A zebrafish sox9 gene required for cartilage morphogenesis

Yi Lin Yan, Craig T. Miller, Robert Nissen, Amy Singer, Dong Liu, Anette Kirn, Bruce Draper, John Willoughby, Paul A. Morcos, Adam Amsterdam, Bon Chu Chung, Monte Westerfield, Pascal Haffter, Nancy Hopkins, Charles Kimmel, John H. Postlethwait

Research output: Contribution to journalArticle

215 Citations (Scopus)

Abstract

The molecular genetic mechanisms of cartilage construction are incompletely understood. Zebrafish embryos homozygous for jellyfish (jef) mutations show craniofacial defects and lack cartilage elements of the neurocranium, pharyngeal arches, and pectoral girdle similar to humans with campomelic dysplasia. We show that two alleles of jef contain mutations in sox9a, one of two zebrafish orthologs of the human transcription factor SOX9. A mutation induced by ethyl nitrosourea changed a conserved nucleotide at a splice junction and severely reduced splicing of sox9a transcript. A retrovirus insertion into sox9a disrupted its DNA-binding domain. Inhibiting splicing of the sox9a transcript in wild-type embryos with splice site-directed morpholino antisense oligonucleotides produced a phenotype like jef mutant larvae, and caused sox9a transcript to accumulate in the nucleus; this accumulation can serve as an assay for the efficacy of a morpholino independent of phenotype. RNase-protection assays showed that in morpholino-injected animals, the percent of splicing inhibition decreased from 80% at 28 hours post fertilization to 45% by 4 days. Homozygous mutant embryos had greatly reduced quantities of col2a1 message, the major collagen of cartilage. Analysis of dlx2 expression showed that neural crest specification and migration was normal in jef (sox9a) embryos. Confocal images of living embryos stained with BODIPY-ceramide revealed at single-cell resolution the formation of precartilage condensations in mutant embryos. Besides the lack of overt cartilage differentiation, pharyngeal arch condensations in jef (sox9a) mutants lacked three specific morphogenetic behaviors: the stacking of chondrocytes into orderly arrays, the individuation of pharyngeal cartilage organs and the proper shaping of individual cartilages. Despite the severe reduction of cartilages, analysis of titin expression showed normal muscle patterning in jef (sox9a) mutants. Likewise, calcein labeling revealed that early bone formation was largely unaffected in jef (sox9a) mutants. These studies show that jef (sox9a) is essential for both morphogenesis of condensations and overt cartilage differentiation.

Original languageEnglish (US)
Pages (from-to)5065-5079
Number of pages15
JournalDevelopment
Volume129
Issue number21
StatePublished - Nov 2002
Externally publishedYes

Fingerprint

Zebrafish
Morphogenesis
Cartilage
Embryonic Structures
Morpholinos
Genes
Branchial Region
Mutation
SOX9 Transcription Factor
Campomelic Dysplasia
Individuation
Connectin
Phenotype
Neural Crest
Antisense Oligonucleotides
Ceramides
Retroviridae
Ribonucleases
Chondrocytes
Fertilization

Keywords

  • Campomelic dysplasia
  • Cartilage
  • Chondrogenesis
  • Col2al
  • Pharyngeal arches
  • Sox9a
  • Titin
  • Zebrafish

ASJC Scopus subject areas

  • Anatomy
  • Cell Biology

Cite this

Yan, Y. L., Miller, C. T., Nissen, R., Singer, A., Liu, D., Kirn, A., ... Postlethwait, J. H. (2002). A zebrafish sox9 gene required for cartilage morphogenesis. Development, 129(21), 5065-5079.

A zebrafish sox9 gene required for cartilage morphogenesis. / Yan, Yi Lin; Miller, Craig T.; Nissen, Robert; Singer, Amy; Liu, Dong; Kirn, Anette; Draper, Bruce; Willoughby, John; Morcos, Paul A.; Amsterdam, Adam; Chung, Bon Chu; Westerfield, Monte; Haffter, Pascal; Hopkins, Nancy; Kimmel, Charles; Postlethwait, John H.

In: Development, Vol. 129, No. 21, 11.2002, p. 5065-5079.

