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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.

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Wdr68 is required for craniofacial development between the 17 somites and prim-5 stages.A) Ventral view of 5dpf alcian blue stained cartilages in wild type zebrafish. M: Meckel’s PQ: Palatoquadrate. B) Mild mutant phenotype resulting from rearing of embryos at 24°C, characterized by joint fusions (arrowhead) between M and PQ. C) Severe mutant phenotype resulting from rearing of embryos at 32°C, characterized by the loss of M and PQ. D) Immunohistochemistry readily detects maternal Wdr68 protein up to the 17 somites stage in wdr68hi3812/hi3812 mutants raised at the permissive 24°C temperature (blue line). Maternal Wdr68 is lost by the 17 somites stage in wdr68hi3812/hi3812 mutants raised at the non-permissive 32°C temperature (red line). E) wdr68-MO injected non-transgenic (Non-Tg) animals display jaw defects regardless of heat shock. wdr68-MO injected Tg(hsp70l:GFP-Wdr68)csu9 animals that are heat shocked by the prim-5 stage show rescue from jaw defects. Error bars indicate standard deviation. Additional abbreviations: Shield (Sh), 15 somites (15s), 17 somites (17s), 20 somites (20s), 25 somites (25s), prim-5 (p5), prim-25 (p25), no heat shock (-HS).
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pone.0166984.g001: Wdr68 is required for craniofacial development between the 17 somites and prim-5 stages.A) Ventral view of 5dpf alcian blue stained cartilages in wild type zebrafish. M: Meckel’s PQ: Palatoquadrate. B) Mild mutant phenotype resulting from rearing of embryos at 24°C, characterized by joint fusions (arrowhead) between M and PQ. C) Severe mutant phenotype resulting from rearing of embryos at 32°C, characterized by the loss of M and PQ. D) Immunohistochemistry readily detects maternal Wdr68 protein up to the 17 somites stage in wdr68hi3812/hi3812 mutants raised at the permissive 24°C temperature (blue line). Maternal Wdr68 is lost by the 17 somites stage in wdr68hi3812/hi3812 mutants raised at the non-permissive 32°C temperature (red line). E) wdr68-MO injected non-transgenic (Non-Tg) animals display jaw defects regardless of heat shock. wdr68-MO injected Tg(hsp70l:GFP-Wdr68)csu9 animals that are heat shocked by the prim-5 stage show rescue from jaw defects. Error bars indicate standard deviation. Additional abbreviations: Shield (Sh), 15 somites (15s), 17 somites (17s), 20 somites (20s), 25 somites (25s), prim-5 (p5), prim-25 (p25), no heat shock (-HS).

Mentions: The wdr68hi3812/hi3812 mutant was originally described as presenting a range of jaw defects that varied from only mild joint fusions to near complete losses of the palatoquadrate (PQ) and Meckel’s (M) cartilages [36]. The initial characterization was completed using embryos raised at the standard 28.5°C. We have since found that rearing the animals at 24°C yielded mutant animals that mostly present only the mild M-PQ joint fusion defects (Fig 1B red arrowhead, compare to 1A). Specifically at 24°C, 79% of mutants presented only mild M-PQ fusions versus 21% displaying severe loss of the M and/or PQ cartilage. In contrast, rearing embryos at 32°C yielded mutant animals that mostly present severe losses of the M and PQ cartilages (Fig 1C compare to 1A, 1B). Specifically at 32°C, 6% of mutants presented only mild M-PQ fusions versus 94% displaying severe loss of the M and/or PQ cartilage. Because the wdr68hi3812 allele is a retroviral insertion within exon-1 near a splicing junction [36], and insertional mutations can cause temperature-dependent splicing defects [56], we examined animals reared at 24°C versus 32°C by RT-PCR in an effort to detect potential alternative splicing products but found no evidence to support such a mechanism (data not shown).


