ZnT8 Loss of Function Mutation Increases Resistance of Human Embryonic Stem Cell-Derived Beta Cells to Apoptosis in Low Zinc Condition.

The rare SLC30A8 mutation encoding a truncating p.Arg138* variant (R138X) in zinc transporter 8 (ZnT8) is associated with a 65% reduced risk for type 2 diabetes. To determine whether ZnT8 is required for beta cell development and function, we derived human pluripotent stem cells carrying the R138X mutation and differentiated them into insulin-producing cells. We found that human pluripotent stem cells with homozygous or heterozygous R138X mutation and the null (KO) mutation have normal efficiency of differentiation towards insulin-producing cells, but these cells show diffuse granules that lack crystalline zinc-containing insulin granules. Insulin secretion is not compromised in vitro by KO or R138X mutations in human embryonic stem cell-derived beta cells (sc-beta cells). Likewise, the ability of sc-beta cells to secrete insulin and maintain glucose homeostasis after transplantation into mice was comparable across different genotypes. Interestingly, sc-beta cells with the SLC30A8 KO mutation showed increased cytoplasmic zinc, and cells with either KO or R138X mutation were resistant to apoptosis when extracellular zinc was limiting. These findings are consistent with a protective role of zinc in cell death and with the protective role of zinc in T2D.

Murine Perinatal ß-Cell Proliferation and the Differentiation of Human Stem Cell-Derived Insulin-Expressing Cells Require NEUROD1.

Inactivation of the ß-cell transcription factor NEUROD1 causes diabetes in mice and humans. In this study, we uncovered novel functions of NEUROD1 during murine islet cell development and during the differentiation of human embryonic stem cells (HESCs) into insulin-producing cells. In mice, we determined that Neurod1 is required for perinatal proliferation of α- and ß-cells. Surprisingly, apoptosis only makes a minor contribution to ß-cell loss when Neurod1 is deleted. Inactivation of NEUROD1 in HESCs severely impaired their differentiation from pancreatic progenitors into insulin-expressing (HESC-ß) cells; however, survival or proliferation was not affected at the time points analyzed. NEUROD1 was also required in HESC-ß cells for the full activation of an essential ß-cell transcription factor network. These data reveal conserved and distinct functions of NEUROD1 during mouse and human ß-cell development and maturation, with important implications about the function of NEUROD1 in diabetes.

Embracing change: striated-for-smooth muscle replacement in esophagus development.

The esophagus functions to transport food from the oropharyngeal region to the stomach via waves of peristalsis and transient relaxation of the lower esophageal sphincter. The gastrointestinal tract, including the esophagus, is ensheathed by the muscularis externa (ME). However, while the ME of the gastrointestinal tract distal to the esophagus is exclusively smooth muscle, the esophageal ME of many vertebrate species comprises a variable amount of striated muscle. The esophageal ME is initially composed only of smooth muscle, but its developmental maturation involves proximal-to-distal replacement of smooth muscle with striated muscle. This fascinating phenomenon raises two important questions: what is the developmental origin of the striated muscle precursor cells, and what are the cellular and morphogenetic mechanisms underlying the process? Studies addressing these questions have provided controversial answers. In this review, we discuss the development of ideas in this area and recent work that has shed light on these issues. A working model has emerged that should permit deeper understanding of the role of ME development and maturation in esophageal disorders and in the functional and evolutionary underpinnings of the variable degree of esophageal striated myogenesis in vertebrate species.

PAX7 is required for patterning the esophageal musculature.

BACKGROUND: The mammalian esophageal musculature is unique in that it makes a transition from smooth to skeletal muscle, with most of this process occurring after birth. In order to better understand the mechanisms that control esophageal musculature development, we investigated the roles in this process of the paired box transcription factor, PAX7, a principal regulator of skeletal myogenic progenitor cells. Previous studies showed that Pax7 is important for determining the esophageal muscle composition. RESULTS: We characterized the postnatal development of the esophageal musculature in Pax7 (-/-) mice by analyzing morphology, muscle composition, and the expression of markers of myogenesis, cell proliferation, and apoptosis. Pax7 (-/-) mice displayed megaesophagus with a severe defect in the postnatal developmental process whereby esophageal smooth muscle is replaced by skeletal muscle. Pax7 (-/-) esophagi have substantially reduced skeletal muscle, most likely due to diminished proliferation and premature differentiation of skeletal muscle precursor cells. This impaired the proximal-to-distal progression of skeletal myogenesis and indirectly affected the patterning of the smooth muscle-containing portion of the esophageal musculature. CONCLUSIONS: Postnatal patterning of the esophageal musculature appears to require robust, PAX7-dependent cell proliferation to drive the proximal-to-distal progression of skeletal myogenesis. This process in turn influences distal smooth muscle morphogenesis and development of the mature pattern of the esophageal musculature.

Pancreatic islet cell development and regeneration.

PURPOSE OF REVIEW: This review will discuss recent advances in understanding mouse and human pancreatic islet cell development, novel concepts related to ß cell dysfunction and improved approaches for replenishing ß cells to treat diabetes. RECENT FINDINGS: Considerable knowledge about pancreatic islet development and function has been gained using model systems with subsequent validation in human tissues. Recently, several rodent studies have revealed that differentiated adult islet cells retain remarkable plasticity and can be converted to other islet cell types by perturbing their transcription factor profiles. Furthermore, significant advances have been made in the generation of ß-like cells from stem cell populations. Therefore, the generation of functionally mature ß cells by the in-situ conversion of non-ß cell populations or by the directed differentiation of human pluripotent stem cells could represent novel mechanisms for replenishing ß cells in diabetic patients. SUMMARY: The overall conservation between mouse and human pancreatic development, islet physiology and etiology of diabetes encourages the translation of novel ß cell replacement therapies to humans. Further deciphering the molecular mechanisms that direct islet cell regeneration, plasticity and function could improve and expand the ß cell replacement strategies for treating diabetes.

