Immunohistochemical analysis of Pax6 and Pax7 expression in the CNS of adult Xenopus laevis

Journal of Chemical Neuroanatomy 57–58 (2014) 24–41

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Immunohistochemical analysis of Pax6 and Pax7 expression in the CNS of adult Xenopus laevis Sandra Bandı´n, Ruth Morona, Jesu´s M. Lo´pez, Nerea Moreno, Agustı´n Gonza´lez * Department of Cell Biology, Faculty of Biology, University Complutense, 28040 Madrid, Spain

A R T I C L E I N F O

A B S T R A C T

Article history: Received 30 January 2014 Received in revised form 26 March 2014 Accepted 27 March 2014 Available online 6 April 2014

Pax6 and Pax7 are transcription factors essential for the development of the CNS. In addition, increasing data, mainly obtained in amniotes, support that they are expressed in subsets of neurons in the adult, likely playing a role in maintaining neuron type identity. In the present study we analyzed the detailed distribution of Pax6 and Pax7 cells in the adult CNS of Xenopus laevis. Immunohistochemistry with antibodies that are required for high-resolution analysis of Pax-expressing cells was conducted. A wide distribution of Pax6 and Pax7 cells throughout the CNS was detected, with distinct patterns that showed only slight overlapping. Only Pax6 was expressed in the telencephalon, including the olfactory bulbs, septum, striatum and amygdaloid complex. In the diencephalon, Pax6 and Pax7 were distinct in the alar and basal parts, respectively, of prosomere 3. Large numbers of Pax6 and Pax7 cells were distributed in the pretectal region (alar plate of prosomere 1) but only Pax6 cells extended into basal plate. Pax7 specifically labeled cells in the optic tectum, including the ventricular zone, and Pax6 cells were the only cells found in the tegmentum. Pax6 was found in most granule cells of the cerebellum and Pax7 expression was found only in the ventricular zone. In the rostral rhombomere 1, Pax7 labeling was detected in cells of the ventricular zone of the alar plate, but numerous migrated cells were located in the basal plate, including the griseum centrale and the interpeduncular nucleus. Pax6 cells also formed a column of scattered neurons in the reticular formation and were found in the octavolateral area. The rhombencephalic ventricular zone of the alar plate expressed Pax7. Dorsal Pax7 cells and ventral Pax6 cells were found along the spinal cord separated from the ventricle, which did not show immunoreactivity. Our results show that the expression of Pax6 and Pax7 is widely maintained in the adult brain of Xenopus, like in urodele amphibians and in contrast to the situation described in amniotes. Therefore, in amphibians these transcription factors seem to be needed to maintain specific entities of subpopulations of neurons in the adult CNS. ß 2014 Elsevier B.V. All rights reserved.

Keywords: Pax genes Immunohistochemistry Segmental organization Telencephalon Diencephalon Brain evolution

Abbreviations: A, anterior nucleus of the thalamus; ABB, alar-basal boundary; Acc, accumbens nucleus; al, anterior lobe of the hypophysis; aol, area octavolateralis; ap, alar plate; APt, anterior pretectal nucleus; Av, anteroventral tegmental nucleus; b, basal band of the mesencephalon; bp, basal plate; BSM, bed nucleus of the stria medullaris; BST, bed nucleus of the stria terminalis; CB, calbindin D-28k; Cb, cerebellum; cc, central canal; CeA, central amygdala; ChAT, choline acetyltransferase; chp, choroid plexus; CoP, commissural pretectum; d, dorsal band of the mesencephalon; dh, dorsal horn of the spinal cord; DMB, diencephalo-mesencephalic boundary; DMN, dorsal medullary nucleus; Dp, dorsal pallium; fr, fasciculus retroflexus; Gc, griseum centrale; gl, glomerular layer of the olfactory bulb; GT, griseum tectale; H, hypothalamus; Hd, dorsal habenular nucleus; igl, internal granular layer of the olfactory bulb; III, oculomotor nucleus; il, intermediate lobe of the hypophysis; Ip, interpeduncular nucleus; Ipn, interpeduncular neuropil; Is, isthmic nucleus; ISc, inner nucleus of the subcommissural organ; Isl1, islet 1; IV, trochlear nucleus; JcP, juxtacommissural pretectum; l, lateral band of the mesencephalon; LA, lateral amygdala; Lc, locus coeruleus; LDT, laterodorsal tegmental nucleus; Lp, lateral pallium; LR, laterorostral mesencephalic nucleus; m, medial band of the mesencephalon; Ma, mammillary region; MeA, medial amygdala; Mes, mesencephalon; MOB, main olfactory bulb; Mp, medial pallium; nl, neural lobe of the hypophysis; NOS, nitric oxide synthase; NPv, nucleus of the periventricular hypothalamic organ; Nsol, nucleus of the solitary tract; nIII, oculomor nerve; nV, trigeminal nerve; nVI, abducens nerve; on, olfactory nerve; OPT, olivary pretectal nucleus; OSc, outer nucleus of the subcommissural organ; OT, optic tectum; 0tp, orthopedia; p1–p3, prosomeres 1–3; pc, posterior commissure; PcP, precommissural pretectum; PcPC, parvocellular nucleus of the posterior commissure; Pdi, posterodorsal tegmental nucleus, isthmic part; pe, postolfactory eminence; PO, preoptic area; po, pineal organ; PPN, pedunculopontine tegmental nucleus; PrPt, principal pretectal nucleus; PTh, prethalamus; PThE, prethalamic eminence; r0, isthmus (rhombomere r0); r1–r8, rhombomeres 1–8; Ras, superior raphe nucleus; Rh, rhombencephalon; Ri, nucleus reticularis inferior; Rm, nucleus reticularis medius; Rs, nucleus reticularis superior; SC, suprachiasmatic nucleus; ScO, subcommissural organ; Sd, septum dorsalis; sgr, stratum granulare of the cerebellum; Sl, septum lateralis; Sm, septum medialis; sm, stria medullaris; smn, spinal motor neurons; smol, stratum moleculare of the cerebellum; sol, solitary tract; sP, stratum of Purkinje cells; SpL, nucleus spiriformis lateralis; Str, striatum; tc, tectal commissure; Teg, mesencephalic tegmentum; Tel, telencephalon; TH, tyrosine hydroxylase; Th, thalamus; TP, nucleus of the tuberculum posterior; Ts, torus semicircularis; Tub, tuberal hypothalamic region; v, ventricle; vh, ventral horn of the spinal cord; VIa, accessory abducens nucleus; VIm, main abducens nucleus; VIIm, facial motor nucleus; VL, ventrolateral nucleus of the prethalamus; VJc, ventral juxtacommissural nucleus; VM, ventromedial nucleus of the prethalamus; Vm, trigeminal motor nucleus; vz, ventricular zone. * Corresponding author at: Departamento de Biologı´a Celular, Facultad de Biologı´a, Universidad Complutense, Calle Jose Antonio Novais, 2, 28040 Madrid, Spain. Tel.: +34 913944972; fax: +34 913944981. E-mail address: [email protected] (A. Gonza´lez). http://dx.doi.org/10.1016/j.jchemneu.2014.03.006 0891-0618/ß 2014 Elsevier B.V. All rights reserved.

S. Bandı´n et al. / Journal of Chemical Neuroanatomy 57–58 (2014) 24–41

1. Introduction Pax genes encode a set of transcription factors that are involved in a wide range of developmental processes in metazoans, acting as tissue-specific regulators of organogenesis (cell fate and patterning), cell proliferation and disease (Chalepakis et al., 1993; Noll, 1993; Stuart et al., 1994; Wehr and Gruss, 1996; Balczarek et al., 1997; Mansouri et al., 1999; Chi and Epstein, 2002; Haubst et al., 2004; Buckingham and Relaix, 2007; Lang et al., 2007; Blake et al., 2008; Wang et al., 2008). In particular, among the Pax genes, Pax6 and Pax7 are expressed in regionally restricted patterns in the developing brain, controlling neuronal proliferation, brain regionalization, cell differentiation and neuronal survival (Wehr and Gruss, 1996; Lang et al., 2007; Thompson et al., 2007; Osumi et al., 2008; Wang et al., 2008). Interestingly, they are highly conserved across vertebrates and the proteins encoded by these two genes are also conserved (Goulding et al., 1993; Matsuo et al., 1993; Epstein et al., 1994; Li et al., 1994, 1997; Kallur et al., 2008), and the deduced amino acid sequences show more than 85% overall identity across vertebrates (Callaerts et al., 1997; Hirsch and Harris, 1997; Seo et al., 1998). Pax6 and Pax7 also appear to be involved in maintaining pluripotency throughout adulthood, in subsets of cell populations characterized as stem/progenitor cells, as well as subpopulations of mature nerve cells within certain brain regions, with ability to respond to environmental signals (Chi and Epstein, 2002; Maekawa et al., 2005; Thomas et al., 2007; Thompson et al., 2007, 2008; Blake et al., 2008; Fedtsova et al., 2008; Osumi et al., 2008). Thus, Pax6 and Pax7 expressing neurons were reported in the adult brain of rodents. In general, Pax6 is expressed in retinal cells, telencephalon, diencephalon, ventral mesencephalon, cerebellum and pons/medulla (Stoykova and Gruss, 1994; Kohwi et al., 2005; Maekawa et al., 2005; Nacher et al., 2005; Stanescu et al., 2007; Duan et al., 2013), whereas Pax7 is expressed in the superior colliculus and in specific nuclei of the pons/medulla and thalamus (Stoykova and Gruss, 1994; Shin et al., 2003; Thomas et al., 2007; Thompson et al., 2007, 2008). In all these regions, Pax6 and/or Pax7 seem to be required for maintaining distinct neuronal identity (Ninkovic et al., 2010) and physiological functions in mature neurons (Stoykova and Gruss, 1994; Shin et al., 2003). Research during development of the CNS in some representatives of all major vertebrate classes has shown that the Pax6 and Pax7 expression patterns are highly comparable across species (Stoykova and Gruss, 1994; Stuart et al., 1994; Kawakami et al., 1997; Murakami et al., 2001; Derobert et al., 2002; Haubst et al., 2004; Pritz and Ruan, 2009; Moreno and Gonza´lez, 2011; Duan et al., 2013). Moreover, detailed distribution maps of Pax6 and Pax7 expressing cell in the brain of amphibians have been recently obtained by means of immunohistochemical procedures (Bandı´n et al., 2013; Joven et al., 2013b). The use of these highly sensitive techniques also revealed that the expression of Pax6 and Pax7 is widely maintained in the brains of adult urodeles, in contrast to the situation observed in other tetrapods, and this discrepancy was proposed to be related to the pedomorphic features of urodele brains (Joven et al., 2013a). In contrast, a previous study based on in situ hybridization techniques, found that Pax6 expression continued in the brain of juvenile and reproductive adults Xenopus only in very restricted regions of the forebrain, including the dorsal portion of the septum and the prethalamus (Moreno et al., 2008a). In the present study we have analyzed the distribution patterns of Pax6- and Pax7-immunoreactive cells (Pax6 and Pax7 cells, respectively) in the adult brain of the anuran Xenopus laevis using antibodies that have been proven to be highly sensitive to unravel these transcription factors (Hitchcock et al., 1996; Wullimann and Rink, 2001; Gonza´lez and Northcutt, 2009; Ferreiro-Galve et al., 2012; Bandı´n et al., 2013; Joven et al., 2013a,b; Gonza´lez et al., 2014).

