Jonathan M. Graff

White Fat Progenitor Cells Reside in the Adipose Vasculature

White adipose (fat) tissues regulate metabolism, reproduction, and life span. Adipocytes form throughout life, with the most marked expansion of the lineage occurring during the postnatal period. Adipocytes develop in coordination with the vasculature, but the identity and location of white adipocyte progenitor cells in vivo are unknown. We used genetically marked mice to isolate proliferating and renewing adipogenic progenitors. We found that most adipocytes descend from a pool of these proliferating progenitors that are already committed, either prenatally or early in postnatal life. These progenitors reside in the mural cell compartment of the adipose vasculature, but not in the vasculature of other tissues. Thus, the adipose vasculature appears to function as a progenitor niche and may provide signals for adipocyte development.

Smad3 Mutant Mice Develop Metastatic Colorectal Cancer

TGFbeta-related growth factors have been implicated in a variety of developmental and physiological processes in organisms ranging from nematodes to mammals. TGFbeta transduces its signal to the interior of the cell via Smad2, Smad3, and Smad4. We report the cloning and targeted disruption of the mouse Smad3 gene. Smad3 mutant mice are viable and fertile. Between 4 and 6 months of age, the Smad3 mutant mice become moribund with colorectal adenocarcinomas. The neoplasms penetrate through the intestinal wall and metastasize to lymph nodes. These results directly implicate TGFbeta signaling in the pathogenesis of colorectal cancer and provide a compelling animal model for the study of human colorectal cancer.

Casein kinase I transduces Wnt signals.

The Wnt signalling cascade is essential for the development of both invertebrates and vertebrates, and is altered during tumorigenesis. Although a general framework for Wnt signalling has been elucidated, not all of the components have been identified. Here we describe a serine kinase, casein kinase I (CKI), which was isolated by expression cloning in Xenopus embryos. CKI reproduces several properties of Wnt signals, including generation of complete dorsal axes, stabilization of β-catenin and induction of genes that are direct targets of Wnt signals. Dominant-negative forms of CKI and a pharmacological blocker of CKI inhibited Wnt signals in Xenopus. Inhibiting CKI in Caenorhabditis elegans generated worms with a mom phenotype, indicative of a loss of Wnt signals. In addition, CKI bound to and increased the phosphorylation of dishevelled, a known component of the Wnt pathway. These data indicate that CKI may be a conserved component of the Wnt pathway.

C. elegans: A Model for Exploring the Genetics of Fat Storage

To gain insights into the genetic cascades that regulate fat biology, we evaluated C. elegans as an appropriate model organism. We generated worms that lack two transcription factors, SREBP and C/EBP, crucial for formation of mammalian fat. Worms deficient in either of these genes displayed a lipid-depleted phenotype-pale, skinny, larval-arrested worms that lack fat stores. On the basis of this phenotype, we used a reverse genetic screen to identify several additional genes that play a role in worm lipid storage. Two of the genes encode components of the mitochondrial respiratory chain (MRC). When the MRC was inhibited chemically in worms or in a mammalian adipocyte model, fat accumulation was markedly reduced. A third encodes lpd-3, whose homolog is also required for fat storage in a mammalian model. These data suggest that C. elegans is a genetically tractable model to study the mechanisms that underlie the biology of fat-storing tissues.

