Prosomeric organization of the hypothalamus in an elasmobranch, the catshark Scyliorhinus canicula

ORIGINAL RESEARCH published: 08 April 2015 doi: 10.3389/fnana.2015.00037 Edited by: Luis Puelles, Universidad de Murcia, Spain Reviewed by: Manuel A. Pombal, University of Vigo, Spain Alino Martinez-Marcos, Universidad de Castilla, Spain *Correspondence: Eva Candal, Centro de Investigaciones Biológicas, Department of Cell Biology and Ecology, University of Santiago de Compostela, Campus Vida, Avenida Lope Gómez de Marzoa, s/n, Santiago de Compostela E-15782, Spain [email protected] Received: 01 December 2014 Paper pending published: 12 January 2015 Accepted: 09 March 2015 Published: 08 April 2015 Citation: Santos-Durán GN, Menuet A, Lagadec R, Mayeur H, Ferreiro-Galve S, Mazan S, Rodríguez-Moldes I and Candal E (2015) Prosomeric organization of the hypothalamus in an elasmobranch, the catshark Scyliorhinus canicula. Front. Neuroanat. 9:37. doi: 10.3389/fnana.2015.00037 Prosomeric organization of the hypothalamus in an elasmobranch, the catshark Scyliorhinus canicula Gabriel N. Santos-Durán1,Arnaud Menuet2, Ronan Lagadec3,Hélène Mayeur3, Susana Ferreiro-Galve3,Sylvie Mazan3,Isabel Rodríguez-Moldes1and Eva Candal1* 1Centro de Investigaciones Biológicas, Department of Cell Biology and Ecology, University of Santiago de Compostela, Santiago de Compostela, Spain, 2Centre National de la Recherche Scientifique, Experimental and Molecular Immunology and Neurogenetics, University of Orleans, UMR7355, Orleans, France, 3Centre National de la Recherche Scientifique, FR2424, Development and Evolution of Vertebrates Group, Sorbonne Universités – Université Pierre et Marie Curie, Roscoff, France The hypothalamus has been a central topic in neuroanatomy because of its important physiological functions, but its mature organization remains elusive. Deciphering its embryonic and adult organization is crucial in an evolutionary approach of the organization of the vertebrate forebrain. Here we studied the molecular organization of the hypothalamus and neighboring telencephalic domains in a cartilaginous fish, the catshark, Scyliorhinus canicula,focusingonScFoxg1a,ScShh, ScNkx2.1, ScDlx2/5,ScOtp, and ScTbr1 expression profiles and on the identification αacetylated-tubulin-immunoreactive (ir), TH-ir, 5-HT-ir, and GFAP-ir structures by means of immunohistochemistry. Analysis of the results within the updated prosomeric model framework support the existence of alar and basal histogenetic compartments in the hypothalamus similar to those described in the mouse, suggesting the ancestrality of these subdivisions in jawed vertebrates. These data provide new insights into hypothalamic organization in cartilaginous fishes and highlight the generality of key features of the prosomeric model in jawed vertebrates. Keywords: chondrichthyan, forebrain patterning, evolution, development, prosomeric model, Shh,Nkx2.1, Otp Introduction Biological diversity emerges, at least in part, through changes in development. Organisms are different because their developmental process differ and, what is more, because their developmental process also evolve (Kutschera and Niklas, 2004;Müller, 2007;Medina et al., 2011). Thus, the Abbreviations: ABB, alar-basal boundary; ac, anterior commissure; AHy, alar hypothalamus; At, acroterminal region; AP, alar plate; BHy, basal hypothalamus; BP, basal plate; CAHy, caudal part of the alar hypothalamus; CBHy, caudal part of the basal hypothalamus; D, diencephalon; F, forebrain; FP, floor plate; HDB, hypothalamo-diencephalic boundary; hp1, prosomere hp1 or peduncular hypothalamus; hp2, prosomere hp2 or terminal hypothalamus; IHB, intrahypothalamic boundary; MM, mammillary area; MTT, mammillo-tegmental tract; mz, marginal zone; os, optic stalk; p1, prosomere 1; p2, prosomere 2; p2Tg, tegmental part of prosomere 2; p3, prosomere 3; p3Tg, tegmental part of prosomere 3; P, pallium; PM, perimammillary area; POA, preoptic area; PPa, peduncular paraventricular area; PRM, periretromammillary area; PSPa, peduncular subparaventricular area; PThE, prethalamic eminence; RAHy, rostral part of the alar hypothalamus; RBHy, rostral part of the basal hypothalamus; Rh, rhombencephalon; RM, retromammillary area; rmc, retromammillary commissure; RP, roof plate; RTu, retrotuberal domain; sot, supraoptic tract; Sp, subpallium; T, telencephalon; TPa, terminal paraventricular area; TPOC, tract of the postoptic commissure; TSPa, terminal subparaventricular area; Tu, tuberal domain; vz, ventricular zone. Frontiers in Neuroanatomy | www.frontiersin.org 1April 2015 | Volume 9 | Article 37 Santos-Durán et al. Prosomeric organization of the catshark hypothalamus understanding of the development of the vertebrate brain becomes fundamental to comprehend its structure and evolution. In this context, the hypothalamus has been both a central and elusive topic. The hypothalamus is a conserved integrative center that coordinates autonomic, endocrine, and limbic responses (Sarnat and Netsky, 1981;Kandel and Schwartz, 2001; Butler and Hodos, 2005).Itsdevelopment,atthebaseofthe