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Luo T
,
Xu Y
,
Hoffman TL
,
Zhang T
,
Schilling T
,
Sargent TD
.
???displayArticle.abstract??? Inca (induced in neural crest by AP2) is a novel protein discovered in a microarray screen for genes that are upregulated in Xenopus embryos by the transcriptional activator protein Tfap2a. It has no significant similarity to any known protein, but is conserved among vertebrates. In Xenopus, zebrafish and mouse embryos, Inca is expressed predominantly in the premigratory and migrating neural crest (NC). Knockdown experiments in frog and fish using antisense morpholinos reveal essential functions for Inca in a subset of NC cells that form craniofacial cartilage. Cells lacking Inca migrate successfully but fail to condense into skeletal primordia. Overexpression of Inca disrupts cortical actin and prevents formation of actin "purse strings", which are required for wound healing in Xenopus embryos. We show that Inca physically interacts with p21-activated kinase 5 (PAK5), a known regulator of the actin cytoskeleton that is co-expressed with Inca in embryonic ectoderm, including in the NC. These results suggest that Inca and PAK5 cooperate in restructuring cytoskeletal organization and in the regulation of cell adhesion in the early embryo and in NC cells during craniofacial development.
Fig. 2. Expression patterns in Xenopus, zebrafish and mouse embryos. (A) Developmental northern blot of Xenopus Inca, with 18S RNA as a loading control. Nieuwkoop-Faber stages are indicated. (B-H) Whole-mount in situ hybridization for Inca in Xenopus embryos at stages 10.5-32. (B) Stage 10.5, sagittal section. d, dorsal. v, ventral. (C) Stage 14, dorsal view of expression in cranial NC (nc) and notochord (n). a, anterior. p, posterior. (D) Stage 19, dorsal view of expression in cranial NC migration streams, labeled as 1-3. (E) Stage 25, lateral view; arrowheads indicate approximate section levels in F and G. NC migration streams into pharyngeal arches 1-3 as indicated. (F,G) Transverse sections of embryo in E show Inca expression in headmesenchyme (hm) and NC (nc). (H) Stage 32, lateral view of the head. White and red arrowheads indicate expression in trigeminal ganglion and eye, respectively. (I-N) Whole-mount in situ hybridization for inca1 in zebrafish embryos. (I) 6 hpf, lateral view, dorsal to the right. Expression is restricted away from the margin (arrows), in future ectoderm (ecto). (J) 8 hpf, dorsal view. noto, notochord. (K,L) 13 hpf, lateral and dorsal views, respectively, of inca1 expression in premigratory NC. Numbers indicate presumptive pharyngeal arches 1-3. (M,N) 36 hpf, dorsal and face-on views, showing expression in the pharyngeal arches (1,2), diencephalon (di), pituitary (pi) and olfactory epithelia (oe). (O,P) Inca expression in mouse embryos at E9.5 (O) and E11.5 (P) in pharyngeal arches (numbered 1-3), somites (s) and fore-limb (fl) and hind-limb buds (hl). (Q) Northern analysis of adult mouse tissue RNAs probed with Inca and beta-actin as control. Scale bars: 500 μm in O; 1 mm in P.
Fig. 5. Inca is required for cranial NC specification and survival after migration into the arches in Xenopus and zebrafish. Embryos were co-injected into one cell at the two-cell stage with Inca MO, together with lacZ RNA as a lineage tracer and assayed for gene expression by whole-mount in situ hybridization at stage 14 (A-D, injected sides are on the left) and stage 32 (E-H; E,G, injected sides) with probes indicated in the panels. In no case was any change in gene expression observed on the injected side [(A) Slug, n=83; (B) Sox9, n=42; (C) Sox2, n=47; (D) AP2a, n=54; (E) Dlx2, n=63; (G) Sox9, n=78]. At tadpole stage, cranial cartilage was eliminated on the injected side (left side in I). Control MO injection had no effect (J). (K-R) TUNEL staining of embryos injected with control MO (K,M,O) or Inca MO (L,N,P-R), cultured to stage 21 (K,L), stage 30 (M,N) or stage 45 (O-R). Q and R are dorsal and ventral views, respectively, of the tadpole shown in P. Black arrowheads indicate strong TUNEL signal in neurocranium (Q) and jaw cartilage (R). (S-Z) Confocal images of sox10:egfp expression in cranial NC cells of controls (S,U,W,Y) and inca1 morphants (T,V,X,Z). Lateral views, anterior to the left. Stages are indicated in the panels. First and second pharyngeal arches are indicated in S,T, and derived cartilage elements by arrowheads in Y,Z. Asterisks indicate expression of sox10:eGFP in the otic vesicle.
Fig. 7. Inca misexpression disrupts gastrulation, pigmentation and wound healing in Xenopus. (A) Uninjected stage 25 control embryo. (B) Siblings injected with 250 pg IncaA mRNA have shorter body axes and numerous protuberances. (C,D) High-magnification views of ectoderm at stage 8 from uninjected (C) and IncaA mRNA-injected (250 pg; D) embryos showing relocation of cortical pigment to the cell periphery. Scale bar: 100 μm. (E,F) Vegetal explants after animal cap removal and culture for 5 (E) or 25 (F) minutes in 0.3×MMR. Upper row, uninjected; lower row injected with 250 pg IncaA mRNA, showing delayed healing. (G,H) Confocal z-stack images of animal caps from uninjected (G) and IncaA mRNA-injected (H) embryos after 5 minutes of healing in 0.3×MMR. White arrowheads in G indicate purse-string actin-filament structures missing in embryos overexpressing Inca. (I-O) Synergistic effect on wound healing of PAK5 and Inca. Embryos injected with mRNA encoding wild-type PAK5-GFP (J), kinase-dead PAK5-GFP (PAK5/KR; K), constitutively active PAK5-GFP (PAK5/EN; L), IncaA-GFP (M), a mixture of IncaA-GFP and PAK5-GFP (N) or a mixture of IncaA-GFP and PAK5/KR-GFP (O). All PAK5-GFP mRNAs injected at 1 ng, IncaA-GFP mRNA at 250 pg. Vegetal explants were allowed to heal for 25 minutes. Combining Inca+wild-type PAK5 greatly delays wound healing and leads to dissociation (N). PAK5/KR does not exhibit this synergistic effect (O). (I) Uninjected control embryo. Results from these experiments are summarized in Table 1. (P) PAK5-GFP mRNAs were equally translated, as judged by western blot using antibody to GFP. (Q) Co-expression with Inca does not increase PAK5 phosphorylation. Western blots of extracts made from HEK293 cells transfected with 0.5 μg FLAG-tagged XPAK5 plasmid and 0, 0.5 or 1.0μ g of Myc-tagged Xenopus Inca. The ratio of total XPAK5 signal (FLAG antibody) to the level of phosphorylation of regulatory serine residue 474 (phospho-PAK4) was not significantly different in any of the samples. (R) Xenopus PAK5 expression. Whole-mount in situ hybridization at stages 10, 13 and 25. Stage 10 embryos were bisected in the sagittal plane prior to hybridization. PAK5 expression is broad in ectoderm and mesoderm at early stages and essentially ubiquitous later in development.
