Identification, conservation, and expression of tiered pharmacogenes in zebrafish.

The number of adverse drug events in the United States is critically high, with annual rates exceeding 1 million cases over the last nine years. One cause of adverse drug events is the underlying genetic variation that can alter drug responses. Pharmacogenomics is a growing field that seeks to better understand the relationship between a patient's genetics and drug efficacy. Currently, pharmacogenomics relies largely on human trials, as there is not a well-developed animal model for studying preventative measures and alternative treatments. Here, we analyzed pharmacogene expression at two developmental time points in zebrafish to demonstrate the potential of using this model organism for high-throughput pharmacogenomics research. We found that 76% of tiered human pharmacogenes have a zebrafish ortholog, and of these, many have highly conserved amino acid sequences. Additional gene ontology analysis was used to classify pharmacogenes and identify candidate pathways for future modeling in zebrafish. As precision medicine burgeons, adopting a high-throughput in vivo model such as the zebrafish could greatly increase our understanding of the molecular pathology underlying adverse drug events.

Advances in Understanding the Genetic Mechanisms of Zebrafish Renal Multiciliated Cell Development.

Cilia are microtubule-based organelles that project from the cell surface. In humans and other vertebrates, possession of a single cilium structure enables an assortment of cellular processes ranging from mechanosensation to fluid propulsion and locomotion. Interestingly, cells can possess a single cilium or many more, where so-called multiciliated cells (MCCs) possess apical membrane complexes with several dozen or even hundreds of motile cilia that beat in a coordinated fashion. Development of MCCs is, therefore, integral to control fluid flow and/or cellular movement in various physiological processes. As such, MCC dysfunction is associated with numerous pathological states. Understanding MCC ontogeny can be used to address congenital birth defects as well as acquired disease conditions. Today, researchers used both in vitro and in vivo experimental models to address our knowledge gaps about MCC specification and differentiation. In this review, we summarize recent discoveries from our lab and others that have illuminated new insights regarding the genetic pathways that direct MCC ontogeny in the embryonic kidney using the power of the zebrafish animal model.

Ppargc1a Controls Ciliated Cell Development by Regulating Prostaglandin Biosynthesis.

Cilia are microtubule-based organelles that function in a multitude of physiological contexts to perform chemosensing, mechanosensing, and fluid propulsion. The process of ciliogenesis is highly regulated, and disruptions result in disease states termed ciliopathies. Here, we report that peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (ppargc1a) is essential for ciliogenesis in nodal, mono-, and multiciliated cells (MCCs) and for discernment of renal tubule ciliated cell fate during embryogenesis. ppargc1a performs these functions by affecting prostaglandin signaling, whereby cilia formation and renal MCC fate are restored with prostaglandin E2 (PGE2) treatment in ppargc1a-deficient animals. Genetic disruption of ppargc1a specifically reduces expression of the prostanoid biosynthesis gene prostaglandin-endoperoxide synthase 1 (ptgs1), and suboptimal knockdown of both genes shows this synergistic effect. Furthermore, ptgs1 overexpression rescues ciliogenesis and renal MCCs in ppargc1a-deficient embryos. These findings position Ppargc1a as a key genetic regulator of prostaglandin signaling during ciliated cell ontogeny.

PGC-1α in Disease: Recent Renal Insights into a Versatile Metabolic Regulator.

Peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α) is perhaps best known as a master regulator of mitochondrial biogenesis and function. However, by virtue of its interactions as a coactivator for numerous nuclear receptors and transcription factors, PGC-1α also regulates many tissue-specific tasks that include adipogenesis, angiogenesis, gluconeogenesis, heme biosynthesis, thermogenesis, and cellular protection against degeneration. Knowledge about these functions continue to be discovered with ongoing research. Unsurprisingly, alterations in PGC-1α expression lead to a range of deleterious outcomes. In this review, we provide a brief background on the PGC-1 family with an overview of PGC-1α's roles as an adaptive link to meet cellular needs and its pathological consequences in several organ contexts. Among the latter, kidney health is especially reliant on PGC-1α. Thus, we discuss here at length how changes in PGC-1α function impact the states of renal cancer, acute kidney injury (AKI) and chronic kidney disease (CKD), as well as emerging data that illuminate pivotal roles for PGC-1α during renal development. We survey a new intriguing association of PGC-1α function with ciliogenesis and polycystic kidney disease (PKD), where recent animal studies revealed that embryonic renal cyst formation can occur in the context of PGC-1α deficiency. Finally, we explore future prospects for PGC-1α research and therapeutic implications for this multifaceted coactivator.

