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Principal Investigator: Mariano A Loza Coll
Organization: CALIFORNIA STATE UNIVERSITY NORTHRIDGE
Fiscal Year: 2024
Award: $147,500
Funding agency: National Institute of General Medical Sciences
PROJECT SUMMARY
Several master regulator (MR) genes have been characterized in tissue stem cells across organs and
species. However, they remain poor candidates for therapeutic manipulation because they are highly
pleiotropic, affecting hundreds of targets and operating across many organ systems. Therefore, only a
better understanding of how less pleiotropic downstream MR targets coordinate stem cell proliferation,
self-renewal and differentiation will unlock the full potential of stem cells in regenerative medicine. The
long-term goal of this project is to map the regulatory landscape established by MR genes and their
targets in intestinal stem cells (ISCs), using the fruit fly Drosophila melanogaster as a model system.
Drosophila ISCs divide asymmetrically, giving rise to a new ISC and a sister that will become an
absorptive enterocyte (EC) or a hormone-secreting enteroendocrine cell (EE). The specific hypothesis
driving this proposal is that cross-regulatory interactions between MR target pathways can lead to non-
linear, unpredictable outcomes on ISC behavior when they are manipulated simultaneously. To test this
hypothesis, CAP, Klaroid and Indy, three experimentally validated targets of the ISC MR genes Escargot
and STAT, will be manipulated alone or in combination within ISCs using an inducible Gal4/UAS system.
The effect of their individual vs. combined manipulations on intestinal homeostasis will be assessed via
three separate but complementary approaches. In Aim 1, immunofluorescence microscopy will be used
to compare ISC number, morphology, mitotic rate, and differentiation potential, based on well-established
cell type markers (esg-GFP for ISCs, Su(H) activation for EBs, Pdm1 and Pros staining for ECs and EEs,
respectively). Automated image analysis through ImageJ and CellProfiler will be used to analyze multiple
images per group, allowing a robust statistical analysis of the data. In Aim 2, fluorescent activity reporters
will be used to compare the effect of single vs. dual MR target manipulations on key ISC signal
transduction pathways (EGFR, Notch, Wnt, STAT and JNK). In Aim 3, lifespan and intestinal barrier
integrity (Smurf) assays will be used to compare the effect that individual vs. combined MR target
manipulations have on the regenerative capacity of intestinal tissue following chemical injury or
pathogenic infection. These research aims may generate evidence that challenges the widely held
premise that combination therapies can only improve outcome due to additive complementation of
positive effects. If so, this project will have a significant impact on our conceptual approach to stem cell
manipulation for regenerative medicine. In addition, this project was specifically designed to engage a
large number of students from underrepresented backgrounds in biomedical research, satisfying another
important mission of the NIH: to diversify the scientific workforce, and thus foster innovation, improve
research quality and enhance the public trust and investment in science.
Terms: <Address><Affect><Assay><Automobile Driving><Basal Transcription Factor><Basal transcription factor genes><Behavior><Binding><Bioassay><Biologic Models><Biological Assay><Biological Models><Biomedical Research><Body System><Body Tissues><C-jun Amino-Terminal Kinase><C-jun Kinase-1><C-jun N-Terminal Kinase 1><Cell Communication and Signaling><Cell Count><Cell Differentiation><Cell Differentiation process><Cell Number><Cell Signaling><Cell Therapy><Chemical Injury><Combined Modality Therapy><Complement><Complement Proteins><Complex><Critical Paths><Critical Pathways><DNA><Deoxyribonucleic Acid><Development><Drosophila><Drosophila genus><Drosophila melanogaster><EGF Receptor><EGFR><ERBB Protein><Enterocytes><Enteroendocrine Cell><Epidermal Growth Factor Receptor><Epidermal Growth Factor Receptor Kinase><Epidermal Growth Factor Receptor Protein-Tyrosine Kinase><Epidermal Growth Factor-Urogastrone Receptors><Experimental Models><Family><Fostering><Future Generations><Gastrointestinal Diseases><Gene Targeting><Gene Transcription><General Transcription Factor Gene><General Transcription Factors><Genetic><Genetic Transcription><Goals><HER1><Hormone secretion><Image><Immunofluorescence