Abstract:

After using morpholino to knockdown TNNT2 or VEGFA gene in the early life stages of zebrafish, ammonia and urea excretion rates, mRNA expression of ammonia (Rhag and Rhbg) and urea transporter (UT), oxygen consumption rates were measured in the mutant and control zebrafish at 1-4 days post-fertilization. The phenotypes of the mutants and controls were also recorded.

Suggested Citation:

Wang, Y., Grosell, M.. 2020. The effects of reduced cardiac output on ammonia excretion during the early life stages of Zebrafish (Danio Rerio). Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/31N5DBRH

Publications:

Wang, Y., Pasparakis, C., & Grosell, M. (2021). Role of the cardiovascular system in ammonia excretion in early life stages of zebrafish (Danio rerio). American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. doi:10.1152/ajpregu.00284.2020

Purpose:

To see if reduced cardiac output might limit the delivery of ammonia from the site of production to the site of excretion.

Data Parameters and Units:

The folder "NW Excretion Compiled Data" contains 1 Excel file "Raw and Corrected Nitrogenous Waste Excretion", and includes ammonia and urea excretion rates of TNNT2 and controls (pmol N indiv-1 min -1) at different time points (1, 2, 4 dpf) and ammonia and urea excretion rates of VEGFA and controls at 4 dpf. The folder "qPCR" contains 1 Excel file "qPCR.xlsx", and includes mRNA expression of different transporters (Rhag, Rhbg and UT) in TNNT2 and controls were quantified in zebrafish embryos and larvae in different time points (1, 2, 3, 4 dpf), mRNA expression of different transporters (Rhag, Rhbg and UT) in VEGFA and controls were quantified in zebrafish larvae at 4 dpf. The curve data was calculated by the software and included under each gene file. The folder "Phenotypes" contains the TIFF image files and videos taken to record the phenotypes of zebrafish fish larvae after morpholino knockdown of TNNT2 and VEGFA at 4 dpf. The folder "Oxygen Consumption Data" contains the oxygen consumption data for TNNT2 and VEGFA. Data is presented as umol L-1 by elapsed time (seconds).  The Excel files "qPCR", "oxygen consumption data_tnnt2", and "oxygen consumption data_VEGFA" also includes a separate ReadMe worksheet with details on the experiment, color formatting, and data included in the other worksheets.

Methods:

Morpholino was used to knockdown TNNT2 and VEGFA gene. Ammonia and urea excretion rates: Micromodified Indophenol Blue and Diacetyl Monoxime methods. mRNA expression raw data analysis was performed with the qPCR Miner software (Zhao and Fernald, 2005) and normalized to EF1a mRNA expression. Loligo Microplate was used to measure oxygen consumption in developing mahi-mahi.

Instruments:

Oxygen Consumption- Loligo Microplate (24 well optical-fluorescence, oxygen-sensing system). Microinjection was performed using FemtoJet 4i microinjector (Eppendorf, Germany) Bright-field photography was performed on a Leica dissection microscope (MZFLIII; Leica, Germany) using a digital camera (Q-Color 3; Olympus, Japan). For fluorescent photography and videos, an inverted microscope (IX70; Olympus, Japan) was used with the same digital camera.

Provenance and Historical References:

Zhao, S., & Fernald, R. D. (2005). Comprehensive Algorithm for Quantitative Real-Time Polymerase Chain Reaction. Journal of Computational Biology, 12(8), 1047–1064. doi:10.1089/cmb.2005.12.1047

Individual Files: