First observations of intersex development in paddlefish Polyodon spathula

This study reports the first cases of intersex (abnormal development of both male and female reproductive tissues in a gonad) paddlefish Polyodon spathula, an Acipenseriform species of large rivers in the central U.S. and in aquaculture worldwide. Despite a large, multi-decadal data set in Oklahoma, Montana, and North Dakota, intersex development was not observed until 2019, when two individuals were harvested from the Grand Lake/Neosho River stock in Oklahoma. This suggests that intersex development in mid-water, zooplanktivorous paddlefish is rarer than in bottom-dwelling sturgeons for which intersex development is regularly observed. Although contaminants are implicated in causing intersex development in other Acipenseriformes, more investigation is needed.     

Isolation and characterization of marsupial IL5 genes

The genomic nucleotide sequence and chromosomal position of the interleukin 5 (IL5) gene has been described for the model marsupial Macropus eugenii (tammar wallaby). A 272 base pair genomic IL5 polymerase chain reaction (PCR) product spanning exon 3, intron 3, and exon 4 was generated using stripe-faced dunnart (Sminthopsis macroura) DNA. This PCR product was used to isolate a genomic lambda clone containing the complete IL5 gene from a tammar wallaby EMBL3 lambda library. Sequencing revealed that the tammar wallaby IL5 gene consists of four exons separated by three introns. Comparison of the marsupial coding sequence with coding sequences from eutherian species revealed 61 to 69% identity at the nucleotide level and 48 to 63% identity at the amino acid (aa) level. A polymorphic complex compound microsatellite was identified within intron 2 of the tammar wallaby IL5 gene. This microsatellite was also found in other marsupials including the swamp wallaby, tree kangaroo, stripe-faced dunnart, South American opossum, brushtail possum, and koala. Fluorescence in situ hybridization using DNA from the IL5 clone on tammar wallaby chromosomes indicated that the IL5 gene is located on Chromosome 1.     

Asymmetry in sexual development of gonads in intersex rainbow trout

Large‐scale sampling of spontaneous rainbow trout Oncorhynchus mykiss intersexes indicated a strong asymmetry of gonad differentiation in XX females; the right gonad was more sensitive to the mutation‐induced masculinization than the left one.     

High-resolution association mapping of number of teats in pigs reveals regions controlling vertebral development

Background

Selection pressure on the number of teats has been applied to be able to provide enough teats for the increase in litter size in pigs. Although many QTL were reported, they cover large chromosomal regions and the functional mutations and their underlying biological mechanisms have not yet been identified. To gain a better insight in the genetic architecture of the trait number of teats, we performed a genome-wide association study by genotyping 936 Large White pigs using the Illumina PorcineSNP60 Beadchip. The analysis is based on deregressed breeding values to account for the dense family structure and a Bayesian approach for estimation of the SNP effects.

Results

The genome-wide association study resulted in 212 significant SNPs. In total, 39 QTL regions were defined including 170 SNPs on 13 Sus scrofa chromosomes (SSC) of which 5 regions on SSC7, 9, 10, 12 and 14 were highly significant. All significantly associated regions together explain 9.5% of the genetic variance where a QTL on SSC7 explains the most genetic variance (2.5%). For the five highly significant QTL regions, a search for candidate genes was performed. The most convincing candidate genes were VRTN and Prox2 on SSC7, MPP7, ARMC4, and MKX on SSC10, and vertebrae δ-EF1 on SSC12. All three QTL contain candidate genes which are known to be associated with vertebral development. In the new QTL regions on SSC9 and SSC14, no obvious candidate genes were identified.

Conclusions

Five major QTL were found at high resolution on SSC7, 9, 10, 12, and 14 of which the QTL on SSC9 and SSC14 are the first ones to be reported on these chromosomes. The significant SNPs found in this study could be used in selection to increase number of teats in pigs, so that the increasing number of live-born piglets can be nursed by the sow. This study points to common genetic mechanisms regulating number of vertebrae and number of teats.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-542) contains supplementary material, which is available to authorized users.     

High resolution mapping of chromosomal regions controlling resistance to gastrointestinal nematode infections in an advanced intercross line of mice

Fine mapping of quantitative trait loci (QTL) associated with resistance to the gastrointestinal parasite Heligmosomoides polygyrus was achieved on F₆/F₇ offspring (1076 mice) from resistant (SWR) and susceptible (CBA) mouse strains by selective genotyping (top and bottom 20% selected on total worm count in week 6). Fecal egg counts were recorded at weeks 2, 4, and 6, and the average was also analyzed. Blood packed cell volume in weeks 3 and 6 and five immunological traits (mucosal mast cell protease 1, granuloma score, IgG1 against adult worm, IgG1, and IgE to L4 antigen) were also recorded. On Chromosome 1 single-trait analyses identified a QTL with effects on eight traits located at about 24 cM on the F₂ mouse genome database (MGD) linkage map, with a 95% confidence interval (CI) of 20-32 cM established from a multitrait analysis. On Chromosome 17 a QTL with effects on nine traits was located at about 18 cM on the MGD map (CI 17.9-18.4 cM). Strong candidate genes for the QTL position on Chromosome 1 include genes known to be involved in regulating immune responses and on Chromosome 17 genes within the MHC, notably the Class II molecules and tumor necrosis factor.     