Overweight in mice and enhanced adipogenesis in vitro are associated with lack of the hedgehog coreceptor boc.

Obesity arises from a combination of genetic, environmental, and behavioral factors. However, the processes that regulate white adipose tissue (WAT) expansion at the level of the adipocyte are not well understood. The Hedgehog (HH) pathway plays a conserved role in adipogenesis, inhibiting fat formation in vivo and in vitro, but it has not been shown that mice with reduced HH pathway activity have enhanced adiposity. We report that mice lacking the HH coreceptor BOC displayed age-related overweight and excess WAT. They also displayed alterations in some metabolic parameters but normal food intake. Furthermore, they had an exacerbated response to a high-fat diet, including enhanced weight gain and adipocyte hypertrophy, livers with greater fat accumulation, and elevated expression of genes related to adipogenesis, lipid metabolism, and adipokine production. Cultured Boc(-/-) mouse embryo fibroblasts showed enhanced adipogenesis relative to Boc(+/+) cells, and they expressed reduced levels of HH pathway target genes. Therefore, a loss-of-function mutation in an HH pathway component is associated with WAT accumulation and overweight in mice. Variant alleles of such HH regulators may contribute to WAT accumulation in human individuals with additional genetic or lifestyle-based predisposition to obesity.

Smooth muscle fascicular reorientation is required for esophageal morphogenesis and dependent on Cdo.

Postnatal maturation of esophageal musculature involves proximal-to-distal replacement of smooth muscle with skeletal muscle by elusive mechanisms. We report that this process is impaired in mice lacking the cell surface receptor Cdo and identify the underlying developmental mechanism. A myogenic transition zone containing proliferative skeletal muscle precursor cells migrated in a proximal-distal direction, leaving differentiated myofibers in its wake. Distal to the transition zone, smooth muscle fascicles underwent a morphogenetic process whereby they changed their orientation relative to each other and to the lumen. Consequently, a path was cleared for the transition zone, and smooth muscle ultimately occupied only the distal-most esophagus; there was no loss of smooth muscle. Cdo(-/-) mice were specifically defective in fascicular reorientation, resulting in an aberrantly proximal skeletal-smooth muscle boundary. Furthermore, Cdo(-/-) mice displayed megaesophagus and achalasia, and their lower esophageal sphincter was resistant to nitric oxide-induced relaxation, suggesting a developmental linkage between patterning and sphincter function. Collectively, these results illuminate mechanisms of esophageal morphogenesis and motility disorders.

Requirement of the tissue-restricted homeodomain transcription factor Nkx6.3 in differentiation of gastrin-producing G cells in the stomach antrum.

Many homeodomain transcription factors function in organogenesis and cell differentiation. The Nkx family illustrates these functions especially well, and the Nkx6 subfamily controls differentiation in the central nervous system and pancreas. Nkx6.3, a recent addition to this subfamily, overlaps Nkx6.1 and Nkx6.2 in expression in the hindbrain and stomach. Nkx6.3 transcripts localize in the epithelium of the most distal stomach region, the antrum and pylorus; expression in the adult intestine is lower and confined to the proximal duodenum. Nkx6.3(-)(/)(-) mice develop and grow normally, with a grossly intact stomach and duodenum. These mice show markedly reduced gastrin mRNA, many fewer gastrin-producing (G) cells in the stomach antrum, hypogastrinemia, and increased stomach luminal pH, with a corresponding increase in somatostatin mRNA levels and antral somatostatin-producing (D) cells. They express normal levels of other transcription factors required for gastric endocrine cell differentiation, Pdx1, Pax6, and Ngn3; conversely, Ngn3(-)(/)(-) mice, which also show reduced gastrin levels, express Nkx6.3 normally. These studies implicate Nkx6.3 as a selective regulator of G- and D-cell lineages, which are believed to derive from a common progenitor, and suggest that it operates in parallel with Ngn3.

A dynamic expression survey identifies transcription factors relevant in mouse digestive tract development.

Tissue-restricted transcription factors (TFs), which confer specialized cellular properties, are usually identified through sequence homology or cis-element analysis of lineage-specific genes; conventional modes of mRNA profiling often fail to report non-abundant TF transcripts. We evaluated the dynamic expression during mouse gut organogenesis of 1381 transcripts, covering nearly every known and predicted TF, and documented the expression of approximately 1000 TF genes in gastrointestinal development. Despite distinctive structures and functions, the stomach and intestine exhibit limited differences in TF genes. Among differentially expressed transcripts, a few are virtually restricted to the digestive tract, including Nr2e3, previously regarded as a photoreceptor-specific product. TFs that are enriched in digestive organs commonly serve essential tissue-specific functions, hence justifying a search for other tissue-restricted TFs. Computational data mining and experimental investigation focused interest on a novel homeobox TF, Isx, which appears selectively in gut epithelium and mirrors expression of the intestinal TF Cdx2. Isx-deficient mice carry a specific defect in intestinal gene expression: dysregulation of the high density lipoprotein (HDL) receptor and cholesterol transporter scavenger receptor class B, type I (Scarb1). Thus, integration of developmental gene expression with biological assessment, as described here for TFs, represents a powerful tool to investigate control of tissue differentiation.