Genomic screen for genes involved in mammalian craniofacial development

Abstract

Using a subtractive hybridisation approach, we enriched for genes likely to play a role in embryonic development of the mammalian face and other structures. This was achieved by subtracting cDNA derived from adult mouse liver from that derived from 10.5 dpc mouse embryonic branchial arches 1 and 2. Random sequencing of clones from the resultant library revealed that a high percentage correspond to genes with a previously established role in embryonic development and disease, while 15% represent novel or uncharacterised genes. Whole mount in situ hybridisation analysis of novel genes revealed that approximately 50% have restricted expression during embryonic development. In addition to expression in branchial arches, these genes showed a range of expression domains commonly including neural tube and somites. Notably, all genes analysed were found to be expressed not only in the branchial arches but also in the developing limb buds, providing support for the hypothesis that development of the limbs and face is likely to involve analogous molecular processes.

The metalloendopeptidase gene Pitrm1 is regulated by hedgehog signaling in the developing mouse limb and is expressed in muscle progenitors

“…The Pax3 riboprobe was generated from 1,320 bp of genbank clone AK147919 and represents the previously published 516-bp probe between the HindIII and Pst1 restriction sites within the ORF (Bober et al, 1994) plus an additional 804 bp of 3 0 UTR. Embryos for in situ analyses were dissected in cold PBS and fixed overnight in 4% paraformaldehyde at 4 C. Whole mount and DIG section in situ hybridisation was carried out essentially as described previously (Fowles et al, 2003;Wilhelm et al, 2007, respectively). At least 3 embryos were examined at each stage (n > 3).…”

The coding region of TP53INP2, a gene expressed in the developing nervous system, is not altered in a family with autosomal recessive non‐progressive infantile ataxia on chromosome 20q11‐q13

“…We first identified the Tp53inp2 gene from a subtracted cDNA library derived from mouse pharyngeal arch tissue (Fowles et al, 2003;Bennetts et al, 2006). At that time, it had not been annotated, nor had a putative fulllength sequence been established in publicly available databases.…”

“…The Tp53inp2 probe used for in situ hybridization experiments was synthesized from a plasmid containing 485 bp of 3ЈUTR sequence (nt 2516-3000; NM_178111). Whole mount in situ hybridisation was performed as described previously (Christiansen et al, 1995;Fowles et al, 2003). Briefly, embryos were dissected from pregnant CD1 mice at a range of mid-gestational stages and were fixed overnight in 4% paraformaldehyde/PBS at 4°C, dehydrated, and then rehydrated through a methanol series.…”

“…Analysis of gene and protein sequences for homologies to known genes and for the presence of functional protein domains was performed as described previously (Fowles et al, 2003). Homology searches were performed using BLAST algorithms (Altschul et al, 1990(Altschul et al, , 1997 to identify homologous nucleotide and amino acid sequences through the National Center for Biotechnology Information (NCBI) public database (http://www.…”

Whole genome microarray analysis of chicken embryo facial prominences

“…Toward this end, several studies have performed expression analysis of facial regions using methods ranging from subtractive hybridization against a nonfacial tissue (liver; Fowles et al, 2003) to microarrays (Brown et al, 2003;Cai et al, 2005;Ivins et al, 2005;Handrigan et al, 2007) and serial analysis of gene expression (SAGE, Cai et al, 2005). Previously, two studies on avian frontonasal mass gene expression were carried out using custom cDNA arrays from older stage embryos when the skeletal condensations are present.…”