25

For the correct identification of the cell groups expressing Pax6 and/ or Pax7, we used combined immunofluorescence to reveal simultaneously several transcription factors and neuronal markers, which in turn served as landmarks for brain regions, because their distribution is well established in the brain of Xenopus, as previously reported (Gonza´lez et al., 1993; Marı´n et al., 1997; Lo´pez and Gonza´lez, 2002; Gonza´lez et al., 2002; Moreno et al., 2008b; Morona and Gonza´lez, 2008, 2009; Domı´nguez et al., 2011, 2013, 2014). These markers included the g-aminobutyric acid (GABA), calbindin D-28k (CB), choline acetyltransferase (ChAT), nitric oxide synthase (NOS), serotonin (5-HT), tyrosine hydroxylase (TH), and the transcription factors Nkx2.1, Nkx2.2, Islet 1 (Isl1), and orthopedia (Otp). For comparisons across species, the currently adopted paradigm of brain segmentation based on spatially restricted gene expression patterns has proven extremely useful (Gilland and Baker, 1993; Marı´n and Puelles, 1995; Puelles et al., 1996; Fritzsch, 1998; Cambronero and Puelles, 2000; Dı´az et al., 2000; Puelles and Rubenstein, 2003; Straka et al., 2006). Our results show a highly conserved pattern of Pax6 and Pax7 expression across vertebrates and demonstrate that the expression of these transcriptions factors is widely maintained in adult Xenopus brains, supporting their importance throughout life. 2. Materials and methods 2.1. Animals and tissue processing Adult specimens of the anuran amphibian X. laevis (n = 26) were purchased from commercial suppliers (XenopusOne, Dexter, MI), and kept in tap water at 20–25 8C. The animals were treated according to the regulations and laws of the European Union (2010/63/EU) and Spain (Royal Decree 53/2013) for care and handling of animals in research, after approval from the University Complutense to conduct the experiments described. The animals were anesthetized by immersion in a 0.4 mg/ml solution of tricaine methanesulfonate (MS222, Sigma Chemical Co., St. Louis, MO) and perfused transcardially with 100 ml 0.9% NaCl, followed by 200 ml 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4), or the fixative MEMFA (0.1 M MOPS [4morpholinopropanesulfonic acid], 2 mM EGTA [ethylene glycol tetraacetic acid], 1 mM MgSO4, 3.7% formaldehyde). The brain and spinal cord were dissected out and postfixed approximately 24 h in the same fixative solution at 4 8C. Subsequently, they were immersed in a solution of 30% sucrose in 0.1 M phosphate buffer (PB; pH 7.4) for 4–6 h at 4 8C until they sank. For sectioning on a freezing microtome (Thermo Scientific Microm HM 450) the tissue was embedded in a solution of 20% gelatin with 30% sucrose in PB, and stored overnight in formaldehyde diluted 1:10 in 30% sucrose in PB at 4 8C. Sections were obtained at 30–40 mm thickness in the transverse or sagittal plane, and collected in PB in four series of adjacent sections. 2.2. Immunohistochemistry Immunohistofluorescence procedures were conducted for different primary antibodies, all of which were diluted in 5–10% normal goat or mouse serum (depending on the source of the primary antibody) in PB with 0.1% Triton X-100 (Sigma, St. Louis, MO) and 2% bovine serum albumin (BSA, Sigma). Different protocols were carried out on free-floating sections, with incubation in the primary antibodies for 72 h at 4 8C, or for 16–24 h at room temperature in the antigen retrieval pre-treated slides. The dilution of each primary antibody used is detailed in Table 1. Single-staining protocols for the detection of Pax6 and Pax7 were carried out on the free-floating sections as follows: (1) incubation for 72 h at 4 8C in the dilution of each primary serum (see Table 1). (2) According to the species in which the primary antibody was raised, the second incubations were conducted with the appropriately labeled secondary antibody diluted 1:500 for 90 min at room temperature: Alexa 594-conjugated goat anti-rabbit (red fluorescence; Molecular Probes, Eugene, OR; catalog reference: A11037), Alexa 488-conjugated goat anti-mouse (green fluorescence; Molecular Probes; catalog reference: A21042). For bright field immunohistochemistry, free-floating sections were rinsed twice in PB, treated with 1% H2O2 in PB for 20 min to reduce endogenous peroxidase activity, rinsed again three times in PB, incubated in the primary antibody dilution (mouse anti-Pax6 or mouse anti-Pax7) with 0.025% Triton in PB, revealed with biotinylated horse anti-mouse (1:100; Vector, Burlingame, CA; catalog reference: BA-2000), rinsed three times in PB, and visualized by the ABC-DAB kit method (Vector, SK4100). To study the relative distribution of two proteins in the same sections, the twostep protocol for immunohistofluorescence was used, with cocktails of pairs of primary antibodies (always developed in different species), at the same dilutions specified in Table 1 and following the conditions detailed above for the

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Table 1 List of primary antibodies used in the present study. Name

Immunogen

Commercial supplier

CB

E. coli-produced recombinant rat calbindin D-28k E. coli-produced recombinant rat calbindin D-28k Human placental choline acetyltransferase g-Aminobutyric acid (GABA) conjugated to BSA Amino acids 247–349 at the C-terminus of rat Isl1 Amino acids 110–122 from the amino terminus E. coli-derived recombinant chick NKX2.2 transcription factor related Recombinant rat NOS

Monoclonal mouse anti-calbindin D-28k; Swant, Bellinzona, Switzerland; catalog No. 300 Polyclonal rabbit anti-calbindin D-28k; Swant, Bellinzona, Switzerland; catalog No. CB-38a Polyclonal goat anti-ChAT; Chemicon; catalog No. AB144P Polyclonal rabbit anti-g-aminobutyric acid; Sigma, St. Louis MO; catalog No. A2052 Monoclonal mouse anti-Isl 1; Developmental Studies Hybridoma Bank, catalog reference: 39.4D5 Polyclonal rabbit anti-TTF; Biopat Immunotechnologies, Caserta, Italy; catalog No. PA 0100 Monoclonal mouse anti-Pax6; Developmental Studies Hybridoma Bank, Iowa City, IA; catalog No. 74.5A5

28

1:500

28

1:500

Polyclonal sheep-anti-NOS K205 antibody; Dr. P.C. Emson, The Babraham Institute Polyclonal rabbit anti-Otp; Pikcell Laboratories, Kruislaan, Amsterdam, The Netherlands Monoclonal mouse anti-Pax6; Developmental Studies Hybridoma Bank, Iowa City, IA; catalog No. PAX6 Polyclonal rabbit anti-Pax6; Covance, California, USA; catalog No. PBR-278

CB ChAT GABA Isl1 Nkx2.1 Nkx2.2

NOS Otp PAX6 PAX6

PAX7

TH TH 5-HT

Amino acid sequence: RKALEHTVSMSFT of the C-terminal OTP E. coli-derived recombinant chick PAX6. aa 1–223 of the chick Pax6 Peptide sequence: QVPGSEPDMSQYWPRLQ of the C-terminus of the mouse PAX6 protein E. coli-derived recombinant chick PAX7. aa 352–523 of the chick Pax7 Protein purified from rat pheochromocytoma TH purified from rat PC12 cells Serotonin conjugated to BSA with paraformaldehyde

MW (kDa)

68 0.0103

Dilution

1:100 1:3000

39

1:500

42–37

1:500

30

1:500

155

1:20,000

34

1:1000

46

1:250

46

1:300

Monoclonal mouse anti-Pax7; Developmental Studies Hybridoma Bank, Iowa City, IA; catalog No. PAX7

