Roth M, Bonev B, Lindsay J, Lea R, Panagiotaki N, Houart C, Papalopulu N.

057819/Z/05 Wellcome Trust , G0601064 Medical Research Council , MRC_G0601064 Medical Research Council

	Fig. 3. Expression of X. tropicalis TLEs. Expression patterns of TLE1, TLE2, AES and FoxG1 as shown by whole-mount in situ hybridisation of X. tropicalis embryos at neural plate stage (st. 16; frontal view), early tailbud (st. 22-23; frontal view), and late tailbud (st. 28; lateral view). TLE1 and TLE2 show expression throughout the anterior neural plate (st. 16), but TLE1 is strongest in the anterior neural ridge (black arrow), whereas TLE2 is strongest in a more medial ridge (black arrow). Note the expression of TLE1, but not TLE2, in the forebrain of st. 28 embryos, which largely overlaps with the telencephalic expression of FoxG1 (telencephalon indicated by red arrowheads). Ey, eye; m, midbrain; h, hindbrain; ba, brancial arches; di, diencephalon; ov, otic vesicle.
	Fig. 4. Region-specific expression of TLE1 and TLE2 in the developing telencephalon. (A-M) Whole-mount in situ hybridisation for FoxG1 (A-E), TLE1 (F-I) and TLE2 (E,J-M) in X. tropicalis. All images except E (st. 28) represent st. 22 embryos. (B,C,G,H,K,L) Transverse sections (20 μm) of whole-mount stained embryos. The neural tissue is outlined by black dashed lines, and the dorsal border of the telencephalon is indicated by blue dashed lines. The plane of the sections is shown in cleared embryos (D,I,M, anterior to the left, eyes removed). Black dashed lines in G and K indicate the telencephalon and the whole brain tissue. (E) A double in situ stained for FoxG1 (purple) and TLE2 (blue) at st. 28; the plane of section is similar to C. di, diencephalon; dt, dorsal telencephalon; e, eye; oc, optic chiasm; os, optic stalk; m, mesencephalon; t, telencephalon; vt, ventral telencephalon. Annotation based on Bachy et al. (Bachy et al., 2002).
	Fig. 7. The effect of FoxG1 and TLE2 MO on forebrain development. (A) Transverse sections of forebrain of FoxG1 MO-injected (7.5 ng) embryos (st. 28) stained with DAPI (blue), anti-pH3 (green) and anti-FoxG1 (red). Note the reduction of FoxG1 staining and in pH3-positive cells on the MO-injected (left) side in the presumptive FoxG1 expression region. Dashed white line indicates the midline. (B) Schematic representation of the TLE2 splice MO (red) and location of PCR primers in relation to the exon-intron arrangement of TLE2. (C) Semi-quantitative PCR from embryos injected with 10 ng Control MO (lanes 1, 4), 10 ng TLE2 MO (lanes 2, 5) or 20 ng TLE2 MO (lanes 3, 6). Primers used were ornithine decarboxylase (ODC) as control (lanes 4-6) and TLE2 exon 1 and TLE2 exon 3 (lanes 1-3) (see B). (D-G) Nkx2.1 expression in the forebrain of st. 34 control embryos and embryos injected at the one-cell stage with 10 ng Control MO (E), 15 ng FoxG1 MO (F) and 15 ng TLE2 MO (G). The expression in the ventral telencephalon (red arrowhead) is reduced or missing in TLE2 and FoxG1 morphant embryos (F,G). di, diencephalon, dt, dorsal telencephalon, vt, ventral telencephalon. (H-N) Embryos injected in one cell at the two-cell stage with FoxG1 MO (7.5 ng) and TLE2 MO (7.5 ng) processed for in situ hybridisation for elrC (H-K) and TUNEL staining (red nuclei; L-N). Injected side is to the right, as marked by X-Gal staining (H-K) or FITC (L-N). (H,J) Frontal views; (I,K-N) transverse sections through the forebrain of st. 19 (H-K) or st. 32 (L-N) embryos. Reduced forebrain elrC expression on the MO-injected side is indicated by red arrows. There was no statistically significant increase in TUNEL staining. (O) In FoxG1 MO-injected forebrains there was a 40% decrease (P=0.003, paired t-test, n=12 embryos) in pH3-positive cells. (P-T) Transverse sections through the forebrain of FoxG1 MO-injected embryos, subsequently electroporated with wild-type FoxG1 and various FoxG1 mutated versions, as indicated, together with lacZ DNA and analysed for Nkx2.1 expression. Light blue (X-Gal staining) indicates cells that have taken up the electroporated DNA (red arrowheads indicate examples of electroporated cells); dark purple indicates cells expressing Nkx2.1. The experiment was repeated four times with similar results. See Materials and methods for details. Scale bars: 100 μm.
