Hepatic gene expression in multiparous Holstein cows treated with bovine somatotropin and fed n-3 fatty acids in early lactation.

Multiparous cows were fed supplemental dietary fat and treated with bST to assess effects of n-3 fatty acid supply, bovine somatotropin (bST), and stage of lactation on hepatic gene expression. Cows were blocked by expected calving date and previous milk yield and assigned randomly to treatment. Supplemental dietary fat was provided from calving as either whole high-oil sunflower seeds (SS; 10% of dietary dry matter; n-6/n-3 ratio of 4.6) as a source of linoleic acid or a mixture of Alifet-High Energy and Alifet-Repro (AF; 3.5 and 1.5% of dietary dry matter, respectively; n-6/n-3 ratio of 2.6) as a source of protected n-3 fatty acids. Cows were treated with 0 (SSN, AFN) or 500 (SSY, AFY) mg of bST every 10 d from 12 to 70 d in milk (DIM) and at 14-d intervals thereafter. Liver biopsies were collected on -12, 10, 24, and 136 DIM for gene expression analysis. Growth hormone receptor (GHR), insulin-like growth factor-I (IGF-I), IGF-binding protein-3 (IGFBP3), hepatic nuclear factor 4alpha (HNF4alpha), fibroblast growth factor-21 (FGF-21), and peroxisome proliferator-activated receptor alpha (PPARalpha) were the target genes and hypoxanthine phosphoribosyltransferase (HPRT) was used as an endogenous control gene. Expression was measured by quantitative real-time reverse transcription-PCR analyses of 4 samples from each of 32 cows (8 complete blocks). Amounts of hepatic HPRT mRNA were not affected by bST or diet but were increased by approximately 3.8% in early lactation (3.42, 3.52, 3.54, and 3.41 x 10(4) message copies for -12, 10, 24, and 136 DIM, respectively). This small change had little detectable impact on the ability of HPRT to serve as an internal control gene. Amounts of hepatic GHR, IGF-I, and IGFBP3 mRNA were reduced by 1.5 to 2-fold after calving. Expression of GHR and IGF-I increased and IGFBP3 tended to increase within 12 d (by 24 DIM) of bST administration. These effects of bST persisted through 136 DIM. Hepatic HNF4alpha mRNA was not altered by DIM or any of the treatments. Abundance of PPARalpha mRNA was unchanged through 24 DIM but increased by 136 DIM. There was a trend for an interaction of bST, diet, and DIM on PPARalpha mRNA abundance from 24 to 136 DIM because the amount of PPARalpha mRNA increased in SSN, SSY, and AFN cows but was not altered in AFY cows. The amount of FGF-21 mRNA increased markedly in early lactation but, like HNF4alpha mRNA, was not affected by bST, diet, or their interactions. These results indicate 1) that bST induced increases in hepatic expression of GHR, IGF-I, and IGFBP3 when cows were in negative energy balance in early lactation, 2) there was no effect of reduced dietary n-6/n-3 content on hepatic gene expression, and 3) there was support for a potential homeorhetic role of hepatic FGF-21 via uncoupling the somatotropin-IGF-axis in early lactation.

Application of the Sleeping Beauty transposon system to avian cells.

We have for the first time assessed the ability of the Sleeping Beauty (SB) transposon system to enhance transgenesis in chicken and turkey cells. The efficiency of transgenesis with a transposon encoding an antibiotic resistance gene was dramatically enhanced 15- to 35-fold when transposase was supplied by co-transfection of immortalized chicken and turkey cells with a construct encoding SB. In contrast, transgenesis of primary chicken embryo fibroblast (CEF) cells was not significantly increased by providing transposase, suggesting that the benefits of transposon-transgenesis in primary avian cells will require the application of more efficient transfection methods, further enhanced SB transposase or an alternative transposon system.

Characterization of expressed sequence tags from turkey skeletal muscle.

