2010 — 2014 |
Reiner, David John |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Ral Small Gtpase in C. Elegans Development @ Texas a&M University Health Science Ctr
DESCRIPTION (provided by applicant: Ras is the most frequently mutated oncogene in human cancers and the Raf-MEK-ERK protein kinase cascade is the best characterized downstream effector function of Ras. However, it is now clear that Ras function cannot be ascribed simply to one effector pathway, and that Ras utilizes a spectrum of functionally diverse effectors, with at least five implicated in oncogenesis. What remains unclear is how effector utilization is regulated. Recent studies have implicated a critical role for guanine nucleotide exchange factors for the Ral small GTPases (RalGEFs) as effectors of Ras in cancer. Yet we know little about the interplay between RalGEF-Ral and other effector pathways, and we know even less about the normal functions of RalGEF-Ral activity during development. Genetic analyses of the Ras homolog in C. elegans, LET-60, have elucidated the role of the Raf-MEK-ERK cascade in development and provided critical clues for understanding mechanisms of Ras signaling in mammalian cells. Since essentially all known components of RalGEF-Ral signaling are conserved in C. elegans, this prompted our interest in elucidating a role for the evolutionarily conserved C. elegans RGL-1 (RalGEF) and RAL-1 (Ral) in LET-60-regulated development. Our preliminary results indicate that, unexpectedly, RGL-1-RAL-1 signaling antagonized rather than cooperated with the Raf-MEK-ERK signal in inducing 10 cell fate in the vulva, and does so in parallel or downstream to the ERK substrate, the LIN- 31/HNF transcription factor. Additionally, our results suggest that RAL-1 is expressed only in 20 cells neighboring 10 vulval cells, suggesting that LET-60/Ras switches from the use of one effector (LIN-45/Raf) in 10 cells to another (RGL-1/RalGEF) in 20 cells, and that RAL-1 controls the balance of Ras-dependent cell fates. These observations provide the rationale for our analysis of the role of RGL-1-RAL-1 signaling in cell fate induction, a model for RalGEF-Ral oncogenic functions, and to our knowledge the first developmental model to study the potentially critical regulatory mechanism of differential effector usage by Ras. We propose studies that will apply a multi-faceted approach that utilizes genetic, biochemical and cell biological methods to elucidate the mechanisms and roles by which LET-60 dynamically regulates these two distinct effector- signaling networks. We propose aims to (1) determine the signaling properties of the RGL-1-RAL-1 module relative to the Ras-Raf-MEK-ERK pathway, (2) determine the developmental mechanisms by which RGL-1- RAL-1 controls equivalence group fate decisions, and (3) determine the genetic and molecular mechanism of RGL-1-RAL-1 function in 20 cells. Our studies will address outstanding questions of developmental patterning in response to a morphogen gradient, balance between Ras and Notch signaling, and differential use of effectors in development and cancer, all of which lie at a critical intersection between developmental biology and cancer.
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0.921 |
2015 — 2016 |
Reiner, David |
F31Activity Code Description: To provide predoctoral individuals with supervised research training in specified health and health-related areas leading toward the research degree (e.g., Ph.D.). |
Amylin Receptors in the Lateral Dorsal Tegmental Area Regulate Food Intake @ University of Pennsylvania
? DESCRIPTION (provided by applicant): Amylin is a pancreatic-derived hormone that acts in the CNS to reduce food intake. While historically the hypophagic effects of amylin have been attributed to processing by the area postrema, recently the ventral tegmental area (VTA), a nucleus in the mesolimbic reward system, was highlighted as a pharmacologically and physiologically relevant site of action for amylin-mediated control of energy balance and food reward. These discoveries encourage a broader assessment of the CNS circuitry mediating amylin's effects on food intake and reward. Given that the lateral dorsal tegmental area (LDTg) of the brainstem: [1] receives input from feeding-relevant nuclei of the hindbrain and forebrain, [2] binds amylin, and [3] sends input to the VTA to modulate rewarding behavior, the proposed aims will test the hypothesis that amylin receptor signaling in the LDTg is integral for the regulation of energy balance and food reward. Specifically, we will test the physiological requirement of LDTg amylin receptor signaling in regulation of energy balance, the role of LDTg amylin receptor signaling in rewarding value of food, and the potential interaction with gut-to-brain glucagon- like peptide-1 (GLP-1) within-meal satiation signaling as a putative mechanism contributing to intake suppression. Together the proposed aims will highlight the amylin receptor system, as well as LDTg neural processing as critical to the regulation of food intake and food motivated behaviors.