55

1:500

Polyclonal rabbit anti-TH; Chemicon International, Inc, USA; catalog No. AB152 Monoclonal mouse anti-TH; ImmunoStar, Hudson, WI; catalog No. 22941 Polyclonal rabbit anti-5-HT; Incstar, Stillwater, USA; catalog No. 20080

62

1:1000

62 0.176

1:1000 1:1000

single-staining protocol. According to the species in which the primary antibody was raised, the second incubations were conducted with the appropriate fluorescent-labeled secondary antibody cocktails diluted 1:500 in PB for 90 min at room temperature: Alexa 594-conjugated goat anti-rabbit, Alexa 488-conjugated goat anti-mouse, Alexa 594-conjugated donkey anti-goat (Molecular Probes; catalog reference: A11058), Alexa 594-conjugated chicken anti-rabbit (Molecular Probes; catalog reference: A21442), or fluoresceinconjugated rabbit anti-sheep (Vector; catalog reference: FI-6000). In all cases, sections were counterstained with the nuclear marker Ho¨echst (blue fluorescence; Sigma-Aldrich, St. Louis, MO; Ho¨echst 33258) to facilitate interpretation of the results. In all cases, after being rinsed the sections were mounted on glass slides and coverslipped with Vectashield mounting medium (Vector; catalog reference: H1000). 2.3. Controls and specificity of the antibodies General controls for the immunohistochemical reaction included: (1) staining some selected sections with preimmune mouse, rabbit or goat serum instead of the primary antibody, and (2) controls in which either the primary and/or the secondary antibody was omitted. In all these negative controls, the immunostaining was eliminated. In addition, all the antibodies used have been tested, under identical conditions, in tissues devoid of antigen (rat brain slices at levels revealing no expression), as negative control, and in tissues positive for the antigen (rat brain slices at levels expressing the antigen). In all cases, the controls were satisfactory. The specificity of the antibodies used has been assessed by the commercial companies (Table 1), and, in addition, the immunoblotting conducted in our previous studies with X. laevis showed that all antibodies used labeled a single band, which corresponded well (with minor variations) to the bands labeled in the rat lanes (see Morona and Gonza´lez, 2008, 2009; Morona et al., 2011; Moreno et al., 2012; Domı´nguez et al., 2013). In the cases of Pax6, CB and TH, monoclonal and polyclonal antibodies were used (Table 1) with fully comparable results in the pattern of immunostaining. 2.4. Imaging The sections were analyzed with an Olympus BX51 microscope equipped for fluorescence with appropriate filter combinations. Selected sections were photographed using a digital camera (Olympus DP70). Photomicrographs were adjusted for contrast and brightness with Adobe PhotoShop CS4 (Adobe Systems, San Jose, CA) and were mounted on plates using Canvas 11 (ACS Systems International, Santa Clara, CA).

3. Results Pax6 and Pax7 immunoreactivity was clearly visible in subpopulations of neurons in most main regions of the adult Xenopus brain, and the immunoreaction product was present in the cell nuclei. The distinct patterns of immunolabeling for each protein were constant from animal to animal and showed mostly segregated distributions for Pax6 and Pax7, although in some areas codistribution/colocalization of both proteins in some neurons was detected by the double immunohistofluorescence technique. In spite of the fact that only adult specimens were analyzed, labeling was observed in cells of the ventricular zone and migrated into the mantle zone, depending on the particular brain region, as will be described below. The patterns of labeling for Pax6 and Pax7 are described from rostral to caudal levels and attending to the main subdivisions of the brain (Table 2). The drawing in Fig. 1 corresponds to series of transverse sections and is intended to aid in the description. The data are further presented as photomicrographs of the single-labeled sections for Pax6 or Pax7 (Figs. 2 and 3), in which bright field or inverse pictures from the fluorescence material are presented. We will comment on the codistribution/colocalization of Pax6 and Pax7, and between these and the other markers used (Figs. 4 and 5), describing the precise location and identification of the cells that contain these transcription factors. The results were analyzed primarily within the context of recently proposed subdivisions of the telencephalon (Marı´n et al., 1998; Brox et al., 2003; Moreno and Gonza´lez, 2006) and the neuromeric organization of the brain, following the current model validated for many vertebrates, including amphibians (prosencephalon: Puelles and Rubenstein, 1993, 2003; midbrain: Dı´az et al., 2000; rhombencephalon: Gilland and Baker, 1993; Marı´n and Puelles, 1995; Cambronero and Puelles, 2000; Aroca and Puelles, 2005; Straka et al., 2006).

S. Bandı´n et al. / Journal of Chemical Neuroanatomy 57–58 (2014) 24–41 Table 2 Comparative localization of Pax6 and Pax7 cells in the CNS of adult Xenopus laevis. Pax6 Forebrain Olfactory bulb Septum Striatum Amygdaloid complex Paraphysis Alar part of p3 Basal part of p3 Roof (dorsal) p2 Alar part of p1 (pretectal region) Basal part of p1 Hypothalamic mammillary region Intermediate lobe of the hypophysis Midbrain Optic tectum Torus semicircularis Mesencephalic tegmentum Hindbrain Alar part of r1 Cerebellum, ventricular zone Cerebellum, granule cell layer Interpeduncular nucleus Central gray (r1–r2) Dorsal medullary nucleus Reticular nuclei, basal rhombencephalic vz Alar rhombencephalic vz, nucleus of the solitary tract Spinal cord Dorsal gray Ventral gray

Pax7

+ + + + + + +* +*

+* + +* + + +

+ + +

+ + + + + +

+ +

+

+ +

+, cells present; +*, location with doubly labeled cells.

3.1. Forebrain The primary prosencephalic vesicle gives rise through development to the diencephalon (caudally) and the secondary prosencephalon (rostrally), the latter formed by the telencephalon and the hypothalamus, and all these regions constitute the forebrain (see Puelles and Rubenstein, 2003). Only Pax6 cells were detected in the rostral parts of the forebrain of Xenopus, mainly in the olfactory bulbs (secondarily evaginated at the rostral tip of the telencephalon) and ventral (subpallial) regions of the telencephalic hemispheres (Figs. 1a–e, 2a–d, f and 4a–e). Within the olfactory bulbs, Pax6 cells were found in large number in the internal granule cell layer, forming a compact cell population, whereas scattered labeled cells extended peripherally, mainly around the glomeruli that define the glomerular layer of the bulb (Figs. 1a and 2a). Double labeling experiments demonstrated that Pax6 cells were distinct from those labeled for CB and NOS, whereas virtually all neurons labeled for TH (putative dopaminergic cells) were Pax6 cells, most notably those located in periglomerular positions (Fig. 4a). The Pax6 cells located most ventrally in the olfactory bulb extended caudally into the ventromedial part of the rostral telencephalic hemisphere, where they constituted a conspicuous population in the region of the postolfactory eminence (name after Northcutt and Kicliter, 1980; Fig. 2a and b). Slightly more caudally, Pax6 cells were found in the nucleus accumbens, also in the ventromedial part of the hemisphere, identified by the dense plexus formed by TH positive fibers (Gonza´lez et al., 1993; Figs. 1b and 4b). This population of Pax6 cells diminished caudally along the hemisphere, remaining only few labeled cells close to the ventral tip of the lateral ventricle, in the region of the bed nucleus of the stria terminalis (Moreno et al., 2012; Figs. 1c and 2c). Also in the medial wall of the telencephalic hemispheres, a striking rostrocaudal band of Pax6 cells extended from ventromedial

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(rostrally) to dorsomedial (caudally) locations (Fig. 2a). Thus, at rostral telencephalic levels, these cells occupied the region of the ventral medial septum (Fig. 1b), whereas caudally they were distributed in the medial septal nucleus that was identified by ChAT immunohistochemistry (Gonza´lez and Lo´pez, 2002) and was distinct from the lateral septal nucleus, labeled with TH positive fibers (Figs. 1b and c and 4b). At caudal telencephalic levels, these Pax6 cells were located at the most dorsal part of the medial septum, within the named dorsal septal nucleus, just beneath the medial pallium (Figs. 1d and 2a and c). Most Pax6 cells in the ventrolateral wall of the telencephalic hemisphere were labeled in the striatum (Fig. 1b and c). They formed a band located close to the ventricle, medial to the striatal neuropil formed by TH positive fibers (Fig. 4b). The precise localization of these Pax6 cells within the striatum was assessed by double labeling for Isl1, which labeled the subpallial striatal region (Moreno et al., 2008b; Fig. 4c). However, in these double labeled sections it was observed that the Pax6 cell population extended above the striatal region (Fig. 4c) in the area identified as the ventral pallium, which contains the lateral amygdala of anurans that can be identified, among other features, by its intense labeling for NOS (see Moreno and Gonza´lez, 2004). Apart from the lateral amygdala, the only pallial amygdaloid component in amphibians, Pax6 cells were also observed in the other two main amygdaloid nuclei, the central and the medial nuclei (Figs. 1d and e and 2d and f). The central amygdala constitutes a striatal component in the caudal pole of the telencephalon that is also labeled for Isl1 (Fig. 4d) and was characterized by its large NOS positive neurons (Fig. 4d0 ), which extend their dendritic processes caudally (Fig. 4e). Double labeling experiments revealed that the Pax6 neurons in the central amygdala formed a cell population located medial to the cells labeled for Isl1 and NOS, which were more separated from the ventricle. The medial amygdala, or vomeronasal amygdala, receives the bulk of the projection from the accessory olfactory bulb that forms a rounded neuropil in the caudal telencephalon (asterisk in Fig. 4e). The cells in this region form a thick band close to the ventricle and a subpopulation was clearly labeled for Pax6 (Figs. 1e, 2d and 4e). The adjacent preoptic area, which is currently considered a telencephalic region (see Domı´nguez et al., 2013) was devoid of Pax6 cells, as it was also the case of the whole hypothalamus (Fig. 1e–k). Regarding the presence of Pax7 cells in the rostral prosencephalon, only a conspicuous labeling was identified in the paraphysis, in relation to the choroid plexus that extended between the telencephalic hemispheres (Figs. 1e and 3a). In addition, a few Pax7 cells were located in the caudobasal hypothalamus within the mammillary region, in close relation to the diencephalic cell population (see below). Of note, conspicuous Pax7 cells formed a dense population in the intermediate lobe of the hypophysis (Figs. 3f, i and j and 5m), whereas only scattered, weakly immunoreactive Pax6 cells were observed in the anterior lobe (Fig. 4n). Distinct patterns of Pax6 and Pax7 cell distribution were observed in the diencephalon. Three segments form the diencephalon, which area named prosomeres 1–3 (p1–p3, from caudal to rostral). It is worth mentioning that these three segments are bent due to the cephalic flexure, so that in conventional ‘‘transverse’’ sections they are observed one at the top of the other, with p1 ‘‘dorsal’’ to p2, and p2 ‘‘dorsal’’ to p3 (see Fig. 1f–k). The caudal p1 contains in its dorsal part (alar part) the pretectal region, whereas p2 contains the thalamus (former dorsal thalamus) and p3 the prethalamus (former ventral thalamus). These three prosomeres posses smaller basal (tegmental) regions that are rostrally continuous with the basal hypothalamus. Starting from rostral levels, Pax6 cells were strikingly abundant in the dorsal part of p3. This cell population was formed by scattered and intensely Pax6