Wdr68 Mediates Dorsal and Ventral Patterning Events for Craniofacial Development
Wdr68 is required for craniofacial development between the 17 somites and prim-5 stages.A) Ventral view of 5dpf alcian blue stained cartilages in wild type zebrafish. M: Meckel’s PQ: Palatoquadrate. B) Mild mutant phenotype resulting from rearing of embryos at 24°C, characterized by joint fusions (arrowhead) between M and PQ. C) Severe mutant phenotype resulting from rearing of embryos at 32°C, characterized by the loss of M and PQ. D) Immunohistochemistry readily detects maternal Wdr68 protein up to the 17 somites stage in wdr68hi3812/hi3812 mutants raised at the permissive 24°C temperature (blue line). Maternal Wdr68 is lost by the 17 somites stage in wdr68hi3812/hi3812 mutants raised at the non-permissive 32°C temperature (red line). E) wdr68-MO injected non-transgenic (Non-Tg) animals display jaw defects regardless of heat shock. wdr68-MO injected Tg(hsp70l:GFP-Wdr68)csu9 animals that are heat shocked by the prim-5 stage show rescue from jaw defects. Error bars indicate standard deviation. Additional abbreviations: Shield (Sh), 15 somites (15s), 17 somites (17s), 20 somites (20s), 25 somites (25s), prim-5 (p5), prim-25 (p25), no heat shock (-HS).
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pone.0166984.g001: Wdr68 is required for craniofacial development between the 17 somites and prim-5 stages.A) Ventral view of 5dpf alcian blue stained cartilages in wild type zebrafish. M: Meckel’s PQ: Palatoquadrate. B) Mild mutant phenotype resulting from rearing of embryos at 24°C, characterized by joint fusions (arrowhead) between M and PQ. C) Severe mutant phenotype resulting from rearing of embryos at 32°C, characterized by the loss of M and PQ. D) Immunohistochemistry readily detects maternal Wdr68 protein up to the 17 somites stage in wdr68hi3812/hi3812 mutants raised at the permissive 24°C temperature (blue line). Maternal Wdr68 is lost by the 17 somites stage in wdr68hi3812/hi3812 mutants raised at the non-permissive 32°C temperature (red line). E) wdr68-MO injected non-transgenic (Non-Tg) animals display jaw defects regardless of heat shock. wdr68-MO injected Tg(hsp70l:GFP-Wdr68)csu9 animals that are heat shocked by the prim-5 stage show rescue from jaw defects. Error bars indicate standard deviation. Additional abbreviations: Shield (Sh), 15 somites (15s), 17 somites (17s), 20 somites (20s), 25 somites (25s), prim-5 (p5), prim-25 (p25), no heat shock (-HS).
Mentions: The wdr68hi3812/hi3812 mutant was originally described as presenting a range of jaw defects that varied from only mild joint fusions to near complete losses of the palatoquadrate (PQ) and Meckel’s (M) cartilages [36]. The initial characterization was completed using embryos raised at the standard 28.5°C. We have since found that rearing the animals at 24°C yielded mutant animals that mostly present only the mild M-PQ joint fusion defects (Fig 1B red arrowhead, compare to 1A). Specifically at 24°C, 79% of mutants presented only mild M-PQ fusions versus 21% displaying severe loss of the M and/or PQ cartilage. In contrast, rearing embryos at 32°C yielded mutant animals that mostly present severe losses of the M and PQ cartilages (Fig 1C compare to 1A, 1B). Specifically at 32°C, 6% of mutants presented only mild M-PQ fusions versus 94% displaying severe loss of the M and/or PQ cartilage. Because the wdr68hi3812 allele is a retroviral insertion within exon-1 near a splicing junction [36], and insertional mutations can cause temperature-dependent splicing defects [56], we examined animals reared at 24°C versus 32°C by RT-PCR in an effort to detect potential alternative splicing products but found no evidence to support such a mechanism (data not shown).

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.


Related in: MedlinePlus