Limits...
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.

Show MeSH

Related in: MedlinePlus

p18ahub mutants exhibit delayed progression of epiboly and severe dorsalization.Late blastula stage WT embryo (A), and age-matched p18ahub embryo (B). WT embryo at an early gastrula stage (C) with the shield (organizer) on the dorsal side (arrow). A p18ahub embryo at an early gastrula stage (D) displaying apparent radial hyperconvergence with no definitive dorsal side. (A,B) lateral views; (C,D) animal pole views. WT (E) and p18ahub (F) embryos at 24 hours post fertilization (hpf) (lateral views). (G) Pie chart depicting proportion of embryos with indicated phenotypes at 24 hpf sampled over a total of 2308 embryos. (H and I), in situ hybridization on 3–5-somite stage WT (H, n = 15) and age-matched p18ahub (I, n = 13) embryos for six3 (s) expression in presumptive forebrain, pax2.1 expression in the midbrain-hindbrain boundary (p) and pronephros (p*), krox20 (k) in hindbrain rhombomeres 3 and 5, and myod (m) in paraxial mesoderm; dorsal view in (H), lateral view in (I), anterior to top. (J and K), 10-somite stage WT (J, n = 17, dorsal view), and age-matched p18ahub embryos (K, n = 18, anterior view) processed for krox20 in situ hybridization. (L–Q) in situ hybridization on mid gastrula stage WT and equivalent stage p18ahub embryos shown for: otx2, WT (L, n = 18) and p18ahub (M, n = 10); cyp26a, WT (N, n = 16) and p18ahub (O, n = 15); and hoxb1b, WT (P, n = 22) and p18ahub (Q, n = 21). Lateral views, anterior at top, dorsal to right.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3814294&req=5

pgen-1003822-g001: p18ahub mutants exhibit delayed progression of epiboly and severe dorsalization.Late blastula stage WT embryo (A), and age-matched p18ahub embryo (B). WT embryo at an early gastrula stage (C) with the shield (organizer) on the dorsal side (arrow). A p18ahub embryo at an early gastrula stage (D) displaying apparent radial hyperconvergence with no definitive dorsal side. (A,B) lateral views; (C,D) animal pole views. WT (E) and p18ahub (F) embryos at 24 hours post fertilization (hpf) (lateral views). (G) Pie chart depicting proportion of embryos with indicated phenotypes at 24 hpf sampled over a total of 2308 embryos. (H and I), in situ hybridization on 3–5-somite stage WT (H, n = 15) and age-matched p18ahub (I, n = 13) embryos for six3 (s) expression in presumptive forebrain, pax2.1 expression in the midbrain-hindbrain boundary (p) and pronephros (p*), krox20 (k) in hindbrain rhombomeres 3 and 5, and myod (m) in paraxial mesoderm; dorsal view in (H), lateral view in (I), anterior to top. (J and K), 10-somite stage WT (J, n = 17, dorsal view), and age-matched p18ahub embryos (K, n = 18, anterior view) processed for krox20 in situ hybridization. (L–Q) in situ hybridization on mid gastrula stage WT and equivalent stage p18ahub embryos shown for: otx2, WT (L, n = 18) and p18ahub (M, n = 10); cyp26a, WT (N, n = 16) and p18ahub (O, n = 15); and hoxb1b, WT (P, n = 22) and p18ahub (Q, n = 21). Lateral views, anterior at top, dorsal to right.

Mentions: We isolated p18ahub, a recessive maternal-effect dorsalizing mutation, in an ENU-induced mutagenesis screen designed to identify novel maternal factors required for early embryonic development and patterning in zebrafish (similar to that described in [41], [42]). Mutant females yielded embryos with similar phenotypes whether they were crossed to mutant or wild-type (WT) males, indicating a strictly maternal-effect defect with no zygotic contribution. The first defect evident in embryos from p18ahub mutant mothers (henceforth referred to as p18ahub mutant embryos) was a delay in the initiation of epiboly, the morphogenetic process by which the blastoderm cells move over and encompass the yolk cell [43]. As WT embryos reached the late blastula/50% epiboly stage (Figure 1A), mutant embryos typically had not initiated epiboly movements (Figure 1B). In early gastrulation stages, WT embryos displayed a single dorsal thickening corresponding to the embryonic axis (Figure 1C), whereas mutant embryos often developed a radial thickening possibly due to hyper convergence of cells around the entire embryonic margin (Figure 1D). Approximately 50% of mutant embryos (Figure 1G) lysed prior to 24 hpf.


