Vestigial-like 3 is a novel Ets1 interacting partner and regulates trigeminal nerve formation and cranial neural crest migration

Drosophila Vestigial is the founding member of a protein family containing a highly conserved domain, called Tondu, which mediates their interaction with members of the TEAD family of transcription factors (Scalloped in Drosophila). In Drosophila, the Vestigial/Scalloped complex controls wing development by regulating the expression of target genes through binding to MCAT sequences. In vertebrates, there are four Vestigial-like genes, the functions of which are still not well understood. Here, we describe the regulation and function of vestigial-like 3 (vgll3) during Xenopus early development. A combination of signals, including FGF8, Wnt8a, Hoxa2, Hoxb2 and retinoic acid, limits vgll3 expression to hindbrain rhombomere 2. We show that vgll3 regulates trigeminal placode and nerve formation and is required for normal neural crest development by affecting their migration and adhesion properties. At the molecular level, vgll3 is a potent activator of pax3, zic1, Wnt and FGF, which are important for brain patterning and neural crest cell formation. Vgll3 interacts in the embryo with Tead proteins but unexpectedly with Ets1, with which it is able to stimulate a MCAT driven luciferase reporter gene. Our findings highlight a critical function for vgll3 in vertebrate early development

Vestigial-like 3 is a novel Ets1 interacting partner and regulates trigeminal nerve formation and cranial neural crest migration

International audienceDrosophila Vestigial is the founding member of a protein family containing a highly conserved domain, called Tondu that mediates their interaction with members of the TEAD family of transcription factors (Scalloped in Drosophila). In Drosophila, the Vestigial/Scalloped complex controls wing development by regulating the expression of target genes through binding to MCAT sequences. In vertebrates, there are four Vestigial-like genes whose functions are still not well understood. Here we describe the regulation and function of vestigial-like 3 (vgll3) during Xenopus early development. A combination of signals including FGF8, Wnt8a, Hoxa2, Hoxb2 and retinoic acid limits vgll3 expression to hindbrain rhombomere 2. We show that vgll3 regulates trigeminal placode and nerve formation and is required for normal neural crest development by affecting their migration and adhesion properties. At the molecular level, vgll3 is a potent activator of pax3, zic1, Wnt and FGF that are important for brain patterning and neural crest cell formation. Vgll3 interacts in the embryo with Tead proteins but unexpectedly with Ets1 with which it is able to stimulate a MCAT driven luciferase reporter gene. Our findings highlight a critical function for vgll3 in vertebrate early development

Comparative Functional Analysis of <em>ZFP36</em> Genes during <em>Xenopus</em> Development

<div><p>ZFP36 constitutes a small family of RNA binding proteins (formerly known as the TIS11 family) that target mRNA and promote their degradation. In mammals, ZFP36 proteins are encoded by four genes and, although they show similar activities in a cellular RNA destabilization assay, there is still a limited knowledge of their mRNA targets and it is not known whether or not they have redundant functions. In the present work, we have used the <em>Xenopus</em> embryo, a model system allowing gain- and loss-of-function studies, to investigate, whether individual ZFP36 proteins had distinct or redundant functions. We show that overexpression of individual amphibian zfp36 proteins leads to embryos having the same defects, with alteration in somites segmentation and pronephros formation. In these embryos, members of the Notch signalling pathway such as <em>hairy2a</em> or <em>esr5</em> mRNA are down-regulated, suggesting common targets for the different proteins. We also show that mouse Zfp36 protein overexpression gives the same phenotype, indicating an evolutionary conserved property among ZFP36 vertebrate proteins. Morpholino oligonucleotide-induced loss-of-function leads to defects in pronephros formation, reduction in tubule size and duct coiling alterations for both <em>zfp36</em> and <em>zfp36l1</em>, indicating no functional redundancy between these two genes. Given the conservation in gene structure and function between the amphibian and mammalian proteins and the conserved mechanisms for pronephros development, our study highlights a potential and hitherto unreported role of <em>ZFP36</em> gene in kidney morphogenesis.</p> </div

mRNA expression of <i>Xenopus zfp36</i> genes during development.

