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

Segregation of brain and organizer precursors is differentially regulated by Nodal signaling at blastula stage

The blastula Chordin- A nd Noggin-expressing (BCNE) center comprises animal-dorsal and marginal-dorsal cells of the amphibian blastula and contains the precursors of the brain and the gastrula organizer. Previous findings suggested that the BCNE behaves as a homogeneous cell population that only depends on nuclear β-catenin activity but does not require Nodal and later segregates into its descendants during gastrulation. In contrast to previous findings, in this work, we show that the BCNE does not behave as a homogeneous cell population in response to Nodal antagonists. In fact, we found that chordin.1 expression in a marginal subpopulation of notochordal precursors indeed requires Nodal input. We also establish that an animal BCNE subpopulation of cells that express both, chordin.1 and sox2 (a marker of pluripotent neuroectodermal cells), and gives rise to most of the brain, persisted at blastula stage after blocking Nodal. Therefore, Nodal signaling is required to define a population of chordin.1+ cells and to restrict the recruitment of brain precursors within the BCNE as early as at blastula stage.We discuss our findings in Xenopus in comparison to other vertebrate models, uncovering similitudes in early brain induction and delimitation through Nodal signaling.Fil: 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; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Biología Celular e Histología; ArgentinaFil: Tavella, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Boyadjián López, Laura Elena. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Biología Celular e Histología; Argentina. 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: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Franchini, Lucia Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Lopez, Silvia Liliana. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Biología Celular e Histología; Argentina. 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

Notch1 is asymmetrically distributed from the beginning of embryogenesis and controls the ventral center

Based on functional evidence, we have previously demonstrated that early ventral Notch1 activity restricts dorsoanterior development in Xenopus. We found that Notch1 has ventralizing properties and abolishes the dorsalizing activity of β-catenin by reducing its steady state levels, in a process that does not require β-catenin phosphorylation by glycogen synthase kinase 3β. In the present work, we demonstrate that Notch1 mRNA and protein are enriched in the ventral region from the beginning of embryogenesis in Xenopus. This is the earliest sign of ventral development, preceding the localized expression of wnt8a, bmp4 and Ventx genes in the ventral center and the dorsal accumulation of nuclear β-catenin. Knockdown experiments indicate that Notch1 is necessary for the normal expression of genes essential for ventral-posterior development. These results indicate that during early embryogenesis ventrally located Notch1 promotes the development of the ventral center. Together with our previous evidence, these results suggest that ventral enrichment of Notch1 underlies the process by which Notch1 participates in restricting nuclear accumulation of β-catenin to the dorsal side.Fil: 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: Revinski, Diego Raul. 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; Argentina. Aix Marseille Université, CNRS, IBDM; France; ArgentinaFil: Encinas, Paula Inés. 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: Baez, María Verónica. 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: Rodríguez Abinal, Mateo. 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: Kodjabachian, Laurent. Aix Marseille Université, CNRS, IBDM; France; ArgentinaFil: Franchini, Lucia Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas; 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

<i>FoxA4</i> overexpression produced complementary effects on <i>Xanf1</i> and <i>otx2</i> and disfavoured anterior development.

