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«Zebrafish Kidney Development Iain A. Drummond* and Alan J. Davidson† * Departments of Medicine and Genetics, Harvard Medical School and Nephrology ...»

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Zebrafish Kidney Development

Iain A. Drummond* and Alan J. Davidson†


Departments of Medicine and Genetics, Harvard Medical School and Nephrology Division, Massachusetts

General Hospital, Charlestown, Massachusetts

Center for Regenerative Medicine, Massachusetts General Hospital, Boston, Massachusetts


I. Introduction

II. Structure of the Zebrafish Pronephros

III. Formation of the Pronephros

A. Origin of the Nephrogenic Mesoderm

B. Early Subdomains within the Nephrogenic Mesoderm C. Role of Retinoic Acid Signaling D. Role of Pax2a E. Role of the Endoderm F. Differentiation of the Tubular Epithelium G. Formation of the Glomerulus H. Formation of the Cloaca IV. Methods to Study Pronephros Function A. Embryo Dissociation B. Isolation of Fluorescently Labeled Cells by Fluorescence Activated Cell Sorting (FACS) C. A Simple Assay for Glomerular Filtration D. Gentamicin Induced Kidney Tubule Injury in Embryos and Adults E. Adult Kidney Isolation F. Non-Lethal Surgical Access to the Adult Kidney G. Detecting and Imaging Zebrafish Cilia H. Histological Sectioning of Whole mount Stained Embryos I. Electron Microscopy Methods for Zebrafish 234 Iain A. Drummond and Alan J. Davidson Abstract The zebrafish pronephric kidney provides a useful and relevant model of kidney development and function. It is composed of cell types common to all vertebrate kidneys and pronephric organogenesis is regulated by transcription factors that have highly conserved functions in mammalian kidney development. Pronephric nephrons are a good model of tubule segmentation and differentiation of epithelial cell types. The pronephric glomerulus provides a simple model to assay gene function in regulating cell structure and cell interactions that form the blood filtration apparatus. The relative simplicity of the pronephric kidney combined with the ease of genetic manipulation in zebrafish makes it well suited for mutation analysis and gene discovery, in vivo imaging, functional screens of candidate genes from other species, and cell isolation by FACS. In addition, the larval and adult zebrafish kidneys have emerged as systems to study kidney regeneration after injury. This chapter provides a review of pronephric structure and development as well as current methods to study the pronephros.

I. Introduction

The kidney has two principal functions: to remove waste from the blood and to balance ion and metabolite concentrations in the blood within physiological ranges that support proper functioning of all other cells (Vize et al., 2002). Kidney function is achieved largely by first filtering the blood and then recovering useful ions and small molecules by directed epithelial transport. This work is performed by nephrons, the functional units of the kidney (Fig. 1). The nephron is comprised of a blood filter, called the glomerulus, attached to a tubular epithelium (Fig. 1C and D). The glomer­ ulus contains specialized epithelial cells called podocytes that form a basket-like extension of cellular processes around a capillary tuft. The basement membrane between podocytes and capillary endothelial cells together with the specialized junc­ tions between the podocyte cell processes (slit diaphragms) function as a blood filtration barrier, allowing passage of small molecules, ions, and blood fluid into the urinary space, while retaining high molecular weight proteins in the vascular system (Fig. 1; see also Fig. 12D). The blood filtrate travels down the lumen of the kidney tubule, encountering distinct proximal and distal tubule segments that modify the composition of the urine via specific solute transport activities. The urine is drained by the collecting ducts, which further modify its salt and water composition, until eventually being voided outside the body (Fig. 1; Vize et al., 2002).

In the course of vertebrate evolution, three distinct forms of kidneys of increasing complexity have been generated: the pronephros, mesonephros, and metanephros (Saxén, 1987). The pronephros is the first kidney to form during embryogenesis. In vertebrates with free-swimming larvae, including amphibians and teleost fish, the pronephros is the func­ tional kidney of early larval life (Howland, 1921; Tytler, 1988; Tytler et al., 1996; Vize et al.,

1997) and is required for proper osmoregulation (Howland, 1921). Later, in juvenile stages of fish and frog development, a mesonephros forms around and along the length of the pronephros and later serves as the final adult kidney. The metanephric kidney forms (A) Vertebrate nephron Zebrafish pronephric nephrons

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Fig. 1 The zebrafish pronephros. (A) Functional features of the vertebrate nephron and the zebrafish pronephric nephrons. See text for details. (B) Stages in zebrafish pronephric kidney development.