This study was designed to identify important muscle gene homologues in the turkey. Three skeletal muscle cDNA libraries representing distinct muscle developmental stages were constructed. A total of 20,042 clones were sequenced resulting in 13,023 finished high-quality sequences (trimmed, quality scored and masked) for analysis. Sequence clustering produced 1113 contigs and 4144 singletons (5257 putative transcripts). Sequences were compared by blastn to the chicken whole-genome sequence and to the Ensembl and NCBI databases to identify homologous sequences. These surveys indicated that most of the important muscle genes are included in the sequence collection. Examination of contigs identified 1288 single nucleotide polymorphisms and in 320 of those the minor allele was observed to be present in more than one sequence. This resource provides sequence variants for numerous genes in the turkey, as demonstrated by the SNP haplotypes that were constructed for 10 genes. Sequences obtained in this study provide the basis for constructing a skeletal muscle-focused microarray, a tool that will facilitate the analysis of genes expressed during turkey muscle development, as well as the expression of genes underlying the genetic basis of muscle characteristics associated with meat quality.

Comparative and physical mapping of 111 previously reported and 105 new porcine microsatellites.

Here we report radiation hybrid mapping of 105 new porcine microsatellite markers on the IMpRH(7000) radiation hybrid panel. In addition, we searched flanking sequences of these markers, as well as 673 previously reported RH-mapped microsatellite markers, for orthology to human sequences. Eighty-seven new and 111 previously mapped sequences exhibited orthology to human sequences. Using a stringent sequence alignment, 25 microsatellite-flanking sequences were found to be highly similar to genic sequences, whereas 173 were similar to non-genic sequences in the human genome. Five markers were located near known breakpoints of synteny between human and swine.

Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system.

The recently developed engineered nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9, provide new opportunities for gene editing in a straightforward manner. However, few reports are available regarding CRISPR application and efficiency in cattle. Here, the CRISPR/Cas9 system was used with the aim of inducing knockout and knock-in alleles of the bovine PRNP gene, responsible for mad cow disease, both in bovine fetal fibroblasts and in IVF embryos. Five single-guide RNAs were designed to target 875 bp of PRNP exon 3, and all five were codelivered with Cas9. The feasibility of inducing homologous recombination (HR) was evaluated with a reporter vector carrying EGFP flanked by 1 kbp PRNP regions (pHRegfp). For somatic cells, plasmids coding for Cas9 and for each of the five single-guide RNAs (pCMVCas9 and pSPgRNAs) were transfected under two different conditions (1X and 2X). For IVF zygotes, cytoplasmic injection was conducted with either plasmids or mRNA. For plasmid injection groups, 1 pg pCMVCas9 + 0.1 pg of each pSPgRNA (DNA2X) was used per zygote. In the case of RNA, two amounts (RNA1X and RNA2X) were compared. To assess the occurrence of HR, a group additionally cotransfected or coinjected with pHRegfp plasmid was included. Somatic cell lysates were analyzed by polymerase chain reaction and surveyor assay. In the case of embryos, the in vitro development and the genotype of blastocysts were evaluated by polymerase chain reaction and sequencing. In somatic cells, 2X transfection resulted in indels and large deletions of the targeted PRNP region. Regarding embryo injection, higher blastocyst rates were obtained for RNA injected groups (46/103 [44.6%] and 55/116 [47.4%] for RNA1X and RNA2X) than for the DNA2X group (26/140 [18.6%], P < 0.05). In 46% (26/56) of the total sequenced blastocysts, specific gene editing was detected. The total number of genetic modifications (29) was higher than the total number of gene-edited embryos, as three blastocysts from the group RNA2X reported more than one type of modification. The modifications included indels (10/56; 17.9%) and large deletions (19/56; 33.9%). Moreover, it was possible to detect HR in 1/8 (12.5%) embryos treated with RNA2X. These results report that the CRISPR/Cas9 system can be applied for site-specific edition of the bovine genome, which could have a great impact on the development of large animals resistant to important zoonotic diseases.

