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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.


Inhibition of TGF-β signaling partially rescues M cartilage and edn1 expression in wdr68hi3812/hi3812 zebrafish.(A-D) Ventral views of 5dpf Alcian stained craniofacial cartilages of zebrafish raised at 32°C and treated with DMSO or 10μM SB431542 at 14- to 15-somites stage. A) Wildtype zebrafish treated with DMSO control. Red arrow indicates M-PQ joint region. B) wdr68hi3812/hi3812 mutants treated with DMSO control show a lack of M cartilage. C) Wildtype zebrafish treated with 10μM SB431542 show normal craniofacial cartilage formation. D) wdr68hi3812/hi3812 mutants treated with 10μM SB431542 show a partial rescue of M. E) SB431542-treated mutants show a significantly reduced fraction of severe defects compared to the control group (p<0.012). (F-I) Dorsal views of edn1 ISH analysis on 22-somites stage embryos treated with DMSO or 10μM SB431542 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 SB431542 show similar expression compared to wild type. I) wdr68hi3812/hi3812 mutants treated with SB431542 show partial restoration of edn1 expression.
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pone.0166984.g007: Inhibition of TGF-β signaling partially rescues M cartilage and edn1 expression in wdr68hi3812/hi3812 zebrafish.(A-D) Ventral views of 5dpf Alcian stained craniofacial cartilages of zebrafish raised at 32°C and treated with DMSO or 10μM SB431542 at 14- to 15-somites stage. A) Wildtype zebrafish treated with DMSO control. Red arrow indicates M-PQ joint region. B) wdr68hi3812/hi3812 mutants treated with DMSO control show a lack of M cartilage. C) Wildtype zebrafish treated with 10μM SB431542 show normal craniofacial cartilage formation. D) wdr68hi3812/hi3812 mutants treated with 10μM SB431542 show a partial rescue of M. E) SB431542-treated mutants show a significantly reduced fraction of severe defects compared to the control group (p<0.012). (F-I) Dorsal views of edn1 ISH analysis on 22-somites stage embryos treated with DMSO or 10μM SB431542 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 SB431542 show similar expression compared to wild type. I) wdr68hi3812/hi3812 mutants treated with SB431542 show partial restoration of edn1 expression.

Mentions: If enhanced TGF-β interference with BMP signaling contributes to the wdr68 mutant phenotype in zebrafish, then inhibiting TGF-β pathway function should at least partially restore cartilage development in wdr68 mutants. SB431542 is a well-characterized inhibitor of the TGF-β family receptor kinases Alk4/5 [61], that is also known to inhibit TGF-β signaling activity in the zebrafish [62–64]. To avoid perturbing earlier developmental roles of TGF-β family signaling, embryos were treated with 10μM SB431542 starting at the 14- to 15-somites stages. 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 (Fig 1C). At 32°C, we found that DMSO-treated wildtype zebrafish developed the M cartilage as expected (Fig 7A, 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 7B, red arrowhead). 10μM SB431542-treated wildtype embryos were indistinguishable from DMSO-treated wildtypes (compare Fig 7C to 7A). In contrast, SB431542-treated mutants raised at 32°C exhibited a partial restoration of the M and PQ cartilages (compare Fig 7D to 7B, red arrowheads). Quantitation of the changes in phenotypic severity revealed a significant shift in the number of mutants displaying severe versus mild phenotypes. Under DMSO treatment, mild wdr68hi3812/hi3812 embryos were 8.6% of the total sample population whereas severe wdr68hi3812/hi3812 embryos were 14.3% of the total population (Fig 7E, left). Under SB431542 treatment, mild wdr68hi3812/hi3812 embryos were 19.2% of the total population whereas severe mutants were 8.0% of the total population (Fig 7E, right). Thus, SB431542 treatment decreased the fraction of severe mutants relative to the DMSO control (Fig 7E, red bars, p < 0.012).


