Teresa L. Mastracci

Krt19+/Lgr5− Cells Are Radioresistant Cancer-Initiating Stem Cells in the Colon and Intestine

Epithelium of the colon and intestine are renewed every 3 days. In the intestine there are at least two principal stem cell pools. The first contains rapid cycling crypt-based columnar (CBC) Lgr5(+) cells, and the second is composed of slower cycling Bmi1-expressing cells at the +4 position above the crypt base. In the colon, however, the identification of Lgr5(-) stem cell pools has proven more challenging. Here, we demonstrate that the intermediate filament keratin-19 (Krt19) marks long-lived, radiation-resistant cells above the crypt base that generate Lgr5(+) CBCs in the colon and intestine. In colorectal cancer models, Krt19(+) cancer-initiating cells are also radioresistant, while Lgr5(+) stem cells are radiosensitive. Moreover, Lgr5(+) stem cells are dispensable in both the normal and neoplastic colonic epithelium, as ablation of Lgr5(+) stem cells results in their regeneration from Krt19-expressing cells. Thus, Krt19(+) stem cells are a discrete target relevant for cancer therapy.

GABAergic Differentiation Induced by Mash1 Is Compromised by the bHLH Proteins Neurogenin2, NeuroD1, and NeuroD2

During forebrain development, Mash1 directs gamma-aminobutyric acid (GABA)ergic neuron differentiation ventrally in the ganglionic eminences. Repression of Mash1 in the cortex is necessary to prevent the formation of GABAergic interneurons. Negative regulation of Mash1 has been attributed to members of the Neurogenin family; the genetic ablation of Neurogenin2 (Ngn2) leads to the derepression of Mash1 and the formation of ectopic GABAergic neurons in the cortex. We have developed an in vitro system to clarify the importance of NeuroD proteins in the Mash1 regulatory pathway. Using a neurosphere culture system, we show that the downstream effectors of the Ngn2 pathway NeuroD1 and NeuroD2 can abrogate GABAergic differentiation directed by Mash1. The ectopic expression of either of these genes in Mash1-expressing cells derived from the lateral ganglionic eminence, independently downregulate Mash1 expression without affecting expression of distal less homeodomain genes. This results in a complete loss of the GABAergic phenotype. Moreover, we demonstrate that ectopic expression of Mash1 in cortical progenitors is sufficient to phenocopy the loss of Ngn2 and strongly enhances ectopic GABAergic differentiation. Collectively, our results define the compensatory and cross-regulatory mechanisms that exist among basic helix-loop-helix transcription factors during neuronal fate specification.

The Endocrine Pancreas: insights into development, differentiation and diabetes.

In the developing embryo, appropriate patterning of the endoderm fated to become pancreas requires the spatial and temporal coordination of soluble factors secreted by the surrounding tissues. Once pancreatic progenitor cells are specified in the developing gut tube epithelium, epithelial–mesenchymal interactions, as well as a cascade of transcription factors, subsequently delineate three distinct lineages, including endocrine, exocrine, and ductal cells. Simultaneous morphological changes, including branching, vascularization, and proximal organ development, also influence the process of specification and differentiation. Decades of research using mouse genetics have uncovered many of the key factors involved in pancreatic cell fate decisions. When pancreas development or islet cell functions go awry, due to mutations in genes important for proper organogenesis and development, the result can lead to a common pancreatic affliction, diabetes mellitus. Current treatments for diabetes are adequate but not curative. Therefore, researchers are utilizing the current understanding of normal embryonic pancreas development in vivo, to direct embryonic stem cells toward a pancreatic fate with the goal of transplanting these in vitro generated ‘islets’ into patients. Mimicking development in vitro has proven difficult; however, significant progress has been made and the current differentiation protocols are becoming more efficient. The continued partnership between developmental biologists and stem cell researchers will guarantee that the in vitro generation of insulin‐producing β cells is a possible therapeutic option for the treatment of diabetes. WIREs Dev Biol 2011. doi: 10.1002/wdev.44

Nkx2.2 and Arx genetically interact to regulate pancreatic endocrine cell development and endocrine hormone expression

