INHIBITION OF ENERGY METABOLISM BY BENZOXAZOLIN-2-ONE

Abstract--l. The effects of the title compound (BOA) on energy-linked reactions in mitochondria were studied. 2. BOA inhibited electron transfer between the flavin and ubiquinone in Complex I, and ATP synthesis at the F~ moiety of the ATPase complex. 3. These results are discussed in relation to the toxicity of BOA towards a wide range of aerobic organisms

Conservation of Zebrafish MicroRNA-145 and Its Role during Neural Crest Cell Development

The neural crest is a multipotent cell population that develops from the dorsal neural fold of vertebrate embryos in order to migrate extensively and differentiate into a variety of tissues. A number of gene regulatory networks coordinating neural crest cell specification and differentiation have been extensively studied to date. Although several publications suggest a common role for microRNA-145 (miR-145) in molecular reprogramming for cell cycle regulation and/or cellular differentiation, little is known about its role during in vivo cranial neural crest development. By modifying miR-145 levels in zebrafish embryos, abnormal craniofacial development and aberrant pigmentation phenotypes were detected. By whole-mount in situ hybridization, changes in expression patterns of col2a1a and Sry-related HMG box (Sox) transcription factors sox9a and sox9b were observed in overexpressed miR-145 embryos. In agreement, zebrafish sox9b expression was downregulated by miR-145 overexpression. In silico and in vivo analysis of the sox9b 3′UTR revealed a conserved potential miR-145 binding site likely involved in its post-transcriptional regulation. Based on these findings, we speculate that miR-145 participates in the gene regulatory network governing zebrafish chondrocyte differentiation by controlling sox9b expressionThis research was funded by a CONICET External Grant (March 2018 to A.M.J.W.), an ANPCyT PICT Grant (PICT-2017-0509 to A.M.J.W.), and a CONICET PIP Grant (PIP-2015-0719 to N.B.C.)S

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

<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> 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