Advances in understanding vertebrate nephrogenesis.

The kidney is a complex organ that performs essential functions such as blood filtration and fluid homeostasis, among others. Recent years have heralded significant advancements in our knowledge of the mechanisms that control kidney formation. Here, we provide an overview of vertebrate renal development with a focus on nephrogenesis, the process of generating the epithelialized functional units of the kidney. These steps begin with intermediate mesoderm specification and proceed all the way to the terminally differentiated nephron cell, with many detailed stages in between. The establishment of nephron architecture with proper cellular barriers is vital throughout these processes. Continuously striving to gain further insights into nephrogenesis can ultimately lead to a better understanding and potential treatments for developmental maladies such as Congenital Anomalies of the Kidney and Urinary Tract (CAKUT).

New zebrafish model for monitoring proximal tubule physiology in genetic and acquired renal Fanconi syndromes.

Inherited and acquired disorders that affect proximal tubule endocytosis and lysosomal processing manifest with improper loss of solutes and proteins. The zebrafish pronephros is conserved with humans and is used to model numerous renal conditions, but has few quantitative measures for proximal tubule function. Here, Chen et al. developed a high-throughput assay to quantify proteinuria and lysosomal processing in transgenic zebrafish by labeling vitamin D protein, allowing for precise reporting of proximal tubule function.

Visualizing gene expression during zebrafish pronephros development and regeneration.

The vertebrate kidney is comprised of functional units known as nephrons. Defects in nephron development or activity are a common feature of kidney disease. Current medical treatments are unable to ameliorate the dire consequences of nephron deficit or injury. Although there have been tremendous advancements in our understanding of nephron ontogeny and the response to damage, many significant knowledge gaps still remain. The zebrafish embryo kidney, or pronephros, is an ideal model for many renal development and regeneration studies because it is comprised of nephrons that share conserved features with the nephron units that comprise the mammalian metanephric kidney. In this chapter, we provide an overview about the benefits of using the zebrafish pronephros to study the mechanisms underlying nephrogenesis as well as epithelial repair and regeneration. We subsequently detail methods for the spatiotemporal assessment of gene and protein expression in zebrafish embryos that can be used to extend the understanding of nephron development and disease, and thereby create new opportunities to identify therapeutic strategies for regenerative medicine.

Prostaglandin signaling regulates renal multiciliated cell specification and maturation.

Multiciliated cells (MCCs) are specialized epithelia with apical bundles of motile cilia that direct fluid flow. MCC dysfunction is associated with human diseases of the respiratory, reproductive, and central nervous systems. Further, the appearance of renal MCCs has been cataloged in several kidney conditions, where their function is unknown. Despite their pivotal health importance, many aspects of MCC development remain poorly understood. Here, we utilized a chemical screen to identify molecules that affect MCC ontogeny in the zebrafish embryo kidney, and found prostaglandin signaling is essential both for renal MCC progenitor formation and terminal differentiation. Moreover, we show that prostaglandin activity is required downstream of the transcription factor ets variant 5a (etv5a) during MCC fate choice, where modulating prostaglandin E2 (PGE2) levels rescued MCC number. The discovery that prostaglandin signaling mediates renal MCC development has broad implications for other tissues, and could provide insight into a multitude of pathological states.

Iroquois transcription factor irx2a is required for multiciliated and transporter cell fate decisions during zebrafish pronephros development.

The genetic regulation of nephron patterning during kidney organogenesis remains poorly understood. Nephron tubules in zebrafish are composed of segment populations that have unique absorptive and secretory roles, as well as multiciliated cells (MCCs) that govern fluid flow. Here, we report that the transcription factor iroquois 2a (irx2a) is requisite for zebrafish nephrogenesis. irx2a transcripts localized to the developing pronephros and maturing MCCs, and loss of function altered formation of two segment populations and reduced MCC number. Interestingly, irx2a deficient embryos had reduced expression of an essential MCC gene ets variant 5a (etv5a), and were rescued by etv5a overexpression, supporting the conclusion that etv5a acts downstream of irx2a to control MCC ontogeny. Finally, we found that retinoic acid (RA) signaling affects the irx2a expression domain in renal progenitors, positioning irx2a downstream of RA. In sum, this work reveals new roles for irx2a during nephrogenesis, identifying irx2a as a crucial connection between RA signaling, segmentation, and the control of etv5a mediated MCC formation. Further investigation of the genetic players involved in these events will enhance our understanding of the molecular pathways that govern renal development, which can be used help create therapeutics to treat congenital and acquired kidney diseases.