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S. Bandı´n et al. / Journal of Chemical Neuroanatomy 57–58 (2014) 24–41

Fig. 1. Diagrams (a–p) of transverse sections through the brain of Xenopus laevis (levels indicated in the upper scheme of a lateral view of the brain) showing the distribution of immunoreactive cells for Pax6 and Pax7. For abbreviations, see list. Scale bar = 500 mm.

S. Bandı´n et al. / Journal of Chemical Neuroanatomy 57–58 (2014) 24–41

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Fig. 2. Photomicrographs of single-stained sagittal (a, f, h, n) and transverse (b–e, g, i–m, o–r) sections showing the localization of Pax6 cells in the telencephalon (a–d), the diencephalon (e–j), the mesencephalic tegmentum (k, l), the central gray in rhombomere 1 and cerebellum (m, n), at the level of the nucleus reticularis medius (o), the dorsal medullary nucleus of the area octavolateralis (p), the nucleus reticularis inferior (q), and in the ventral gray close to the obex (r). In the sagittal sections the orientation is indicated in each photomicrograph. The vertical bars in a, f, and m indicate the levels of the sections shown in the photomicrographs indicated. For abbreviations, see list. Scale bars = 200 mm (a, b, d, e, g, i–r), 500 mm (c, f, h).

labeled cells in the most dorsal part of the segment, within the currently named ‘‘prethalamic eminence’’, in particular close to the fibers of the stria medullaris, in the cell group named bed nucleus of the stria medullaris (Figs. 1f, 2e and f and 4e). Pax6 cells located most dorsally within the prethalamus were also scattered (Figs. 1f, 2e and f and 4e), whereas more ventrally the labeled neurons were

more closely packed in the medial part of p3, within the traditionally named ventromedial and ventrolateral nuclei (Figs. 1g and h and 2g–i), with only a few cells extending laterally into the regions of the lateral geniculate and Bellonci’s nuclei (see Puelles et al., 1996). The most ventral part of the Pax6 cell population in the prethalamus formed parallel bands of cells that

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Fig. 3. Photomicrographs of single-stained transverse (a–e, g, h, j–m, o–s) and sagittal (f, i, n) sections showing the localization of Pax7 cells in the paraphysis (a), the diencephalon and hypothalamus (b–h), the mesencephalon (i–k), the rostral rhombencephalon (l–n), the rostral area octavolateralis (o), the region dorsal to the nucleus reticularis medius (p), the nucleus of the solitary tract (q), in the dorsal ventricular zone and gray close to the obex (r), and in the cervical spinal cord (s). In the sagittal sections the orientation is indicated in each photomicrograph. The vertical bars in f, i, l, and m indicate the levels of the sections shown in the photomicrographs indicated. The arrow in photomicrograph k indicates to the point where the ventricular expression of Pax7 ends. For abbreviations, see list. Scale bars = 200 mm (a–h, k, n, o), 500 mm (i, j, l), 100 mm (p–s).

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Fig. 4. Photomicrographs of double labeled transverse (a–d0 , f–j00 , l–p0 , s–y, z0 ) and sagittal (e, k, q, r, z) sections through the brain. The detected molecules are indicated on each photomicrograph. (a) Pax6 labeling in the olfactory bulbs, showing that those most closely located to the glomeruli are catecholaminergic cells (TH-immunoreactive); the inset is a higher magnification to show double labeled cells. (b) Localization of the Pax6 cells in the septum and caudal part of the nucleus accumbens. (c) Pax6 cells in the striatum and lateral amygdala in relation to the Isl1 stained cells. (d and d0 ) Pax6 cells in the central and medial amygdala. (e) Pax6 cells in the amygdaloid complex and the dorsal part of p3 (asterisk marks the neuropil of the medial amygdala). (f and f0 ) Pax6 labeled cells in the prethalamus and the precise boundaries with the thalamus and

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were clearly distinct from the adjacent thalamus (labeled for example for CB or Nkx2.2, Fig. 4f and h) or hypothalamus (labeled for TH, Fig. 4g). Double labeling experiments demonstrated that a subpopulation of the most ventrally located Pax6 cells in the alar region of p3 were also TH and Nkx2.2 positive (Fig. 4g and h), whereas double labeling for NOS demonstrated that the Pax6 cells constituted a separate population in p3, located lateral to the nitrergic cells (Fig. 4i). The simultaneous staining for Pax6 and Pax7 demonstrated a certain degree of codistribution (but not colocalization) of both cell populations in a manner that Pax6 cells entered the basal part of p3 but remained located lateral to the Pax7 cells (Figs. 1i and j and 4j– j00 ). Actually, among the most conspicuous Pax7 cell populations in the brain of Xenopus was the group of neurons labeled in the basal part of p3 (Figs. 1i and j and 3b–f). These cells formed a band of packed neurons in the rostral part of the basal p3, close to the boundary with the hypothalamus, which is topologically rostral (Figs. 3b, c and f, 4j–j00 and 5b–h). This Pax7 cell population was identified in the basal part of p3 by double labeling for TH, Nkx2.2, Nkx2.1, NOS, Otp, and 5-HT (Fig. 5b–h). As previously mentioned, double Pax6/Pax7 labeling demonstrated distinct cell populations in p3, where Pax7 neurons were mainly located medial to the Pax6 cells (Fig. 4j–j00 ). Of note, the Pax7 cells located topologically more ventral in p3 extended into regions of the adjacent basal hypothalamus, in particular within the mammillary region, which contained the nucleus of the periventricular organ (labeled for 5HT, Fig. 5d) and abundant TH cells (Fig. 5i). Actually, the Pax7 cells in the basal p3 characteristically expressed Nkx2.1, whereas those located within the hypothalamus did not express this transcription factor, making these cells distinct (Fig. 5e, g and j). In striking contrast with the abundant Pax6 and Pax7 cell populations found in p3 (and also in p1), the second diencephalic prosomere, p2, was virtually devoid of labeling. Only the roof plate of p2 was intensely labeled for Pax7 (Figs. 1g and 3b), identified rostrally between the two habenular dorsal components, labeled for CB or ChAT (Fig. 5a). In addition, the pineal organ also showed Pax6 and Pax7 staining (Figs. 3d and 5d). Actually, the lack of labeling in p2 served to identify, in many cases, the boundaries between the three diencephalic prosomeres (Fig. 1i and j). A large mixed population of Pax6 and Pax7 cells was localized in the dorsal part of p1 (Figs. 1i–k, 2j, 3c, d, g–i, 4j–l and 5k–m). The roof plate of p1, which contains the subcommissural organ, was intensely labeled for both Pax6 and Pax7 (Figs. 1j and k, 2j, 3d, 4j and j0 and 5d). The alar derivatives of p1 that form the complex pretectal region, which in Xenopus contains distinct nuclei identified through development (Morona et al., 2011). Three main pretectal parts are distinguished that, from rostral to caudal, are named precommissural (PcP), juxtacommissural (JcP), and commissural (CoP) in relation to the posterior commissure. While the PcP was virtually devoid of Pax6 and Pax7 cells, abundant cells were labeled in the other two parts. In particular, only a few Pax7 cells were located rostrally in the PcP within the anterior pretectal nucleus (Figs. 3g and h and 4k and l). More abundant Pax7 cells were found throughout the intermediate JcP, where the labeled cells were specially observed in the ventral juxtacommissural