<p>(<b>A, B, F, G, K, L</b>) Anterior views of early neural plate stage embryos analysed by WMISH with the following markers: <i>Xanf1</i> and <i>en2</i> (A, F, K), <i>otx2</i> and <i>krox20</i> (B, G, L). Embryos were injected with 0.25 ng (F, K) or 1 ng (G, L) of <i>foxA4FL</i> mRNA before the first cleavage, or were left uninjected (A, B). <i>FoxA4</i> overexpression affected <i>Xanf1</i> expression in 83% of the injected embryos (n = 23) as follows: it was reduced in the ANR but not in the SHA (48%, n = 23) (F), or it was reduced in the whole domain (35%, n = 23) (K). The expression hole of the <i>otx2</i> domain was filled with <i>otx2</i> transcripts (42%, n = 19) (G); the <i>otx2</i> domain was reduced, but the caudal diencephalic/mesencephalic stripes were expanded (89%, n = 19) (L). (<b>C–E, H–J, M–O</b>) Anterior views of tailbuds (stage 27/28) injected with 0.25 ng (H, I, M, N) or 0.5 ng (J, O) of <i>foxA4FL</i> mRNA before the first cleavage (I, J, M–O) or into one cell at the 2-cell stage (H), or left uninjected (C, D, E). Expression of <i>otx2</i> (C,H,M), <i>Xanf1</i> and <i>en2</i> (D, I, N), <i>Xag1</i>/<i>N-tubulin</i> (E, J, O). After overexpression of <i>foxA4</i>, the caudal diencephalic <i>otx2</i> subdomain was expanded anteriorly (54%, n = 13) (H) or the telencephalic <i>otx2</i> subdomain disappeared (40% n = 10) (M). The pituitary <i>Xanf1</i> domain was expanded (33%, n = 15) (I). <i>N-tubulin</i> expression in the rostral forebrain (rectangle in E) was deleted and the <i>Xag1</i> + cement and hatching glands were reduced (J) or the cement gland was absent (O) (54%, n = 24). The most severe phenotypes presented head truncations (23%, n = 13) (M,N,O). Red arrowhead, anterior limit of the caudal diencephalic <i>otx2</i> subdomain; white arrowhead, posterior limit of the ventral telencephalic <i>otx2</i> subdomain; vtel, ventral telencephalic <i>otx2</i>; cdi, caudal diencephalic <i>otx2</i>; mes, mesencephalic <i>otx2</i>; op, olfactory placode; epiph, epiphisis; ev, eye vesicle; cg, cement gland; hg, hatching gland cells; pit, pituitary anlage; is, injected side; nis, non-injected side.</p

FoxA4MO reproduced the effects on anterior neural markers in embryos in which mesoderm induction was blocked with Cer-S.

<p>(<b>A–D</b>) Expression of <i>foxA4</i> in sibling controls (A, C) or in embryos injected with Cer-S mRNA (B, D), analysed at stage 9 (A, B) or at stage 11 (C, D). After blocking mesoderm induction, <i>foxa4</i> expression persisted in the BCNE (B) (100%, n = 15) but was suppressed from the axial mesoderm (D) (100%, n = 7). (<b>E, F</b>) <i>MyoD</i> expression at neural plate stage in a sibling control (E) and in an embryo injected with Cer-S. Mesoderm was suppressed in 100% of the injected embryos analyzed with <i>myoD</i> (100%, n = 8). (<b>G–I</b>) Expression of <i>Xanf1</i> and <i>en2</i> at neural plate stage in a sibling control (G), and in embryos in which mesoderm induction was blocked with Cer-S and were injected at the 1-cell stage with either 20 ng of CtrlMO (H) or 20 ng of FoxA4MO (I). Embryos injected with CtrlMO + Cer-S showed expression of <i>Xanf1</i> (yellow arrow) and <i>en2</i> (black arrow) (H, 100%, n = 8). In embryos injected with FoxA4MO + Cer-S, <i>Xanf1</i> was expanded and up-regulated (yellow arrow) and <i>en2</i> was down-regulated (black arrow) (I, 100%, n = 10) in comparison to embryos injected with CtrlMO + Cer-S (H). (J–L) Expression of <i>otx2</i> at neural plate stage in a sibling control (J), and in embryos in which mesoderm induction was blocked with Cer-S and were injected at the 1-cell stage with either 20 ng of Ctrl MO (K) or 20 ng of FoxA4MO (L). Embryos injected with CtrlMO + Cer-S showed expression of <i>otx2</i> (K, 100%, n = 4). In embryos injected with FoxA4MO + Cer-S, <i>otx2</i> was down-regulated (L, 100%, n = 20) in comparison to embryos injected with CtrlMO + Cer-S (K).</p

FoxA4 depletion altered neurogenesis along the A–P axis.

