This work was supported by funding to RAW from the following: National Institutes of Health grants K01 DK083512, DP2 OD008470, and R01 DK100237; March of Dimes Basil O&#8217;Connor Starter Scholar grant award #5-FY12-75; start up funds from the University of Notre Dame College of Science and Department of Biological Sciences; and a generous gift to the University of Notre Dame from Elizabeth and Michael Gallagher on behalf of the Gallagher Family to foster stem&#160;cell research. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank the staffs of the Department of Biological Sciences for their support, and the Center for Zebrafish Research at Notre Dame for their outstanding dedication in the care and welfare of our zebrafish colony. Finally, we thank the members of our research lab for their comments, discussions and insights about this work.

The authors have nothing to disclose.

Here, we have described methods that enable the visualization of nephron segment components in the adult zebrafish. The injection of fluorescent dextran conjugates enables preferential PCT labeling due the endocytic properties of these proximal tubule cells30,38. This method can be performed with great flexibility due to the existence of dextrans that can be obtained with a bevy of different fluorescent conjugates. In addition, the use of lysine-fixable dextrans is an especially convenient way to label PCT segments, as secondary labeling procedures are not required. This enables relatively straightforward signal detection and is compatible with many other fluorescent labels, immunolabeling, and use of transgenic reporter lines. Alkaline phosphatase labeling also marks the proximal tubule, with strong reactivity in the PCT and slightly weaker reactivity in the PST. Finally, the DBA staining enables labeling of distal tubule segments, which display a characteristic branched morphology. Various lectin molecules, which are sugar-binding proteins of nonimmune origin, have been used extensively to distinguish mammalian nephron segments47. It is interesting that DBA in the zebrafish correlates with distal tubule segments, as it is utilized to mark collecting ducts in mammalian kidneys47.
Taken together, these methods can be utilized in various combinations to document renal composition and function. Since all of these methods can be used in whole kidney preparations, they provide tools to evaluate nephron structure and function throughout the organ without entirely relying on the use of more time-consuming, tedious methods, e.g., cryosection work and immunolabeling. However, the stains described in this methods article are viable in cryosection. Cryosection analysis generates samples (compared to whole mount) that are better suited for immunohistochemistry to detect specific peptides, though of course this type of analysis requires the availability of appropriate primary antibodies. Unfortunately one major limitation in working with the zebrafish animal model remains the poor availability of antibodies. Thus WISH remains the most widely used, i.e., &#8216;go-to,&#8217; procedure for the analysis of gene expression. WISH using BCIP, NBT, and/or INT substrate reactions to produce purple or red precipitates is not compatible with fluorescent staining on cryosections. One option would be to invoke the use of fluorescent WISH detection, a procedure that has been optimized for zebrafish embryonic samples and could be tailored for use with the adult kidney. The description of the methods in this video article should allow for further experimentation with techniques like fluorescent WISH in combination with transgenic zebrafish lines in which individual segments are labeled with a reporter such as eGFP or mCherry. Alternatively, the methods described here could be tested in combination with other vital dyes, e.g., other lectins. Thus, further adaptation and expansion of the methods described here could be invoked to suit the researcher&#8217;s needs for whole mount kidney preparations and analysis.
