In Chondrichthyans, the paraventricular hypothalamus (TPa/PPa) has been identified as a domain expressing ScOtp bounded ventrally by the ScDlx2-expressing domain of the subparaventricular hypothalamus (TSPa/PSPa) and caudally by the ScDlx2-expressing domain of p3a (present results; Santos-Durán et al., 2015, 2016).

Roughly speaking, the ScOtp-expressing TPa/PPa domain develops as the hypothalamic region in which well-conserved vasotocin-like populations can be found in the adult (see Vallarino et al., 1990a). Since vasotocin is produced from a prohormone that, after post-translational modifications, also yields the carrier protein neurophysin (e.g., Rodriguez-Santiago et al., 2017), these populations must have belonged to the neurophysin-positive nucleus reported by Meurling et al. (1996), which was termed magnocellular preoptic nucleus. This nucleus, together with its projections to the hypothalamus–hypophyseal tract and the neurointermediate lobe of the hypophysis, form the classic neurosecretory system. Oxytocin-like peptides (asvatocin and phasvatocin) isolated from the S. canicula pituitary (Chauvet et al., 1994) are likely to be expressed in cells of this domain, although this has not been assessed. It is noteworthy that the paraventricular hypothalamus (TPa/PPa) is probably the progenitor domain of other peptidergic cells found in adjacent territories (e.g., the subparaventricular hypothalamus; see below), given that Otp is involved in the development of such phenotypes in the hypothalamus in different vertebrates (Simeone et al., 1994; Acampora et al., 1999, 2000; Wang and Lufkin, 2000; Del Giacco et al., 2006; Bardet et al., 2008; Morales-Delgado et al., 2011, 2014; Puelles et al., 2012; Affaticati et al., 2015; Díaz et al., 2015), and that many migratory pathways of peptidergic neuronal populations have been described from the paraventricular hypothalamus toward neighboring regions (García-Moreno et al., 2010; Morales-Delgado et al., 2011, 2014; Puelles et al., 2012; Díaz et al., 2015). Indeed, we cannot discard that some peptidergic cells observed in the basal hypothalamus or diencephalon in catshark originated from the TPa/PPa since extensive migrations of neurons from the TPa/PPa to these territories are observed in mammals (Morales-Delgado et al., 2011, 2014; Puelles et al., 2012; Díaz et al., 2015). The TPa/PPa domain also seems to present a conserved glutamatergic neuronal phenotype (Villar-Cerviño et al., 2011; Puelles et al., 2012), which has not been studied in Chondrichthyans.

Of note, in sharks, ScLhx5 is distinctly expressed in the TPa (but not in the PPa), supporting the existence of terminal and peduncular paraventricular subdomains. The boundary between TPa and PPa is further supported by the course of the 5-HT-ir fibers in the mfb and/or Pax6-ir cells in the PPa, the latter being the only useful landmark for the IHB at late stages (Figure 6; see also Santos-Durán et al., 2015, 2016). Moreover, two subdivisions of what had been termed the pre-optic nucleus (i.e., the parvicellular and magnocellular preoptic nuclei in Smeets et al., 1983) can be distinguished in the paraventricular domain since they present different peptides and different spatial distribution, which supports the existence of TPa and PPa subdivisions (Figures 6–8). Considering these nuclei are not preoptic (telencephalic) but paraventricular (hypothalamic), they should be renamed as parvicellular and magnocellular paraventricular nuclei (NPP; NPM). In mammals, rostrocaudal subdivisions can be identified by combinations of various molecular markers (Morales-Delgado et al., 2011, 2014; Puelles et al., 2012; Díaz et al., 2015; Ferran et al., 2015) and Otp-expressing cell clusters can be grouped into rostral and caudal (terminal and peduncular) paraventricular domains (Simeone et al., 1994; Acampora et al., 1999, 2000; Wang and Lufkin, 2000; Del Giacco et al., 2006; Bardet et al., 2008; Puelles et al., 2012).

The ScDlx2-expressing TSPa/PSPa is located ventrally to the ScOtp-expressing TPa/PPa (Figures 2, 3, 6–8). In both chondrichthyans and mammals, this domain is also characterized by a GABAergic neuronal phenotype associated with the expression of Dlx genes (Carrera et al., 2008a; Ferreiro-Galve et al., 2008; Puelles et al., 2012; Santos-Durán, 2015; Santos-Durán et al., 2015, 2016). The TSPa/PSPa domain contains ScOtp-expressing cells probably migrated from the TPa/PPa (present results). These cells could give rise to different peptidergic cell types that have been described near the catshark optic chiasm (Met-Enkephalin, Vallarino et al., 1994; Tyrotropin Releasing Hormone, Teijido et al., 2002; Somatostatin, Sobrido-Cameán et al., 2020). Of note, Growth Hormone releasing hormone (Ghrh)-positive cells described in the PSPa of mammals have presumably migrated from the PPa (Morales-Delgado et al., 2014).

