Generation of mice with functional inactivation oftalpid3, a gene first identified in chicken

Abstract

SUMMARYSpecification of digit number and identity is central to digit pattern in vertebrate limbs. The classical talpid 3 chicken mutant has many unpatterned digits together with defects in other regions, depending on hedgehog (Hh) signalling, and exhibits embryonic lethality. The talpid 3 chicken has a mutation in KIAA0586, which encodes a centrosomal protein required for the formation of primary cilia, which are sites of vertebrate Hh signalling. The highly conserved exons 11 and 12 of KIAA0586 are essential to rescue cilia in talpid 3 chicken mutants. We constitutively deleted these two exons to make a talpid3 -/-mouse. Mutant mouse embryos lack primary cilia and, like talpid 3 chicken embryos, have face and neural tube defects but also defects in left/right asymmetry. Conditional deletion in mouse limb mesenchyme results in polydactyly and in brachydactyly and a failure of subperisoteal bone formation, defects that are attributable to abnormal sonic hedgehog and Indian hedgehog signalling, respectively. Like talpid 3 chicken limbs, the mutant mouse limbs are syndactylous with uneven digit spacing as reflected in altered Raldh2 expression, which is normally associated with interdigital mesenchyme. Both mouse and chicken mutant limb buds are broad and short. talpid3 -/-mouse cells migrate more slowly than wild-type mouse cells, a change in cell behaviour that possibly contributes to altered limb bud morphogenesis. This genetic mouse model will facilitate further conditional approaches, epistatic experiments and open up investigation into the function of the novel talpid3 gene using the many resources available for mice.

Mutations in KIAA0586 Cause Lethal Ciliopathies Ranging from a Hydrolethalus Phenotype to Short-Rib Polydactyly Syndrome

“…KIAA0586, the ortholog of chicken KIAA0586, was considered an excellent candidate gene given that its disruption in animal models causes defects, including polydactyly and abnormal dorsoventral patterning of the neural tube, attributed to abnormal hedgehog signaling. [8][9][10][11] The c.230C>G (p.Ser77*) variant (GenBank: NM_001244189.1) segregated with the expected patterns of autosomal-recessive inheritance in all available family members and is absent from dbSNP, the NHLBI Exome Sequencing Project (ESP) Exome Variant Server (EVS), the Exome Aggregation Consortium (ExAC) Browser, and 300 Lebanese control chromosomes. This homozygous nonsense variation is located in KIAA0586 exon 2, and mRNAs produced are predicted to be targeted for nonsense-mediated decay (NMD).…”

“…Thus, KIAA0586 mutant cells exhibit abnormal SHH responsiveness, suggesting that at least some of the defects in affected individuals with KIAA0586 variations might be secondary to abnormal SHH signaling, as observed in animal models. [8][9][10][11]17 In this study, using a targeted high-throughput sequencing strategy for candidate ciliary genes, we identified homozygous nonsense and splicing KIAA0586 mutations as responsible for lethal ciliopathies. This candidate-gene strategy previously succeeded in identifying ciliopathy genes, including TCTN3 (MIM: 613847), which is associated with Mohr-Majewski syndrome (OFD4 [MIM: 258860]).…”

“…in KIAA0586 animal models. [8][9][10][11]17 In particular, the KIAA0586 chicken with a frameshift mutation in exon 7 (corresponding to human exon 9) closely models the human SRP ciliopathy phenotype, including a small rib cage and polydactyly, as well as lung anomalies. 17 Interestingly, the c.1815G>A variation identified in families 2-4 leads to the deletion of human exon 14, corresponding to mouse exon 12 ( Figure S2), which has been shown to be essential for the function of the protein.…”

Cilia in vertebrate left–right patterning

“…In the chicken embryo, Hensen's node is site of the first asymmetric gene expression, but this structure is thought to lack motile cilia. Chicken Talpid3 mutants with cilia defects develop normal LR asymmetry [78][79][80], indicating a cilia-independent mechanism is used for breaking symmetry. Indeed, cell-tracking experiments revealed that asymmetric cell movements around Hensen's node set up asymmetric signalling in the chicken embryo [81].…”

Dimeric combinations of MafB, cFos and cJun control the apoptosis-survival balance in limb morphogenesis

“…Fluorescent immunostaining was performed on cryosections of limb buds at stage 30 as described previously (Bangs et al, 2011). Primary antibodies used were anti-MafB (Santa Cruz; sc-10022), anti-Fos, anti-Jun and antimacrophage (Acris Antibodies; AM08143PU-N).…”