Morona R et al. (2020), Amphibian thalamic nuclear organization during ...

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J Comp Neurol 2020 Oct 01;52814:2361-2403. doi: 10.1002/cne.24899.

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Amphibian thalamic nuclear organization during larval development and in the adult frog Xenopus laevis: Genoarchitecture and hodological analysis.

Morona R , Bandín S , López JM , Moreno N , González A .

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The early patterning of the thalamus during embryonic development defines rostral and caudal progenitor domains, which are conserved from fishes to mammals. However, the subsequent developmental mechanisms that lead to the adult thalamic configuration have only been investigated for mammals and other amniotes. In this study, we have analyzed in the anuran amphibian Xenopus laevis (an anamniote vertebrate), through larval and postmetamorphic development, the progressive regional expression of specific markers for the rostral (GABA, GAD67, Lhx1, and Nkx2.2) and caudal (Gbx2, VGlut2, Lhx2, Lhx9, and Sox2) domains. In addition, the regional distributions at different developmental stages of other markers such as calcium binding proteins and neuropeptides, helped the identification of thalamic nuclei. It was observed that the two embryonic domains were progressively specified and compartmentalized during premetamorphosis, and cell subpopulations characterized by particular gene expression combinations were located in periventricular, intermediate and superficial strata. During prometamorphosis, three dorsoventral tiers formed from the caudal domain and most pronuclei were defined, which were modified into the definitive nuclear configuration through the metamorphic climax. Mixed cell populations originated from the rostral and caudal domains constitute most of the final nuclei and allowed us to propose additional subdivisions in the adult thalamus, whose main afferent and efferent connections were assessed by tracing techniques under in vitro conditions. This study corroborates shared features of early gene expression patterns in the thalamus between Xenopus and mouse, however, the dynamic changes in gene expression observed at later stages in the amphibian support mechanisms different from those of mammals.

???displayArticle.pubmedLink??? 32162311
???displayArticle.link??? J Comp Neurol
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Species referenced: Xenopus laevis
Genes referenced: chat cth gad1.1 gbx2.2 igl lhx1 lhx2 lhx9 neurog2 nkx2-5 npy pax7 psmd6 serpine2 shh slc17a6 sox2 tcf4 tcf7l2
GO keywords: larval development [+]
???displayArticle.antibodies??? Calb2 Ab4 Calbindin Ab6 Chat Ab1 GABA Ab2 Lhx2 Ab2 Nkx2-2 Ab1 Pax7 Ab4 Sox2 Ab1

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	Figure 1 Western blot of Xenopus brain extract stained with the mouse anti-Lhx2 (a) and rabbit anti Sox2 antibodies (b). A single band above 46 and 43 KDa, respectively are observed, which are compared with the band stained for rat brain extract in each case. The expected molecular weight is indicated for each transcription factor or protein and the molecular weight standard is represented on the right of each photograph
	Figure 2 The scheme in (a) represents a sagittal view of the diencephalon of Xenopus that illustrates the thalamic nuclei (shaded in gray) considered in this study. The main nuclei of the adjacent pretectum (in p1) and prethalamus (in p3) are indicated, as well as the diencephalic prosomeric boundaries and the alar/basal boundary. Note that the coordinate system is sharply rotated because the longitudinal axis of the brain bends in the diencephalon and, therefore, classically interpreted ventral regions in the diencephalon and hypothalamus are actually rostral regions, whereas caudal regions are dorsal (according to the current prosomeric model). For clarity, the nuclei located laterally in the thalamus (La, LPv, and GdN) are not drawn. The photomicrographs (b–i) are a rostrocaudal series of Nissl-stained transverse sections through an adult Xenopus brain illustrating the general cytoarchitecture of the thalamic region, which is enclosed by dashed lines. The levels of the sections are indicated in (a). The abbreviations refer to the nomenclature classically used for the cell groups in the thalamus of anurans. For abbreviations, see list. Scale bars = 200 μm
	Figure 3 Photomicrographs of Nissl-stained transverse sections through the thalamus of Xenopus larvae at premetamorphic (a,d–g), prometamorphic (b,h–k), and metamorphic (c,l-o) stages. The approximate levels of the sections are indicated on the respective schemes of the first line (a–c). The photomicrographs illustrate the progressive compartmentalization of the main thalamic domains during development. Scale bars = 100 μm (d–g); 200 μm h–o)
	Figure 4 Set of photomicrographs of sagittal (a,c,e,h,j,n,q) and transverse (b,d,f,g,i,k–m,o) Xenopus brain sections illustrating the main markers used to define the rTh and cTh domains as well as the rostral and caudal boundaries of the thalamus during premetamorphosis. In each panel, the larval stage is indicated in the upper right corner. ISH for Shh, Tcf4, Gbx2, Ngn2, GAD67, NPY, VGlut2VGlut2, and Lhx2 (in blue or orange for bright field or red in the case of fluorescence images) and immunohistochemistry (IHC) for Nkx2.2, Sox2, Lhx2, CB, or CR (in brown for bright field or green/red for fluorescence images) are shown. The markers and the color code are indicated in the upper left corner. In panels (e) and (h) the levels of sections shown in other panels are indicated by bars. (a) Sagittal section showing by double ISH the expression of Shh and Tcf4 that delimitates the rostral thalamo-prethalamic boundary. (b) Double ISH showing the expression of Gbx2 in combination with Shh. (c) Sagittal section showing by double ISH the expression of Shh in combination with Ngn2. (d) Expression of Ngn2 (ISH) in combination with Nkx2.2 (IHC) showing the rTh and cTh subdivisions and the compartmentalization of cTh revealed by Ngn2 ISH in the cTh1. (e–g) Sections illustrating the expression of Gbx2 (ISH) in combination with Nkx2.2 (IHC) (the levels of the transverse sections