As such, these methods have broad potential for implementation with the zebrafish model for ongoing regeneration studies and also in genetic disease modeling. There is a pressing need for innovative research and treatments for renal afflictions. Millions of people suffer from some form of kidney disease each year that results from congenital, acute, and/or chronic causes. Further, the prevalence of kidney disease continues to rise worldwide, making these diseases a global public health issue14,15. Treatments such as hemodialysis are available whereby an external dialysis machine serves to rid a patient&#8217;s blood of metabolic waste if their kidneys are not able to perform this role. Unfortunately, this intervention has limitations, as ongoing treatment is necessary for survival. Moreover, patients will eventually require a kidney transplant, which can take years to receive once a patient is placed on a waitlist. Even after obtaining a kidney transplant, many patients suffer adverse effects as a result of the procedure and must battle these effects for the rest of their lives. These difficulties demonstrate the serious need for the development of novel treatments to either help prevent kidney disease or to perhaps augment tissue regeneration8,16,52,53. Given the obvious ethical limitations of the use of human test subjects in biomedical laboratories, animal models are essential for the study of human disease pathogenesis and for the testing of new treatments. As a result of its anatomical similarity and evolutionary proximity, the mouse has become the most widely used model of human disease45. However, there are certain limitations with this animal, including inability to visualize organ development in vivo and practicality of completing large-scale genetic screens. Zebrafish are a relevant and useful model organism for the study of organ development and modeling of human disease45,46. In regards to human disease, functional homologs in zebrafish exist for nearly 70% of all human genes54,55. 
Recent work has highlighted the utility of zebrafish for many areas of kidney research31,37,56. Studies of regeneration and repair in zebrafish after AKI suggest that tubule epithelial regeneration and neonephrogenesis are two processes that occur and overlap throughout the regeneration timecourse30,37. The use of an aminoglycoside antibiotic called gentamicin has been utilized as an injury paradigm through which to study the outcomes of AKI in adult zebrafish29,30,37. Over an approximately two-week period following injury from gentamicin, the proximal tubules regenerate, and meanwhile new nephrons form throughout the organ29,30,37. In general, the mechanisms that regulate epithelial regeneration remain highly controversial. In one proposed mechanism of the repair process, there is the initial acute injury event followed by a sloughing of dead cells into the lumen. Next, there is a series of de-differentiation, proliferation, and migration events, after which new cells differentiate, repopulating the injured basement membrane and restoring function to the injured site. Alternatively, other studies have suggested that replacement cells arise from stem/progenitor cells that reside within nephron tubules. As regards the process of neonephrogenesis in zebrafish, cellular aggregates have been observed following AKI by gentamicin injection29,30. These aggregates later mature into new nephrons that plumb into already existing tubules29,30. The common thread that unites nephron epithelial regeneration and neonephrogenesis is their sheer mystery factor: at present there are exponentially more questions about how these regenerative feats occur than there are available insights.
To date, however, several exciting lines of evidence support the notion that renal regeneration research using zebrafish can indeed provide relevant comparative insights into human AKI that may also have direct translational potential56-58. Chemical screening to identify small molecules that increase the proliferation of renal progenitor cells in the zebrafish embryo recently led to the identification of the histone deacetylase inhibitor methyl-4-(phenylthio)-butanoate (m4PTB)56,57. Administration of this compound to zebrafish larvae with gentamicin-induced AKI revealed that larval survival increased and that renal tubular proliferation was enhanced56,58. When mice with moderate ischemia-induced AKI were treated with m4PTB, their recovery was accelerated in association with a reduction in both tubular atrophy and cell cycle arrest of proliferating renal tubular cells58. These findings suggest that the pathways responsible for normal zebrafish renal development and/or the regenerative response to AKI will be conserved with mammals56.
Thus, while further studies are needed to tease apart the mechanisms of regeneration&#8212;namely to identify the signaling pathways that are involved and understand their interactions&#8212;the zebrafish provides one promising avenue to pursue this important goal in the nephrology field. Tools such as the nephron cell labels described in this protocol represent one group of assays that can be utilized to evaluate renal composition and functionality in AKI models. Further, they can be applied to characterize transgenic models of kidney disease and may provide ways to identify chemical therapeutics capable of promoting restoration of nephron structure after renal damage.

