Lori Sussel

Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling.

During vertebrate development, the specification of distinct cell types is thought to be controlled by inductive signals acting at different concentration thresholds. The degree of receptor activation in response to these signals is a known determinant of cell fate, but the later steps at which graded signals are converted into all-or-none distinctions in cell identity remain poorly resolved. In the ventral neural tube, motor neuron and interneuron generation depends on the graded activity of the signalling protein Sonic hedgehog (Shh). These neuronal subtypes derive from distinct progenitor cell populations that express the homeodomain proteins Nkx2.2 or Pax6 in response to graded Shh signalling,. In mice lacking Pax6, progenitor cells generate neurons characteristic of exposure to greater Shh activity,. However, Nkx2.2 expression expands dosally in Pax6 mutants, raising the possibility that Pax6 controls neuronal pattern indirectly. Here we provide evidence that Nkx2.2 has a primary role in ventral neuronal patterning. In Nkx2.2 mutants, Pax6 expression is unchanged but cells undergo a ventral-to-dorsal transformation in fate and generate motor neurons rather than interneurons. Thus, Nkx2.2 has an essential role in interpreting graded Shh signals and selecting neuronal identity.

Pancreatic β Cell Dedifferentiation as a Mechanism of Diabetic β Cell Failure

Diabetes is associated with β cell failure. But it remains unclear whether the latter results from reduced β cell number or function. FoxO1 integrates β cell proliferation with adaptive β cell function. We interrogated the contribution of these two processes to β cell dysfunction, using mice lacking FoxO1 in β cells. FoxO1 ablation caused hyperglycemia with reduced β cell mass following physiologic stress, such as multiparity and aging. Surprisingly, lineage-tracing experiments demonstrated that loss of β cell mass was due to β cell dedifferentiation, not death. Dedifferentiated β cells reverted to progenitor-like cells expressing Neurogenin3, Oct4, Nanog, and L-Myc. A subset of FoxO1-deficient β cells adopted the α cell fate, resulting in hyperglucagonemia. Strikingly, we identify the same sequence of events as a feature of different models of murine diabetes. We propose that dedifferentiation trumps endocrine cell death in the natural history of β cell failure and suggest that treatment of β cell dysfunction should restore differentiation, rather than promoting β cell replication.

A Wnt1-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo

Midbrain neurons synthesizing the neurotransmitter dopamine play a central role in the modulation of different brain functions and are associated with major neurological and psychiatric disorders. Despite the importance of these cells, the molecular mechanisms controlling their development are still poorly understood. The secreted glycoprotein Wnt1 is expressed in close vicinity to developing midbrain dopaminergic neurons. Here, we show that Wnt1 regulates the genetic network, including Otx2 and Nkx2-2, that is required for the establishment of the midbrain dopaminergic progenitor domain during embryonic development. In addition, Wnt1 is required for the terminal differentiation of midbrain dopaminergic neurons at later stages of embryogenesis. These results identify Wnt1 as a key molecule in the development of midbrain dopaminergic neurons in vivo. They also suggest the Wnt1-controlled signaling pathway as a promising target for new therapeutic strategies in the treatment of Parkinsons disease.

Human β Cell Transcriptome Analysis Uncovers lncRNAs That Are Tissue-Specific, Dynamically Regulated, and Abnormally Expressed in Type 2 Diabetes

A significant portion of the genome is transcribed as long noncoding RNAs (lncRNAs), several of which are known to control gene expression. The repertoire and regulation of lncRNAs in disease-relevant tissues, however, has not been systematically explored. We report a comprehensive strand-specific transcriptome map of human pancreatic islets and β cells, and uncover >1100 intergenic and antisense islet-cell lncRNA genes. We find islet lncRNAs that are dynamically regulated and show that they are an integral component of the β cell differentiation and maturation program. We sequenced the mouse islet transcriptome and identify lncRNA orthologs that are regulated like their human counterparts. Depletion of HI-LNC25, a β cell-specific lncRNA, downregulated GLIS3 mRNA, thus exemplifying a gene regulatory function of islet lncRNAs. Finally, selected islet lncRNAs were dysregulated in type 2 diabetes or mapped to genetic loci underlying diabetes susceptibility. These findings reveal a new class of islet-cell genes relevant to β cell programming and diabetes pathophysiology.