vertebrate forebrain (prosencephalon), involves complex patterning processes dependent on different signaling events that converge at this point. It also undergoes a complex morphological deformation during development, which misleads its topological (vs. topographic) location (Shimamura et al., 1995;Puelles and Rubenstein, 2003;Puelles, 2009;Puelles et al., 2012). As a result, the hypothalamic organization remains a matter of debate (Figdor and Stern, 1993;Puelles and Rubenstein, 2003; Shimogori et al., 2010;Diez-Roux et al., 2011;Puelles et al., 2012). Cross-species comparisons can be important to resolve this issue, and an important effort to understand the underlying unity of hypothalamic embryonic and adult organization across vertebrates has been made recently (Shimogori et al., 2010; Domínguez, 2011;Morales-Delgado et al., 2011, 2014;Moreno et al., 2012;Domínguez et al., 2013, 2014;Herget et al., 2014). The prosomeric model (Puelles and Rubenstein, 2003;Puelles et al., 2004, 2012;Medina, 2008;Puelles, 2009)hasbecomeakey reference in such comparative studies, since it offers a mechanistic paradigm of the vertebrate brain structure and organization. Initially based on analyses of amniotes, this model defines for the first time anatomical structures as developmental hierarchical units based on specification mechanisms that determine longitudinal and transverse axis orientation, segmental structure, transcription factor expression profiles and the emergence of differential histogenetic domains (Puelles and Rubenstein, 2003; Puelles, 2009;Martínez et al., 2012). A major interest and novelty of this model is that it puts emphasis on developmental criteria (including topological relationships among certain morphological landmarks, regulatory gene expression patterns and signaling molecules). Testing their conservation across vertebrates is a powerful approach for the correct establishment of homologies between embryonic territories beyond amniotes (Puelles and Medina, 2002). The underlying notion is that formation of the vertebrate brain involves a conserved core of highly constrained, invariant mechanisms and genetic networks, which are the basis for homology establishment. This in no way excludes the emergence of diversifications through evolution, which are the source of the neuroanatomic diversity observed among vertebrates. Latest updates of the model provide novel views on the organization of the rostral-most (secondary) prosencephalon, and its telencephalic and hypothalamic moieties (Puelles and Rubenstein, 2003;Pombal et al., 2009;Puelles et al., 2012). Detailed studies in different vertebrate groups are necessary to validate the model assumptions. Cartilaginous fishes or chondrichthyans are crucial in this task because they are among the most basal extant groups of gnathostomes (jawed vertebrates). Because of its phylogenetic position as the closest outgroup to osteichthyans (the other major phylum of gnathostomes, which includes bony fish and tetrapods), chondrichthyans are essential to reconstruct gnathostome ancestral characteristics through comparisons with other vertebrate models. Here we studied the molecular histogenetic organization of the hypothalamus and directly adjoining territories of an elasmobranch representative of one of the most basal extant gnathostome lineage, the catshark Scyliorhinus canicula, and analyzed them under the updated prosomeric framework. We have integrated data from neuroepithelial specification codes (based on the expression of catshark orthologues of Foxg1a,Shh,Nkx2.1,Dlx2/5,Otp, and Tbr1), and from the distribution of α-acetylated-tubulinimmunoreactive (-ir) and TH-ir cell groups, neuron-fiber tracts (5-HT-ir) and glial-processes (GFAP-ir). In the search of conserved traits among jawed vertebrates, we compared our data in S. canicula with that obtained in murine models. Our analysis reveals a strikingly high degree in the conservation of hypothalamic histogenetic compartments between chondrichthyan and murine models. Furthermore, we identified some of the boundaries and confirmed some of the assumptions predicted by the prosomeric model. However, some differences and discrepancies also exist mainly concerning the neuroepithelial specification genetic codes of the basal hypothalamus (BHy). Similar studies are required in other basal species to figure out if these differences should prompt the model update or they are the consequence of shark specialization. Materials and Methods Phylogenetic Reconstructions Sequence alignments of the sequences listed in Table S1 were constructed using the alignment editor Seaview 3.0 and the MUSCLE algorithm. S. canicula sequences were retrieved by tblastn searches in transcriptomic databases obtained by Sanger and Illumina sequencing of embryonic and adult cDNA libraries (stages 8–25 and mixed adult tissues). Maximum-likelihood trees were inferred using the PhyML program version 3, the LGF+12+I substitution model and the SPR algorithm. Posterior probabilities (PPs) supporting groupings were calculated using the aLRT