Terminal (TSPa) and peduncular (PSPa) subdivisions were identified in sharks in early developmental stages (up to stage 31) by the differential presence of Pax-6 ir cells in the mantle of PSPa (Santos-Durán et al., 2016), though a scarce number of Pax6-ir cells has been observed to invade TSPa at later stages (e.g., Figures 4A,E).

In sharks, the terminal subdivision, the TSPa, harbors the suprachiasmatic nucleus (NSC), an unpaired GABAergic and catecholaminergic cell group located caudal and ventral to the optic chiasm (Carrera de Figueiredo, 2008; Carrera et al., 2012). Considering its topological location and neurochemical markers, this nucleus could be equivalent to the suprachiasmatic and the anterior hypothalamic nuclei of mammals together (being the former free of TH-ir cells, and the latter corresponding to the ventral part of the catecholaminergic neuronal group identified alphanumerically as A14; Bilbao et al., 2022). In mammals, the suprachiasmatic nucleus receives projections from the retina and is involved in pacemaker circadian functions (Puelles et al., 2012; Gotlieb et al., 2020; Kim and Blackshaw, 2020). In Chondrichthyans comparable retinal connections have been described in the NSC (Smeets et al., 1983), suggesting similar functions. Dispersed ScOtp-expressing cells found here can explain the peptidergic and/or catecholaminergic cellular phenotypes of this nucleus (Vallarino et al., 1994; Teijido et al., 2002; Carrera et al., 2005, 2012; Sobrido-Cameán et al., 2020). Of note, catshark NSC catecholaminergic cells have homolog counterparts in mammals and may have been involved in neuroendocrine function by sending projections to the neurohypophysis (Carrera et al., 2012).

Finally, the entopeduncular nucleus of mammals, which originates from PPa (Otp) and PSPa (Dlx) subdivisions (Puelles et al., 2012), presents dispersed Pax6-positive cells (Stoykova et al., 1996). In Chondrichthyans, similar Pax6-ir cells can be recognized in the PSPa (Figure 4), suggesting homology with those described in mammals (see also Santos-Durán et al., 2016). Of note, in chondrichthyans, the nucleus named entopeduncular nucleus (Smeets et al., 1983) does not belong to the hypothalamus and, therefore, is not homologous to the nucleus of the same name in tetrapods.

The Tu/RTu is mainly, but not exclusively, characterized by the expression of Dlx genes and cells with a GABAergic phenotype in both chondrichthyans and mammals (Carrera de Figueiredo, 2008; Puelles et al., 2012; Santos-Durán, 2015; Santos-Durán et al., 2015, 2018). The Tu/RTu comprises territories extending from the optic chiasm to the ventral end of the Sv (Figures 6, 7). The chondrichthyan Tu/RTu harbors several unpaired and paired structures with specific dorso-ventral locations, which, considered together with the differential expression of various molecular markers, support further dorso-ventral and rostrocaudal subdivisions, as with mammals (Morales-Delgado et al., 2011, 2014; Puelles et al., 2012; Díaz et al., 2015; Ferran et al., 2015).

The dorsalmost cell group in the catshark tuberal hypothalamus is the hypothalamic nucleus medius (NMH; Figures 6, 7D–F, 8). The territory that harbors the NMH expresses ScOtp (but not ScDlx2) and is coextensive with the region of the adenohypophysis expressing ScPitx2 and Pax6 (data not shown). It has been described in adults as a rostrally (topologically dorsally) unpaired and caudally (topologically ventrally) paired nucleus that partially overlaps the median eminence, a well-known neurohemal organ (Smeets et al., 1983). The mammalian counterparts of these unpaired and paired portions could be the anterobasal nucleus and the arcuate nucleus, respectively. The mammalian anterobasal nucleus is an unpaired structure that expresses Otp at the early stages, although, later, these Otp-expressing cells migrate tangentially to the paired arcuate nucleus (Joly et al., 2007; Bardet et al., 2008; Morales-Delgado et al., 2011; Puelles et al., 2012). The arcuate nucleus consists of diverse neuroendocrine cell types and is tightly connected to the neurosecretory median eminence (Joly et al., 2007).

