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The integrator complex subunit 6 (Ints6) confines the dorsal organizer in vertebrate embryogenesis.

Kapp LD, Abrams EW, Marlow FL, Mullins MC - PLoS Genet. (2013)

Bottom Line: We have isolated a recessive dorsalizing maternal-effect mutation disrupting the gene encoding Integrator Complex Subunit 6 (Ints6).Limiting Nodal signaling or restoring BMP signaling restores wild-type patterning to affected embryos.Our results are consistent with a novel role for Ints6 in restricting the vertebrate organizer to a dorsal domain in embryonic patterning.

View Article: PubMed Central - PubMed

Affiliation: Perelman School of Medicine at the University of Pennsylvania, Department of Cell and Developmental Biology, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Dorsoventral patterning of the embryonic axis relies upon the mutual antagonism of competing signaling pathways to establish a balance between ventralizing BMP signaling and dorsal cell fate specification mediated by the organizer. In zebrafish, the initial embryo-wide domain of BMP signaling is refined into a morphogenetic gradient following activation dorsally of a maternal Wnt pathway. The accumulation of β-catenin in nuclei on the dorsal side of the embryo then leads to repression of BMP signaling dorsally and the induction of dorsal cell fates mediated by Nodal and FGF signaling. A separate Wnt pathway operates zygotically via Wnt8a to limit dorsal cell fate specification and maintain the expression of ventralizing genes in ventrolateral domains. We have isolated a recessive dorsalizing maternal-effect mutation disrupting the gene encoding Integrator Complex Subunit 6 (Ints6). Due to widespread de-repression of dorsal organizer genes, embryos from mutant mothers fail to maintain expression of BMP ligands, fail to fully express vox and ved, two mediators of Wnt8a, display delayed cell movements during gastrulation, and severe dorsalization. Consistent with radial dorsalization, affected embryos display multiple independent axial domains along with ectopic dorsal forerunner cells. Limiting Nodal signaling or restoring BMP signaling restores wild-type patterning to affected embryos. Our results are consistent with a novel role for Ints6 in restricting the vertebrate organizer to a dorsal domain in embryonic patterning.

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The Wnt8a pathway is repressed but mechanistically intact in p18ahub embryos.(A–J) In situ hybridization for Wnt8a pathway components or downstream genes. Animal pole views, dorsal at right, except (C, D) are lateral views, dorsal at right, insets show animal pole views. At an early gastrula stage in WT (A, n = 14) wnt8a was expressed around the margin but was absent from the axial mesoderm (arrows). In p18ahub embryos at a late blastula stage (B, n = 20) wnt8a expression was absent from the presumptive dorsal half of the embryo. The expression of vox was reduced in late blastula p18ahub embryos (D, 11/13 embryos) compared to WT embryos (C, n = 18). By early gastrulation, WT embryos (E, n = 20) showed prominent ventrolateral vox expression, while vox was nearly absent from equivalent stage p18ahub embryos (F, n = 12). ved was expressed similarly to vox in WT (G, n = 10) and was also nearly undetectable in p18ahub embryos (H, n = 10) at early gastrulation. eve1 in WT (I, n = 17) marks developing ventroposterior mesoderm and was nearly absent from p18ahub embryos by early gastrulation (J, n = 12). Splice blocking wnt8a MOs variably dorsalized WT embryos (K). (L) Injection of MO against chd ventralized both WT and p18ahub embryos (compare to p18ahub uninjected, K). (M) Simultaneous reduction of Chd and Wnt8a causes dorsalization in p18ahub embryos, indicating that endogenous Wnt8a signal transduction is intact and functional in p18ahub embryos that are depleted of Chd. Representative embryos at 1 dpf are shown in N–Q.
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pgen-1003822-g004: The Wnt8a pathway is repressed but mechanistically intact in p18ahub embryos.(A–J) In situ hybridization for Wnt8a pathway components or downstream genes. Animal pole views, dorsal at right, except (C, D) are lateral views, dorsal at right, insets show animal pole views. At an early gastrula stage in WT (A, n = 14) wnt8a was expressed around the margin but was absent from the axial mesoderm (arrows). In p18ahub embryos at a late blastula stage (B, n = 20) wnt8a expression was absent from the presumptive dorsal half of the embryo. The expression of vox was reduced in late blastula p18ahub embryos (D, 11/13 embryos) compared to WT embryos (C, n = 18). By early gastrulation, WT embryos (E, n = 20) showed prominent ventrolateral vox expression, while vox was nearly absent from equivalent stage p18ahub embryos (F, n = 12). ved was expressed similarly to vox in WT (G, n = 10) and was also nearly undetectable in p18ahub embryos (H, n = 10) at early gastrulation. eve1 in WT (I, n = 17) marks developing ventroposterior mesoderm and was nearly absent from p18ahub embryos by early gastrulation (J, n = 12). Splice blocking wnt8a MOs variably dorsalized WT embryos (K). (L) Injection of MO against chd ventralized both WT and p18ahub embryos (compare to p18ahub uninjected, K). (M) Simultaneous reduction of Chd and Wnt8a causes dorsalization in p18ahub embryos, indicating that endogenous Wnt8a signal transduction is intact and functional in p18ahub embryos that are depleted of Chd. Representative embryos at 1 dpf are shown in N–Q.

