Deletion of long-range regulatory elements upstream of SOX9 causes campomelic dysplasia

Abstract

Campomelic dysplasia (CD) is a rare, neonatal human chondrodysplasia characterized by bowing of the long bones and often associated with male-to-female sex-reversal. Patients present with either heterozygous mutations in the SOX9 gene or chromosome rearrangements mapping at least 50 kb upstream of SOX9. Whereas mutations in SOX9 ORF cause haploinsufficiency, the effects of translocations 5' to SOX9 are unclear. To test whether these rearrangements also cause haploinsufficiency by altering spatial and temporal expression of SOX9, we generated mice transgenic for human SOX9-lacZ yeast artificial chromosomes containing variable amounts of DNA sequences upstream of SOX9. We show that elements necessary for SOX9 expression during skeletal development are highly conserved between mouse and human and reveal that a rearrangement upstream of SOX9, similar to those observed in CD patients, leads to a substantial reduction of SOX9 expression, particularly in chondrogenic tissues. These data demonstrate that important regulatory elements are scattered over a large region upstream of SOX9 and explain how particular aspects of the CD phenotype are caused by chromosomal rearrangements 5' to SOX9.

Figure 1

Molecular analysis of CD patients and construction of the SOX9/lacZ YACs. The YAC ICRF-946e12 was used as probe on metaphase spreads prepared from lymphoblastoid cell lines from sex-reversed patient cu002: 46,XY, inv17(q11.2;q24.3–25.1) (a) and patient cu004: 46,XY, t(9;17)(p13;q23.3–24.1) (b). (c) Deletion of YAC sequences was performed by using chromosome fragmentation vectors, and YAC derivatives still positive for SOX9 were selected. Fragmented YACs are depicted as vertical lines, and the names of identified markers, including their distances in kb, relative to SOX9, are given on a schematic representation of human chromosome 17q. Fluorescence in situ hybridization analysis performed on both patients and hybridization using the YAC derivative panel mapped the two cytogenetic probes λ11H, λ11I, to 75–150 kb and 200–350 kb 5′ of SOX9, respectively. (d) Schematic representation of the yeast-targeting vector 5′3′S9β-neoSVpA after homologous recombination with the YACs. The vector 5′3′S9β-neoSVpA was designed to integrate a lacZ reporter gene in frame with SOX9 coding sequence and to delete SOX9 HMG box (large, solid box) and transactivation domain (open box) via one event of homologous recombination with the 108.15 and 108.43 YACs.

Figure 2

(a) Analysis of transgenic offspring. After microinjection of purified YAC DNA, integrity and copy number of the transgenes were tested in the transgenic mice. Copy numbers are indicated between brackets below the names of each transgenic line. Markers used for testing YAC transgene integrity are listed, and their positions, relative to SOX9, are indicated. +, Marker present in the tested mice; −, absent marker. Tissues showing modifications in SOX9/lacZ expression are also reported. +, Normal tissue; −, altered tissue. (b–e) Comparison between expression derived from mouse endogenous Sox9 gene and the 600-kb human YAC transgene. (b and d) In situ hybridization using a Sox9 RNA probe on wild-type embryos. (c and e) β-Galactosidase staining of embryos from line A46 transgenic for one copy of the 600-kb YAC. (b and c) Embryos (10.5 dpc). (d and e) Embryos (11.5 dpc). SOX9/lacZ expression is detected at the correct time in the mesenchyme of the head, the otic vesicles, and branchial arches. Expression is also found in the neural tube, notochord, migrating sclerotomal cells, and developing ribs. Whereas expression in the limb buds is highly similar at 10.5 dpc, differences in the patterns occur from 11.5 dpc. At that stage, SOX9/lacZ is strongly expressed in the mesenchymal condensation of the long bones and expression in the hand and foot plates is detected only from 12.5 dpc. In contrast, from 11.5 dpc, mouse Sox9 expression is strongly expressed in the hand and foot plates, the developing long bones only expressing weakly Sox9. This difference may be caused by species-specific elements or by the lack, on the YAC, of control elements involved in SOX9/Sox9 expression during limb development.

Figure 3

Comparison between SOX9/lacZ expression derived from the 350-kb and 600-kb YACs. (a–f) β-Galactosidase staining of embryos from line A46 (one copy of the 600-kb YAC), line A45.1 (one copy of the short construct), and line A75 (multiple copies of the 350-kb YAC). (g–n) Microtome sections performed on 10.5- to 12.5-dpc embryos from lines A46 and A75. ba, branchial arches; ov, otic vesicle; lb, long bones; hp, hand plate; fp, foot plate; r, ribs; ea, endolymphatic duct; ut, utriculus; md, mandibular process of the first branchial arch; mx, maxillary process; h, humeral mesenchymal condensation; r/u, radio-ulna mesenchymal condensation. Throughout development, expression in line A45.1 is found dramatically decreased in all tissues normally expressing SOX9/lacZ (line A46). Expression in the neuroectoderm, however, is only mildly affected. (g, h, k, and l) Expression derived from the 350-kb YAC occurs at the correct location in the developing ear, but a significant delay in the establishment of the expression pattern takes place: SOX9/lacZ expression in the endolymphatic and utricular portions of the otocyst is only detected from 12.5 dpc instead of 11.5 dpc with the 600-kb YAC. This delay may correspond to the time necessary for SOX9/LacZ proteins to accumulate in the cells to obtain detectable levels of β-galactosidase activity. (i and m) In the developing branchial arches, the deletion of SOX9 upstream sequences results in SOX9/lacZ expression only in the maxillary process of the first branchial arch. This suggests the involvement of multiple elements in the control of SOX9 expression in distinct regions of the branchial arches. (j and n) In contrast to line A46, where expression in the developing long bones precedes expression in the hand and foot plates, expression derived from the 350-kb YAC is induced from 11.5 dpc in the hand and foot plates, with the expression in the long bones remaining weak. The 5′ deletion therefore alters onset and level of expression in particular regions of the limb bud.