An Intact Brachyury Function Is Necessary to Prevent Spurious Axial Development in Xenopus laevis

We have previously shown that the member of the HES family hairy2 induces the ectopic expression of dorsal markers when it is overexpressed in the ventral side of Xenopus embryos. Intriguingly, hairy2 represses the mesoderm transcription factor brachyury (bra) throughout its domain in the marginal zone. Here we show that in early gastrula, bra and hairy2 are expressed in complementary domains. Overexpression of bra repressed hairy2. Interference of bra function with a dominant-negative construct expanded the hairy2 domain and, like hairy2 overexpression, promoted ectopic expression of dorsal axial markers in the ventral side and induced secondary axes without head and notochord. Hairy2 depletion rescued the ectopic dorsal development induced by interference of bra function. We concluded that an intact bra function is necessary to exclude hairy2 expression from the non-organiser field, to impede the ectopic specification of dorsal axial fates and the appearance of incomplete secondary axes. This evidence supports a previously unrecognised role for bra in maintaining the dorsal fates inhibited in the ventral marginal zone, preventing the appearance of trunk duplications.Fil: Aguirre, Cecilia Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencias "Profesor Eduardo de Robertis"; ArgentinaFil: Murgan, Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencias "Profesor Eduardo de Robertis"; ArgentinaFil: Carrasco, Andres Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencias "Profesor Eduardo de Robertis"; ArgentinaFil: Lopez, Silvia Liliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencias "Profesor Eduardo de Robertis"; Argentin

Foxa4 favours notochord formation by inhibiting contiguous mesodermal fates and restricts anterior neural development in Xenopus embryos

In vertebrates, the embryonic dorsal midline is a crucial signalling centre that patterns the surrounding tissues during development. Members of the FoxA subfamily of transcription factors are expressed in the structures that compose this centre. Foxa2 is essential for dorsal midline development in mammals, since knock-out mouse embryos lack a definitive node, notochord and floor plate. The related gene foxA4 is only present in amphibians. Expression begins in the blastula –chordin and –noggin expressing centre (BCNE) and is later restricted to the dorsal midline derivatives of the Spemann's organiser. It was suggested that the early functions of mammalian foxa2 are carried out by foxA4 in frogs, but functional experiments were needed to test this hypothesis. Here, we show that some important dorsal midline functions of mammalian foxa2 are exerted by foxA4 in Xenopus. We provide new evidence that the latter prevents the respecification of dorsal midline precursors towards contiguous fates, inhibiting prechordal and paraxial mesoderm development in favour of the notochord. In addition, we show that foxA4 is required for the correct regionalisation and maintenance of the central nervous system. FoxA4 participates in constraining the prospective rostral forebrain territory during neural specification and is necessary for the correct segregation of the most anterior ectodermal derivatives, such as the cement gland and the pituitary anlagen. Moreover, the early expression of foxA4 in the BCNE (which contains precursors of the whole forebrain and most of the midbrain and hindbrain) is directly required to restrict anterior neural development.Fil: Murgan, Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Castro Colabianchi, Aitana Manuela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Monti, Renato José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Boyadjián López, Laura Elena. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencias; ArgentinaFil: Aguirre, Cecilia Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Gonzalez Stivala, Ernesto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Carrasco, Andres Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Lopez, Silvia Liliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentin

Wnt ligands regulate the asymmetric divisions of neuronal progenitors in C. elegans embryos

International audienceWnt/β-catenin signalling has been implicated in the terminal asymmetric divisions of neuronal progenitors in vertebrates and invertebrates. However, the role of Wnt ligands in this process remains poorly characterized. Here, we used the terminal divisions of the embryonic neuronal progenitors in C. elegans to characterize the role of Wnt ligands during this process, focusing on a lineage that produces the cholinergic interneuron AIY. We observed that, during interphase, the neuronal progenitor is elongated along the anteroposterior axis, then divides along its major axis, generating an anterior and a posterior daughter with different fates. Using time-controlled perturbations, we show that three Wnt ligands, which are transcribed at higher levels at the posterior of the embryo, regulate the orientation of the neuronal progenitor and its asymmetric division. We also identify a role for a Wnt receptor (MOM-5) and a cortical transducer APC (APR-1), which are, respectively, enriched at the posterior and anterior poles of the neuronal progenitor. Our study establishes a role for Wnt ligands in the regulation of the shape and terminal asymmetric divisions of neuronal progenitors, and identifies downstream components

<i>Hairy2</i> restricts the <i>bra</i> domain.