Wdr68 Mediates Dorsal and Ventral Patterning Events for Craniofacial Development
Inhibition of TGF-β signaling partially rescues M cartilage and edn1 expression in wdr68hi3812/hi3812 zebrafish.(A-D) Ventral views of 5dpf Alcian stained craniofacial cartilages of zebrafish raised at 32°C and treated with DMSO or 10μM SB431542 at 14- to 15-somites stage. A) Wildtype zebrafish treated with DMSO control. Red arrow indicates M-PQ joint region. B) wdr68hi3812/hi3812 mutants treated with DMSO control show a lack of M cartilage. C) Wildtype zebrafish treated with 10μM SB431542 show normal craniofacial cartilage formation. D) wdr68hi3812/hi3812 mutants treated with 10μM SB431542 show a partial rescue of M. E) SB431542-treated mutants show a significantly reduced fraction of severe defects compared to the control group (p<0.012). (F-I) Dorsal views of edn1 ISH analysis on 22-somites stage embryos treated with DMSO or 10μM SB431542 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 SB431542 show similar expression compared to wild type. I) wdr68hi3812/hi3812 mutants treated with SB431542 show partial restoration of edn1 expression.
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pone.0166984.g007: Inhibition of TGF-β signaling partially rescues M cartilage and edn1 expression in wdr68hi3812/hi3812 zebrafish.(A-D) Ventral views of 5dpf Alcian stained craniofacial cartilages of zebrafish raised at 32°C and treated with DMSO or 10μM SB431542 at 14- to 15-somites stage. A) Wildtype zebrafish treated with DMSO control. Red arrow indicates M-PQ joint region. B) wdr68hi3812/hi3812 mutants treated with DMSO control show a lack of M cartilage. C) Wildtype zebrafish treated with 10μM SB431542 show normal craniofacial cartilage formation. D) wdr68hi3812/hi3812 mutants treated with 10μM SB431542 show a partial rescue of M. E) SB431542-treated mutants show a significantly reduced fraction of severe defects compared to the control group (p<0.012). (F-I) Dorsal views of edn1 ISH analysis on 22-somites stage embryos treated with DMSO or 10μM SB431542 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 SB431542 show similar expression compared to wild type. I) wdr68hi3812/hi3812 mutants treated with SB431542 show partial restoration of edn1 expression.
Mentions: If enhanced TGF-β interference with BMP signaling contributes to the wdr68 mutant phenotype in zebrafish, then inhibiting TGF-β pathway function should at least partially restore cartilage development in wdr68 mutants. SB431542 is a well-characterized inhibitor of the TGF-β family receptor kinases Alk4/5 [61], that is also known to inhibit TGF-β signaling activity in the zebrafish [62–64]. To avoid perturbing earlier developmental roles of TGF-β family signaling, embryos were treated with 10μM SB431542 starting at the 14- to 15-somites stages. 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 (Fig 1C). At 32°C, we found that DMSO-treated wildtype zebrafish developed the M cartilage as expected (Fig 7A, 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 7B, red arrowhead). 10μM SB431542-treated wildtype embryos were indistinguishable from DMSO-treated wildtypes (compare Fig 7C to 7A). In contrast, SB431542-treated mutants raised at 32°C exhibited a partial restoration of the M and PQ cartilages (compare Fig 7D to 7B, red arrowheads). Quantitation of the changes in phenotypic severity revealed a significant shift in the number of mutants displaying severe versus mild phenotypes. Under DMSO treatment, mild wdr68hi3812/hi3812 embryos were 8.6% of the total sample population whereas severe wdr68hi3812/hi3812 embryos were 14.3% of the total population (Fig 7E, left). Under SB431542 treatment, mild wdr68hi3812/hi3812 embryos were 19.2% of the total population whereas severe mutants were 8.0% of the total population (Fig 7E, right). Thus, SB431542 treatment decreased the fraction of severe mutants relative to the DMSO control (Fig 7E, red bars, p < 0.012).

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.