nucleus and the nucleus spiriformis lateralis (Fig. 3g and h). Of note, the latter nucleus also contained Pax6 cells (Fig. 4k). Finally, a large population of intermingled Pax6 and Pax7 cells were seen in the CoP, extending from the ventricular zone to the most superficial zones including the inner and outer nuclei of the subcommissural organ and the parvocellular nucleus of the posterior commissure in the dorsal part of CoP (Figs. 1i–k, 2j, 3c and d and 4j–j00 ). More ventrally, Pax7 cells were also located in the principal pretectal nucleus, also in CoP (Figs. 3g and 4l). The particular distribution of Pax6 and Pax7 cells in the pretectum was assessed in the double labeling sections for CB and TH, primarily, which specifically labeled pretectal nuclei. Thus, for example, the olivary pretectal nucleus was the only one labeled for TH with high degree of colocalization for Pax7 (Fig. 5l–n), whereas CB labeled the parvocellular pretectal nucleus and colocalized Pax6 in CoP (Fig. 4l and l0 ), and the magnocellular pretectal nucleus in JcP that lacked both Pax6 and Pax7 expression (Fig. 5k). Apart from the conspicuous Pax6 and Pax7 cell populations in the alar region of p1, the Pax6 cells extended into the rostral basal territory of p1, where they formed a band of cells that was caudally continued by the Pax6 cells located in the mesencephalic tegmentum (Figs. 1k and 4k). 3.2. Midbrain The localization of Pax6 cells in the caudal CoP domain and the lack of Pax6 expression in the dorsal midbrain (mesencephalon) highlighted the diencephalo-mesencephalic boundary (Figs. 2h and 4k). The mesencephalon is considered here subdivided into four longitudinal columns or bands, named dorsal and lateral in the alar region, and basal and medial in the basal region (Dı´az et al., 2000). Significantly, the most outstanding labeling in the mesencephalon was found in the optic tectum where Pax7 cells were abundant in all cell layers, including intensely labeled cells in the ventricular zone (Figs. 1k–m, 3h–k, 4k and 5m). The extent of this patent labeling abruptly ended caudally at the border between the optic tectum and the torus semicircularis, both structures located in the dorsal band of the mesencephalon (Figs. 1l, 3i–k and 4k). Double labeling experiments showed that the Pax7 cells in the optic tectum included subpopulations of GABA and CB containing neurons, whereas the NOS positive cells in the tectum were Pax7 negative (Fig. 5o–q). The lateral band of the mesencephalon corresponds to the ventral part of the alar region and contained CB and, particularly, NOS immunoreactive cells, but it characteristically lacked Pax6 and Px7 cells (Figs. 1l and 4m and p). In contrast, a conspicuous Pax6 cell population extended along the basal band of the mesencephalic tegmentum (Figs. 1l, 2k and l and 4m–o). These cells represented a caudal continuation of the basal Pax6 cell population in p1 (Fig. 4k). They formed a small group rostrally at the level of the rostral pole of the oculomotor nucleus (Figs. 2k and 4m and n), whereas caudally they were more widely distributed into cell bands in the anteroventral tegmental nucleus, with scattered cells invading the dorsal part of the medial band of the mesencephalon, above the caudal part of the oculomotor nucleus

hypothalamus are shown by the combined staining. (g–i) Distribution of the Pax6 cells in the prethalamus in relation to the indicated markers, showing colocalization and codistribution. (j–j00 ) Three rostro-caudal sections, with 100 mm of separation between each section, showing the relative distribution of Pax6 and Pax7 cells in p1 and p3. (k) Distribution of Pax6 and Pax7 cells in the caudal forebrain, midbrain and upper hindbrain. (l and l0 ) Abundant Pax6 cells in the commissural region of the pretectum where CBcontaining cells are also located. (m–q) Pax6 cells in the mesencephalic tegmentum in relation to different markers. (r) Pax6 cells in the mesencephalic tegmentum and Pax7 cells in r1, leaving r0 virtually devoid of labeling. (s) Distinct Pax6 and Pax7 labeling in the rostral rhombencephalon and cerebellum. (t and u) Abundant Pax6 cells in the cerebellar granule cell layer at rostral (t) and caudal (u) levels. (v and w) Localization of Pax6 cells in the rhombencephalon in relation to the motor neurons of the facial and abducens nuclei. (x) Colocalization of Pax6 and NOS in neurons of the nucleus reticularis inferior. (y) Pax6 cells at the obex region. (z and z0 ) Sagittal and transverse sections at the obex region showing the relative distribution of Pax6 and Pax7 cells. In the sagittal sections the orientation is indicated in each photomicrograph. The vertical bars in d0 and z indicate the levels of the sections shown in the photomicrographs indicated. For abbreviations, see list. Scale bars = 200 mm (a–c, d0 –f0 , j–k, m–s, u–w), 100 mm (d, g–i, l, l0 , t, x).

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Fig. 5. Photomicrographs of double-labeled transverse (a–l, o–w, y) and sagittal (m, n, x) sections through the brain. The detected molecules are indicated on each photomicrograph. (a) Pax7 labeling in the roof plate of p2 between the two dorsal habenular nuclei. (b–j) Distribution of Pax7 cells in the basal p3 in relation to different markers and the scattered cells entering the mammillary region of the hypothalamus (the inset in panel d is a higher magnification of the nucleus of the periventricular organ in the mammillary region). (k and l) Pax7 cells in the pretectum in relation to CB and TH cells. (m and n) Localization of the Pax7 cells in p3 and pretectum in relation to the TH immunoreactive cells (photomicrograph n is a higher magnification of the pretectum in m to illustrate double labeled cells). (o–q) Pax7 labeled cells in the optic tectum are

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(Figs. 2l and 4o). This band of Pax6 cells ended abruptly at the boundary with the isthmus (r0; Fig. 4k, q and r) and the double labeling experiments demonstrated that notable colocalization with CB was present in the internal rows of Pax6 cells of this group (Fig. 4p), whereas the staining for Nkx2.2 form a longitudinal band that marks the alar-basal boundary throughout the mesencephalon (Fig. 4p0 ). 3.3. Hindbrain We here considered the hindbrain formed by the rostral segment of the isthmus, or rhombomere 0 (r0) and the transverse rhombencephalic subdivisions of rhombomeres 1–7 (r1–r7) and the long r8, which is not clearly defined and probably represents more than one segment (Cambronero and Puelles, 2000). The cerebellum is considered in the rostral rhombencephalon as a derivative of the alar region of r1. We evaluated the distribution pattern of Pax6 and Pax7 cells under this paradigm because in anurans the segmentation can be inferred by examining the organization of the segmentally patterned motor nuclei (Marı´n et al., 1997; Straka et al., 2006). Therefore, double labeling for ChAT was commonly conducted to analyze the precise localization of the labeled structures for Pax6 and Pax7 (Fig. 4u–w). Pax6 and Pax7 were practically absent from segment r0, which, given the obliquity of the isthmomesencephalic boundary, is severely curved (Figs. 3i, 4k, r and 5x). Only some scattered Pax7 cells could be identified in the isthmic part of the posterodorsal tegmental nucleus and in the rostral part of the interpeduncular nucleus (Figs. 1m, 3l and n, 4r and 5r), which most likely correspond to a rostral extent of the very numerous populations found in r1. In particular, Pax7 cells were abundant in r1, which is a large rhombomere that extends from the caudal pole of the trochlear nucleus in r0 to the rostral pole of the trigeminal motor nucleus in r2. Due to the wedge shape of r0, the rostral pole of r1 seems to extend more rostrally at its ventral part than at the dorsal part (Figs. 3i, 4k and r and 5x). Pax7 cells were weakly labeled in the ventricular zone of the alar plate and numerous cells extended laterally and ventrally within r1 (Figs. 1m and 3l). The laterally located Pax7 cells in r1 were distinct from the cholinergic cells in the pedunculopontine and laterodorsal tegmental nuclei (Fig. 5r and s) or the noradrenergic cells of the locus coeruleus (Fig. 5u and v), which are characteristically located in r1. Interestingly, abundant Pax7 cells occupied ventromedial positions, mainly in the interpeduncular nucleus, although Pax7 cell labeling was not observed in the ventricular zone of the basal plate (Figs. 3m and n and 5t, u and w). The cells in the interpeduncular nucleus extended rostrally into r0 (marked by the presence of the trochlear nucleus), but the bulk of the population was found in the rostral part of r1, whereas in the caudal part of r1 the Pax7 cells located ventromedially were gradually more dispersed (Fig. 3n). Some Pax7 cells were codistributed with 5-HT cells of the superior raphe nucleus, although actual colocalization of both markers in the same neurons was not observed (Fig. 5t). To help in the analysis of the Pax7 cell population in r1, the double labeling with the transcription factor Otp was very useful. Thus, distinct Otp/Pax7 cells were seen intermingled in the interpeduncular nucleus and in the region of the rostral part of the central gray (Fig. 5w and x), whereas clearly distinct domains were labeled in the alar (Pax7) and basal (Otp) ventricular zone at this level (Fig. 5w). In addition, a more medial Pax6 cell population was