Research output: Contribution to journalArticle

Yan, YL, Miller, CT, Nissen, R, Singer, A, Liu, D, Kirn, A, Draper, B, Willoughby, J, Morcos, PA, Amsterdam, A, Chung, BC, Westerfield, M, Haffter, P, Hopkins, N, Kimmel, C & Postlethwait, JH 2002, 'A zebrafish sox9 gene required for cartilage morphogenesis', Development, vol. 129, no. 21, pp. 5065-5079.
Yan YL, Miller CT, Nissen R, Singer A, Liu D, Kirn A et al. A zebrafish sox9 gene required for cartilage morphogenesis. Development. 2002 Nov;129(21):5065-5079.
Yan, Yi Lin ; Miller, Craig T. ; Nissen, Robert ; Singer, Amy ; Liu, Dong ; Kirn, Anette ; Draper, Bruce ; Willoughby, John ; Morcos, Paul A. ; Amsterdam, Adam ; Chung, Bon Chu ; Westerfield, Monte ; Haffter, Pascal ; Hopkins, Nancy ; Kimmel, Charles ; Postlethwait, John H. / A zebrafish sox9 gene required for cartilage morphogenesis. In: Development. 2002 ; Vol. 129, No. 21. pp. 5065-5079.
@article{abb8e39697b84203a2409cdb0a1eaa24,
title = "A zebrafish sox9 gene required for cartilage morphogenesis",
abstract = "The molecular genetic mechanisms of cartilage construction are incompletely understood. Zebrafish embryos homozygous for jellyfish (jef) mutations show craniofacial defects and lack cartilage elements of the neurocranium, pharyngeal arches, and pectoral girdle similar to humans with campomelic dysplasia. We show that two alleles of jef contain mutations in sox9a, one of two zebrafish orthologs of the human transcription factor SOX9. A mutation induced by ethyl nitrosourea changed a conserved nucleotide at a splice junction and severely reduced splicing of sox9a transcript. A retrovirus insertion into sox9a disrupted its DNA-binding domain. Inhibiting splicing of the sox9a transcript in wild-type embryos with splice site-directed morpholino antisense oligonucleotides produced a phenotype like jef mutant larvae, and caused sox9a transcript to accumulate in the nucleus; this accumulation can serve as an assay for the efficacy of a morpholino independent of phenotype. RNase-protection assays showed that in morpholino-injected animals, the percent of splicing inhibition decreased from 80{\%} at 28 hours post fertilization to 45{\%} by 4 days. Homozygous mutant embryos had greatly reduced quantities of col2a1 message, the major collagen of cartilage. Analysis of dlx2 expression showed that neural crest specification and migration was normal in jef (sox9a) embryos. Confocal images of living embryos stained with BODIPY-ceramide revealed at single-cell resolution the formation of precartilage condensations in mutant embryos. Besides the lack of overt cartilage differentiation, pharyngeal arch condensations in jef (sox9a) mutants lacked three specific morphogenetic behaviors: the stacking of chondrocytes into orderly arrays, the individuation of pharyngeal cartilage organs and the proper shaping of individual cartilages. Despite the severe reduction of cartilages, analysis of titin expression showed normal muscle patterning in jef (sox9a) mutants. Likewise, calcein labeling revealed that early bone formation was largely unaffected in jef (sox9a) mutants. These studies show that jef (sox9a) is essential for both morphogenesis of condensations and overt cartilage differentiation.",
keywords = "Campomelic dysplasia, Cartilage, Chondrogenesis, Col2al, Pharyngeal arches, Sox9a, Titin, Zebrafish",
author = "Yan, {Yi Lin} and Miller, {Craig T.} and Robert Nissen and Amy Singer and Dong Liu and Anette Kirn and Bruce Draper and John Willoughby and Morcos, {Paul A.} and Adam Amsterdam and Chung, {Bon Chu} and Monte Westerfield and Pascal Haffter and Nancy Hopkins and Charles Kimmel and Postlethwait, {John H.}",
year = "2002",
month = "11",
language = "English (US)",
volume = "129",
pages = "5065--5079",
journal = "Development (Cambridge)",
issn = "0950-1991",
publisher = "Company of Biologists Ltd",
number = "21",

}

TY - JOUR

T1 - A zebrafish sox9 gene required for cartilage morphogenesis

AU - Yan, Yi Lin

AU - Miller, Craig T.

AU - Nissen, Robert

AU - Singer, Amy

AU - Liu, Dong

AU - Kirn, Anette

AU - Draper, Bruce

AU - Willoughby, John

AU - Morcos, Paul A.

AU - Amsterdam, Adam

AU - Chung, Bon Chu

AU - Westerfield, Monte

AU - Haffter, Pascal

AU - Hopkins, Nancy

AU - Kimmel, Charles

AU - Postlethwait, John H.