Differential expression of WNT4 in testicular and ovarian development in a marsupial

Background  

WNT4 is a key regulator of gonadal differentiation in humans and mice, playing a pivotal role in early embryogenesis. Using a marsupial, the tammar wallaby, in which most gonadal differentiation occurs after birth whilst the young is in the pouch, we show by quantitative PCR during early testicular and ovarian development that WNT4 is differentially expressed ingonads.     

Isolation and chromosomal localization of a human ATP-regulated potassium channel

K_ATP channels are K⁺ channels whose activity is inhibited by the presence of and enhanced by the absence of cytosolic ATP. This property allows K_ATP channels to sense cellular intermediary metabolism and directly couple this information to the modulation of membrane excitability. Indeed, recent studies from our laboratory and others have suggested that activation of K_ATP channels during anoxia is important in the response and adaptation of central neurons to hypoxia. In order to identify K_ATP channels from human brain, we performed a polymerase chain reaction (PCR) using human cerebral cortex mRNA and primers derived from the ROMK1 sequence, a cDNA clone encoding an ATP-regulated potassium channel, recently isolated from rat kidney. We thus identified a novel 308-bp PCR product, pKCNJ1, whose expression was found to be restricted to a 3.0-kb band in the kidney by probing a human multiple tissue northern blot. pKCNJ1 was then used to isolate genomic clones and, using fluorescence in situ hybridization (FISH) to human metaphase chromosomes, was mapped to chromosome 11q.     

Semelparity in a large marsupial

Complete mortality of males after mating is known in several small dasyurid and didelphid species (up to 300g) and has previously been suggested to be a consequence of their small size and their inability to sequester sufficient fat reserves for an intense rut in the winter. Males of these species use increased corticosteroid levels to allow protein catabolism, enabling them to support their mating effort with other body reserves. However, increased corticosteroid levels have negative consequences such as anaemia, gastrointestinal ulceration, immune suppression and disease. The Australian dasyurid Dasyurus hallucatus shows complete male die off after mating in tropical savannah, yet males of this species may weigh as much as 1120 g and continue to eat during the rut. Die off in D. hallucatus shows many similarities to that in the smaller species including weight loss, fur loss, parasite infestation, increased testosterone levels and anaemia. However, in contrast to smaller species, there is no evidence of elevated corticosteroid levels or gastrointestinal ulceration. Consequently, the phenomenon of male die off after mating lacks a universal explanation.     

Intrauterine development after diapause in the marsupial Macropus eugenii

Three-quarters of adult female Macropus eugenii carry a diapausing blastocyst for up to 10 months of the year. For the first half of the year the diapause is due to the presence of a pouch young, but it continues through the subsequent anestrus. Spontaneous resumption occurs 1–15 days after the summer solstice. Development can be initiated experimentally during the first half of the year by removing the pouch young (RPY) and during anestrus by 10 daily injections of 10 mg progesterone.All blastocysts completed development after RPY, but only about half successfully completed intrauterine development after progesterone treatment. After RPY the corpus luteum grew and influenced growth of the endometrium and blastocyst. After day 13, however, the endometrium was further stimulated by the presence of the embryo or fetal membranes, probably the latter. Most of the failure after progesterone treatment occurred during the first 10 days and after the blastocyst had resumed development. It is suggested that these embryos failed to reach the stage where they could stimulate the endometrium directly. The later failure of progesterone-treated animals to give birth to full term fetuses was probably due to lack of stimulation from the anestrous ovaries.     

Isolation and chromosomal localization of ecdysterone-responsive genes in a Drosophila cell line

Cultured Kc 0% cells of Drosophila melanogaster are responsive to ecdysterone treatment. A library of lambda phages carrying segments of Drosophila genomic DNA was differential screened using poly(A)⁺RNAs from control and ecdysterone-treated cells. Nine independent recombinant phages that hybridized more intensely with poly(A)⁺ RNA from treated cells and six that hybridized most strongly with poly(A)⁺RNA from untreated cells were selected. Genomic localization of these inducible and repressible sequences was determined by hybridization in situ. These results suggest that expression of several unique genes is increased by the hormone. The six repressible sequences each contained DNA that hybridized to multiple chromosomal sites and appeared to be mobile elements, suggesting that the steroid hormone might be acting on the transposable elements. These probes will be useful for the study of positive and negative steroid regulation within the same cell.     

