Myostatin (MSTN) negatively regulates muscle growth and development through inhibiting myoblast proliferation and differentiation. Five alternative splicing isoforms of MSTN (MSTN-A to MSTN-E) have been discovered in domestic avian species. MSTN-A has high expression in skeletal muscle and encodes the full-length peptide with anti-myogenic activity. Another isoform, MSTN-B, is also highly expressed in skeletal muscle and encodes a truncated peptide that has pro-myogenic capabilities in vitro, which include promoting the proliferation and differentiation of quail muscle precursor cells. The objective of this study was to investigate overexpression of MSTN-B in vivo by using two independent lines of transgenic Japanese quail with expression directed in the skeletal muscle. Unexpectedly, the chicken skeletal muscle alpha actin 1 (cACTA1) promoter resulted in restricted exogenous MSTN-B protein expression to certain skeletal muscles, such as the gastrocnemius and tibialis anterior, but not the pectoralis major muscle. Gastrocnemius weight as a percentage of body weight in transgenic quail was increased compared to non-transgenic quail at posthatch day 21 (D21) and posthatch D42. An increase in the size of the gastrocnemius in transgenic quail was attributed to an increase in fiber number but not fiber cross-sectional area (CSA). During embryonic development, paired box 7 (PAX7) expression was prolonged in the transgenic embryos, but other myogenic regulatory factors (MRFs) were unchanged after MSTN-B overexpression. Taken together, these data provide novel insights into the regulation of skeletal muscle development by alternative splicing mechanisms in avians.

Identification of tissue- and stage-specific gene promoters is valuable for delineating the functional roles of specific genes in genetically engineered animals. Here, through the comparison of gene expression in different tissues by analysis of a microarray database, the intestinal specificity of mucin 2 (MUC2) expression was identified in mice and humans, and further confirmed in chickens by RT-PCR (reverse transcription-PCR) analysis. An analysis of cis-acting elements in avian MUC2 gene promoters revealed conservation of binding sites, within a 2.9 kb proximal promoter region, for transcription factors such as caudal type homeobox 2 (CDX2), GATA binding protein 4 (GATA4), hepatocyte nuclear factor 4 α (HNF4A), and transcription factor 4 (TCF4) that are important for maintaining intestinal homeostasis and functional integrity. By generating transgenic quail, we demonstrated that the 2.9 kb chicken MUC2 promoter could drive green fluorescent protein (GFP) reporter expression exclusively in the small intestine, large intestine, and ceca. Fluorescence image analysis further revealed GFP expression in intestine epithelial cells. The GFP expression was barely detectable in the embryonic intestine, but increased during post-hatch development. The spatiotemporal expression pattern of the reporter gene confirmed that the 2.9 kb MUC2 promoter could retain the regulatory element to drive expression of target genes in intestinal tissues after hatching. This new transgene expression system, using the MUC2 promoter, will provide a new method of overexpressing target genes to study gene function in the avian intestine.

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Antibiotics are extensively used worldwide for the treatment of common infections by agents such as E. coli and Salmonella. They also represent the most common cause of alteration of the microbiota in people. We addressed whether broad-spectrum and Gram-negative-targeting antibiotics differentially regulate systemic and mucosal immune responses to vaccines. Antibiotics treatment enhances serum IgG1 responses in mice immunized systemically with a model polyvalent vaccine. This increase was not seen for other IgG subclasses and was dependent on the immunogenicity of vaccine antigens. The broad-spectrum antibiotic cocktail also enhanced serum IgA responses. Interestingly, both the broad spectrum and the antibiotic targeting Gram-negative bacteria enhanced the number of IgA antibody secreting cells in the intestinal lamina propria. This effect was unlikely to be due to an increase in cells expressing gut-homing receptors (i.e., CCR9 and α4β7) in peripheral tissues. On the other hand, the microbiome in mice treated with antibiotics was characterized by an overall reduction of the number of firmicutes. Furthermore, Bacteroidetes were increased by either treatment, and Proteobacteria were increased by the broad-spectrum antibiotics cocktail. Thus, immunoglobulin isotype and subclass responses are differentially regulated by oral antibiotics treatment and the gut microbiota shapes mucosal antibody responses after systemic immunization.

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