shown in f and g are indicated in the sagittal section in e). (h,i) Sagittal (h) and transverse (i) sections (the level of section i is indicated in h) showing the expression of GAD67 (ISH) in combination with Nkx2.2 (IHC). (j) NPY expression (ISH) in the rTh at premetamorphic stages shown in a sagittal section in combination with PAX7 (IHC). (k) Combination of NPY (ISH) and Nkx2.2 (IHC) in a transverse section showing double labeled cells in the rTh and only a few in the cTh. (l,m) Transverse sections at rostral (l) and caudal (m) thalamic levels showing the expression of VGlut2VGlut2 (ISH) in cTh2 with Nkx2.2 (IHC) in the rTh. (n,o) Sagittal (n) and transverse (o) sections showing the Nkx2.2 and Sox2 immunoreactive cells in the rTh and cTh, respectively. VGlut2 (l) cells in cTh domain. (p) Transverse section showing Lhx2 and Nkx2.2 in the cTh and rTh, respectively. (q,r) Sagittal (q) and transverse (r) sections showing Lhx2 and Sox2 immunoreactive cells in cTh with a segregated pattern dorsally. (s) Combined IHC for CB and CR in a transverse section. For abbreviations, see list. A magenta-green version of this figure is provided as Figure S1. Scale bars = 100 μm (a–d); 50 μm (e–s) [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 5 Set of photomicrographs of sagittal (a–c,h,k,m,q,u–w) and transverse (d–g,i,j,l,n–p,r–t) Xenopus brain sections illustrating the main markers for the rTh domain and its derivatives, at different larval stages. In each panel, the stage is indicated in the upper right corner. The markers and the color code are indicated in the upper left corner. (a–g) Sagittal (a–c) and transverse (d–g) sections at premetamorphic (a,d,e), prometamorphic (b,f), and metamorphic (c,g) larval stages stained for Nkx2.2 and GABA. (a,d,e). Colocalization (arrowheads) in the rostral rim of rTh next to the Zli and single Nhx2.2 cells caudally. (b,f) Nkx2.2 expression in the Ar and Ac at prometamorphic stages. The Ac does not contain double GABA/Nkx2.2 cells, which are intensely detected in the rostroventral rim. (c,g) Nkx2.2 and GABA expressions in the thalamic region at the metamorphic climax. Double GABA/Nkx2.2 cells occupy the Ar2 and GdN laterally. Note Nkx2.2 positive cells also in the La nucleus of the superficial stratum. (h–j) Distribution of Nkx2.2 cells in comparison with CB cells at premetamorphic stages. CB in the periventricular stratum of the rostral part of rTh and in the superficial stratum of the cTh. (k,l) Distribution of Nkx2.2 cells in comparison with CR cells at premetamorphic stages. (m–p) Sagittal (m), and transverse (n–p) sections though the thalamic region showing combined CB and Nkx2.2 staining at prometamorphic stages. CB marks intensely the Ar and is absent in La and GdN. Note a subpopulation of Nkx2.2/CB double labeled cells in the IGL (p1, arrowhead). (q–t) Sagittal (q) and transverse (r–t) sections though the thalamic region showing combined CR and Nkx2.2 staining at prometamorphic stages. CR marks intensely the Ar, La and GdN as well as most caudal nuclear derivatives, but do not colocalize with Nkx2.2. (u) At the metamorphic climax, CB cells are intensely labeled in the Ar1 and codistributed with Nkx2.2 expressing cells in the Ar2. (v) CR labeled intensely the Hb, Ar1, Ac1, and Ar2 leaving a gap in the SH, with scarce cells in DC. (w) Pseudo color generated image showing the subdivisions observed by the overlapping of adjacent sections labeled for Nkx2.2/GABA and CB/CR. Note the extensive GABA/Nkx2.2 domain in the IGL, the absence of Nkx2.2 or GABA in the Ar1 and Ac1, the exclusive staining for CB in the Ar and the displaced Nkx2.2 cell population in the C. For abbreviations, see list. A magenta-green version of this figure is provided as Figure S2. Scale bars = 100 μm (a–w) [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 6 Set of photomicrographs of sagittal (a,e,h,i,l,p) and transverse (b–d, f,g,j, k,m-o,q-s) Xenopus brain sections illustrating some additional markers for the rTh domain and its derivatives, at different larval stages. In each panel, the stage is indicated in the upper right corner. The markers and the color code are indicated in the upper left corner. (a-d) Sagittal (a) and transverse (b–d) sections at prometamorphic stages stained for CB and GABA. CB and GABA are codistributed in Ar without colocalization. Single GABAergic cells are located in the GdN. (e–g) Sagittal (e) and transverse (f,g) sections at metamorphic climax stained for CB and GABA. Note the intensive CB staining in cells of the Ar and the absence GABA in the Ar1 and Ac1. (h-k) Sagittal (h,i) and transverse (j,k) sections at prometamorphic larval stages stained for CR and GABA. Both markers are codistributed in Ar and GdN without colocalization. Double-labeled cells appear transiently in the IGL, rostral part of C and LPv2. Single GABAergic cells are located medially to the IGL. (l–o) Sagittal (l), and transverse (m–o) sections though the thalamic region showing combined CR and GABA staining at the metamorphic climax. CR and GABA are extensively codistributed in the Ar2 and Ac2 and also in the rostral part of C and LPv2, without colocalization. In contrast to previous stages, cells labeled for CR do not appear in the GdN, IGL, and Z. (p–s) Sagittal (p), and transverse (q–s) sections though the thalamic region showing combined CR and CB staining at prometamorphic stages. Double labeling is abundant in the Ar and in some cells of the LPv. For abbreviations, see list. A magenta-green version of this figure is provided as Figure S3. Scale bars = 100 μm (a–s) [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 7 Set of photomicrographs of sagittal (a,c,k,o) and transverse (b,d-j,l-n,p-s) Xenopus brain sections illustrating the combination of markers for the rTh domain (Nkx2.2 and CB) with markers for the cTh domain (Sox2) and its derivatives, at different larval stages. In each panel, the stage is indicated in the upper right corner. The markers and the color code are indicated in the upper left corner. (a,b) Combined Nkx2.2 and Sox2 staining at premetamorphic stages showing the distribution of Nkx2.2 in the rTh and Sox2 in the cTh. (c–f) Sagittal (c) and transverse (d–f) sections of the thalamic region at prometamorphic stages showing the