Koebernick K et al. (2006), Xenopus Teashirt1 regulates posterior identity ...

XB-ART-40

Dev Biol 2006 Oct 01;2981:312-26. doi: 10.1016/j.ydbio.2006.06.041.

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Xenopus Teashirt1 regulates posterior identity in brain and cranial neural crest.

Koebernick K , Kashef J , Pieler T , Wedlich D .

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We have isolated two related Xenopus homologues of the homeotic zinc finger protein Teashirt1 (Tsh1), XTsh1a and XTsh1b. While Drosophila teashirt specifies trunk identity in the fly, the developmental relevance of vertebrate Tsh homologues is unknown. XTsh1a/b are expressed in prospective trunk CNS throughout early neurula stages and later in the migrating cranial neural crest (CNC) of the third arch. In postmigratory CNC, XTsh1a/b is uniformly activated in the posterior arches. Gain- and loss-of-function experiments reveal that reduction or increase of XTsh1 levels selectively inhibits specification of the hindbrain and mid/hindbrain boundary in Xenopus embryos. In addition, both overexpression and depletion of XTsh1 interfere with the determination of CNC segment identity. In transplantation assays, ectopic XTsh1a inhibits the routing of posterior, but not of mandibular CNC streams. The loss of function phenotype could be rescued with low amounts either of XTsh1a or murine Tsh3. Our results demonstrate that proper expression of XTsh1 is essential for segmentally restricted gene expression in the posterior brain and CNC and suggest for the first time that teashirt genes act as positional factors also in vertebrate development.

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Species referenced: Xenopus laevis
Genes referenced: adam13 cdh11 chrd egr2 en2 epha4 hoxa2 hoxb9 meis3 nkx3-2 pax6 pcdh8 slc5a5 tshb tshz1 tshz3 twist1
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Phenotypes: Xla Wt + tshz1 (Fig 3 E) [+]