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

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

p18ahub mutants exhibit delayed progression of epiboly and severe dorsalization.Late blastula stage WT embryo (A), and age-matched p18ahub embryo (B). WT embryo at an early gastrula stage (C) with the shield (organizer) on the dorsal side (arrow). A p18ahub embryo at an early gastrula stage (D) displaying apparent radial hyperconvergence with no definitive dorsal side. (A,B) lateral views; (C,D) animal pole views. WT (E) and p18ahub (F) embryos at 24 hours post fertilization (hpf) (lateral views). (G) Pie chart depicting proportion of embryos with indicated phenotypes at 24 hpf sampled over a total of 2308 embryos. (H and I), in situ hybridization on 3–5-somite stage WT (H, n = 15) and age-matched p18ahub (I, n = 13) embryos for six3 (s) expression in presumptive forebrain, pax2.1 expression in the midbrain-hindbrain boundary (p) and pronephros (p*), krox20 (k) in hindbrain rhombomeres 3 and 5, and myod (m) in paraxial mesoderm; dorsal view in (H), lateral view in (I), anterior to top. (J and K), 10-somite stage WT (J, n = 17, dorsal view), and age-matched p18ahub embryos (K, n = 18, anterior view) processed for krox20 in situ hybridization. (L–Q) in situ hybridization on mid gastrula stage WT and equivalent stage p18ahub embryos shown for: otx2, WT (L, n = 18) and p18ahub (M, n = 10); cyp26a, WT (N, n = 16) and p18ahub (O, n = 15); and hoxb1b, WT (P, n = 22) and p18ahub (Q, n = 21). Lateral views, anterior at top, dorsal to right.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003822-g001: p18ahub mutants exhibit delayed progression of epiboly and severe dorsalization.Late blastula stage WT embryo (A), and age-matched p18ahub embryo (B). WT embryo at an early gastrula stage (C) with the shield (organizer) on the dorsal side (arrow). A p18ahub embryo at an early gastrula stage (D) displaying apparent radial hyperconvergence with no definitive dorsal side. (A,B) lateral views; (C,D) animal pole views. WT (E) and p18ahub (F) embryos at 24 hours post fertilization (hpf) (lateral views). (G) Pie chart depicting proportion of embryos with indicated phenotypes at 24 hpf sampled over a total of 2308 embryos. (H and I), in situ hybridization on 3–5-somite stage WT (H, n = 15) and age-matched p18ahub (I, n = 13) embryos for six3 (s) expression in presumptive forebrain, pax2.1 expression in the midbrain-hindbrain boundary (p) and pronephros (p*), krox20 (k) in hindbrain rhombomeres 3 and 5, and myod (m) in paraxial mesoderm; dorsal view in (H), lateral view in (I), anterior to top. (J and K), 10-somite stage WT (J, n = 17, dorsal view), and age-matched p18ahub embryos (K, n = 18, anterior view) processed for krox20 in situ hybridization. (L–Q) in situ hybridization on mid gastrula stage WT and equivalent stage p18ahub embryos shown for: otx2, WT (L, n = 18) and p18ahub (M, n = 10); cyp26a, WT (N, n = 16) and p18ahub (O, n = 15); and hoxb1b, WT (P, n = 22) and p18ahub (Q, n = 21). Lateral views, anterior at top, dorsal to right.
Mentions: We isolated p18ahub, a recessive maternal-effect dorsalizing mutation, in an ENU-induced mutagenesis screen designed to identify novel maternal factors required for early embryonic development and patterning in zebrafish (similar to that described in [41], [42]). Mutant females yielded embryos with similar phenotypes whether they were crossed to mutant or wild-type (WT) males, indicating a strictly maternal-effect defect with no zygotic contribution. The first defect evident in embryos from p18ahub mutant mothers (henceforth referred to as p18ahub mutant embryos) was a delay in the initiation of epiboly, the morphogenetic process by which the blastoderm cells move over and encompass the yolk cell [43]. As WT embryos reached the late blastula/50% epiboly stage (Figure 1A), mutant embryos typically had not initiated epiboly movements (Figure 1B). In early gastrulation stages, WT embryos displayed a single dorsal thickening corresponding to the embryonic axis (Figure 1C), whereas mutant embryos often developed a radial thickening possibly due to hyper convergence of cells around the entire embryonic margin (Figure 1D). Approximately 50% of mutant embryos (Figure 1G) lysed prior to 24 hpf.

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