More ventrally in the Tu/RTu, we can find the median eminence, the neurointermediate lobe of the Nh (which maintains ScDlx2-expression as in former stages; Figures 3, 7, 8; see also Santos-Durán et al., 2015) and the Sv, an enigmatic circumventricular organ found in jawed fishes associated to circadian functions (Figures 6, 7E,F, 8; see also Sueiro et al., 2007; Nakane et al., 2013). The Sv is considered part of the Tu/RTu because it originates from the ventral portion of the infundibular walls (i.e., rostral- and ventral-most Tu; van de Kamer and Shuurmans, 1953; Sueiro et al., 2007) and presents dispersed ScDlx2- (Santos-Durán et al., 2015, 2018) and ScPitx2-expressing cells (present results). In mammals, it has been speculated that a thin underdeveloped territory of the rostralmost Tu could be homologous to the Sv since it presents a similar topology and ependymal character (Puelles et al., 2012). However, the ventral part of the Tu/RTu in mammals has been recently proposed as the hypothalamic ventricular organ, a linear longitudinal ependymal specialization of suggested organizer properties (Díaz and Puelles, 2020). Worth to note that at stage 32, the Sv presents abundant proliferating cells (PCNA-ir; see Figure 3H in Santos-Durán et al., 2018) compared with other hypothalamic regions, supporting its large expansion at later stages (van de Kamer and Shuurmans, 1953; Sueiro et al., 2007).

In more ventral portions of the chondrichthyan Tu, we found paired structures defined by the lack or the presence of expression of ScDlx2, which harbor the lateral tuberal nucleus (NLT) and the nucleus of the lateral lobes (NLL) in the IHL, respectively (Figures 7E,F, 8). In mammals, the ventromedial and dorsomedial hypothalamic nuclei (the latter lying topologically ventral to the former; Puelles and Rubenstein, 2015) present similar expression patterns (Dlx-negative and Dlx-positive, respectively; Morales-Delgado et al., 2011, 2014; Puelles et al., 2012), suggesting homologies with chondrichthyans. It is noteworthy that, at stage 32 (present results), the NLT is negative for the markers considered here, although it is positive for ScLhx5 at previous stages (Santos-Durán et al., 2018). Similarly, the mammalian ventromedial nucleus seems to be positive for Lhx5 and abuts Dlx-expressing territories at early stages (Sheng et al., 1997; Abellán et al., 2010), but it is negative at later stages (Szabó et al., 2009; Heide et al., 2015; Miquelajáuregui et al., 2015). The catshark NLT presents several types of peptidergic cells (somatostatin, Sobrido-Cameán et al., 2020; met-enkephalin and leu-enkephalin, Vallarino et al., 1994; melanin-concentrating hormone, Vallarino et al., 1989a; atrial natriuretic factor, Vallarino et al., 1990b; delta sleep-inducing peptide, Vallarino et al., 1992; beta-endorphin, Vallarino et al., 1989b; galanin, Vallarino et al., 1991; neuropeptide Y, Vallarino et al., 1988; corticotropin-releasing factor, Vallarino et al., 1989c; adrenocorticotropic hormone, Vallarino and Ottonello, 1987, Vallarino et al., 1989b), and is thought to be associated with the hypophysis, as reported in actinopterygian fishes (reviewed in Butler and Hodos, 2005). The mammalian ventromedial hypothalamus is involved in feeding, fear, thermoregulation, and sexual activity (Harding and McGinnis, 2005; King, 2006; Silva et al., 2016).

The NLL is located in the inferior hypothalamic lobes (IHL), which are distinctive structures of the hypothalamus of chondrichthyans and actinopterygian fishes. In chondrichthyans, the IHL constitutes the largest part of the adult hypothalamus. They are involved in feeding and aggression-related behavioral responses (Demski, 1977) and have been considered a major relay center between the telencephalon and brainstem because of their widespread ascending and descending connections (Smeets and Boord, 1985). At stage 32, the IHL walls present an intense proliferative activity, although the differential density of proliferating cells along these walls suggests that different subdivisions could emerge late in development, as pointed out in studies in a teleost (Corujo and Anadón, 1990). Interestingly, at this stage, the distribution of proliferating cells matches that of ScDlx2-expressing cells (compare Figures 3G,H, 4G,H). Of note, parts of the IHL of chondrichthyans have been reported to regulate feeding behavior, while the Dlx2-expressing mammalian basal hypothalamus has been involved in feeding-dependent circadian rhythms, wakefulness rhythms, temperature regulation, and energy expenditure (Mieda et al., 2006; Shimogori et al., 2010; Zhao et al., 2017; Pulix and Plagge, 2020; Kim and Blackshaw, 2020), which support some kind of homology between these nuclei. Moreover, the NLL could also harbor counterparts of the mammalian lateral hypothalamus (LH) expressing hypocretins/orexins. These peptidergic cells, involved in feeding behavior and sleep-wake cycle, originated from progenitors in Lhx9-expressing RTu territories that migrate to the LH (Shimogori et al., 2010; Dalal et al., 2013; Díaz et al., 2015). Homolog ScLhx9-expressing domains have been described in the catshark (Santos-Durán et al., 2016) and preliminary data from our lab suggest the presence of cells migrating toward the IHL (data not shown). Despite data supporting the role of the IHL in the feeding behavior of sharks (Evan et al., 1976; Demski, 1977), recent studies in zebrafish point out that the IHL could rather be involved in sensory integration (Bloch et al., 2019). However, the teleost IHL has a more complex origin, receiving a substantial population of projection neurons originating from the midbrain that is hardly comparable to any shark population (Bloch et al., 2019).