<p>(<b>A–J</b>) <i>N-tubulin</i> and <i>Xag1</i> expression in stage 27/28 embryos injected with 40 ng of CtrlMO (A, C, E–G) or FoxA4MO (B, D, H–J) into one cell at the 2-cell stage. (A, B) Dorsal views. (C, D) Magnified images of the trunk region of another pair of CtrlMO (C) and FoxA4MO (D) injected embryos, shown in dorso-lateral views to facilitate comparisons. The red arrow points to an <i>N-tubulin</i> + projection on the injected side. (E, F, H, I) Lateral views of the head region. (G, J) Anterior views. <i>N-tubulin</i> is expressed in the trigeminal (tn) and vestibulocochlear (vn) nerves. In FoxA4MO-injected embryos, the tn and vn were disturbed (red arrowhead) (H), as revealed by <i>N-tubulin</i>; the cement gland (cg) was bent and closer to the forebrain, and the hatching gland (hg) was shortened on the FoxA4-depleted side, as revealed by <i>Xag1</i> (green arrowhead) (J) (90%, n = 21). (<b>K–N</b>) Transverse sections at the levels indicated by dotted black lines in C,D, shown in bright field (K, M) and their corresponding nuclear Hoescht fluorescence (L, N). Bright field images were processed with Adobe Photoshop CS2 in order to superimpose the <i>N-tubulin</i> expression (yellow) to the Hoescht fluorescence. Yellow arrows point to the projection emerging from the dorsal neural tube; cyan arrows point to both sides of the ventral neural tube; green arrows point to the place left by the disorganised or absent notochord; no, notochord; is, injected side; nis, non-injected side. (O, P) Summary of the results shown in A–J.</p

<i>Xanf1</i> and <i>otx2</i> progressively establish complementary domains.

<p>Normal expression of <i>Xanf1</i>, <i>en2</i> and <i>hoxb7</i> (A, B, C) and <i>otx2</i> and <i>krox20</i> (D–F) in progressively older neural plate stage embryos in anterior views. Yellow asterisks, posterior border of <i>Xanf1</i> in the ANR; red asterisks, anterior border of <i>Xanf1</i> in the ANR; white asterisks, SHA; black asterisks, expression hole in the neural <i>otx2</i> subdomain; black arrows, caudal diencephalic-mesencephalic stripes demarcating the posterior border of the <i>otx2</i> neural subdomain; yellow arrows, stripe corresponding to the anterior border of <i>otx2</i> in the ANR; red arrows, SHA; white arrows, CGA. (G) Diagram summarising the expression patterns of <i>otx2</i>, <i>Xanf1</i>, <i>en2</i>, and <i>krox20</i> in an anterior view of embryos at the stages shown in B, C, E, F. ANR, anterior neural ridge; r3, third rhombomere; m/h, midbrain/hindbrain boundary.</p

<i>FoxA4</i> inhibition affected anterior ectodermal/neural specification.

<p>Embryos at early neural plate stage injected with 20 ng of CtrlMO or of FoxA4MO before the first cleavage (A, B, F, G, K–N, P–S) or unilaterally injected into 1 cell at the 2-cell stage (C, D, H, I, O, T). The injected side was determined by DOG fluorescence (insets). Embryos were hybridised with the following probes: <i>Xanf1</i>, <i>en2</i>, and <i>hoxb7</i> (A–C, F–H); <i>hoxc6</i> (D, I); <i>otx2</i> and <i>krox20</i> (K, L, P, Q); <i>Xag1</i> and <i>N-tubulin</i> (M, N, R, S); <i>en2</i> and <i>hoxb1</i> (O, T). Green arrows in (A, F) point to the anterior limit of <i>hoxb7</i>. At neural plate stage, <i>Xag1</i> marks the cement gland anlage (indicated between red arrowheads in N), and the hatching gland primordia (red arrows in N) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110559#pone.0110559-Wardle1" target="_blank">[98]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110559#pone.0110559-Theveneau1" target="_blank">[90]</a>. Black arrowhead in (H), caudal shift of <i>Xanf1</i> and fusion with the <i>en2</i> stripe. Black arrow in (H), fusion of the SHA and the ANR. Red arrow in (I), down-regulation of <i>hoxc6</i> on the injected side. Yellow arrowhead in (Q), diffuse CGA and anterior border of the ANR. Notice the down-regulation and the caudal shift of <i>en2</i> (red arrows) and <i>hoxb1</i> (green arrows) by comparing the injected side (right) with the non-injected side (left) in the FoxA4 morphant in (T) and with the CtrlMO-injected embryo in (O). (E) Ratio between the distance from the posterior limit of <i>Xanf1</i> to the blastopore and the total length of the embryo in the groups shown in A, B, F, G (r-Xanf1). The ratio was significantly lower in FoxA4 morphants; <i>P</i><0.0001, two-tailed t-test. (J) The ratio between the length of the <i>hoxb7</i> domain and the embryo’s length was significantly lower in FoxA4 morphants, as measured in the groups shown in A, B, F, G; <i>P</i><0.0001, two-tailed t-test. ANR, anterior neural ridge; CGA, cement gland anlage; SHA, stomodeal-adenohypophyseal anlage; m/h, midbrain/hindbrain boundary; m, d, presumptive mesencephalic and caudal diencephalic regions expressing <i>otx2</i>; r3, third rhombomere; r4, fourth rhombomere. (A, D, F, I, K, M, O, P, Q, R, T) are dorsal views; (B, C, G, H, L, N, Q, S) are anterior views.</p

