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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.


ISL treatment partially rescues M cartilage and edn1 expression in wdr68hi3812/hi3812 zebrafish.(A-D) Flatmounts of 5dpf ventral cartilages of Alcian stained zebrafish raised at 32°C and treated with DMSO or 5μM ISL starting at the 14- to 15-somites stage. A) Wildtype zebrafish treated with DMSO control. Red arrow indicate M. B) wdr68hi3812/hi3812 mutants treated with DMSO control show a lack of M cartilage. C) Wildtype zebrafish treated with 5μM ISL show normal craniofacial cartilage formation. D) wdr68hi3812/hi3812 mutants treated with 5μM ISL show a partial rescue of M. E) Fraction of mutant embryos with partial M is significantly greater in the ISL treated group (p<0.006). (F-I) Dorsal views of edn1 ISH analysis on 20-somites stage embryos treated with DMSO or 5μM ISL starting at the 14- to 15-somites stage. F) Wildtype embryos treated with DMSO control. G) wdr68hi3812/hi3812 mutants treated with DMSO control show lack of edn1 expression. H) Wildtype embryos treated with ISL show similar expression compared to wild type. I) wdr68hi3812/hi3812 mutants treated with ISL are indistinguishable from that of wildtype.
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pone.0166984.g005: ISL treatment partially rescues M cartilage and edn1 expression in wdr68hi3812/hi3812 zebrafish.(A-D) Flatmounts of 5dpf ventral cartilages of Alcian stained zebrafish raised at 32°C and treated with DMSO or 5μM ISL starting at the 14- to 15-somites stage. A) Wildtype zebrafish treated with DMSO control. Red arrow indicate M. B) wdr68hi3812/hi3812 mutants treated with DMSO control show a lack of M cartilage. C) Wildtype zebrafish treated with 5μM ISL show normal craniofacial cartilage formation. D) wdr68hi3812/hi3812 mutants treated with 5μM ISL show a partial rescue of M. E) Fraction of mutant embryos with partial M is significantly greater in the ISL treated group (p<0.006). (F-I) Dorsal views of edn1 ISH analysis on 20-somites stage embryos treated with DMSO or 5μM ISL starting at the 14- to 15-somites stage. F) Wildtype embryos treated with DMSO control. G) wdr68hi3812/hi3812 mutants treated with DMSO control show lack of edn1 expression. H) Wildtype embryos treated with ISL show similar expression compared to wild type. I) wdr68hi3812/hi3812 mutants treated with ISL are indistinguishable from that of wildtype.

Mentions: If wdr68 facilitates BMP induction of edn1, then treating wdr68 mutants with a BMP agonist should at least partially rescue M cartilage formation and edn1 expression. To test this, we treated embryos raised at 32°C with the BMP signaling agonist isoliquiritigenin (ISL) starting at the 14–15 somites stages [57]. Embryos were raised at 32°C so that mutants would exhibit severe loss of the M cartilage from which potential rescue of M cartilage formation could be most clearly assessed (see Fig 1C). Alcian blue stained zebrafish cartilages were dissected and flat mounted on slides for additional clarity. At 32°C, we found that DMSO-treated wildtype zebrafish developed the M cartilage as expected (Fig 5A, red arrowhead). Also at 32°C, we found that most DMSO-treated mutants exhibited complete loss of M and reduction of PQ as previously described (Fig 5B, red arrowhead). ISL-treated wildtype embryos were indistinguishable from DMSO-treated wildtype (compare Fig 5C to 5A). In contrast, ISL-treated mutants raised at 32°C exhibited a partial restoration of the M cartilage (compare Fig 5D to 5B, red arrowheads). However, ISL treatment did not appear to significantly restore formation of the PQ (compare Fig 5D to 5A and 5C). We quantified the fraction of mutants in each treatment exhibiting a discernible M-like cartilage element and found that 59% of ISL-treated animals displayed an M-like cartilage versus only 17% of DMSO-treated animals (Fig 5E, p<0.006).