Gene discovery and expression profiling in porcine Peyer's patch.

Peyer's patches of the intestinal mucosa are essential for host defense and immune regulation in the enteric system. To better understand molecular mechanisms of Peyer's patch function, we have screened for differentially expressed genes specific to Peyer's patch. cDNA libraries were created from normal Peyer's patch, immune stimulated Peyer's patch, and pooled cDNA subtracted with fibroblast RNA. From the subtracted library, 3687 expressed sequence tags (ESTs), representing 2414 unique nucleotide sequences, were isolated, identified by BLAST searches against public databases, and spotted onto a microarray for gene expression profiling. Approximately 30% of these ESTs BLAST to genes of unknown function and 20% have no known homology in the public databases (novel genes). Of the novel genes, 70% are expressed in normal immune tissues by microarray analysis, suggesting that at least 371 of the unidentified EST sequences from the subtracted library are novel porcine genes and can now be further characterized to determine their function in the porcine Peyer's patch. We surmise that the products of these genes participate in biochemical and cellular functions related to the unique immunological and gastroenterological functions of the small intestine. The BLAST and gene ontology information for each of the subtracted library EST sequences, the normal and immune stimulated libraries, and the microarray are all valuable resources that will facilitate further examination of the biological function of porcine Peyer's patch tissue.

Structure of the gene for uteroferrin.

The published structure of the gene for uteroferrin differs from that of the human and mouse tartrate-resistant acid phosphatase (TRAP) genes. Polymerase chain reaction using genomic DNA as template and primers designed from exon 2 of the porcine uteroferrin gene amplified a product containing two previously undescribed introns. Because of these discrepancies, we cloned an EcoRI fragment from a porcine genomic BAC library containing the uteroferrin gene, and the region containing the uteroferrin gene was completely sequenced. The uteroferrin gene spanned 2.5 kb and contained five exons, which is similar to the structure previously reported for human and mouse TRAP genes but different from the published structure of the uteroferrin gene. Southern blotting of porcine genomic DNA digested with a variety of enzymes was consistent with the sequence that we obtained. The most likely explanation for the differing results is that the previously reported structure for the uteroferrin gene was the result of artifactual elimination of introns 2 and 3 by bacteria and artifactual recombination of the region upstream of the transcription start site of this gene.

Tracheal epithelial permeability to nonelectrolytes: species differences.

We developed a new excised tracheal preparation to measure the epithelial permeability of large lipid-insoluble nonelectrolytes and macromolecules. Tracheae were suspended vertically in a Ringer solution bath, and a solution containing labeled test solutes was positioned in the center of the tracheal segment, away from damaged ends. Permeability coefficients, calculated from solute fluxes into the bath, were constant for > or = 2 h at 37 degrees C, and no histological changes were observed. Measurements after epithelial removal with detergent indicate that in the intact trachea the epithelium represents > 90% of the resistance to transport. For the rat trachea, permeability coefficients for sucrose, inulin, and Dextran 20 were 9.22, 2.20, and 0.214 x 10(-7) cm/s, respectively. Values for cat tracheae were similar, those for rabbit tracheae were lower, and those for guinea pig tracheae were markedly greater. With the assumption of transport by diffusion through thin rectangular slits between epithelial cells, the rat and guinea pig data fit a slit width of 7-8 nm, whereas the rabbit and cat data cannot be explained by a model with slits of a single size.

Proteinase-free myeloperoxidase increases airway epithelial permeability in a whole trachea model.