intermingled populations of Nkx2.2 and Sox2 cells in the Ar, Ac, and the rostral rim of the C. Nkx2.2 cells seem to origin in the rTh ventricle (asterisk in d,e,f) and occupy the Ar, Ac, GdN, and La in the superficial strata. Sox2 cells occupy the periventricular stratum in the Ac and are abundant in the Ar. (g–j) Rostrocaudal series of transverse sections through the thalamic region at metamorphic climax stages showing the intense Sox2 expression in the Ar, Ac, IGL, C, and LPv, with only weak expression in the La, GdN, and Z. (k–n) Sagittal (k) and transverse (l–n) sections showing the combination of CB and Sox2 staining at prometamorphic stages. Double labeled cells mark the extension of the Ar. (o) Distinct NPY expression in the IGL and colocalization with Nkx2.2 at prometamorphic stages, illustrated in the higher magnification in (o1). (p–r) Rostrocaudal series of transverse sections through the thalamic region at metamorphic climax stages showing the colocalization of CB and Sox2 in the Ar and single Sox2 positive cells in the Ac, IGL, C, and LPv; and weak expression in the La, GdN, and Z. (s) NPY expression in the IGL at metamorphic climax in comparison with Gbx2. Extensive codistribution of these markers is observed in the IGL. For abbreviations, see list. A magenta-green version of this figure is provided as Figure S4. Scale bars = 25 (o1); 50 μm (o); 100 μm (p–s) [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 8 Set of photomicrographs of sagittal (a-c,i,k,m) and transverse (d-h, j,l) Xenopus brain sections illustrating the combination of markers for the rTh domain (GABA and CB) with markers for the cTh domain (Lhx2) and its derivatives at different larval stages. In each panel, the stage is indicated in the upper right corner. The markers and the color code are indicated in the upper left corner. (a–h) Sagittal (a–c) and transverse (d–h) sections at premetamorphic (a,d,e), prometamorphic (b,e,f), and metamorphic (c,g,h) larval stages, stained for Lhx2 and GABA. (a,d) GABA expression in the Ar and codistribution with Lhx2 periventricularly in the Ac are shown at premetamorphic stages. No colocalization is observed. (b,e,f) Extensive codistribution of Lhx2 and GABA is shown in the caudal and ventral part of the Ac at prometamorphic stages. (e) GABA expression extends in the superficial stratum. (f) Lhx2 expression occupies the lateral part of the IGL. (c,g,h) Distribution of Lhx2 in comparison with GABA at metamorphic climax. The Lhx2 positive domain is reduced and codistribution of Lhx2 and GABA remains in a small part of the Ac ventrally, and in the C. (g) both markers are codistributed in the Ac, IGL and C with scarce expression in the La. (h) some Lhx2 cells remain laterally in the IGL. (i,j) Sagittal (i) and transverse (j) sections at premetamorphic stages showing CB labeling in cells in the Ar and LPv, but not in the ac. Lhx2 is expressed in the Hb and DC as well as in the Ac and C, periventricularly. No colocalization is observed. (k,l) Sagittal (k) and transverse (l) sections at prometamorphic stages illustrating the downregulation of Lhx2 rostrally in the Ar and Hb. CB labeled cells are abundant in the Ar and scarce in the LPv. (m) Sagittal section at metamorphic climax showing the virtual absence of Lhx2 expression in the Ar and Ac and its presence in the C and Cv. For abbreviations, see list. A magenta-green version of this figure is provided as Figure S5. Scale bars = 100 μm (a–m) [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 9 Set of photomicrographs of sagittal (a–c,h–j) and transverse (d–g,k) Xenopus brain sections showing the expression of cTh markers (Gbx2 and Lhx9) in blue by ISH and the rTh marker Nkx2.2 in brown by IHC. In each panel, the stage is indicated in the upper right corner. The markers and the color code are indicated in the upper left corner. (a,d) Sagittal (a) and transverse (d) sections through the thalamic region at early prometamorphic stages showing the distribution of Gbx2 in the cTh, excluding the habenula, combined with Nkx2.2 in the rTh. Note (in d) the main periventricular distribution of Gbx2 in cTh but also in the intermediate strata. (b,e) Sagittal (b) and transverse sections at late prometamorphic stages showing different levels of expression of Gbx2 in the cTh along the three dorsoventral tiers (dt, it and vt). Observe also the intense Gbx2 expression dorsally in the Ar and Ac, within the Nkx2.2 positive region, as seen in the transverse section (e). (c,f,g) Sagittal (c) and transverse (f,g) sections at metamorphic climax stages showing distinct expression of Gbx2 in the LPv, C, Ac, and Ar, in contrast to its absence in the La and GdN. The higher magnification shown in f1 illustrates the segregation of Gbx2 and Nkx2.2 cells in the Ar. (h) Sagittal section at premetamorphic stages showing the exclusive pattern of Lhx9 expression in the cTh, while the Nkx2.2 cells are in the rTh. (i) Distinct levels of expression of Lhx9 is shown in the cTh along the three dorsoventral tiers at late prometamorphic stages, and the intense expression in the dorsal part of the Ar and Ac, within the Nkx2.2 positive region. (j,k) Sagittal (j) and transverse (k) sections at metamorphic climax stages showing the widespread expression of Lhx9 in the C, Ac, and Ar and its absence in the La, LPv, IGL, and Z. For abbreviations, see list. Scale bars = 50 μm (f1); 100 μm (a–s) [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 10 Set of photomicrographs of sagittal (a–c) and transverse (d–r) Xenopus brain sections showing the combination of the markers Sox2 and Lhx2 in the cTh domain. In each panel, the stage is indicated in the upper right corner. The markers and the color code are indicated in the upper left corner. In the sagittal sections (a–c), the levels of the sections shown in the other panels are indicated by bars. (a,d–h) Sagittal (a) section at premetamorphic stages showing the Lhx2 and Sox2 positive domains extensively overlapping in the cTh, and segregated populations in the rTh and Hb. The series of rostrocaudal transverse sections at premetamorphic stages (d-h) shows segregated populations in the Hb, Ar and, Ac (d,e), while in the cTh numerous double labeled cells are found in the C with segregated populations