β-Cell Failure in Type 2 Diabetes: Postulated Mechanisms and Prospects for Prevention and Treatment

OBJECTIVE This article examines the foundation of β-cell failure in type 2 diabetes (T2D) and suggests areas for future research on the underlying mechanisms that may lead to improved prevention and treatment. RESEARCH DESIGN AND METHODS A group of experts participated in a conference on 14–16 October 2013 cosponsored by the Endocrine Society and the American Diabetes Association. A writing group prepared this summary and recommendations. RESULTS The writing group based this article on conference presentations, discussion, and debate. Topics covered include genetic predisposition, foundations of β-cell failure, natural history of β-cell failure, and impact of therapeutic interventions. CONCLUSIONS β-Cell failure is central to the development and progression of T2D. It antedates and predicts diabetes onset and progression, is in part genetically determined, and often can be identified with accuracy even though current tests are cumbersome and not well standardized. Multiple pathways underlie decreased β-cell function and mass, some of which may be shared and may also be a consequence of processes that initially caused dysfunction. Goals for future research include to 1) impact the natural history of β-cell failure; 2) identify and characterize genetic loci for T2D; 3) target β-cell signaling, metabolic, and genetic pathways to improve function/mass; 4) develop alternative sources of β-cells for cell-based therapy; 5) focus on metabolic environment to provide indirect benefit to β-cells; 6) improve understanding of the physiology of responses to bypass surgery; and 7) identify circulating factors and neuronal circuits underlying the axis of communication between the brain and β-cells.

Control of anteroposterior and dorsoventral domains of Nkx-6.1 gene expression relative to other Nkx genes during vertebrate CNS development

Here we report the isolation, sequence and developmental expression in the central nervous system of several members of the chicken and mouse Nkx gene family. These are among the earliest genes to be regionally expressed in the neural plate; they are expressed just above the axial mesendoderm (prechordal mesendoderm and notochord). Each Nkx gene has a distinct spatial pattern of expression along the anterior-posterior axis of the ventral central nervous system: Nkx-2. 2 is expressed along the entire axis, whereas Nkx-2.1 is restricted to the forebrain, and Nkx-6.1 and Nkx-6.2 are largely excluded from the forebrain. They are also expressed in distinct patterns along the dorsal-ventral axis. These genes are expressed in both the ventricular and mantle zones; in the mantle zone Nkx-6.1 is co-expressed with Islet-1 in a subset of motor neurons. Like other Nkx genes, expression of Nkx-6.1 is induced by the axial mesendoderm and by sonic hedgehog protein. BMP-7 represses Nkx-6.1 expression. While the notochord can induce Nkx-6.1 expression in the anterior neural plate, sonic hedgehog protein does not, suggesting that the notochord produces additional molecules that can regulate ventral patterning.

Pancreatic β Cells Require NeuroD to Achieve and Maintain Functional Maturity

NeuroD, a transactivator of the insulin gene, is critical for development of the endocrine pancreas, and NeuroD mutations cause MODY6 in humans. To investigate the role of NeuroD in differentiated beta cells, we generated mice in which neuroD is deleted in insulin-expressing cells. These mice exhibit severe glucose intolerance. Islets lacking NeuroD respond poorly to glucose and display a glucose metabolic profile similar to immature beta cells, featuring increased expression of glycolytic genes and LDHA, elevated basal insulin secretion and O2 consumption, and overexpression of NPY. Moreover, the mutant islets appear to have defective K(ATP) channel-mediated insulin secretion. Unexpectedly, virtually all insulin in the mutant mice is derived from ins2, whereas ins1 expression is almost extinguished. Overall, these results indicate that NeuroD is required for beta cell maturation and demonstrate the importance of NeuroD in the acquisition and maintenance of fully functional glucose-responsive beta cells.