In cystic fibrosis the bronchiectatic conducting airways have large numbers of neutrophils in their walls and in their luminal contents. The neutrophil's primary granule enzyme activities of elastase and peroxidase are increased in the sputum of these patients. It has been postulated that these enzymes--together or individually--act to damage the airway epithelium. However, only peroxidase activity has consistently correlated with the degree of structural and functional airway disease in these patients with leakage of plasma protein into the airway lumen (Regelmann et al., Pediatr Pulmonol, 1995; 19:1-9). The present study was designed to test whether human neutrophil-derived myeloperoxidase can independently produce bronchial epithelial damage without the presence of proteases, as measured by increased permeability of the airway epithelium. Human peripheral blood neutrophils were purified, their primary granules isolated, and their peroxidase purified using affinity and ion exchange column chromatography. Activity of the proteinase-free peroxidase was measured using a chromogenic substrate. The effect of this peroxidase on the permeability of excised rat tracheas was measured using radioactive and fluorescent-labeled non-ionic molecules of varying molecular weight. Rat tracheas exposed to 15 minute treatments with either 130 U of peroxidase or hydrogen peroxide (10(-5) M) did not show a significant increase in the permeability of the epithelium to [3H]inulin, [14C]sucrose, and fluorescein isothiocyanate dextran 20 compared with control tracheas. However, those tracheas exposed to 130 U peroxidase followed by 10(-5) M hydrogen peroxide showed an increased permeability to each of the three test solutes. We conclude that proteinase-free myeloperoxidase, in the presence of non-toxic concentrations of its substrates, hydrogen peroxide and halide, produced increases in permeability to non-ionic molecules in the rat trachea within 15 minutes.

Structure of the genes for porcine endometrial secreted and membrane folate binding proteins.

The endometrium of the pig produces two types of folate binding proteins (FBP) which, based on their sequences, are likely to be membrane (m) and secreted (s) forms. A clone containing both a gene coding for the sFBP cDNA and a gene coding for the mFBP was isolated from a yeast artificial chromosome (YAC) library. Each gene was subcloned and sequenced. The gene for sFBP spanned 4.4 kbp and included 5 exons. The mFBP gene spanned 7.0 kbp and also contained 5 exons. Structures of the genes were very similar for the last three exons, and this similarity was shared with other known FBP/folate receptor (FR) gene sequences. Unexpectedly, portions of introns 3 and 4 of both genes were highly homologous, suggesting the possibility that sequences within these introns served some as yet unknown function. In contrast, the structures of the 5' exons differed between the two genes and other known FBP/FR genes. Comparison of putative promoter regions for the two genes with promoter regions for human FBP/FR genes revealed significant sequence homology between sFBP and human gammaFBP and between mFBP and human alphaFR. These regions of homology may play a role in control of transcription of each gene.

Optimum mating systems for the myostatin locus in cattle.

Inactive myostatin (one or two copies) results in increased muscularity, increased yield of closely trimmed retail product, reduced fat content, increased lean growth efficiency, reduced quality grade, increased birth weight, and increased dystocia. Even though one or two copies of inactive myostatin reduces quality grade or marbling compared to zero copies, there is no decrease in meat tenderness. It may be possible to use mating systems to make the most of the advantages of inactive myostatin while minimizing the disadvantages. The objective of this study was to develop a method to compare mating systems among genotypes at the myostatin locus. Economic variables that influence the profitability of alternative mating systems are prices per unit of retail product for USDA quality grades Standard, Select, and Choice; cost of an assisted calving; and cost of genotyping. Because of variation in both economic variables and biological parameters, a single mating system is not expected to universally maximize profit. We identified seven mating systems that each yield maximum profit for different combinations of values for biological parameters and economic variables. Use of inactive myostatin was profitable as long as the price for Select was at least 80% of the Choice price and the price for Standard at least 60%. As the price for Select and Standard increase up to the Choice price, mating systems that produce a higher proportion of inactive myostatin alleles become more profitable. Profitable use of inactive myostatin depends either on retaining ownership of beef until it is fabricated into retail product or the development of specialty markets that place greater value on lean yield and less on marbling, unlike conventional U. S. markets.

Technical note: direct genotyping of the double-muscling locus (mh) in Piedmontese and Belgian Blue cattle by fluorescent PCR.