in the rostral periventricular rim, ventral portion, and mostly Sox2 single labeled cells are located in the LPv and Z (f–h). (b,i–m) Sagittal section (b) at prometamorphic stages showing the downregulation of Lhx2 in the Ar and Ac (positive for Sox2). Extensive colocalization is found in the central part of the C and segregated populations in Cr and Cv. The series of rostrocaudal transverse sections at prometamorphic stages (i–m) illustrates the progressive definition of the Cr at the rostral and periventricular limits of the C and the segregated Cv. (c, n–r) Sagittal (c) and transverse (n–r) sections at metamorphic climax showing distinct expression of Sox2 single labeled cells in the Ar, Ac, and IGL. (o,p) Lhx2/Sox2 double labeled cells are numerous in the developed C, whereas segregated populations are observed in Cr. (q–r) Widespread expression of Sox2 in the IGL and LPv2 with more abundant expression of Lhx2 in the Cv and LPv1, and colocalization in the C, with very low levels of expression in the GdN and Z. For abbreviations, see list. A magenta-green version of this figure is provided as Figure S6. Scale bars = 100 μm (a–r) [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 12 Set of photomicrographs of sagittal (a–c) and transverse (d–l) Xenopus brain sections showing the combination of the markers CR and Lhx2 in the cTh domain. In each panel, the stage is indicated in the upper right corner. The markers and the color code are indicated in the upper left corner. In the sagittal sections (a–c), the levels of the sections shown in the other panels are indicated by bars. (a,d–h) Sagittal (a) and transverse (d–h) sections at premetamorphic stages showing the Lhx2 and CR positive domains extensively overlapping in the intermediate tier of the cTh, and segregated cell populations in the dorsal and ventral tiers of the cTh and rTh. The series of rostrocaudal transverse sections show mostly segregated populations at premetamorphic stages. Lhx2 is selectively distributed in the periventricular stratum and CR is located in the outer periventricular, intermediate and superficial strata. Both markers colocalize in the outer periventricular stratum of the cTh. (b,i–k) The sagittal section (b) shows the downregulation of Lhx2 in the Ar and Ac (positive for CR), at prometamorphic stages. Extensive colocalization in the central part of the C and segregated populations in Cr and Cv. The series of rostrocaudal transverse sections (i–k) illustrates the progressive definition of the Cr at the rostral and periventricular limits of the C and the segregated Cv during prometamorphosis. (c,l) Sagittal (c) and transverse (l) sections at the metamorphic climax showing distinct expression of CR single labeled cells in the Ar, Ac, IGL, and LPv. Many Lhx2 single labeled cells intermingled with CRir cells and occupy the periventricular and rostral rim of C (Cr), and exclusive Lhx2 expression is found in the Cv. Lhx2/CR double labeled cells marked the C. For abbreviations, see list. A magenta-green version of this figure is provided as Figure S8. Scale bars = 100 μm (a–l) [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 13 Series of sagittal topologic schemata representing the model of thalamic molecular regionalization along the anteroposterior and dorsoventral axis during premetamorphosis (stages 46–52) (a), prometamorphosis (stages 53–59) (d) and metamorphic climax (stages 60–66) (g) summarizing the expression patterns observed for Nkx2.2, Lhx1, GABA, CB, NPY, Sox2, Gbx2, Lhx2, Lhx9, CR, and Ngn2 in the thalamus of Xenopus. Rostral is oriented to the left and dorsal is up. The radial subdivisions observed in each period are also represented in ideal sectioning planes across the rostral and caudal thalamic levels. The topological specification map showed the subdivisions obtained as a result of combined gene expression patterns at premetamorphic (a–c), prometamorphic (d–f) and the definitive nuclear configuration at metamorphic climax (g–i) stages. (j,k) These schemata represents ideal parasagittal sections at periventricular (j) and lateral (k) levels with a thalamic map of the main nuclear subdivisions, in a color code of the most plausible origin for each nuclear subdivision. For abbreviations, see list [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 14 Photomicrographs of transverse (a,b,d-f,h-j,l,m) and sagittal (c,g,k,n) sections showing the labeled cells and fibers after BDA injection in the A of juvenile Xenopus, combined with the markers CB, CR, GABA, or Nkx2.2 to corroborate that the tracer was delivered into this particular area and to establish the actual location of the labeled somata. The markers and the color code are indicated in the upper left corner. (a–c) Photomicrographs of small injection sites. Retrogradely labeled cells are shown in the pallium (d), BST and prethalamic eminence (e), the preoptic area (f), the subparaventricular area of the hypothalamus (g), the tuberal hypothalamus (h), the prethalamus (i), the caudal C (j), the pretectal nuclei (k), the griseum tectale (l), the mesencephalic tegmentum (m), and superior reticular nucleus (n). For abbreviations, see list. A magenta-green version of this figure is provided as Figure S9. Scale bars = 100 μm (b,f, i–m); 200 μm (a,c–e, g,h,n) [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 15 Photomicrographs of transverse sections showing the labeled cells and fibers after BDA injection in the C of juvenile Xenopus, combined with the markers CB, CR, Sox2, or ChAT to corroborate that the tracer was delivered into this particular area and to establish the actual location of the labeled cells and fibers. The markers and the color code are indicated in the upper left corner. (a–c) Photomicrographs of small injection sites. Labeling after the injections are shown for the nucleus accumbens (d) and striatum (e), the rostral thalamic region (f,g), the mid-caudal thalamic region (h–k), the pretectal region (l, m), the optic tectum (n), mesencephalic (o–r), and isthmic (s) tegmentum, torus semicircularis (t), and isthmic nucleus (u). Note that photomicrographs p1 and r1 are higher magnifications of the squared areas in the corresponding panoramic figures. For abbreviations, see list. A magenta-green