Assembling the adipose organ: adipocyte lineage segregation and adipogenesis in vivo.

Adipose tissue is composed of anatomically distinct depots that mediate several important aspects of energy homeostasis. The past two decades have witnessed increased research effort to elucidate the ontogenetic basis of adipose form and function. In this Review, we discuss advances in our understanding of adipose tissue development with particular emphasis on the embryonic patterning of depot-specific adipocyte lineages and adipocyte differentiation in vivo Micro-environmental cues and other factors that influence cell identity and cell behavior at various junctures in the adipocyte lineage hierarchy are also considered.

A mesodermal fate map for adipose tissue.

The embryonic origin of distinct fat depots and the role for ontogeny in specifying the functional differences among adipocyte lineages between and within depots is unclear. Using a Cre/Lox-based strategy to track the fate of major mesodermal subcompartments in mice we present evidence that <50% of interscapular brown adipocytes are derived from progenitors of the central dermomyotome. Furthermore, we demonstrate that depot-specific adipocyte lineages spatially diverge as early as gastrulation, and that perigonadal adipocytes arise from separate mesodermal subcompartments in males and females. Last, we show adipocyte precursors (APs) of distinct lineages within the same depot exhibit indistinguishable responses to a high fat diet, indicating that ontogenetic differences between APs do not necessarily correspond to functional differences in this context. Altogether, these findings shed light on adipose tissue patterning and suggest that the behavior of adipocyte lineage cells is not strictly determined by developmental history.

Prepubertal androgen signaling is required to establish male fat distribution.

Fat distribution is sexually dimorphic and is associated with metabolic disease risk. It is unknown if prepubertal sex-hormone signaling influences adult fat distribution. Here, we show that karyotypically male androgen-insensitive mice exhibit pronounced subcutaneous adiposity compared with wild-type males and females. This subcutaneous adipose bias emerges prior to puberty and is not due to differences in adipocyte size or rates of adipogenesis between visceral and subcutaneous fat. Instead, we find that androgen-insensitive mice lack an adequate progenitor pool for normal visceral-fat expansion during development, thus increasing the subcutaneous-to-visceral-fat ratio. Obesogenic visceral-fat expansion is likewise inhibited in these mice, yet their metabolic health is similar to wild-type animals with comparable total fat mass. Taken together, these data show that adult fat distribution can be determined prior to the onset of puberty by the relative number of progenitors that seed nascent adipose depots.

Testosterone metabolites differentially regulate obesogenesis and fat distribution.

OBJECTIVE: Low testosterone in men (hypogonadism) is associated with obesity and type II diabetes. Testosterone replacement therapy has been shown to reverse these effects. However, the mechanisms by which testosterone regulates total fat mass, fat distribution, and metabolic health are unclear. In this study, we clarify the impact of hypogonadism on these parameters, as well as parse the role of testosterone from its downstream metabolites, dihydrotestosterone (DHT), and estradiol, in the regulation of depot-specific adipose tissue mass. METHODS: To achieve this objective, we utilized mouse models of male hypogonadism coupled with hormone replacement therapy, magnetic resonance imaging (MRI), glucose tolerance tests, flow cytometry, and immunohistochemical techniques. RESULTS: We observed that castrated mice develop increased fat mass, reduced muscle mass, and impaired glucose metabolism compared with gonadally intact males. Interestingly, obesity is further accelerated in castrated mice fed a high-fat diet, suggesting hypogonadism increases susceptibility to obesogenesis when dietary consumption of fat is elevated. By performing hormone replacement therapy in castrated mice, we show that testosterone impedes visceral and subcutaneous fat mass expansion. Testosterone-derived estradiol selectively blocks visceral fat growth, and DHT selectively blocks the growth of subcutaneous fat. These effects are mediated by depot-specific alterations in adipocyte size. We also show that high-fat diet-induced adipogenesis is elevated in castrated mice and that this can be rescued by androgen treatment. Obesogenic adipogenesis is also elevated in mice where androgen receptor activity is inhibited. CONCLUSIONS: These data indicate that hypogonadism impairs glucose metabolism and increases obesogenic fat mass expansion through adipocyte hypertrophy and adipogenesis. In addition, our findings highlight distinct roles for testosterone, DHT, and estradiol in the regulation of total fat mass and fat distribution and reveal that androgen signaling blocks obesogenic adipogenesis in vivo.

Bone Marrow Adiposity: Basic and Clinical Implications.

The presence of adipocytes in mammalian bone marrow (BM) has been recognized histologically for decades, yet, until recently, these cells have received little attention from the research community. Advancements in mouse transgenics and imaging methods, particularly in the last 10 years, have permitted more detailed examinations of marrow adipocytes than ever before and yielded data that show these cells are critical regulators of the BM microenvironment and whole-body metabolism. Indeed, marrow adipocytes are anatomically and functionally separate from brown, beige, and classic white adipocytes. Thus, areas of BM space populated by adipocytes can be considered distinct fat depots and are collectively referred to as marrow adipose tissue (MAT) in this review. In the proceeding text, we focus on the developmental origin and physiologic functions of MAT. We also discuss the signals that cause the accumulation and loss of marrow adipocytes and the ability of these cells to regulate other cell lineages in the BM. Last, we consider roles for MAT in human physiology and disease.

An optimized TALEN application for mutagenesis and screening in Drosophila melanogaster.

Transcription activator-like effector nucleases (TALENs) emerged as powerful tools for locus-specific genome engineering. Due to the ease of TALEN assembly, the key to streamlining TALEN-induced mutagenesis lies in identifying efficient TALEN pairs and optimizing TALEN mRNA injection concentrations to minimize the effort to screen for mutant offspring. Here we present a simple methodology to quantitatively assess bi-allelic TALEN cutting, as well as approaches that permit accurate measures of somatic and germline mutation rates in Drosophila melanogaster. We report that percent lethality from pilot injection of candidate TALEN mRNAs into Lig4 null embryos can be used to effectively gauge bi-allelic TALEN cutting efficiency and occurs in a dose-dependent manner. This timely Lig4-dependent embryonic survival assay also applies to CRISPR/Cas9-mediated targeting. Moreover, the somatic mutation rate of individual G0 flies can be rapidly quantitated using SURVEYOR nuclease and capillary electrophoresis, and germline transmission rate determined by scoring progeny of G0 outcrosses. Together, these optimized methods provide an effective step-wise guide for routine TALEN-mediated gene editing in the fly.

A simplified and efficient germline-specific CRISPR/Cas9 system for Drosophila genomic engineering.

The type II CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated) has recently emerged as an efficient and simple tool for site-specific engineering of eukaryotic genomes. To improve its applications in Drosophila genome engineering, we simplified the standard two-component CRISPR/Cas9 system by generating a stable transgenic fly line expressing the Cas9 endonuclease in the germline (Vasa-Cas9 line). By injecting vectors expressing engineered target-specific guide RNAs into Vasa-Cas9 fly embryos, mutations were generated from site-specific DNA cleavages and efficiently transmitted into progenies. Because Cas9 endonuclease is the universal component of the type II CRISPR/Cas9 system, site-specific genomic engineering based on this improved platform can be achieved with lower complexity and toxicity, greater consistency, and excellent versatility.