The kidney is a complex organ that fulfills a multitude of physiological functions in the body. The foremost function of the kidney is metabolic waste excretion, and this task is closely intertwined with the maintenance of fluid homeostasis. The kidney performs these jobs by filtering the blood and forming urine, while concomitantly regulating blood pressure, electrolyte levels, and acid-base balance. Highly specialized epithelial tubules called nephrons serve as the basic functional unit of the kidney1,2. In adult vertebrates, nephrons are typically organized in a tightly coiled arrangement surrounding a centralized drainage system, thus allowing for many tubules to pack into the fairly small organ. For example, each human kidney can contain over 1 million nephrons3. Other mammals like the mouse possess on the order of 8-10 thousand nephrons per kidney4. The degree of renal complexity differs between these mammals and other vertebrates due to variations in total nephron number and their architectural layout within the kidney. Vertebrate species form as many as three successive kidney structures during development, and each form displays increasing complexity with regard to nephron endowment and arrangement: the pronephros, mesonephros, and metanephros. The last renal structure to be retained serves as the adult kidney organ&#8212;typically a mesonephros or a metanephros2. Each kidney form, however, has a nephron-based composition2.
The nephron is the workhorse of the kidney and is responsible for blood filtration along with metabolite secretion and reabsorption. Nephrons are simple tubular structures comprised of epithelial cells and have a segmental organization, in which discrete, highly specialized domains of differentiated epithelia perform specific tasks. In the order of filtrate flow through the nephron, there are typically three major parts that comprise each unit: (1) the renal corpuscle, (2) a tubule with proximal, intermediate, and distal segments, and (3) a collecting duct1. The proximal tubule is responsible for the reabsorption of organic solutes, most notably glucose and amino acids1. The intermediate tubule (or loop of Henle) is a major site of salt and water regulation1. Finally, fine-tuning of salt and other ions occurs in the distal tubule and collecting duct and is highly regulated by the endocrine system1. From there, the filtrate travels through the ureter and bladder, ultimately exiting the body as a waste product1.
The loss of renal function can stem from a multitude of causes that prevent normal nephron activity. These causes can occur during kidney development, e.g.,&#160;congenital defects that lead to the malformation of nephrons5, or causes from different types of injury (stemming from both genetic and environmental origins). Acquired injuries are broadly categorized as acute kidney injury (AKI) or chronic kidney injury (CKD). AKI involves an abrupt loss of renal function, often resulting from ischemia or toxin exposure6,7. In mammals, nephron epithelial repair is possible after such injuries8, and many people exhibit full restoration of renal function after AKI. However, AKI is also associated with high morbidity and mortality that have been reported across a broad 30 - 70% incidence range6,7. CKD, in contrast, is associated with progressive loss of renal function that results from years of prolonged damage, typically associated with fibrosis8,9. Further, an interplay exists between AKI and CKD, as either one can predispose an individual to the other10-12. For instance, those who have suffered from AKI are more susceptible to CKD and even death13. The incidence of kidney disease, both in the US and worldwide, has reached epidemic proportions and is predicted to rise as the aged population expands&#8212;thus there is a growing need to identify regenerative medicine interventions for the kidney14,15.
Despite the knowledge that AKI patients can recover, it has long been thought that the kidney is an organ without innate regenerative powers. This traditional view has been dramatically revised in recent years16. Studies investigating renal damage in mice and rats after AKI have shown that nephron tubule epithelial cells can proliferate and rebuild functional nephrons17-20. Although mammals possess this adaptive ability to regenerate nephrons, there are notable limitations and maladaptive responses can be triggered which lead to renal fibrosis and CKD21. For example, a recent study demonstrated that repeated epithelial cell ablation in murine nephrons was associated with diminished regeneration and renal fibrosis&#8212;suggesting nephrons have a limited regeneration threshold22. There is no evidence that the adult mammalian kidney forms new nephrons to replace lost or damaged ones, thus their destruction leads to a permanent nephron deficit8,9. Interestingly, some vertebrates do respond to AKI by regenerating nephron epithelia and by also producing new nephrons, a process referred to as neonephrogenesis or nephron neogenesis23. Neonephrogenesis occurs in fish after toxin exposure or partial resection, and injury models have historically included the goldfish24,25, tilapia26, skate27, medaka28, and more recently, the zebrafish29,30. Of these, zebrafish provide a viable genetic research model to discover the cellular and molecular pathways responsible for renal regeneration.