A simple PCR-based allele detection system has been developed to assist in the management of the two most prevalent double-muscled (mh) breeds in the U.S. Application of this assay will permit the implementation of structured mating systems dependent on precise genotypes at the mh locus. The genetic assay uses standard fluorescent genotyping technology and relies on the unique nucleotide composition of wild-type and mutant alleles of myostatin, the gene underlying the double-muscled phenotype. We present data demonstrating the efficacy of this fluorescent primer-based PCR assay in genotyping animal populations carrying normal and(or) mutant alleles of the myostatin gene.

Quantitative analysis of birth, weaning, and yearling weights and calving difficulty in Piedmontese crossbreds segregating an inactive myostatin allele.

The Piedmontese breed has a high frequency of double-muscling. Animals tested in this breed are homozygous for a guanine to adenine transition in exon 3 (C313Y) of the myostatin (MSTN) gene. This transition seems to be responsible for the double-muscling phenotype. The objective of this study was to compare effects of alternative MSTN genotypes on proportion of assisted calving and weights at birth, weaning, and 1 yr of age. Reciprocal backcross and F2 calves out of Piedmontese-Angus (PA) and Piedmontese-Hereford (PH) dams born in 1995 (n = 82), 1996 (n = 75), and 1997 (n = 144) were evaluated for birth (BWT, kg), adjusted weaning (W200, kg), and yearling (W365, kg) weights and calving difficulty expressed as a proportion of assisted calving (CD). The number of copies of C313Y was assessed in each calf. Data were analyzed with a model that included effects of year, sex, subclasses of proportion Piedmontese (.25, .5, .75) by number of C313Y copies (0 = +/+, 1 = mh/+, 2 = mh/mh), and age of dam as covariate. For BWT, heterozygous mh/+ animals were 3.2 +/- .8 kg heavier than +/+ animals. Homozygous mh/mh animals increased .19 +/- .06 in proportion of CD compared with mh/+ animals. Differences between homozygous animals (mh/mh - +/+) were 5.2 +/- 1 kg for BWT and .21 +/- .06 for CD. Heterozygous mh/+ animals were 9.1 +/- 4 kg heavier at W200 than homozygous +/+ animals. Homozygous +/+ and heterozygous animals were 20 +/- 8 and 24.5 +/- 8 kg, respectively, heavier at W365 than mh/mh animals. Differences between mh/+ and the mean of mh/mh and +/+ genotypes for W200 and W365 were 8.8 +/- 3 and 18 +/- 5 kg, respectively, suggesting dominance effects on postnatal growth. Production of heterozygous animals, to take advantage of the positive impact of one copy of C313Y on carcass traits, may be a viable option when the value of increased retail product yield is greater than the increased cost associated with calving difficulty.

Differential gene expression of ewes varying in tolerance to dietary nitrate.

Ruminants consuming diets with increased concentrations of nitrate (NO(3)(-)) can accumulate nitrite (NO(2)(-)) in the blood, resulting in toxicity. In a previous experiment, ewes identified as highly tolerant to subacute dietary NO(3)(-) were able to consume greater amounts of NO(3)(-) than lowly tolerant ewes without exhibiting signs of toxicity. We hypothesized that highly tolerant and lowly tolerant ewes differ in their ability to metabolize NO(3)(-) and thereby differ in the expression of hepatic genes involved in NO(3)(-) metabolism. Therefore, our objective was to identify hepatic genes differentially expressed between ewes classified as lowly tolerant and highly tolerant after administration of a subacute quantity of dietary NO(3)(-). Analysis of the Bovine Oligonucleotide Microarray data identified 100 oligonucleotides as differentially expressed (P < 0.05) between lowly tolerant and highly tolerant ewes. Functional analysis of the genes associated with these oligonucleotides revealed 2 response clusters of interest: metabolic and stress. Genes of interest within these 2 clusters (n = 17) and nonclustered genes with the greatest fold changes (FC; n = 5) were selected for validation by real-time reverse-transcription PCR. Relative expression, genomic regulation, and FC agreed between microarray and real-time reverse-transcription-PCR analyses, and FC differences (P < 0.05) between lowly tolerant and highly tolerant ewes were confirmed for 12 genes. Metabolic genes that were downregulated (P ≤ 0.032) in lowly tolerant ewes vs. highly tolerant ewes included aldehyde oxidase 1, argininosuccinate lyase, putative steroid dehydrogenase, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase1, and sterol carrier protein 2. In contrast, the metabolic gene homeobox was upregulated (P = 0.037) in lowly tolerant ewes. The glutathione peroxidase 3 and inter-α (globulin) inhibitor H4 genes in the stress response cluster were upregulated (P ≤ 0.045) in lowly tolerant ewes. Genes with the greatest FC, but did not cluster within the functional analysis included haptoglobin, which was upregulated (P = 0.024) in lowly tolerant ewes, and fatty acid desaturase 2 and thyroid hormone responsive, both of which were downregulated (P ≤ 0.019) in lowly tolerant ewes. Results from this study indicate that hepatic gene expression differs in ewes identified as lowly tolerant and highly tolerant to increased dietary NO(3)(-).