version of this figure is provided as Figure S10. Scale bars = 25 μm (r1); 100 μm (e–i, l–u); 200 μm (s–d,j,k) [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 16 Photomicrographs of transverse sections showing the labeled cells and fibers after BDA injection in the LPv of juvenile Xenopus, combined with the markers CB, CR or Sox2 to corroborate that the tracer was delivered into this particular area and to establish the actual location of the labeled cells and fibers. The markers and the color code are indicated in the upper left corner. (a) Photomicrographs of small injection sites. Labeling after the injections are shown for the pallidum (b), the preoptic area (c), the tuberal hypothalamus (d), the anterior thalamus (e,f,h,i), the prethalamus (g), the mid-caudal thalamus (j,k), the pretectum (l), the optic tectum (m), and the superior reticular nucleus (n). For abbreviations, see list. A magenta-green version of this figure is provided as Figure S11. Scale bars = 100 μm (a–n) [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 17 Photomicrographs of transverse sections (a–k) through the brain of Xenopus showing retrogradely labeled cells and anterogradely labeled fibers in the Ar, Ac, La, C, and LPv after BDA injections in the different brain regions indicated in each photograph. For abbreviations, see list. Scale bars = 100 μm (a–k)
	Figure 18 Photomicrographs of transverse (a–c) and sagittal (d–g) sections showing the retrogradely labeled cells after BDA injection in the pallium (a–c) and striatum (d-g) of juvenile Xenopus, combined with the markers CB, CR, Sox2, or Lhx2 to corroborate that the tracer was delivered into these particular areas and the location of the retrogradely labeled cells. The markers and the color code are indicated in the upper left corner. The dorsoventral and rostrocaudal axis orientation are indicated with arrows down in the left corner in the sagittal sections. For abbreviations, see list. A magenta-green version of this figure is provided as Figure S12. Scale bars = 25 μm (e1,e2,f1); 100 μm (b–g); 500 μm (a) [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 19 Photomicrographs of sagittal sections showing the retrogradely labeled cells after BDA injection in the hypothalamus (a–d), prethalamus (e,f), optic tectum (g–j), and area octavolateralis (k–m) of juvenile Xenopus, combined with the markers CB, CR, Nkx2.2, Lhx2, or Sox2 to corroborate that the tracer was delivered into these particular areas and the location of the retrogradely labeled cells. The markers and the color code are indicated in the upper left corner. The dorsoventral and rostrocaudal axis orientation are indicated with arrows down in the left corner. For abbreviations, see list. A magenta-green version of this figure is provided as Figure S13. Scale bars = 25 μm (a1,b1,c1,d1,j1); 100 μm (a–m) [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 20 Summary diagrams showing in a lateral view of the brain the afferent and efferent connections of the Xenopus thalamic nuclei Ar, Ac, La, C, and LPv found in the present study. For abbreviations, see list [Color figure can be viewed at wileyonlinelibrary.com]
	Figure S1 Set of photomicrographs of sagittal (a,c,e,h,j,n,q) and transverse (b,d,f,g,i,k–m,o) sections illustrating the main markers used to define the rTh and cTh domains as well as the rostral and caudal boundaries of the thalamus during premetamorphosis. In each panel, the larval stage is indicated in the upper right corner. In situ hybridization (ISH) for Shh, Tcf4, Gbx2, Ngn2, GAD67, NPY, VGlut2, and Lhx9 (in blue or orange for bright field or magenta in the case of fluorescence images) and immunohistochemistry (IHC) for Nkx2.2, Sox2, Lhx2, CB, or CR (in brown for bright field or green/magenta for fluorescence images) are shown. The markers and the color code are indicated in the upper left corner. In Panels (e) and (h) the levels of sections shown in other panels are indicated by bars. (a) Sagittal section showing by double ISH the expression of Shh and Tcf4 that delimitates the rostral thalamo-prethalamic boundary. (b) Double ISH showing the expression of Gbx2 in combination with Shh, highlighting the caudal boundary of the thalamus defined by Gbx2. (c) Sagittal section showing by double ISH the expression of Shh in combination with Ngn2 showing the rostral limit of the thalamus defined by Shh, and the caudal limit defined by Ngn2. (d) Expression of Ngn1 (ISH) in combination with Nkx2.2 (IHC) showing the rTh and cTh subdivisions and the compartmentalization of cTh revealed by Ngn2 ISH in the cTh1. (e–g) Sections illustrating the expression of Gbx2 (ISH) in combination with Nkx2.2 (IHC) showing the extension and shape of the rTh and cTh subdivisions (the levels of the transverse sections shown in f and g are indicated in the sagittal section in e). (h,i) Sagittal (h) and transverse (i) sections (the level of section i is indicated in h) showing the expression of GAD67 (ISH) in combination with Nkx2.2 (IHC). Note their colocalization in the rostral part of the rTh and a single Nkx2.2 positive region located caudally. (j) NPY expression (ISH) in the rTh at premetamorphic stages shown in a sagittal section in combination with PAX7 (IHC). (k) Combination of NPY (ISH) and Nkx2.2 (IHC) in a transverse section showing double labeled cells in the rTh and only a few in the cTh. (l,m) Transverse sections at rostral (l) and caudal (m) thalamic levels showing the expression of VGlut2 (ISH) in the cTh2 compartment, in combination with Nkx2.2 (IHC) in the rTh. (n,o) Sagittal (n) and transverse (o) sections showing the Nkx2.2 and Sox2 immunoreactive cells in the rTh and cTh, respectively. Note the overlapping pattern of Sox2 with VGlut2 (l) cells in cTh domain. (p) Transverse section showing the exclusive pattern of Lhx2 and Nkx2.2 in the cTh and rTh, respectively. (q,r) Sagittal (q) and transverse (r) sections showing the overlapping patterns and colocalization of Lhx2 and Sox2 immunoreactive cells in cTh and the segregated pattern dorsally. (s) Combined IHC for CB and CR showing in a transverse section the complementary pattern in the rTh and cTh and their overlapping in the habenula. For abbreviations, see list. Scale bars = 100 μm (a–d); 50 μm (e–v).