Homeogene emx1 is required for nephron distal segment development in zebrafish.

Vertebrate kidneys contain nephron functional units where specialized epithelial cell types are organized into segments with discrete physiological roles. Many gaps remain in our understanding of how segment regions develop. Here, we report that the transcription factor empty spiracles homeobox gene 1 (emx1) is a novel nephron segment regulator during embryonic kidney development in zebrafish. emx1 loss of function altered the domains of distal segments without changes in cell turnover or traits like size and morphology, indicating that emx1 directs distal segment fates during nephrogenesis. In exploring how emx1 influences nephron patterning, we found that retinoic acid (RA), a morphogen that induces proximal and represses distal segments, negatively regulates emx1 expression. Next, through a series of genetic studies, we found that emx1 acts downstream of a cascade involving mecom and tbx2b, which encode essential distal segment transcription factors. Finally, we determined that emx1 regulates the expression domains of irx3b and irx1a to control distal segmentation, and sim1a to control corpuscle of Stannius formation. Taken together, our work reveals for the first time that emx1 is a key component of the pronephros segmentation network, which has implications for understanding the genetic regulatory cascades that orchestrate vertebrate nephron patterning.

ppargc1a controls nephron segmentation during zebrafish embryonic kidney ontogeny.

Nephron segmentation involves a concert of genetic and molecular signals that are not fully understood. Through a chemical screen, we discovered that alteration of peroxisome proliferator-activated receptor (PPAR) signaling disrupts nephron segmentation in the zebrafish embryonic kidney (Poureetezadi et al., 2016). Here, we show that the PPAR co-activator ppargc1a directs renal progenitor fate. ppargc1a mutants form a small distal late (DL) segment and an expanded proximal straight tubule (PST) segment. ppargc1a promotes DL fate by regulating the transcription factor tbx2b, and restricts expression of the transcription factor sim1a to inhibit PST fate. Interestingly, sim1a restricts ppargc1a expression to promote the PST, and PST development is fully restored in ppargc1a/sim1a-deficient embryos, suggesting Ppargc1a and Sim1a counterbalance each other in an antagonistic fashion to delineate the PST segment boundary during nephrogenesis. Taken together, our data reveal new roles for Ppargc1a during development, which have implications for understanding renal birth defects.

Prostaglandin signaling regulates nephron segment patterning of renal progenitors during zebrafish kidney development.

Kidney formation involves patterning events that induce renal progenitors to form nephrons with an intricate composition of multiple segments. Here, we performed a chemical genetic screen using zebrafish and discovered that prostaglandins, lipid mediators involved in many physiological functions, influenced pronephros segmentation. Modulating levels of prostaglandin E2 (PGE2) or PGB2 restricted distal segment formation and expanded a proximal segment lineage. Perturbation of prostaglandin synthesis by manipulating Cox1 or Cox2 activity altered distal segment formation and was rescued by exogenous PGE2. Disruption of the PGE2 receptors Ptger2a and Ptger4a similarly affected the distal segments. Further, changes in Cox activity or PGE2 levels affected expression of the transcription factors irx3b and sim1a that mitigate pronephros segment patterning. These findings show for the first time that PGE2 is a regulator of nephron formation in the zebrafish embryonic kidney, thus revealing that prostaglandin signaling may have implications for renal birth defects and other diseases.

Evolving technology: creating kidney organoids from stem cells.

The kidney is a complex organ whose excretory and regulatory functions are vital for maintaining homeostasis. Previous techniques used to study the kidney, including various animal models and 2D cell culture systems to investigate the mechanisms of renal development and regeneration have many benefits but also possess inherent shortcomings. Some of those limitations can be addressed using the emerging technology of 3D organoids. An organoid is a 3D cluster of differentiated cells that are developed ex vivo by addition of various growth factors that result in a miniature organ containing structures present in the tissue of origin. Here, we discuss renal organoids, their development, and how they can be employed to further understand kidney development and disease.