Wdr68 Mediates Dorsal and Ventral Patterning Events for Craniofacial Development
ISL treatment partially rescues M cartilage and edn1 expression in wdr68hi3812/hi3812 zebrafish.(A-D) Flatmounts of 5dpf ventral cartilages of Alcian stained zebrafish raised at 32°C and treated with DMSO or 5μM ISL starting at the 14- to 15-somites stage. A) Wildtype zebrafish treated with DMSO control. Red arrow indicate M. B) wdr68hi3812/hi3812 mutants treated with DMSO control show a lack of M cartilage. C) Wildtype zebrafish treated with 5μM ISL show normal craniofacial cartilage formation. D) wdr68hi3812/hi3812 mutants treated with 5μM ISL show a partial rescue of M. E) Fraction of mutant embryos with partial M is significantly greater in the ISL treated group (p<0.006). (F-I) Dorsal views of edn1 ISH analysis on 20-somites stage embryos treated with DMSO or 5μM ISL starting at the 14- to 15-somites stage. F) Wildtype embryos treated with DMSO control. G) wdr68hi3812/hi3812 mutants treated with DMSO control show lack of edn1 expression. H) Wildtype embryos treated with ISL show similar expression compared to wild type. I) wdr68hi3812/hi3812 mutants treated with ISL are indistinguishable from that of wildtype.
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pone.0166984.g005: ISL treatment partially rescues M cartilage and edn1 expression in wdr68hi3812/hi3812 zebrafish.(A-D) Flatmounts of 5dpf ventral cartilages of Alcian stained zebrafish raised at 32°C and treated with DMSO or 5μM ISL starting at the 14- to 15-somites stage. A) Wildtype zebrafish treated with DMSO control. Red arrow indicate M. B) wdr68hi3812/hi3812 mutants treated with DMSO control show a lack of M cartilage. C) Wildtype zebrafish treated with 5μM ISL show normal craniofacial cartilage formation. D) wdr68hi3812/hi3812 mutants treated with 5μM ISL show a partial rescue of M. E) Fraction of mutant embryos with partial M is significantly greater in the ISL treated group (p<0.006). (F-I) Dorsal views of edn1 ISH analysis on 20-somites stage embryos treated with DMSO or 5μM ISL starting at the 14- to 15-somites stage. F) Wildtype embryos treated with DMSO control. G) wdr68hi3812/hi3812 mutants treated with DMSO control show lack of edn1 expression. H) Wildtype embryos treated with ISL show similar expression compared to wild type. I) wdr68hi3812/hi3812 mutants treated with ISL are indistinguishable from that of wildtype.
Mentions: If wdr68 facilitates BMP induction of edn1, then treating wdr68 mutants with a BMP agonist should at least partially rescue M cartilage formation and edn1 expression. To test this, we treated embryos raised at 32°C with the BMP signaling agonist isoliquiritigenin (ISL) starting at the 14–15 somites stages [57]. Embryos were raised at 32°C so that mutants would exhibit severe loss of the M cartilage from which potential rescue of M cartilage formation could be most clearly assessed (see Fig 1C). Alcian blue stained zebrafish cartilages were dissected and flat mounted on slides for additional clarity. At 32°C, we found that DMSO-treated wildtype zebrafish developed the M cartilage as expected (Fig 5A, red arrowhead). Also at 32°C, we found that most DMSO-treated mutants exhibited complete loss of M and reduction of PQ as previously described (Fig 5B, red arrowhead). ISL-treated wildtype embryos were indistinguishable from DMSO-treated wildtype (compare Fig 5C to 5A). In contrast, ISL-treated mutants raised at 32°C exhibited a partial restoration of the M cartilage (compare Fig 5D to 5B, red arrowheads). However, ISL treatment did not appear to significantly restore formation of the PQ (compare Fig 5D to 5A and 5C). We quantified the fraction of mutants in each treatment exhibiting a discernible M-like cartilage element and found that 59% of ISL-treated animals displayed an M-like cartilage versus only 17% of DMSO-treated animals (Fig 5E, p<0.006).

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&rsquo;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-&beta;) signaling pathway is also known to be important for craniofacial development and can interfere with BMP signaling. Here we further report that TGF-&beta; 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-&beta; 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.