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). Additional embryos were kindly provided by the Aquaria of Gijón (Asturias, Spain), O Grove (Pontevedra, Spain) and Finisterrae (A Coruña, Spain). Embryos were staged by their external features according to Ballard et al. (1993). For more information about the relationship of embryonic stages with body size, gestation and birth, see Table 1 in Ferreiro-Galve et al. (2010). Sixty-nine embryos from stages 28 to 32 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 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.

Embryos were deeply anesthetized with 0.5% tricaine methane sulfonate (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 planes on a cryostat and mounted on Superfrost Plus (Menzel-Glasser, Madison, WI, USA) slides.

For heat-induced epitope retrieval, sections were pre-treated with 0.01 M citrate buffer (pH 6.0) for 30 min at 95°C and 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 (polyclonal rabbit anti-Sonic Hedgehog [anti-Shh], Santa Cruz Biotechnology, Santa Cruz, CA, USA, diluted 1:300; monoclonal mouse anti-proliferating cell nuclear antigen [anti-PCNA] Sigma, St. Louis, MO, 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. The immunoreaction was developed with 0.005% diaminobenzidine (DAB; Sigma) and 0.003% H₂O₂ for 20–40 min. Sections were rinsed in distilled water (twice for 30 min), allowed to dry for 2 h at 37°C and mounted in MOWIOL 4-88 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).

Whole mounts embryos were prepared for IHC as previously described in Santos-Durán et al. (2015). After fixation with 4% PFA in 0.01 M PBS at 4°C for 2 days, embryos were washed in 0.9% NaCl in distilled water, dehydrated in graded series of methanol solutions (50%, 80%, 100%) and stored at −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), samples were sequentially blocked using spin-clarified aqueous 1% periodic acid and 5% non-fat dried milk in TST (TSTM). Primary antibody (polyclonal rabbit anti-Sonic Hedgehog [anti-Shh], Santa Cruz Biotechnology, Santa Cruz, CA, USA, diluted 1:300) 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 (horseradish peroxidase [HRP]-conjugated goat anti-rabbit, BIORAD, diluted 1:200 in TSTM) was incubated overnight. After a final washing in TST, the embryos were pre-incubated with 0.25 mg/mL diaminobenzidinetetrahydrochloride (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% H₂O₂ for 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%) and observed under the stereomicroscope.

No immunostaining was detected when primary or secondary antibodies were omitted during incubations. The monoclonal anti-PCNA antibody specifically labels proliferating cells in the brain, retina and olfactory epithelium of this species (Rodríguez-Moldes et al., 2008; Ferrando et al., 2010; Ferreiro-Galve et al., 2010; Quintana-Urzainqui et al., 2014). The polyclonal anti-Shh antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was raised in rabbit against the amino acids 41–200 of the human Shh protein. We previously reported that 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 (Santos-Durán et al., 2015).

We applied in situ hybridization for ScOtp (Quintana-Urzainqui, 2013; Santos-Durán et al., 2015, 2016), ScDlx2 (Quintana-Urzainqui et al., 2012, 2015; Compagnucci et al., 2013; Debiais-Thibaud et al., 2013; Quintana-Urzainqui, 2013; Santos-Durán et al., 2015, 2016), ScDlx5 (Compagnucci et al., 2013; Debiais-Thibaud et al., 2013; Santos-Durán et al., 2015, 2016), ScNkx2.1 (Quintana-Urzainqui et al., 2012; Quintana-Urzainqui, 2013; Santos-Durán et al., 2015, 2016), ScLhx5 (Santos-Durán et al., 2016), ScEmx2 (Derobert et al., 2002), ScLmx1b (Pose-Méndez et al., 2016), ScPitx2 (Lagadec et al., 2015), ScPitx3a, ScFoxa1, ScFoxa2 and ScNeurog2 (Santos-Durán et al., 2016) genes. These probes were selected from a collection of S. canicula embryonic cDNA library (mixed stages S9–S22), constructed in pSPORT1, and submitted to high throughput EST sequencing. Selected 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. In situ hybridization 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 and incubated with the alkaline 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.

Light field images were obtained with an Olympus BX51 microscope equipped with an Olympus DP71 color digital camera. Photographs were adjusted for brightness and contrast and plates were prepared using Adobe Photoshop CS4 (Adobe, San Jose, CA, USA).

An overview of the expression of ScShh, ScNkx2.1, ScOtp and ScDlx2/ScDlx5 in the basal hypothalamus of S. canicula, mainly in early stages of development, has been previously described in Santos-Durán et al. (2015). The location of the ABB was re-examined in Santos-Durán et al. (2016).