In this video article, we demonstrate how to label nephron segments in adult zebrafish. Knowledge of nephron anatomy, molecular features, and functional characteristics are essential for successful future renal studies using zebrafish. The adult zebrafish kidney is a mesonephros structure and is inherently less complex in terms of nephron number and organization than the metanephros structure found in adult mammals, avians, and reptiles31. However, zebrafish nephron segment organization is analogous to mammals, and was first documented in the embryonic kidney based on gene expression studies31-35. Zebrafish embryo nephrons contain linear tubule segments that include the proximal convoluted tubule (PCT), proximal straight tubule (PST), distal early (DE), and distal late (DL)31-35 (Figure 1). Zebrafish adult nephrons possess similar tubular segments30,31,36,37 (Figure 1). The PCT can also be identified by a functional phenotype: epithelial cells in the PCT will incorporate fluorescently labeled dextran compounds (10-70 kDA in size) present in the circulation, which has been used in both the embryonic and adult zebrafish kidney to assess&#160;endocytic uptake by these proximal tubule cells38-41 (Figure 1). One difference in nephron segments between zebrafish and mammals is that zebrafish lack an intermediate tubule, or loop of Henle30-37; since this segment functions in mammals to conserve water, and zebrafish are a freshwater species without an urgency to concentrate their urine, it is not surprising that zebrafish lack this segment32,33. Thus, the overall nephron composition is largely conserved between the zebrafish and mammalian kidney32,33. These similarities have enabled zebrafish to be an effective research tool to investigate the functions of renal-expressed genes during developmental nephrogenesis32-35,42 and to model numerous kidney diseases43,44, thereby adding to the substantial list of human conditions that can be investigated using zebrafish45,46.
Today, the adult zebrafish kidney is an attractive model for comparative studies of renal regeneration due to its genetic tractability and the expanding palate of technological resources available to interrogate gene function and signaling pathways in this species. Several studies have used the zebrafish mesonephros to investigate the mechanisms of regeneration after AKI29,30,37. For continued AKI research using the adult zebrafish kidney, it is essential to have exquisite methods to label different nephron regions and methods to survey nephron functionality. Several methods for adult kidney analysis have been adapted from embryonic kidney protocols. Intraperitoneal injection of fluorescently labeled dextran in the adult zebrafish labels the PCT epithelium in mesonephros nephrons via endocytosis30, as in zebrafish embryos38-41 &#160; (Figure 1). This method has been used to visualize when proximal tubules regain their reabsorbing property following AKI, which enables one assessment of restored physiological function30 (Figure 1). Another feature of the nephron proximal tubule is that the epithelial cells in this segment possess a brush border37 that shows high levels of endogenous alkaline phosphatase activity. Thus, the proximal epithelium can be localized visually in the adult zebrafish kidney using a fluorescence-based technique known as ELF-(Enzyme-Labeled Fluorescence)-9747.
Here, we describe how to perform fluorescent dextran uptake assays30,48 in the adult zebrafish kidney, so as to obtain a functional assessment of the proximal tubule and map the size and contours of this tubule segment. Next, we describe techniques to label the proximal tubule regions of the adult nephron with the fluorescent label alkaline phosphatase. Third, we show for the first time that the rhodamine-tagged lectin Dolichos biflorus agglutinin (DBA), which has been used as a general marker of the collecting ducts in the mammalian kidney49, marks the distal tubules of the adult zebrafish kidney. As DBA is mutually exclusive to alkaline phosphatase staining, these labels provide a way to broadly distinguish pan-proximal versus pan-distal stretches of the adult zebrafish nephron. Throughout the implementation of these fluorescent stains in both whole mount and cryostat histological sections, we correlate these labels with expression domains of solute transporter genes that uniquely identify each nephron segment. Therefore this protocol also contains a guide to the modified tissue processing procedures for adult tissue whole mount in situ hybridization (WISH) analysis based on our embryo WISH protocol50. These procedures can be used in various combinations (Figure 2)&#160;to document morphological and functional attributes of adult zebrafish kidney nephrons. Thus, these protocols can be applied to regeneration studies, the phenotypic characterization of other renal disease models, and even used to study formation of the adult kidney.