	Fig. 5. FoxG1 interacts physically and cooperates functionally with TLE2. (A-F) X. laevis embryos were injected at the two-cell stage and processed for X-Gal staining (light blue) and whole-mount in situ hybridisation for N-tubulin (purple) at st. 16. Low-dose injection of X. laevis FoxG1 (50 pg) gives rise to ectopic neurogenesis throughout the lateral ectoderm (A), whereas high-dose injection (250 pg) causes a complete suppression of endogenous neurogenesis on the injected site and ectopic neuronogenesis outside the X-Gal staining area (B,C). Injection of X. tropicalis FoxG1 mRNA gave rise to similar phenotypes (not shown). Co-expression of 200 pg X. tropicalis TLE2 with 50 pg X. laevis FoxG1 (E,F) gives rise to a phenotype that is similar to high-dose injections (250 pg) of FoxG1 alone (B,C). TLE2 alone has no effect (D). (A,B,D,E) Dorsal views; (C,F) lateral views. In all panels, anterior is to the left. (G) Percentage of low-dose and high-dose phenotypes resulting from each injection. (H) Co-immunoprecipitation of X. laevis FoxG1-HA and X. tropicalis TLE-Flag with anti-Flag antibody followed by western blotting with anti-HA. The bottom panel shows loading of equal amounts of lysates followed by western blotting with anti-Flag and anti-HA. TLE2-Flag strongly interacts with FoxG1-HA, whereas TLE1- Flag expressed at similar levels shows slightly weaker binding.
	Fig. 6. Motifs in FoxG1 that are responsible for the binding and functional cooperativity of FoxG1 and TLE2. (A)Amino acid sequence of X. laevis FoxG1 and potential TLE binding motifs. The DNA-binding domain is shown in blue, the N-terminal eh1 motif is highlighted in red, the C- terminal WRPW-like motif in green and the cryptic eh1 motif in purple. (B)Schematic representation of the FoxG1 mutants. The N-terminal domain is shown in dark grey, the DNA-binding domain in blue, the potential TLE1 interaction domain in orange and the HA epitope in red. Beneath is shown the sequence of mutated motifs (same colour coding as in A). (C)Immunoprecipitation (IP) of HEK 293T cells transfected with FoxG1-HA or FoxG1 mutants together with TLE2-Flag, showing that mutation of the N-terminal eh1 motif (FoxG1-N3A-HA), either alone or simultaneously with the C-terminal motif (FoxG1-N3A-C-HA), leads to loss of binding to TLE2. The top two panels show IP with anti-Flag followed by western blotting with anti-HA and anti-Flag. Lower panels show the input (pre-IP lysates) analysed by western blotting with anti-Flag and anti-HA. (D)The phenotypes of embryos injected with the indicated amounts of X. tropicalis TLE2 and X. laevis FoxG1 or FoxG1 mutant mRNA were scored as low- dose or high-dose as in Fig. 5. TLE2 does not cooperate with any of the three mutants to convert a low-dose FoxG1 injection into a high-dose phenotype. (E)HEK 293T cells transfected with either FoxG1-HA or FoxG1-N3A-HA. Both proteins are nuclear.