Here we revisited these data to deepen in the genoarchitectonic profile of the basal hypothalamus and further characterize possible dorso-ventral and rostro-caudal subdomains of this territory. A detailed comparative analysis of the expression of such genes in sagittal and transverse sections is presented from stages 29 to 32, when the basic mature cytoarchitecture and organization of the adult hypothalamus are clearly recognized.

The expression of ScEmx2 has been analyzed by Derobert et al. (2002) in the brain and related tissues from early stages of development (stage 19) until mid-gestation stages (stages 28–30). Here we analyze in detail the expression of ScEmx2 in the basal hypothalamus from stage 29 until stage 31. From stage 29 onwards, ScEmx2 is expressed in the basal hypothalamus in a well-defined domain spreading into part of rostral and ventral-most Tu domain, the PM/PRM and the MM domains (Figures 3A–D). Of note, its expression lacks in midline domains of the Tu (acroterminal territory) such as the neurohypophysis (Figure 3A) and saccus vasculosus (Figure 3B) but is present immediately caudal to the last (Figures 3C,D).

A comparison of ScEmx2 expression with other genes reveals several correlations. We compared ScEmx2 expression patterns with the presence of PCNA-immunoreactive (-ir) cells (Figures 3E–H). PCNA-ir cells define proliferative zones that are separated by non-proliferative (PCNA-immunonegative) ventricular regions, which are believed to define important segmental boundaries (reviewed in Candal et al., 2005). Of note, the caudal border of ScEmx2 expression in the MM domain (arrowheads in Figures 3A,E) correlates with a domain of reduced PCNA immunoreactivity in the ventricular zone (arrowheads in Figures 3E–G). The caudal border of ScEmx2 expression in the PRM domain also correspond with a domain of restricted PCNA immunoreactivity (compare arrowheads in Figures 3D,H). Thus, a band of reduced or negative proliferation seems to spread from the rostral and dorsal border of the RM (Figures 3F–H), p3Tg and zona limitans intrathalamica (zli; not shown).

Finally, we compared ScEmx2 expression with that of other genes usually expressed in the basal hypothalamus to better understand its organization. A comparison with Shh immunoreactivity (Figure 3I) revealed that the ScEmx2-expressing domain in the PRM is fairly complementary to Shh in the RM domain (compare Figures 3A,I). Besides, this ScEmx2-expressing domain includes the caudal domain expressing ScLhx5 in the PM/PRM and MM (compare Figures 3A,J) and ScOtp in the PM/PRM (compare Figures 3B,L). Of note, ScEmx2, ScLhx5 and ScOtp define consecutively more restricted domains (compare Figures 3B,K,L). Moreover, the expression of ScEmx2 abuts that of ScDlx2/5 in dorsal and caudal positions (RTu domain) while they co-distribute in more rostral and ventral positions (not shown).

ScFoxa1 and ScFoxa2 are expressed in fairly the same spatial and temporal patters in the regions and stages considered in this study and thus are conjointly referred as ScFoxa1/2.

At stage 29 ScLmx1b, ScPitx2, ScPitx3a, ScFoxa1/2 and ScNeurog2 are expressed in a similar pattern spreading caudally from RM into the diencephalon including the zli in the case of ScPitx2, ScPitx3a, ScFoxa1/2 and ScNeurog2 (Figures 4A–D,B′,C′,O). On transverse sections the expression of these genes dorsally abuts the PRM (Figures 4E–H,F′). Rostrally these genes also abut the MM (arrowheads in Figures 4E–H). From stage 29 onwards this general pattern persists although some differences emerge. At stage 31, ScLmx1b becomes downregulated being restricted to the floor plate (Figure 4I) while Shh immunoreactivity is still found in the basal plate (arrowheads in Figure 4I). At stage 31 ScPitx2 (Figure 4J) is still expressed in the pattern observed at stage 29, while ScPitx3a is restrictedly expressed in the caudal-most diencephalon (not shown). In the case of ScFoxa1/2, there is slight dorsal and ventral downregulation but the main pattern persists through RM and diencephalon (Figure 4K). From stage 30 onwards, ScNeurog2 becomes downregulated in the mentioned territories although it can be recognized in the zli and habenulae (data not shown). Finally, ScLmx1b, ScPitx2 and ScFoxa1/2 still present a sharp border of expression that abuts the MM domain at this developmental stage (arrowheads in Figures 4L–N).

