Effects of hmgb family genes on mesoderm gene expression and early development in zebrafish. Embryos were injected with different mRNAs as indicated on the left and in situ hybridization was performed at the 50% epiboly stage using the markers indicated on the top. (A-D) Dorsal view of gsc expression pattern. (E-H) Lateral view of ntl expression. (I-L) Animal pole view of eve1 expression. (M-P) Lateral view of live images of control and injected embryos at 48 hpf with anterior to the left.

HMGB3 up-regulates gsc expression at different stages and inhibits ntl expression at the injection site. Embryos were injected with hmgb3 mRNA alone or coinjected with membrane GFP mRNA, they were cultured to various stages for in situ hybridization using gsc or ntl probe. (A-C) gsc expression in control embryos at the 50% epiboly (A), shield (B) and bud (C) stages. (D-F) gsc expression in hmgb3-injected embryos at the 50% epiboly (D), shield (E) and bud (F) stages. (G and H) ntl expression in GFP-injected (G) and GFP and hmgb3 coinjected (H) embryos. Brown color indicates membrane GFP-labeled cells. (I) Summary of the down-regulated ntl expression domain in hmgb3-injected embryos, as analyzed by double in situ hybridizations using ntl and gsc probes (see Fig. S2). The numbers on the top indicate total embryos analyzed from two independent experiments

Effects of different hmgb3 mutants on mesoderm gene expression and on early development in zebrafish. Embryos were injected with different mRNAs corresponding to different hmgb3 mutants as indicated on the left and in situ hybridization was performed using the markers indicated on the top. (A-D) Dorsal view (expect B, which is animal pole view) of gsc expression at 50% epiboly. (A) A control embryo. (B) A hmgb3EnR-injected embryo shows expansion of gsc expression. (C) A hmgb3VP16-injected embryo shows weakly inhibited gsc expression. (D) A hmgb3ΔC-injected embryo with expansion of gsc expression. (E–H) Animal pole view of ntl expression at 50% epiboly. The uninjected control embryo (E) shows circular ntl expression at the margin, while the hmgb3EnR-injected embryo (F) shows strong inhibition of ntl expression. hmgb3VP16 (G) has no obvious effect on ntl expression and hmgb3ΔC (H) moderately inhibits ntl expression. (I-L) Animal pole view of eve1 expression at 50% epiboly. (I) An uninjected embryo. (J) hmgb3EnR blocks eve1 expression. (K) hmgb3VP16 does not affect eve1 expression pattern. (L) hmgb3ΔC inhibits eve1 expression to a lesser extent than hmgb3EnR. (M–P) Lateral view of live images of control (M) and injected embryos at 48 hpf with anterior to the left. The embryos injected with hmgb3EnR (N) or hmgb3ΔC (P) mRNA show trunk and posterior deficiency, while the hmgb3VP16-injected embryo (O) shows weak anterior deficiency. (Q) Summary of the phenotypes following overexpression of different hmgb mRNAs and hmgb3 mutants. Severely affected embryos are as shown in N with absence of trunk and posterior regions, mild phenotypes are intermediate between normal and severely affected, as shown in Fig. 1N-P. Numbers on the top indicate total embryos scored from four independent experiments. (R) Summary of the phenotypes following hmgb3VP16 overexpression. Severely affected embryos show absence of eyes, while mild phenotypes are as shown in O. Numbers on the top indicate total embryos scored from three independent experiments.

HMGB3 differentially regulates dorsoventral mesoderm gene expression. (A1–B5) Animal pole view of chordin (A1-A5) and flh (B1-B5) expression at the shield stage in control and embryos injected with wild-type hmgb3 or its mutants, as indicated on the top. Dorsal region is to the right. The expression of these genes is expanded in hmgb3-, hmgb3ΔC- and hmgb3EnR-injected embryos. (C1-C5) Lateral view of sqt expression which is reduced in hmgb3-, hmgb3ΔC- and hmgb3EnR-injected embryos. (D1-I5) Animal pole view of fgf8 (D1-D5), wnt11 (E1-E5), wnt8a (F1-F5), tbx6 (G1-G5), vent1 (H1-H5) and vox (I1-I5) expression at 50% epiboly. For F1-I5, dorsal is to the right. Note that HMGB3, HMGB3ΔC and HMGB3EnR inhibit vox expression in the ventrolateral region of mesoderm, but expand its expression in the ectoderm.

Expression of ectoderm, neuroectoderm and endoderm markers in embryos overexpressing hmgb3 and different mutants. (A-E) Animal pole view of otx2 expression at 80% epiboly in control and injected embryos, as indicated on the top. (A′-E′) Lateral view of the same embryos as in A–E. (F-J) Dorsal view of hoxb1b expression at 80% epiboly in control and injected embryos. (F′-J′) Lateral view of the same embryos as in F–J, dorsal region is to the right. (K-L) Animal pole view of gata2a expression at 50% epiboly in control and injected embryos as indicated on the top, dorsal is to the right. (P-T) Dorsal view of sox32 expression at 75% epiboly in control and injected embryos as indicated on the top.

Wild-type HMGB3 and HMGB3 mutants inhibit the mesoderm-inducing activity of exogenous factors in zebrafish and Xenopus embryos. (A–K) Animal pole view of ntl (A–G) and gsc (H–K) expression at the 50% epiboly stage of zebrafish embryos previously injected with the mRNAs as indicated on the top. (L) RT-PCR analysis of Xbra and wnt8 gene expression in Xenopus ectoderm explants previously injected with the indicated mRNAs. Fibronectin (FN) was used as an input control. (M) Quantification of Xbra and wnt8 expression level in L. The intensity of Xbra and wnt8 was normalized to that of FN.

HMGB3 and HMGB3ΔC inhibit mesoderm gene expression and mesoderm formation in Xenopus whole embryo. Synthetic mRNA was injected along with the Lac Z mRNA as a cell lineage tracer at the 4-cell stage and in situ hybridization was performed using the markers as indicated on the top at the early gastrula (A-I), late gastrula (J-Q) or tail-bud (P-R) stage in control or injected embryos as indicated on the left. (A-C) Vegetal view of Xbra expression. (D-F) Dorso-vegetal view of type II cytokeratin expression. (G-I) Dorso-vegetal view of Xema expression. (J-L) Dorso-vegetal view of sox2 expression. (M-O) Dorsal view of sox3 expression. (P-R) Lateral view of myosin light chain (MLC) expression. Arrows indicate the dorsal lip of blastopore.

Double in situ hybridizations of ntl and gsc expression in hmgb3-injected embryos. (A) A control embryo. (B–D) hmgb3-injected embryos with ntl expression down-regulated in the dorsal (B), ventral (C) or lateral (D) region. The expression domain of gsc is stained in red (arrowheads).

Acknowledgments
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Reprinted from Mechanisms of Development, 129(9-12), Cao, J.M., Li, S.Q., Zhang, H.W., and Shi, D.L., High mobility group B proteins regulate mesoderm formation and dorsoventral patterning during zebrafish and Xenopus early development, 263-274, Copyright (2012) with permission from Elsevier. Full text @ Mech. Dev.