algorithm implemented in PhyML and are displayed as percentages at the corresponding nodes. Only PP >80% are indicated. The trees were viewed and edited using Mega6. Experimental Animals Some embryos of the catshark (lesser spotted dogfish; S. canicula) were supplied by the Marine Biological Model Supply Service of the CNRS UPMC Roscoff Biological Station (France) and the Estación de Bioloxía Mariña da Graña (Galicia, Spain). Additional embryos were kindly provided by the Aquaria of Gijón (Asturias, Spain), O Grove (Pontevedra, Spain) and the Aquarium Finisterrae (A Coruña, Spain). Embryos were staged by their external features according to Ballard et al. (1993). For more information about the relationship of the embryonic stages with body size, gestation and birth, see Table 1 in Ferreiro-Galve et al. (2010). Thirty-seven embryos from stages 12 to 31 were used in this study. Eggs from different broods were raised in seawater tanks in standard conditions of temperature (15–16◦C), pH (7.5–8.5) and salinity (35 g/L). Adequate measures were taken to Frontiers in Neuroanatomy | www.frontiersin.org 2April 2015 | Volume 9 | Article 37 Santos-Durán et al. Prosomeric organization of the catshark hypothalamus minimize animal pain or discomfort. All procedures conformed to the guidelines established by the European Communities Council Directive of 22 September 2010 (2010/63/UE) and by the Spanish Royal Decree 53/2013 for animal experimentation and were approved by the Ethics Committee of the University of Santiago de Compostela. Tissue Processing Embryos were deeply anesthetized with 0.5% tricaine methanesulfonate (MS-222; Sigma, St. Louis, MO, USA) in seawater and separated from the yolk before fixation in 4% paraformaldehyde (PFA) in elasmobranch’s phosphate buffer [EPB: 0.1 M phosphate buffer (PB) containing 1,75% urea, pH 7.4] for 48–72 h depending on the stage of development. Subsequently, they were rinsed in phosphate buffer saline (PBS), cryoprotected with 30% sucrose in PB, embedded in OCT compound (Tissue Tek, Torrance, CA, USA), and frozen with liquid nitrogen-cooled isopentane. Parallel series of sections (12–20 μm thick) were obtained in transverse and sagittal planes on a cryostat and mounted on Superfrost Plus (Menzel-Glasser, Madison, WI, USA) slides. Single and Double Immunohistochemistry on Sections and Whole Mounts For heat-induced epitope retrieval, sections were pre-treated with 0.01 M citrate buffer (pH 6.0) for 30 min at 95◦Cand allowed to cool for 20–30 min at room temperature (RT). Sections were then rinsed twice in 0.05 M Tris-buffered saline (TBS; pH 7.4) for 5 min each and incubated overnight with the primary antibody (rabbit anti-serotonin [anti-5-HT] polyclonal antiserum, DiaSorin, Immunostar, Hudson, WI, USA, diluted 1:5000; polyclonal rabbit anti-Sonic Hedgehog [anti-Shh], Sta. Cruz Biotechnology, Santa Cruz, CA, USA, diluted 1:300; polyclonal rabbit anti-glial fibrillary acidic protein [anti-GFAP], Dako, Glostrup, Denmark, diluted 1:500; and monoclonal mouse antityrosine hydroxilase [anti-TH], Millipore, Billerica, MA, USA, diluted 1:500). Appropriate secondary antibodies [horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse, BIORAD, diluted 1:200] were incubated for 2 h at RT. For double immunohistochemistry (IHC) experiments, cocktails of primary antibodies were mixed at optimal dilutions and subsequently detected by using mixtures of appropriate secondary antibodies. Sections were rinsed in distilled water (twice for 30 min), allowed to dry for 2 h at 37◦CandmountedinMOWIOL488 Reagent (Calbiochem, MerkKGaA, Darmstadt, Germany). All dilutions were made with TBS containing 15% donkey normal serum (DNS; Millipore, Billerica, MA, USA), 0.2% Triton X-100 (Sigma) and 2% bovine serum albumin (BSA, Sigma). Double IHC with primary antibodies raised in the same species was performed as described in Tornehave et al. (2000). For whole mounts embryos were prepared as previously described in Kuratani and Horigome (2000) with minor modifications. After fixation with 4% PFA in 0.01 M PBS at 4◦Cfor 2 days, embryos were washed in 0.9% NaCl in distilled water, dehydrated in graded series of methanol solutions (50, 80, 100%) andstoredat−20◦C. Samples to be stained were placed on ice in 2 mL of dimethyl sulfoxide (DMSO)/methanol (1/1) until they sank. Then, 0.5 mL of 10% Triton X-100/distilled water was added, and the embryos were incubated for 30 min at RT. After washing in 0.05 M TBS with 0.1% Triton X-100 (TST, pH 7.4) the samples were sequentially blocked using spin-clarified aqueous 1% periodic acid and 5% non-fat dried milk in TST (TSTM). Primary antibody (monoclonal mouse anti-α-acetylated-tubulin, Sigma, 1:1000) was diluted in TSTM containing 0.1% sodium azide for 2–4 days at RT with gently agitation on a shaking platform. The secondary antibody HRP-conjugated goat anti-rabbit, BIORAD, dilution 1:200 in TSTM) was incubated overnight. After a final washing in TST, the embryos were pre-incubated with 0.25 mg/mL diaminobenzidine tetrahydrochloride (DAB, Sigma) in TST with 2.5 mg/mL nickel ammonium