The ventralmost portion of the catshark Tu/RTu is characterized by a low ScDlx2-expression and will give rise to the paired portion of a circumventricular organ known in S. canicula as paraventricular organ (PVO; Rodríguez-Moldes, 2011). The ventricular surface of this organ is characteristically folded and contains a high density of cerebrospinal fluid (CSF)-contacting neurons of catecholaminergic, serotoninergic, and peptidergic nature (see Rodriguez-Moldes and Anadon, 1987; Meurling and Rodríguez, 1990; Molist et al., 1993; Carrera et al., 2012; Sobrido-Cameán et al., 2020). In mammals, the ventralmost Tu/Rtu is also Dlx-positive (Morales-Delgado et al., 2011, 2014; Puelles et al., 2012). A homologous circumventricular organ has been described in the same region (i.e., hypothalamic ventricular organ), extending through its rostro-caudal extension from the HDB to the rostralmost Tu (Puelles et al., 2012), as it happens in the catshark. In mammals, this ventralmost Tu/RTu has also been described to contain a progenitor domain of hypothalamic histaminergic neurons (Puelles et al., 2012). Histaminergic neurons are only present at the transition between tuberal and mammillary hypothalamus (Puelles et al., 2012), whose most characteristic cell group is the tuberomammillary nucleus involved in promoting sleep/wake transitions (Kim and Blackshaw, 2020). Histaminergic cells have not been, so far, investigated in sharks despite the relevant role of histaminergic neurons in teleosts (Sundvik and Panula, 2012).

Finally, in mammals, the A13 catecholaminergic group has been ascribed to the dorsal and caudalmost part of the RTu (Puelles et al., 2012; Bilbao et al., 2022), while these cells seem to arise from Dlx- and Pax6-positive cells originated from prosomere 3 (Mastick and Andrews, 2001; Andrews et al., 2003; Puelles et al., 2012). The A13 equivalent, which was interpreted as a dorsomedial hypothalamic (topologically caudal) TH-ir group observed in sharks by Carrera et al. (2012), can be interpreted to belong to the RTu domain. The presence of Pax6-ir cells in the catshark RTu area suggests that their dopaminergic cell groups (Carrera et al., 2012) have the same origin.

Ventral to the Dlx-expressing Tu/RTu, Otp expression defines the PM/PRM hypothalamus of both chondrichthyans and mammals (Morales-Delgado et al., 2011, 2014; Puelles et al., 2012; Santos-Durán et al., 2015, 2018). In chondrichthyans, ScOtp expression can be recognized in the territory where an unpaired circumventricular organ named posterior recess organ (Meurling and Rodríguez, 1990) develops (Figures 6, 7D–F). Of note, according to the prosomeric model, this organ is not the caudalmost structure of the hypothalamus, nor does it derive from the mammillary region, and, therefore, the use of terms such as posterior recess or mamillary recess, can be misleading. Accordingly, we suggest the name of perimamillary recess and/or perimamillary recess organ (PRO) to refer to this structure derived from the PM/PRM. In adult catshark, a functional and structural continuity has been described between the PVO and the PRO-based on the common expression of peptides and monoamines (Meurling and Rodríguez, 1990; Rodríguez-Moldes, 2011). Previous studies revealed that ScEmx2 expression roughly corresponds to the 5-HT-ir neuron-bearing region of the PVO and PRO (see Santos-Durán et al., 2018). However, differential expression of ScDlx2 and ScOtp in domains in which the PVO and PRO will develop, respectively, suggests that these organs have a different origin. Whether the differential expression of ScDlx2 and ScOtp is related to the development of the CSF-contacting aminergic and peptidergic neurons of these circumventricular organs remains to be investigated.

