Bibonne A et al. (2013), Three calcium-sensitive genes, fus, brd3 and wd...

XB-ART-46473

Biochim Biophys Acta 2013 Jul 01;18337:1665-71. doi: 10.1016/j.bbamcr.2012.12.015.

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Three calcium-sensitive genes, fus, brd3 and wdr5, are highly expressed in neural and renal territories during amphibian development.

Bibonne A , Néant I , Batut J , Leclerc C , Moreau M , Gilbert T .

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Numerous Ca(2+) signaling events have been associated with early development of vertebrate embryo, from fertilization to organogenesis. In Xenopus laevis, Ca(2+) signals are key regulators in the earliest steps of the nervous system development. If neural determination is one of the best-characterized examples of the role of Ca(2+) during embryogenesis, increasing literature supports a determining role of organogenesis and differentiation. In blastula the cells of the presumptive ectoderm (animal caps) are pluripotent and can be induced toward neural fate with an intracellular increase of free Ca(2+) triggered by caffeine. To identify genes that are transcribed early upon Ca(2+) stimuli and involved in neural determination, we have constructed a subtractive cDNA library between neuralized and non-neuralized animal caps. Here we present the expression pattern of three new Ca(2+)-sensitive genes: fus (fused in sarcoma), brd3 (bromodomain containing 3) and wdr5 (WD repeat domain 5) as they all represent potential regulators of the transcriptional machinery. Using in situ hybridization we illustrated the spatial expression pattern of fus, brd3 and wdr5 during early developmental stages of Xenopus embryos. Strikingly, their domains of expression are not restricted to neural territories. They all share a specific expression throughout renal organogenesis which has been found to rely also on Ca(2+) signaling. This therefore highlights the key function of Ca(2+) target genes in specific territories during early development. We propose that Ca(2+) signaling through modulation of fus, brd3 and wdr5 expressions can control the transcription machinery to achieve proper embryogenesis. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

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Species referenced: Xenopus laevis
Genes referenced: brd3 fus nono odc1 wdr5

