RESUMEN
Human disorders of the post-squalene cholesterol biosynthesis pathway frequently result in skeletal abnormalities, yet our understanding of the mechanisms involved is limited. In a forward-genetic approach, we have found that a late-onset skeletal mutant, named kolibernu7 , is the result of a cis-acting regulatory mutation leading to loss of methylsterol monooxygenase 1 (msmo1) expression within pre-hypertrophic chondrocytes. Generated msmo1nu81 knockdown mutation resulted in lethality at larval stage. We demonstrated that this is a result of both cholesterol deprivation and sterol intermediate accumulation by creating a mutation eliminating activity of Lanosterol synthase (Lss). Our results indicate that double lssnu60;msmo1nu81 and single lssnu60 mutants survive significantly longer than msmo1nu81 homozygotes. Liver-specific restoration of either Msmo1 or Lss in corresponding mutant backgrounds suppresses larval lethality. Rescued mutants develop dramatic skeletal abnormalities, with a loss of Msmo1 activity resulting in a more-severe patterning defect of a near-complete loss of hypertrophic chondrocytes marked by col10a1a expression. Our analysis suggests that hypertrophic chondrocytes depend on endogenous cholesterol synthesis, and blocking C4 demethylation exacerbates the cholesterol deficiency phenotype. Our findings offer new insight into the genetic control of bone development and provide new zebrafish models for human disorders of the cholesterol biosynthesis pathway.
Asunto(s)
Enfermedades del Desarrollo Óseo/metabolismo , Huesos/metabolismo , Colesterol/biosíntesis , Condrocitos/metabolismo , Hígado/metabolismo , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Enfermedades del Desarrollo Óseo/genética , Enfermedades del Desarrollo Óseo/patología , Huesos/patología , Condrocitos/patología , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Transferasas Intramoleculares/genética , Transferasas Intramoleculares/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Mutación , Fenotipo , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The Wnt/Planar Cell Polarity (PCP) pathway controls cell morphology and behavior during animal development. Several zebrafish mutants were identified as having perturbed Wnt/PCP signaling. Many of these mutants have defects in craniofacial formation. To better understand the role that Wnt/PCP plays in craniofacial development we set out to identify which of the mutants, known to be associated with the Wnt/PCP pathway, perturb head cartilage formation by disrupting chondrocyte morphology. Here we demonstrate that while vang-like 2 (vangl2), wnt11 and scribbled (scrib) mutants have severe craniofacial morphogenesis defects they do not display the chondrocyte stacking and intercalation problems seen in glypican 4 (gpc4) and wnt5b mutants. The function of Gpc4 or Wnt5b appears to be important for chondrocyte organization, as the neural crest in both mutants is specified, undergoes migration, and differentiates into the same number of cells to compose the craniofacial cartilage elements. We demonstrate that Gpc4 activity is required cell autonomously in the chondrocytes and that the phenotype of single heterozygous mutants is slightly enhanced in embryos double heterozygous for wnt5b and gpc4. This data suggests a novel mechanism for Wnt5b and Gpc4 regulation of chondrocyte behavior that is independent of the core Wnt/PCP molecules and differs from their collaborative action of controlling cell movements during gastrulation.
Asunto(s)
Condrocitos/metabolismo , Condrogénesis/genética , Glipicanos/genética , Proteínas Wnt/genética , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Región Branquial/embriología , Región Branquial/metabolismo , Recuento de Células , Movimiento Celular/genética , Tamaño de la Célula , Condrocitos/citología , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica , Glipicanos/deficiencia , Mutación , Cresta Neural/embriología , Cresta Neural/metabolismo , Fenotipo , Proteínas Wnt/deficiencia , Vía de Señalización Wnt/genética , Proteína Wnt-5a , Pez Cebra/metabolismo , Proteínas de Pez Cebra/deficienciaRESUMEN
The heparan sulfate proteoglycan Glypican 4 (Gpc4) is part of the Wnt/planar cell polarity pathway, which is required for convergence and extension during zebrafish gastrulation. To observe Glypican 4-deficient phenotypes at later stages, we rescued gpc4(-/-) (knypek) homozygotes and raised them for more than one year. Adult mutants showed diverse cranial malformations of both dermal and endochondral bones, ranging from shortening of the rostral-most skull to loss of the symplectic. Additionally, the adult palatoquadrate cartilage was disorganized, with abnormal chondrocyte orientation. To understand how the palatoquadrate cartilage normally develops, we examined a juvenile series of wild type and mutant specimens. This identified two novel domains of elongated chondrocytes in the larval palatoquadrate, which normally form prior to endochondral ossification. In contrast, gpc4(-/-) larvae never form these domains, suggesting a failure of chondrocyte orientation, though not differentiation. Our findings implicate Gpc4 in the regulation of zebrafish cartilage and bone morphogenesis.