sulfate for 1 h, and then allowed to react with DAB in TST containing 2.5 mg/mL nickel ammonium sulfate and 0.00075% H2O2for 20–40 min at RT. The reaction was stopped using Tris-HCL buffered saline and specimens were post-fixed with 4% PFA overnight at 4◦C. Epidermis and mesodermic derivatives were carefully removed and specimens were rinsed in graded series of glycerol (25, 50, 75, and 100%) in order to directly observe the neural tube under the stereomicroscope. Controls and Specificity of the Antibodies No immunostaining was detected when primary or secondary antibodies were omitted during incubations. Controls and specificity of anti-TH and anti-5-HT were performed as described in Pose-Méndez et al. (2014). The primary anti-α-acetylated-tubulin antibody has been shown to label early differentiated neurons and their processes in the embryonic nervous system (Piperno and Fuller, 1985;Chitnis and Kuwada, 1990). The polyclonal anti-Shh antibody (Santa Cruz Biotechnology Inc, CA, USA) was raised in rabbit against the amino acids 41–200 of Shh human protein. The in situ hybridization (ISH) results were similar to those obtained by IHC, and therefore validate the specificity of the anti-Shh antibody used here. In Situ Hybridization on Whole Mount Embryos and on Sections We applied ISH for ScFoxg1a,ScShh (Compagnucci et al., 2013; Quintana-Urzainqui, 2013), ScNkx2.1 (Quintana-Urzainqui et al., 2012;Quintana-Urzainqui, 2013), ScDlx5 (Compagnucci et al., 2013;Debiais-Thibaud et al., 2013), ScOtp (QuintanaUrzainqui, 2013), ScTbr1 (Quintana-Urzainqui, 2013), and ScDlx2 (Quintana-Urzainqui et al., 2012;Compagnucci et al., 2013;Debiais-Thibaud et al., 2013;Quintana-Urzainqui, 2013) genes. These probes were selected from a collection of S. canicula embryonic cDNA library (mixed stages 9–22), constructed in pSPORT1,andsubmittedtohighthroughputESTsequencing. cDNA fragments were cloned in pSPORT vectors. Sense and antisense digoxigenin-UTP-labeled and fluorescein-UTP-labeled probes were synthesized directly by in vitro transcription using as templates linearized recombinant plasmid DNA or cDNA fragments prepared by PCR amplification of the recombinant plasmids. ISH in whole mount and on cryostat sections was carried out following standard protocols (Coolen et al., 2009). Briefly, sections were permeabilized with proteinase K, hybridized with sense or antisense probes overnight at 65◦C(in sections) or 70◦C (whole mount) and incubated with the alkaline Frontiers in Neuroanatomy | www.frontiersin.org 3April 2015 | Volume 9 | Article 37 Santos-Durán et al. Prosomeric organization of the catshark hypothalamus phosphatase-coupled anti-digoxigenin and anti-fluorescein antibody (1:2000, Roche Applied Science, Manheim, Germany) overnight at 4◦C. The color reaction was performed in the presence of BM-Purple (Roche). Control sense probes did not produce any detectable signal. Inhibition of the Shh Pathway Inhibition of the Shh pathway was performed by in ovo injection of the pharmacological inhibitor cyclopamine in order to test whether, as in osteichthyans, the initiation of ScNkx2.1 expression in the forebrain is dependent on Shh. First, 200 μLofasolution containing 1x PBS, 500 μMcyclopamineand5%DMSOwere injected through the shell of stage 15–16 S. canicula eggs. This solution was replaced by the same volume of 5% DMSO in 1x PBS for control embryos. The eggs were maintained for 3 days in oxygenated sea water at 17◦C, with viabilities higher than 90%. Embryos reached stage 18 in these conditions. They were dissected, fixed in PFA 4%, dehydrated and stored in methanol 100% prior to ISH. Image Acquisition and Analysis Light field images were obtained with an Olympus BX51 microscope equipped with an Olympus DP71 color digital camera. In toto embryos were analyzed in the Olympus SZX12 stereomicroscope fitted to an Olympus DP12 color digital camera. Photographs were adjusted for brightness and contrast and plates were prepared using Adobe Photoshop CS4 (Adobe, San Jose, CA, USA). Results Identification of Catshark Orthologues of the Genes Studied Exhaustive phylogenetic characterizations of the catshark Dlx gene repertoire have been previously published (Debiais-Thibaud et al., 2013), confirming the identity of ScDlx2 and ScDlx5.In order to unambiguously identify the catshark orthologues of Foxg1,Shh,Nkx2.1,Otp, and Tbr1, we conducted systematic phylogenetic analyses of the corresponding vertebrate gene families, including all the vertebrate classes derived from duplications of a single ancestral chordate orthologue (Figure 1). In each case, phylogenies were constructed from alignments containing deduced amino acid sequences of all paralogous sequences retrieved from catshark transcriptomic databases and from a representative sampling of actinopterygians and sarcopterygians. The trees were rooted using a Branchiostoma floridae sequence, except in the case of Otp which could not be found in the amphioxus Ensembl database. In the case of Foxg1, three strongly supported classes (posterior probability or PP >90%), each containing a catshark and