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Wdr68 Mediates Dorsal and Ventral Patterning Events for Craniofacial Development

View Article: PubMed Central - PubMed

ABSTRACT

Birth defects are among the leading causes of infant mortality and contribute substantially to illness and long-term disability. Defects in Bone Morphogenetic Protein (BMP) signaling are associated with cleft lip/palate. Many craniofacial syndromes are caused by defects in signaling pathways that pattern the cranial neural crest cells (CNCCs) along the dorsal-ventral axis. For example, auriculocondylar syndrome is caused by impaired Endothelin-1 (Edn1) signaling, and Alagille syndrome is caused by defects in Jagged-Notch signaling. The BMP, Edn1, and Jag1b pathways intersect because BMP signaling is required for ventral edn1 expression that, in turn, restricts jag1b to dorsal CNCC territory. In zebrafish, the scaffolding protein Wdr68 is required for edn1 expression and subsequent formation of the ventral Meckel’s cartilage as well as the dorsal Palatoquadrate. Here we report that wdr68 activity is required between the 17-somites and prim-5 stages, that edn1 functions downstream of wdr68, and that wdr68 activity restricts jag1b, hey1, and grem2 expression from ventral CNCC territory. Expression of dlx1a and dlx2a was also severely reduced in anterior dorsal and ventral 1st arch CNCC territory in wdr68 mutants. We also found that the BMP agonist isoliquiritigenin (ISL) can partially rescue lower jaw formation and edn1 expression in wdr68 mutants. However, we found no significant defects in BMP reporter induction or pSmad1/5 accumulation in wdr68 mutant cells or zebrafish. The Transforming Growth Factor Beta (TGF-β) signaling pathway is also known to be important for craniofacial development and can interfere with BMP signaling. Here we further report that TGF-β interference with BMP signaling was greater in wdr68 mutant cells relative to control cells. To determine whether interference might also act in vivo, we treated wdr68 mutant zebrafish embryos with the TGF-β signaling inhibitor SB431542 and found partial rescue of edn1 expression and craniofacial development. While ISL treatment failed, SB431542 partially rescued dlx2a expression in wdr68 mutants. Together these findings reveal an indirect role for Wdr68 in the BMP-Edn1-Jag1b signaling hierarchy and dorso-anterior expression of dlx1a/2a.

No MeSH data available.


dlx1a and dlx2a expression is wdr68-dependent and responsive to inhibition of TGF-β signaling in wdr68hi3812/hi3812 zebrafish.(A-H) ISH analysis of prim-12 stage embryos raised at 32°C. A-F) dlx2a expression with red underline for anterior portion of 1st arch and blue underline for 2nd arch. A, C, E) lateral view. B, D, F) dorso-lateral view. A, B) DMSO-treated wildtype sibling showing normal dlx2a. C, D) DMSO-treated wdr68hi3812/hi3812 mutant showing loss of anterior 1st arch dlx2a. E, F) SB431542-treated wdr68hi3812/hi3812 mutant showing partial rescue of anterior 1st arch dlx2a. G, H) dlx1a expression. G) wildtype sibling showing normal dlx1a. H) wdr68hi3812/hi3812 mutant showing loss of anterior 1st arch dlx1a.
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pone.0166984.g008: dlx1a and dlx2a expression is wdr68-dependent and responsive to inhibition of TGF-β signaling in wdr68hi3812/hi3812 zebrafish.(A-H) ISH analysis of prim-12 stage embryos raised at 32°C. A-F) dlx2a expression with red underline for anterior portion of 1st arch and blue underline for 2nd arch. A, C, E) lateral view. B, D, F) dorso-lateral view. A, B) DMSO-treated wildtype sibling showing normal dlx2a. C, D) DMSO-treated wdr68hi3812/hi3812 mutant showing loss of anterior 1st arch dlx2a. E, F) SB431542-treated wdr68hi3812/hi3812 mutant showing partial rescue of anterior 1st arch dlx2a. G, H) dlx1a expression. G) wildtype sibling showing normal dlx1a. H) wdr68hi3812/hi3812 mutant showing loss of anterior 1st arch dlx1a.