(1) Specification of mesoderm to a nephric fate: expression patterns of pax2.1 and lim-1 define a posterior region of the intermediate mesoderm (im) and suggest that a nephrogenic field is established in early development. (2) Epithelialization of the pronephros (pn) follows somitogenesis and is complete by 24 hpf.

(3) Patterning of the nephron gives rise to the pronephric glomerulus (gl) and pronephric tubules (pt).

(4) Angiogenic sprouts from the dorsal aorta (da) invade the glomerulus and form the capillary loop. The cardinal vein (cv) is apposed to the tubules and receives recovered solutes. (C) Diagram of the mature zebrafish pronephric kidney in 3-day larva. A midline compound glomerulus connects to the segmented pronephric tubules that run laterally. The nephrons are joined at the cloaca where they communicate with the exterior.

(D) Patterning of the pronephric nephron generates discrete segments: neck (N), proximal convoluted tubule (PCT), proximal straight tubule (PST), distal early (DE), late distal (DL), and collecting duct (CD).

236 Iain A. Drummond and Alan J. Davidson exclusively in the amniotes (mammals, birds, and reptiles) and, in the case of mammals, is adapted for water retention and producing concentrated urine. Despite some differences in organ morphology between the various kidney forms, many common elements exist at the cellular and molecular level that can be exploited to further our general understanding of renal development and biology. In particular, the zebrafish pronephros has provided a useful model of nephrogenic mesoderm differentiation, kidney cell type differentiation, nephron patterning, kidney, vasculature interactions, glomerular function, and diseases affecting glomerular filtration and tubule lumen size, i.e., cystic kidney disease. While much remains to be done, the basic features of zebrafish pronephric development and patterning have emerged from studies using simple histology, cell lineage tracing, gene expression patterns, and analysis of zebrafish mutants affecting this process.

II. Structure of the Zebrafish Pronephros

The zebrafish pronephros consists of only two nephrons with glomeruli fused at the embryo midline just ventral to the dorsal aorta (Fig. 1C) (Agarwal and John, 1988;

Armstrong, 1932; Balfour, 1880; Drummond, 2000; Drummond et al., 1998; Goodrich, 1930; Hentschel and Elger, 1996; Marshall and Smith, 1930; Newstead and Ford, 1960;

Tytler, 1988; Tytler et al., 1996). Historically, much of the tubular epithelium extending from the glomerulus to the cloaca has been referred to as pronephric duct. This nomenclature was based on the similar anatomical location of the pronephric or Wolffian ducts in amphibians, chickens, and mammals (Vize et al., 2002). However, based on new molecular data there is now a consensus that the tubular epithelium of the zebrafish pronephros is actually subdivided into two proximal tubule segments (proximal con­ voluted tubule (PCT) and proximal straight tubule (PST)) and two distal tubule segments (distal early (DE) and distal late (DL)) that are homologous in many ways to the segments of the mammalian nephron (Wingert and Davidson, 2008). What was pre­ viously considered “tubule” is now believed to represent a “neck” segment, such as that described in the adult kidneys of other teleosts (Kamunde and Kisia, 1994).

The PCT segment is structurally similar to the proximal tubules of the mammalian kidney, displaying a well-developed brush border and high columnar epithelial cells (Seldin and Giebisch, 1992). The proximal tubule in other vertebrates plays a major role in reabsorbing the bulk of the salts, sugars, and small proteins that pass through the glomerular filtration barrier (Vize et al., 2002). The zebrafish PCT expresses the endocytic receptors megalin and cubalin and takes up small fluorescent dextrans that pass through the glomerulus, consistent with a conserved absorptive function (Anzenberger et al., 2006). The PCT also expresses the chloride/bicarbonate anion exchanger AE2, the sodium/bicarbonate cotransporter NBC1, and the sodium/hydrogen exchanger NHE (Fig. 2A) (Nichane et al., 2006; Shmukler et al., 2005; Wingert et al., 2007), indicating a role in acid/base homeostasis which is also shared with proximal tubules in mammals.

