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Gene/Clone	Species	Stage	Anatomy Item	Experimenter
irx2	xenopus		forebrain

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Experiment details for irx2

Juraver-Geslin HA et al. (2014) Assay

The conserved barH-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by Sonic Hedgehog.

Gene	Clone	Species	Stages	Anatomy
irx2.L		laevis	NF stage 29 and 30	forebrain

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Fig. 1. The ZLI develops inside the rostral alar p2 territory that expresses barhl2, otx2, irx3 and does not express irx1 and irx2. ISH or Double ISH on wt embryos, shown as dissected neural tubes from a side view, dorsal up, anterior left. The markers and stages are indicated. (D)–(P) Enlargement views centered on p2 as indicated by the red square on A. The pineal gland (red star) located on top of p2 is used as a morphological landmark. The black dashed lines are indicative of the putative anterior and posterior borders of p2. The scale bar stands for 0.5 mm. p: prosomere; MDF: Mid Diencephalic Furrow. (A–C) Time course analysis of shh expression in the Xenopus forebrain: (A) at st. 29 and st. 30 shh is only expressed in the forebrain floor and basal plates; (B) at st. 32 shh is detected in the forming ZLI; (C) at st. 37 development of the ZLI is mostly achieved. (D–L) At st. 30 and st. 31 the p2 alar plate contains two subdomains. (D) The ZLI forms in a domain that expresses barhl2. (E) The p2 anterior limit of barhl2 abuts that of fezf2, which marks the p3/p2 boundary; in the p2 alar plate barhl2 is co-expressed with (F) otx2 and (G) irx3. (H) pax6 marked p1 and p3 but is excluded from p2, except for the most dorsal part, which gives rise to the epithalamus. barhl2 is expressed in the part of the p2 domain devoid of pax6 expression, i.e. the mid-diencephalic furrow. A comparative analysis of barhl2 and (I–L) iroquois expression revealed two subdomains inside the alar p2: a rostral p2 domain that expresses barhl2 (I, K), otx2 (F), irx3 (G, J, L) and a caudal p2 domain that expresses barhl2 (I, K), otx2 (F), irx3 (G, J, L), irx1 (I, J) and irx2 (K, L). (M–P) The ZLI develops inside the rostral p2 domain. Double ISH on st. 32 and st. 36 dissected neural tubes with shh and iroquois genes that mark either the rostral p2 territory (M, N) irx3, or the caudal p2 territory (O, P) irx1 as probes. The alar progression of shh expression occurs in a domain that expresses irx3 (M, N) but not irx1 (O, P). (Q) Schematic of diencephalic markers at st. 31. Prosomere p2 is indicated in blue; Areas of expression are shown for fezf2 (green) a marker of the p3–p2 border; nkx2.1 (purple) that marks basal p2; shh (red) marks the p2 basal plate and the ZLI; p1, p3 and the epithalamus are revealed using pax6 (yellow). (R) Schematic of dynamics of p2 alar plate markers expression: The basal plate expresses shh (light blue). Within the p2 territory devoid of pax6 but expressing barhl2 and otx2: At stage 30 the rostral domain expresses irx3 and not irx1/2 (orange) the caudal domain expresses irx1/2/3 (green). At stage 37 the rostral domain expressed the same markers and shh (light blue) and the caudal domain expresses irx1/2 (green).

Gene	Clone	Species	Stages	Anatomy
irx2.L		laevis	NF stage 32	forebrain

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Fig. 2. Barhl2-depleted embryos exhibit defects in p2 alar plate patterning. Gene expression profiles of forebrain markers in Barhl2 morphants (MObarhl2) are shown at st. 27, st. 32 and st. 37 as indicated. Both the control (CT) and the injected (INJ) sides of a representative neural tube are shown, anterior left, dorsal up, allowing direct comparison of expression territories. n: number of embryos analyzed. The scale bar stands for 0.4 mm. In Barhl2-depleted embryos (A) the p3–p2 limit, marked by the caudal limit of the dorsal prethalamic marker fezf2 (n=24), or (B) arx2 (n=24) is established properly. The p2-like cells in MObarhl2-injected embryos are present and, at least in part, specified as shown by (C) the expression of the p2 basal plate marker nkx2.1 (n=16), (D) that of otx2 (n=24) and (E) tcf4 (n=20). However, the p2 alar plate is misspecified: (F) the domain in which pax6 is expressed spreads ventrally inside the mid-diencephalic furrow (n=20); In the rostral p2 (G) irx3 expression remains unchanged compared to the CT side (n=24); Conversely (G) irx3 expression is expanded in the caudal p2 of barhl2-depleted embryos (n=24), and expression levels of both caudal p2 markers (H) irx1 (n=24) and (I) irx2 (n=36) were down-regulated upon depletion of Barhl2 activity. (J) At st. 37 the progression of shh inside the p2 alar plate is inhibited (n=16).