Introduction
The sense of smell is essential for a variety of behaviors in vertebrates like mating, feeding, fear and aggression. The organization of the olfactory system is well conserved in vertebrates not only in terms of function, but also in connectivity and also concerning the developmental origin of different structures within the peripheral and central olfactory system. Numerous investigations in mammals indicate that the olfactory system constitutes an excellent model to study various developmental aspects of the nervous system such as neurogenesis, neuronal migration, and axon guidance (Blanchart et al.
2011; Díaz-Guerra et al.
2013; Lim and Alvarez-Buylla
2016).
In rodents, the main olfactory system is related with the olfactory chemoreception of odorants and comprises a primary olfactory pathway consisting of the nasal olfactory epithelium (OE) and the main olfactory bulb (MOB), and a secondary olfactory pathway that includes all cortical regions directly innervated by MOB projection neurons (known together as the olfactory cortex); the olfactory cortex in turn releases signals to higher cortical areas involved in conscious perception and to limbic areas that control basic drives and emotions (reviewed in Boehm
2006; Treloar et al.
2010).
Besides, olfactory chemoreception of pheromones depends on an accessory olfactory system called vomeronasal system (VNS) that comprises the neuroepithelium of the vomeronasal organ (VNO or Jacobson’s organ) and the accessory olfactory bulb (AOB); signals from the AOB are relayed to regions of the amygdala and hypothalamus implicated in behavioral and physiological effects of pheromones (for review see Boehm
2006; Huilgol and Tole
2016).
In rodents, projecting axons of the olfactory receptor neurons located in the OE reach the telencephalic vesicle and induce the growth of the olfactory bulb primordium (OP). The MOB becomes evident macroscopically around the day 12 of embryonic development (E12; Gong and Shipley
1995). During the morphogenesis of the mouse MOB, projection neurons (mitral cells) are born from pallial progenitor cells and, then, interneurons (granule and periglomerular cells) that arise from the subpallium migrate tangentially toward their destination within the MOB (Blanchart et al.
2006; Vergaño-Vera et al.
2006; Imamura and Greer
2013; Huilgol and Tole
2016). The cellular organization of the MOB and AOB is similar. Projection neurons of the MOB and anterior AOB arise from the same region; however, differences in the domains of origin and migration routes of projection neurons are present between the anterior and posterior regions of the AOB. Interneurons of the MOB and AOB are born in the same region (for review see: Huilgol et al.
2013; Huilgol and Tole
2016).
Despite most studies about the olfactory system are focused on mammals, other animal models are necessary nowadays to understand different embryological aspects occurring during development of the olfactory system. Cartilaginous fishes represent one of the three living lineages of vertebrates (cyclostomes, cartilaginous fishes and bony vertebrates). Cartilaginous fishes diverged from bony vertebrates about 450 million years ago. Embryological studies in cartilaginous fishes reveal a conserved pattern of gene expression in diverse developmental process across gnathostome vertebrates (Gillis and Shubin
2009), and also morphogenetic processes and regionalization patterns are strikingly similar to mammals (Rodríguez-Moldes et al.
2017). More recently, the whole-genome analysis of three elasmobranch species has shown the presence of genes related with homeostasis, reproduction, and mechanisms for the generation of neuronal cell diversity homologous to that found in mammals (Hara et al.
2018). These studies indicate that cartilaginous fishes represent a key model for better knowledge of evolution of gnathostome brain development.
Moreover, cartilaginous fishes possess a well-developed sense of smell that is important for survival, localizing preys, avoiding predators, and chemosensory communication (for review see: Yopak et al.
2015). Numerous investigations have been referred to the adult olfactory system in elasmobranchs fishes. Several studies have been referred to the cell organization of the OE of the catshark, where sensory ciliated neurons, which in tetrapod vertebrates project to the MOB, are lacking; however, in the OE, microvillous olfactory receptor neurons and crypt sensory neurons are clearly recognizable (Theisen et al.
1986; Ferrando et al.
2006a,
b,
2009,
2010,
2012; Zaccone et al.
2011). In addition, genomic studies show that the predominant olfactory receptor type in the catshark and the elephant shark is the vomeronasal type2 receptor (V2R) (Sharma et al.
2019). These evidences together with ultrastructural and immunohistochemical data indicate that in the catshark the olfaction could mainly rely on a VNS (Ferrando and Gallus
2013). Interestingly, anatomical and molecular data show a primordial accessory olfactory system in the sea lamprey (Chang et al.
2013) and an accessory olfactory system was also identified in the African lungfish (González et al.
2010) and zebrafish (Biechl et al.
2017).
Though the OBs are laminated structures, in sharks, they do not present the six cell layers described in mammals. The cytoarchitectonic organization of the OB in the catshark has been investigated by classic staining techniques and summarized by Smeets et al. (
1983). Three main layers can be observed in this species: the olfactory nerve layer, the glomerular layer and the granular layer. Two main types of cells have been described in the OB: interneurons and projection neurons. The ultrastructure of the OB (Dryer and Graziadei
1996) and the arrangement of the primary and secondary olfactory projections have been described in different elasmobranchs species (Dryer and Graziadei
1993,
1994; Yáñez et al.
2011). Besides, in juveniles and/or adults of
S. canicula, the OB have been characterized using antibodies against enzymes like tyrosine hydroxylase (TH) (Carrera et al.
2012), glutamate acid decarboxylase (GAD) (Sueiro
2003), neuronal nitric oxide synthase (nNOS) (Ferrando et al.
2012) and choline acetyltransferase (ChAT) (Anadón et al.
2000); other neuroactive substances such as glycine (Anadón et al.
2013), serotonin (Carrera et al.