<table border="0" cellpadding="0" cellspacing="0" width="754">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1X Pbs</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td style="width:321px;">Made by diluting 10 X Pbs in distilled water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X Pbst</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td style="width:321px;">0.1% Tween-20 detergent in 1 X Pbs.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X Pbs with 0.05 % Tween</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td style="width:321px;">0.05% Tween-20 detergent in 1 X Pbs.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Tween 20</td>
			<td style="width:99px;">American Bioanalytical</td>
			<td style="width:119px;">AB02038</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;">4% PFA/1X Pbs</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td style="width:321px;">Dissolve 4% PFA (w/v) (Electron Microscopy Sciences, Cat # 19210) in 1 X Pbs, bring to boil on a hot plate in a fume hood. Cool and freeze the aliquots for storage in the freezer at -20&#176;C. Thaw just before use and do not refreeze stocks.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine Forceps</td>
			<td style="width:99px;">Roboz</td>
			<td style="width:119px;">RS-1050</td>
			<td style="width:321px;">Dumont Tweezers Pattern #55&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Glass Slide</td>
			<td style="width:99px;">Thermo-Fisher</td>
			<td style="width:119px;">4445</td>
			<td style="width:321px;">White Frost&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Glass Coverslip</td>
			<td style="width:99px;">Thermo-Fisher</td>
			<td style="width:119px;">12-540A</td>
			<td style="width:321px;">18 x 18 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Modeling Clay</td>
			<td style="width:99px;">Hasbro</td>
			<td style="width:119px;">Playdoh</td>
			<td style="width:321px;">Other modeling clays can be substituted and work similarly</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Slide Holder</td>
			<td style="width:99px;">Thermo-Fisher</td>
			<td style="width:119px;">12-587</td>
			<td style="width:321px;">Optional: cardboard tray to store slides flat</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;">Bleaching solution</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td style="width:321px;">Bleach Mix Formula (for a 20 mL solution): 
			1.6 mL of 10% Potassium hydroxide solution 
			0.6 mL of 30% Hydrogen peroxide solution 
			100 &#956;l of 20% Tween 
			Fill to 20 mL with distilled water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Hydroxide</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">221473</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrogen Peroxide</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">H1009</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;">Blocking solution</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td style="width:321px;">Blocking Solution (for a 10 mL solution): 
			8 mL of 1X Pbs with 0.05% Tween 
			2 mL of fetal calf serum 
			150 &#956;l of DMSO</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Alkaline phosphatase activity detection</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:119px;">E6601</td>
			<td style="width:321px;">We utilize the ELF 97 Endogenous Phosphatase Detection Kit.&#160; To prepare the working substrate solution, dilute the substrate 20-fold in the detection buffer, according to the manufacturer&#39;s instructions.&#160;</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Alkaline phosphatase wash solution</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td style="width:321px;">Recipe for Wash Buffer Solution (for a 100 mL solution): 
			5 mL of 0.5M EDTA 
			120 mg of Levamisole 
			95 mL of 1X PBS</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dextran, fluorescein, 40,000 MW</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:119px;">D1844</td>
			<td style="width:321px;">Dilute with distilled water to make a 50 mg/ml stock solution, and store aliquots in the freezer at -20&#176;C.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dextran, cascade blue, 10,000 MW</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:119px;">D1976</td>
			<td style="width:321px;">Dilute with distilled water to make a 50 mg/ml stock solution, and store aliquots in the freezer at -20&#176;C.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dextran, lucifer yellow, 10,000 MW</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:119px;">D1825</td>