Embryonic Patterning: To BMP or Not to BMP, That Is the Question

Although some progress in understanding the molecular mechanisms of early embryogenesis has been made, a few outstanding issues remain to be clarified. Of particular interest is whether dorsal–ventral patterning of the endoderm is also controlled by BMP signaling. It appears quite likely that TGFβ signals are essential for endodermal patterning (3xGamer, L and Wright, C. Dev. Biol. 1995; 171: 240–251Crossref | PubMed | Scopus (107)See all References, 7xHenry, G, Brivanlou, I, Kessler, D, Hemmati-Brivanlou, A, and Melton, D. Development. 1996; 122: 1007–1015PubMedSee all References, 13xSasai, Y, Lu, B, Piccolo, S, and De Robertis, E. EMBO J. 1996; 15: 4547–4555PubMedSee all References). Preliminary results suggest that BMP4, chordin, and noggin, the same molecules used in the ectoderm and the mesoderm, also pattern the endoderm (Sasai et al. 1996xSasai, Y, Lu, B, Piccolo, S, and De Robertis, E. EMBO J. 1996; 15: 4547–4555PubMedSee all ReferencesSasai et al. 1996). This suggests that conserved mechanisms of patterning are employed in all three germ layers (Figure 4Figure 4). BMPs are active in many other important biological processes, and it remains to be established whether noggin and chordin are important in BMP action at other times and places. It is unclear why two factors, noggin and chordin, with essentially identical activity are needed when only one might suffice. Relatedly, if there are two, there might be other proteins with similar activity either for BMPs or for other TGFβs. Although it appears that BMP signaling is essential for formation of the ventral side of the embryo, it is unclear what establishes the restricted pattern of BMP expression on the ventral side of the embryo. In addition, it remains unknown what signals establish formation of the organizer or restrict expression of chordin and noggin to the organizer. The best data supports a role for the Wnt signaling pathway in organizer formation, although how this is established is poorly understood (Carnac et al. 1996xCarnac, G, Kodjabachian, L, Gurdon, J, and Lemaire, P. Development. 1996; 122: 3055–3065PubMedSee all ReferencesCarnac et al. 1996references therein). Finally, although we have begun to understand what patterns the ectoderm, mesoderm, and endoderm, we still do not know the nature of the molecule(s) that initially induces formation of the mesoderm.

Transcriptional recapitulation and subversion of embryonic colon development by mouse colon tumor models and human colon cancer

BackgroundThe expression of carcino-embryonic antigen by colorectal cancer is an example of oncogenic activation of embryonic gene expression. Hypothesizing that oncogenesis-recapitulating-ontogenesis may represent a broad programmatic commitment, we compared gene expression patterns of human colorectal cancers (CRCs) and mouse colon tumor models to those of mouse colon development embryonic days 13.5-18.5.ResultsWe report here that 39 colon tumors from four independent mouse models and 100 human CRCs encompassing all clinical stages shared a striking recapitulation of embryonic colon gene expression. Compared to normal adult colon, all mouse and human tumors over-expressed a large cluster of genes highly enriched for functional association to the control of cell cycle progression, proliferation, and migration, including those encoding MYC, AKT2, PLK1 and SPARC. Mouse tumors positive for nuclear β-catenin shifted the shared embryonic pattern to that of early development. Human and mouse tumors differed from normal embryonic colon by their loss of expression modules enriched for tumor suppressors (EDNRB, HSPE, KIT and LSP1). Human CRC adenocarcinomas lost an additional suppressor module (IGFBP4, MAP4K1, PDGFRA, STAB1 and WNT4). Many human tumor samples also gained expression of a coordinately regulated module associated with advanced malignancy (ABCC1, FOXO3A, LIF, PIK3R1, PRNP, TNC, TIMP3 and VEGF).ConclusionCross-species, developmental, and multi-model gene expression patterning comparisons provide an integrated and versatile framework for definition of transcriptional programs associated with oncogenesis. This approach also provides a general method for identifying pattern-specific biomarkers and therapeutic targets. This delineation and categorization of developmental and non-developmental activator and suppressor gene modules can thus facilitate the formulation of sophisticated hypotheses to evaluate potential synergistic effects of targeting within- and between-modules for next-generation combinatorial therapeutics and improved mouse models.

The transcription factor ATF4 regulates glucose metabolism in mice through its expression in osteoblasts

The recent demonstration that osteoblasts have a role in controlling energy metabolism suggests that they express cell-specific regulatory genes involved in this process. Activating transcription factor 4 (ATF4) is a transcription factor that accumulates predominantly in osteoblasts, where it regulates virtually all functions linked to the maintenance of bone mass. Since Atf4-/- mice have smaller fat pads than littermate controls, we investigated whether ATF4 also influences energy metabolism. Here, we have shown, through analysis of Atf4-/- mice, that ATF4 inhibits insulin secretion and decreases insulin sensitivity in liver, fat, and muscle. Several lines of evidence indicated that this function of ATF4 occurred through its osteoblastic expression. First, insulin sensitivity is enhanced in the liver of Atf4-/- mice, but not in cultured hepatocytes from these mice. Second, mice overexpressing ATF4 in osteoblasts only [termed here alpha1(I)Collagen-Atf4 mice] displayed a decrease in insulin secretion and were insulin insensitive. Third, the alpha1(I)Collagen-Atf4 transgene corrected the energy metabolism phenotype of Atf4-/- mice. Fourth, and more definitely, mice lacking ATF4 only in osteoblasts presented the same metabolic abnormalities as Atf4-/- mice. Molecularly, ATF4 favored expression in osteoblasts of Esp, which encodes a product that decreases the bioactivity of osteocalcin, an osteoblast-specific secreted molecule that enhances secretion of and sensitivity to insulin. These results provide a transcriptional basis to the observation that osteoblasts fulfill endocrine functions and identify ATF4 as a regulator of most functions of osteoblasts.