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	Fig. 1. Structure and expression pattern of XTsh1a. (A) Schematic representation of XTsh1a protein structure. Boxes representing unconventional Teashirt-type Zn finger motifs (Zn) are marked in dark gray, classical Zn finger motifs in light gray, the predicted homeodomain (HD) is indicated in white. (B) Real-time RT-PCR analysis of XTsh1a expression in embryonic stages. Samples were normalized to levels of ornithine decarboxylase (ODC). For better comparison, transcript amounts of the stages are shown relative to stage 6 which was set to 100%. (C–E) Spatiotemporal expression of XTsh1a in neurula stages. Dorsal view, anterior is right. (F) Transverse vibratome section corresponding to the dashed line indicated in panel D. (G, H) Double whole-mount in situ hybridization with XTsh1a probe (brown) in combination with Krox-20 (G, red) or En-2 (H, red) probes. Dorsoanterior view, anterior is down. (I–K) Expression of XTsh1a in tailbud embryos. (I) Transverse vibratome section at the hindbrain level (stage 26). (J, K) Lateral view, anterior is right. mhb; midbrain–hindbrain boundary, nc; notochord, ne; neuroectoderm, nt; neural tube, pn; pronephros, r3/r5; rhombomeres 3/5, dn; diencephalon, o; olfactory placode.
	Fig. 2. XTsh1a expression during CNC migration. Whole-mount in situ hybridization of XTsh1a alone (A, B) or XTsh1a (brown) together with Krox-20 (red, C–F). (A, B) Dorsal view, anterior is down. (B, D, E) Lateral view of head region, anterior is right. (F) Horizontal vibratome section of a stage 23 embryo (corresponding to D) stained for Krox-20 (red) and XTsh1a (purple) expression. The white arrow in panel F marks the XTsh1a-positive cell population.
	Fig. 3. Anteroposterior neural patterning in XTsh1a-injected embryos and XTsh1 morphants. After injection of XTsh1a mRNA (A, G; 500 pg, C, E; 125 pg) or XTsh1-MO (B, D; 2.5 pmol, F, H; 2 pmol) into one of two blastomeres, embryos were fixed at stages 14–16 and analyzed for the expression of different AP marker genes. Repression or complete loss of En-2 expression (A) appeared in 53% of XTsh1a-injected embryos (n = 26) and in 36% of XTsh1 morphants (n = 66). Reduction or absence of Krox-20 expression (B) was detected in 86% of XTsh1a-injected embryos (n = 27) and in 63% of XTsh1 morphants (n = 119) (C). XPax-6 expression was found to be reduced in the hindbrain rhombomeres (r3 and r5, red arrowheads) of 57% of the XTsh1a-injected embryos (n = 14) and in 45% of XTsh1 morphants (n = 104). (G, H) Otx-2 expression was still detectable in the manipulated side with a slight reduction of the signal intensity occurring in 40% of XTsh1a-injected embryos (G, stage 14, n = 20) and 50% of XTsh1a morphants (H, stage 14, n = 24). In XTsh1a mRNA injection experiments, only those embryos were scored, in which the region of marker gene expression overlapped with the staining of the β-galactosidase lineage tracer (light blue). (I, J) Sox-2 (pan-neural marker) expression remains unchanged. (K, L) XPAPC (paraxial mesoderm marker) expression was not altered. In panels I–L, fluorescein dextran was used as lineage tracer. is: injected side, nis: non-injected side. Anterior pole of the embryos is orientated to the lower edge of the frame.
	Fig. 4. Analysis of genes regulating brain patterning in XTsh1a-injected embryos and XTsh1 morphants. (A, B) XWnt-4 expression is unchanged in XTsh1a-injected embryos but repressed in XTsh1 morphants. (C, D) FGF-8 expression remains unaffected. (E–G) XMeis3 expression is unchanged in XTsh1a-injected embryos but shifts posteriorly in XTsh1 morphants. A–F: albino embryos of stage 16 were used for in situ hybridization, G: wild-type embryo of stage 19 is shown. Embryos were injected with XTsh1a mRNA (500 pg) or XTsh1-MO (2.5 pmol) into one of two cells. Fluorescein dextran was used as lineage tracer. is: injected side, nis: non-injected side. Anterior pole of the embryos is orientated to the upper edge of the frame.
	Fig. 5. CNC development in XTsh1a-injected embryos and XTsh1 morphants. Embryos were injected with XTsh1a mRNA (500 pg) or XTsh1-MO (2.5 pmol) into one of two cells, fixed at stage 16 (A), 19 (G) or 26 (B–F, H–J) and stained for the expression of the NC marker genes Twist (A–D, G–J) and AP-2 (E, F). At neurula stage, Twist expression was not altered in 88% (n = 17) of XTsh1a-injected embryos (A). Although manipulated embryos still express Twist at tailbud stage (stage 26), the expression pattern appears severely disorganized in the hyoid and branchial arches in 68% (C, n = 62) of the XTsh1a-injected embryos. Disruption of AP-2 expression occurred with similar frequencies (E, F). In XTsh1 morphants, Twist expression was also not reduced at late neurula stage (G). However, segmental patterning of the CNC streams appeared abnormal in 52% (n = 25) of the XTsh1-MO-injected embryos (G). At tailbud stage (H, I, stage 26), Twist was reduced in 48% and detected in a slightly irregular shape in 31% of XTsh1 morphants (n = 29). (D, J) Horizontal vibratome sections of the branchial arch region of Twist-stained embryos (stage 26) injected with XTsh1a (D, 500 pg) or XTsh1-MO (J, 2.5 pmol). Defective segmentation of the pharyngeal pouches is revealed in the injected side of XTsh1a-injected embryos (D) but not evident in XTsh1-MO-injected embryos at this stage (J), which exhibit reduced numbers of Twist-expressing cells in the CNC streams. is: injected side, nis: non-injected side.
	Fig. 6. Analysis of segmental CNC identities in Xenopus embryos upon overexpression and knockdown of XTsh1. Following injection of XTsh1a mRNA (A, B, E, F, I–L; 500 pg) or XTsh1-MO (C, D; 2.5 pmol, G, H; 2 pmol) into one of two blastomeres, embryos were fixed at stage 26 and analyzed for the expression of marker genes for the different pharyngeal arch segments. XTsh1-injected embryos exhibit strongly or completely reduced expression of the third arch marker genes Krox-20 (A, B, 84%; n = 19) and EphA4 (C, D, 60%; n = 20). Similar effects were observed in XTsh1a morphants, where CNC domains of Krox-20 (C, D) and EphA4 (G, H) were reduced in 56% (n = 18) and 8% (n = 26), respectively. XTsh1a-injected embryos also revealed a reduced expression of the second arch marker gene Hoxa2 (arrow, I, J, 55%, n = 20). Although reduced Xbap expression was observed in posterior pharyngeal arches of the XTsh1a-injected embryos (25%; n = 20), Xbap expression in the first pharyngeal arch (arrowheads in K and L) was not affected. is: injected side, nis: non-injected side, pa; pharyngeal arch.
	Fig. 7. XTsh1 misexpression inhibits branchial CNC migration. GFP-labeled CNC grafts were taken from anterior (A–D) or posterior (E–J) levels of control embryos (A, B, E, F), XTsh1a-injected embryos (500 pg, C, D, G, H) or XTsh1 morphants (2.5 pmol, I, J) and transplanted to uninjected host embryos. (K) Transplantation of a GFP-labeled control CNC graft to the posterior region of a host embryo, which had been co-injected with XTsh1a (1 ng) and dsRED2 (1 ng) mRNA into both blastomeres of the two-cell stage. Transplantations were performed at neurula stage (stage 14), and migration patterns were evaluated at stage 26. Normal light images and corresponding fluorescent images of the head region of representative examples are shown (anterior is right).
	Fig. 8. Selective influence of XTsh1a on posterior CNC migration pathways. For double transplantation experiments, control CNC grafts were labeled with GFP (500 pg, 1/2 cells) and XTsh1a-expressing grafts (1 ng, 2/2 cells) were labeled with dsRED2 (1 ng, 2/2 cells). The grafts were either placed to the axial position corresponding to their origin (homotopic; A, C) or interchanged to more posterior or anterior positions (heterotopic; B, D) of uninjected host embryos. Merged fluorescent images show typical results.
	tshz1 (teashirt zinc finger homeobox 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior down.
	tshz1 (teashirt zinc finger homeobox 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, lateral view, head region, anterior right, dorsal up.
	tshz1 (teashirt zinc finger homeobox 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior right, dorsal up.

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