2008a) and diverse neuropeptides (Rodríguez-Moldes et al.
1993; Molist et al.
1995; Teijido et al.
2002) have also been detected in the OB of the catshark.
In contrast, studies about development of the olfactory system are scarce and mainly focused on the peripheral olfactory system (Ferrando et al.
2012; Ferreiro-Galve et al.
2012; Quintana-Urzainqui et al.
2014). Using tract-tracing and immunohistochemical techniques, the development of the peripheral olfactory system of the catshark
Scyliorhinus canicula has been described, and numerous Pax6-expressing cells have been observed in the OE, and along the developing olfactory nerve (Ferreiro-Galve et al.
2012; Quintana-Urzainqui et al.
2014). In addition, numerous Pax6-positive cells have been also reported in the ventricular zone of the ventrolateral pallium of embryos before the appearance of the OP (Ferreiro-Galve et al.
2012). However, the phenotype of these cells is unknown and their relationship with OB development has not been addressed so far. On the other hand, information about the origin, specification and differentiation of the OB cell types in the catshark is scarce and restricted to dopaminergic cells. These cells originate in a subpallial ventricular domain in late embryos and reach the OB following a route named lateral stream. The presence of TH-ir cells in this stream, as well as in the OB of stage 32 embryos, indicate that these cells may be the source of the granular and periglomerular cells of the mature OB (Ferreiro-Galve et al.
2012; Carrera et al.
2012; Quintana-Urzainqui et al.
2015). However, information about the development of OB glutamatergic cells (mitral cells) is lacking in sharks.
The transcriptional program involved in the specification and differentiation of mitral cells is well known in mammals and, curiously, is the same that operates on the specification/differentiation of pallial glutamatergic neurons before the mature organization of the OB is achieved (for review see: Bulfone et al.
1998; Englund et al.
2005; Díaz-Guerra et al.
2013; Imamura and Greer
2013; Kahoud et al.
2014; Roybon et al.
2015; Mihalas and Hevner
2017). Different experimental approaches carried out in mammals have shown that the transcription factor-paired homeobox 6 (Pax6), together with Sox2 (a transcription factor expressed in stem cells), is involved in neural stem cell self-renewal, neurogenesis and differentiation of specific neural cell types (Dellovade et al.
1998; Nomura and Osumi
2004; Kohwi et al.
2005; Sansom et al.
2009; Gómez-López et al.
2011; Curto et al.
2014). Pax6-expressing cells have been reported in the ventricular zone of the embryonic pallium as well as in the developing OB in mammals (Stoykova and Gruss
1994; Puelles et al.
2000), amphibians (Franco et al.
2001) and elasmobranch fishes, even though in the catshark, Pax6 is expressed throughout the VZ of the pallium, as in other vertebrates, the gradient described in mammals (lateral high, medial low and anterior high, and posterior low) is not evident in sharks (Ferreiro-Galve et al.
2012; Quintana-Urzainqui et al.
2012; Rodríguez-Moldes et al.
2017). In the MOB of rodents, projection neurons (mitral and tufted cells) derive from Pax6-positive radial glia progenitors located in the ventricular zone of the dorsal pallium (Winpenny et al.
2011; Imamura et al.
2011; Imamura and Greer
2013). Besides, Pax6 regulates the expression of two T-box genes (Tbr1 and Tbr2, the latter also known as Eomes) (Bulfone et al.
1995; Méndez-Gómez et al.
2011; Mizuguchi et al.
2012; Imamura and Greer
2013), which are also expressed along mitral cell development in the OB in rodents (Bulfone et al.
1999; Faedo et al.
2002; Winpenny et al.
2011; Mizuguchi et al.
2012; Roybon et al.
2015), birds (Bulfone et al.
1999), amphibians (Moreno et al.
2003; Brox et al.
2004) and zebrafish (Mione et al.
2001; Mueller and Wulliman
2016). In addition, the basic helix–loop–helix transcription factor NeuroD is also implicated in the terminal differentiation of mitral cells (Boutin et al.
2010; Osorio et al.
2010; Roybon et al.
2015). At the end of the embryonic period in mammals, mitral cells begin to express the glutamate vesicular transporter 1 (Vglut1; Ohmomo et al.
2011), which is also a marker of mitral cells in other groups of vertebrates such as reptiles (Sarkar and Atoji
2018). Mutant mice, where expression of Pax6, Tbr1 and Tbr2 is altered, show a disrupted OB morphogenesis indicating that this transcriptional cascade plays a key role in the correct morphogenesis of the OB (Bulfone et al.
1998; Nomura and Osumi
2004; Kahoud et al.
2014). Curiously, in mammals Tbr1 is also implicated in the specification of the anterior AOB; however, the specification of the posterior AOB is under the control of different genes (for review see: Huilgol and Tole
2016).
With the purpose of shedding light into the development of the central olfactory system in an evo–devo context, we have characterized Pax6 immunoreactive cells present in the ventricular zone of the ventrolateral pallium embryos of catshark with different progenitor markers such as Sox2 (stem cells), GFAP and BLBP (radial glia cells) and PCNA (proliferating cells). Then, we have studied the expression pattern of Pax6, once the OP emerges, and we have analyzed the expression pattern of transcription factors (such as Tbr2, NeuroD and Tbr1) and the vesicular transporter of glutamate 1 (vGlut1) related to differentiation of glutamatergic cells in the developing OB using immunocytochemistry and in situ hybridization techniques. In addition, we have carried out a BrdU pulse-chase study to determine the developmental period where these cells are generated. Finally, we have discussed our results in an evo–devo context and also at the light of the possibility of the existence of an AOB in the catshark.
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