Sequence analysis
The models analyzed by NCBI BLAST PROTEIN were Homo sapiens (taxid: 9606) as a query, and Trachemys scripta elegans (taxid: 31138), Xenopus laevis (taxid: 8355), Protopterus annectens (taxid: 7888), Lepisosteus oculatus (taxid: 7918), Danio rerio (taxid: 7955) and Polypterus senegalus (taxid: 55291), to establish comparisons of the Satb2 protein sequence under analysis among these species. In all cases, only the BLAST results with a percentage above 60% has been considered. In addition, two sequences [Xenopus laevis-isoform X3 (XP_041432137.1) and Polypterus senegalus-isoform X6 (XP_039611916.1)] were discarded from the analysis because they had large gaps in their sequences.
The analysis was carried out based on the whole sequence (Figure 1a) or the epitope region, located between amino acid 613-673 (Figure 1b). The query, located starting at amino acid 601 of C-terminal of human Satb2 protein (NP_001165980) showed a 95.09% of identity with Trachemys scripta elegans (XP_034642297.1) (see Fig. 1). In the case for Xenopus laevis three isoforms were obtained (isoform X1: XP_041432133.1, 85.16% of identity; isoform X2: XP_041432136.1, 85.62% of identity, and isoform X3: XP_041432137.1, 85.10% of identity, but this sequence was not included in the analysis because it presented some gaps). In the sarcopterygian fish, Protopterus annectens, three isoforms of Satb2 sequence were found (isoform X1: XP_043916573.1, 88.06% of identity; isoform X2: XP_043916574.1, 88.18% of identity; isoform X3: XP_043916575.1, 74.44% of identity) (see Fig. 1). In actinopterygian fishes, two isoforms were detected in Danio rerio (isoform X1: XP_005167766.1, 65.12% of identity and isoform X2: XP_017213189.1, 65.20% of identity) and six different isoforms of Satb2 protein were found in Polypterus senegalus (isoform X1: XP_039611906.1, 75.97% of identity; isoform X2: XP_039611912.1, 75.97% of identity; isoform X3: XP_039611913.1, 74.45% of identity; isoform X4: XP_039611914.1, 74.62% of identity; isoform X5: XP_039611915.1, 74.86% of identity; isoform X6: XP_039611916.1, 64.63% of identity).
In the analysis of the epitope sequence (from amino acid 601 of C-terminal human Satb2 protein, Uniprot: Q9UPW6), we considered the region with at least 95% of identity between all species (epitope sequence between amino acids 613-673). All species analyzed showed 100% identity, except Danio rerio sequences (XP_005167766.1, XP_017213189.1, NP_001122004.1), which presented a 96.72% of identity (showing only two differences: one glutamine for one histidine at position 696 and one valine for one isoleucine at position 722).
Expression pattern
The nomenclature used follow previous studies in similar species (Moreno et al. 2008, 2010, 2012; Domínguez et al. 2015; López et al. 2019, 2020, 2022; Lozano et al. 2019; Jiménez et al. 2020; Jiménez and Moreno 2022). The distribution of the immunoreactive labeling for Satb2, Otp, TH and 5-HT (Figures 2-9) was analyzed in relation to the main prosencephalic regions (Puelles and Rubenstein 2015), adapted for the brain of each model. It is represented in a sagittal drawing, considering the neuromeric regionalization of the brain (Figure 10) and summarized in Tables 3 and 4.
Distribution of Satb2 in the brain of sarcopterygian species
Telencephalon: Satb2 expression was analyzed in the telencephalon of sarcopterygians in combination with markers such as TH and Otp (Figs. 2 and 3). The olfactory bulbs of sarcopterygian species studied were devoid of Satb2 labeling. The presence of Satb2 in the pallium of the turtle Trachemys scripta was intensely detected in the dorsal cortex. Many of the cells in the main layer of this region expressed Satb2 (Fig. 2a), whereas additional Satb2-immunoreactive (-ir) cells were found scattered in the medial cortex (see arrowheads in MCx in Fig. 2a). In the lateral derivatives, in particular in the pallial thickening, Satb2 expression was also intense (PT; Fig. 2a). In the ventral pallial derivatives, the lateral cortex (see arrowhead in Fig. 2b) and the dorsal ventricular ridge (DVR), numerous Satb2-ir cells were found (Fig. 2a). The boundary of this region with the adjacent subpallial domain was clearly observed by the combination of Satb2 with TH (Fig. 2b). In the preoptic region of turtle, numerous Satb2-ir cells were observed (Figs. 2c, 3a, b). Moreover, the combination at this level of Satb2 with Otp allowed the identification of the boundary between the preoptic domain and the Otp-rich paraventricular hypothalamic domain (Figs. 2c, 3b). In addition, a subpopulation of doubly labeled Satb2/Otp cells was observed (see empty arrowheads in Fig. 2d and Fig. 3b). Similarly, in the medial amygdala, where scattered Satb2-ir cells were also observed, some of them co-expressed Otp (see empty arrowheads in Fig. 