Precision genetics for complex objectives in animal agriculture.

Indirect modification of animal genomes by interspecific hybridization, cross-breeding, and selection has produced an enormous spectrum of phenotypic diversity over more than 10,000 yr of animal domestication. Using these established technologies, the farming community has successfully increased the yield and efficiency of production in most agricultural species while utilizing land resources that are often unsuitable for other agricultural purposes. Moving forward, animal well-being and agricultural sustainability are moral and economic priorities of consumers and producers alike. Therefore, these considerations will be included in any strategy designed to meet the challenges produced by global climate change and an expanding world population. Improvements in the efficiency and precision of genetic technologies will enable a timely response to meet the multifaceted food requirements of a rapidly increasing world population.

Transcriptional profiling of stress response in cultured porcine islets.

Cell-based diabetes therapy may be achieved through xenotransplantation of adult porcine islets, but tissue quality and immunoreactivity barriers need to be overcome. Early identification and exclusion of irreversibly stressed and dying islets may improve transplant outcomes. We used oligonucleotide microarray and quantitative RT-PCR to identify molecular markers of physiological and immunological stress in porcine islets cultured under stress conditions of elevated glucose (16.7 mM), inflammatory cytokine addition (IL-1beta, TNF-alpha, and IFN-gamma), or both, for 48 h. Hyperglycemic conditions were associated with increased thioredoxin interacting protein and metabolic process mRNAs, as observed in rodent and primate species. Cytokine treatment increased expression of JAK-STAT pathway components, oxidative stress (transglutaminase 2), and beta cell dysfunction genes. Transglutaminase 2 induction is unique to porcine islets. Biomarkers involved in hyperglycemia and islet inflammation may serve as novel targets for improving and monitoring isolated porcine islet function and viability.

Mapping of genes expressed in activated porcine Peyer's patch.

To determine the chromosomal locations for genes expressed in porcine Peyer's patches, polymerase chain reaction-based mapping of expressed sequence tags (ESTs) isolated from a porcine Peyer's patch-specific cDNA library was performed across a 6500-rad swine radiation hybrid panel. A total of 116 ESTs were mapped with LOD scores >6.0, and another 11 ESTs had LOD scores between 5.0 and 6.0. Of these 127 ESTs, 63% matched known genes (

Genomic organization and genetic mapping of the bovine PREF-1 gene.

As a potential regulator of nutrient partitioning in beef cattle, we have cloned and genetically mapped the bovine PREF-1 gene. A full-length PREF-1 cDNA was isolated by iterative purification from a mixed-tissue cDNA library to which adipose contributed mRNA. Analysis of partial cDNAs from this library revealed that the 3'-terminal exon of the bovine PREF-1 mRNA is spliced in a manner analogous to its murine ortholog. However, we have also detected a PREF-1 splice form apparently unique to cattle. Aside from this alternative selection of a splice donor in the bovine fifth exon, the exon/intron junctions of the bovine PREF-1 gene recapitulate those observed for mice. The sequences proximal to the bovine PREF-1 transcription start site are homologous to the mouse PREF-1 promoter. Importantly, the sequence experimentally identified as critical to PREF-1 "suppression in adipocyte differentiation" is conserved in the bovine gene. The bovine PREF-1 gene was mapped to the telomeric end of BTA 21 by virtue of a physically linked microsatellite with seven alleles and 285 informative meiosis.