In mammals, Otp expression has not been described in the HVO (the equivalent of the PVO of anamniotes) but rather ventrally to this organ, thus, defining the PM/PRM (Puelles et al., 2012). Like with other Otp-expressing domains, the PM/PRM seems to be a rich glutamatergic domain that gives rise to the classic dorsal premamillary nucleus (Puelles et al., 2012). This nucleus has been involved in fear behavior (Canteras et al., 2020).

According to previous studies in mammals, markers expressed in both the MM and RM are not known, except for those related to the glutamatergic phenotype (Szabó et al., 2009; Puelles et al., 2012). The MM is characterized by Nkx2.1, Emx2, or Lhx5 expression and the RM by Shh, Foxa1, or Pitx2 expression, although the limit between both areas can also be recognized by the 5-HT-ir retro- or supramamillary commissure (Szabó et al., 2009; Puelles et al., 2012; Santos-Durán et al., 2015, 2018). Moreover, markers expressed in the MM are typically expressed in other domains of the Nkx2.1-expressing hypothalamus, while markers expressed in the Pitx2-expressing RM are typically continuously expressed in the basal diencephalic prosomeres, also known as rich glutamatergic domains (Puelles et al., 2012). These data also suggest a different histogenetic hypothalamic organization that has been discussed in previous studies (Santos-Durán et al., 2018). In mammals, the caudal limit of the RM can be identified by the expression of Nr5a1 in the basal plate of prosomere 3 (p3Tg; Puelles et al., 2012). It is noteworthy that conserved Dlx- and/or Pax6-positive postmitotic cells in prosomere 3 from chondrichthyans to mammals could also be useful markers (Figures 4, 6; see also Stoykova et al., 1996; Mastick and Andrews, 2001; Andrews et al., 2003; Ferreiro-Galve et al., 2008; Santos-Durán et al., 2016).

Classical studies of chondrichthyans consider the posterior recess organ (now named PRO; see above) as a caudalmost part of the hypothalamus (Molist et al., 1993; Teijido et al., 2002; Rodríguez-Moldes, 2011; Anadón et al., 2013). The present observations support a subdivision of the posterior tubercle in a rostral part derived from the MM/RM and a caudal part derived from p3Tg (tegmentum or basal plate of prosomere 3) (Figure 6; see also Santos-Durán et al., 2015, 2018). In fishes, the posterior tubercle is known by harboring important catecholaminergic populations involved in sensory-motor control (revised in Carrera et al., 2012). Genes like Shh, Otp, Neurogenin, Foxa, and Pitx seem to be involved in their origin (reviewed in Vernier and Wullimann, 2009), most of them being detected in sharks (Santos-Durán et al., 2015, 2018). Based on the differential expression of ScNkx2.1, ScShh, and ScPitx2, the hypothalamic part of the posterior tuberculum could be divided into a rostral domain (the MM, expressing ScNkx2.1 but not ScShh or ScPitx2) and a caudal domain (RM, expressing ScShh and ScPitx2 but not ScNkx2.1), harboring the nucleus of the saccus vasculosus (NSV) and the nucleus of the posterior tuberculum (NPTu), respectively (present results; see also Santos-Durán et al., 2015).

The NSV, which receives projections from neurons of the Sv, has been described rostral to the postinfundibular commissure (Smeets, 1998), likely homolog of the retromamillary commissure (see also Santos-Durán et al., 2015). Since the Sv is a structure found exclusively in jawed fishes, an equivalent nucleus cannot be recognized in tetrapods. Therefore, a possible relation of the NSV with the classic medial and lateral mamillary nucleus harbored in the mammalian MM (Puelles et al., 2012) is difficult to sustain.

The NPTu is located caudally to the retromamillary commissure and presents peptidergic cells (Molist et al., 1992) likely migrated from the ScOtp-expressing PM/PRM. The NPTu has been described as rich in catecholamines and peptidergic neuron phenotypes involved in sensorimotor control also associated with the basal ganglia (Figures 6, 7D,E, 8; Molist et al., 1992; Carrera et al., 2012; Quintana-Urzainqui et al., 2012; Santos-Durán et al., 2015, 2018). In mammals, the classic subthalamic nucleus that is involved in similar functions also emerges from the RM and expresses Pitx2 (Martin et al., 2004; Puelles et al., 2012). It is noteworthy that the mammalian subthalamic nucleus has been described as an RM derivative containing migrated Pitx2-expressing cells that are finally located in the RTu (Puelles et al., 2012). Strikingly, in the catshark, there is a ScPitx2-expressing population that fits with this description (Figures 6, 7D,E), whose homology needs to be further investigated. The mammalian RM also gives rise to the classical lateral, medial retromamillary nucleus besides the migrated parasubthalamic nucleus and lateral tuberal nucleus.