Sarah Barbey

Attenuated sensing of SHH by Ptch1 underlies evolution of bovine limbs

The large spectrum of limb morphologies reflects the wide evolutionary diversification of the basic pentadactyl pattern in tetrapods. In even-toed ungulates (artiodactyls, including cattle), limbs are adapted for running as a consequence of progressive reduction of their distal skeleton to symmetrical and elongated middle digits with hoofed phalanges. Here we analyse bovine embryos to establish that polarized gene expression is progressively lost during limb development in comparison to the mouse. Notably, the transcriptional upregulation of the Ptch1 gene, which encodes a Sonic hedgehog (SHH) receptor, is disrupted specifically in the bovine limb bud mesenchyme. This is due to evolutionary alteration of a Ptch1 cis-regulatory module, which no longer responds to graded SHH signalling during bovine handplate development. Our study provides a molecular explanation for the loss of digit asymmetry in bovine limb buds and suggests that modifications affecting the Ptch1 cis-regulatory landscape have contributed to evolutionary diversification of artiodactyl limbs.

Novel Insights into the Bovine Polled Phenotype and Horn Ontogenesis in Bovidae

Despite massive research efforts, the molecular etiology of bovine polledness and the developmental pathways involved in horn ontogenesis are still poorly understood. In a recent article, we provided evidence for the existence of at least two different alleles at the Polled locus and identified candidate mutations for each of them. None of these mutations was located in known coding or regulatory regions, thus adding to the complexity of understanding the molecular basis of polledness. We confirm previous results here and exhaustively identify the causative mutation for the Celtic allele (PC) and four candidate mutations for the Friesian allele (PF). We describe a previously unreported eyelash-and-eyelid phenotype associated with regular polledness, and present unique histological and gene expression data on bovine horn bud differentiation in fetuses affected by three different horn defect syndromes, as well as in wild-type controls. We propose the ectopic expression of a lincRNA in PC/p horn buds as a probable cause of horn bud agenesis. In addition, we provide evidence for an involvement of OLIG2, FOXL2 and RXFP2 in horn bud differentiation, and draw a first link between bovine, ovine and caprine Polled loci. Our results represent a first and important step in understanding the genetic pathways and key process involved in horn bud differentiation in Bovidae.

Anti-Müllerian hormone: a predictive marker of embryo production in cattle?

In cattle, the embryo production rate after superovulation varies between individuals and is difficult to predict. Recently, we proposed that anti-Müllerian hormone (AMH) plasma levels measured before treatment can help predict superovulatory responses. To establish whether blood measurement of AMH can help predict the number of embryos produced by a given cow after superovulation, data collected over 4 years from 45 dairy cows submitted to repeated embryo production were analysed in a retrospective study. A high within-animal repeatability (0.38 and 0.36) and a strong effect of the father of the donor cow (P < 0.01) were observed for the numbers of collected and transferable embryos, respectively. AMH concentration, measured in the plasma of donor cows during first lactation and several months before the start of the embryo production campaigns, was found to be highly correlated with the maximal number of collected (P < 0.0001) and transferable (P < 0.01) embryos per cow. In conclusion, the capacity of embryo production is a repeatable and probably heritable trait in the cow, and blood measurement of AMH in potential donor cows could be of value in determining a cows intrinsic capacity to produce transferable embryos.

A 3.7 Mb Deletion Encompassing ZEB2 Causes a Novel Polled and Multisystemic Syndrome in the Progeny of a Somatic Mosaic Bull

Polled and Multisystemic Syndrome (PMS) is a novel developmental disorder occurring in the progeny of a single bull. Its clinical spectrum includes polledness (complete agenesis of horns), facial dysmorphism, growth delay, chronic diarrhea, premature ovarian failure, and variable neurological and cardiac anomalies. PMS is also characterized by a deviation of the sex-ratio, suggesting male lethality during pregnancy. Using Mendelian error mapping and whole-genome sequencing, we identified a 3.7 Mb deletion on the paternal bovine chromosome 2 encompassing ARHGAP15, GTDC1 and ZEB2 genes. We then produced control and affected 90-day old fetuses to characterize this syndrome by histological and expression analyses. Compared to wild type individuals, affected animals showed a decreased expression of the three deleted genes. Based on a comparison with human Mowat-Wilson syndrome, we suggest that deletion of ZEB2, is responsible for most of the effects of the mutation. Finally sperm-FISH, embryo genotyping and analysis of reproduction records confirmed somatic mosaicism in the founder bull and male-specific lethality during the first third of gestation. In conclusion, we identified a novel locus involved in bovid horn ontogenesis and suggest that epithelial-to-mesenchymal transition plays a critical role in horn bud differentiation. We also provide new insights into the pathogenicity of ZEB2 loss of heterozygosity in bovine and humans and describe the first case of male-specific lethality associated with an autosomal locus in a non-murine mammalian species. This result sets PMS as a unique model to study sex-specific gene expression/regulation.

C-Nap1 mutation affects centriole cohesion and is associated with a Seckel-like syndrome in cattle

Caprine-like Generalized Hypoplasia Syndrome (SHGC) is an autosomal-recessive disorder in Montbéliarde cattle. Affected animals present a wide range of clinical features that include the following: delayed development with low birth weight, hind limb muscular hypoplasia, caprine-like thin head and partial coat depigmentation. Here we show that SHGC is caused by a truncating mutation in the CEP250 gene that encodes the centrosomal protein C-Nap1. This mutation results in centrosome splitting, which neither affects centriole ultrastructure and duplication in dividing cells nor centriole function in cilium assembly and mitotic spindle organization. Loss of C-Nap1-mediated centriole cohesion leads to an altered cell migration phenotype. This discovery extends the range of loci that constitute the spectrum of autosomal primary recessive microcephaly (MCPH) and Seckel-like syndromes.

Sensing of Escherichia coli and LPS by mammary epithelial cells is modulated by O-antigen chain and CD14

Escherichia coli is one of the major pathogens causing mastitis in dairy cattle. Yet, the factors which mediate the ability for E. coli to develop in the bovine mammary gland remain poorly elucidated. In a mouse model, infections induced by the reference mastitis E. coli P4 showed a strong colonisation of the mammary gland, while this strain had a low stimulating power on cells of the PS bovine mammary epithelial cell line. In order to understand if such a reduced response contributes to the severity of infection, a library of random mutants of P4 strain was screened to identify mutants inducing stronger response of PS cells. Among hyper-stimulating mutants, six were altered in genes involved in biosynthesis of lipopolysaccharide (LPS) and had lost their O-polysaccharide region, suggesting that the presence of O-antigen impairs the response of PS cells to LPS. Using purified smooth (S) and rough (R) fractions of LPS, we showed that the R-LPS fraction induced a stronger response from PS cells than the smooth LPS fraction. Biological activity of the S-LPS fraction could be restored by the addition of recombinant bovine CD14 (rbCD14), indicating a crucial role of CD14 in the recognition of S-LPS by Mammary Epithelial Cells (MEC). When S-LPS and R-LPS were injected in udder quarters of healthy lactating cows, an inflammation developed in all infused quarters, but the S-LPS induced a more intense pro-inflammatory response, possibly in relation to sizeable concentrations of CD14 in milk. Altogether, our results demonstrate that the O-antigen modulates the pro-inflammatory response of MEC to LPS, that S-LPS and R-LPS trigger different responses of MEC and that these responses depend on the presence of CD14.