<p>(A) RT-PCR analyses showed that all <i>zfp36</i> genes are maternally expressed. <i>zfp36, zfp36l1</i> and <i>zfp36l2</i> mRNAs are expressed at a constant level throughout development from stage 2 to stage 33 while <i>zfp36l4</i> mRNA level decreases after the mid-blastula transition (MBT, arrow). (B) <i>In situ</i> hybridization showed that all four <i>zfp36</i> mRNA are localized at the animal pole in 4-cell stage (a–d) and morula stage (i–l) embyos. e–h correspond to histological sections from embryos shown in a–d. (C) RT-PCR analysis showed that <i>zfp36</i> mRNAs are preferentially expressed in the animal pole region of blastula embryos. (D) RT-PCR analysis showed that <i>zfp36</i> mRNAs are expressed throughout the embryo at the gastrula stage. An, animal pole; DM, dorsal marginal zone; Emb, whole embryo; Ve, vegetal pole; VM, ventral marginal zone. A control embryo (Emb) assayed by RT-PCR for the expression of control genes <i>chordin</i> and <i>wnt8</i>. <i>odc</i> was used as control of loading and a reaction was performed in the absence of reverse transcriptase to check for genomic DNA contamination (-).</p

<i>Zfp36</i> mRNA overexpression does not prevent mesoderm induction nor myogenic factor expression.

<p>(A) Two-cell stage embryos were injected with 250 pg of the different <i>Xenopus zfp36</i> mRNAs or mouse <i>zfp36</i> mRNA (<i>mzfp36</i>) and animal caps were explanted at stage 8.5–9 then treated with 10 ng/ml of activin before analysis by RT-PCR for <i>xbra</i> expression when control embryos reached stage 12. Stage 12 embryo (Emb) or untreated animal caps (-) were assayed by RT-PCR in parallel. <i>Odc</i> was used as control of loading and a reaction was performed in the absence of reverse transcriptase to check for genomic DNA contamination (-RT). (B) 250 pg of <i>Xenopus zfp36</i> (a, b) and <i>zfp36l1</i> (c, d) or mouse <i>Zfp36</i> (<i>mZfp36</i>, e, f) mRNAs were injected in one blastomere of two-cell stage embryos and developing embryos were fixed at stage 28 and analyzed by <i>in situ</i> hybridization for <i>myod</i> expression.</p

Overexpression of Leap2 impairs Xenopus embryonic development and modulates FGF and activin signals

Besides its widely described function in the innate immune response, no other clear physiological function has been attributed so far to the Liver-Expressed-Antimicrobial-Peptide 2 (LEAP2). We used the Xenopus embryo model to investigate potentially new functions for this peptide. We identified the amphibian leap2 gene which is highly related to its mammalian orthologues at both structural and sequence levels. The gene is expressed in the embryo mostly in the endoderm-derived tissues. Accordingly it is induced in pluripotent animal cap cells by FGF, activin or a combination of vegT/beta-catenin. Modulating leap2 expression level by gain-of-function strategy impaired normal embryonic development. When over-expressed in pluripotent embryonic cells derived from blastula animal cap explant, leap2 stimulated FGF while it reduced the activin response. Finally, we demonstrate that LEAP2 blocks FGF-induced migration of HUman Vascular Endothelial Cells (HUVEC). Altogether these findings suggest a model in which LEAP2 could act at the extracellular level as a modulator of FGF and activin signals, thus opening new avenues to explore it in relation with cellular processes such as cell differentiation and migration. (C) 2016 Elsevier Inc. All rights reserved

<i>Zfp36</i> mRNA overexpression alters the formation of pronephros and affects pronephric marker genes expression.