2e). In the Xenopus laevis pallium, Satb2 expression was very intense in the lateral pallium (Fig. 2f, q). In addition, scattered cells were seen in the medial pallium (see arrowhead in Fig. 2f). Combining Satb2 with TH at these telencephalic levels allowed us to identify scattered Satb2-ir cells in the ventral pallium (Fig. 2g, h) and in higher number in the medial amygdala (MeA; see arrowhead in Fig. 2g, h) and central amygdala (Fig. 2g, h). In the medial region, Satb2-ir cells were observed in the bed nucleus of the stria terminalis (BST; Fig. 2g, h) and to a much lesser extent in the medial septum (Fig. 2g). Caudally, at the level of the optic recess, Satb2 labeling was detected in scattered but numerous cells in the amygdala and densely in the preoptic area (Figs. 2i, l, 3c-e). The combination of Satb2 with Otp at these levels made it possible to identify that a subpopulation of Satb2-ir cells in the most dorsal locations of MeA co-expressed Otp (see empty arrowhead in Fig. 2j-l). At these caudal levels, Satb2 expression was observed in the ventral portion of the medial pallium (see arrowhead in Fig. 2g-k). In addition, at the most caudal levels, Satb2 labeling was observed in a region adjacent to the MeA, described as the pallial amygdala (pA; see full arrowhead in Fig. 2l). In the most caudal region of the telencephalon, coinciding with the anterior portion of the alar hypothalamus, Satb2 and Otp populations were observed intermingled in this area (Pa; Fig. 2m). Some of these cells in the paraventricular area co-expressed both markers (see empty arrowhead in Fig. 2o) as in the MeA (see empty arrowhead in Fig. 2n).
The labeling of Satb2 in the telencephalon of the lungfish Protopterus dolloi was observed scattered in the ventral and lateral regions of the pallium (see arrowhead in Fig. 2p, q). At caudal levels, scattered Satb2-ir cells were detected in the region described as medial amygdala (Fig. 2r), a region that was clearly identified in combination with TH (Fig. 2s) and Otp (Fig. 2t), but no double-labeled cells were detected. The preoptic area of Protopterus also showed a prominent Satb2 labeling like the rest of sarcopterygians analyzed. Moreover, the combination with TH and Otp revealed the boundaries with the paraventricular domain of the alar hypothalamus (Fig. 3f, g). In the case of the combination with TH in the preoptic region of Trachemys (Fig. 3a), Xenopus (Fig. 3d) and Protopterus, no doubly labeled cells were identified, except in the most rostral domain in the acroterminal portion, where numerous Satb2-ir cells were also TH-ir (POAr; see empty arrowhead in Fig. 3a, d).
Hypothalamus and diencephalon: In the turtle diencephalon, both the thalamus and the prethalamus housed scattered Satb2-ir cells (Fig. 4a). In the thalamus, these cells were found in the nucleus reuniens (Fig. 4b). At these levels, the combination with Otp (Fig. 4a, c) and TH (Fig. 4e, f) made it possible to see Satb2 expression in scattered cells in the thalamus. At these levels, in the alar hypothalamus a similar combination (Fig. 4e) allowed us to identify the Satb2 expression in scattered cells in the subparaventricular area, which did not co-express TH (Fig. 4f). In addition, in the basal hypothalamus Satb2 expression was restricted to the caudal domain within the tuberal region (4d, f), avoiding the Otp-ir region (Fig. 4d), and with no coexpression of TH (Fig. 4f). In the mamillary region, where a significant serotonergic population was located, no Satb2 expression was found (Fig. 4g).
In the alar hypothalamus of Xenopus, Satb2 expression was scarce in the paraventricular area, but some of these cells co-expressed Otp (see empty arrowhead in Fig. 4h), and although they were found near the dopaminergic populations present in this area, no double-labeled cells were found (Fig. 4i). In the subparaventricular region Satb2-ir cells were also found (Fig. 4j-l) in the region where TH-ir (Fig. 4j) and 5-HT-ir cells were present (Fig. 4k), but in none of the cases those cells were doubly labeled. In the basal hypothalamus, numerous Satb2-ir cells were observed in its caudal portion (Fig. 4l), whereas in the rostral portion only scattered cells can be observed (Fig. 4m). In the diencephalon of Xenopus, a few scattered cells were observed in the prethalamus (Fig. 4k, l). In the lungfish brain, Satb2-ir cells were also found in the subparaventricular region, but these cells were not TH-ir (Fig. 4n-p). As for the basal hypothalamus, in this model Satb2-ir cells were also found in the tuberal region (Fig. 4s, t), but in this case, some double Otp-labeled cells were found in the rostral portion, but only at caudal hypothalamic levels (Fig. 4s-u). In the lungfish diencephalon, small groups of Satb2-ir cells were also observed in the thalamus and prethalamus (Fig. 4o, q, r).
