Zorn AM, Krieg PA.

HD25179 NICHD NIH HHS , Wellcome Trust

	Figure 6.  Embryonic expression of Xqua mRNA. Whole-mount in situ hybridization using an RNA probe specific to the 5-kb embryonic form of Xqua. (A) Lateral view of a stage 11 gastrula embryo shows that transcripts are first detected in the chordamesoderm of the dorsal blastopore lip. (B) By late gastrula, Xqua is strongly expressed in the notochord with weak expression in the circumblastoporal region (B, embryo on the right, dorsal view). Embryos hybridized with sense strand control probes (B, embryo on the left) show no staining. In neurula embryos (stage 18) (C, dorsal view;D, posterior view; E, anterior view), the expression domain has expanded to include the notochord, paraxial mesoderm, circumblastoporal region, and the neural plate. In tailbud embryos (stages 24–30) (F,G), Xqua is strongly expressed in the brain and neural tube, the developing heart, tail blastema, and branchial arches. (G) By late tailbud (stage 30), the expression in the notochord and somites is declining. (H) Cross sections through the head of the stage 30 whole-mount embryo show that expression in the brain is largely restricted to the ventricular and marginal zones and the floor plate. (I) Later in development at hatched tadpole stage, Xqua expression is almost exclusively in the head and heart. (anp) Anterior neural plate; (bp) blastopore; (dl) dorsal lip; (h) heart; (n) notochord.
	Figure 7.  Overexpression of Xqua and δD causes defects in anterior development. Synthetic mRNA encoding either pXqua357, the truncated pXqua protein δD (which lacks the KH motif), or control rRNA was overexpressed in embryos by microinjection into the equatorial region of each of the two dorsal blastomeres at the four-cell stage. (A) Overexpression of rRNA (2.5 ng/blastomere) has no effect on development. (B) Dorsal injection of Xqua mRNA (2.5 ng/blastomere) resulted in exaggerated anterior development including elongation of the hindbrain region and reduced cement glands. (C) Schematic representation of pXqua357 and δD proteins. Representative embryos illustrating the phenotypes are shown. (D) The δD protein is able to form dimers with wild-type pXqua. 35S-Labeled pXqua357 or δD was incubated with either 2 μg of GST alone or a GST–Xqua fusion protein coupled to glutathione–Sepharose. The bound proteins were resolved on 12% SDS-PAGE and visualized by fluorography. The lane marked T represents 20% of the input translation. (E) δD/pXqua heterodimers have reduced RNA-binding activity. 35S-Labeled pXqua was synthesized in vitro, either alone or by cotranslation with a fivefold excess of δD mRNA. The RNA-binding activity of the wild-type pXqua, or the δD/pXqua mixture, was assayed by binding to poly(G)–agarose. Bound proteins were resolved on 12.5% SDS-PAGE and quantitated by PhosphorImager analysis. The results, averaged from three separate experiments, are presented as a histograph that indicates that δD/pXqua heterodimers exhibit impaired RNA binding. Dorsal overexpression of δD resulted in a dose-dependent deletion of anterior structures ranging from (F) microcephalic and (G) acephalic embryos at low mRNA levels (0.5–1.25 ng/blastomere) to (H) severe anterior-half truncations at higher doses (2.5 ng/blastomere). (I) Lineage tracing of embryos coinjected with a high dose of δD and β-galactosidase mRNA shows that the phenotypic effects of δD are specific to dorsal tissues. The tissue expressing the injected mRNA is stained blue.
	Figure 8.  Histological and immunostaining analysis of abnormal development resulting from dorsal Xqua and δD overexpression. Control rRNA, Xqua, or δD was microinjected into the equatorial region of each of the two dorsal blastomeres at the four-cell stage (2.5 ng/blastomere, except embryo in C that was 1.25 ng/blastomere). Representative embryos were examined by (A–D) histological sectioning and hemotoxylin/eosin staining or by whole-mount immunostaining with the monoclonal antibodies (E,H,K) Anti-NCAM for neural tissue, (F,I,L) 12/101 for somites, and (G,J,M) MZ15 for notochord. Control embryos injected with rRNA are shown in A, E, F, and G; embryos overexpressing Xqua are shown in B, H, I, and J; and embryos overexpressing δD are shown in C, D, K, L, and M. In all panels, anterior is left and a lateral view is shown, except thebottom image in G and J, which are dorsal views. (A,G) In control embryos the notochord ends anteriorly behind the forebrain. (B) Dorsal injection of Xquaresults in an elongation of the notochord into