<p>(A) 250 pg of <i>Xenopus zfp36</i> mRNA were injected into one ventral blastomere of 8-cell stage embryos and developing embryos were fixed at stage 39 before immunhistochemistry analysis with the pronephros specific markers 3G8 and 4A6. Arrows in b and d mark the pronephros (pn) alteration on the injected side. (B) 250 pg of <i>Xenopus zfp36</i> (a–d) or <i>zfp36l1</i> (e–h) mRNA was injected into one ventral blastomere of 8-cell stage embryos and developing embryos were fixed at stage 22 (a, b and e–h) or stage 26 (c, d) before <i>in situ</i> hybridization analysis for <i>pax8</i> or <i>lim1</i> expression. Arrows in b, d, f and h mark the pronephros (pn) alteration on the injected side. (C) Two-cell stage embryos were injected or not (NI) with 250 pg of the different <i>Xenopus zfp36</i> mRNAs. Animal caps were explanted at stage 8.5–9 and treated with activin plus retinoic acid (RA) before analysis by RT-PCR for <i>smp30</i> expression when control embryos reached stage 35. Stage 35 embryo (Emb) or untreated animal caps (-) were assayed by RT-PCR in parallel. <i>Odc</i> was used as control of loading and a reaction was performed in the absence of reverse transcriptase to check for genomic DNA contamination (-RT).</p

<i>Zfp36</i> mRNA overexpression induces somites segmentation defects.

<p>250 pg of mouse <i>zfp36</i> mRNA (a, b) or <i>Xenopus zfp36</i> (d, e), <i>zfp36l1</i> (I, j), <i>zfp36l2</i> (k, l) <i>or zfp36l4</i> (m, n) mRNA were injected into one blastomere of two-cell stage embryos and developing embryos were fixed at stage 28 before immunhistochemistry analysis with the somite specific marker 12/101. Embryos were embedded in paraffin then sectioned longitudinally (c, f) or treated for scanning electronic microscopy (g, h). The arrows mark the alteration of segmentation on the injected side (Inj) by comparison with the uninjected side (Uninj). no, notochord; so, somite.</p

<i>Zfp36</i> and <i>zfp36l1</i> morpholino knock down induces pronephros alterations.

<p>20 ng of morpholinos directed against <i>zfp36</i> (a, b) or <i>zfp36l1</i> (c, d) mRNAs or control morpholinos (e, f) were injected into one ventral blastomere of 8-cell stage embryos with 250 pg of <i>lacZ</i> mRNA. In rescue experiments, 100–200 pg of mouse <i>zfp36</i> mRNA were co-injected with 20 ng of MO <i>zfp36</i> (g, h). Developing embryos were fixed at stage 40 before <i>lacZ</i> staining and immunohistochemistry analysis to reveal the expression of pronephros specific markers, 3G8 and 4A6. Arrows and arrowheads in b, d, f and h, mark the pronephros proximal tubule (tu) and duct (du) respectively on injected sides of the embryos. I–p, Close up views of anterior region showing uninjected or injected sides of representative phenotypes for <i>zfp36</i> morphants (i–l) and <i>zfp36l1</i> morphants (m–p).</p

Conservative evolution of vertebrate <i>zfp36</i> genes.

<p>(A) Conserved syntenic regions between human (<i>Hsa</i>), mouse (<i>Mmu</i>) and <i>Xenopus tropicalis</i> (<i>Xtr</i>) chromosome regions containing <i>zfp36</i>, <i>zfp36l1</i> and <i>zfp36l2</i>. Gene names symbols are according to HUGO. Boxes with the same colour correspond to the same gene; white boxes correspond to genes without annotation or without orthologues in the species shown here. The drawing is not to scale to avoid complexity and dashes represent long chromosome regions. (B) Conserved structural organization of vertebrates <i>zfp36</i> genes between evolutionary distant animals. Exons (1, 2) are figured in open boxes and intron as a solid line respectively. Shaded box, untranslated region. TZF, Tandem Zing Finger domain.</p