Temporal and spatial localization patterns of Gata4 during porcine gonadogenesis.

The zinc finger transcription factor Gata4, is associated with gonadal development in many species. The present study characterizes temporal and spatial localization of Gata4 throughout gonadogenesis in porcine embryos. Immunohistochemical studies illustrated that Gata4 protein is present in the coelomic epithelium prior to histological differentiation of the nascent bipotential gonad, marking the future site of both XX and XY porcine gonads. Many somatic cells of both XX and XY bipotential gonads continue to retain Gata4 immunoreactivity throughout sexual differentiation and subsequent gonadal development. Testicular cords were evident by 26 days postcoitum. Gata4 was present in Sertoli cells, identified by virtue of coexpression with Müllerian inhibiting substance and also interstitial cells including Leydig cells throughout fetal and postnatal life. Many somatic cells of the differentiating ovary including follicular cells also contained Gata4 protein throughout fetal and postnatal life. Gata4 was not present in germ cells, endothelial cells, or other undifferentiated mesenchymal cells of both XX and XY gonads. A population of Gata4-positive cells in the dorsal mesentery was continuous with the coelomic epithelium of the gonad. This localization pattern led to the hypothesis that a subpopulation of somatic cells in the dorsal mesentery moves toward the gonad. An in vitro cell migration assay demonstrated that Gata4-positive cells preferentially migrate toward explanted gonadal tissue, and morphological features of the developing gonad supported this hypothesis. This study illustrates that Gata4 is a very early marker for gonad formation, highlights species differences in temporal and spatial localization patterns, and suggests a potential role for Gata4 in the development of both XX and XY porcine gonads. Further, we suggest that mesenchymal cells of the dorsal mesentery may provide a source of somatic cells that migrate and incorporate into the gonad and contribute to various somatic cell lineages. Overall, the spatial and temporal localization patterns of Gata4 during porcine gonadogenesis implies a much earlier and wider role for Gata4 than previously reported in other species.

Alternative transcriptional initiation and splicing define the translational efficiencies of zebrafish mRNAs encoding eukaryotic initiation factor 4E.

Translation initiation factor 4E (eIF4E) binds to the m7GTP cap structure of eukaryotic mRNAs and influences the overall rates of translation. The eIF4E protein is subject to regulation at a number of levels that allow it to modulate translation of maternal mRNAs in early embryos before the onset of zygotic transcription. In zebrafish eIF4E (zeIF4E) mRNA levels are elevated in specific tissues and at specific times during embryogenesis. We have characterized the organization of the zeIF4E gene to facilitate elucidation of the molecular mechanisms that influence its expression. The zeIF4E gene spans about 14 kb and like its human counterpart is comprised of seven exons. Alternative splicing between the first and second exon generates two mRNA splice-forms called SF1 and SF2. Nuclease-S1-protection and primer-extension reveal two zeIF4E transcriptional start-sites. Transcripts initiating from the distal start-site during oogenesis are exclusively SF1, while initiation from the proximal start-site generates both splice-forms. Although translation in vitro of SF1 mRNA gives rise to a protein consistent in mass with affinity-purified zeIF4E, SF2 mRNA does not. Instead, SF2 mRNA inhibits in vitro protein synthesis in a concentration-dependent manner, suggesting it functions as a translational attenuator. Thus, specific transcriptional activation from the distal start-site may provide a unique mechanism for transcriptional regulation of the levels, as well as the function of zeIF4E mRNAs.