Juraver-Geslin HA et al. (2014), The conserved barH-like homeobox-2 gene barhl2 ...

XB-ART-50448

Dev Biol 2014 Dec 01;3961:107-20. doi: 10.1016/j.ydbio.2014.09.027.

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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.

Juraver-Geslin HA , Gómez-Skarmeta JL , Durand BC .

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In this study, we investigated the gene regulatory network that governs formation of the Zona limitans intrathalamica (ZLI), a signaling center that secretes Sonic Hedgehog (Shh) to control the growth and regionalization of the caudal forebrain. Using loss- and gain-of-function, explants and grafting experiments in amphibians, we demonstrate that barhl2 acts downstream of otx2 and together with the iroquois (irx)-3 gene in establishment of the ZLI compartment initiated by Shh influence. We find that the presumptive (pre)-ZLI domain expresses barhl2, otx2 and irx3, whereas the thalamus territory caudally bordering the pre-ZLI expresses barhl2, otx2 and irx1/2 and early on irx3. We demonstrate that Barhl2 activity is required for determination of the ZLI and thalamus fates and that within the p2 alar plate the ratio of Irx3 to Irx1/2 contributes to ZLI specification and size determination. We show that when continuously exposed to Shh, neuroepithelial cells coexpressing barhl2, otx2 and irx3 acquire two characteristics of the ZLI compartment-the competence to express shh and the ability to segregate from anterior neural plate cells. In contrast, neuroepithelial cells expressing barhl2, otx2 and irx1/2, are not competent to express shh. Noteworthy in explants, under Shh influence, ZLI-like cells segregate from thalamic-like cells. Our study establishes that Barhl2 activity plays a key role in p2 alar plate patterning, specifically ZLI formation, and provides new insights on establishment of the signaling center of the caudal forebrain.