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0.915 |
2017 — 2018 |
Mangone, Marco Reiner, David John |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Global Changes in the 3'Utrome Toggle Responsiveness to Growth Factors @ Texas a&M University Health Science Ctr
SUMMARY RNA binding proteins like the Pumilio family (PUFs) exert repression on 3'UTRs, and hence mRNA stability and translation. Alternative polyadenylation (APA) causes 3'UTR length to vary. Coordinated large-scale shortening of 3'UTRs through APA can lead to evasion from 3'UTR- mediated repressive signals, and may be a key regulatory regime during development. Yet most identified cases of 3'UTR-mediated PUF repression - and APA to evade repression - were found ad hoc, between interacting gene pairs. Thus a gap exists in our understanding of how these interactions are orchestrated at the global level of the 3'UTRome and APA. EGF patterns the C. elegans vulva. Of six equipotent vulval precursor cells (VPCs), the three closest to the EGF source are induced to form the vulva, while the distal three remain uninduced. Remarkably, this event occurs with 99.8% fidelity. Three redundant PUFs are expressed specifically in the uninduced cells, suggesting that distal VPCs enact a PUF- and 3'UTR-dependent program to become non-responsive to signal. In the germline, the same PUFs repress ERK/MAP kinase; this same mechanism may be adopted by non-responsive VPCs. Germline immunoprecipitation (IP) of one PUF identified many potential target 3'UTRs. From this dataset, we identified multiple target mRNAs from genes in each vulval signaling cascade (EGFR?Ras?Raf?MEK?ERK, Notch?CSL, PI3K?PDK?Akt, and EGFR?Ras? RalGEF?Ral). We hypothesize that the redundant PUFs collectively repress mRNAs of all four identified signaling cascades to demarcate signal-non-responsive from signal-responsive cells. Our central hypothesis is that switching from proximal (short 3'UTR) to distal (long 3'UTR) polyadenylation sequence (PAS) usage governs switching from signal-responsiveness to non- responsiveness. We will systematically test this hypothesis by joining bottom-up and top-down specific aims. We propose to: 1) test 3'UTRs from the PUF IP list of vulval signaling genes for ability to mediate PUF-dependent reporter repression in non-responsive cells, 2) survey the VPC 3'UTRome and global proximal-to-distal PAS switching, and 3) validate select candidates by altering endogenous 3'UTRs and polyadenylation signals via CRISPR/Cas9 genome editing, and deleting associated PUFs. We will ascertain the contribution to developmental fidelity by APA, and the changes in repressive access points in the 3'UTRs caused by APA.
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0.921 |
2017 — 2020 |
Reiner, David John |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Novel Signals Regulate Cell Fate Patterning @ Texas a&M University Health Science Ctr
Cell fate patterning of the C. elegans vulva is an ideal system for the study of signal transduction mechanisms. EGF induces six equipotent vulval precursor cells (VPCs) to assume cell fates in a 3?-3?-2?-1?-2?-3? pattern with 99.8% accuracy. In the ?Morphogen Gradient? model for vulval patterning, distance from the EGF source dictates the fate of each VPC. In the ?Sequential Induction? model, the Ras?Raf?MEK?ERK MAP kinase cascade induces 1? fate, elicits DSL ligand production, and thus, via the LIN-12/Notch receptor, induces its two neighboring VPCs to become 2?. Because of the absence of key molecular details, these two models were debated for 16 years. We resolved this debate: to interpret the EGF gradient, presumptive 2? cells use Ras?RalGEF?Ral rather than the canonical Ras?Raf used in 1?s. Mutating RalGEF or Ral (a cousin of Ras) does not confer strong patterning defects, suggesting that sequential induction is the dominant patterning mechanism. These are clinically relevant molecules: Ras is the most mutated oncoprotein. Ras?RalGEF?Ral is thought to be equally important for oncogenesis as the canonical Ras?Raf. Our central hypothesis is that the Ras?RalGEF?Ral story justifies pursuing unexplored areas of VPC patterning because clinically important signals are found as positive and negative regulators. The objectives of this proposal are to unveil new facets of the molecular basis for the 1?/2? fate choice and its high reproducibility in vivo, exploiting the strengths of this system for dissection of signaling mechanisms. Our preliminary results support the feasibility of three aims, each with a hypothesis focused on distinct molecular mechanisms. Aim 1: Our CRISPR-generated activating mutation in endogenous Rap1 (Ras proximal) induced ectopic 1? cells. Lack of Rap1 reduces 1? cell induction. We will test the hypothesis that Rap1 is the nexus of two opposed regulatory inputs that promote and repress ERK activation in presumptive 1? vs 2? cells, respectively. Aim 2: We identified a novel Ral effector, GCK-2/MAP4 kinase, that possibly signals via p38 MAP kinase to promote 2? fate. We will test the hypothesis that Ral?GCK-2 triggers a p38 cascade. We will also test whether Ral?GCK-2 signal regulates CCCH RNA binding proteins to stabilize 3?UTRs of 2?-promoting genes. Aim 3: MIG-15/MAP4K, the sole paralog of GCK-2/MAP4K, paradoxically inhibits 2? fate. We will test the hypothesis that MIG-15 triggers a JNK MAP kinase cascade, which may be the missing signal that represses the Notch receptor in presumptive 1° cells. MIG-15 defines a novel class of vulval-specific Notch repressors. We will make use of the results of a screen we have completed for MIG-15-like targets to identify new players. For all three aims we will use CRISPR-engineered endogenous fluorescent reporters to deconvolute specific signals embedded within the vulval signaling network. Successful completion of these aims will define components and organizational principles of the vulval signaling network that leads to the exceptional fidelity of patterning, which are likely to apply both broadly and specifically to other developmental systems.