FoxA4MO disrupted DML formation.

<p>(<b>A–H</b>) Stage 15 embryos left uninjected (A) or injected with 0.5 ng of <i>foxA4FL</i> mRNA (E), 40 ng of CtrlMO (B–D) or of FoxA4MO (F–H) before the first cleavage. (<b>I–T</b>) Tailbud stage embryos injected into two dorsal cells at the 4-cell stage with 20 ng of CtrlMO or of FoxA4MO. The descendants of these cells give rise to the DML and the cephalic region. Expression of <i>chd</i> (A, B, E, F, I, J, M, N, Q, R), <i>shh</i> (C, G, K, L, O, P, S, T), <i>hairy2</i> (D, H). The inset in K shows a magnified view of the area depicted by the rectangle; fp, floor plate; no, notochord; h, hypochord. Embyos injected with <i>foxA4FL</i> mRNA showed a thicker notochord than control siblings, as revealed by <i>chd</i> expression (87%, n = 15) (E). The percentage of embryos injected with FoxA4MO showing the indicated changes in the corresponding markers is indicated between parentheses, as follows. At neural plate stage, the expression of <i>chd</i> (90%, n = 20) (F) and <i>shh</i> (54%, n = 22) (G) was patchy, and <i>hairy2</i> expression was reduced in the FP domain (60%, n = 23) (H). At stage 26, the <i>chd</i> domain was patchy and/or thinner (80%, n = 21) (M, N, Q, R). Arrowheads point to <i>chd</i> + cells in dorsal and ventral positions with respect to a notochordal gap. <i>Shh</i> was also patchy (arrowheads, O, P) or completely abolished (S,T) (86%, n = 22). In overall, tailbuds injected with FoxA4MO showed reduced heads (89%, n = 51) (M, Q, O, S). (A–H, J, L, N, P, R, T) dorsal views; (I, K, M, O, Q, S) lateral views.</p

The DML is invaded by paraxial mesoderm and neuroectoderm in FoxA4 morphants.

<p>Dorsal views of embryos at neural plate stage hybridised with <i>myf5</i> (A, D), <i>myoD</i> (B, E, G, H), or <i>sox2</i> probes (C, F). They were injected before the first cleavage with 20 ng CtrlMO (A–C) or of FoxA4MO (D–F), 0.25 ng of <i>foxA4FL</i> mRNA (H), or left uninjected (G). In FoxA4 morphants, <i>myf5</i> (D) and <i>myoD</i> expression (E) was found in the DML (40%, n = 23; 56%, n = 23, respectively). The left embryo in E shows that <i>myoD</i> expression invaded the DML along the A–P axis. The right embryo in E shows stretches of the <i>myoD</i> domains fused at the midline (arrowheads). <i>Sox2</i> transcripts obliterated the prospective FP (79%, n = 19) (F). In embryos injected with <i>foxA4FL</i> mRNA, <i>myoD</i> expression was reduced, and the medial borders of the bilateral domains were more separated (H, 42%, n = 26) than in control siblings (G).</p