Functional Analysis of Conserved Non-Coding Regions Around the Short Stature hox Gene (shox) in Whole Zebrafish Embryos

Emma J Kenyon,Greg Elgar,Gayle K Mcewen,Heather Callaway

doi:10.1371/journal.pone.0021498

Abstract

BackgroundMutations in the SHOX gene are responsible for Leri-Weill Dyschondrosteosis, a disorder characterised by mesomelic limb shortening. Recent investigations into regulatory elements surrounding SHOX have shown that deletions of conserved non-coding elements (CNEs) downstream of the SHOX gene produce a phenotype indistinguishable from Leri-Weill Dyschondrosteosis. As this gene is not found in rodents, we used zebrafish as a model to characterise the expression pattern of the shox gene across the whole embryo and characterise the enhancer domains of different CNEs associated with this gene.Methodology/Principal FindingsExpression of the shox gene in zebrafish was identified using in situ hybridization, with embryos showing expression in the blood, putative heart, hatching gland, brain pharyngeal arch, olfactory epithelium, and fin bud apical ectodermal ridge. By identifying sequences showing 65% identity over at least 40 nucleotides between Fugu, human, dog and opossum we uncovered 35 CNEs around the shox gene. These CNEs were compared with CNEs previously discovered by Sabherwal et al., resulting in the identification of smaller more deeply conserved sub-sequence. Sabherwal et al.'s CNEs were assayed for regulatory function in whole zebrafish embryos resulting in the identification of additional tissues under the regulatory control of these CNEs.Conclusion/SignificanceOur results using whole zebrafish embryos have provided a more comprehensive picture of the expression pattern of the shox gene, and a better understanding of its regulation via deeply conserved noncoding elements. In particular, we identify additional tissues under the regulatory control of previously identified SHOX CNEs. We also demonstrate the importance of these CNEs in evolution by identifying duplicated shox CNEs and more deeply conserved sub-sequences within already identified CNEs.

Highlights

Mutations in the short stature HOX gene, SHOX have been shown to be responsible for the dominantly inherited skeletal dysplasia Leri-Weill Dyschondrosteosis (LWD)
Identification of CNEs at the shox locus Conserved non-coding elements (CNEs) around the shox gene were identified from a multiple alignment of the human, dog, opossum and Fugu loci using MLAGAN [11]
By comparing Sabherwal’s human CNE4 with the our smaller CNE hCRCNE00011095, we found that our hCRCNE000111095 drives less expression (28 out of 276 embryos at day 2, 18 out of 249 at day 3) than Sabherwal’s hCNE4 (49 out of 243 embryos at day 2, 42 out of 236 embryos at day 3)

Summary

Introduction

Mutations in the short stature HOX gene, SHOX have been shown to be responsible for the dominantly inherited skeletal dysplasia Leri-Weill Dyschondrosteosis (LWD). LWD is characterised by disproportionate short stature with mesomelic limb shortening [1] This disorder is the result of haploinsufficiency of the SHOX gene, which is found in the pseudoautosomal region at the telomere of the short arm of the X and Y chromosomes. SHOX was discovered when looking for a Turner syndrome short-stature gene in the Xp-Yp pseudoautosomal region (PAR1) Genes within this region escape X-inactivation in females and participate in obligate recombination during male meiosis. Recent investigations into regulatory elements surrounding SHOX have shown that deletions of conserved non-coding elements (CNEs) downstream of the SHOX gene produce a phenotype indistinguishable from LeriWeill Dyschondrosteosis. As this gene is not found in rodents, we used zebrafish as a model to characterise the expression pattern of the shox gene across the whole embryo and characterise the enhancer domains of different CNEs associated with this gene

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