Nkx2.2 and Arx are essential pancreatic transcription factors. Nkx2.2 is necessary for the appropriate specification of the islet alpha, beta, PP and epsilon cell lineages, whereas Arx is required to form the correct ratio of alpha, beta, delta and PP cells. To begin to understand the cooperative functions of Nkx2.2 and Arx in the development of endocrine cell lineages, we generated progenitor cell-specific deletions of Arx on the Nkx2.2 null background. The analysis of these mutants demonstrates that expansion of the ghrelin cell population in the Nkx2.2 null pancreas is not dependent on Arx; however, Arx is necessary for the upregulation of ghrelin mRNA levels in Nkx2.2 mutant epsilon cells. Alternatively, in the absence of Arx, delta cell numbers are increased and Nkx2.2 becomes essential for the repression of somatostatin gene expression. Interestingly, the dysregulation of ghrelin and somatostatin expression in the Nkx2.2/Arx compound mutant (Nkx2.2(null);Arx(Δpanc)) results in the appearance of ghrelin+/somatostatin+ co-expressing cells. These compound mutants also revealed a genetic interaction between Nkx2.2 and Arx in the regulation of the PP cell lineage; the PP cell population is reduced when Nkx2.2 is deleted but is restored back to wildtype numbers in the Nkx2.2(null);Arx(Δpanc) mutant. Moreover, conditional deletion of Arx in specific pancreatic cell populations established that the functions of Arx are necessary in the Neurog3+ endocrine progenitors. Together, these experiments identify novel genetic interactions between Nkx2.2 and Arx within the endocrine progenitor cells that ensure the correct specification and regulation of endocrine hormone-producing cells.

Genetic evidence that Nkx2.2 and Pdgfra are major determinants of the timing of oligodendrocyte differentiation in the developing CNS

In the central nervous system (CNS), oligodendrocyte maturation and axonal myelination occur on a predictable schedule, but the underlying timing mechanisms are largely unknown. In the present study, we demonstrate that Nkx2.2 homeodomain transcription factor is a key regulator for the timing of oligodendrocyte differentiation during development. Whereas induced expression of Nkx2.2 in early oligodendrocyte precursor cells (OPCs) causes precocious differentiation of oligodendrocytes, conditional ablation of Nkx2.2 temporally delays oligodendrocyte maturation. Moreover, Nkx2.2 can directly bind to the promoter of platelet-derived growth factor receptor alpha (Pdgfra) and repress its gene expression. Genetic ablation of Pdgfra mimics the effect of Nkx2.2 overexpression in accelerating OPC differentiation in the developing spinal cord. Together, our findings strongly suggest that Nkx2.2 functions as a major ‘switch’ to turn off Pdgfra signaling in OPCs and initiate the intrinsic program for oligodendrocyte differentiation.

Regulation of Neurod1 contributes to the lineage potential of Neurogenin3+ endocrine precursor cells in the pancreas.

During pancreatic development, transcription factor cascades gradually commit precursor populations to the different endocrine cell fate pathways. Although mutational analyses have defined the functions of many individual pancreatic transcription factors, the integrative transcription factor networks required to regulate lineage specification, as well as their sites of action, are poorly understood. In this study, we investigated where and how the transcription factors Nkx2.2 and Neurod1 genetically interact to differentially regulate endocrine cell specification. In an Nkx2.2 null background, we conditionally deleted Neurod1 in the Pdx1+ pancreatic progenitor cells, the Neurog3+ endocrine progenitor cells, or the glucagon+ alpha cells. These studies determined that, in the absence of Nkx2.2 activity, removal of Neurod1 from the Pdx1+ or Neurog3+ progenitor populations is sufficient to reestablish the specification of the PP and epsilon cell lineages. Alternatively, in the absence of Nkx2.2, removal of Neurod1 from the Pdx1+ pancreatic progenitor population, but not the Neurog3+ endocrine progenitor cells, restores alpha cell specification. Subsequent in vitro reporter assays demonstrated that Nkx2.2 represses Neurod1 in alpha cells. Based on these findings, we conclude that, although Nkx2.2 and Neurod1 are both necessary to promote beta cell differentiation, Nkx2.2 must repress Neurod1 in a Pdx1+ pancreatic progenitor population to appropriately commit a subset of Neurog3+ endocrine progenitor cells to the alpha cell lineage. These results are consistent with the proposed idea that Neurog3+ endocrine progenitor cells represent a heterogeneous population of unipotent cells, each restricted to a particular endocrine lineage.