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0.921 |
2018 |
Reiner, David John |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Novel Signals Regulate Cell Fate Patterning - Equipment Supplement @ Texas a&M University Health Science Ctr
Cell fate patterning of the C. elegans vulva is an ideal system for the study of signal transduction mechanisms. EGF induces six equipotent vulval precursor cells (VPCs) to assume cell fates in a 3?-3?-2?-1?-2?-3? pattern with 99.8% accuracy. In the ?Morphogen Gradient? model for vulval patterning, distance from the EGF source dictates the fate of each VPC. In the ?Sequential Induction? model, the Ras?Raf?MEK?ERK MAP kinase cascade induces 1? fate, elicits DSL ligand production, and thus, via the LIN-12/Notch receptor, induces its two neighboring VPCs to become 2?. Because of the absence of key molecular details, these two models were debated for 16 years. We resolved this debate: to interpret the EGF gradient, presumptive 2? cells use Ras?RalGEF?Ral rather than the canonical Ras?Raf used in 1?s. Mutating RalGEF or Ral (a cousin of Ras) does not confer strong patterning defects, suggesting that sequential induction is the dominant patterning mechanism. These are clinically relevant molecules: Ras is the most mutated oncoprotein. Ras?RalGEF?Ral is thought to be equally important for oncogenesis as the canonical Ras?Raf. Our central hypothesis is that the Ras?RalGEF?Ral story justifies pursuing unexplored areas of VPC patterning because clinically important signals are found as positive and negative regulators. The objectives of this proposal are to unveil new facets of the molecular basis for the 1?/2? fate choice and its high reproducibility in vivo, exploiting the strengths of this system for dissection of signaling mechanisms. Our preliminary results support the feasibility of three aims, each with a hypothesis focused on distinct molecular mechanisms. Aim 1: Our CRISPR-generated activating mutation in endogenous Rap1 (Ras proximal) induced ectopic 1? cells. Lack of Rap1 reduces 1? cell induction. We will test the hypothesis that Rap1 is the nexus of two opposed regulatory inputs that promote and repress ERK activation in presumptive 1? vs 2? cells, respectively. Aim 2: We identified a novel Ral effector, GCK-2/MAP4 kinase, that possibly signals via p38 MAP kinase to promote 2? fate. We will test the hypothesis that Ral?GCK-2 triggers a p38 cascade. We will also test whether Ral?GCK-2 signal regulates CCCH RNA binding proteins to stabilize 3?UTRs of 2?-promoting genes. Aim 3: MIG-15/MAP4K, the sole paralog of GCK-2/MAP4K, paradoxically inhibits 2? fate. We will test the hypothesis that MIG-15 triggers a JNK MAP kinase cascade, which may be the missing signal that represses the Notch receptor in presumptive 1° cells. MIG-15 defines a novel class of vulval-specific Notch repressors. We will make use of the results of a screen we have completed for MIG-15-like targets to identify new players. For all three aims we will use CRISPR-engineered endogenous fluorescent reporters to deconvolute specific signals embedded within the vulval signaling network. Successful completion of these aims will define components and organizational principles of the vulval signaling network that leads to the exceptional fidelity of patterning, which are likely to apply both broadly and specifically to other developmental systems.
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0.921 |