Growing Kidney in the Frog Free

Organogenesis is an important and attractive subject for developmental biologists. In the past decade, there has been a great deal of focus on understanding organogenesis, and many investigations of the mechanisms underlying organogenesis are reported in the literature. The kidney represents a popular and interesting model for investigating organogenesis. Vertebrates use three different kidney forms to control drainage and excrete waste throughout their lifetime: the pronephros, mesonephros and metanephros. Development of the kidney is based on successive induction of the different kidney forms. All kidneys are derived from intermediate mesoderm and develop according to an ordered temporospatial process. These unique features make the kidney an attractive model for analyzing organogenesis. In this article, we focus on Xenopus laevis, an ideal animal model for investigating vertebrate development, and review the advantages of Xenopus for analyzing kidney organogenesis, and highlight the latest research in this field.

X. laevis, a popular animal model for developmental and cell biologists, has been studied and analyzed for many decades. Xenopus can be induced by the hormone injection to lay thousands of eggs and this can be repeated after a few months. The fertilized Xenopus eggs develop to swimming tadpoles in 4 days and commence metamorphosis within 2 months. The eggs are an ideal size to allow micromanipulation and observation of embryogenesis. In addition, a simple in vitro induction assay (fig. 1C–E) also provides an efficient and elegant system to allow investigation of the developmental process [1, 2, 3]. The presumptive ectoderm can be isolated from the late Xenopus blastula (animal cap) for the study of cell induction in an isolated environment. These features make Xenopus an excellent model for analyzing vertebrate embryogenesis.

All three vertebrate kidneys consist of a basic unit, the nephron, but differ with respect to the number and organization of the nephron within the kidney. In higher vertebrates, the pronephros and mesonephros are transient structures in embryogenesis. The nephric duct situated in the intermediate mesoderm induces a series of tubules surrounded by glomeruli in the adjacent mesoderm to form the mesonephros. Development of the metanephros is initiated by the interaction between the nephric mesenchyme and the ureteric bud on the caudal portion of the duct. Unlike the mesonephros and metanephros, development of the pronephros is not dependent on induction from the nephric duct, and the determination of tubule differentiation occurs earlier than determination of duct differentiation. Furthermore, the pronephros is the simplest and earliest form of kidney during development (fig. 1A, B). For this reason, investigations of pronephros development can provide an understanding of induction and differentiation not possible from an analysis of the metanephros.

The pronephros is the main excretory organ of Xenopus at the larval stage. The Xenopus pronephros is derived from somatic and splanchnic intermediate mesoderm and the anlage of the pronephros is formed at the late gastrula stage. Differentiation of the pronephric tubule is determined first, at the late gastrula stage, and the onset of pronephric duct differentiation proceeds from stage 14. At stage 20/21, the anlage of the pronephros becomes morphologically distinguishable. The pronephros anlage is distinctly separated from lateral mesoderm and the cells are arranged in a radical pattern from tail-bud stage. Lumenization of the tubules and anterior portion of the duct occurs at the late tail-bud stage. From stage 35/36, the entire pronephros is lumenized and starts to function. Coinciding with this stage in development is the appearance of the glomus, which can be first identified by histology on the splanchnic side of the coelom. The mesonephric cells are observed at the caudal region from stage 39. The pronephros begins to degenerate at stage 53, coinciding with initiation of mesonephros function, and has disappeared by stage 64 [4, 5, 6].

Recent reports outline the molecular basis of pronephros development (fig. 2). The activation of expression of molecules involved in pronephros development occurs successively and is accompanied by morphologic changes. Lim1 and Pax8 are the first genes expressed in the pronephros anlage and are detected from stage 12/13. Expression of Lim1 and Pax8 is thought to induce pronephros development. Overexpression of Lim1, Pax8, and Pax2 leading to enlargement of the pronephros in the lateral mesoderm supports this hypothesis [7]. Another report suggests that Lim1 is necessary for determination of tubule development and not for induction of pronephros development [8].

From the late neurula/early tail-bud stage, changes in the shape of cells in the pronephros anlage are accompanied by expression of Pax2, Wnt4, and other molecular signals. Wnts signals are known to be important for nephrogenesis. Overexpression of Wnt4 leads to fused pronephric tubules in Xenopus, and antisense morpholino oligonucleotide-mediated knockdown of Wnt4 results in complete loss of pronephric tubules, although the ducts are unaffected [9]. In Xenopus, morpholino-mediated knockdown of frizzled-8(Xfz8),a receptor of the Wnt protein and regulator of the activity of Wnt signals, causes a significant reduction in duct differentiation, but does not affect the expression of early pronephric maker genes in the duct region [10]. Xfz8 is involved in both the Wnt/β-catenin signaling pathway and the PCP pathway [11, 12, 13]. It is also possible that Xfz8functions in duct differentiation without ligand activation. Secondary axis formation caused by overexpression of Xfz8 is not inhibited by Nxfz8, a secretory form of N-terminal extracellular domain of Xfz8[12]. Wnt-11 might be a ligand for Xfz8 in pronephric duct development, because Wnt-11 transcripts are observed around the somites adjacent to the pronephric duct. Like Xfz8, Wnt-11 is also reported to regulate convergent extension movement [14, 15]. Wnt11 is also reported to play a role in kidney development [16], although there are no reports of cooperation between Wnt-11 and Xfz8.