Microscopy><Individual><Infection><Injury><Intervention><Intervention Strategies><Intestinal><Intestines><Intracellular Communication and Signaling><Investigators><Investments><JN Kinase><JNK><JNK Mitogen-Activated Protein Kinases><JNK1><JNK1 Kinase><JNK1 protein><JNK1A2><JNK21B1/2><Knowledge><MAP Kinase 8><MAP Kinase 8 Gene><MAPK8><MAPK8 Mitogen-Activated Protein Kinase><MAPK8 gene><Maps><Midgut><Minority><Mission><Mitogen-Activated Protein Kinase 8><Mitotic><Model System><Molecular><Molecular Interaction><Morphology><Multimodal Therapy><Multimodal Treatment><NIH><National Institutes of Health><Organ><Organ System><Outcome><PRKM8><Pathogenicity><Pathway interactions><Phenotype><Progenitor Cells><R-Series Research Projects><R01 Mechanism><R01 Program><RNA Expression><Regenerative Medicine><Regenerative capacity><Regulation><Regulator Genes><Regulatory Pathway><Reporter><Research><Research Grants><Research Personnel><Research Project Grants><Research Projects><Researchers><SAP Kinase-1><SAPK/JNK><SAPK1 Mitogen-Activated Protein Kinase><SAPK1/JNK><STAT protein><Science><Signal Pathway><Signal Transducer and Activator of Transcription><Signal Transduction><Signal Transduction Pathway><Signal Transduction Systems><Signaling><Sister><Snails><Staining method><Stains><Statistical Data Analyses><Statistical Data Analysis><Statistical Data Interpretation><Stress-Activated Protein Kinase JNK1><Stress-Activated Protein Kinase gamma><Students><System><TGF-alpha Receptor><Testing><Therapeutic><Tissues><Toxic effect><Toxicities><Transcription><Transcription Factor Proto-Oncogene><Transcription factor genes><Transcriptional Regulatory Elements><Transforming Growth Factor alpha Receptor><Underrepresented Students><United States National Institutes of Health><Universities><Urogastrone Receptor><adult progenitor><adult stem cell><automated image analysis><biological signal transduction><bowel><c-erbB-1><c-erbB-1 Protein><c-jun N-Terminal Kinase><cell behavior><cell mediated therapies><cell type><cell-based therapeutic><cell-based therapy><cellular behavior><cellular differentiation><cellular therapeutic><cellular therapy><chemical trauma><classroom environment><college atmosphere><collegial atmosphere><collegiate atmosphere><combination therapy><combinatorial><combined modality treatment><combined treatment><complementation><design><designing><develop therapy><developmental><driving><education atmosphere><educational atmosphere><educational environment><erbB-1><erbB-1 Proto-Oncogene Protein><erbBl><fruit fly><gastrointestinal disorder><gastrointestinal homeostasis><gene conservation><genome scale><genome-wide><genomewide><hormonal secretion><imaging><improved><improved outcome><in vivo><injuries><innovate><innovation><innovative><intellectual atmosphere><intervention development><interventional strategy><intestinal barrier><intestinal homeostasis><intestinal mucosal barrier><jun-NH2-Terminal Kinase><learning atmosphere><learning environment><life span><lifespan><model organism><multi-modal therapy><multi-modal treatment><new approaches><notch><notch protein><notch receptors><novel approaches><novel strategies><novel strategy><pathway><premature><prematurity><prevent><preventing><progenitor cell based therapy><progenitor cell function><progenitor cell proliferation><progenitor cell therapy><progenitor cell treatment><progenitor function><progenitor proliferation><progenitor therapy><progenitor treatment><proto-oncogene protein c-erbB-1><public trust><regeneration ability><regeneration capacity><regeneration function><regeneration potential><regenerative function><regenerative functionality><regenerative potential><regulatory gene><school atmosphere><school climate><self-renew><self-renewal><side effect><somatic progenitor><somatic stem cell><statistical analysis><stem and progenitor cell function><stem and progenitor cell proliferation><stem and progenitor cell therapy><stem and progenitor function><stem cell based therapy><stem cell function><stem cell mediated therapy><stem cell proliferation><stem cell therapeutics><stem cell therapy><stem cell treatment><stem cell-based therapeutic><stem cell-based treatment><stem cells><stress-activated protein kinase 1><therapeutic candidate><therapeutic target><therapy development><tissue progenitor><tissue stem cells><training atmosphere><training opportunity><trans acting element><transcription factor><treatment development><undergrad><undergraduate><undergraduate student><university atmosphere>