	Figure S2 Set of photomicrographs of sagittal (a–c,h,k,m,q,u–w) and transverse (d–g,i,j,l,n–p,r–t) sections illustrating the main markers for the rTh domain and its derivatives, at different larval stages. In each panel, the stage is indicated in the upper right corner. The markers and the color code are indicated in the upper left corner. (a–g) Sagittal (a–c), and transverse (d–g) sections at premetamorphic (a,d,e), prometamorphic (b,f), and metamorphic (c,g) larval stages stained for Nkx2.2 and GABA. (a,d,e) Note the intense colocalization (arrowheads) in the rostral rim of rTh next to the Zli and a caudal subdivision of single Nhx2.2 cells. (b,f) Nkx2.2 expression in the Ar and Ac at prometamorphic stages. The Ac does not contain double GABA/Nkx2.2 cells, which are intensely detected in Ar and the rostroventral rim. (c,g) Nkx2.2 and GABA expressions in the thalamic region at the metamorphic climax expand caudally to the C and were reduced dorsally in Ar1 and Ac1 subdivisions. Double GABA/Nkx2.2 cells occupy the Ar2 and GdN laterally. Note Nkx2.2 positive cells also in the La nucleus of the superficial stratum. (h–j) Distribution of Nkx2.2 cells in comparison with CB cells at premetamorphic stages. CB marks some cells in the periventricular stratum of the rostral part of rTh and laterally in the superficial stratum of the cTh. (k,l) Distribution of Nkx2.2 cells in comparison with CR cells at premetamorphic stages. CR marks intensely the cTh and part of the Hb. Observe also some cells in the rTh. (m–p) Sagittal (m), and transverse (n–p) sections though the thalamic region showing combined CB and Nkx2.2 staining at prometamorphic stages. CB marks intensely the Ar and is absent in La and GdN. Note a subpopulation of Nkx2.2/CB double labeled cells ventrally in the rostral rim of rTh in the IGL (p1, arrowhead). (q–t) Sagittal (q), and transverse (r–t) sections though the thalamic region showing combined CR and Nkx2.2 staining at prometamorphic stages. As opposed to CB, CR marks intensely the Ar, La, and GdN as well as most caudal nuclear derivatives, but do not colocalize with Nkx2.2. (u) At the metamorphic climax, CB cells are intensely labeled in the Ar1 and are codistributed with Nkx2.2 expressing cells in the Ar2. (v) CR labeled intensely the Hb, Ar1, Ac1, and Ar2 leaving a gap in the SH, with scarce cells in DC. Some cells are positive also in the Ar2 and are also scarce in the IGL. (w) Pseudo color generated image showing the subdivisions observed by the overlapping of adjacent sections labeled for Nkx2.2/GABA and CB/CR. Note the extensive GABA/Nkx2.2 domain in the IGL, the absence of Nkx2.2 or GABA in the Ar1 and Ac1, the exclusive staining for CB in the Ar and the displaced Nkx2.2 cell population in the C. For abbreviations, see list. Scale bars = 100 μm (a–w).
	Figure S3 Set of photomicrographs of sagittal (a,e,h,i,l,p) and transverse (b–d, f,g,j, k,m–o,q–s) sections illustrating some additional markers for the rTh domain and its derivatives, at different larval stages. In each panel, the stage is indicated in the upper right corner. The markers and the color code are indicated in the upper left corner. (a–d) Sagittal (a) and transverse (b–d) sections at prometamorphic stages stained for CB and GABA. CB and GABA are codistributed in Ar without colocalization. Single GABAergic cells are located in the GdN. (e–g) Sagittal (e) and transverse (f,g) sections at metamorphic climax stained for CB and GABA. Note the intensive CB staining in cells of the Ar and the absence GABA in the Ar1 and Ac1. (h–k) Sagittal (h,i) and transverse (j,k) sections at prometamorphic larval stages stained for CR and GABA. Both markers are codistributed in Ar and GdN without colocalization. Double labeled cells appear transiently in the IGL, rostral part of C and LPv2. Single GABAergic cells are located medially to the IGL. (l–o) Sagittal (l), and transverse (m–o) sections though the thalamic region showing combined CR and GABA staining at the metamorphic climax. CR and GABA are extensively codistributed in the Ar2 and Ac2 and also in the rostral part of C and LPv2, without colocalization. In contrast to previous stages, cells labeled for CR do not appear in the GdN, IGL, and Z. (p–s) Sagittal (p), and transverse (q–s) sections though the thalamic region showing combined CR and CB staining at prometamorphic stages. Double labeling is abundant in the Ar and in some cells of the LPv. For abbreviations, see list. Scale bars = 100 μm (a–s).
	Figure S4 Set of photomicrographs of sagittal (a,c,k,o) and transverse (b,d–j,l–n,p–s) sections illustrating the combination of markers for the rTh domain (Nkx2.2 and CB) with markers for the cTh domain (Sox2) and its derivatives, at different larval stages. In each panel, the stage is indicated in the upper right corner. The markers and the color code are indicated in the upper left corner. (a,b) Combined Nkx2.2 and Sox2 staining at premetamorphic stages showing the distribution of Nkx2.2 in the rTh and Sox2 in the cTh. (c-f) Sagittal (c) and transverse (d-f) sections of the thalamic region at prometamorphic stages showing the intermingled populations of Nkx2.2 and Sox2 cells in the Ar, Ac and the rostral rim of the C. Nkx2.2 cells seem to origin in the rTh ventricle (asterisk in d,e,f) and occupy the Ar, Ac, GdN, and La in the superficial strata. Sox2 cells occupy the periventricular stratum in the Ac and are abundant in the Ar. (g–j) Rostrocaudal series of transverse sections through the thalamic region at metamorphic climax stages showing the intense Sox2 expression in the Ar, Ac, IGL, C, and LPv, with only weak expression in the La, GdN, and Z. (k–n) Sagittal (k) and transverse (l–n) sections showing the combination of CB and Sox2 staining at prometamorphic stages. Double labeled cells mark the extension of the Ar. (o) Distinct NPY expression in the IGL and colocalization with Nkx2.2 at prometamorphic stages, illustrated in the higher magnification in (o1). (p–r) Rostrocaudal series of transverse sections through the thalamic region at metamorphic climax stages showing the colocalization of CB and Sox2 in the Ar and single Sox2 positive cells in the Ac, IGL, C, and LPv; and weak expression in the La, GdN, and Z. (s) NPY expression in the IGL at metamorphic climax in comparison with Gbx2. Extensive codistribution of these markers is observed in the IGL. For abbreviations, see list. Scale bars =25 (o1); 50 μm (o); 100 μm (p-s).