The function of the PST segment is less clear. Markers of this segment include an aspartoacylase homolog (Fig. 2B), the zebrafish starmaker gene (Fig. 2C) (Sollner et al., 2003; Sprague et al., 2008), slc13a1 (a sodium/sulfate symporter), and trpm7

9. Zebrafish Kidney Development

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Fig. 2 Ion transporter mRNA expression defines pronephric nephron segments. (A) The chloride– bicarbonate anion exchanger (AE2) is expressed in the proximal convoluted tubule. The proximal straight segment specifically expresses the zebrafish starmaker gene (B) and an aspartoacylase homolog (C). Early distal segments express the Na–K–Cl symporter slc12a1 (D). Expression of a putative ABC transporter (ibd2207) is observed initially throughout most of the forming pronephric tubules at the 15-somite stage (E and F) but becomes restricted primarily to the late distal segment by 24 hpf (G). Embryos in parts B, C, E, F, and G are counterstained with pax2a probe in red for reference. (A and D, courtesy of Alan Davidson et al.; B, C, E, F, and G, courtesy of Neil Hukreide.)

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A. Origin of the Nephrogenic Mesoderm Cell labeling and lineage tracing in zebrafish gastrula stage embryos have demonstrated that cells destined to form the pronephros arise from the ventral mesoderm, in a region partially overlapping with cells fated to form blood (Fig. 3A) (Kimmel et al., 1990). These cells emerge shortly after the completion of epiboly as a band of tissue, the intermediate mesoderm (IM), at the posterior lateral edge of the paraxial mesoderm (Fig. 3B and C). In zebrafish, unlike other non-teleost vertebrates, the IM gives rise to both kidney and blood cells. The size and positioning of the IM are significantly influenced by dorsoventral and anterior–posterior axis patterning molecules, such as the ventralizing factors bone mor­ phogenetic proteins (BMPs) and their inhibitors, and the Cdx family of homeobox genes (see Table I for a summary of zebrafish mutants with pronephric defects).

B. Early Subdomains within the Nephrogenic Mesoderm By the early stages of somitogenesis, the nephrogenic mesoderm component of the IM is clearly defined by the expression of renal markers such as the transcription factors pax2a, pax8, and lhx1a, which extend from the level of somite 3 to the cloaca (Carroll et al., 1999; Drummond, 2000; Heller and Brandli, 1999; Krauss et al., 1991; Majumdar

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Fig. 3 Origins of the intermediate mesoderm. (A) Approximate positions of cells in a shield stage embryo destined to contribute to the blood/vasculature and pronephric lineages in the ventral (V) germ ring.

(D; dorsal shield). (B) Migration of cells during gastrulation to populate the intermediate mesoderm (im) (C).

9. Zebrafish Kidney Development

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C. Role of Retinoic Acid Signaling Recently it was shown that retinoic acid (RA) signaling plays a major role in establishing the proximo-distal segmentation pattern of the pronephros (Wingert et al., 2007). Exposure of embryos to high RA doses induces the formation of expanded proximal tubule segments at the expense of the distal segments. Conversely, inhibition of RA synthesis with diethylaminobenzaldehyde (DEAB), a competitive inhibitor of the aldehyde dehydrogenase enzymes, favors distal nephron cell fates.

DEAB could elicit these patterning effects when added at the end of gastrulation through to the beginning of somitogenesis, consistent with a requirement for RA during early IM patterning. In support of this, DEAB-treated embryos show a loss of proximally restricted genes such as jagged-2a and delta-c and a concomitant expansion in the distal marker meccom at the 8-somite stage (Wingert et al., 2007). RA may also function later in pronephric development as transgenic RA reporter embryos show significant activity in the pronephric tubules at the 18-somite stage (Perz-Edwards et al., 2001). The source of the RA is presumed to be the paraxial mesoderm, which expresses high levels of aldh1a2 (aka retinaldehyde dehydrogenase-2).

In addition to tubule patterning defects, DEAB-treated embryos are also characterized by a loss of podocytes. Expression of wt1a, implicated in podocyte differentiation (Perner et al., 2007), is absent in DEAB-treated embryos from the subdomain of the IM fated to give rise to the glomerulus. Analysis of the wt1a promoter identified an RAresponsive element consistent with wt1a being a direct target of RA signaling (Bollig et al., 2009). Additional RA target genes that specify podocytes or maintain podocyte function (Vaughan et al., 2005) remain to be explored in zebrafish.

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E. Role of the Endoderm Although pronephric development does not require proper development of the endoderm, overdevelopment of the endoderm can alter pronephric development (Mudumana et al., 2008). Knockdown of odd-skipped related1 gene (osr1), encoding

9. Zebrafish Kidney Development

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Fig. 5 Formation of the glomerulus–tubule boundary is disrupted in no isthmus (noi; pax2a) mutants.

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