The acroterminal domain involves the alar and basal plate spreading from the rostral-most roof plate to the rostral-most floor plate. It has been suggested that specialized structures like the lamina terminalis, the optic chiasm and the neurohypophysis emerge here under particular signaling events (Puelles et al., 2012; Puelles and Rubenstein, 2015). We have identified at least 6 subdomains inside the basal acroterminal region named 1–6 from dorsal to ventral (BAt1–6; Figure 6B). Noteworthy the acroterminal territory (medial) is easily distinguishable from the remaining hypothalamus (lateral) due to genes differentially expressed at these locations (compare Figures 6A,B).

The two dorsal-most domains (BAt1–2) are positive for ScNkx2.1 (Figure 1I) and ScOtp (Figure 2F) but only BAt1 shows expression of ScLhx5 (Figure 3J). Furthermore, ScOtp co-distributes with ScNkx2.8 (Figures 2F,F′). BAt1–2 subdomains are negative for other genes broadly expressed in the Tu such as ScDlx2/5 and Shh (compare Figure 1M with Figure 2F). Of note, at later stages ScLhx5 is absent from the midline (arrowhead in Figure 3K). Noteworthy, BAt1–2 are almost co-extensive with the developing adenohypophysis (Figures 1I, 6B), which in part is co-extensive with negative subdomains for ScDlx2/5-expression and Shh immunoreactivity (Figure 1M). Of note, these gaps are as wide as the adenohypophysis, which has been noted in other vertebrates even for different adenohypophysis sizes (see Figure 2N in Manning et al., 2006), suggesting a role for the adenohypophysis in the local patterning of the hypothalamus. In shark, both the gaps of ScDlx2/5-expression and Shh immunoreactivity and the expression of ScNkx2.8 are wider than the medio-lateral extension of ScOtp-expression (Figures 2F,F′), which suggests the existence of additional medio-lateral subdomains.

BAt3 (the acroterminal region at the level of the neurohypophysis) is also Shh immunonegative and also expresses ScNkx2.1 (Figures 1H,I), but differently from BAt1–2, it expresses ScDlx2/5 (Figures 1L, 6B).

Ventrally to BAt3, we identified BAt4 as a subdomain that corresponds to the primourdium of the saccus vasculosus (Figure 6B; see also Van de Kamer and Shuurmans, 1953; Sueiro et al., 2007). The initial tiny domain expands becoming morphologically distinguishable (stage 29, Figures 1H, 2E; stage 30, Figure 1I). This domain is characterized by the expression of ScNkx2.1 and dispersed ScDlx2/5-expressing cells (Figure 1L). Since ScDlx2/5 is involved in the development of a GABAergic phenotype (Anderson et al., 1999), its expression in the saccus vasculosus could explain the existence of GABAergic cells at this point (Sueiro et al., 2007). Besides, GFAP-immunoreactivity has been described to be restricted to BAt4 (the developing saccus vasculosus), and it is not observable in more caudal subdomains (Sueiro et al., 2007). Finally, BAt4 is also characterized by lack of ScEmx2 (Figure 3B) which, however, is present in more caudal acroterminal subdomains (Figures 3C,D; see also BAt5 in Figure 6B) and in lateral (non-acroterminal) domains (Figures 6A,D). Noteworthy, in S. canicula, the tip of the notochord has been described to reach the primordium of the saccus vasculosus (BAt4; Figure 1 in Van de Kamer and Shuurmans, 1953) suggesting a causal relationship to saccus vasculosus development.

BAt4 shares ScNkx2.1 expression and dispersed ScDlx2/5-expressing cells with the domain ventral to it (BAt5; Figure 6B). However, as commented above, BAt5 differentially presents a lack GFAP-immunoreactivity and the presence of ScEmx2 expression (Figures 3C,D; see also Figure 6B) at late stages of development.

The ventral-most acroterminal domain is BAt6 which express ScNkx2.1, ScEmx2, ScOtp and ScLhx5, but not ScDlx2/5 (see Figure 6B). However, we cannot discard that this territory could be interpreted as the floor plate of the MM (Figure 6D) that in mouse (but not in shark; compare Figures 7E,F) differentially expresses Shh (see Figure 8.9B in Puelles et al., 2012).