The endocrine pancreas: insights into development, differentiation, and diabetes

In the developing embryo, appropriate patterning of the endoderm fated to become pancreas requires the spatial and temporal coordination of soluble factors secreted by the surrounding tissues. Once pancreatic progenitor cells are specified in the developing gut tube epithelium, epithelial–mesenchymal interactions, as well as a cascade of transcription factors, subsequently delineate three distinct lineages, including endocrine, exocrine, and ductal cells. Simultaneous morphological changes, including branching, vascularization, and proximal organ development, also influence the process of specification and differentiation. Decades of research using mouse genetics have uncovered many of the key factors involved in pancreatic cell fate decisions. When pancreas development or islet cell functions go awry, due to mutations in genes important for proper organogenesis and development, the result can lead to a common pancreatic affliction, diabetes mellitus. Current treatments for diabetes are adequate but not curative. Therefore, researchers are utilizing the current understanding of normal embryonic pancreas development in vivo, to direct embryonic stem cells toward a pancreatic fate with the goal of transplanting these in vitro generated ‘islets’ into patients. Mimicking development in vitro has proven difficult; however, significant progress has been made and the current differentiation protocols are becoming more efficient. The continued partnership between developmental biologists and stem cell researchers will guarantee that the in vitro generation of insulin‐producing β cells is a possible therapeutic option for the treatment of diabetes. WIREs Dev Biol 2011. doi: 10.1002/wdev.44

Genomics and premalignant breast lesions: clues to the development and progression of lobular breast cancer

Advances in genomic technology have improved our understanding of the genetic events that parallel breast cancer development. Because almost all mammary carcinomas develop in the terminal duct lobular units of the breast, understanding the events involved in mammary gland development make it possible to recognize those events that, when altered, contribute to breast neoplasia. In this review we focus on lobular carcinomas, discussing the pathology, development, and progression of premalignant lobular lesions from a genomic point of view. We highlight studies utilizing genomic approaches and describe how these investigations have furthered our understanding of the complexity of premalignant breast lesions.

Pancreatic β cell identity requires continual repression of non–β cell programs

Loss of &bgr; cell identity, the presence of polyhormonal cells, and reprogramming are emerging as important features of &bgr; cell dysfunction in patients with type 1 and type 2 diabetes. In this study, we have demonstrated that the transcription factor NKX2.2 is essential for the active maintenance of adult &bgr; cell identity as well as function. Deletion of Nkx2.2 in &bgr; cells caused rapid onset of a diabetic phenotype in mice that was attributed to loss of insulin and downregulation of many &bgr; cell functional genes. Concomitantly, NKX2.2-deficient murine &bgr; cells acquired non–&bgr; cell endocrine features, resulting in populations of completely reprogrammed cells and bihormonal cells that displayed hybrid endocrine cell morphological characteristics. Molecular analysis in mouse and human islets revealed that NKX2.2 is a conserved master regulatory protein that controls the acquisition and maintenance of a functional, monohormonal &bgr; cell identity by directly activating critical &bgr; cell genes and actively repressing genes that specify the alternative islet endocrine cell lineages. This study demonstrates the highly volatile nature of the &bgr; cell, indicating that acquiring and sustaining &bgr; cell identity and function requires not only active maintaining of the expression of genes involved in &bgr; cell function, but also continual repression of closely related endocrine gene programs.

Ghrelin is dispensable for embryonic pancreatic islet development and differentiation

Ghrelin is a peptide hormone that has been implicated in the regulation of food intake and energy homeostasis. Ghrelin is predominantly produced in the stomach, but is also expressed in many other tissues where its functions are not well characterized. In the rodent and human pancreas, ghrelin levels peak at late gestation and gradually decline postnatally. Several studies have suggested that ghrelin regulates beta cell function during embryonic development and in the adult. In addition, in a number of mouse models, ghrelin cells appear to replace insulin- and glucagon-producing cells in the islet. In this analysis, we investigated whether the absence or overexpression of ghrelin influenced the development and differentiation of the pancreatic islet during embryonic development. These studies revealed that ghrelin is dispensable for normal pancreas development during gestation. Conversely, we demonstrated that elevated ghrelin in the Nkx2.2 null islets is not responsible for the absence of insulin- and glucagon-producing cells. Finally, we have also determined that in the absence of insulin, ghrelin cells form in their normal numbers and ghrelin is expressed at wild type levels.