the head until it reaches the ventral pharyngeal endoderm and (B,H) a reduction in forebrain and anterior facial structures with an enlarged hindbrain vesicle. (J, dorsal view) Xqua overexpression occasionally results in duplications of the notochord and facial structures; notice two notochords lying side by side and two cement glands. (I) The somites are relatively normal in Xquaoverexpressing embryos. (C) Histological section of embryos with a low-level overexpression of δD (1.25 ng/blastomere) shows a truncation of the anterior notochord with a loss of forebrain and anterior head structures. (D,K,L,M) High-level overexpression of δD (2.5 ng/blastomere) causes a complete truncation of the anterior half of the embryos. (K) These embryos exhibit little neural tissue, (L) reduced somites, and (M) only small amounts of posterior notochord tissue. δD embryos show a high frequency of spina bifida resulting in a splitting of the residual notochord tissue; notice the duplicated notochord in the tail. (cg) Cement gland; (fb) forebrain; (hb) hindbrain; (n) notochord; (o) otic vesicle; (pe) pharyngeal endoderm.
	Figure 9.  Overexpression of the pXqua mutant δD inhibits notochord differentiation. rRNA (2.5 ng), Xqua, or δD mRNA was injected into each of the two dorsal blastomeres at the four-cell stage. Dorsal mesoderm explants containing presumptive notochord and somite tissue were dissected from injected embryos at early gastrula (stage 10–10.5) and cultured in isolation to examine notochord differentiation directly. (A,C,E,G) External morphology of the explants is shown when sibling embryos (WE) reached early neurula stage. (B,D,F,H,I). At later tailbud stage, explants were fixed, sectioned, and immunostained with MZ15 for notochord (black) and 12/101 for somite (red). From a total of 12 explant experiments, a section through a typical explant is presented. (C) Control rRNA explants exhibit considerable elongation resulting from convergent extention movements of the developing notochord, and (D) immunostaining of control explants shows a substantial amount of notochord and somite tissue. (E)Xqua overexpressing explants exhibit more dramatic morphological elongation and (F) contain moderately more notochord and somite tissue than controls (cf. amount of unstained tissue to that in D). (G) Explants overexpressing δD exhibit very little morphological elongation compared with controls (cf. to C) and (H) show dramatically less notochord tissue but little change in the amount of somite tissue. (I) Inhibition of notochord differentiation owing to δD overexpression is rescued by coinjection of an equal amount of synthetic Xqua mRNA (5 ng of δD + 5 ng ofXqua) (cf. amount of notochord in I to H andD), indicating that the biological effects of δD overexpression are specific to the Xqua pathway.
	Figure 10. Inhibition of Xqua function results in down-regulation of early notochord marker mRNAs. mRNA (2.5 ng) encoding the dominant-negative pXqua protein δD or δD plus β-galactosidase was microinjected into the equatorial region of each of the two dorsal blastomeres at the four-cell stage. The resulting embryos, together with uninjected control embryos, were fixed at gastrula stages and analyzed by whole-mount in situ hybridization for expression of early dorsal mesoderm and notochord mRNA markers:Xnot-2 (A–C), Xbra (D–I), andgsc (J–L). Arrows indicate the location of the dorsal lip in early stage embryos. In C, F, I, and L,embryos were stained with X-gal (blue) prior to in situ hybridization (purple/brown), to indicate the cells that received the δD mutant. Overexpression of δD in the dorsal mesoderm results in disruption of gastrulation and a loss of detectable Xnot-2, Xbra, and gsc transcripts in recipient cells.
	qki ( QKI, KH domain containing, RNA binding ) gene expression in Xenopus laevis embryos, NF stage 24, as assayed by in situ hybridization, lateral view, anterior right, dorsal up.
	qki ( QKI, KH domain containing, RNA binding ) gene expression in Xenopus laevis embryos, NF stage 30, as assayed by in situ hybridization, lateral view, anterior right, dorsal up.
	qki ( QKI, KH domain containing, RNA binding ) gene expression in Xenopus laevis embryos, NF stage 30, as assayed by in situ hybridization, cross section through the head, anterior up.
	qki ( QKI, KH domain containing, RNA binding ) gene expression in Xenopus laevis embryos, NF stage 30, as assayed by in situ hybridization, lateral view, anterior right, dorsal up.

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