The dorsal and rostral-most domain is Tu1 and expresses genes like ScNkx2.1 and ScLhx5 (Figures 1H, 2A, 6C). Caudally to it, we have distinguished a similar domain lacking ScLhx5 but expressing ScDlx2/5, named as Tu2 (compare 1L and Figure 2A; see Figure 6C). These two subdomains belong to the subliminal part of the basal hypothalamus and so, they lack ScShh (compare Figures 2I,J; see Figure 6C) and they express ScNkx2.8 and ScLhx9 (see Figure 5B in Santos-Durán et al., 2016). More ventrally, two subdomains, Tu3 and Tu4, appear as the ventral extension of Tu1 and Tu2 respectively, since they share with them either ScLhx5 or ScDlx2/5 expression. However, they additionally express ScShh (Figure 6C) but lack ScNkx2.8 and ScLhx9 expression (see Figure 5B in Santos-Durán et al., 2016). The expression of ScLhx5 in the Tu1/Tu3 appears complementary to that of ScDlx2/5 in Tu2/Tu4 (compare Figure 1L with Figure 2A; see Figure 6C). Complementary patterns between Dlx and Lhx5 have been previously described in the mouse forebrain (Sheng et al., 1997), which suggests a conserved inhibitory relationship between both genes. Moreover, a small domain ventral (and caudal) to Tu4, which expressed ScNkx2.1 and ScDlx2/5 but was negative to Shh immunoreactivity, was referred as Tu5 (Figure 6C). A more ventral subdomain, referred as Tu6 is characterized by the expression of ScEmx2 and a dispersed distribution of ScDlx2/5- and ScLhx5-expressing cells (compare Figure 1L with Figures 2D and Figure 3A; see also 6C).The dorsal and caudal-most subdomain identified is RTu1 (Figure 6C), which expresses the same genes as Tu2 and Tu5 (ScNkx2.1, ScDlx2/5; see Figure 6C).

Ventral to Tu6, we identified PM1 a domain where ScNkx2.1/ScEmx2/ScLhx5 are co-expressed (compare Figures 1K, 3C,K; see Figure 6D). We term PM2 (PM-like in Santos-Durán et al., 2015) the subdomain expressing ScNkx2.1/ScEmx2/ScLhx5/ScOtp (compare Figures 1K, 3C,K,L; see Figure 6D). The caudal continuation of PM1 and PM2 are referred as PRM1 and PRM2 (PRM-like in Santos-Durán et al., 2015) and express the same genes (Figure 6D).

MM1 (MM-like in Santos-Durán et al., 2015) expresses ScNkx2.1/ScEmx2/ScLhx5 (Figures 1K, 2D, 3A) but not ScOtp (Figures 2H, 6D). Of note, the genes expressed in the MM1 show a clear-cut border with those expressed in the RM domain (RM-like in Santos-Durán et al., 2015; see Figures 6D,E).

We have identified three dorso-ventral subdomains in the ventral and caudal-most point of the basal hypothalamus here referred as RM1, RM2 and RM3 (Figure 6E), which together fairly correspond to the previously defined RM-like territory (Santos-Durán et al., 2015; see also Figure 5). The dorsal-most domain, RM1, may be defined based on lack of Shh immunoreactivity at stage 29 (as does not reaches the ABB; see Santos-Durán et al., 2016) and the expression of ScLmx1b/ScPitx2/ScPitx3a/ScFoxa1/ScFoxa2/ScNeurog2 (compare Figure 1A with Figures 3A–D; see Figure 6E). In the RM2, these genes co-distribute with Shh immunoreactivity while the ventral-most domain, RM3, is again characterized by the lack of Shh immunoreactivity (compare Figure 1F with Figures 1E–H; see Figure 6E). Of note, from stage 30 onwards, ScPitx2 is expressed in the whole RM (Figures 4J,M) but the downregulation of other genes suggest the existence of even more dorso-ventral subdomains. Finally, RM3 also presents ScLhx5- and ScOtp-expressing cells likely born in neighbor domains (Figures 2D,H, 6E; Table 1).

The prosomeric model offers a key tool to study homologies and brain evolution (Puelles and Rubenstein, 2003, 2015). Counterparts of the genes here considered have been also studied in other vertebrates. Nevertheless, the lack of detailed data in prosomeric terms makes difficult to perform comparisons with most groups, except for mammals, at the level of microdomains. Below, detailed comparisons are made with mammals and we assume they are mostly transferable to other amniotes (Figure 7) due to the similarity of several patterns observed between mice and birds (Manning et al., 2006; Bardet et al., 2008, 2010; García-Calero et al., 2008; Abellán et al., 2010; also reviewed in Domínguez et al., 2014). Gross comparisons are also made with anamniotes that are, however, still informative (Figure 8).