Atf4 Regulates Obesity, Glucose Homeostasis, and Energy Expenditure

OBJECTIVE We evaluate a potential role of activating transcription factor 4 (Atf4) in invertebrate and mammalian metabolism. RESEARCH DESIGN AND METHODS With two parallel approaches—a fat body–specific green fluorescent protein enhancer trap screen in D. melanogaster and expression profiling of developing murine fat tissues—we identified Atf4 as expressed in invertebrate and vertebrate metabolic tissues. We assessed the functional relevance of the evolutionarily conserved expression by analyzing Atf4 mutant flies and Atf4 mutant mice for possible metabolic phenotypes. RESULTS Flies with insertions at the Atf4 locus have reduced fat content, increased starvation sensitivity, and lower levels of circulating carbohydrate. Atf4 null mice are also lean, and they resist age-related and diet-induced obesity. Atf4 null mice have increased energy expenditure potentially accounting for the lean phenotype. Atf4 null mice are hypoglycemic, even before substantial changes in fat content, indicating that Atf4 regulates mammalian carbohydrate metabolism. In addition, the Atf4 mutation blunts diet-induced diabetes as well as hyperlipidemia and hepatosteatosis. Several aspects of the Atf4 mutant phenotype resemble mice with mutations in components of the target of rapamycin (TOR) pathway. Consistent with the phenotypic similarities, Atf4 null mice have reduced expression of genes that regulate intracellular amino acid concentrations and lower intracellular concentration of amino acids, a key TOR input. Further, Atf4 mutants have reduced S6K activity in liver and adipose tissues. CONCLUSIONS Atf4 regulates age-related and diet-induced obesity as well as glucose homeostasis in mammals and has conserved metabolic functions in flies.

The developmental origins of adipose tissue

Adipose tissue is formed at stereotypic times and locations in a diverse array of organisms. Once formed, the tissue is dynamic, responding to homeostatic and external cues and capable of a 15-fold expansion. The formation and maintenance of adipose tissue is essential to many biological processes and when perturbed leads to significant diseases. Despite this basic and clinical significance, understanding of the developmental biology of adipose tissue has languished. In this Review, we highlight recent efforts to unveil adipose developmental cues, adipose stem cell biology and the regulators of adipose tissue homeostasis and dynamism.

Redundant roles of SMAD2 and SMAD3 in ovarian granulosa cells in vivo.

ABSTRACT Transforming growth factor β (TGF-β) superfamily members are critical in maintaining cell growth and differentiation in the ovary. Although signaling of activins, TGF-βs, growth differentiation factor 9, and nodal converge preferentially to SMAD2 and SMAD3, the in vivo functions and redundancy of these SMADs in the ovary and female reproduction remain largely unidentified. To circumvent the deleterious phenotypic aspects of ubiquitous deletion of Smad2 and Smad3, a conditional knockout strategy was formulated to selectively inactivate Smad2, Smad3, or both Smad2 and Smad3 in ovarian granulosa cells. While granulosa cell ablation of individual Smad2 or Smad3 caused insignificant changes in female fertility, deletion of both Smad2 and Smad3 led to dramatically reduced female fertility and fecundity. These defects were associated with the disruption of multiple ovarian processes, including follicular development, ovulation, and cumulus cell expansion. Furthermore, the impaired expansion of cumulus cells may be partially associated with altered cumulus expansion-related transcripts that are regulated by SMAD2/3 signaling. Our results indicate that SMAD2 and SMAD3 function redundantly in vivo to maintain normal female fertility and further support the involvement of an intraovarian SMAD2/3 pathway in mediating oocyte-produced signals essential for coordinating key events of the ovulatory process.