several osteichthyan sequences, were retrieved, highlighting for the first time the presence of three gnathostome Foxg1 classes (Figure 1A). These classes were termed Foxg1a, Foxg1b, and Foxg1c, respectively. One coelacanth and several actinopterygian sequences, but no amphibian or amniote sequence, were found in the Foxg1b and Foxg1c classes, suggesting a loss of their representatives in tetrapods. We focused the expression analysis on ScFoxg1a, the catshark FIGURE 1 | Phylogenetic analysis of the Scyliorhinus canicula genes analyzed in this study. Phylogenetic trees for the Foxg1, Hedgehog, Nkx2.1/Nkx2.4, Otp and Tbr1/Tbx21/Eomes families are shown in (A–E) respectively. The number of substitutions per site is indicated at the bottom of each tree, on the left. S. canicula genes are displayed in red. Abbreviations used: Hs, Homo sapiens (human); Gg, Gallus gallus (chick); Ac, Anolis carolinensis (anole lizard); Xt, Xenopus tropicalis (African clawed frog); Lc, Latimeria chalumnae (coelacanth); Lo, Lepisosteus oculatus (spotted gar); Ol, Oryzias latipes (medaka); Dr, Danio rerio (zebrafish); Cm, Callorhinchus milii (elephant shark); Sc, Scyliorhinus canicula (catshark or lesser spotted dogfish); Bf, Branchiostoma floridae (amphioxus). Frontiers in Neuroanatomy | www.frontiersin.org 4April 2015 | Volume 9 | Article 37 Santos-Durán et al. Prosomeric organization of the catshark hypothalamus orthologue of the only Foxg1 gene retained in all major gnathostome lineages including tetrapods. The tree topology obtained for the Hedgehog family confirmed the presence of the three gnathostome classes, corresponding to the Indian Hedgehog, Desert Hedgehog and Sonic Hedgehog classes already reported in osteichthyans, and confirmed ScShh as the representative of the latter (Figure 1B). Concerning the Nkx2.1/Nkx2.4 family, a single catshark gene could be identified and it was unambiguously assigned to the Nkx2.1 class based on the strongly supported grouping of its deduced amino acid sequence with teleost, chick, and human Nkx2.1 sequences (PP =97%; Figure 1C). This gene is therefore referred to as ScNkx2.1 hereafter. A single catshark Otp related sequence, termed ScOtp, could be found and as expected, it clustered with the elephant shark sequence annotated as Otp in the reconstruction shown in Figure 1D. Finally, the Tbr1, Tbx21, and Eomes classes were retrieved with high statistical support (PP =83, 100, and 99%, respectively) within the Tbr1/Tbx21/Eomes family. Each class contained a single catshark sequence at the expected position, allowing an unambiguous identification of the ScTbr1 gene analyzed in this study (Figure 1E). Preliminar Considerations Concerning Vertebrate Segmental Prosencephalic Organization The organization of the shark hypothalamus has been analyzed in the framework of the updated prosomeric model (Puelles et al., 2012). Figure 2 summarizes the general architecture of the hypothalamus in mouse according to the updated prosomeric model (Puelles et al., 2012). This model is mainly inspired in murine data though it is usually assumed that it can be extrapolated to all vertebrates because it also integrates information from other vertebrates (Puelles and Rubenstein, 2003;Pombal et al., 2009;Puelles, 2009). Indeed, this model represents a useful developmental and comparative framework since it makes use of concepts, nomenclature and topological references that can be used across different vertebrate species. The prosomeric model establishes that hypothalamus and telencephalon are part of the secondary prosencephalon, which is understood as a segmental unit at the rostral-most point of the neural tube, the hypothalamus being located ventral to the telencephalon and rostral to the diencephalon (see Figure 2A). The model also postulates that the rostral-most point of the brain, referred as the acroterminal region (At), lies at the rostral border of the secondary prosencephalon. This region is restricted to the frontal border of the neural tube where left and right alar and basal plates meet. This border expands dorso-ventrally from the rostral-most roof plate (which is telencephalic) to the rostralmost floor plate (which is hypothalamic). Thus, every structure classically considered being dorsal or ventral to these points (see arrowheads in Figure 2A), should be considered as caudal in this framework. Of note, the anterior commissure, located in the rostral-most roof plate, is a clear landmark of both the dorso-ventral and rostro-caudal axis (Puelles et al., 2012;seealso Figures 2A,B). FIGURE 2 | Squematic representations of the prosencephalon of early (A) and late (B,C) mouse embryo to show correspondence of longitudinal and tranverse domains in the secondary prosencephalon under the updated prosomeric model. Domains in (A) are illustrated according to Figure 1.1C in Martínez et al. (2012). Domains in (B,C) are illustrated according to Figure 8.5B in Puelles et al. (2012). (A) Longitudinal domains in early embryos. The arrowheads mark both the dorso-caudal and ventro-caudal limits of the acroterminal territory (At). This territory is considered the rostral-most domain of the neural tube. The dorso-caudal limit of the At can be identified caudal to the anterior commissure. (B) Longitudinal and transverse organization in late embryos. (C) Segmental organization of the secondary prosencephalon according to the prosomeric model. For abbreviations, see list. Frontiers in Neuroanatomy | www.frontiersin.org 5April 2015 | Volume 9 | Article 37 Santos-Durán et al. Prosomeric organization of the catshark hypothalamus The secondary prosencephalon presents two true segments rostro-caudally arranged (Figure 2B): hp2 (rostral or terminal) and hp1 (caudal or peduncular). Each segment harbors telencephalic and hypothalamic derivatives (Figures 2B,C). However, the telencephalon harbors only roof and alar plates while the hypothalamus harbors alar, basal, and floor plate derivatives. The existence of these segments is supported by several genes differentially expressed in the rostro-caudal axis, the location of commissures in the roof and floor plates (anterior and retromammillary commissures, respectively), and the course of important tracts [medial forebrain bundle (mfb); lateral forebrain bundle (lfb); and fornix (fx)] running by a common path at the rostral border of hp1, through alar and basal plates. These data, in turn, support the existence of an intersegmental boundary that separates terminal and peduncular subdivisions of both telencephalon and hypothalamus, which is referred as the intrahypothalamic boundary (IHB; Figures 2B,C). Caudally, the secondary prosencephalon limits with the diencephalon at the hypothalamic diencephalic border (HDB), another intersegmental limit among hp1 and p3, though it should be noticed that part of the caudal limit of the secondary prosencephalon does correspond to the telencephalon (Puelles et al., 2012;seealsoFigure 2C). The model considers the adult hypothalamic organization arranged in different histogenetic territories defined by neuroepithelial specification codes and radial units (Puelles and Medina, 2002;Puelles et al., 2012). These codes reveal that telencephalon and hypothalamus belong to different histogenetic territories being the preoptic area (POA) the unique terminal territory of the telencephalon (Figure 2C). Of note, the POA also harbors the anterior commissure (Puelles et al., 2012;seealsoFigures 2B,C). ScFoxg1a Expression In mice, Foxg1 is one of the earliest transcription factors expressed specifically in the part of the neural plate that gives rise to the telencephalon and it remains expressed throughout the telencephalon during embryonic development (see Manuel et al., 2011). In an attempt to discriminate telencephalic and underlying hypothalamic domains throughout S. canicula development, we have analyzed the expression of ScFoxg1a in the developing nervoussystemofthisspecies.Atstage18,ScFoxg1a expression was found in the dorsal-most portion of the secondary prosencephalon including the optic cup, extending from the level of the optic stalk (which is located rostrally, within the At) up to a caudal point in the roof plate, which has been tentatively identified as the dorsal border between the telencephalon and the diencephalon (Figure 3A). At stage 22, ScFoxg1a was observed in the telencephalon and in the nasal part of the optic cup (Figure 3B). The expression in the telencephalon was maintained until late stages of development (Figure 3C), which allowed identifying the border between the telencephalon and the hypothalamus. ScShh Expression ScShh expression was detected during gastrulation (stage 12) in the caudal midline of the embryo (data not shown). At stage 14, during early neurulation, it has been detected in the axial mesoderm of the notochord and the prechordal plate and in the ectoderm of the caudal midline (data not shown). After the closure of the neural tube (stage 17), the signal was detected as a ventral longitudinal continuous band that extends from the caudal end of the spinal cord to the At of the forebrain, roughly at the level of the optic stalk (Figure 3D). As in other vertebrates (Shimamura et al., 1995), the expression of ScShh can be used to define the alar-basal boundary (ABB; Figures 3E–H). At stage 19, ScShh expression became downregulated in the forebrain to progressively give rise to a caudal and a rostral domain (arrow in Figures 3E–H). The narrow transverse and dorsally directed stripe of ScShh-expressing cells within the caudal domain was identified as the developing zona limitans intrathalamica (zli; arrowhead in Figure 3G). The rostral border of the ScShh caudal domain, in turn, was somewhat extended rostral to the HDB (Puelles et al., 2012), which at this stage was identified as the point where the neural tube expands to acquire the distinctive shape of the ventral hypothalamus. Therefore, the BHy appeared to be divided in three domains: two positive for ScShh (one rostral and other caudal) and one (intermediate) negative for ScShh (arrow in Figures 3G,H;seealsoFigure5HinCompagnucci et al., 2013). Of note, the dorsal border of the rostral