, incomplete eye maturation, and dramatic increases in apoptotic eye progenitors. We propose that mab21l2 is an early downstream effector of rx3 and is critical for survival of eye progenitors.
mab21l1 and mab21l2 paralogs have widespread and dynamic expression patterns during vertebrate development. Both genes are expressed in the developing eye, midbrain, neural tube, and branchial arches. Our goal was to identify promoter regions with activity in mab21l2 expression domains. Assays of mab21l2 promoter-EGFP constructs in zebrafish embryos confirm that constructs containing 7.2 or 4.9 kb of mab21l2 promoter region are sufficient to drive expression in known (e.g., tectum, branchial arches) and unexpected domains (e.g., lens and retinal amacrine cells). A comparative analysis identifies complementary and novel expression domains of endogenous mab21l2 (e.g., lens and ventral iridocorneal canal) and mab21l1 (e.g., retinal amacrine and ganglion cells). Interestingly, therefore, despite the absence of conserved non-coding elements, a 4.9-kb mab21l2 promoter is sufficient to recapitulate expression in tissues unique to mab21l1 or mab21l2. Developmental Dynamics 240:745-754,
The ␣ subunit of cone transducin (T␣C) is expressed exclusively in cone photoreceptors of the eye and pineal. T␣C is a key phototransduction protein, and inherited mutations in T␣C cause total color blindness in humans. We use transgenic zebrafish to identify and characterize cone photoreceptor regulatory element 1 (CPRE-1) a novel 20-bp enhancer element in the T␣C promoter (T␣CP). CPRE-1 is located ϳ2.5 kb upstream of the translation start site and is necessary for strong cone photoreceptor-specific expression in vivo. CPRE-1 comprises of a modular arrangement of two 10-bp elements that have separate, but co-dependent transcriptional activities. In vitro, CPRE-1 specifically binds nuclear factors that are enriched in ocular tissue. Bioinformatic alignments reveal that CPRE-1 sites are evolutionarily conserved in the promoter regions of fish, rodent, and mammalian T␣C orthologues and identify a 5-CTGGAGTG(A/T)TGGA(G/A)G-CAGGG(G/C)T-3 consensus sequence.The vertebrate retina contains distinct cone and rod photoreceptors that mediate scotopic and photopic vision, respectively (1). Although cones and rods originate from the same population of retinal progenitor cells, they have unique gene expression profiles that account for their differential cell fate, morphology, and signal transduction mechanisms. For example, many components of the G protein-coupled receptor phototransduction cascade are encoded by cone-or rod-specific genes (1). The molecular genetics underpinning cone photoreceptor-specific gene expression remain poorly defined.Several transcription factors, including Crx, Nr2e3, Nrl, and Tr2, regulate photoreceptor-specific gene expression. Cone rod homeobox is expressed in photoreceptors, and cone rod homeobox depletion leads to a developmental loss of both cone and rod photoreceptors in mice and zebrafish and to blindness in humans (2-4). Nr2e3 and Nrl are expressed in rods where they repress expression of cone-specific genes (5-10). Mutations in Nr2e3 or Nrl lead to rods inappropriately expressing cone-specific markers and to the human retinal disease enhanced S-cone syndrome (5-12). Thyroid hormone receptor 2 is a nuclear receptor required for M-cone development (13,14). Targeted deletion of thyroid hormone receptor 2 leads to a loss of M-cone function with an increase in functional S-cones (13,14). Although these transcription factors are known to regulate photoreceptor genes, the molecular regulators of conespecific gene expression are not well defined.One method of deciphering the molecular mechanisms regulating cone-specific expression is to characterize the cis-elements in a cone-specific promoter. Several studies have identified large promoter regions sufficient to direct transgene expression in cones (15-23). However, many of these promoter regions have weak activity, exhibit ectopic, non-cone expression, or require a heterologous enhancer element (16 -19, 21-23). With the exception of a cone rod homeobox-binding element, none of these studies have characterized individual cis-elements that d...
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