Maternal Microbiota Modulate a Fragile X-like Syndrome in Offspring Mice.

Maternal microbial dysbiosis has been implicated in adverse postnatal health conditions in offspring, such as obesity, cancer, and neurological disorders. We observed that the progeny of mice fed a Westernized diet (WD) with low fiber and extra fat exhibited higher frequencies of stereotypy, hyperactivity, cranial features and lower FMRP protein expression, similar to what is typically observed in Fragile X Syndrome (FXS) in humans. We hypothesized that gut dysbiosis and inflammation during pregnancy influenced the prenatal uterine environment, leading to abnormal phenotypes in offspring. We found that oral in utero supplementation with a beneficial anti-inflammatory probiotic microbe, Lactobacillus reuteri, was sufficient to inhibit FXS-like phenotypes in offspring mice. Cytokine profiles in the pregnant WD females showed that their circulating levels of pro-inflammatory cytokine interleukin (Il)-17 were increased relative to matched gravid mice and to those given supplementary L. reuteri probiotic. To test our hypothesis of prenatal contributions to this neurodevelopmental phenotype, we performed Caesarian (C-section) births using dissimilar foster mothers to eliminate effects of maternal microbiota transferred during vaginal delivery or nursing after birth. We found that foster-reared offspring still displayed a high frequency of these FXS-like features, indicating significant in utero contributions. In contrast, matched foster-reared progeny of L. reuteri-treated mothers did not exhibit the FXS-like typical features, supporting a key role for microbiota during pregnancy. Our findings suggest that diet-induced dysbiosis in the prenatal uterine environment is strongly associated with the incidence of this neurological phenotype in progeny but can be alleviated by addressing gut dysbiosis through probiotic supplementation.

An EMT-primary cilium-GLIS2 signaling axis regulates mammogenesis and claudin-low breast tumorigenesis.

The epithelial-mesenchymal transition (EMT) and primary ciliogenesis induce stem cell properties in basal mammary stem cells (MaSCs) to promote mammogenesis, but the underlying mechanisms remain incompletely understood. Here, we show that EMT transcription factors promote ciliogenesis upon entry into intermediate EMT states by activating ciliogenesis inducers, including FGFR1. The resulting primary cilia promote ubiquitination and inactivation of a transcriptional repressor, GLIS2, which localizes to the ciliary base. We show that GLIS2 inactivation promotes MaSC stemness, and GLIS2 is required for normal mammary gland development. Moreover, GLIS2 inactivation is required to induce the proliferative and tumorigenic capacities of the mammary tumor­initiating cells (MaTICs) of claudin-low breast cancers. Claudin-low breast tumors can be segregated from other breast tumor subtypes based on a GLIS2-dependent gene expression signature. Collectively, our findings establish molecular mechanisms by which EMT programs induce ciliogenesis to control MaSC and MaTIC stemness, mammary gland development, and claudin-low breast cancer formation.

Phosphocholine accumulation and PHOSPHO1 depletion promote adipose tissue thermogenesis.

Phosphocholine phosphatase-1 (PHOSPHO1) is a phosphocholine phosphatase that catalyzes the hydrolysis of phosphocholine (PC) to choline. Here we demonstrate that the PHOSPHO1 transcript is highly enriched in mature brown adipose tissue (BAT) and is further induced by cold and isoproterenol treatments of BAT and primary brown adipocytes. In defining the functional relevance of PHOPSPHO1 in BAT thermogenesis and energy metabolism, we show that PHOSPHO1 knockout mice are cold-tolerant, with higher expression of thermogenic genes in BAT, and are protected from high-fat diet-induced obesity and development of insulin resistance. Treatment of mice with the PHOSPHO1 substrate phosphocholine is sufficient to induce cold tolerance, thermogenic gene expression, and allied metabolic benefits. Our results reveal a role of PHOSPHO1 as a negative regulator of BAT thermogenesis, and inhibition of PHOSPHO1 or enhancement of phosphocholine represent innovative approaches to manage the metabolic syndrome.

