Belinda J. Norris

Identifying the germline in an equally cleaving mollusc: Vasa and Nanos expression during embryonic and larval development of the vetigastropod Haliotis asinina

Members of the Vasa and Nanos gene families are important for the specification and development of the germline in diverse animals. Here, we determine spatial and temporal expression of Vasa and Nanos to investigate germline development in the vetigastropod Haliotis asinina. This is the first time these genes have been examined in an equally cleaving lophotrochozoan species. We find that HasVasa and HasNanos have largely overlapping, but not identical, expression patterns during embryonic and larval development, with both being maternally expressed and localized to the micromere cell lineages during cleavage. As embryonic development continues, HasVasa and HasNanos become progressively more enriched in the dorsal quadrant of the embryo. By the trochophore stage, both HasVasa and HasNanos are expressed in the putative mesodermal bands of the larva. This differs from the unequally cleaving gastropod Illyanasa obsoleta, in which IoVasa and IoNanos expression is detectable only in the early embryo and not during gastrulation and larval development. Our results suggest that the H. asinina germline arises from the 4d cell lineage and that primordial germ cells (PGCs) are not specified exclusively by maternally inherited determinants (preformation). As such, we infer that inductive signals (epigenesis) play an important role in specifying PGCs in H. asinina. We hypothesize that HasVasa is expressed in a population of undifferentiated multipotent cells, from which the PGCs are segregated later during development.

A genomics-informed, SNP association study reveals FBLN1 and FABP4 as contributing to resistance to fleece rot in Australian Merino sheep

BackgroundFleece rot (FR) and body-strike of Merino sheep by the sheep blowfly Lucilia cuprina are major problems for the Australian wool industry, causing significant losses as a result of increased management costs coupled with reduced wool productivity and quality. In addition to direct effects on fleece quality, fleece rot is a major predisposing factor to blowfly strike on the body of sheep. In order to investigate the genetic drivers of resistance to fleece rot, we constructed a combined ovine-bovine cDNA microarray of almost 12,000 probes including 6,125 skin expressed sequence tags and 5,760 anonymous clones obtained from skin subtracted libraries derived from fleece rot resistant and susceptible animals. This microarray platform was used to profile the gene expression changes between skin samples of six resistant and six susceptible animals taken immediately before, during and after FR induction. Mixed-model equations were employed to normalize the data and 155 genes were found to be differentially expressed (DE). Ten DE genes were selected for validation using real-time PCR on independent skin samples. The genomic regions of a further 5 DE genes were surveyed to identify single nucleotide polymorphisms (SNP) that were genotyped across three populations for their associations with fleece rot resistance.ResultsThe majority of the DE genes originated from the fleece rot subtracted libraries and over-representing gene ontology terms included defense response to bacterium and epidermis development, indicating a role of these processes in modulating the sheeps response to fleece rot. We focused on genes that contribute to the physical barrier function of skin, including keratins, collagens, fibulin and lipid proteins, to identify SNPs that were associated to fleece rot scores.ConclusionsWe identified FBLN1 (fibulin) and FABP4 (fatty acid binding protein 4) as key factors in sheeps resistance to fleece rot. Validation of these markers in other populations could lead to vital tests for marker assisted selection that will ultimately increase the natural fleece rot resistance of Merino sheep.

Gene expression profiling of ovine skin and wool follicle development using a combined ovine–bovine skin cDNA microarray

Low fibre diameter and high fleece weight are important determinants of the economic value of the Merino fleece. The combination of these traits is found in Merino sheep with high follicle densities resulting from a high secondary to primary follicle ratio. Morphological stages in the development of primary and secondary follicles of fetal sheep skin have been well described. We have used gene expression profiling of fetal skin to identify genes that may be important in controlling these follicle developmental processes. A combined ovine (2.3 K) and bovine (6.14 K) cDNA microarray of 2 fetal and 1 adult stage skin tissues was constructed to compare gene expression levels between fetal day 82, day 105, day 120 and adult sheep skin developmental stages. The transcript profile resulted in 238 differentially expressed array elements relative to the adult expression, which represented 132 unique genes. These clustered into 50 up- and 82 down-regulated genes and distinct gene ontologies including structural constituents, phosphate transport, signal transduction and organogenesis. Northern blot analysis of 2 selected genes, S100A7LI and TAGLN, validated the microarray results. This list of genes contains candidates of interest for further investigation into the molecular control of wool follicle development.

