Dicer1 is required for pigment cell and craniofacial development in zebrafish.

The multidomain RNase III endoribonuclease DICER is required for the generation of most functional microRNAs (miRNAs). Loss of Dicer affects developmental processes at different levels. Here, we characterized the zebrafish Dicer1 mutant, dicer1sa9205, which has a single point mutation induced by N-ethyl-N-nitrosourea mutagenesis. Heterozygous dicer1sa9205 developed normally, being phenotypically indistinguishable from wild-type siblings. Homozygous dicer1sa9205 mutants display smaller eyes, abnormal craniofacial development and aberrant pigmentation. Reduced numbers of both iridophores and melanocytes were observed in the head and ventral trunk of dicer1sa9205 homozygotes; the effect on melanocytes was stronger and detectable earlier in development. The expression of microphthalmia-associated transcription factor a (mitfa), the master gene for melanocytes differentiation, was enhanced in dicer1-depleted fish. Similarly, the expression of SRY-box containing gene 10 (sox10), required for mitfa activation, was higher in mutants than in wild types. In silico and in vivo analyses of either sox10 or mitfa 3'UTRs revealed conserved potential miRNA binding sites likely involved in the post-transcriptional regulation of both genes. Based on these findings, we propose that dicer1 participates in the gene regulatory network governing zebrafish melanocyte differentiation by controlling the expression of mitfa and sox10

Fishing the Molecular Bases of Treacher Collins Syndrome

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development, and mutations in the TCOF1 gene are responsible for over 90% of TCS cases. The knowledge about the molecular mechanisms responsible for this syndrome is relatively scant, probably due to the difficulty of reproducing the pathology in experimental animals. Zebrafish is an emerging model for human disease studies, and we therefore assessed it as a model for studying TCS. We identified in silico the putative zebrafish TCOF1 ortholog and cloned the corresponding cDNA. The derived polypeptide shares the main structural domains found in mammals and amphibians. Tcof1 expression is restricted to the anterior-most regions of zebrafish developing embryos, similar to what happens in mouse embryos. Tcof1 loss-of-function resulted in fish showing phenotypes similar to those observed in TCS patients, and enabled a further characterization of the mechanisms underlying craniofacial malformation. Besides, we initiated the identification of potential molecular targets of treacle in zebrafish. We found that Tcof1 loss-of-function led to a decrease in the expression of cellular proliferation and craniofacial development. Together, results presented here strongly suggest that it is possible to achieve fish with TCS-like phenotype by knocking down the expression of the TCOF1 ortholog in zebrafish. This experimental condition may facilitate the study of the disease etiology during embryonic development

Zebrafish <i>tcof1</i> morphants showed reduced NC-specifier gene expression patterns.

<p>Lateral (<b>G</b>, <b>I</b>, <b>K</b>, and <b>M</b>) and dorsal (<b>A–F</b>, <b>H</b>, <b>J</b>, <b>L</b>, and <b>N</b>) views of control (<b>A</b>, <b>C</b>, <b>E</b>, and <b>G</b>–<b>J</b>) and in6:ex7-MO+ex7:in7-MO-treated (<b>B</b>, <b>D</b>, <b>F</b>, <b>K</b>–<b>N</b>) embryos. Embryos at 14 hpf were analyzed for <i>foxD3</i> (<b>A</b>–<b>B</b> and <b>E</b>–<b>F</b>) and <i>sox9b</i> (<b>C</b>–<b>D</b> and <b>G</b>–<b>H</b>) expression patterns using whole-mount <i>in situ</i> hybridization. Abbreviations: de, diencephalic; hb, hindbrain; mb, midbrain; mhb, mid-hindbrain; ov, otic vesicles; s, somites. Scale bar: 185 µm for <b>A</b>–<b>N</b>.</p

Relative expression levels of putative off-target activated genes in MO-treated embryos using qRT-PCR.

<p>The x-axis shows different genes for each sample of MO-treated embryos and the y-axis shows gene expression levels relative to control embryos. Gene expression levels were normalized with <i>ef1α</i> and <i>rpl14</i> expression. Significant differences are labeled with an asterisk and the p-values are indicated between parentheses for each gene.</p

<i>tcof1</i> loss-of-function adversely affects zebrafish craniofacial cartilage development.

