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Development of posterior hypothalamic neurons enlightens a switch in the prosencephalic basic plan.

In rats and mice, ascending and descending axons from neurons producing melanin-concentrating hormone (MCH) reach the cerebral cortex and spinal cord. However, these ascending and descending projections originate from distinct sub-populations expressing or not "Cocaine-and-Amphetamine-Regulated-Transcript" (CART) peptide. Using a BrdU approach, MCH cell bodies are among the very first generated in the hypothalamus, within a longitudinal cell cord made of earliest delaminating neuroblasts in the diencephalon and extending from the chiasmatic region to the ventral midbrain. This region also specifically expresses the regulatory genes Sonic hedgehog (Shh) and Nkx2.2. First MCH axons run through the tractus postopticus (tpoc) which gathers pioneer axons from the cell cord and courses parallel to the Shh/Nkx2.2 expression domain. Subsequently generated MCH neurons and ascending MCH axons differentiate while neurogenesis and mantle layer differentiation are generalized in the prosencephalon, including telencephalon. Ascending MCH axons follow dopaminergic axons of the mesotelencephalic tract, both being an initial component of the medial forebrain bundle (mfb). Netrin1 and Slit2 proteins that are involved in the establishment of the tpoc and mfb, respectively attract or repulse MCH axons.We conclude that first generated MCH neurons develop in a diencephalic segment of a longitudinal Shh/Nkx2.2 domain. This region can be seen as a prosencephalic segment of a medial neurogenic column extending from the chiasmatic region through the ventral neural tube. However, as the telencephalon expends, it exerts a trophic action and the mfb expands, inducing a switch in the longitudinal axial organization of the prosencephalon

Mesencephalic or telencephalic influences on MCH axonal growth.

<p>(A) Series of line drawings of co-culture experiments. Red dots in hypothalamic explants represent MCH perikarya; red lines represent MCH fibers revealed by immunofluorescence. Note that the innervation of the telencephalic and/or mesencephalic explants is independent of the size of these explants and of the presence of MCH perikarya close to the corresponding edge in the hypothalamic explants. (B) Newborn mice caudal hypothalamic explants were co-cultured with E11 to E14 embryonic basal midbrain and basal telencephalic explants. Data were obtained from at least 3 independent experiments for each embryonic stage. 12 to 19 basal midbrain and basal telencephalic explants were analyzed per stages and 9 to 17 tectal explants that were used as controls. Data indicate mean ±SEM. aaa: p≤0,001 mesencephalon vs tectum; bbb: p≤0,001 - b: p≤0,05 telencephalon vs tectum; **: p≤0,01 mesencephalon vs telencephalon; n.s.: not significant, Kruskal Wallis's test. (C–E) Photomicrographs of slices after two days <i>in vitro</i> and MCH immunofluorescence. Low (C) and higher (D,E) magnifications to illustrate that MCH neurons survived very well, and that perikarya and axons are clearly labeled and can easily be numbered. Scale bar in E: C = 60 µm; D, E = 30 µm.</p

MCH-GFP perikarya and axons and TH axons at E14–15.

<p>Photomicrographs of the GFP labeling on horizontal sections of a E14 (A–C) and E15 (D) MCH-GFP embryos. (A–C) At E14, many MCH-GFP perikarya (A) are observed in the tuberal hypothalamus and some fibers take a rostral direction toward the telencephalon. On the ventrally adjacent section (B), MCH-AS labels perikarya, but axons are not detected. (C) On the section dorsally adjacent to A, a tyrosine hydroxylase (TH)-AS intensely labeled mesostriatal axons in the medial forebrain bundle (mfb). (D) At E15, rostrally directed axons are very abundant in the mfb. Scale bar: A, D = 200 µm; B, C = 500 µm. ANT: anterior level, hypothalamus; MAM: mammillary level hypothalamus; V3: third ventricle.</p

Netrin1 attracts and Slit2 repulses MCH axons.

<p>(A) Tridimensional culture experiments. E16 embryonic brains were dissected out. The ventral midline was incised to expose the ventricular surfaces. Posterior hypothalamic regions were isolated, cut into 200 µm thick pieces and deposited around cell aggregates. Quantification was performed as follow: after two days <i>in vitro</i>, each explant was divided into four quadrants. TUJ1 or GFP positive fibers were counted in the proximal and distal quadrants. The ratio between proximal and distal (p/d) fiber number was then calculated for each explant. (B) Statistical analysis: caudal hypothalamic axons were significantly attracted by Netrin1-transfected cells aggregates, but not by control cells. Addition of an anti-DCC antibody blocks the effect of Netrin1 on axons. (C–F): Netrin1 attracts axons of caudal lateral hypothalamus. Aggregates of control or Netrin1-expressing HEK-293 cells were confronted to caudal hypothalamic explants containing interneurons. Axons growth is visualized with the TUJ1 (anti-β3 tubulin) antibody. Netrin1 exerts a clear attractive effect compared to control cases. This effect is abolished by incubation with the anti-DCC antibody. (G, H) Photomicrographs of two explants labeled by the GFP-AS and cultured cell aggregates expressing Netrin1and incubated with control IgG or anti-DCC IgG. Netrin1 attracts GFP axons, but this effect is inhibited by anti-DCC IgG (I) Statistical analysis: MCH-GFP axons are attracted by Netrin1 but not by controls. (J, K) Slit2 repelled MCH-GFP axons. Aggregates of control or Slit2-expressing HEK-293 cells were confronted to caudal hypothalamic explants containing interneurons. Axons growth is visualized with the GFP antibody. Slit2 exerts a clear repulsive effect compared to control cases. This effect is abolished by incubation with the anti-Robo2 antibody. (L) Statistical analysis: MCH-GFP axons are repelled by Slit2 but not by controls. Data indicate mean ±SEM. ***: p≤0,001; *: p≤0.05; n.s.: not significant, Mann Whitney's test. Scale bar (C–F): C = 200 µm; D–F = 400 µm; Scale bar (G, H, J, K): G, H = 200 µm; J, K = 400 µm.</p

MCH-GFP perikarya and axons at E11.

<p>(A–B) On a sagittal section of E12 brain, MCH-GFP fibers follow the <i>tpoc</i>. Most MCH-GFP axons course dorsally/caudally within this tract in direction of the mesencephalon. B shows a higher magnification. Scale bar: A = 150 µm; B = 75 µm. DOR: thalamus dorsal; Tel: telencephalon; <i>tpoc</i>: <i>tractus postopticus</i>; Mes: mesencephalon; VNT: thalamus ventral.</p

Expression of ROBO2, DCC and MCH.

<p>(A–D) Photomicrographs of adjacent horizontal sections passing through the hypothalamus of E15 rat embryos and labeled by <i>in situ</i> hybridization for MCH, DCC and Robo2. The MCH region contains intense DCC and Robo2 <i>in situ</i> signals. Scale bar = 300 µm.</p

GFP and MCH are expressed by the same cells in MCH-GFP mice.

<p>(A, B) Photomicrographs of two adjacent sections passing through the caudal lateral hypothalamus of an adult mouse brain and labeled by the GFP- or MCH-AS using the standard peroxidase anti-peroxydase procedure. Both AS labeled neurons showing similar distribution patterns in the perifornical region (fx: fornix) and adjacent to the cerebral peduncle (cpd). (C, D): A double immunohistochemical procedure (peroxidase – GFP, immunofluresecnce - MCH), both AS revealed the same neurons. Scale bar: A, B = 500 µm; C, D = 50 µm.</p