16S rRNA gene microbial analysis of the skin of fleece rot resistant and susceptible sheep

Fleece rot is a bacterial dermatitis that follows prolonged wetting of the sheep’s skin, and a major pre-disposing condition to body strike in the Australian Merino. Several studies have examined bacterial load of the fleece in relation to fleece rot using traditional culture-based techniques focussing on only a few bacterial species. We examined the natural bacterial diversity of the healthy sheep skin and changes that occurred in fleece-rot resistant and susceptible animals during fleece rot development. Presented is a preliminary molecular genetic analysis of the bacterial ecology of the sheep skin. Eight 16S rRNA gene libraries were constructed from susceptible and resistant sheep both before and after onset of the disease following induction by simulated rainfall. Approximately 75% of the sequences obtained in this study have not been previously identified in fleece-rot studies. Four operational taxonomic units (OTU; groups of >97% sequence similarity) of major interest were present on susceptible animals and absent from resistant animals. Data on these OTU expand current knowledge of bacteria involved in inflammation and wounding of sheep skin tissue, and provide direction for future research that may lead to new treatment options for fleece rot and body strike.

Reconstructing CNV genotypes using segregation analysis: combining pedigree information with CNV assay.

BackgroundRepeated blocks of genome sequence have been shown to be associated with genetic diversity and disease risk in humans, and with phenotypic diversity in model organisms and domestic animals. Reliable tests are desirable to determine whether individuals are carriers of copy number variants associated with disease risk in humans and livestock, or associated with economically important traits in livestock. In some cases, copy number variants affect the phenotype through a dosage effect but in other cases, allele combinations have non-additive effects. In the latter cases, it has been difficult to develop tests because assays typically return an estimate of the sum of the copy number counts on the maternally and paternally inherited chromosome segments, and this sum does not uniquely determine the allele configuration. In this study, we show that there is an old solution to this new problem: segregation analysis, which has been used for many years to infer alleles in pedigreed populations.MethodsSegregation analysis was used to estimate copy number alleles from assay data on simulated half-sib sheep populations. Copy number variation at the Agouti locus, known to be responsible for the recessive self-colour black phenotype, was used as a model for the simulation and an appropriate penetrance function was derived. The precision with which carriers and non-carriers of the undesirable single copy allele could be identified, was used to evaluate the method for various family sizes, assay strategies and assay accuracies.ResultsUsing relationship data and segregation analysis, the probabilities of carrying the copy number alleles responsible for black or white fleece were estimated with much greater precision than by analyzing assay results for animals individually. The proportion of lambs correctly identified as non-carriers of the undesirable allele increased from 7% when the lambs were analysed alone to 80% when the lambs were analysed in half-sib families.ConclusionsWhen a quantitative assay is used to estimate copy number alleles, segregation analysis of related individuals can greatly improve the precision of the estimates. Existing software for segregation analysis would require little if any change to accommodate the penetrance function for copy number assay data.