The development of the immune tissues in marsupial pouch young

Current knowledge of the development of the marsupial immune system, particularly in the context of lymphoid tissue development and the appearance of lymphocytes, has been examined and limitations identified. While primary lymphoid tissues like the thymus have been extensively studied, secondary lymphoid tissues such as the spleen and lymph nodes have been examined to a lesser extent, partly due to the difficulty of macroscopically identifying these structures, particularly in very small neonates. In addition, little research has been conducted on the mucosal‐associated lymphoid tissues; tissues that directly trap antigens and play an important role in the maturity of adaptive immune responses. Research on the development of the marsupial immune tissues to date serves as a solid foundation for further research, particularly on the mechanisms behind the development of the immune system of marsupials. With the recent sequencing and annotation of whole marsupial genomes, the current wealth of sequence data will be essential in the development of marsupial specific reagents, including antibodies, that are required to widen our specific knowledge of the complex marsupial immune system and its development. J. Morphol. 275:822–839, 2014. © 2014 Wiley Periodicals, Inc.     

Identifying regions of the wheat genome controlling seed development by mapping expression quantitative trait loci

Statistical methods established for the genetic analysis of quantitative traits can be applied to gene expression data. Quantitative trait locus (QTL) analysis can associate the expression of genes or groups of genes with particular genomic regions, and thereby identify regions regulating gene expression. A segregating population of 41 doubled haploid (DH) lines from the hard red spring wheat cross RL4452 x 'AC Domain' was used to map expression level polymorphisms. This population had previously been mapped with microsatellites, and includes a full QTL analysis for agronomic and seed quality traits. Expression analysis on mRNA from developing seed grown in two field locations was conducted on 39 of the 41 DH lines using the Affymetrix GeneChip Wheat Genome Array. Analysis of the hybridization intensity identified 1484 Affymetrix probe sets in the first location and 10,280 probe sets in the second location, where the hybridization intensity varied significantly between genotypes of the population. A common set of 1455 probe sets differing in intensity between genotypes in both locations was used for mapping, and 542 QTLs were identified that each mapped to a single chromosome interval, illustrating that major gene expression QTLs could be found in wheat. Genomic regions corresponding to multiple gene expression QTLs were identified. Comparison of expression mapping data with physical mapping of wheat expressed sequence tag (EST) sequences using rice synteny, as well as logarithm of odds (LOD) score analysis, showed that both cis- and trans-acting expression QTLs were present. Chromosomes 1D and 4B may contain significant trans-regulatory regions in this population.     

Desert hedgehog is a mammal-specific gene expressed during testicular and ovarian development in a marsupial

Background

Sphingosine-1-phosophate (S1P) is a biologically active sphingolipid metabolite that influences cellular events including differentiation, proliferation, and migration. S1P acts through five distinct cell surface receptors designated S1P_1-5R, with S1P₁R having the highest expression level in the developing heart. S1P₁R is critical for vascular maturation, with its loss leading to embryonic death by E14.5; however, its function during early cardiac development is not well known. Our previous studies demonstrated that altered S1P levels adversely affects atrioventricular (AV) canal development in vitro, with reduced levels leading to cell death and elevated levels inhibiting cell migration and endothelial to mesenchymal cell transformation (EMT).

Results

We determined, by real-time PCR analysis, that S1P₁R was expressed at least 10-fold higher than other S1P receptors in the developing heart. Immunohistochemical analysis revealed S1P₁R protein expression in both endothelial and myocardial cells in the developing atrium and ventricle. Using AV canal cultures, we observed that treatment with either FTY720 (an S1P_1,3,4,5R agonist) or KRP203 (an S1P₁R-specific agonist) caused similar effects on AV canal cultures as S1P treatment, including induction of cell rounding, inhibition of cell migration, and inhibition of EMT. In vivo, morphological analysis of embryonic hearts at E10.5 revealed that S1P₁R-/- hearts were malformed with reduced myocardial tissue. In addition to reduced myocardial tissue, E12.5 S1P₁R-/- hearts had disrupted morphology of the heart wall and trabeculae, with thickened and disorganized outer compact layer and reduced fibronectin (FN) deposition compared to S1P₁R+/+ littermates. The reduced myocardium was accompanied by a decrease in cell proliferation but not an increase in apoptosis.

Conclusions

These data indicate that S1P₁R is the primary mediator of S1P action in AV canal cultures and that loss of S1P₁R expression in vivo leads to malformed embryonic hearts, in part due to reduced fibronectin expression and reduced cell proliferation.