Generation of transgenic zebrafish to study podocyte biology
Date
26/11/2022Author
Brown, Cara Michelle
Metadata
Abstract
The Global Burden of Disease Study has shown that kidney disease is one of the
fastest rising causes of mortality worldwide and claims 1.1 million lives per year. A
range of factors can cause kidney damage. The kidneys filter approximately 180 litres
of blood per day, and a decline in kidney function is quickly followed by a severe
decline in health and eventually death. A late-stage indicator of kidney disease is
proteinuria, and a common feature connecting many kidney diseases is damage to
podocyte cells. Podocyte cells are a vital cell type involved in blood filtration and the
maintenance of kidney function. Podocytes are highly specialised cells that envelope
the glomerular capillaries and form part of a selective barrier, ensuring the retention
of larger proteins within the lumen of the capillaries they encapsulate. Increasing
the understanding of podocyte cell physiology will allow us to gain novel insight,
improve our knowledge of cellular interactions within podocytes, and hopefully lead
to novel therapeutic techniques. Elucidating the physiology of the kidney will enable
the development of improved therapies and treatments for managing kidney diseases.
In this study, novel transgenic zebrafish podocyte reporter lines have been
created as informative tools in analysing podocyte cell function and changes within
podocytes. The rapid external development of larvae and the optical clarity of zebrafish made it an ideal model for this project, where observation and analysis of
podocyte cells in vivo and the ability to image the transparent larvae was an essential
aspect of this work. In this study, using a range of advanced imaging techniques and
the analysis of the reporter lines, it has been possible to capture changes to podocyte
cells.
The overarching aim of this work was to gain greater insight into podocyte
physiology. Using gateway cloning, informative, podocyte-specific, transgenic zebrafish lines were generated and used as tools to study podocyte cells. These lines
used podocyte-specific fluorescent proteins to identify and locate podocytes within
larvae and adults, allowing these cells to be targeted, isolated, and monitored in experimental use. Using SPIM (Single/ Selective Plane Illumination Microscopy), it was
possible to induce injury within podocytes of larval fish and visualise changes in calcium ion dynamics in real-time. Through the microinjection of the known nephrotoxic
agent Puromycin aminonucleoside, it was possible to induce oedema in larval fish and
assess the morphological changes to podocyte cells through analysis via transmission
electron microscopy. Using the novel GCaMP6s LifeActTagRFP-T zebrafish line the
structure of the actin cytoskeleton was assessed in closer detail. Bioinformatics approaches were used to develop a pathway diagram elucidating the interactions within
podocyte cells through manual curation of literature, the use of current datasets,
and incorporating novel scRNA seq data. This detailed diagram presents an interactive network that allows the visualisation of podocyte cell interactions in a way that
is more accessible and permits visualisation of the data in ways that would not be
possible with graphs and images alone. It allows for the identification of discrepancies in metabolic pathways and intracellular communications to be displayed; it also
contributes to what is currently known about podocyte physiology.
Finally, using GCaMP6s and LifeActTagRFP-T tagged podocyte cells disassociated from adult fish, it was possible to generate a single-cell RNA sequencing (scRNA-seq) library, detailing active transcripts present within healthy zebrafish
podocytes and the identification of distinct cell populations and podocyte cell markers,
adding to and allowing comparisons with libraries generated from zebrafish podocytes
in alternative states, such as having undergone injury, or in alternative species. The
combination of these techniques and outcomes of this work has allowed the presentation of a project which has contributed innovative transgenic zebrafish reporter lines,
the novel display of an increase of calcium ion concentration in injured podocyte cells,
the production of a detailed, novel scRNA seq library and a pathway diagram utilising
bioinformatics techniques, that is interactive and informative. These contributions
can add to the generation of new therapies for treating cardiovascular diseases and
have provided additional transgenic zebrafish lines to be used as a model to study
kidney disease development, injury, and repair. The Global Burden of Disease Study has shown that kidney disease is one of the
fastest rising causes of mortality worldwide and claims 1.1 million lives per year. A
range of factors can cause kidney damage. The kidneys filter approximately 180 litres
of blood per day, and a decline in kidney function is quickly followed by a severe
decline in health and eventually death. A late-stage indicator of kidney disease is
proteinuria, and a common feature connecting many kidney diseases is damage to
podocyte cells. Podocyte cells are a vital cell type involved in blood filtration and the
maintenance of kidney function. Podocytes are highly specialised cells that envelope
the glomerular capillaries and form part of a selective barrier, ensuring the retention
of larger proteins within the lumen of the capillaries they encapsulate. Increasing
the understanding of podocyte cell physiology will allow us to gain novel insight,
improve our knowledge of cellular interactions within podocytes, and hopefully lead
to novel therapeutic techniques. Elucidating the physiology of the kidney will enable
the development of improved therapies and treatments for managing kidney diseases.
In this study, novel transgenic zebrafish podocyte reporter lines have been
created as informative tools in analysing podocyte cell function and changes within
podocytes. The rapid external development of larvae and the optical clarity of zebrafish
made it an ideal model for this project, where observation and analysis of
podocyte cells in vivo and the ability to image the transparent larvae was an essential
aspect of this work. In this study, using a range of advanced imaging techniques and
the analysis of the reporter lines, it has been possible to capture changes to podocyte
cells.
The overarching aim of this work was to gain greater insight into podocyte
physiology. Using gateway cloning, informative, podocyte-specific, transgenic zebrafish
lines were generated and used as tools to study podocyte cells. These lines
used podocyte-specific fluorescent proteins to identify and locate podocytes within
larvae and adults, allowing these cells to be targeted, isolated, and monitored in
experimental use. Using SPIM (Single/ Selective Plane Illumination Microscopy), it was
possible to induce injury within podocytes of larval fish and visualise changes in calcium
ion dynamics in real-time. Through the microinjection of the known nephrotoxic
agent Puromycin aminonucleoside, it was possible to induce oedema in larval fish and
assess the morphological changes to podocyte cells through analysis via transmission
electron microscopy. Using the novel GCaMP6s LifeActTagRFP-T zebrafish line the
structure of the actin cytoskeleton was assessed in closer detail. Bioinformatics approaches
were used to develop a pathway diagram elucidating the interactions within
podocyte cells through manual curation of literature, the use of current datasets,
and incorporating novel scRNA seq data. This detailed diagram presents an interactive
network that allows the visualisation of podocyte cell interactions in a way that
is more accessible and permits visualisation of the data in ways that would not be
possible with graphs and images alone. It allows for the identification of discrepancies
in metabolic pathways and intracellular communications to be displayed; it also
contributes to what is currently known about podocyte physiology.
Finally, using GCaMP6s and LifeActTagRFP-T tagged podocyte cells disassociated
from adult fish, it was possible to generate a single-cell RNA sequencing
(scRNA-seq) library, detailing active transcripts present within healthy zebrafish
podocytes and the identification of distinct cell populations and podocyte cell markers,
adding to and allowing comparisons with libraries generated from zebrafish podocytes
in alternative states, such as having undergone injury, or in alternative species. The
combination of these techniques and outcomes of this work has allowed the presentation
of a project which has contributed innovative transgenic zebrafish reporter lines,
the novel display of an increase of calcium ion concentration in injured podocyte cells,
the production of a detailed, novel scRNA seq library and a pathway diagram utilising
bioinformatics techniques, that is interactive and informative. These contributions
can add to the generation of new therapies for treating cardiovascular diseases and
have provided additional transgenic zebrafish lines to be used as a model to study
kidney disease development, injury, and repair.