Butyrophilin-like 2 regulates site-specific adaptations of intestinal γδ intraepithelial lymphocytes.

Tissue-resident γδ intraepithelial lymphocytes (IELs) orchestrate innate and adaptive immune responses to maintain intestinal epithelial barrier integrity. Epithelia-specific butyrophilin-like (Btnl) molecules induce perinatal development of distinct Vγ TCR+ IELs, however, the mechanisms that control γδ IEL maintenance within discrete intestinal segments are unclear. Here, we show that Btnl2 suppressed homeostatic proliferation of γδ IELs preferentially in the ileum. High throughput transcriptomic characterization of site-specific Btnl2-KO γδ IELs reveals that Btnl2 regulated the antimicrobial response module of ileal γδ IELs. Btnl2 deficiency shapes the TCR specificities and TCRγ/δ repertoire diversity of ileal γδ IELs. During DSS-induced colitis, Btnl2-KO mice exhibit increased inflammation and delayed mucosal repair in the colon. Collectively, these data suggest that Btnl2 fine-tunes γδ IEL frequencies and TCR specificities in response to site-specific homeostatic and inflammatory cues. Hence, Btnl-mediated targeting of γδ IEL development and maintenance may help dissect their immunological functions in intestinal diseases with segment-specific manifestations.

Deletion of Adam6 in Mus musculus leads to male subfertility and deficits in sperm ascent into the oviduct.

The Adisintegrin and metalloprotease domain-containing (ADAM) family of proteins is involved in cell adhesion, migration, proteolysis, and signaling. Many ADAMs are required for reproduction; however, the role of Adam6 has remained largely unknown. In the course of humanizing the mouse immunoglobulin heavy chain (IgH) locus, we generated Adam6-deficient mice that demonstrate severe subfertility. We decided to elucidate the role of ADAM6 in fertility and explore the underlying mechanisms. Despite normal sperm development and motility, Adam6-deficient mice display diminished male fertility, have abnormal sperm adhesion, and most importantly cannot transition from uterus to oviduct. To test whether ADAM6 is required for sperm's binding to extracellular matrix (ECM) components, we used a panel of ECM components and showed that unlike normal sperm, Adam6-deficient sperm cannot bind fibronectin, laminin, and tenascin. Reintroduction of Adam6 into these deficient mice repaired sperm interaction with ECM, restored male fertility, and corrected the sperm transport deficit. Together, our data suggest that ADAM6, either alone or in complex with other proteins, aids sperm transport through the female reproductive tract by providing a temporary site of attachment of sperm to ECM components prior to ascent into the oviduct.

Loss of SFRP4 Alters Body Size, Food Intake, and Energy Expenditure in Diet-Induced Obese Male Mice.

Secreted frizzled-related protein 4 (SFRP4) is an extracellular regulator of the wingless-type mouse mammary tumor virus integration site family (WNT) pathway. SFRP4 has been implicated in adipocyte dysfunction, obesity, insulin resistance, and impaired insulin secretion in patients with type 2 diabetes. However, the exact role of SFRP4 in regulating whole-body metabolism and glucose homeostasis is unknown. We show here that male Sfrp4(-/-) mice have increased spine length and gain more weight when fed a high-fat diet. The body composition and body mass per spine length of diet-induced obese Sfrp4(-/-) mice is similar to wild-type littermates, suggesting that the increase in body weight can be accounted for by their longer body size. The diet-induced obese Sfrp4(-/-) mice have reduced energy expenditure, food intake, and bone mineral density. Sfrp4(-/-) mice have normal glucose and insulin tolerance and ß-cell mass. Diet-induced obese Sfrp4(-/-) and control mice show similar impairments of glucose tolerance and a 5-fold compensatory expansion of their ß-cell mass. In summary, our data suggest that loss of SFRP4 alters body length and bone mineral density as well as energy expenditure and food intake. However, SFRP4 does not control glucose homeostasis and ß-cell mass in mice.

Mouse ghrelin-O-acyltransferase (GOAT) plays a critical role in bile acid reabsorption.

Ghrelin is a unique peptide gut hormone that requires post-translational modification to stimulate both feeding and growth hormone release. Ghrelin O-acyltransferase (GOAT) was identified as a specific acyl-transferase for ghrelin, and recent genetic deletion studies of the Goat gene (Goat(-/-)) uncovered the role of ghrelin in the regulation of glucose homeostasis. To further understand the physiological functions of the GOAT/ghrelin system, we have conducted a metabolomic and microarray profile of Goat-null mice, as well as determined Goat expression in different tissues using the lacZ reporter gene. Serum metabolite profile analysis revealed that Goat(-/-) mice exhibited increased secondary bile acids >2.5-fold. This was attributed to increased mRNA and protein expression of the ileal sodium-dependent bile acid transporter (ISBT) in the intestinal and biliary tract. Increased expression of additional solute carrier proteins, including Slc5a12 (>10-fold) were also detected in the small intestine and bile duct. Goat staining was consistently observed in the pituitary glands, stomach, and intestines, and to a lesser extent in the gallbladder and pancreatic duct. This is the first report that the GOAT/ghrelin system regulates bile acid metabolism, and these findings suggest a novel function of GOAT in the regulation of intestinal bile acid reabsorption..