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Species referenced: Xenopus laevis
Genes referenced: aacs arx barhl2 cat.2 ctnnb1 emx2 fezf2 irx1 irx2 irx3 nkx2-1 nog otx2 pax6 shh tcf4
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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).
	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).
	Fig. 3. Within alar p2, depletion of Irx1 and Irx2 activities, or an increase in Irx3 activity, promotes ZLI specification. Double ISH or ISH on embryos (A–D) either depleted for both Irx1 and Irx2 activities or (E–H) overexpressing irx3, or (I) overexpressing irx3-GR using barhl2 (A, E), otx2 (B, F), irx3 (C), irx1 (G), shh (D, H, I) probes as indicated. The scale bar stands for 0.5 mm. RNA encoding for irx3, or irx3-GR were co-injected with a MO control used as tracer, coupled to fluorescein detected by immunohistochemistry (red). (A–D) Co-depletion of Irx1/2 shifts the ZLI caudal border. In st. 32 embryos depleted for irx1/2 (A) barhl2 (n=24) expression is not significantly affected, and (B) otx2 (n=20) expression is slightly decreased. We observed (C) a weak increase of irx3 in the thalamic domain (n=36). (D) The ZLI territory, characterized by shh expression, is enlarged (n=36); DISH using barhl2 together with shh probes shows that the barhl2 anterior border and the ZLI anterior border coincided, indicating that the ZLI posterior boundary is caudally shifted. (E–I)irx3 or irx3-GR overexpression promotes ZLI specification. At st. 32 in irx3 overexpressing embryos (E) barhl2 (n=40) expression is modified compared with the CT sides whereas (F) otx2 (n=40) and (G) irx1 (n=40) expression are weakly decreased in the caudal p2 domain. (H) In irx3 overexpressing embryos the surface of the ZLI territory marked by shh is enlarged (n=22). DISH using barhl2 together with shh as probes demonstrates that the ZLI anterior border is not affected. Note that in embryos overexpressing Irx3 the expansion of the ZLI territory is associated to a decrease in shh staining intensity. (I) In embryos overexpressing a hormone-inducible form of irx3 (irx3-GR) and exposed to dexamethasone at st. 20, the surface of the ZLI territory marked by shh is enlarged (n=10). (J) The average width of p2 is not modified in embryos depleted of Irx1/2 or overexpressing Irx3. Using barhl2 as a p2 marker, the width of p2 was measured (Image J) on both the non-injected and the injected sides of embryos injected with GFP (CT, n=11), depleted for Irx1/2 (MOirx1/2, n=12) or overexpressing Irx3 (Irx3, n=14). The ratio of the p2 width of the injected side relative to the control side is shown. The error bars indicate the standard deviation. (K and L) The ZLI surface is significantly increased in Irx3 overexpressing embryos. (K) We delimited the alar/basal plate boundary of the diencephalon by drawing a line at the base of the ZLI area. We measure the ZLI area as the alar p2 area expressing Xshh. (L) Using Image J the surface of the ZLI territory was measured on both the non-injected and the injected sides of embryos injected with GFP (CT, n=9), overexpressing Irx3 (Irx3, n=9), or overexpressing a hormone-inducible form of Irx3 (Irx3-GR, n=9). The average ratio of the ZLI surface of the injected size relative to the control size is shown. The error bars indicate the standard deviation. We observed a significant increase of the ZLI surface in both Irx3 overexpressing embryos (R=1.5±0.19, t-test p≤0.004) and Irx3-GR overexpressing embryos (R=1.2±0.17, t-test p≤0.03).
	Fig. 4. In animal cap explants barhl2, otx2 and irx3 co-expression enables induction of shh expression by Shh. (A) Experimental design: AAC were prepared from embryos injected with RNA as indicated. AACs were sandwiched to yield ßGal/GFP conjugates and cultured for 48 h. In experiments shown in (D) the N-Shh-expressing part of the explant was replaced by beads. AACs were analyzed by ISH for expression of Xenopus (X) shh. The % of explants showing the phenotype is indicated. The scale bar stands for 0.5 mm. Representative sandwiched explants are shown. (B) Explants of anterior, or p2-like, neuroepithelial identity cultured in the presence of N-Shh are not competent to express shh. AACs expressing noggin (a) or barhl2 and otx2 (b) were sandwiched with AACs expressing N-Shh. Neither AACs (100%, n=72), nor p2-like explants (barhl2+otx2) (100%, n=24) expressed endogenous Xshh when exposed to N-Shh. (C, D) In the presence of N-Shh pre-ZLI’s cells are competent to express Xshh. Pre-ZLI (barhl2, otx2, irx3) explants expressing or not ßGal (red) as indicated were sandwiched with: (C) (a) AACs (100%, n=72), or (b) AACs expressing N-Shh (b) (55%, n=96), (c) (72%, n=25). Enlarged image are shown in (b). Clusters of cells expressing Xshh appear along the border between the (barhl2, otx2, irx3)- and the Shh-expressing parts of the explants (white arrow). (D) beads (a) without N-Shh (88%, n=25) or (b) with N-Shh (76%, n=25). (c) Enlarged image of (b): clusters of cells expressing Xshh developed in close contact with the N-Shh-impregnated beads. The gray circle indicates the bead’s location. (E) Induction of Xshh occurs within barhl2, otx2, irx3 expressing cells and when any of the transcription factors barhl2 or otx2 or irx3 is absent, Xshh expression is not induced in explants. (a) Pre-ZLI (barhl2, otx2, irx3) explants expressing ßGal (red) were sandwiched with AACs expressing N-Shh (72%, n=25). Representative explants and vibratome sections (30 µm) are shown. The Xshh expressing-cells exhibit ßGal activity. Representative sandwiched AACs are shown for: (b) AACs (barhl2+irx3) with AACs N-Shh (100%, n=24), (c) AACs (otx2+irx3) with AACs N-Shh (92%, n=24). The scale bar stands for 0.5 mm.