detected in r1, in particular in the central gray, where the cells were located more medial than those labeled for Pax7, and Pax6 labeling was found in the ventricular zone of the basal plate (Fig. 4s). As a derivative of the alar r1, the cerebellum showed Pax7 labeling virtually in the whole ventricular zone (Figs. 1n, 3m and 4k, r and s), whereas Pax6 cells were abundant in the most medial part of the cerebellar ventricular zone and in the granule cells of the cerebellar plate and auriculae (Figs. 1n, 2m and n and 4k and s). Labeling for CB, which specifically stains Purkinje cells (Morona and Gonza´lez, 2009), in combination with Pax6 demonstrated that these cells were not Pax6 positive (Fig. 4t). Caudally in the rhombencephalon, the double labeling for Pax6 or Pax7 with ChAT served to identify the correct location of the Pax cells in relation to the motor nuclei (Fig. 4u–w). Along the rhombencephalon, Pax7 labeling was found in the ventricular zone of the alar plate, being more intense in the ventral part of the alar plate than in the dorsal part (Figs. 1o and 3o–q). Separate Pax7 cells from the ventricular zone were scarce, mainly in the rostral area octavolateralis (Fig. 3o), at the level of the nucleus reticularis medius (Figs. 3p and 5y), and the nucleus of the solitary tract (Fig. 3q). In turn, Pax6 cells were more abundant than Pax7 cells, in particular in the region of the nucleus reticularis medius, the dorsal medullary nucleus of the area octavolateralis, and the nucleus reticularis inferior (Fig. 2o–q); and Pax6 labeling in the ventricular zone of the basal plate was seen throughout the rhombencephalon (Figs. 1n and o, 2o, q and 4s, v–x and z). Of note, although Pax6 cells were seen close to several motor neurons, in particular those of the abducens nuclei (Fig. 4w), no double Pax6/ChAT labeled cells were observed. Among the abundant Pax6 cells found in the rhombencephalic reticular formation, a large contingent of the NOScontaining cells located in the nucleus reticularis inferior were double labeled for Pax6 (Fig. 4x). 3.4. Spinal cord Close to the obex, once the central canal is formed, distinct Pax7 (dorsal) and Pax6 (ventral) labeling of the ventricular zone was observed (Figs. 1p, 2r, 3r and 4y, z and z0 ). In addition, Pax6 cells were labeled detached from the ventricular zone into the ventrolateral region of the somatomotor neurons (Fig. 2r), but double labeling for ChAT demonstrated that the Pax6 cells were not cholinergic neurons (Fig. 4y). Similarly, in the dorsal region a compact group of Pax7 cells migrated from the ventricular zone were intensely labeled at the obex region (Fig. 3r). Noteworthy, caudally in the spinal cord the Pax6 and Pax7 cell populations persisted in the ventral and dorsal parts of the cord, respectively. However, the labeling of the ventricular zone in the spinal cord disappeared, starting from the most rostral (cervical) segments (Fig. 3s). 4. Discussion The present study describes the distribution of Pax6 and Pax7 cells in the adult brain of the anuran amphibian X. laevis. An unexpected wide distribution of labeled cells is detected by immunohistochemistry with antibodies that are required for highresolution analysis of Pax-expressing cells. Actually, the distribution of the Pax6 and Pax7 proteins and their mRNA in the brain of developing Xenopus seems to be highly coincident when immunohistochemical and in situ hybridization techniques are per-

not NOS positive (o) but some contain GABA (p) or CB (q). (r–x) Distribution of Pax7 cells in the rostral rhombencephalon in relation to several markers. (y) Detail of the relative distribution of the Otp and Pax7 labeling in the ventricular zone and in the region above the nucleus reticularis medius. In the sagittal sections the orientation is indicated in each photomicrograph. The vertical bars in u, w, and x indicate the levels of the sections shown in the photomicrographs indicated. For abbreviations, see list. Scale bars = 100 mm (a, b, e, f, k, t, y), 200 mm (c, g–j, l, r, s, u, w, x), 50 mm (n–q).

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formed (Moreno et al., 2008a; Bandı´n et al., 2013). However, in the adult CNS the level of expression and the capacity for detecting Pax mRNA appears to decay and, therefore, only restricted distribution was previously observed by means of in situ hybridization, in particular for Pax6 (Moreno et al., 2008a). Our present results show that immunohistochemical techniques allow a detailed neuroanatomical resolution that reveals a much higher degree of Pax6 and Pax7 expression in the adult brain than previously reported. Thus, in studies of different fish species the suitability of the immunohistochemical techniques to reveal transcription factors was strengthened (Hitchcock et al., 1996; Wullimann and Rink, 2001; Gonza´lez and Northcutt, 2009; Ferreiro-Galve et al., 2012; Gonza´lez et al., 2014). A similar situation was observed in adult rodents when in situ hybridization (Stoykova and Gruss, 1994) and immunohistochemistry (Duan et al., 2013) techniques were used to study the adult distribution of Pax transcription factors. In our previous study of the adult brain of the urodele amphibian Pleurodeles waltl we observed by means of immunohistochemistry a extremely wide distribution of Pax proteins in neurons of almost all main brain regions (Joven et al., 2013a). This result was interpreted as a possible reflection of a rather simplified organization of the adult brain of urodeles, which might retain pedomorphic characteristics in the adult (Roth et al., 1993). However, when similar immunohistochemical techniques were applied to the developing brain of Xenopus levis (Domı´nguez et al., 2013, 2014; Bandı´n et al., 2013), it was observed that at the end of the metamorphosis a wide distribution of Pax6 and Pax7 cells still persisted in the brain, and these results prompted the present study in the adult brain. Numerous studies have localized postnatal expression of Pax6 and Pax7 in the brain of amniotes (primarily mammals and birds) in subsets of postmitotic cells in restricted regions, including the telencephalon, diencephalon, mesencephalon, cerebellum and pons/medulla (Walther and Gruss, 1991; Mansouri et al., 1994; Stoykova and Gruss, 1994; Nakatomi et al., 2002; Shin et al., 2003; Hack et al., 2005; Nacher et al., 2005; Stanescu et al., 2007; Thomas et al., 2007; Thompson et al., 2007, 2008). Most of these studies probably reported only partial results, obtained with the in situ hybridization technique, as inferred when comparing with the immunohistochemical procedures (Duan et al., 2013). Despite their well characterized roles in development (Osumi et al., 2008), functions of the transcription factors Pax6 and Pax7 in adult brain still remain elusive. In general, it was suggested that their function in mature subsets of neurons would contribute to maintain specific neuronal subtypes in the adult CNS (Walther and Gruss, 1991; Stoykova and Gruss, 1994). Furthermore, recent data demonstrated that they are required for the maintenance of progenitor cell phenotype (such as Pax6 in adult neurogenesis) or for maintenance of plasticity in mature neurons in response to environmental stimuli (Gerber et al., 2002; Thompson and Ziman, 2011). In our study, the Pax6 and Pax7 expression observed in the ventricular zone, and immediately adjacent to it, in several locations of the brain could be related to the fact that Xenopus possesses adult cell proliferation in ventricular zones throughout the brain (D’Amico et al., 2011, 2013). Actually, in general the brains of adult ectothermic vertebrates are characterized by a greater number of proliferation and neurogenic compartments than previously described in other vertebrates (Raucci et al., 2006; Almli and Wilczynski, 2007). Therefore, it is tempting to correlate adult cell proliferation to the Pax6 and Pax7 expression observed in the ventricular lining of many brain regions in juveniles and adult reptiles (Moreno et al., 2010), amphibians (Joven et al., 2013a; present results), and lungfishes (Gonza´lez and Northcutt, 2009). In the following sections the main features of the distribution of Pax expression in the brain of Xenopus will be discussed from

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rostral to caudal levels in relation to data available for other species. We will generally restrict the discussion to Pax distribution in adult brains, and where data are not available for adults we offer insights gained from studies where Pax expression has been reported only for certain developmental stages. The combined localization of Pax6 and Pax7 cells and the cells containing TH, ChAT, NOS, GABA, CB, Nkx2.1, Nkx2.2, and Otp has been a useful tool for identifying the precise signature of diverse cell groups not distinguishable on the basis of cytoarchitecture. The use of the neuromeric model of brain organization is extremely useful to frame our results and compare homologous regions of Pax expression across different vertebrate classes (Stoykova and Gruss, 1994; Stuart et al., 1994; Kawakami et al., 1997; Murakami et al., 2001; Derobert et al., 2002; Haubst et al., 2004; Pritz and Ruan, 2009; Duan et al., 2013). 4.1. Telencephalon The expression of Pax6 in the rostral portions of the forebrain of adult Xenopus is circumscribed to subsets of cells in the olfactory bulbs, septal region, basal ganglia and amygdaloid complex. The large olfactory bulbs of the adult possess a numerous population of Pax6 cells mainly located in the inner granule cell layer, but they are also distinct in periglomerular locations. Comparatively, in the adult olfactory bulb of rodents (Baltana´s et al., 2009), Pax6 is present in different types of interneurons and is abundantly expressed in periglomerular neurons (Dellovade et al., 1998). Actually, recent studies have demonstrated that Pax6 is essential for the formation of granule cells and periglomerular cells (Hack et al., 2005; Kohwi et al., 2005; Roybon et al., 2009). In line with our results in the adult amphibians, in which the dopaminergic cells of the olfactory bulb (TH positive) specifically express Pax6 (Joven et al., 2013a; present results), in the adult bulb of the rat all the TH reactive periglomerular cells were seen to contain Pax6 (Baltana´s et al., 2009), regulating survival of these cells by inhibiting programmed cell death in the mature bulb (Ninkovic et al., 2010). Only recently a mechanism involving Pax6 and co-factors, as Meis2, has been demonstrated to act in neurogenesis and dopaminergic periglomerular fate specification in the olfactory bulb (Agoston et al., 2014). In the telencephalic development Pax6 play crucial roles in the regionalization of the major divisions, mainly restricting cell movements across the pallio-subpallial boundary and regulating appropriate gene expression in pallial and subpallial territories (Puelles et al., 2000). Throughout development, Pax6 selective regional expression in the ventricular zone cells has identified it as a general pallial marker in all species studied (Walther and Gruss, 1991; Smith-Ferna´ndez et al., 1998; Puelles et al., 2000; Murakami et al., 2001; Wullimann and Rink, 2001; Derobert et al., 2002; Moreno et al., 2008a, 2010). However, no Pax6 expression persists in the adult pallium of rodents (Stoykova and Gruss, 1994) with the exception of the hippocampal dentate gyrus and subventricular zone/rostral migratory stream, regions in which neuronal precursors exist during adult life (Nacher et al., 2005). The only pallial Pax6 expression noted in adult Xenopus corresponds to the region of the pallio-subpallial boundary in the dorsal septum, medially, and in the ventral part of the ventral pallium, laterally. Identification of Pax6 expression in this boundary zone has been reported in most vertebrates studied (Puelles et al., 2000; Wullimann and Rink, 2002; Ferreiro-Galve et al., 2008; Moreno et al., 2008a; Georgala et al., 2011; Joven et al., 2013a,b), including humans (Kerwin et al., 2004; Lindsay et al., 2005). The ventral pallial region adjacent to the pallio-subpallial boundary that contains Pax6 cells in all vertebrates has been demonstrated to include the lateral amygdala in anuran amphibians (Brox et al., 2004; Moreno and Gonza´lez, 2004) and it will be discussed below together with other