domain (presumably corresponding to the ABB) seems to codistribute with α-acetylatedtubulin-immunoreactive (-ir) longitudinal tracts (arrowheads in Figure 3I). At stage 24 (Figure 3H), a new domain emerged within the telencephalon. This short domain (arrowhead in Figure 3H) extended from a region located dorsally to the optic stalk without reaching the prospective territory of the anterior commissure (that can be identified at early development by means of α-tubulin-immunoreactivity; asterisk in Figures 3H,I). A clear gap of expression was observed between this telencephalic domain and the rostral hypothalamic one (Figure 3H). The telencephalic domain was located medially while the hypothalamic one also expanded laterally (not shown). From stage 27 onward the zli expanded dorsally toward the roof plate (arrowhead in Figure 4A). At stage 29 the medio-lateral histologic organization of the developing walls of the forebrain become more evident. As in previous developmental stages, Shh immunoreactivity was clearly identified in the basal plate of the diencephalon entering the caudo-ventral part of the BHy (arrow in Figures 4A,B,B) and in the rostro-dorsal part of the BHy (Figures 4A,B), so that a clear negative gap of Shh-immunoreactivity occupied most of the caudal BHy (CBHy; Figures 4A,B) and part of the rostral BHy (RBHy). In the telencephalon, Shh-immunoreactivity expanded caudally beyond the prospective territory of the anterior commissure (arrowhead in Figure 4B; compare with Figure 3H). Of note, from late stage 30 onward, Shh-immunoreactivity is downregulated in the CBHy and basal diencephalon, except in the zli (data not shown). ScNkx2.1 Expression The expression of ScNkx2.1 was first detected at stage 18 in the rostro-ventral portion of the forebrain, in a longitudinal band which extended ventral to the optic stalk (arrowhead in Figure 3J). At stage 23 ScNkx2.1 was expressed in most of the BHy (Figure 3K). Differently from ScShh, ScNkx2.1 delimited the ABB even in the CBHy (Figure 3K; Frontiers in Neuroanatomy | www.frontiersin.org 6April 2015 | Volume 9 | Article 37 Santos-Durán et al. Prosomeric organization of the catshark hypothalamus FIGURE 3 | Continued Frontiers in Neuroanatomy | www.frontiersin.org 7April 2015 | Volume 9 | Article 37 Santos-Durán et al. Prosomeric organization of the catshark hypothalamus FIGURE 3 |Continued Regionalization of the hypothalamus and neighbor territories in embryos of S. canicula from stages 18–29 based on the expression pattern of ScFoxg1a (A–C), ScShh (D–H), ScNkx2.1 (J–L), ScDlx5 (M–O), ScOtp (P,Q), ScTbr1 (R) genes and α-acetylated-tubulin immunoreactivity (I). In all panels, dotted lines define the hypothalamo-telencephalic boundary (HTB), dashed lines indicate the caudal border of the secondary prosencephalon and red lines indicate the ABB. (A–C) ScFoxg1a expression in the secondary prosencephalon at indicated stages. The arrowheads in (A) mark the caudo-dorsal and rostro-ventral limit of ScFoxg1a expression. (D–H) ScShh expression at the indicated stages. The arrowhead in (D) marks the rostral-most point of ScShh expression in the forebrain. The arrows in (E–H) indicate the downregulation of ScShh expression in the hypothalamus. The arrowhead in (G) points to the developing zli. The arrowhead in (H) points to a novel domain in the telencephalon. The asterisk in (H) marks the prospective territory of the anterior commissure. (I) Anti-α-acetylated-tubulin IHC to show three sets of tracts at stage 25. These tracts are classically referred as sot, TPOC and MTT. The asterisk indicates the territory of the developing anterior commissure. The arrowheads point to the longitudinal TPOC. The arrow points to the rostral-most extension of the MTT. (J–L) ScNkx2.1 expression at the indicated stages. The arrowhead in (J) points to the rostral-most point of ScNkx2.1 expression at stage 18, which was restricted to a short longitudinal domain ventrally to the optic stalk. The arrow in (K,L) points to a small ScNkx2.1-negative domain at the most caudo-ventral BHy. The asterisk in (K,L) marks the prospective territory of the anterior commissure. The arrowhead in (K,L) points to a domain in the telencephalon that spread rostro-caudally. (M–O) ScDlx5 expression at the indicated stages. The arrowheads in (M,N) indicate ScDlx5 expression in the olfactory placode and the anterior part of the telencephalon. The asterisk in (N) indicates the prospective territory of the anterior commissure. The arrowheads in (O) point to the ventral and caudal expansion of ScDlx5 expression in the telencephalon. This domain was fairly continuous with a longitudinal band of ScDlx5 over the ABB. The arrows in (O) point to ScDlx5-expressing domains that spread into the BHy. (P,Q) ScOtp expression at the indicated stages. The arrowhead in (P) indicates a restricted domain of ScOtp expression ventrally located with respect to the optic stalk. The expression of ScOtp in the hypothalamus was faint compared to that of the Rh. The white arrowhead in (Q) points to ScOtp expression in the AHy. Two additional