Mentions: Zygotic Wnt8a signaling in ventrolateral regions represses the expression of the organizer genes, gsc and chd, thus restricting the size of the organizer [7], [8], [9], [10], [11], [18], [19], [26]. Loss of Wnt8a or its mediators Vox, Vent, and Ved causes the expression of some organizer genes to expand and dorsalizes the embryo [7], [8], [9], [10], [11], [19]. Accordingly, we investigated the status of the Wnt8a pathway in mutant embryos. In WT late blastula and early gastrula stage embryos, wnt8a is expressed at the margin in a large domain extending from ventral to dorsolateral regions (Figure 4A) [18], [57]. However, in p18ahub late blastula stage embryos, wnt8a expression was restricted ventrally to a smaller domain (Figure 4B). Furthermore, wnt8a expression was undetectable in mutant embryos age-matched to their WT counterparts at early gastrula stage (not shown), perhaps reflecting a loss in the competence of marginal cells to express wnt8a. Since wnt8a is required for anteroposterior (AP) patterning of neural tissue [18] and a defect is not evident in AP patterning in p18ahub mutants, it suggests that the early, transient expression of wnt8a may be sufficient for AP patterning in p18ahub mutant embryos.


The integrator complex subunit 6 (Ints6) confines the dorsal organizer in vertebrate embryogenesis.

Kapp LD, Abrams EW, Marlow FL, Mullins MC - PLoS Genet. (2013)

The Wnt8a pathway is repressed but mechanistically intact in p18ahub embryos.(A–J) In situ hybridization for Wnt8a pathway components or downstream genes. Animal pole views, dorsal at right, except (C, D) are lateral views, dorsal at right, insets show animal pole views. At an early gastrula stage in WT (A, n = 14) wnt8a was expressed around the margin but was absent from the axial mesoderm (arrows). In p18ahub embryos at a late blastula stage (B, n = 20) wnt8a expression was absent from the presumptive dorsal half of the embryo. The expression of vox was reduced in late blastula p18ahub embryos (D, 11/13 embryos) compared to WT embryos (C, n = 18). By early gastrulation, WT embryos (E, n = 20) showed prominent ventrolateral vox expression, while vox was nearly absent from equivalent stage p18ahub embryos (F, n = 12). ved was expressed similarly to vox in WT (G, n = 10) and was also nearly undetectable in p18ahub embryos (H, n = 10) at early gastrulation. eve1 in WT (I, n = 17) marks developing ventroposterior mesoderm and was nearly absent from p18ahub embryos by early gastrulation (J, n = 12). Splice blocking wnt8a MOs variably dorsalized WT embryos (K). (L) Injection of MO against chd ventralized both WT and p18ahub embryos (compare to p18ahub uninjected, K). (M) Simultaneous reduction of Chd and Wnt8a causes dorsalization in p18ahub embryos, indicating that endogenous Wnt8a signal transduction is intact and functional in p18ahub embryos that are depleted of Chd. Representative embryos at 1 dpf are shown in N–Q.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814294&req=5