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amygdaloid territories. Within the septal region, Pax6 expression was described in the dorsal septum during chicken and mouse development (Puelles et al., 2000) and in the medial and lateral nuclei (in addition to the diagonal band of Broca) after four weeks in mouse (Stoykova and Gruss, 1994; Duan et al., 2013). Noteworthy, by means of in situ hybridization only Pax6 expression in the dorsal septum was previously detected in adult Xenopus (Moreno et al., 2008a), although in the present study a larger population of septal cells along the medial (and not the lateral) septal region has been observed, in agreement with the situation found in the adult brain of urodeles (Joven et al., 2013a). Most Pax6 cells in he subpallium of adult Xenopus appear in the striatal portion (including the nucleus accumbens). During development, Pax6 cells were not detected in the ventricular zone of the chicken and mouse striatum, but migrated cells in the striatal mantle were abundant and persisted postnatally at the lateral edge of the ventral striatum (Puelles et al., 2000). However, the primordium of the nucleus accumbens at the rostromedial wall of the telencephalon showed Pax6 expression in the proliferative zone and in migrated cells into the subpial mantle (Puelles et al., 2000). In Xenopus, both during development and in the adult (Moreno et al., 2008a; present results), the double labeling for Pax6/TH identified the Pax6 cells just outside and adjacent to the TH neuropil that characterizes the nucleus accumbens (Gonza´lez et al., 1993; Marı´n et al., 1998). Regarding the pallidal component of the basal ganglia, in the developing chicken Nkx2.1 positive postmitotic cells (of pallidal origin) in the subpallium partly intermingle with Pax6 cells, but Pax6 is virtually absent in the primordium of the pallidum (Puelles et al., 2000). In adult mice, Pax6 cells were identified in the ventral pallidum and in the entopeduncular nucleus (Duan et al., 2013). In contrast, both in anuran and urodele amphibians the double labeling for Pax6 and Nkx2.1 (marker of the pallidal derivatives; Gonza´lez et al., 2002; Moreno and Gonza´lez, 2007; Moreno et al., 2012) reveal the lack of Pax6 expression in pallidal territories (Moreno et al., 2008a; Joven et al., 2013a,b; present results). In the amygdaloid complex of Xenopus three main components are distinguished, lateral, central, and medial (Moreno and Gonza´lez, 2006). The Pax6 cells detected in the lateral amygdala would correspond to ventral pallial neurons, located just above the striatal region, as revealed with the Isl1 labeling (Moreno et al., 2008a,b). The central amygdala, which represents a striatal component, and the medial amygdala (or vomeronasal amygdala) also show abundant Pax6 cells in the adult Xenopus. This result contrast with the situation found in amniotes where only small numbers of Pax6 cells were reported, mainly related to the basolateral complex (Duan et al., 2013) and the medial amygdala (Puelles et al., 2000; Bupesh et al., 2011; Abella´n et al., 2013). In the telencephalon, Pax7 acts early in development to establish dorsal polarity (Jostes et al., 1990) but is soon restricted to the roof plate of the telencephalon (Thompson et al., 2004). In the chicken, Pax7 was expressed at late stages of development only in the anterior roof plate covering the midline of the telencephalon (Matsunaga et al., 2001). Of note, both in anuran and urodele amphibians during development and in the adult a strong Pax7 expression in the telencephalon is present only in the paraphysis, a membranous formation of the telencephalic roof plate that might correspond to the expression noted in mammals and birds (Bandı´n et al., 2013; Joven et al., 2013a,b; present results). Similar Pax7 expression in the paraphysis has been reported only during development in the chick (Nomura et al., 1998). 4.2. Diencephalon Caudally, in the Xenopus forebrain, Pax6 cells characteristically occupy the dorsal part of the diencephalic p3 segment, i.e. the

prethalamic eminence and prethalamus. In mammals, this represents one of the major Pax6 expression territories in the forebrain during development (Stoykova and Gruss, 1994; Puelles et al., 2000) that is gradually reduced, and in the adult is limited to the zona incerta (Duan et al., 2013). Similar Pax6 expression in the alar derivatives of p3 has been described for all vertebrates studied (Puelles et al., 2000; Murakami et al., 2001; Wullimann and Rink, 2001; Derobert et al., 2002; Ferreiro-Galve et al., 2008; Moreno et al., 2008a; Joven et al., 2013a,b). Interestingly, the Pax6 cells in the zona incerta develop into dopaminergic neurons (Vitalis et al., 2000; Mastick and Andrews, 2001). In line with this might be the observation in teleosts (Wullimann and Rink, 2001), urodeles (Joven et al., 2013a), and anurans (present results) of colocalization of TH and Pax6 in numerous cells of the alar p3 in the adult. In addition, the ventral extent of the Pax6 cells in p3 of Xenopus reaches the basal plate, as it was also reported for the zebrafish (Wullimann and Rink, 2001). The dorsalmost part of p3 is currently interpreted as the prethalamic eminence (after Puelles and Rubenstein, 2003). The observed Pax6 cells in the prethalamic eminence of Xenopus concur with descriptions through development of the mouse and chicken diencephalon (Puelles et al., 2000). Furthermore, it has been suggested that a contingent of Pax6 cells observed in the medial amygdala is produced in the prethalamic eminence (Bupesh et al., 2011; Abella´n et al., 2013). Given the proximity of the medial amygdala and the prethalamic eminence in Xenopus, the same situation might exist, although cell-tracking experiments during development are needed to corroborate this migration. The Pax6 cells of the prethalamic eminence localized in relation to the stria medullaris, a tract that includes olfactory axons toward the contralateral medial amygdala (Moreno and Gonza´lez, 2003), in non-mammalian vertebrates might constitute a permissive pathway for growing olfactory axons in their way to the habenular commissure that is lost in mammals (Abella´n et al., 2013). In adult Xenopus, the basal p3 is particularly rich in Pax7 cells but complementary Pax6/Pax7 expression pattern exist mainly in the dorsal part of the basal plate. A Pax7 cell group is present in the p3 basal plate and defines the alar/basal portions of this segment in all vertebrates studied so far (Joven et al., 2013a,b). Supporting the basal nature of this subpopulation, we found double Pax7+/ Nkx2.1+ cells, in contrast to the neighboring hypothalamus, which lacks Pax7 expression (Domı´nguez et al., 2014). However, a small number of Pax7 cells can be identified in the adjacent mammillary region of the basal hypothalamus that most likely are originated in the basal part of p3, as previously proposed for anuran amphibians and chelonians (Moreno et al., 2012; Domı´nguez et al., 2014). Pax6 is expressed in the embryonic thalamus (Grindley et al., 1997; Kawano et al., 1999; Pratt et al., 2000) but it is lost during development, with the exception of the pineal organ, an evagination of the epithalamus in the dorsal part of p2 (Walther and Gruss, 1991; Estivill-Torrus et al., 2001). In addition, Pax7 postmitotic cells were observed in the pineal organ of the chicken at late developmental stages (Kawakami et al., 1997). These observations are in line with the results obtained in Xenopus (Bandı´n et al., 2013; present results). The Pax6 immunoreactive pinealocytes maintained in the adult have been tentatively considered to represent a reminiscent population of pinealocyte precursors (Rath et al., 2013). Strikingly, Pax6 and Pax7 are consistently located in the pretectal region (alar part of p1) of all vertebrates studied (Ferran et al., 2007, 2008; Ferreiro-Galve et al., 2008; Mercha´n et al., 2011; Morona et al., 2011). In mammals, their expression is severely restricted through development (Stoykova and Gruss, 1994; Duan et al., 2013), in contrast to the situation found in amphibians (Bandı´n et al., 2013; Joven et al., 2013a,b; present results). The distinct distribution of Pax6 and Pax7 cells, among others, in the