Mentions: A molecular explanation for the absence of the PQ in wdr68hi3812/hi3812 mutants is still lacking. Interestingly, SB431542-treated wdr68hi3812/hi3812 mutants not only displayed partial rescue of the ventral M cartilage, but also a consistent partial rescue of the dorsal PQ cartilage (Fig 7D). This was in contrast to either ectopic expression of edn1 (Fig 2C), or ISL-treatment (Fig 5D), both of which failed to restore the dorsal PQ cartilage in wdr68hi3812/hi3812 mutants. It has been previously reported that simultaneous antisense knockdown of dlx1a and dlx2a causes loss of the PQ while retaining the M and PTP [65]. The reported dlx1a/2a-MO phenotype bears similarities to the residual defects we observed for both ectopic edn1 (Fig 2C) and ISL-treatment (Fig 5D) in wdr68hi3812/hi3812 mutants. Therefore, we re-examined the expression of the dlx1a and dlx2a genes at the prim-12 stage in wdr68hi3812/hi3812 mutant embryos raised at 32°C (Fig 8). In wildtype sibling embryos, we found robust expression of dlx2a in both dorsal and ventral CNCCs of the 1st and 2nd arches (Fig 8A and 8B). In contrast, we found severely reduced dlx2a expression in the anterior portion of the 1st arch in wdr68hi3812/hi3812 mutants (Fig 8C and 8D, compare red underline between 8D and 8B). Consistent with the relatively minor 2nd arch-derived cartilage defects in wdr68hi3812/hi3812 mutants (Figs 1C, 2B, 5B and 7B), expression of dlx2a in the 2nd arch was relatively unchanged in wdr68hi3812/hi3812 mutants (Fig 8D compare blue underline to 8B). To determine whether inhibiting TGF-β signaling in severe wdr68hi3812/hi3812 embryos would partially rescue dlx2a expression, we again treated embryos raised at 32°C with 10μM SB431542 starting at the 14- to 15-somites stages. While SB431542 treatment yielded no discernible changes in wildtype siblings, we found partial rescue of anterior dorsal and ventral 1st arch expression of dlx2a in wdr68hi3812/hi3812 mutants (Fig 8E and 8F, compare red underline to Fig 8C and 8D). Analysis of dlx1a expression revealed a similar loss of anterior dorsal and ventral 1st arch expression in wdr68hi3812/hi3812 mutants (Fig 8H, compare red underline to Fig 8G).


Wdr68 Mediates Dorsal and Ventral Patterning Events for Craniofacial Development
dlx1a and dlx2a expression is wdr68-dependent and responsive to inhibition of TGF-β signaling in wdr68hi3812/hi3812 zebrafish.(A-H) ISH analysis of prim-12 stage embryos raised at 32°C. A-F) dlx2a expression with red underline for anterior portion of 1st arch and blue underline for 2nd arch. A, C, E) lateral view. B, D, F) dorso-lateral view. A, B) DMSO-treated wildtype sibling showing normal dlx2a. C, D) DMSO-treated wdr68hi3812/hi3812 mutant showing loss of anterior 1st arch dlx2a. E, F) SB431542-treated wdr68hi3812/hi3812 mutant showing partial rescue of anterior 1st arch dlx2a. G, H) dlx1a expression. G) wildtype sibling showing normal dlx1a. H) wdr68hi3812/hi3812 mutant showing loss of anterior 1st arch dlx1a.
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pone.0166984.g008: dlx1a and dlx2a expression is wdr68-dependent and responsive to inhibition of TGF-β signaling in wdr68hi3812/hi3812 zebrafish.(A-H) ISH analysis of prim-12 stage embryos raised at 32°C. A-F) dlx2a expression with red underline for anterior portion of 1st arch and blue underline for 2nd arch. A, C, E) lateral view. B, D, F) dorso-lateral view. A, B) DMSO-treated wildtype sibling showing normal dlx2a. C, D) DMSO-treated wdr68hi3812/hi3812 mutant showing loss of anterior 1st arch dlx2a. E, F) SB431542-treated wdr68hi3812/hi3812 mutant showing partial rescue of anterior 1st arch dlx2a. G, H) dlx1a expression. G) wildtype sibling showing normal dlx1a. H) wdr68hi3812/hi3812 mutant showing loss of anterior 1st arch dlx1a.
Mentions: A molecular explanation for the absence of the PQ in wdr68hi3812/hi3812 mutants is still lacking. Interestingly, SB431542-treated wdr68hi3812/hi3812 mutants not only displayed partial rescue of the ventral M cartilage, but also a consistent partial rescue of the dorsal PQ cartilage (Fig 7D). This was in contrast to either ectopic expression of edn1 (Fig 2C), or ISL-treatment (Fig 5D), both of which failed to restore the dorsal PQ cartilage in wdr68hi3812/hi3812 mutants. It has been previously reported that simultaneous antisense knockdown of dlx1a and dlx2a causes loss of the PQ while retaining the M and PTP [65]. The reported dlx1a/2a-MO phenotype bears similarities to the residual defects we observed for both ectopic edn1 (Fig 2C) and ISL-treatment (Fig 5D) in wdr68hi3812/hi3812 mutants. Therefore, we re-examined the expression of the dlx1a and dlx2a genes at the prim-12 stage in wdr68hi3812/hi3812 mutant embryos raised at 32°C (Fig 8). In wildtype sibling embryos, we found robust expression of dlx2a in both dorsal and ventral CNCCs of the 1st and 2nd arches (Fig 8A and 8B). In contrast, we found severely reduced dlx2a expression in the anterior portion of the 1st arch in wdr68hi3812/hi3812 mutants (Fig 8C and 8D, compare red underline between 8D and 8B). Consistent with the relatively minor 2nd arch-derived cartilage defects in wdr68hi3812/hi3812 mutants (Figs 1C, 2B, 5B and 7B), expression of dlx2a in the 2nd arch was relatively unchanged in wdr68hi3812/hi3812 mutants (Fig 8D compare blue underline to 8B). To determine whether inhibiting TGF-β signaling in severe wdr68hi3812/hi3812 embryos would partially rescue dlx2a expression, we again treated embryos raised at 32°C with 10μM SB431542 starting at the 14- to 15-somites stages. While SB431542 treatment yielded no discernible changes in wildtype siblings, we found partial rescue of anterior dorsal and ventral 1st arch expression of dlx2a in wdr68hi3812/hi3812 mutants (Fig 8E and 8F, compare red underline to Fig 8C and 8D). Analysis of dlx1a expression revealed a similar loss of anterior dorsal and ventral 1st arch expression in wdr68hi3812/hi3812 mutants (Fig 8H, compare red underline to Fig 8G).