PY - 2002/11

Y1 - 2002/11

N2 - The molecular genetic mechanisms of cartilage construction are incompletely understood. Zebrafish embryos homozygous for jellyfish (jef) mutations show craniofacial defects and lack cartilage elements of the neurocranium, pharyngeal arches, and pectoral girdle similar to humans with campomelic dysplasia. We show that two alleles of jef contain mutations in sox9a, one of two zebrafish orthologs of the human transcription factor SOX9. A mutation induced by ethyl nitrosourea changed a conserved nucleotide at a splice junction and severely reduced splicing of sox9a transcript. A retrovirus insertion into sox9a disrupted its DNA-binding domain. Inhibiting splicing of the sox9a transcript in wild-type embryos with splice site-directed morpholino antisense oligonucleotides produced a phenotype like jef mutant larvae, and caused sox9a transcript to accumulate in the nucleus; this accumulation can serve as an assay for the efficacy of a morpholino independent of phenotype. RNase-protection assays showed that in morpholino-injected animals, the percent of splicing inhibition decreased from 80% at 28 hours post fertilization to 45% by 4 days. Homozygous mutant embryos had greatly reduced quantities of col2a1 message, the major collagen of cartilage. Analysis of dlx2 expression showed that neural crest specification and migration was normal in jef (sox9a) embryos. Confocal images of living embryos stained with BODIPY-ceramide revealed at single-cell resolution the formation of precartilage condensations in mutant embryos. Besides the lack of overt cartilage differentiation, pharyngeal arch condensations in jef (sox9a) mutants lacked three specific morphogenetic behaviors: the stacking of chondrocytes into orderly arrays, the individuation of pharyngeal cartilage organs and the proper shaping of individual cartilages. Despite the severe reduction of cartilages, analysis of titin expression showed normal muscle patterning in jef (sox9a) mutants. Likewise, calcein labeling revealed that early bone formation was largely unaffected in jef (sox9a) mutants. These studies show that jef (sox9a) is essential for both morphogenesis of condensations and overt cartilage differentiation.

AB - The molecular genetic mechanisms of cartilage construction are incompletely understood. Zebrafish embryos homozygous for jellyfish (jef) mutations show craniofacial defects and lack cartilage elements of the neurocranium, pharyngeal arches, and pectoral girdle similar to humans with campomelic dysplasia. We show that two alleles of jef contain mutations in sox9a, one of two zebrafish orthologs of the human transcription factor SOX9. A mutation induced by ethyl nitrosourea changed a conserved nucleotide at a splice junction and severely reduced splicing of sox9a transcript. A retrovirus insertion into sox9a disrupted its DNA-binding domain. Inhibiting splicing of the sox9a transcript in wild-type embryos with splice site-directed morpholino antisense oligonucleotides produced a phenotype like jef mutant larvae, and caused sox9a transcript to accumulate in the nucleus; this accumulation can serve as an assay for the efficacy of a morpholino independent of phenotype. RNase-protection assays showed that in morpholino-injected animals, the percent of splicing inhibition decreased from 80% at 28 hours post fertilization to 45% by 4 days. Homozygous mutant embryos had greatly reduced quantities of col2a1 message, the major collagen of cartilage. Analysis of dlx2 expression showed that neural crest specification and migration was normal in jef (sox9a) embryos. Confocal images of living embryos stained with BODIPY-ceramide revealed at single-cell resolution the formation of precartilage condensations in mutant embryos. Besides the lack of overt cartilage differentiation, pharyngeal arch condensations in jef (sox9a) mutants lacked three specific morphogenetic behaviors: the stacking of chondrocytes into orderly arrays, the individuation of pharyngeal cartilage organs and the proper shaping of individual cartilages. Despite the severe reduction of cartilages, analysis of titin expression showed normal muscle patterning in jef (sox9a) mutants. Likewise, calcein labeling revealed that early bone formation was largely unaffected in jef (sox9a) mutants. These studies show that jef (sox9a) is essential for both morphogenesis of condensations and overt cartilage differentiation.

KW - Campomelic dysplasia

KW - Cartilage

KW - Chondrogenesis

KW - Col2al

KW - Pharyngeal arches

KW - Sox9a

KW - Titin

KW - Zebrafish

UR - http://www.scopus.com/inward/record.url?scp=0036848704&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036848704&partnerID=8YFLogxK

M3 - Article

C2 - 12397114

AN - SCOPUS:0036848704

VL - 129

SP - 5065

EP - 5079

JO - Development (Cambridge)

JF - Development (Cambridge)

SN - 0950-1991

IS - 21

ER -