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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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Related in: MedlinePlus

p18ahub affects the Integrator Complex Subunit 6 (Ints6).(A) p18ahub was narrowed to an approximately 1.35 Mb interval between SSLP marker z7120 and an SSLP marker that we generated in BAC CR545476.14 (Zv9) (right marker) by examining meiotic recombination between markers flanking the mutation. The recombinants found in the number of meioses examined at each marker is shown and the positions of markers are approximate. (B) p18ahub is a T to A transition in an exon of ints6 that converts valine 375 to an aspartate. An alignment of several Ints6 proteins was generated using ClustalW and reveals that V375 is nearly invariant among widely divergent species. (C and D) In situ hybridization for ints6 transcripts on WT embryos at (C) 128-cell (n = 8) and (D) late blastula stage (n = 19); lateral views, animal to top. (E–G) Rescue experiments: pie charts show fractions of embryos evaluated at 1 dpf with indicated phenotypes. Numbers of embryos are shown in the center of each chart. (H) Uninjected p18ahub mutant embryos and (I) mutant embryos rescued by injection of 50 pg ints6 mRNA, shown at 1 dpf.
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pgen-1003822-g007: p18ahub affects the Integrator Complex Subunit 6 (Ints6).(A) p18ahub was narrowed to an approximately 1.35 Mb interval between SSLP marker z7120 and an SSLP marker that we generated in BAC CR545476.14 (Zv9) (right marker) by examining meiotic recombination between markers flanking the mutation. The recombinants found in the number of meioses examined at each marker is shown and the positions of markers are approximate. (B) p18ahub is a T to A transition in an exon of ints6 that converts valine 375 to an aspartate. An alignment of several Ints6 proteins was generated using ClustalW and reveals that V375 is nearly invariant among widely divergent species. (C and D) In situ hybridization for ints6 transcripts on WT embryos at (C) 128-cell (n = 8) and (D) late blastula stage (n = 19); lateral views, animal to top. (E–G) Rescue experiments: pie charts show fractions of embryos evaluated at 1 dpf with indicated phenotypes. Numbers of embryos are shown in the center of each chart. (H) Uninjected p18ahub mutant embryos and (I) mutant embryos rescued by injection of 50 pg ints6 mRNA, shown at 1 dpf.

Mentions: To identify the molecular nature of p18ahub, we mapped the mutation to a chromosomal position by examining linkage to simple sequence length polymorphic (SSLP) markers. We first found linkage of p18ahub to z1660 on chromosome 9. Further fine mapping examining over 1100 meioses placed p18ahub within a 1.35 Mb interval between the SSLP marker z7120 and an SSLP marker that we generated for BAC CR545476.14 (Zv9). (Figure 7A). The interval displays synteny with human chromosome 13. It contains just over one dozen genes, none of which were known to function in DV patterning. We proceeded to sequence the open reading frames of cDNAs of genes within the interval.


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 affects the Integrator Complex Subunit 6 (Ints6).(A) p18ahub was narrowed to an approximately 1.35 Mb interval between SSLP marker z7120 and an SSLP marker that we generated in BAC CR545476.14 (Zv9) (right marker) by examining meiotic recombination between markers flanking the mutation. The recombinants found in the number of meioses examined at each marker is shown and the positions of markers are approximate. (B) p18ahub is a T to A transition in an exon of ints6 that converts valine 375 to an aspartate. An alignment of several Ints6 proteins was generated using ClustalW and reveals that V375 is nearly invariant among widely divergent species. (C and D) In situ hybridization for ints6 transcripts on WT embryos at (C) 128-cell (n = 8) and (D) late blastula stage (n = 19); lateral views, animal to top. (E–G) Rescue experiments: pie charts show fractions of embryos evaluated at 1 dpf with indicated phenotypes. Numbers of embryos are shown in the center of each chart. (H) Uninjected p18ahub mutant embryos and (I) mutant embryos rescued by injection of 50 pg ints6 mRNA, shown at 1 dpf.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003822-g007: p18ahub affects the Integrator Complex Subunit 6 (Ints6).(A) p18ahub was narrowed to an approximately 1.35 Mb interval between SSLP marker z7120 and an SSLP marker that we generated in BAC CR545476.14 (Zv9) (right marker) by examining meiotic recombination between markers flanking the mutation. The recombinants found in the number of meioses examined at each marker is shown and the positions of markers are approximate. (B) p18ahub is a T to A transition in an exon of ints6 that converts valine 375 to an aspartate. An alignment of several Ints6 proteins was generated using ClustalW and reveals that V375 is nearly invariant among widely divergent species. (C and D) In situ hybridization for ints6 transcripts on WT embryos at (C) 128-cell (n = 8) and (D) late blastula stage (n = 19); lateral views, animal to top. (E–G) Rescue experiments: pie charts show fractions of embryos evaluated at 1 dpf with indicated phenotypes. Numbers of embryos are shown in the center of each chart. (H) Uninjected p18ahub mutant embryos and (I) mutant embryos rescued by injection of 50 pg ints6 mRNA, shown at 1 dpf.
Mentions: To identify the molecular nature of p18ahub, we mapped the mutation to a chromosomal position by examining linkage to simple sequence length polymorphic (SSLP) markers. We first found linkage of p18ahub to z1660 on chromosome 9. Further fine mapping examining over 1100 meioses placed p18ahub within a 1.35 Mb interval between the SSLP marker z7120 and an SSLP marker that we generated for BAC CR545476.14 (Zv9). (Figure 7A). The interval displays synteny with human chromosome 13. It contains just over one dozen genes, none of which were known to function in DV patterning. We proceeded to sequence the open reading frames of cDNAs of genes within the interval.

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