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. 2015 Apr 8;9:37. doi: 10.3389/fnana.2015.00037

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2015 Santos-Durán, Menuet, Lagadec, Mayeur, Ferreiro-Galve, Mazan, Rodríguez-Moldes and Candal.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Regionalization of the shark hypothalamus from stages 29–31 based on immunoreactivity to Shh (A,B,B′,D,I–N), TH (B′), 5-HT (I–L), GFAP (O, P) and expression of ScNkx2.1 (C,C′,D,G″), ScDlx2/5(E,E′,F,M) and ScOtp(G,G′, G″, H, H′, N) genes by means of single immunohistochemistry (IHC; A,B,O,P), double IHC (B′,I–L), single ISH (C,E,E′,F,G, G′,H,H′) and/or combined with IHC (D,M,N) on sagittal (A,B,B′,C–G,G″,H–J,M,N) or transverse sections (C′,E′,G′,H′,K,L,O,P). Image in (G″) results from the overlapping of two parallel sections respectively hybridized with ScOtp and ScNkx2.1 probes. Color for ScNkx2.1 was digitally converted to brown to ease comparison. Dotted lines define the hypothalamo-telencephalic boundary (HTB), dashed lines indicate the caudal border of the secondary prosencephalon, red lines indicate the ABB and continuous black lines represent the path followed by 5-HT-ir fibers. (A) Shh-immunoreactivity was observed in the most caudo-ventral part of the CBHy (arrow) and in the RBHy. The arrowhead points to the zli. (B) Shh-immunoreactivity was observed in the telencephalon (arrowhead) beyond the territory of the anterior commissure (asterisk). The arrow points to Shh-immunoreactivity in the caudal-most CBHy. (B′) Detail of the squared area in (B) to show Shh- and TH-immunoreactivity in its most caudo-ventral part (arrow). (C,D) ScNkx2.1 expression was observed in the hypothalamus and telencephalon. A detail of a transverse section at the level indicated in (C) is shown in (C′). The arrows in (C,C′,D) point to ScNkx2.1-expressing cells in the mantle zone of the most caudo-ventral CBHy. The arrowheads in (C,D) indicate a wedge-shaped domain lacking ScNkx2.1 expression. The asterisk in (D) indicates the territory of the anterior commissure. (E,F) ScDlx2/5 expression was observed in p3 and in the secondary prosencephalon. A detail of a transverse section at the level indicated in (D) is shown in (D′). The arrow in (E,E′) points to ScDlx2/5-expressing cells in the mantle of the p3Tg. The star in (E,E′) indicates the prospective territory of the PThE. The black asterisks in (E,F) indicate a gap of ScDlx2/5 expression in the telencephalon. The arrowheads in (E,F) point to ScDlx2/5 expression in the BHy. (G,H) ScOtp expression in the hypothalamus. Details in (G′,H′) correspond to transverse sections at the levels indicated in (G,H). Detail in (G″) correspond to the squared area in (G). The arrows in (G,G″) point to the ventricular domain expressing ScOtp in the caudal CBHy. The black arrowheads in (G,G″, H′) point to ScOtp-expressing cells in the mantle of the most caudo-ventral part of CBHy and p3Tg. The arrow in (G″) indicates a domain expressing ScNkx2.1 alone. The white asterisks in (G′,H) indicate ScOtp-expressing cells in the mantle zone. The black arrowhead in (H) points to ScOtp-expressing cells in the AHy and the white arrowhead in (H) points to ScOtp-expressing cells between the alar and basal domains. (I–L) Double Shh- and 5-HT-immunoreactivity. (K,L) correspond to trasverse sections at the level indicated in (J). 5-HT-ir fibers in (I) are observed in the basal plate of the secondary prosencephalon. In the rostral hypothalamus such fibers coursed among RAHy and RBHy, and were located dorsally to Shh-immunoreactivity (I,J). Note the presence of 5-HT-ir fibers in the Sp (I,J,L). The white arrowhead in (J,K) points to 5-HT-ir fibers that course in the rostral CAHy. The black arrowhead in (J,L) points to 5-HT-ir fibers in the telencephalon. The arrow in (J,K) points to 5-HT-ir fibers decussating in the CBHy. The arrow in (L) points to the faint Shh immunoreactivity in the zli. (M,N) Shh IHC combined with ScDlx2/5 expression (M) or ScOtp expression (N). A gap of expression is observed between the rostral and caudal domains of ScDlx2/5 and ScOtp expression, which appeared to coincide with the path followed by 5-HT-ir fibers. (O) GFAP-ir processes at the level shown in (J). The arrowhead points to the GFAP-ir processes among RAHy and Sp. The arrow points to the GFAP-ir processes in the CBHy. (P) GFAP-ir processes at the level shown in (J). The arrow points to GFAP-ir processes in the Sp. For abbreviations, see list.