A Chicken Model for DGCR6 as a Modifier Gene in the DiGeorge Critical Region

Abstract

DGCR6 is the most centromeric gene in the human DiGeorge critical region and is the only gene in the region with a second functional copy on a repeat localized more distally on chromosome 22. We isolated the chicken ortholog of DGCR6 and showed an embryonic expression pattern that is initially broad but becomes gradually restricted to neural crest cell derivatives of the cardiovasculature. Retrovirus based gene transduction was used to deliver sense and antisense messages to premigrating neural crest cells in vivo. Embryos in which DGCR6 expression was attenuated revealed cardiovascular anomalies reminiscent of those found in DiGeorge syndrome. Moreover, the expression profiles of three other genes from the DiGeorge critical region, TBX-1, UFD1L, and HIRA, were shown to be altered in this model. TBX-1 and UFD1L levels were increased, whereas HIRA was decreased in the hearts and pharyngeal arches of embryos treated with antisense or partial sense constructs, but not with sense constructs for DGCR6. The expression changes were transient and followed the normal DGCR6 expression profile. These data show that neural crest cells might have a role in the distribution of modulator signals to the heart and pharyngeal arches. Moreover, it shows a repressor function for DGCR6 on the expression of TBX-1 and UFD1L. For the first time, DiGeorge syndrome is shown to be a contiguous gene syndrome in which not only several genes from the critical region, but also different cell types within the embryo, interact in the development of the phenotype. Congenital anomalies can provide important clues for the impact of specific processes in embryonic development. With respect to the chromosome 22 deletion syndrome or DGS (OMIM 188400), a patient's phenotype is clearly related to disturbance of migration or differentiation of neural crest. This might also be evoked by an inadequate interaction with surrounding cells like the endo-and mesoderm in the pharyngeal arches. In DGS patients, cardiovascular anomalies often coincide with thymic, thyroid, and parathyroid defects (1). All these areas are populated by cephalic and cardiac NCC that delaminate from the dorsal neural tube during neurulation and enter the mesodermal compartment of the embryo (2-5). The chicken NCC ablation model (6) shows that these neural crest areas are preferentially disturbed in DGS. In this respect, it is remarkable that, of the cardiovasculature in DGS patients, primarily the cardiac outflow tract and the fourth pharyngeal arch arteries are affected, although all arch arteries as well as outflow and inflow tracts of the heart are invaded by NCC (5, 7). The DGS phenotype in general presents with IAA-B, PTA, VSD, and tetralogy of Fallot (8,9).Although the affected chromosomal region has been known for over a decade, pinpointing the gene(s) involved in DGS is difficult. Genomic analysis of patients revealed a hemizygously typical deleted region of 1.5-to 3.0-Mb on chromosome 22q1.1 carrying over 30 genes. This region appears highly unstable due to the presence of A...

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“…RNA was treated with DNAse-I, and 500 ng was reverse transcribed into cDNA with M-MuLV Reverse Transcriptase (Amersham). Equal amounts of cDNA were subjected in duplicate to QRT-PCR, using Brilliant SYBR Green QPCR Master Mix (Stratagene) on an MX3000P PCR machine (Stratagene) [22]. The reactions contained 1× PCR Master Mix, 2 µl cDNA template and 10 pmol of each primer.…”

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