	Figure S5 Set of photomicrographs of sagittal (a–c,i,k,m) and transverse (d–h, j,l) sections illustrating the combination of markers for the rTh domain (GABA and CB) with markers for the cTh domain (Lhx2) and its derivatives at different larval stages. In each panel, the stage is indicated in the upper right corner. The markers and the color code are indicated in the upper left corner. (a–h) Sagittal (a–c) and transverse (d–h) sections at premetamorphic (a,d,e), prometamorphic (b,e,f), and metamorphic (c,g,h) larval stages, stained for Lhx2 and GABA. (a,d) GABA expression in the Ar and codistribution with Lhx2 periventricularly in the Ac are shown at premetamorphic stages. No colocalization is observed. (b,e,f) Extensive codistribution of Lhx2 and GABA is shown in the caudal and ventral part of the Ac at prometamorphic stages. (e) GABA expression extends in the superficial stratum. (f) Lhx2 expression occupies the lateral part of the IGL. (c,g,h) Distribution of Lhx2 in comparison with GABA at metamorphic climax. The Lhx2 positive domain is reduced and codistribution of Lhx2 and GABA remains in a small part of the Ac ventrally, and in the C. (g) Both markers are codistributed in the Ac, IGL and C with scarce expression in the La. (h) Some Lhx2 cells remain laterally in the IGL. (i,j) Sagittal (i) and transverse (j) sections at premetamorphic stages showing CB labeling in cells in the Ar and LPv, but not in the Ac. Lhx2 is expressed in the Hb and DC as well as in the Ac and C, periventricularly. No colocalization is observed. (k,l) Sagittal (k) and transverse (l) sections at prometamorphic stages illustrating the downregulation of Lhx2 rostrally in the Ar and Hb. CB labeled cells are abundant in the Ar and scarce in the LPv. (m) Sagittal section at metamorphic climax showing the virtual absence of Lhx2 expression in the Ar and Ac and its presence in the C and Cv. For abbreviations, see list. Scale bars =100 μm (a–m).
	Figure S6 Set of photomicrographs of sagittal (a–c) and transverse (d–m) sections showing the combination of the markers Sox2 and Lhx2 in the cTh domain. In each panel, the stage is indicated in the upper right corner. The markers and the color code are indicated in the upper left corner. In the sagittal sections (a–c), the levels of the sections shown in the other panels are indicated by bars. (a,d–h) Sagittal (a) section at premetamorphic stages showing the Lhx2 and Sox2 positive domains extensively overlapping in the cTh, and segregated populations in the rTh and Hb. The series of rostrocaudal transverse sections at premetamorphic stages (d–h) shows segregated populations in the Hb, Ar and Ac (d,e), while in the cTh numerous double labeled cells are found in the C with segregated populations in the rostral periventricular rim, ventral portion, and mostly Sox2 single labeled cells are located in the LPv and Z (f–h). (b,i–m) Sagittal section (b) at prometamorphic stages showing the downregulation of Lhx2 in the Ar and Ac (positive for Sox2). Extensive colocalization is found in the central part of the C and segregated populations in Cr and Cv. The series of rostrocaudal transverse sections at prometamorphic stages (i–m) illustrates the progressive definition of the Cr at the rostral and periventricular limits of the C and the segregated Cv. (c, n–r) Sagittal (c) and transverse (n–r) sections at metamorphic climax showing distinct expression of Sox2 single labeled cells in the Ar, Ac, and IGL. (o,p) Lhx2/Sox2 double labeled cells are numerous in the developed C, whereas segregated populations are observed in Cr. (q–r) Widespread expression of Sox2 in the IGL and LPv2 with more abundant expression of Lhx2 in the Cv and LPv1, and colocalization in the C, with very low levels of expression in the GdN and Z. For abbreviations, see list. Scale bars = 100 μm (a–r).
	Figure S7 Set of photomicrographs of sagittal (a,e,i) and transverse (b–d,f–h,j–l) sections showing the combination of the markers CR and Sox2 in the cTh domain. In each panel, the stage is indicated in the upper right corner. The markers and the color code are indicated in the upper left corner. In the sagittal sections (a,e,i), the levels of the sections shown in the other panels are indicated by bars. (a–d) Sagittal (a) and transverse (b–d) sections at premetamorphic stages showing segregated CR and Sox2 cell populations in the rTh, with only partial overlapping in the cTh. Sox2 expression occupies the periventricular stratum, whereas CR is found in the outer part of the periventricular stratum and in the intermediate and superficial strata. Both markers colocalize in the outer periventricular stratum. (e–h) Sagittal (e) and transverse (f–h) sections at prometamorphic stages showing development of the double CR/Sox2 area in the Ar, Ac, and C. Extensive colocalization is observed in the central part of the C, and segregated cell populations in the La and LPv (mostly cells labeled for CR) as well as in the C, Cv, IGL, and Z (single Sox2 expression). (i–l) Sagittal (i) and transverse (j–l) sections at metamorphic climax. Double CR/Sox2 cells occupy all subdivisions of the Ar and Ac and the central part of the C. Single CRir cells are abundant in the La and LPv1 but they are absent in the SH, LPv2 and Cv. Sox2 expressing cells are downregulated in the La and Z, remaining some intensely labeled cells in the IGL. For abbreviations, see list. Scale bars = 50 μm (a–d); 100 μm (e–l).
