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The neural crest is a dynamic embryonic structure that plays a major role in the formation of the vertebrate craniofacial skeleton. Neural crest formation is regulated by a complex sequence of events directed by a network of transcription factors working in concert with chromatin modifiers. The high mobility group nucleosome binding protein 1 (Hmgn1) is a nonhistone chromatin architectural protein, associated with transcriptionally active chromatin. Here we report the expression and function of Hmgn1 during Xenopus neural crest and craniofacial development. Hmgn1 is broadly expressed at the gastrula and neurula stages, and is enriched in the head region at the tailbud stage, especially in the eyes and the pharyngeal arches. Hmgn1 knockdown affected the expression of several neural crest specifiers, including sox8, sox10, foxd3, and twist1, while other genes (sox9 and snai2) were only marginally affected. The specificity of this phenotype was confirmed by rescue, where injection of Hmgn1 mRNA was able to restore sox10 expression in morphant embryos. The reduction in neural crest gene expression at the neurula stage in Hmgn1 morphant embryos correlated with a decreased number of sox10- and twist1-positive cells in the pharyngeal arches at the tailbud stage, and hypoplastic craniofacial cartilages at the tadpole stage. These results point to a novel role for Hmgn1 in the control of gene expression essential for neural crest and craniofacial development. Future work will investigate the precise mode of action of Hmgn1 in this context.
Figure 1 Developmental expression of hmgn1 in Xenopus laevis embryos. (a) At gastrula stages (NF stage 10–12), hmgn1 transcripts(hmgn1-as; upper panels) are detected throughout the embryonic ectoderm. At neurula stages (NF stage 15–18) hmgn1 appears to be enriched atthe neural plate and the neural plate border (stage 18; arrowheads). At stage 23 and 33, hmgn1 is still broadly expressed with stronger expressionin the pharyngeal arches (brackets) and the developing eyes (arrows). Stages 10–18 are dorsal views, anterior to top. Stages 23–33 are lateralviews, dorsal to top, anterior to right. Staining with a control sense probe (hmgn1-s; lower panels) is shown for comparison. (b) Transversesections of a stage 33 embryo at the forebrain (f; left panel) and hindbrain (h; right panel) levels, highlight hmgn1 expression in the skin (arrows),brain, retina of the eye (r) and to a lesser extent in the otic vesicle (o)
FIGURE 2 Hmgn1 knockdown affects sox10 expression. (a) The sequence targeted by the Hmgn1 translation blocking morpholino antisenseoligonucleotide (Hmgn1MO) is depicted in red. Hmgn1MO targets the ATG start site (blue) of both hmgn1.S and hmgn1.L. The start sequence ofthe rescuing hmgn1 mRNA is shown for comparison, it lacks the 8 bp upstream of the ATG targeted by Hmgn1MO. (b) Western blot analysis fromlysates of embryos injected with hmgn1-Myc mRNA (100 pg) alone, or in combination with increasing doses of Hmgn1MO, 10 ng (+) and 100 ng(++). α-tubulin is shown as a loading control. (c) Unilateral injection of wild-type hmgn1 mRNA (Hmgn1WT; 10 pg) restores sox10 expression inHmgn1-depleted embryos (Hmgn1MO; 20 ng). The injected side is indicated by an asterisk. (d) Quantification of the phenotypes from fourindependent experiments. The number of embryos showing a given phenotype is indicated in each bar
FIGURE 3 Hmgn1 knockdown affects neural crest formation. (a) Phenotype of Hmgn1 MO-injected embryos (20 ng) on neural crest (upper row) and placode (lower row) gene expression at stage 15. The injected side is indicated by an asterisk. Dorsal views, anterior totop, except for dmrta1 and foxi4.1for which anterior views are shown. (b) Quantification of thephenotypes. The number ofembryos showing a givenphenotype is indicated ineach bar
FIGURE 4 Hmgn1 depletion does not affect eye and lens development. (a) Hmgn1 knockdown does not affect the expression of pax6 in the developing eye, prospective lens and forebrain at NF stage 23. Injected side (asterisk) isshowing the lineage tracer Red-Gal.Frontal view, dorsal to top. (b) The expression of foxe3 in the lens vesicle is not affected in Hmgn1 morphant embryos at NF stage 30. Injected side is showingthe lineage tracer Red-Gal. Lateral views,dorsal to top, anterior to left.(c) Quantification of the phenotypes. Thenumber of embryos showing a givenphenotype is indicated in each bar
FIGURE 5 Hmgn1 knockdown does not affect cell death or proliferation in the ectoderm. (a) TUNEL staining of representative Hmgn1 and Sf3b4 morphant embryos at stage 15. The injected side is indicated by an asterisk. Dorsal view, anterior to top. (b) Quantification of the number of TUNEL-positive cells in control and injected sides of Hmgn1MO (n = 40; left graph) and Sf3b4MO (n = 47; right graph) injected embryos at stage15. (c) pHH3 immunostaining of a representative Hmgn1 morphant embryos at stage 15. (d) Quantification of the number of pHH3-positive cells in control and injected sides of Hmgn1MO-injected embryos (n = 26). (b, d) Each dot represents one embryo. p-values were calculated using unpaired t-test with Welch's correction, **** p < .0001; ns: not significant
FIGURE 6 Hmgn1 knockdown affects neural crest streams formation. (a) Phenotype of CoMO (20 ng) and Hmgn1MO (20 ng) injected embryos at NF stage 23–25 analyzed for sox10 and twist1 expression. The red brackets indicate the extent of dorso-ventral extension of neural crest streams in control and MO-injected sides. (b) Higher magnification of an embryo stained with sox10 showing the position of the three neural crest streams. Lateral view, dorsal to top, anterior to left. (c,d) Graph plotting the distance migrated by neural crest cells in stream 1, 2, and3 of CoMO- and Hmgn1MO-injected embryos stained by sox10 (c) or twist1 (d). A similar number of embryos were analyzed for each gene(CoMO, n = 21 and Hmgn1MO, n = 30). Brown–Forsyth and welch analysis of variance test, with multiple comparisons test (Dunnett) with individual variances computed for each comparison. *, p = .0141; **, p = .0013; ***, p = .0003; **** p < .0001; ns: not significant
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