<p>(A–D) Distribution of <i>bra</i> transcripts (blue-purplish) at stage 10.5. Turquoise staining corresponds to <i>chordin</i> (<i>chd</i>) transcripts in the organiser. Embryos were injected into a ventral (A,B,E,G) or dorsal (C,D,E–G) cell at the 4-cell stage with 1.25 ng (C,E), 2.5 ng (A,E–G), or 5 ng (E,G) of control MO; 1.25 ng (D,E), 2.5 ng (B,E–G), or 5 ng (E,G) of <i>hairy2a</i> MO; 1.25 ng (E), 2.5 ng (E,G), or 5 ng (E,G) of <i>hairy2b</i> MO, or with a mix containing 2.5 ng of <i>hairy2a</i> MO +2.5 ng of <i>hairy2b</i> MO (G). DOG fluorescence (insets) indicates the injected side. (A,B) Whole embryos in vegetal views. Dorsal is oriented to the top. (C,D) Dorsal views of gastrulae showing <i>bra</i> and <i>chd</i> expression in the MZ. The green arrows in B,D point to up-regulations or domain expansions of <i>bra</i>. White bars in B,D indicate the width of the <i>bra</i> domain. (E) Percentage of embryos with <i>bra</i> expansion in the MZ. Comparison between different doses of control MO (blue line), <i>hairy2b</i> MO (pink line) and <i>hairy2a</i> MO (orange line). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054777#pone-0054777-t001" target="_blank">Table 1</a>. (F) Percentage of embryos displaying the arc of low <i>bra</i> expression that is normally observed in the organiser region (blue) or with an increase of <i>bra</i> expression in the organiser (light orange). (G) Percentage of embryos with <i>bra</i> expansion in the MZ. Comparison between the simultaneous knock-down of <i>hairy2a</i> and <i>hairy2b</i> and different doses of control MO, <i>hairy2a</i> MO, and <i>hairy2b</i> MO. n indicates the total number of injected embryos.</p

Effects of <i>braEnR</i> on morphogenesis and on DML markers at neurula stage.

<p>Ventral injections in one cell at the 4-cell stage with 1 ng of <i>braEnR</i> mRNA +10 ng of DOG (B–C’,E–F’,H–I’,K–L’). Expression of <i>foxA4a</i> (A–C), <i>shh</i> (D–F), <i>not I</i> (G–I), and <i>chd</i> (J–L), when control embryos reached stage 14 (A,D,G,J). At this stage, these markers are normally expressed in the DML <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054777#pone.0054777-Sasai1" target="_blank">[63] </a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054777#pone.0054777-RuiziAltaba1" target="_blank">[112]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054777#pone.0054777-Ekker1" target="_blank">[114]</a>. Arrows point to the dorsal axis. Yellow arrowheads point to cells expressing ectopic DML markers. Injected embryos are shown with the corresponding fluorescence image at the right (DOG) to identify the descendants of the injected cells. Embryos were classified into phenotypes A and B according to the grade of anterior-posterior extension of the dorsal axis. Embryos are shown in dorsal (A,D,G,J), posterior-dorsal (B,E,H) or posterior (C,F,I,K,L) views. The percentages of embryos displaying each phenotype are indicated in B,C,E,F,H,I,K,L. For each marker, % A+B indicates the percentage of affected embryos (A plus B phenotypes) and N, the total number of embryos analysed.</p

Ectopic induction of DML markers after interfering with <i>bra</i> function or overexpressing <i>hairy2a</i>.

<p>(A,C,E) Uninjected sibling controls. Embryos were ventrally injected into one cell at the 4-cell stage with 1 ng of <i>braEnR</i> (B,B’,D,D’,F,F’,G) or of <i>hairy2a</i> mRNAs (G). Expression of <i>chd</i> (A,B,G), <i>foxA4a</i> (C,D,G) and <i>not I</i> (E–G) at stage 10.5. The injected side was detected by DOG fluorescence (B’,D’,F’). Green arrows point to the ectopic expression of the analysed markers. At this stage, the markers analysed are normally expressed only in the organiser <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054777#pone.0054777-Sasai1" target="_blank">[63] </a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054777#pone.0054777-RuiziAltaba1" target="_blank">[112]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054777#pone.0054777-VonDassow1" target="_blank">[113]</a>. (G) Percentage of gastrulae ventrally injected with <i>braEnR</i> (blue bars) or <i>hairy2a</i> mRNAs (light orange bars) expressing ectopic <i>chd</i>, <i>foxA4a</i>, and <i>not I</i> (<i>chd</i>: 100%, n = 26; <i>foxA4a</i>: 100%, n = 14; <i>not</i> I: 79%, n = 11/14, for <i>braEnR</i>; <i>chd</i>: 92%, n = 23/25; <i>foxA4a</i>: 80%, n = 8/10; <i>not I</i>: 60%, n = 3/5, for <i>hairy2a</i>).</p

Opposite regulation of <i>chd</i> by <i>hairy2a</i> or <i>braEnR</i> depending on the D