The PDGF signaling pathway controls multiple steroid-producing lineages.

The platelet-derived growth factor (PDGF) signaling pathway regulates numerous lineages of mesenchymal cell origin during development and in the adult. The transcriptional targets of this pathway have been shown to be required in several PDGF-dependent processes, but the roles of these targets in specific tissues is just beginning to be identified. In this study, we show that five different PDGF target genes are essential for male and/or female fertility. Mutations in each of these five different genes lead to defects in the steroid-producing cells in the testis and/or ovary and altered hormone production, suggesting that the PDGF pathway controls steroidogenesis through these genes in both sexes. Furthermore, conditional mutations of both PDGF receptors revealed a requirement in steroid-producing cells in multiple organs, including the testis, ovary, and adrenal cortex. Therefore, PDGF signaling may constitute a common mechanism in the control of multiple steroidogenic lineages.

PDGF signaling specificity is mediated through multiple immediate early genes.

Growth factor signaling leads to the induction or repression of immediate early genes, but how these genes act collectively as effectors of downstream processes remains unresolved. We have used gene trap-coupled microarray analysis to identify and mutate multiple platelet-derived growth factor (PDGF) intermediate early genes in mice. Mutations in these genes lead to a high frequency of phenotypes that affect the same cell types and processes as those controlled by the PDGF pathway. We conclude that these genes form a network that controls specific processes downstream of PDGF signaling.

Fgf9 induces proliferation and nuclear localization of FGFR2 in Sertoli precursors during male sex determination.

Recently, we demonstrated that loss of Fgf9 results in a block of testis development and a male to female sex-reversed phenotype; however, the function of Fgf9 in sex determination was unknown. We now show that Fgf9 is necessary for two steps of testis development just downstream of the male sex-determining gene, Sry: (1) for the proliferation of a population of cells that give rise to Sertoli progenitors; and (2) for the nuclear localization of an FGF receptor (FGFR2) in Sertoli cell precursors. The nuclear localization of FGFR2 coincides with the initiation of Sry expression and the nuclear localization of SOX9 during the early differentiation of Sertoli cells and the determination of male fate.

Cell proliferation is necessary for the determination of male fate in the gonad.

Cell proliferation has been shown to have multiple functions in development and pattern formation, including roles in growth, morphogenesis, and gene expression. Previously, we determined that the earliest known morphological event downstream of the male sex determining gene, Sry, is the induction of proliferation. In this study, we used proliferation inhibitors to block cell division during early gonad development, at stages before the XY gonad has committed to the testis pathway. Using the expression of sex-specific genes and the formation of testis morphology as markers of testis determination, we found that proliferation within a specific 8-h window was critical for the establishment of the male pathway and the formation of the testis. Inhibition of proliferation before or after this critical period led to smaller gonads, but did not block testis formation. The critical period of proliferation coincides with the initiation of Sry expression and is essential for the differentiation of Sertoli cells, suggesting that proliferation is a vital component of the initiation of the male pathway by Sry. We believe these studies suggest that proliferation is involved not only in the elaboration of organ pattern, but also in the choice between patterns (male and female) in the bipotential gonad.

Colocalization of WT1 and cell proliferation reveals conserved mechanisms in temperature-dependent sex determination.

During vertebrate development the gonad has two possible fates, the testis or the ovary. The choice between these fates is made by a variety of sex-determining mechanisms, from the sex-determining gene on the Y chromosome (Sry) in mammals, to nongenetic temperature-dependent systems in many reptiles. Despite the differences in the mechanisms at the top of the sex-determining cascade, the resulting morphology and many genes involved in early testis and ovarian development are common to most vertebrates, leading to the hypothesis that the underlying processes of sex determination are conserved. In this study, we examined the early steps of gonad development in the red-eared slider turtle (Trachemys scripta), a species that uses the temperature of egg incubation to determine sex. A dramatic increase in cell proliferation was observed in the male gonad during the earliest stages of sex determination. Using the localization of Wilms' Tumor suppressor 1 (WT1), we determined that this proliferation increase occurred in a population that contained pre-Sertoli cells. The proliferation of pre-Sertoli cells has been documented during sex determination in both mice and alligators, suggesting that proliferation of this cell type has an important role in vertebrate testis organogenesis and the determination of male fate.