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Figure 1 Schematic drawings of Xenopus developing brain. Schematic drawings of Xenopus
brains illustrating the morphology at different developing stages (late embryonic, premetamorphic,
and prometamorphic). Main regions are shown on lateral views of the brain (a–c), sagittal sections
(d–f), and transversal sections at rostral and caudal levels (g–l). Abbreviations for this and subsequent
figures—BST, bed nucleus of the stria terminalis; CeA, central amygdala; Dth, dorsal thalamus;
Hyp, hypothalamus; LA, lateral amygdala; LGE, lateral ganglionic eminence; LP, lateral pallium;
MeA, medial amygdala; MGE, medial ganglionic eminence; MP, medial pallium; mz, marginal
zone; OB, olfactory bulb; OT, optic tectum; P1, prosomere 1; P2, prosomere 2; P3,
prosomere 3; P4, prosomere 4; Pa, pallium; pf, postfertilization; POa, anterior preoptic area; S, septum;
Sd, septum dorsalis; Sl, septum lateralis; Str, striatum; SP, subpallium; v, ventricle; VP, sentral
pallium; vz, ventricular zone.
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Figure 2 x-Pax6, x-Lhx9, x-Lhx5, and x-Dll3 expressions are dynamic during Xenopus forebrain
development. Simple and double in situ hybridization photomicrographs of sagittal (a, c, k–m) and
transverse (b, d–j) sections at the indicated stages. (a–f) x-Pax6 expression. Filled arrowheads point to
Pax6-expressing cell masses in the subpallium. In (d), empty arrowheads point to Pax6-expressing
cells in the prospective septum. In (b), dotted lines indicate the x-Pax6 streams of cells. (g–j) Expression
of x-Lhx5 and x-Lhx9, at embryonic (g, h) and larval (i, j) stages. Filled arrowheads in (i) and (j)
point to xLhx5/9-expressing cells in the medial amygdala (i, j). (k–m) Expression of x-Dll3/x-Lhx7 (k),
x-Emx1 (l), and x-Lhx9/7 (m) on sagittal sections. In this and subsequent figures, see Figure 1 for section
levels and orientation and for anatomical abbreviations, and Table 1 for number of animals used
for each experimental procedure. Scale bar ¼ 100 lm (a, f–m) and 50 lm (b–e).
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Figure 3 Nkx2.1 and GABA patterns are dynamic during
Xenopus forebrain development. Transverse sections after
single or double immunofluorescence for Isl1, Nkx2.1, and
GABA at the indicated stages. In the doubly immunostained
sections the use of red or green fluorochromes is indicated
for each case. In (a–c), arrowheads show Nkx2.1+-MGE
originated-positive cells into the Isl1-positive mz of the
LGE. In (d), arrowheads show GABAergic interneurons in
the pallium. In (e), empty and filled arrowheads indicate
Nkx2.1-expressing cells that are detected inside or outside
the Isl1-positive striatum, respectively. In (f) arrowheads
show GABAergic pallial interneurons. Scale bars ¼ 100 lm.
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Figure 4 Isl1 labels both projection neurons and interneurons. Transverse sections through the
developing prosencephalon of Xenopus. (a,b) Retrogradely labeled cells after application of BDA
(red) in the lateral forebrain bundle (a) or the hypothalamus (b), combined with Isl1 immunofluorescence
staining (green). (c–h) Double (c–e, g) and triple (f, h) immunofluorescence with the indicated
markers. Empty arrowheads show single labeled cells and filled arrowheads show doubly
labeled cells. Scale bar ¼ 100 lm (a, b, d–h) and 25 lm (c).
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Figure 5 The striatal Isl1-free-channel contains Nkx2.1
and GABA expressing cells. Transverse sections through
the developing telencephalon at stage 56, after immunoflurescence
for Isl1 (green) and GABA or Nkx2.1 (red). The
boxed areas in (a–c) are shown at higher magnification in
(a0–c0). A ‘‘channel’’ free of Isl1-expressing cells is
delineated by dotted lines in (a0), and contains Nkx2.1-positive
cells (filled arrowheads in c0). Empty arrowheads in
(c0) point to Nkx2.1-expressing cells which enter the Isl1-
expressing striatum. A schematic representation is given in
(d) for comparison with the ‘‘Isl1-positive corridor’’
described in mammals (modified from Lo´pez-Bendito et al.,
2006). Scale bars ¼ 100 lm.
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Figure 6 Doublecortin telencephalic expression: double labeling with Nkx2.1 and GABA.
Transverse sections after single (a, b, d), double (c, f–j) and triple (e) labeling for DCX, Isl1,
Nkx2.1, and GABA at the indicated stages. In case of double/triple immunofluorescence, the use of
red or green fluorochromes is indicated for each case. In (a), arrows point to the DCX chains. In
(b), dotted lines delineate a high DCX expression zone in the internal part of the striatum. In (c),
the white box indicates the area enlarged in (d) and (e). In (e), arrowhead shows a DCX/Nkx2.1
double-labeled cell in the Isl1-negative area, and empty arrowhead shows a Nkx2.1 cell in the Isl1-
positive striatum. In (f), the white boxes indicate the areas enlarged in (g) and (h). In (g–j) the
arrowheads point to doubly labeled cells. Scale bars ¼ 25 lm (b, e, g, f) and 50 lm (a, c, d, f, i, j).
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Figure 7 In vivo electroporation and CMFDA applications demonstrate cell movements in the
telencephalon. (a, h) show the targeted area for cell tracking experiments. (b–c) GFP immunofluorescence
48 h after electroporation. Asterisks indicate the electroporated area and arrowheads show
cells in the subpallium. (d–g) Photomicrographs after small (d, f, g, i, j) or large (e) CMFDA applications
into the ventral pallium (d–g) and the subpallium (i, j) at the indicated stages. Arrowheads
show the presence of labeled cells outside of the zone of injection. In (d), the asterisk indicates the
injection site and the dotted line the two courses followed by labeled cells. Scale bar ¼ 100 lm.
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Figure 8 CMFDA applications in the subpallium demonstrate
tangentially migrating Nkx2.1 and GABAergic cells.
Transverse sections through the developing telencephalon.
After combined CMFDA and immunolabeling for Nkx2.1
(a,d) and GABA (c, e–i), double-labeled cells (arrowheads)
were detected leaving the application area. In (b, e, g) boxes
indicate enlarged area in the next panel. Arrowheads in (b)
and (e) indicate CMFDA cells out of the injection site.
Scale bars ¼ 100 lm (a, b, d, e, g, i) and 25 lm (c, f, h).
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Figure 9 Summary drawing for cell specification and migrations in Xenopus and mouse forebrain.
The markers used are color-coded and represented on transverse schematic sections at developing
stages (the development timing is not compared). In (a), results from the present and previous
studies are summarized: (1) present results; (2) data from Gonza´lez et al., 2002a,b; (3) data from
Endepols et al., 2007; (4) data from Bachy and Re´taux, 2006. The resulting cell types are indicated
in the right column, where the distinction is made between projection neurons and interneuron
types. In (b), the migratory streams observed in this study are compared to those known in mammals.
The Xenopus data are from the present and previous studies (Bachy et al., 2001, 2002a,b;
Brox et al., 2003; Moreno et al., 2003; 2004; Bachy and Re´taux, 2006). The mammalian data are
compiled from Marı´n and Rubenstein (2001), Tole et al. (2005), and Carney et al. (2006).
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