Expression of Notch/Delta is observed in the pronephros from stage 21 [17]. Notch signaling is thought to regulate pronephric duct cell fate. Activation of Notch signaling increases the expression of tubules and glomus markers, but reduces the expression of duct markers and duct formation. Inhibition of Notch signaling also leads to an increase in the proportion of ducts formed. However, the timing of Notch expression is too late in development for it to play a role in determination of duct differentiation. Notch is expressed in the pronephros from stage 21, and the determination of duct differentiation occurs at stage 14. It is likely that still unknown factors are involved in regulating the fate of cells that differentiate into the tubules or ducts in development of the pronephros.

Formation of the glomus starts at the late neurula stage. Accompanied by expression of WT1[18], the glomus anlage separates from the tubule and duct primordium. By stage 25, molecules including annexin IV [19], XTRAP-γ [20] and others start to be expressed. At this stage, patterning of the tubule commences and the duct extends into the cloaca. XTRAP-γis one of four subunits that make up the translocon-associated protein complex. The TRAP complex was thought to aid the translocation of nascent polypeptides into the lumen of the endoplasmic reticulum [21], but this was never demonstrated until now.Morpholino-mediated knockdown of XTRAP-γ causes defects in tubulogenesis and is associated with reduced expression of the pronephros marker genes, Pax2and Wnt4. The expression of Lim1 is enhanced in XTRAP-γ depletion embryos. These results suggest that XTRAP-γ regulates the translocation of molecules that in turn regulate the expression of Pax2, Wnt4, and Lim1. XTRAP-γ expression was observed in the tubules during pronephros developmentfrom stage 25 onward. As already described, the pronephros anlage is distinctly separate from the lateral mesoderm from stage 25, and lumenization is observed in the tubular portion of the pronephros by stage 28. Coincident to these morphogenic changes, expression of Lim1 is reduced and becomes localized to the tips of the pronephric tubules. These observations suggest that a reduction in the expression of Lim1, along with maintenance of Pax2 and Wnt4expression, is necessary for differentiation of the pronephros. It also appears that XTRAP-γ is a regulator of pronephric tubules differentiation. At the late tail-bud stage, the anlage of the tubule is well developed and undergoes further morphogenesis. From stage 30, the expression of gremlin, dullard[22], c-ret[23], Na⁺/K⁺-ATPase [24], and other molecules, is detected in the early-lumenized pronephros.

Mesenchymal-epithelial transition is an important process in tubulogenesis during kidney development. However, the role of the mesenchymal tissue surrounding the pronephros anlage is largely unknown. A recent paper reports that factors in the mesenchymal tissue surrounding the pronephros may regulate pronephros development [25]. One of these factors is XC3H-3b, a member of the TTP/TIS family of CCCH tandem zinc-finger proteins, which are physiological stimulators of instability of the mRNA encoding tumor necrosis factor-α in certain cell types. Expression of XC3H-3b is restricted primarily to the mesodermal tissue surrounding the pronephros. Overexpression and translational inhibition of XC3H-3b markedly and specifically inhibits pronephros development. In both cases, the expression of pronephric marker genes, such as Lim1, Pax2, Pax8, Wnt4, and WT1, is decreased and morphological development of the pronephric tubules is inhibited.

TTP/TIS zinc finger proteins can regulate stability of mRNA by binding to an AU-rich domain in the 3′-UTR [26, 27]. Overexpression of XC3H-3b may also regulate expression of pronephros-specific genes by direct binding, although the expression of XC3H-3b and pronephric marker genes does not overlap and XC3H-3b cannot bind to the mRNA of these pronephric marker genes. Moreover, the phenotype of XC3H-3b depletion suggests that XC3H-3b may regulate the expression of pronephric marker genes without regulating the stability of mRNA of the pronephric marker genes. The TTP/TIS CCCH zinc-finger protein is also reported to play a role in regulating the activity of JNK/MAPK signaling. The TTP/TIS zinc-finger protein affects apoptosis by regulating the level of TNF-α and the activity of JNK and MAPK/ERK [28]. These signals are known to be essential for tubulogenesis during kidney development. FGFs are also known to regulate and specify the fate of mesenchymal cells [29]. Further support for a role of XC3H-3b in regulating tubulogenesis via JNK/MAPK signaling comes from data that shows that deletion of XC3H-3b promotes phosphorylationof ERK and activity of eFGF [Chans, unpubl. data]. Chans also reports that signals from the mesenchyme surrounding the pronephros are essential for pronephros development.

Although many genes are expressed in the entire pronephros, including the duct, the depletion of most of these genes has no effect on pronephric duct development. Differentiation of the pronephric duct is most likely initiated by the interaction of several different molecules. It is also likely that this process involves gene redundancy such that if one of these genes is suppressed, another molecule may rescue its development. The molecular networks underlying early pronephros development have been revealed in the last 10 years, although details of the mechanisms involved are still unknown. In conclusion, Xenopus provides an excellent model for analyzing development of kidney and provides an efficient system to analyze the function of genes involved in this process. Corresponding analyses in higher vertebrate are much more difficult, expensive and time-consuming. The features of Xenopus provide an ideal basis for analyzing the molecular mechanisms underlying early kidney development.