Beyond Mammalian Bladders: A Zebrafish Model of Lower Urinary Tract Function and Development
Duncan Rogers Morhardt, MD, PhD1, Yang Zheng, PhD2, Britton Goodale, PhD2, Camilla Bobrowicz, .3, Nicole Gallien, B.A2, Steven Leach, MD4.
1Dartmouth Health, Lebanon, NH, USA, 2Dartmouth College, Hanover, NH, USA, 3McGill University, Montreal, QC, USA, 4Dartmouth Cancer Center, Dartmouth College, Lebanon, NH, USA.
BACKGROUND: Our knowledge of development has benefited from direct visualization of early ontogenic events. While great strides in understanding bladder and lower urinary tract development have been made using mammalian models, mammals, as in utero amniotes, develop internally and present issues with visualization. Zebrafish offer unique insights because they are transparent during development, possess a host of genetic tools, and allow for excellent assessment of molecular and morphologic changes that result from pharmacological or toxicant exposure. Based largely on larval studies, zebrafish have long been thought to possess only a common cloaca, without separately terminating urologic, genital, anal structures, and a distinct urinary bladder has been assumed absent in zebrafish. These assumptions limited the utility of zebrafish as a model for urinary tract development, but have not been rigorously confirmed. To this end, we sought to characterize the distal pelvic structures in zebrafish.
METHODS: We employed the Tg(Cdh17:mCherry) zebrafish line, which has a fluorescently labelled urinary system, to characterize morphology, function and molecular signatures of pelvic structures in developing juvenile and adult zebrafish. Distal urinary structures were imaged in juvenile zebrafish from 5 to 30 days post fertilization (dpf) and adults, and analyzed for morphology. Distal urinary structures from larval zebrafish at multiple developmental timepoints were dissected and processed for RNA-seq. In adult zebrafish, dextran-conjugated Alexa dye was injected into the pericardium to trace distal urinary structures and excretion, and bladder-like structures and intestine were collected for RNA-seq.
RESULTS: Distal urinary structure morphology (as determined by angle of declination and length of a strong cdh17 signal) remained stable in juveniles until 15-19 dpf, when distal Cdh17+ signal decreased. A second morphological milestone was observed at 19-23 dpf when the same region of the distal urinary tract flattens to an angle to that seen in maturity. Retrograde injection studies and cannulation revealed separate external urinary structures from anal and genital tracts with separate external urinary, genital and anal termination. Just proximal to the urinary outlet, a sacular structure filled and emptied urine with periodic concerted contractions. The emptying of this structure was quantified revealing both short (~30 seconds) and long (~4 minute) intervals. RNA-seq this same contracting region in adult zebrafish revealed high enrichment of orthologs to mammalian bladder-specific genes (including UPK3b), with high level expression in bladder tissue compare to intestine, as well the presence genes critical for urogenital development like orthologs to SHH.
CONCLUSIONS: Zebrafish have evidence of a bladder with compelling similarities to mammalian counterparts. The genitourinary and anorectal anatomy reveals previously unrecognized anatomic similarities to human. Thus zebrafish are a promising tool to investigate bladder physiology, genetics and development and may provide insights into conserved vertebrate mechanisms of micturition and development.
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