ScOtp-expressing domains were observed in the BHy. (R) ScTbr1 expression at stage 25 was found in part of the telencephalon and at the dorsal-most part of the rostral diencephalon (white arrowhead). The asterisk indicates the prospective territory of the anterior commissure. For abbreviations, see list. compare with Figure 3G), though a small gap of expression was observed within the caudo-ventral part of the BHy (arrow in Figure 3K). A second domain emerged in the telencephalon at this stage (arrowhead in Figure 3K). This domain was restricted to the rostral-most portion of the telencephalon and extended from a region located dorsally to the optic stalk to the prospective territory of the anterior commissure (asterisk in Figure 3K). A clear gap of expression was observed between the telencephalic and the hypothalamic domains. At stage 25 (Figure 3L), as in previous developmental stages, ScNkx2.1 expression was lacking in a small domain located within the caudo-ventral BHy (arrow in Figure 3L). This region seems to fit with the rostral border of a basal α-acetylated-tubulin-ir tract [see mammillo-tegmental tract (MTT) in Figure 3I]. In the telencephalon, ScNkx2.1 expression became caudally expanded beyond the prospective territory of the anterior commissure (asterisk in Figure 3L). At stage 29, as in previous developmental stages, ScNkx2.1 was observed throughout most of the BHy, except in a small wedge-shaped domain within the caudo-ventral BHy (arrowhead in Figures 4C,D). Groups of ScNkx2.1-expressing cells in the caudo-ventral portion of the hypothalamus were observed along the marginal zone (arrows in Figures 4C,C,D). In the telencephalon, ScNkx2.1 expression expanded beyond the territory it occupied at previous developmental stages (asterisk in Figure 4D). Of note, Shh immunoreactivity was overlapping with ScNkx2.1 expression beyond the anterior commissure (Figure 4D). In order to test whether, as in osteichthyans, the initiation of ScNkx2.1 expression in the forebrain is dependent on Shh, we used in ovo injections of the Shh inhibitor cyclopamine. All control embryos (n=4) exhibited the expected ScNkx2.1signal in the rostral-most and ventral-most portion of the forebrain (Figure 5). This signal was lost in all embryos dissected following cyclopamine treatment (n=3), supporting the conclusion that Shh signaling is required for the initiation of ScNkx2.1 expression in S. canicula as in osteichthyans. ScDlx2/ScDlx5 Expression We analyzed the expression of ScDlx5 from stage 18 onward and the expression of ScDlx2 from stage 29 onward. Fairly identical results were observed with both markers in the brain of S. canicula from stage 29 onward, so we use ScDlx2/5 at these stages to refer indistinctly to both. General features of ScDlx5 expression and detailed profiles in the developing branchial arches have been previously described from stage 15 to stage 27 in Compagnucci et al. (2013)and from stage 15 to stage 25 in Debiais-Thibaud et al. (2013). We revisited these data focusing on the developing forebrain. At stage 18, ScDlx5 expression was found in the most anterior part of the neural tube (Figure 3M; compare with Figure 3A;see also Figure 5C1 in Debiais-Thibaud et al., 2013). From stage 21 to 25, ScDlx5 becomes mostly restricted to the anteriormost part of the telencephalon and to the olfactory placodes (Figure 3N;seealsoFigure4GinCompagnucci et al., 2013 and Figure 5C’1 in Debiais-Thibaud et al., 2013). However, at later stages (Figure 3O), ScDlx5 expression spread caudally and ventrally (arrowheads in Figure 3O) and reached the rostral-most portion of the optic stalk (see also Figure 9C in Debiais-Thibaud et al., 2013). This domain was fairly continuous with a longitudinal band of ScDlx5 that crossed through the hypothalamus over the ABB (compare with Figures 3H,L) and entered p3 (Figure 3O). Therefore, this domain delineates the ABB along the hypothalamus. Of note, the longitudinal domain appeared to codistribute with α-acetylated-tubulin tracts (arrowheads in Figure 3I). Although both ScDlx5 domains were continuous, a wedge-shaped area of reduced signal intensity was observed between the dorsal (telencephalic) and the ventral (hypothalamic-diencephalic) domains (Figure 3O). Two bands of cells were additionally observed, which were ventrally located with respect to the longitudinal domain (arrows in Figure 3O). One was located at its caudal end and spread ventral-ward at the rostral end of p3. The other spread perpendicularly to the ABB from the caudo-dorsal part of the BHy up to the rostral hypothalamus (Figure 3O). Of note, this ScDlx5-expressing Frontiers in Neuroanatomy | www.frontiersin.org 8April 2015 | Volume 9 | Article 37 Santos-Durán et al. Prosomeric organization of the catshark hypothalamus FIGURE 4 | Continued Frontiers in Neuroanatomy | www.frontiersin.org 9April 2015 | Volume 9 | Article 37 Santos-Durán et al. 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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. Frontiers in Neuroanatomy | www.frontiersin.org 17 April 2015 | Volume 9 | Article 37