View Article: PubMed Central - PubMed

ABSTRACT

Birth defects are among the leading causes of infant mortality and contribute substantially to illness and long-term disability. Defects in Bone Morphogenetic Protein (BMP) signaling are associated with cleft lip/palate. Many craniofacial syndromes are caused by defects in signaling pathways that pattern the cranial neural crest cells (CNCCs) along the dorsal-ventral axis. For example, auriculocondylar syndrome is caused by impaired Endothelin-1 (Edn1) signaling, and Alagille syndrome is caused by defects in Jagged-Notch signaling. The BMP, Edn1, and Jag1b pathways intersect because BMP signaling is required for ventral edn1 expression that, in turn, restricts jag1b to dorsal CNCC territory. In zebrafish, the scaffolding protein Wdr68 is required for edn1 expression and subsequent formation of the ventral Meckel’s cartilage as well as the dorsal Palatoquadrate. Here we report that wdr68 activity is required between the 17-somites and prim-5 stages, that edn1 functions downstream of wdr68, and that wdr68 activity restricts jag1b, hey1, and grem2 expression from ventral CNCC territory. Expression of dlx1a and dlx2a was also severely reduced in anterior dorsal and ventral 1st arch CNCC territory in wdr68 mutants. We also found that the BMP agonist isoliquiritigenin (ISL) can partially rescue lower jaw formation and edn1 expression in wdr68 mutants. However, we found no significant defects in BMP reporter induction or pSmad1/5 accumulation in wdr68 mutant cells or zebrafish. The Transforming Growth Factor Beta (TGF-β) signaling pathway is also known to be important for craniofacial development and can interfere with BMP signaling. Here we further report that TGF-β interference with BMP signaling was greater in wdr68 mutant cells relative to control cells. To determine whether interference might also act in vivo, we treated wdr68 mutant zebrafish embryos with the TGF-β signaling inhibitor SB431542 and found partial rescue of edn1 expression and craniofacial development. While ISL treatment failed, SB431542 partially rescued dlx2a expression in wdr68 mutants. Together these findings reveal an indirect role for Wdr68 in the BMP-Edn1-Jag1b signaling hierarchy and dorso-anterior expression of dlx1a/2a.

No MeSH data available.