<p>–<b>V context.</b> Ventral (A,E) or dorsal (I) injections of 1 ng of <i>braEnR</i> mRNA into one cell at the 4-cell stage. E corresponds to a high-power view of the embryo shown in A. Embryos were fixed when sibling controls reached stage 14 (A) or 11 (I). Red arrows in A,I indicate the descendants of the injected cells. In A, we made use of the myc-tag epitopes fused to the <i>braEnR</i> construct to reveal the distribution of the encoded recombinant protein (brown dots). The injections in I included 10 ng of DOG (inset). Embryos are shown in posterior (A), vegetal (B–D,F–I) or vegetal and dorsal (J) views. (B–D,F–H,J–L) Analysis of <i>chd</i> expression when sibling controls (J) reached stage 11 in ventralised (B–D) or dorsalised (F–H) embryos, which were left uninjected (B,F) or were injected with 1 ng of <i>hairy2a</i> (C,G,K,L) or of <i>braEnR</i> mRNAs (D,H,K,L) before the first cleavage. Yellow asterisks mark the gaps of <i>chd</i>-negative cells. (K) Percentage of embryos expressing (light orange bars) or not expressing (blue bars) <i>chd</i>. UV-irradiated, uninjected embryos (UV) showed complete loss (67%) or very weak expression (37%) of <i>chd</i> (n = 88). <i>Chd</i> was expressed in 80% or 87% of UV-irradiated embryos injected with 1 ng of <i>braEnR</i> (<i>braEnR/</i>UV, n = 55) or of <i>hairy2a</i> mRNAs (<i>hairy2a/</i>UV, n = 96), respectively. (L) Percentage of embryos with asymmetric (blue bars) or radially symmetric <i>chd</i> expression (light orange bars) around the blastopore. The maximum effect of LiCl alone was found in 50% of embryos, which showed complete radial <i>chd</i> expression (LiCl, n = 53). <i>Chd</i> expression decreased in 88% or 100% of dorsalised embryos that were injected with 1 ng of <i>braEnR</i> (<i>braEnR</i>/LiCl; n = 41) or of <i>hairy2a</i> mRNAs (<i>hairy2a</i>/LiCl, n = 23), respectively.</p

Effects of ventral injections of <i>braEnR</i> and <i>hairy2a</i> mRNAs.

<p>(A,B) Ventral injections into one ventral cell at the 4-cell stage produced spina bifida. (C) Mesodermal fate map of the blastomeres of the 16-cell-stage embryo (modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054777#pone.0054777-Kumano1" target="_blank">[58]</a>). Notochord (pink), somites (light blue), ventral blood islands (VBIs, light violet), Spemann’s organiser (SO). Green dots indicate the injection sites. (D–G) Ventral injections as in C occasionally induced small bumps (arrows, D,E) and tail-like structures (arrows, F,G). Embryos shown in D–G were hybridised with a <i>sox2</i> probe. (H) Percentage of embryos with secondary axes+protuberances. Comparison between different doses of <i>braEnR</i> mRNA injected into the AB3 (blue), AB4 (pink), CD3 (yellow) or CD4 (light blue) blastomere. (I) Percentage of embryos with secondary axes classified according to the injected blastomere. The bars correspond to <i>braEnR</i> (blue) or <i>hairy2a</i> (light orange) mRNAs injections. Injections included <i>nuc-lacZ</i> mRNA (A,B) or DOG (D–G) as lineage tracers. Embryos were fixed when sibling controls reached stage 28 and are shown in dorsal (A,B,D,G), dorso-lateral (E) or lateral (F) views. The amounts of mRNA injected are indicated in each figure.</p

<i>Bra</i> and <i>hairy2</i> establish complementary expression domains.

<p> Distribution of <i>hairy2</i> (A,D,G, blue purplish; C,F,J,K, magenta; I,L, turquoise) and <i>bra</i> transcripts (B,E,H, blue-purplish; C,F,J,K, turquoise; I,L, magenta). For double ISHs, the name of each probe is written with the colour corresponding to the substrate with which it was revealed. Stage 9.5 (A–C), 10.25 (D–F), and 10.5 (G–L). (A–F,J,K) ISH in bisected embryos. The animal side is oriented to the top. d: dorsal side, v: ventral side. In C–F and J, the embryos were bisected in the sagittal plane, as represented by the broken black line in L. In K, the embryo was cut in a para-sagittal plane, as represented by the broken white line in L. Two halves of the same embryo are shown in the pairs A,B and D,E. The photographs in A,D were flipped 180° to facilitate comparison with the corresponding contralateral hemisection shown in B,E, respectively. Red arrows in A,B,D,E point to the overlapping of <i>bra</i> and <i>hairy2</i> expression in the MZ. Yellow arrows in D,E mark the incipient blastopore dorsal lip. White asterisks in F,J indicate the overlapping of <i>hairy2</i> and <i>bra</i> expression on the dorsal side. In J, the white, black and yellow arrowheads point to the prechordal mesoderm, the dorsal NIMZ, and the posterior, pre-involuted axial mesoderm, respectively. (G–I) Whole embryos in vegetal views. Dorsal is oriented to the top. The red broken line demarcates the blastopore. Yellow arrowheads in G point to the NIMZ. The arc between green arrows in G–I,L corresponds to the organiser region, with higher levels of <i>hairy2</i> (G,I,L) and lower levels of <i>bra</i> expression (H,I,L). (L) Dorsal view of the same embryo shown in I.</p