Nkx2.2 repressor complex regulates islet β-cell specification and prevents β-to-α-cell reprogramming

Regulation of cell differentiation programs requires complex interactions between transcriptional and epigenetic networks. Elucidating the principal molecular events responsible for the establishment and maintenance of cell fate identities will provide important insights into how cell lineages are specified and maintained and will improve our ability to recapitulate cell differentiation events in vitro. In this study, we demonstrate that Nkx2.2 is part of a large repression complex in pancreatic β cells that includes DNMT3a, Grg3, and HDAC1. Mutation of the endogenous Nkx2.2 tinman (TN) domain in mice abolishes the interaction between Nkx2.2 and Grg3 and disrupts β-cell specification. Furthermore, we demonstrate that Nkx2.2 preferentially recruits Grg3 and HDAC1 to the methylated Aristaless homeobox gene (Arx) promoter in β cells. The Nkx2.2 TN mutation results in ectopic expression of Arx in β cells, causing β-to-α-cell transdifferentiation. A corresponding β-cell-specific deletion of DNMT3a is also sufficient to cause Arx-dependent β-to-α-cell reprogramming. Notably, subsequent removal of Arx in the β cells of Nkx2.2(TNmut/TNmut) mutant mice reverts the β-to-α-cell conversion, indicating that the repressor activities of Nkx2.2 on the methylated Arx promoter in β cells are the primary regulatory events required for maintaining β-cell identity.

FoxA2, Nkx2.2, and PDX-1 Regulate Islet β-Cell-Specific mafA Expression through Conserved Sequences Located between Base Pairs −8118 and −7750 Upstream from the Transcription Start Site

ABSTRACT The MafA transcription factor is both critical to islet β-cell function and has a unique pancreatic cell-type-specific expression pattern. To localize the potential transcriptional regulatory region(s) involved in directing expression to the β cell, areas of identity within the 5′ flanking region of the mouse, human, and rat mafA genes were found between nucleotides −9389 and −9194, −8426 and −8293, −8118 and −7750, −6622 and −6441, −6217 and −6031, and −250 and +56 relative to the transcription start site. The identity between species was greater than 75%, with the highest found between bp −8118 and −7750 (∼94%, termed region 3). Region 3 was the only upstream mammalian conserved region found in chicken mafA (88% identity). In addition, region 3 uniquely displayed β-cell-specific activity in cell-line-based reporter assays. Important regulators of β-cell formation and function, PDX-1, FoxA2, and Nkx2.2, were shown to specifically bind to region 3 in vivo using the chromatin immunoprecipitation assay. Mutational and functional analyses demonstrated that FoxA2 (bp −7943 to −7910), Nkx2.2 (bp −7771 to −7746), and PDX-1 (bp −8087 to −8063) mediated region 3 activation. Consistent with a role in transcription, small interfering RNA-mediated knockdown of PDX-1 led to decreased mafA mRNA production in INS-1-derived β-cell lines (832/13 and 832/3), while MafA expression was undetected in the pancreatic epithelium of Nkx2.2 null animals. These results suggest that β-cell-type-specific mafA transcription is principally controlled by region 3-acting transcription factors that are essential in the formation of functional β cells.

Epigenetic switching of transcriptional states: cis- and trans-acting factors affecting establishment of silencing at the HMR locus in Saccharomyces cerevisiae.

In this study, we used the ADE2 gene in a colony color assay to monitor transcription from the normally silent HMR mating-type locus in Saccharomyces cerevisiae. This sensitive assay reveals that some previously identified cis- and trans-acting mutations destabilize silencing, causing genetically identical cells to switch between repressed and derepressed transcriptional states. Deletion of the autonomously replicating sequence (ARS) consensus element at the HMR-E silencer or mutation of the silencer binding protein RAP1 (rap1s) results in the presence of large sectors within individual colonies of both repressed (Ade-, pink) and derepressed (Ade+, white) cells. These results suggest that both the ARS consensus element and the RAP1 protein play a role in the establishment of repression at HMR. In diploid cells, the two copies of HMR appear to behave identically, suggesting that the switching event, though apparently stochastic, reflects some property of the cell rather than a specific event at each HMR locus. In the ADE2 assay system, silencing depends completely upon the function of the SIR genes, known trans-acting regulators of the silent loci, and is sensitive to the gene dosage of two SIR genes, SIR1 and SIR4. Using the ADE2 colony color assay in a genetic screen for suppressors of rap1s, silencer ARS element deletion double mutants, we have identified a large number of genes that may affect the establishment of repression at the HMR silent mating-type locus.