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Distinct and cooperative roles for Nodal and Hedgehog signals during hypothalamic development

¹ U 368 INSERM, Ecole Normale Supérieure, 46 rue d’Ulm, 75005 Paris, France
² Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK

View larger version (108K): [in a new window]   Fig. 1. shh and emx2 expression sites define complementary domains of the hypothalamus. (A-K) Lateral views of brains of wild-type embryos with anterior towards the left showing expression of nk2.1a, shh and emx2 either in dark blue or red, as indicated (bottom right). Stages are indicated on the far right. Arrowheads in D show additional sites of emx2 expression in the posterior diencephalon and dorsal telencephalon. In J,K, the arrowheads indicate the PV hypothalamic region that expresses nk2.1a and emx2 but not shh. (L-M) Frontal serial sections through the hypothalamus, dorsal towards the top. The dorsal limit of nk2.1a and shh expression domain coincide (arrowheads). (N) Schematic view of nk2.1a, shh and emx2 expression in the 28 hpf brain. nk2.1a expression encompasses both shh and emx2 hypothalamic domains. h, hypothalamus; pd, posterior diencephalon, t, telencephalon; zli, zona limitans intrathalamica.

View larger version (88K): [in a new window]   Fig. 2. Progressive loss of PV and then AD hypothalamus in Nodal pathway mutants. (A-F) Lateral views of hypothalamic gene expression (indicated bottom right) in 28 hpf brains of embryos of various genotypes (indicated top right). Arrowheads in C and D indicate remaining shh and nk2.1a expression in the hypothalamus of MZsqt embryos. The arrow in D indicates posterior diencephalic expression of emx2. e, eye; h, hypothalamus; t, telencephalon; zli, zona limitans intrathalamica.

View larger version (120K): [in a new window]   Fig. 3. Reception of Nodal signals is required for cells to contribute to the PV but not the AD hypothalamus. Embryos in which wild-type and/or MZoep cells, or wild-type cells expressing Fast1SID, were transplanted into the prospective hypothalamus of wild-type hosts are shown. Genotypes are indicated in the top left-hand corner (donor>host). (A-F) Live embryos in which transplanted cells are visualized with GFP (green) or rhodamine-dextran (red). (A) Dorsal view of a shield stage embryo with rostral upwards. The blastoderm margin is indicated by broken line. At this stage, hypothalamic precursors are close to the shield, some distance from the front of the neural plate. (B-C) Lateral views of bud stage embryos with anterior to the left. The anterior limit of the prechordal mesendoderm is indicated by white dots. By this stage, hypothalamic precursors have reached the front of the neural plate. (D-F) Lateral views of brains of 28 hpf embryos with anterior towards the left. (G-L) Frontal sections through the hypothalami of 28 hpf embryos, showing ß-gal+ transplanted cells (brown) in relation to the expression of various hypothalamic markers (indicated in the bottom right-hand corner). AD and PV, anterior dorsal and posterior ventral hypothalamus; or, optic recess. Scale bars: 60 µm.

View larger version (76K): [in a new window]   Fig. 4. Mesendoderm can rescue AD hypothalamus and neurogenesis in MZoep embryos. Views of wild-type embryos, MZoep embryos or MZoep embryos injected in one blastomere at the 16-cell stage with either tar* RNA or tar*+XFD RNA together with nls-GFP or nls-lacZ RNA as lineage tracers. Genotype+injected RNA indicated top right. Probes and antibodies indicated in the bottom right-hand corner. (A-C) Frontal views of live embryos with GFP labeled rescued mesendoderm visible (green). Cyclopia is rescued in B. (D-F) Lateral views of brains with anterior towards the left showing expression of the endodermal marker foxa2. Expression in the brain (arrowhead) is at the zona limitans intrathalamica. (G-I) Lateral views (G,H) and transverse section (I) of brains showing absence/presence of hypothalamic markers. The rescued AD hypothalamus (black arrowheads) and the ß-gal-positive endodermal tar*-expressing cells (arrow) are indicated. Expression of emx2 in G,H (asterisks) is in the dorsal brain (primarily telencephalon). (J) Schematic showing experimental approach in which MZoep cells expressing GFP and lacZ RNA are transplanted into the tar*-expressing domain of a tar* mRNA injected MZoep host. (K) Transverse section of a brain from an embryo that underwent the experimental procedure outlined in J. The transplanted cells (brown nuclei) contribute to the rescued hypothalamus stained with nk2.1a (blue). (L-N) Frontal views of embryos labeled to show Hu immunoreactive neurons in the ventral forebrain (white arrowheads). In N, neurons are restored above the rescued mesendoderm (GFP green). (O,P) Ventral views of brains labeled to show TH immunoreactive ventral diencephalic neurons. These neurons are absent in MZoep embryos (not shown) but are restored in the mesendoderm-rescued embryo shown in P. h, hypothalamus; l, lens; r, retina, t, telencephalon, zli, zona limitans intrathalamica.

View larger version (100K): [in a new window]   Fig. 5. Expression of endodermal and prechordal plate markers in MZoep gastrula following tar* and tar*+XFD RNA injections. Views of MZoep gastrulae (stage indicated above and genes analyzed in the bottom right-hand corner). (A,D,E,H,I,L) Animal pole views with ventral towards the top; (B,C,F,G,J,K) dorsal views with the animal pole towards the top. (A-D) Uninjected embryos. (E-H) tar* RNA-injected embryos. (H) The rescued prechordal plate expression of bmp4 is indicated (arrowhead in H) and the remaining bmp4 expression is in the non-neural ectoderm. (I-L) tar*+XFD RNA injected embryos.

View larger version (104K): [in a new window]   Fig. 6. Hh and Nodal signals have both discrete and cooperative roles in hypothalamus formation. In all panels, genotype + injected RNA/drug treatment is shown in the top right-hand corner, genes/proteins visualized are indicated in the bottom right-hand corner. (A-D) Lateral views of 28 hpf brains. (E-F) Dorsal views of shield stage MZoep embryos showing induction of shh expression upon tar* RNA injection. (G-L) Sections through the hypothalami of wild-type embryos in which ß-gal-labeled cells (brown) were transplanted into the prospective hypothalamus at shield stage. (G-J) Sections through 28 hpf brains. The arrowheads in H,J indicate cells that have been expelled to the mesenchyme around the eyes. This was never observed for MZoep cells or cells expressing Fast1SID and thus does not depend on the depth at which cells were transplanted into the epiblast. (K,L) Sections through bud stage neural plates. Cells were found at various positions along the anteroposterior axis within the nk2.1a expression domain. dd, dorsal diencephalon; h, hypothalamus; t, telencephalon. Scale bars: 25 µm.