RESUMEN
Ciliated cells serve vital functions in the body ranging from mechano- and chemo-sensing to fluid propulsion. Specialized cells with bundles dozens to hundreds of motile cilia known as multiciliated cells (MCCs) are essential as well, where they direct fluid movement in locations such as the respiratory, central nervous and reproductive systems. Intriguingly, the appearance of MCCs has been noted in the kidney in several disease conditions, but knowledge about their contributions to the pathobiology of these states has remained a mystery. As the mechanisms contributing to ciliopathic diseases are not yet fully understood, animal models serve as valuable tools for studying cilia development and how alterations in ciliated cell function impacts disease progression. Like other vertebrates, the zebrafish, Danio rerio, has numerous ciliated tissues. Among these, the embryonic kidney (or pronephros) is comprised of both monociliated cells and MCCs and therefore provides a setting to investigate both ciliated cell fate choice and ciliogenesis. Considering the zebrafish nephron resembles the segmentation and function of human nephrons, the zebrafish provide a tractable model for studying conserved ciliogenesis pathways in vivo. In this chapter, we provide an overview of ciliated cells with a special focus on MCCs, and present a suite of methods that can be used to visualize ciliated cells and their features in the developing zebrafish. Further, these methods enable precise quantification of ciliated cell number and various cilia-related characteristics.
Asunto(s)
Riñón , Pez Cebra , Animales , Humanos , Riñón/metabolismo , Proteínas de Pez Cebra/metabolismo , Cilios/metabolismo , Diferenciación CelularRESUMEN
Despite significant advances in understanding nephron segment patterning, many questions remain about the underlying genes and signaling pathways that orchestrate renal progenitor cell fate choices and regulate differentiation. In an effort to identify elusive regulators of nephron segmentation, our lab conducted a high-throughput drug screen using a bioactive chemical library and developing zebrafish, which are a conserved vertebrate model and particularly conducive to large-scale screening approaches. 17ß-estradiol (E2), which is the dominant form of estrogen in vertebrates, was a particularly interesting hit from this screen. E2 has been extensively studied in the context of gonad development, but roles for E2 in nephron development were unknown. Here, we report that exogenous estrogen treatments affect distal tubule composition, namely, causing an increase in the distal early segment and a decrease in the neighboring distal late. These changes were noted early in development but were not due to changes in cell dynamics. Interestingly, exposure to the xenoestrogens ethinylestradiol and genistein yielded the same changes in distal segments. Further, upon treatment with an estrogen receptor 2 (Esr2) antagonist, PHTPP, we observed the opposite phenotypes. Similarly, genetic deficiency of the Esr2 analog, esr2b, revealed phenotypes consistent with that of PHTPP treatment. Inhibition of E2 signaling also resulted in decreased expression of essential distal transcription factors, irx3b and its target irx1a. These data suggest that estrogenic compounds are essential for distal segment fate during nephrogenesis in the zebrafish pronephros and expand our fundamental understanding of hormone function during kidney organogenesis.
Asunto(s)
Proteínas de Pez Cebra , Pez Cebra , Animales , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Riñón/metabolismo , Nefronas/metabolismo , Estrógenos/metabolismoRESUMEN
A functional vertebrate kidney relies on structural units called nephrons, which are epithelial tubules with a sequence of segments each expressing a distinct repertoire of solute transporters. The transcriptiona`l codes driving regional specification, solute transporter program activation and terminal differentiation of segment populations remain poorly understood. Here, we demonstrate that the KCTD15 paralogs kctd15a and kctd15b function in concert to restrict distal early (DE)/thick ascending limb (TAL) segment lineage assignment in the developing zebrafish pronephros by repressing Tfap2a activity. During renal ontogeny, expression of these factors colocalized with tfap2a in distal tubule precursors. kctd15a/b loss primed nephron cells to adopt distal fates by driving slc12a1, kcnj1a.1 and stc1 expression. These phenotypes were the result of Tfap2a hyperactivity, where kctd15a/b-deficient embryos exhibited increased abundance of this transcription factor. Interestingly, tfap2a reciprocally promoted kctd15a and kctd15b transcription, unveiling a circuit of autoregulation operating in nephron progenitors. Concomitant kctd15b knockdown with tfap2a overexpression further expanded the DE population. Our study reveals that a transcription factor-repressor feedback module employs tight regulation of Tfap2a and Kctd15 kinetics to control nephron segment fate choice and differentiation during kidney development.