Spatial transcriptomic dissection of the vertebrate urinary development program using zebrafish
Principal Investigator: Dr. Duncan Morhardt, MD, PhD
Assistant Professor of Surgery
Project Summary
Congenital malformations of the external genital system (1:150 live born boys) can be devastating. The developmental program of the external urinary system has not been determined. In contrast to mammals, zebrafish offer a means of understanding the conserved, critical mechanisms of external urethral development because of transparency during development and experimentally isolatable genital and urinary systems. We propose to catalogue the expression landscape of urethral development in zebrafish using RNA-seq to inform probes in the new MERFISH multiplexed in situ platform available to the Genomics core. Briefly, In the first aim RNA-seq will be performed over 15-26 days post fertilized fish distal urinary structures and compared to developing GI tract. In the second aim, differential analysis will yield candidate transcripts for probes for the MERFISH platform which will be analyzed to determine relevant pathways within the developing urethra, identify unique cell types over development, and delineate the time course of their transcription events. The results of these studies will inform a larger genetic dissection of critical factors over urethral development using a CRISPR/Cas9 driven deletion system as well as a later comparative development study using MERFISH in human urethra. This study will leverage the substantial new resources in the COBRE. Fred Kolling, PhD, for the multiplexed in situ hybridization technique. Under the mentored direction of H. Robert Frost, PhD, this project will leverage the bioinformatics core for analysis of the MERSCOPE data and interpretation of the results. Together, this is a pivotal pilot study in our understanding of genitourinary development and our understanding of its congenital malformations.
Revealing the impact of tryptophan metabolism on host-microbe and microbe-microbe interactions in the gut
Principal Investigator: Benjamin D Ross, PhD
Assistant Professor of Microbiology and Immunology
Assistant Professor of Orthopaedics
Dr. Benjamin Ross is an Assistant Professor of Microbiology and Immunology and an Assistant Professor of Orthopedics at Geisel. His lab in the Department of Microbiology and Immunology is working to elucidate the mechanism of defense gene acquisition and the ecological impact of T6SS neutralization on gut microbiome assembly and stability. They are also studying the causes and consequences of alterations in the gut microbiome of infants with cystic fibrosis, initially focusing on the large-scale analysis of hundreds of shotgun metagenomic and metabolomic datasets derived from stool samples of healthy infants and infants with CF, and embarking on new studies of the role of tryptophan metabolism in sculpting microbe-microbe and microbe-host interactions in the intestine, using a combination of quantitative assays and gnotobiotic mice colonized with genetically-tractable synthetic microbiota.
Project Summary
The gut microbiota impact human health through the production of metabolites that elicit effects on host physiology. Only a few microbe-metabolite-host relationships are known at the molecular level, due in part to the complexity and intractability of the microbiota. One well-known example is microbial metabolism of tryptophan into diverse signaling molecules that promote immune homeostasis and gastrointestinal barrier function. What mechanisms govern tryptophan flux through different microbial-encoded pathways, how tryptophan metabolism impacts the microbiota, and how tryptophan metabolites act in concert to impact the host are not understood.
This project will establish a 14-member synthetic gut microbiota (SGM) in gnotobiotic mice to reveal mechanistic insight into the dynamics and impact of tryptophan metabolism. The SGM includes 4 species of the genetically-tractable genus Bacteroides, three of which encode the gene tryptophanase (TnaA), which converts tryptophan into indole, as well as several other species predicted to produce other indole-like molecules. We will engineer a variant SGM containing TnaA mutants (SGM-delta) that lack the ability to produce indole, and compare between SM and SM-delta to gain insight into the impact of altered tryptophan metabolites on microbiota composition and on the transcriptome and function of both host intestinal tissue via bulk and single-cell RNA-seq and permeability assays. We will finally perform metabolomics on SGM and SGM-delta to trace tryptophan flux through non-TnaA pathways. This project will help reveal how metabolism of tryptophan impacts microbe-microbe and host-microbe interactions.