Brainstem: Scattered Satb2-ir cells were observed in deep layers of the optic tectum of Trachemys scripta (Fig. 5a). In addition, at these mesencephalic levels, a population of Satb2-ir cells was also observed in the mesencephalic tegmentum (Fig. 5b). The combination with TH at rostral rhombencephalic levels allowed us to observe that there were Satb2 cells around the locus coeruleus, identified by the intense TH immunoreactivity, but the Satb2 cells did not belong to this population (Fig. 5c). In the parabrachial region, a very abundant population of Satb2-ir cells was identified in the parabrachial nucleus (Fig. 5d). In the rhombencephalon, scattered Satb2-ir cells were observed along the reticular formation, but these cells did not co-express 5-HT (Fig. 5e) or TH (Fig. 5f). In the case of Xenopus, the presence of Satb2 in the mesencephalic region is restricted to the torus semicircularis, where scattered cells were found (Fig. 5g), and the tegmentum. In the rhombencephalon the reticular formation also housed Satb2-ir cells, mainly in the superior reticular nucleus (Rs; Fig. 5g). In addition, at these levels a remarkable population of Satb2-ir cells was observed in the parabrachial area (Fig. 5h). Caudally, the reticular population can be found, but no TH double labeled cells were detected. In the mesencephalon of the lungfish Protopterus, Satb2-ir cells were observed in the torus semicircularis and the tegmentum. In the rostral rhombencephalon, Satb2 labeling was detected in the superior reticular nucleus and, at these levels, Satb2-ir cells were also observed in the parabrachial area (Fig. 5i). At more caudal rhombencephalic levels the presence of Satb2-ir cells was maintained in the reticular column, specifically in the median reticular nucleus (Fig. 5j), but neither at these levels (Fig. 5k), nor at more caudal levels, in the inferior reticular nucleus, which continues to maintain Satb2 labeling, did these cells express TH (Fig. 5l).
Distribution of Satb2 in the brain of actinopterygian fishes
Within the telencephalon, the olfactory bulbs and the pallium of actinopterygian fishes studied were devoid of any Satb2 labeling. In contrast, the subpallium houses several populations of Satb2-ir neurons. The most rostral one was observed in the ventral part of the ventral telencephalic area (Vv) of all representatives of the four groups of ray-finned fishes (Fig. 6a, b), in a ventral position to the catecholaminergic population of the dorsal part of the ventral telencephalic area (Vd; Fig. 6c). A few Satb2-ir cells were also detected in the caudal part of Vd of cladistians, chondrosteans and teleosts (Fig. 6b). In more caudal telencephalic levels, the four groups presented two populations of labeled cells in the supracommissural and posterior nuclei of the ventral telencephalic area (Vs and Vp; Figs. 6b, d, g, 7a, b, 8a), except for chondrosteans, which did not present the supracommissural group. These Satb2-ir cells in Vs and Vp co-distributed with the Otp-ir cells of this region (Fig. 6g), whereas some TH-ir cells of Vs co-expressed Satb2 in cladistians (see empty arrowheads in Fig. 6e).
The most conspicuous population of Satb2-ir cells in the brain of all actinopterygian fishes was in the preoptic area (Figs. 6d, g, 7a-e, g, h, 8b). Specifically, two subpopulations can be observed in this region of the four groups of fishes: a caudal one, (following the prosomeric view, dorsal in classical view), more numerous and densely packed, and without colocalization with the TH-ir cells of this region (Figs. 6d, f, g, 7a), and a rostral one (POAr; ventral in classical view), many of whose cells were doubly labeled with TH (Fig. 7b, f-h).
Within the hypothalamus, the alar paraventricular region, identified by the high presence of Otp, was generally devoid of Satb2 labeling (Figs. 7c, 8a), although some scattered Satb2-ir cells can be observed, likely belonging to the adjacent preoptic area (Figs. 6g, 7d, g, 8b, c). These cells were interspersed in the Otp-ir population, none of them being doubly labeled (Figs. 7d, 8b). In the subparaventricular region a grouped population of Satb2-ir cells was detected in the rostral part of the suprachiasmatic nucleus of the four groups of actinopterygian fishes analyzed (Fig. 8a, c). The teleost D. rerio was the only species whose suprachiasmatic population presented some Satb2/TH doubly labeled cells (Fig. 8d). On the other hand, no Satb2-ir cells has been observed in the basal hypothalamus (tuberal and mamillary regions) of ray-finned fishes.
Neither the diencephalon nor the mesencephalon of actinopterygian fishes showed Satb2 labeling. In contrast, some immunoreactive cells can be observed in the rhombencephalon of certain groups. Specifically, in cladistian fishes a restricted population of Satb2-ir cells was detected in the midline of the rostral rhombencephalon, ventral to the serotonergic superior raphe nucleus and dorsal to the interpeduncular neuropile (Fig. 9a). In contrast, holostean fishes presented some scattered cells in the superior reticular nucleus, lateral to the Otp-ir population (Fig. 9b), and in the ventral octavolateral area (Fig. 9c).