SYK kinase mediates brown fat differentiation and activation.

Brown adipose tissue (BAT) metabolism influences glucose homeostasis and metabolic health in mice and humans. Sympathetic stimulation of ß-adrenergic receptors in response to cold induces proliferation, differentiation, and UCP1 expression in pre-adipocytes and mature brown adipocytes. Here we show that spleen tyrosine kinase (SYK) is upregulated during brown adipocyte differentiation and activated by ß-adrenergic stimulation. Deletion or inhibition of SYK, a kinase known for its essential roles in the immune system, blocks brown and white pre-adipocyte proliferation and differentiation in vitro, and results in diminished expression of Ucp1 and other genes regulating brown adipocyte function in response to ß-adrenergic stimulation. Adipocyte-specific SYK deletion in mice reduces BAT mass and BAT that developed consisted of SYK-expressing brown adipocytes that had escaped homozygous Syk deletion. SYK inhibition in vivo represses ß-agonist-induced thermogenesis and oxygen consumption. These results establish SYK as an essential mediator of brown fat formation and function.

EMT programs promote basal mammary stem cell and tumor-initiating cell stemness by inducing primary ciliogenesis and Hedgehog signaling.

Tissue regeneration relies on adult stem cells (SCs) that possess the ability to self-renew and produce differentiating progeny. In an analogous manner, the development of certain carcinomas depends on a small subset of tumor cells, called "tumor-initiating cells" (TICs), with SC-like properties. Mammary SCs (MaSCs) reside in the basal compartment of the mammary epithelium, and their neoplastic counterparts, mammary TICs (MaTICs), are thought to serve as the TICs for the claudin-low subtype of breast cancer. MaSCs and MaTICs both use epithelial-mesenchymal transition (EMT) programs to acquire SC properties, but the mechanism(s) connecting EMT programs to stemness remain unclear. Here we show that this depends on primary cilia, which are nonmotile, cell-surface structures that serve as platforms for receiving cues and enable activation of various signaling pathways. We show that MaSC and MaTIC EMT programs induce primary cilia formation and Hedgehog (Hh) signaling, which has previously been implicated in both MaSC and MaTIC function. Moreover, ablation of these primary cilia is sufficient to repress Hh signaling, the stemness of MaSCs, and the tumor-forming potential of MaTICs. Together, our findings establish primary ciliogenesis and consequent Hh signaling as a key mechanism by which MaSC and MaTIC EMT programs promote stemness and thereby support mammary tissue outgrowth and tumors of basal origin.

Castration eliminates conspecific aggression in group-housed CD1 male surveillance mice (Mus musculus).

Microbiologic surveillance is essential for murine health maintenance. At our institution, female progeny of inhouse-bred CD1 mice are used in both the transgenic facility and health-surveillance program. To reduce overall animal use, the male progeny, otherwise slated for euthanasia due to a lack of utility, also were enrolled as sentinels. However, veterinary technicians noted excessive fighting among cohoused male surveillance mice that was not resolved by environmental enrichment. After review of factors known to influence aggression in male mice, early castration was selected as the most likely approach to eliminate aggressive behavior among cohoused male mice. Male mice were castrated before 1 mo of age and then placed into the surveillance program. Each week, veterinary technicians recorded all incidences of fighting in cages of castrated and noncastrated male surveillance mice to determine differences between groups. Over a 3-mo period, the overall prevalence of fighting in cages of intact male mice was 64% (14 of 22 cages); although all intact male mice were used preferentially for complete necropsy surveillance time points, one of these cages required separation and 4 cages housed mice that incurred severe fight wounds requiring both separation and euthanasia. In comparison, a 0% (0 of 16 cages) prevalence of fighting was observed among castrated male mice. Castration eradicated pain and distress associated with fighting, thereby constituting a refinement, and allowed the use of male mice from the breeding colony for surveillance, thereby reducing the total number of mice bred for surveillance. In conclusion, castration is a minimally invasive, safe, humane, rapid method to eliminate conspecific aggression among male CD1 surveillance mice.