	Figure S8 Set of photomicrographs of sagittal (a–c) and transverse (d–l) sections showing the combination of the markers CR and Lhx2 in the cTh domain. In each panel, the stage is indicated in the upper right corner. The markers and the color code are indicated in the upper left corner. In the sagittal sections (a–c), the levels of the sections shown in the other panels are indicated by bars. (a,d–h) Sagittal (a) and transverse (d–h) sections at premetamorphic stages showing the Lhx2 and CR positive domains extensively overlapping in the intermediate tier of the cTh, and segregated cell populations in the dorsal and ventral tiers of the cTh and rTh. The series of rostrocaudal transverse sections show mostly segregated populations at premetamorphic stages. Lhx2 is selectively distributed in the periventricular stratum and CR is located in the outer periventricular, intermediate and superficial strata. Both markers colocalize in the outer periventricular stratum of the cTh. (b,i–k) The sagittal section (b) shows the downregulation of Lhx2 in the Ar and Ac (positive for CR), at prometamorphic stages. Extensive colocalization in the central part of the C and segregated populations in Cr and Cv. The series of rostrocaudal transverse sections (i–k) illustrates the progressive definition of the Cr at the rostral and periventricular limits of the C and the segregated Cv during prometamorphosis. (c,l) Sagittal (c) and transverse (l) sections at the metamorphic climax showing distinct expression of CR single labeled cells in the Ar, Ac, IGL, and LPv. Many Lhx2 single labeled cells intermingled with CRir cells and occupy the periventricular and rostral rim of C (Cr), and exclusive Lhx2 expression is found in the Cv. Lhx2/CR double labeled cells marked the C. For abbreviations, see list. Scale bars = 100 μm (a–l).
	Figure S9 Photomicrographs of transverse (a,b,d–f,h–j,l,m) and sagittal (c,g,k,n) sections showing the labeled cells and fibers after BDA injection in the A of juvenile Xenopus, combined with the markers CB, CR, GABA, or Nkx2.2 to corroborate that the tracer was delivered into this particular area and to establish the actual location of the labeled somata. The markers and the color code are indicated in the upper left corner. (a–c) Photomicrographs of small injection sites. Retrogradely labeled cells are shown in the pallium (d), BST and prethalamic eminence (e), the preoptic area (f), the subparaventricular area of the hypothalamus (g), the tuberal hypothalamus (h), the prethalamus (i), the caudal C (j), the pretectal nuclei (k), the griseum tectale (l), the mesencephalic tegmentum (m) and superior reticular nucleus (n). For abbreviations, see list. Scale bars = 100 μm (b,f, i–m); 200 μm (a,c–e, g,h,n).
	Figure S10 Photomicrographs of transverse sections showing the labeled cells and fibers after BDA injection in the C of juvenile Xenopus, combined with the markers CB, CR, Sox2, or ChAT to corroborate that the tracer was delivered into this particular area and to establish the actual location of the labeled cells and fibers. The markers and the color code are indicated in the upper left corner. (a–c) Photomicrographs of small injection sites. Labeling after the injections are shown for the nucleus accumbens (d) and striatum (e), the rostral thalamic region (f,g), the mid-caudal thalamic region (h–k), the pretectal region (l, m), the optic tectum (n), mesencephalic (o–r), isthmic (s) tegmentum, torus semicircularis (t), and isthmic nucleus (u). Note that photomicrographs p1 and r1 are higher magnifications of the squared areas in the corresponding panoramic figures. For abbreviations, see list. A magenta-green version of this figure is provided as Figure S10. Scale bars = 25 μm (r1); 100 μm (e–i, l–u); 200 μm (s–d,j,k).
	Figure S11 Photomicrographs of transverse sections showing the labeled cells and fibers after BDA injection in the LPv of juvenile Xenopus, combined with the markers CB, CR, or Sox2 to corroborate that the tracer was delivered into this particular area and to establish the actual location of the labeled cells and fibers. The markers and the color code are indicated in the upper left corner. (a) Photomicrographs of small injection sites. Labeling after the injections are shown for the pallidum (b), the preoptic area (c), the tuberal hypothalamus (d), the anterior thalamus (e,f,h,i), the prethalamus (g), the mid-caudal thalamus (j,k), the pretectum (l), the optic tectum (m), and the superior reticular nucleus (n). For abbreviations, see list. Scale bars = 100 μm (a–n).
	Figure S12 Photomicrographs of transverse (a–c) and sagittal (d–g) sections showing the retrogradely labeled cells after BDA injection in the pallium (a–c) and striatum (d–g) of juvenile Xenopus, combined with the markers CB, CR, Sox2, or Lhx2 to corroborate that the tracer was delivered into these particular areas and the location of the retrogradely labeled cells. The markers and the color code are indicated in the upper left corner. The dorsoventral and rostrocaudal axis orientation are indicated with arrows down in the left corner in the sagittal sections. For abbreviations, see list. Scale bars = 25 μm (e1, e2, f1); 100 μm (b–g); 500 μm (a).
	Figure S13 Photomicrographs of sagittal sections showing the retrogradely labeled cells after BDA injection in the hypothalamus (a–d), prethalamus (e,f), optic tectum (g–j), and octavolateral area (k–m) of juvenile Xenopus, combined with the markers CB, CR, Nkx2.2, Lhx2, or Sox2 to corroborate that the tracer was delivered into these particular areas and the location of the retrogradely labeled cells. The markers and the color code are indicated in the upper left corner. The dorsoventral and rostrocaudal axis orientation are indicated with arrows down in the left corner. For abbreviations, see list. Scale bars = 25 μm (a1,b1,c1,d1,j1); 100 μm (a–m