Riding 'white' horses reveals new insights into pigmentation and melanoma

The genetic basis of human pigmentation traits and cutaneous cancer susceptibility is complex. Individuals with fair skin and hair colour and poor tanning ability have for a long time been known to have an increased incidence of cutaneous cancers. Recent genomewide association studies have identified only a few loci as potentially cancercausing and which explain only a small proportion of cutaneous cancer cases. As with other complex traits, pigmentation and cutaneous cancer risk are determined by multiple, in some cases highly variable, loci which display different population frequencies due partly to strong environmental influences. These features create significant challenges in identifying the genetic loci and molecular pathways of cancer susceptibility and the consequent development of suitable preventative and treatment strategies. The investigation of coat colour in domestic and experimental model animals has provided surprising insights into human pigmentation phenotypes and skin cancer ⁄ melanoma development. Gene variants originally identified as determinants of hair ⁄ wool, eye and skin pigmentation and subsequently associated with cancer susceptibility have provided details of both the fundamental biology of pigmentation and cutaneous cancer development. Recent linkage studies enabled by the sequencing of the human genome are beginning to validate known and identify a small number of novel loci associated with population-specific pigmentation traits and case-specific cutaneous melanoma and non-melanoma cancers. Nevertheless, the study of the molecular genetics and physiology of distinctive animal pigmentation patterns and associated diseases continues to contribute significantly to pinpointing and ⁄ or validating novel candidate gene pathways responsible for human pigmentation variation and skin cancers. In this latest example, the research team of Leif Andersson (Pielberg et al., 2008) has identified the horse Gray-causing mutation, providing new insights into the graying process and melanoma development. Many domestic horses celebrated as ‘white’ coat colour variants are actually genetically Gray horses produced as a result of selection for the causal Graymutation. Born pigmented, Gray horses’ coats become completely white from undergoing a gradual loss of follicular pigmentation while maintaining a dark skin. Gray horses also show a high incidence of vitiligo–like skin depigmentation, dermal melanomas and reduced life span. Pielberg et al. (2008) have identified the horse Gray-causing mutation as a 4.6 kb intronic duplication in the gene for syntaxin-17 (STX17). The duplication comprises a cis-acting regulatory mutation affecting the expression of STX17 and a neighboring gene NR4A3, which encodes nuclear receptor subfamily 4, group A, member 3. A diagnostic PCR-based test developed for the STX17 duplication will likely detect all occurrences of the Gray allele (carried by one in ten horses) as the mutation was completely associated with the Gray phenotype in numerous horses examined representing 14 different breeds (Pielberg et al., 2008). Whilst the functional mechanisms are still to be elucidated, Pielberg et al. (2008) suggest that deregulation of STX17 and ⁄ or NR4A3 leads to hyperproliferation of hair-follicle melanocytes causing a gradual depletion of progenitor stem cells and loss of hair pigmentation with age. At the same time, the deregulation of one or both of these genes leads to proliferation of dermal melanocytes in the glabrous skin causing dark pigmentation and predisposing the Gray horse to melanoma development. These varied effects on stem cell melanocyte populations likely reflect differences in the follicular and epidermal melanocytes and their microenvironment. Hair follicle melanocyte stem cells terminally differentiate and die in pigmenting the hair whereas skin ⁄ dermis melanocytes survive throughout the hair cycle (reviewed by Tobin, 2008). Altogether, this discovery provides further evidence for the distinct compartmentalization of follicular and dermal melanocyte sub-populations. Although it is not yet clear which of the range of Gray horse phenotypic effects is caused by the overexpression of both or one of these genes, the study has provided new perspectives for the possible role(s) of the products of these genes in hair ⁄ skin pigmentation traits and cutaneous cancer development. NR4A3 is a novel member of the steroid ⁄ thyroid superfamily of nuclear transcriptional regulators, the so called ‘‘orphan receptors’’. Smith et al. (2008) recently identified NR4A3 and two other NR4A orphan receptor family members as downstream mediators of MC1R signaling. MC1R expressed in melanocytes is a major regulator of hair, eye and skin colour, is highly polymorphic and variant Red Hair Colour (RHC) alleles have long been recognized as associated with red hair, fair skin, cutaneous UV sensitivity, and increased risk of melanoma and non-melanoma cancers. The study of Smith et al. (2008) showed a-MSH induced expression of NR4A nuclear receptors in melanocytes was impaired by the most common RHC variant alleles indicating a direct association between NR4A expression and MC1R signaling. Coverage on: Pielberg et al. (2008) A cis-acting regulatory mutation causes premature hair graying and susceptibility to melanoma in the horse. Nat. Genet. 40: 1004–1009.