	Fig. 7. The effect of FoxG1 and TLE2 MO on forebrain development. (A)Transverse sections of forebrain of FoxG1 MO-injected (7.5 ng) embryos (st. 28) stained with DAPI (blue), anti-pH3 (green) and anti-FoxG1 (red). Note the reduction of FoxG1 staining and in pH3-positive cells on the MO-injected (left) side in the presumptive FoxG1 expression region. Dashed white line indicates the midline. (B)Schematic representation of the TLE2 splice MO (red) and location of PCR primers in relation to the exon-intron arrangement of TLE2. (C)Semi-quantitative PCR from embryos injected with 10 ng Control MO (lanes 1, 4), 10 ng TLE2 MO (lanes 2, 5) or 20 ng TLE2 MO (lanes 3, 6). Primers used were ornithine decarboxylase (ODC) as control (lanes 4-6) and TLE2 exon 1 and TLE2 exon 3 (lanes 1-3) (see B). (D-G)Nkx2.1 expression in the forebrain of st. 34 control embryos and embryos injected at the one-cell stage with 10 ng Control MO (E), 15 ng FoxG1 MO (F) and 15 ng TLE2 MO (G). The expression in the ventral telencephalon (red arrowhead) is reduced or missing in TLE2 and FoxG1 morphant embryos (F,G). di, diencephalon, dt, dorsal telencephalon, vt, ventral telencephalon. (H-N)Embryos injected in one cell at the two-cell stage with FoxG1 MO (7.5 ng) and TLE2 MO (7.5 ng) processed for in situ hybridisation for elrC (H-K) and TUNEL staining (red nuclei; L-N). Injected side is to the right, as marked by X-Gal staining (H-K) or FITC (L-N). (H,J)Frontal views; (I,K-N) transverse sections through the forebrain of st. 19 (H-K) or st. 32 (L-N) embryos. Reduced forebrain elrC expression on the MO-injected side is indicated by red arrows. There was no statistically significant increase in TUNEL staining. (O)In FoxG1 MO-injected forebrains there was a 40% decrease (P0.003, paired t-test, n12 embryos) in pH3-positive cells. (P-T)Transverse sections through the forebrain of FoxG1 MO-injected embryos, subsequently electroporated with wild-type FoxG1 and various FoxG1 mutated versions, as indicated, together with lacZ DNA and analysed for Nkx2.1 expression. Light blue (X-Gal staining) indicates cells that have taken up the electroporated DNA (red arrowheads indicate examples of electroporated cells); dark purple indicates cells expressing Nkx2.1. The experiment was repeated four times with similar results. See Materials and methods for details. Scale bars: 100m
	Fig S3. Fig. S3. Effect of TLE2 MO and FoxG1 MO on emx1 and TUNEL staining after unilateral injection of X. tropicalis embryos. (A-D) One cell of a two-cell stage embryo was injected with 7.5 ng MO plus lacZ mRNA; the injected side is indicated by light-blue X-Gal staining in st. 32 embryos. (A,B) Frontal views of injected embryos. (C,D) Parasagittal sections of the embryo shown in B, at the levels indicated. emx1 is normally mainly expressed in the dorsal telencephalon (pallium), but the striatum (lge) and olfactory bulb, which are located more ventrally, also contain scattered emx1-positive cells (Bachy et al., 2002), giving the appearance of a 'gap' between dorsal and ventral expression (black arrow in B). In FoxG1 MO-injected embryos this gap does not exist (red arrow in B, 83%, n=12), indicating an expansion of the dorsal expression domain. This is not observed in the TLE2 MO-injected embryos, although the overall emx1 expression domain is often slightly reduced (50%, n=14). Note that the ventral and dorsal parts of the telencephalon are ontogenetically rostral and posterior, respectively; Bachy et al. (Bachy et al., 2002) use the term rostral to describe the subpallium, whereas we use the term ventral for consistency with the zebrafish and mouse literature. (E) Quantification of cell death by TUNEL staining. TUNEL-positive cells were counted on the injected and uninjected sides across two consecutive forebrain sections in at least five embryos and averaged. P-values are as follows: FoxG1 MO, 0.168; TLE2 MO, 0.64; Control MO, 0.1869. P>0.1 and, therefore, not significant in all cases.
	tle1 (transducin-like enhancer of split 1 (E(sp1) homolog)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, anterior view, dorsal up.
	tle1 (transducin-like enhancer of split 1 (E(sp1) homolog)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, anterior view, dorsal up.
	tle1 (transducin-like enhancer of split 1 (E(sp1) homolog)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	tle2 (transducin-like enhancer of split 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, anterior view, dorsal up.
	tle2 (transducin-like enhancer of split 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, anterior view, dorsal up.
	tle2 (transducin-like enhancer of split 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	tle5 (TLE family member 5, transcriptional modulator) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, anterior view, dorsal up.
	tle5 (TLE family member 5, transcriptional modulator) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, anterior view, dorsal up.
	tle5 (TLE family member 5, transcriptional modulator) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 27/28, lateral view, dorsal up, anterior left.
	foxg1 (forkhead box G1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, anterior view, dorsal up.
	foxg1 (forkhead box G1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, anterior view, dorsal up.
	foxg1 (forkhead box G1 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, anterior left, lateral view, dorsal up.

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