	Fig. 5. In reaggregated explants cells expressing pre-ZLI genes exhibit Shh-dependent segregation behaviors: (a) Experimental design: AAC were prepared from embryos injected as indicated. At st. 8/9 ßgal and gfp cells are dissociated and reaggregated to generate explants made of Gfp/ßGal (red) mixed cell types. The explants are cultured for 48 h. (b, c) Representative explants composed of mixed cells expressing the different neuroepithelial genes as indicated are shown (b) without N-Shh (c) with N-Shh (100%, n≥10 for each condition). The % of explants showing the phenotype is indicated. The scale bar stands for 0.5 mm. (A) AAC cells do not segregate from Otx2-expressing cells. AACs cells expressing otx2 spread randomly when mixed with AACs cells (red) (b) in the absence (100%, n=12) and (c) in the presence of N-Shh (100%, n=10). (B) Pre-ZLI-like cells segregate from Otx2-expressing cells. Pre-ZLI cells (red) regrouped and separated from anteriorized neuroepithelial cells in the absence (b) and presence (c) of N-Shh (100%, n=10 for each condition). (F) In the presence of N-Shh pre-ZLI-like cells segregate from thalamus-like cells (b, c) pre-ZLI cells (red) regrouped, and separated from thalamus-like cells in the presence (c) but not in the absence (b) of N-Shh (100%, n=10 for each condition).
	Fig. 6. In pre-ZLI-like/thalamus-like explant sandwiches, cells recapitulate the main features of their in vivo developmental program. (A, B) In sandwiched explants pre-ZLI-like cells segregate and form a boundary with thalamus-like cells in the presence of N-Shh. (a) Experimental design: cRNA were injected as indicated. (b, c) Representative explants of thalamus-like AACs (red) sandwiched with pre-ZLI-like AACs without (A) or with (B) N-Shh. (A) In the absence of N-Shh pre-ZLI-like cells did not express Xshh and intermingled with thalamic-like cells. (c) Enlarged views and (d) section, of a representative explant. In contrast (B) in the presence of N-Shh pre-ZLI-like cells expressed Xshh and formed a boundary with thalamus-like cells (85%, n=15). (c) Enlarged views and (d) section, of a representative explant.
	Fig. 7. When ectopically expressed in the developing neuroepithelium and continuously exposed to Shh, pre-ZLI-like cells recapitulate the characteristic features of their developmental program. (A) Experimental design: AAC were prepared from embryos injected with RNA as indicated. At st. 8/9 cells were dissociated and reaggregated to generate explants made of mixed cell types. Pieces of explants were grafted into sibling’s neural plate at st. 14. Embryos were let to develop until st. 35. (B, C) Grafted cells expressing barhl2, otx2, irx3 develop ectopic ZLI when continuously exposed to Shh. (B) ISH using Xshh as probe on st. 35 embryos grafted with AACs (otx2+N-Shh) (a), or AACs (barhl2, otx2, irx3+N-Shh) (b) mixed explants. The grafted sides of st. 35 representative neural tubes are shown, side view, dorsal up, anterior left. ZLI: zona limitans intrathalamica; fp: floor plate. (c) Enlarged view of grafted cells from (b). (C) Analysis of grafting experiments. In embryos grafted with (otx2+N-Shh) mixed explants no ectopic ZLI develop in ßGal-expressing cell clusters (shown in blue) (100%, n=18). In contrast in embryos grafted with (barhl2, otx2, irx3+N-Shh) mixed explants most embryos developed ectopic ZLI (89%, n=26). From 35 identified ectopic ZLI, 25 developed in contact with an endogenous source of Shh – floor or basal plates, the ZLI – (shown in orange) and 10 developed autonomously (shown in green). (D) Injected pre-ZLI cells develop ectopic ZLI when continuously exposed to Shh. ISH using Xshh as probe on st. 36 embryos injected into the same dorsal blastomere of 16-cell stage embryos with (a) ßgal alone, or together with barhl2, otx2, irx3 genes (b, c). The injected sides of st. 36 neural tubes are shown, side view, dorsal up, anterior left. When in contact with a source of secreted Shh, pre-ZLI-like cells (red) formed cellular clusters inside which Xshh expression is detected (stars). (c) Enlarged view of an “ectopic ZLI”.
	Fig. 8. Some patterning defects observed in Barhl2 depleted embryos are rescued by decreasing ß-catenin levels, but not ZLI development. (A–D) ISH on embryos depleted for barhl2 (a, b) or both barhl2 and ßcatenin (c) using pax6 (A), irx1 (B), irx2 (C), and shh (D) as probes. The CT (MObarhl2) and rescued (MObarhl2/MOßcat) embryos were generated during the same set of experiments, and were coinjected with a MO control used as tracer, coupled to fluorescein detected by immunochemistry (red). For MObarhl2/MOßcat injected embryos, only the injected side is shown. The scale bar stands for 0.2 mm. (E–I) Barhl2 depletion patterning defects are partly rescued by decreasing ß-catenin levels. Barhl2 depletion phenotypic defects, specifically (A) pax6 ectopic expression in the mid-diencephalic furrow (n=25), (B, C) the decrease of irx1 (n=20) and irx2 (n=22) thalamic expression are partly rescued by depletion of ßcat. (D) The loss of ZLI development observed in Barhl2-depleted embryos is not rescued by decreasing ß-catenin levels. At st. 37 the progression of shh inside the p2 alar plate is inhibited in both Barhl2 depleted embryos (b, n=36) and Barhl2/ßCat double morphants embryos (c, n=36).
	Figure S1: Complementary analysis of anterior neural tube patterning defects in Barhl2-depleted embryos. ISH on Barhl2 morphants embryos (MObarhl2) at stages 27, 31 as indicated. The control (CT) and injected sides (MObarhl2) of a representative neural tube are shown anterior left, dorsal up. The red star indicates the pineal gland located on top of p2. The putative anterior and posterior limits of p2 are indicated with black dashed lines. (A) The expression of the p3 prosomeric marker emx2 which marks the pallium and the alar part of p3 at st.27 (n=18) is unaffected by inhibition of Barhl2 activity.
	Figure S2: Time course analysis of irx3 expression in Xenopus forebrain between st.30 and st. 37. (a) At st. 29-30 irx3 transcripts are detected in the alar p2 of the Xenopus forebrain. (b, c) Between st.32 and st.36-37 irx3 expression is restricted to the ZLI and the epithalamus. The stages are indicated.