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pretectum has served to identify the main pretectal subdomains, which are very conserved across vertebrates (Ferran et al., 2007, 2008, 2009; Mercha´n et al., 2011; Morona et al., 2011). In particular, the sharp limit between the caudal extension of prosencephalic Pax6 cells and the Pax6 negative mesencephalic alar plate (optic tectum) defines the diencephalo-mesencephalic boundary (Mastick et al., 1997; Warren and Price, 1997; Kerwin et al., 2004), and Pax6 mutant mice lack this boundary and show fate change of p1 region to the mesencephalon (Mastick et al., 1997). The observation in Xenopus of Pax6 expression in the basal part of p1 in a band continuous with the mesencephalic tegmentum agrees with observations in the chicken diencephalon (Sanders et al., 2002; Schubert and Lumsden, 2005; Ferran et al., 2007) and, in particular, in the developing Alligator (Pritz and Ruan, 2009). 4.3. Hypophysis A strikingly numerous cell population is intensely Pax7 immunoreactive throughout the intermediate lobe of the hypophysis in adult Xenopus. A similar observation was reported for adult urodeles and through development in anurans and urodeles (Bandı´n et al., 2013; Joven et al., 2013a,b), consistent with a previous report that Pax7 is expressed in the precursors of the pituitary gland of the zebrafish (Guner et al., 2008). These cells in Xenopus are melanotrophs that play an important role in the activation of skin melanophores to darken when the animal is placed on a dark background and is controlled from the hypothalamic suprachiasmatic nucleus (Tuinhof et al., 1994a,b; Roubos et al., 2010). In a recent study of the adult mouse pituitary gland it was shown that the majority of the cells in the intermediate lobe expressed Pax7 (78%) and about 60% of the ACTH positive cells in the intermediate lobe also expressed Pax7 (Hosoyama et al., 2010). The same study demonstrated that a small subpopulation of these Pax7 cells are progenitor cells with characteristics between pituitary stem cells and melanotrophs, and this is evolutionary conserved in primates and humans. Our results suggest that the same situation might exist in the intermediate lobe of amphibians. Pax6 cells in Xenopus are restricted to the anterior lobe of the hypophysis. Both in zebrafish and mice Pax6 is initially expressed in Rathke’s pouch and continues to be expressed in the developing anterior lobe (Walther and Gruss, 1991; Puschel et al., 1992; Dasen and Rosenfeld, 2001). A function for Pax6 in the hypophysis development was inferred in the pituitaries of Pax6 mutant mice in which there is an expansion of the ventral cell types, predominantly thymotropes, with a reciprocal loss of the more dorsal somatotrope lineage (Bentley et al., 1999; Kioussi et al., 1999). Therefore, Pax6 would be required for refining the dorsoventral boundaries of the anterior lobe, reminiscent of its role in the neural tube (Ericson et al., 1997). 4.4. Mesencephalon One of the main features of the Pax7 expression in the midbrain of adult Xenopus is the wide distribution of Pax7 cells in almost all cell layers of the optic tectum, with intense expression throughout the ventricular zone, as in developmental stages (Bandı´n et al., 2013). Several experimental data indicate that Pax7 is one of several genes crucial for superior colliculus (mammalian homologous of the optic tectum) boundary formation (Kawakami et al., 1997; Matsunaga et al., 2001; Thompson et al., 2004). During development, Pax7 is an important determinant of polarity within the mouse superior colliculus and a role in retinotopic mapping has been suggested (Thompson et al., 2004, 2007). In addition, important number of Pax7 cells persists in the adult mouse

37

superior colliculus (Stoykova and Gruss, 1994; Thompson et al., 2007, 2008; Duan et al., 2013). In adult chicken, the optic tectum is the brain region showing the most remarkable Pax7 immunoreactivity (Shin et al., 2003). Although the majority of the cells of the early chick tectum express Pax7 (Thomas et al., 2006), they are reduced through development and into adulthood to superficial tectal layers, with much fewer cells in deep layers including the periventricular layer (Shin et al., 2003; Thompson et al., 2004). The comparatively much larger Pax7 expression present in the optic tectum of adult Xenopus, particularly in the ventricular zone, seems to be a shared feature of amphibians (Joven et al., 2013a). In the mesencephalic tegmentum of the adult Xenopus, Pax6 cells are located in the basal band. These cells are clearly distinct from the dopaminergic and cholinergic tegmental cells, as demonstrated by double immunohistochemistry for Pax6/TH and Pax6/ChAT. In adult mice, Pax6 cells were reported in the dorsolateral part of the substantia nigra reticularis (Duan et al., 2013). Comparable Pax6 cell populations have been described in teleosts (Wullimann and Rink, 2001) and urodele amphibians (Joven et al., 2013a). In the reptile Alligator mississipiensis, Pax6 cells in the basal plate of the midbrain were constantly observed during development and they are continuous with their counterparts in the basal p1, but the reactivity decreases as development proceeds (Pritz and Ruan, 2009). However, in juvenile turtle and chicken a similar band of intensely Pax6 immunoreactive cells exist in the midbrain tegmentum, as in adult amphibians (unpublished observations; Moreno et al., 2010). 4.5. Cerebellum The present results reveal intense Pax6 immunoreactivity in the granule cells of the adult cerebellum of Xenopus, whereas practically only the ventricular zone is Pax7 positive. In line with these observations, Pax6 hybridization probes labeled intensely the granule cells in the granular layer of the adult mouse cerebellar cortex (Duan et al., 2013) and no signals were detected in the case of the Pax7 mRNA in rat (Stoykova and Gruss, 1994). In contrast, Pax7 immunoreactivity has been demonstrated in the adult chicken cerebellum only in the Purkinje cell layer, but double labeling techniques demonstrated that the Pax7 immunoreactive cells corresponded to Bergmann glia (Shin et al., 2003). The implication of Pax6 in the regulation of migration and patterning of progenitor cells in the rhombic lip to form the cerebellum seems a conserved feature of vertebrates (Engelkamp et al., 1999; Rodrı´guez-Moldes et al., 2008; Wullimann et al., 2011; Martı´nez et al., 2013). 4.6. Rhombencephalon The numerous cell populations labeled in the rostral rhombencephalon for Pax6 and, primarily, Pax7 in adult Xenopus largely resemble the observations in the adult mammalian pons and medulla, suggesting important conserved roles of these genes in the formation and maintenance of this part of the brain (Stoykova and Gruss, 1994; Duan et al., 2013). The observation of Pax7 labeling in the ventricular zone of the alar plate and abundant disperse cells in the basal plate mantle, particularly in the interpeduncular nucleus, is extremely coincident with the observations in mammals and birds (Lorente-Ca´novas et al., 2012). Taken together, the data of Pax7 expression during development (Bandı´n et al., 2013) and in the adult strongly support that important tangential migration from alar to basal plate locations occur in amphibians as in amniotes (Aroca et al., 2006). Moreover, the combined immunohistochemistry conducted in our study not only supports the origin and final location of the Pax7 cells in the interpeduncular nucleus like in amniotes, but also

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those of the codistributed/colocalized cells expressing other markers, in particular Otp (Lorente-Ca´novas et al., 2012). In Xenopus, Pax7 cells generally characterize the alar ventricular zone throughout the hindbrain in the adult and during development, as previously described in the chicken (Jostes et al., 1990; Ju et al., 2004; Ferran et al., 2007). In addition, Pax6 expression is present in the ventricular zone of the basal plate and in migrated cells of the mantle along the hindbrain, with special relevance in the central gray at rostral levels. Pax6 is currently considered a crucial factor in the segmental organization of the early hindbrain with special significance in regional organization (Kayam et al., 2013). Like in Xenopus, after early development the rhombencephalon of the mouse and chicken show Pax6 cells distributed in a longitudinal ventral stripe along all rhombomenres (Kayam et al., 2013). The pattern of Pax6 expression in the developing hindbrain is shared by all vertebrates studied, including agnathans, chondrichthyans, teleosts, amphibians, birds, and mammals (Murakami et al., 2001; Derobert et al., 2002; Qiu et al., 2009; Rodrı´guez-Moldes et al., 2011; Takahashi and Osumi, 2011; Joven et al., 2013a,b; Kayam et al., 2013). Furthermore, Pax6 has been proposed to play a role in specification of subtypes of motor neurons in the mammalian hindbrain. In particular, Pax6 influence abducent and hypoglossal motoneuron formation, since lack of Pax6 in the rat hindbrain results in the absence of these pools of motoneurons (Osumi et al., 1997). In line with this observation, Pax6 cells have been observed in close relation to abducent motoneurons in Xenopus (present results), an aglossid species that lacks hypoglossal motoneurons and nerve. 4.7. Spinal cord In adult Xenopus, the populations of Pax7 (dorsal) and Pax6 (ventral) cells located in the mantle zone are observed throughout the spinal cord. However, the expression in the ventricular zone observed during embryonic and larval development (Bandı´n et al., 2013) is no longer detectable in the adult. During development, Pax expressing cells in the ventricular zone has been related to the specification of the different progenitor cell identities in similar locations of the spinal cord of all vertebrates studied (Ericson et al., 1997; Diez del Corral et al., 2003; Maczkowiak et al., 2010; Karus et al., 2011; Kuscha et al., 2012). In contrast to mammals, amphibians, such as adult urodeles and anuran larvae (for example, Xenopus) can regenerate their spinal cord after injury (Slack et al., 2008; Tanaka and Ferretti, 2009). The Pax expression in the developing and adult spinal cord in the axolot (urodele amphibian, Ambystoma mexicanum) has been related to the capacity of spinal regeneration (Schnapp and Tanaka, 2005; McHedlishvili et al., 2007, 2012). Interestingly, after spinal cord transection X. laevis can reestablish nerve tracts and achieve functional recovery but this ability is restricted to the larval stages and is lost at the end of metamorphosis (Forehand and Farel, 1982; Beattie et al., 1990; Gibbs et al., 2011), when the Pax6 and Pax7 expression in the ventricular zone of the spinal cord is lost. Acknowledgement Supported by the Spanish MEC, grant BFU2012-31687. References Abella´n, A., Desfilis, E., Medina, L., 2013. The olfactory amygdala in amniotes: an evo–devo approach. Anat. Rec. 296, 1317–1332. Agoston, Z., Heine, P., Brill, M.S., Grebbin, B.M., Hau, A.C., Kallenborn-Gerhardt, W., Schramm, J., Go¨tz, M., Schulte, D., 2014. Meis2 is a Pax6 co-factor in neurogenesis and dopaminergic periglomerular fate specification in the adult olfactory bulb. Development 141, 28–38.

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