			<td style="width:321px;">Dilute with distilled water to make a 50 mg/ml stock solution, and store aliquots in the freezer at -20&#176;C.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dextran, tetramethylrhodamine (fluoro-ruby), 10,000 MW</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:119px;">D1817</td>
			<td style="width:321px;">Dilute with distilled water to make a 50 mg/ml stock solution, and store aliquots in the freezer at -20&#176;C.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Dolichos biflorus agglutinin (DBA)-rhodamine</td>
			<td style="width:99px;">Vector laboratories</td>
			<td style="width:119px;">RL-10-32</td>
			<td style="width:321px;">DBA Staining Solution (for a 100 &#956;l solution): 
			1 &#956;l of DBA 
			99 &#956;l of 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Propidium iodide</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:119px;">E6601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DAPI</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:119px;">P1304MP</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vectashield Hard Set Mounting Medium</td>
			<td style="width:99px;">Vector laboratories</td>
			<td style="width:119px;">H-1400</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micro cover glass</td>
			<td style="width:99px;">VWR</td>
			<td align="right" style="width:119px;">48393081</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Dimethyl Sulfoxide, DMSO</td>
			<td style="width:99px;">American Bioanalytical</td>
			<td style="width:119px;">67-68-5</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">S0289</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">EDTA, 0.5M Solution, pH 8.0</td>
			<td style="width:99px;">American Bioanalytical</td>
			<td style="width:119px;">AB00502</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Levamisole</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">L-9756</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Tissue Freezing Medium</td>
			<td style="width:99px;">Triangle Biomedical Sciences, Inc.</td>
			<td>TFM-C</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tissue-Tek Cryomold Biopsy, 10x10x5mm</td>
			<td>Sakura Finetek</td>
			<td>4565</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TruBond 380 Adhesive Microscope Slides</td>
			<td style="width:99px;">Tru Scientific</td>
			<td style="width:119px;">0380W</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Liquid Blocker &#8211; Super PAP Pen</td>
			<td>Electron Microscopy Sciences</td>
			<td>71312</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Immuno stain moisture chamber</td>
			<td>Evergreen Scientific</td>
			<td>240-9020-Z10&#160;</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cryostat</td>
			<td style="width:99px;">Therm</td>
			<td style="width:119px;">Microm&#8482; HM 525 Cryostat</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:216px;">Stereomicroscope</td>
			<td style="width:99px;">Nikon</td>
			<td style="width:119px;">SMZ645; SMZ1000; 83455 P-Blue GFP/DAPI; 83457 P-Endow GFP/FITC; 83457 P-TRITC</td>
			<td style="width:321px;">Filter set used was as follows:&#160; Hoechst/DAPI filter was used for DAPI, propidium iodide, dextran-cascade blue and alkaline phosphatase detection. GFP/FITC filter was used for dextran-FITC, dextran lucifer yellow, and anti-GFP detection. The TRITC filter was used for dextran-fluoro-ruby, PI, and DBA detection.&#160;</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:216px;">Compound microscope</td>
			<td style="width:99px;">Nikon</td>
			<td style="width:119px;">96310 C-FL UV-2E/C DAPI; 96311 C-FL B-2E/C FITC; 96313 C-FL Y-2E/C Texas Red</td>
			<td style="width:321px;">Filter set used was as follows: Hoechst/DAPI filter was used for DAPI, propidium iodide, dextran-cascade blue and alkaline phosphatase detection. GFP/FITC filter was used for dextran-FITC, dextran lucifer yellow, and anti-GFP detection. The Texas Red filter was used for dextran-fluoro-ruby, PI, and DBA detection.&#160;&#160;</td>
		</tr>
	</tbody>
</table>

analysis of nephron composition and function in the adult zebrafish kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

Watch this Scientific Journal Video about Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney at JoVE.com

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

analysis-nephron-composition-function-adult-zebrafish

Research

JoVE Journal

Biology

43.8K Views.  University of Notre Dame.  Данио взрослых почки является отличной системой для почечных регенерации и болезненных исследований. Важным аспектом таких исследований является оценка структуры нефрона и функции. Этот протокол описывает несколько методик, которые могут быть реализованы...

Video: Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...