<p>Ventral (<b>A</b>–<b>D</b>), and dorsal views (<b>E</b>–<b>F</b>) (anterior to the left) of control (<b>A</b>, <b>C</b>, and <b>E</b>), and in6:ex7-MO+ex7:in7-MO-treated (<b>B</b>, <b>D</b>, and <b>F</b>) 4 dpf larvae stained with Alcian blue. Skeletal staining of control and morphants reveals hypoplasia of numerous craniofacial cartilages. Cranioskeletal hypoplasia is evident in the frontal, premaxillary, and maxillary elements. Abbreviations: cb1–5, ceratobranchial arches 1–5; ch, ceratohyal; ep, ethmoid plate; m, Meckel's cartilage; pc, polar cartilage; pq, palatoquadrate; tr, trabecula.</p

Zebrafish <i>tcof1</i> mRNA developmental expression pattern determined by semi-quantitative RT-PCR.

<p><b>A:</b> Agarose gel electrophoresis of semi-quantitative RT-PCR products amplified for zebrafish <i>tcof1</i> and <i>ef1α</i> mRNAs at the 1-cell stage, 6, 12, 24, 48, and 72 hpf stages. <b>B:</b> Bar graph of zebrafish <i>tcof1</i> mRNA expression profile during embryonic development, normalized to <i>ef1α</i> expression (n = 3).</p

Relative gene expression levels of putative treacle targets using qRT-PCR.

<p>The x-axis shows different gene levels analyzed by qRT-PCR performed on RNA samples obtained from either control or MO-treated embryos. The y-axis shows gene expression levels relative to control embryos. Gene expression levels were normalized with <i>ef1α</i> and <i>rpl14</i> expression. Significant differences are labeled with an asterisk and the p-values are indicated between parentheses for each gene.</p

Localization of <i>B8JIY2</i> in the zebrafish genome.

<p><b>A: </b><i>B8JIY2</i> localize<b>s</b> to chromosome 13, spanning the bases 4,646,686 to 4,674,683. The studied contig is represented with a blue rectangle. Blast hits are labeled in red, showing filled boxes for exons and empty boxes for introns. Immediately below, B8JIY2, <i>nolc1l</i>, and Q7ZUM1 exons are represented with brown boxes and introns with brown broken lines. Near the bottom, the CG % is shown in grey. <b>B: </b><i>B8JIY2 in silico</i> translated amino acid sequence with LisH dimerization (red), TCS treacle like (green), and histone H5 domains (blue) underlined. <b>C:</b> NetPhos 2.0 predicted phosphorylation sites in <i>B8JIY2 in silico</i> translated amino acid sequence.</p

Developmental expression pattern of <i>tcof1</i> by whole mount <i>in situ</i> hybridization.

<p>Lateral (<b>A</b>–<b>I</b>, <b>K</b>, <b>L</b>, <b>N</b>–<b>Q</b>, <b>S</b>) and dorsal (<b>J</b>, <b>M</b>, <b>R</b> and <b>T</b>) views (anterior regions to the left) of zebrafish embryos hybridized with anti-sense (<b>A</b>–<b>D</b>, <b>I</b>, <b>J</b>, <b>L</b>, <b>M</b>, <b>O</b>, <b>P, and R</b>–T) or sense (<b>E</b>–<b>H</b>, <b>K</b>, <b>N</b> and <b>Q</b>) zebrafish <i>tcof1</i> probes. The following stages were analyzed: 2-cell stage (<b>A</b> and <b>E</b>), 6 hpf (<b>B</b> and <b>F</b>), 10 hpf (<b>C</b> and <b>G</b>), 13 hpf (<b>D</b> and <b>H</b>), 24 hpf (<b>I</b>–<b>K</b>), 48 hpf (<b>L</b>–<b>N</b>), 72 hpf (<b>O</b>–<b>R</b>), and 120 hpf (<b>S</b>–<b>T</b>). Abbreviations: cs, craniofacial structures; de, diencephalic region; e, eye; g, gut; mb, midbrain; hb, hindbrain; l, lenses; op, olfactory pits; ot, optic tectum; pa, pharyngeal arches; pf, pectoral fin buds. Scale bar: 180 µm for <b>A</b>–<b>H</b>; 210 µm for <b>I</b>, <b>J</b> and <b>P</b>; 266 µm for <b>K</b>; 320 µm for <b>L</b>, <b>M</b> and <b>R</b>; 380 µm for <b>N</b>, <b>O</b>, and <b>S</b>–<b>T</b>; 530 µm for <b>Q</b>.</p

Percentages of normal, affected, or dead embryos/larvae following microinjection with ex7:in7-MO or both MOs at different developmental stages.

<p>Percentages of normal, affected, or dead embryos/larvae following microinjection with ex7:in7-MO or both MOs at different developmental stages.</p