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	Fig. 1. Temporal expression profiles of fus, brd3 and wdr5 during early development. RT-PCR analysis of RNA extracted from embryos at the indicated stages. PCR on RNA without reverse transcription was performed to check the absence of genomic DNA (lane—RT). odc was used as a loading control. In Xenopus laevis the mid-blastula transition (MBT) occurs 6 h after fertilization, at stage 8 and marks the transition between maternal and zygotic gene expression.
	Fig. 2. Spatial expression patterns of fus, brd3 and wdr5 mRNA during Xenopus laevis' early development. In situ hybridization is performed on embryos and sections at gastrula and neurula stages. Photomicrographs of whole mounts and sections for fus (left panels), brd3 (middle panels) and wdr5 (right panels) are shown. (A–C) At early gastrula (dorsal view, arrows indicate the position of the blastoporal lip) fus, brd3 and wdr5 transcripts are detected in the ectoderm and the mesoderm. (D–F) This pattern is confirmed by section analysis (sagittal section dorsal is on the right, arrows point to the dorsal blastoporal groove) which shows labeled ectoderm (ec) with no difference between the dorsal and the ventral sides and in the dorsal mesoderm (mes). The endoderm (end) is not labeled. (G–H) Whole-mounts at early neurula stage (anterior view, dorsal side up), expression is restricted to neural territories as indicated by the dotted areas. (J–L) Transversal section analysis (section is a trunk level, dorsal is up) shows that fus, brd3 and wdr5 are expressed in the neural tube (nt). Enlargement of respective sections (J’–L’), the epidermal ectoderm (ep) is labeled for fus (J’) but not for brd3 and wdr5 (K’, L’). A strong expression is present in the intermediate mesoderm (im) and in the lateral plate mesoderm (lpm) for fus and wdr5 (J’, L’) while brd3 is clearly expressed in the somitic mesoderm (so). Abbreviations: ec; ectoderm, end; endoderm, ep; epidermal ectoderm, im; intermediate mesoderm, lpm; lateral plate mesoderm, mes; mesoderm, n; notochord, nt; neural tube, so; somitic mesoderm. Scale bars 0.5 mm in (A–L).
	Fig. 3. Spatial expression patterns of fus, brd3 and wdr5 at taibud and tadpole stages in Xenopus laevis. In situ hybridization is performed on embryos and sections at early (A–F), late tailbud (G–L) and tadpole (M–R) stages. Photomicrographs of whole mounts and sections for fus (left panels), brd3 (middle panels) and wdr5 (right panels) are shown. (A–C) At early tailbud fus, brd3 and wdr5 transcripts are expressed in anterior neural structures; telencephalon (Te), mesencephalon (Me) and rhombencephalon (Rh) and in the optic cup (oc). A strong expression is detected in the branchial arches (ba) for fus and wdr5 (A, C). The cement gland (cg) expressed brd3 and wdr5 (B, C). The tail bud (tb) and the pronephros (p) express fus at this stage (A). (D–E) Transversal sections at the level of the pronephros show the expression of all three transcripts in the spinal cord (sp), the pronephric tubule (pt) and the lateral plate mesoderm (lpm), and the endodermal yolk mass (end), notochord (n) and somites (so) are not labeled. (G–I) Late tailbud stage shows similar labeling patterns to the early tailbud. In addition, the pronephric tubule (pt) and duct (pd) are clearly labeled for fus; and the heart territory (h) for fus and wdr5 (G, I). (J–L) Transversal sections at the level of the pronephros confirm these patterns of expression and reveal that the level of brd3 transcripts decreases at this stage. (M–O) Whole-mounts and (P–R) transversal sections at pronephros level tadpole stage. Section analysis at this stage reveals that fus transcripts remain expressed in spinal cord (sp) and pronephric tubules (pt) brd3 and wdr5 are undetectable in these structures. Abbreviations: ba; branchial arches, cg; cement gland, e; eye, end; endodermal yolk mass, h; heart territory, lpm; lateral plate mesoderm, Me; mesencephalon, n; notochord, oc; optic cup, ov; otic vesicle, p; pronephros, pd; pronephric duct, pt; pronephric tubule, Rh; rhombencephalon, so; somite, sp; spinal cord, tb; tail bud, Te; telencephalon.
	Fig. S1. Selection of Ca2 +-dependent genes through subtractive library screening. Autoradiographs of 4 replicates membranes carrying bacterially-amplified fragments corresponding to Ca2 +-enriched cDNA and hybridized with 32P-labeled probes from initial caffeine-induced cDNA population (I), control (non-induced) cDNA population (II), subtracted “sense” (caffeine minus control) cDNA population (III) and subtracted “reverse” (control minus caffeine) cDNA population (IV). The F7 spot (blue tag) corresponds to fus gene and illustrates the enrichment of gene expression as it is labeled by the enriched induced minus non induced probe and is undetectable with the initially induced population (compare I and III replicates, at F7) and absent on II and IV replicates. brd3 was found twice on this spotted membrane, at F6 and G4 spots (red tag) and exhibits the same response than fus. p54nrb gene on F4 deposit (green tag) is revealed with both initial and subtracted “sense” probes and is not labeled with non-induced “reverse” populations. The wdr5 expression in caffeine-treated animal cap explants corresponded to the G1 load in the original report [10]. GFP was spotted on H12 as a negative control.
	brd3 (bromodomain containing 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, anterior view, dorsal up.
	brd3 (bromodomain containing 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up.
	brd3 (bromodomain containing 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	fus (FUS RNA binding protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, anterior view, dorsal up.
	fus (FUS RNA binding protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up.
	fus (FUS RNA binding protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	wdr5 (WD repeat domain 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 11, vegetal view, dorsal up.
	wdr5 (WD repeat domain 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, anterior view, dorsal up.
	wdr5 (WD repeat domain 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up.
	wdr5 (WD repeat domain 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.