pgen-1003822-g004: The Wnt8a pathway is repressed but mechanistically intact in p18ahub embryos.(A–J) In situ hybridization for Wnt8a pathway components or downstream genes. Animal pole views, dorsal at right, except (C, D) are lateral views, dorsal at right, insets show animal pole views. At an early gastrula stage in WT (A, n = 14) wnt8a was expressed around the margin but was absent from the axial mesoderm (arrows). In p18ahub embryos at a late blastula stage (B, n = 20) wnt8a expression was absent from the presumptive dorsal half of the embryo. The expression of vox was reduced in late blastula p18ahub embryos (D, 11/13 embryos) compared to WT embryos (C, n = 18). By early gastrulation, WT embryos (E, n = 20) showed prominent ventrolateral vox expression, while vox was nearly absent from equivalent stage p18ahub embryos (F, n = 12). ved was expressed similarly to vox in WT (G, n = 10) and was also nearly undetectable in p18ahub embryos (H, n = 10) at early gastrulation. eve1 in WT (I, n = 17) marks developing ventroposterior mesoderm and was nearly absent from p18ahub embryos by early gastrulation (J, n = 12). Splice blocking wnt8a MOs variably dorsalized WT embryos (K). (L) Injection of MO against chd ventralized both WT and p18ahub embryos (compare to p18ahub uninjected, K). (M) Simultaneous reduction of Chd and Wnt8a causes dorsalization in p18ahub embryos, indicating that endogenous Wnt8a signal transduction is intact and functional in p18ahub embryos that are depleted of Chd. Representative embryos at 1 dpf are shown in N–Q.
Mentions: Zygotic Wnt8a signaling in ventrolateral regions represses the expression of the organizer genes, gsc and chd, thus restricting the size of the organizer [7], [8], [9], [10], [11], [18], [19], [26]. Loss of Wnt8a or its mediators Vox, Vent, and Ved causes the expression of some organizer genes to expand and dorsalizes the embryo [7], [8], [9], [10], [11], [19]. Accordingly, we investigated the status of the Wnt8a pathway in mutant embryos. In WT late blastula and early gastrula stage embryos, wnt8a is expressed at the margin in a large domain extending from ventral to dorsolateral regions (Figure 4A) [18], [57]. However, in p18ahub late blastula stage embryos, wnt8a expression was restricted ventrally to a smaller domain (Figure 4B). Furthermore, wnt8a expression was undetectable in mutant embryos age-matched to their WT counterparts at early gastrula stage (not shown), perhaps reflecting a loss in the competence of marginal cells to express wnt8a. Since wnt8a is required for anteroposterior (AP) patterning of neural tissue [18] and a defect is not evident in AP patterning in p18ahub mutants, it suggests that the early, transient expression of wnt8a may be sufficient for AP patterning in p18ahub mutant embryos.

Bottom Line: We have isolated a recessive dorsalizing maternal-effect mutation disrupting the gene encoding Integrator Complex Subunit 6 (Ints6).Limiting Nodal signaling or restoring BMP signaling restores wild-type patterning to affected embryos.Our results are consistent with a novel role for Ints6 in restricting the vertebrate organizer to a dorsal domain in embryonic patterning.

View Article: PubMed Central - PubMed

Affiliation: Perelman School of Medicine at the University of Pennsylvania, Department of Cell and Developmental Biology, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Dorsoventral patterning of the embryonic axis relies upon the mutual antagonism of competing signaling pathways to establish a balance between ventralizing BMP signaling and dorsal cell fate specification mediated by the organizer. In zebrafish, the initial embryo-wide domain of BMP signaling is refined into a morphogenetic gradient following activation dorsally of a maternal Wnt pathway. The accumulation of β-catenin in nuclei on the dorsal side of the embryo then leads to repression of BMP signaling dorsally and the induction of dorsal cell fates mediated by Nodal and FGF signaling. A separate Wnt pathway operates zygotically via Wnt8a to limit dorsal cell fate specification and maintain the expression of ventralizing genes in ventrolateral domains. We have isolated a recessive dorsalizing maternal-effect mutation disrupting the gene encoding Integrator Complex Subunit 6 (Ints6). Due to widespread de-repression of dorsal organizer genes, embryos from mutant mothers fail to maintain expression of BMP ligands, fail to fully express vox and ved, two mediators of Wnt8a, display delayed cell movements during gastrulation, and severe dorsalization. Consistent with radial dorsalization, affected embryos display multiple independent axial domains along with ectopic dorsal forerunner cells. Limiting Nodal signaling or restoring BMP signaling restores wild-type patterning to affected embryos. Our results are consistent with a novel role for Ints6 in restricting the vertebrate organizer to a dorsal domain in embryonic patterning.

Show MeSH
Related in: MedlinePlus