The nephrogenic potential of the transcription factors osr1, osr2, hnf1b, lhx1 and pax8 assessed in Xenopus animal caps
© Drews et al; licensee BioMed Central Ltd. 2011
Received: 25 August 2010
Accepted: 31 January 2011
Published: 31 January 2011
The three distinct types of kidneys, pronephros, mesonephros and metanephros, develop consecutively in vertebrates. The earliest form of embryonic kidney, the pronephros, is derived from intermediate mesoderm and the first expressed genes localized in the pronephros anlage are the transcription factors osr1, osr2, hnf1b, lhx1 and pax8, here referred to as the early nephrogenic transcription factors. However, the pathway inducing nephrogenesis and the network of theses factors are poorly understood. Treatment of the undifferentiated animal pole explant (animal cap) of Xenopus with activin A and retinoic acid induces pronephros formation providing a powerful tool to analyze key molecular events in nephrogenesis.
We have investigated the expression kinetics of the early nephrogenic transcription factors in activin A and retinoic acid treated animal caps and their potential to induce pronephric differentiation. In treated animal caps, expression of osr1, osr2, hnf1b and lhx1 are induced early, whereas pax8 expression occurs later implying an indirect activation. Activin A alone is able to induce osr2 and lhx1 after three hours treatment in animal caps while retinoic acid fails to induce any of these nephrogenic transcription factors. The early expression of the five transcription factors and their interference with pronephros development when overexpressed in embryos suggest that these factors potentially induce nephrogenesis upon expression in animal caps. But no pronephros development is achieved by either overexpression of OSR1, by HNF1B injection with activin A treatment, or the combined application of LHX1 and PAX8, although they influenced the expression of several early nephrogenic transcription factors in some cases. In an additional approach we could show that HNF1B induces several genes important in nephrogenesis and regulates lhx1 expression by an HNF1 binding site in the lhx1 promoter.
The early nephrogenic transcription factors play an important role in nephrogenesis, but have no pronephros induction potential upon overexpression in animal caps. They activate transcriptional cascades that partially reflect the gene activation initiated by activin A and retinoic acid. Significantly, HNF1B activates the lhx1 promoter directly, thus extending the known activin A regulation of the lhx1 gene via an activin A responsive element.
During vertebrate development three kidney types of increasing complexity (pronephros, mesonephros and metanephros) form successively from the intermediate mesoderm, located between the paraxial mesoderm (developing somites) and the lateral plate . The pronephros is the simplest, functional form of kidney in larval stages of fish and amphibians and consists of three major components: glomus, tubules and duct. In adults the pronephros is replaced by the mesonephros. In mammals the pronephros is not functional, but required for mesonephros formation that is replaced by the metanephros, the kidney of the adult .
All components of the pronephros arise from intermediate mesoderm, but the signals that direct pattering of the presumptive pronephric mesoderm towards pronephric lineages are unknown. Experiments showed that the anterior somites are crucial for pronephros development and provide an essential first signal. If the anterior somites are removed  or separated from the presumptive pronephros , pronephroi do not form. Anterior somites can also induce pronephric tubules in unspecified intermediate mesoderm . Although the exact timing and nature of the signal provided by the anterior somites are yet unknown, wnt11b expressed throughout the anterior somites has recently been shown as a crucial signal .
Xenopus is a very attractive model organism to analyse key molecular events in nephrogenesis, because most genes essential for pronephros development in Xenopus embryos are also crucial for the formation of the more complex mammalian kidneys [6–9]. A classical method to identify important molecules in Xenopus development is the injection of mRNAs or morpholino oligonucleotides into the fertilized egg or into blastomeres of early cleaving stages [10, 11]. Thus, several pronephric regulators have been functionally identified [7, 8, 12]. An additional experimental tool to study early events of nephrogenesis involves explanting the animal pole of the blastula. These explanted animal caps have pluripotency and differentiate into various tissues upon exposure to inducing substances [13, 14]. Importantly, animal caps treated with activin A and retinoic acid differentiate into pronephros  and in this in vitro system genes are induced with similar kinetics as in vivo [16–18].
In Xenopus the first genes expressed in the pronephros anlage are the transcription factors osr1 and osr2, members of the odd-skipped family of proteins , hnf1b, a member of the homeobox factors , lhx1 (lim1), a lim homeobox factor  as well as pax8, a member of the paired box domain family . We refer to these five transcription factors as the early nephrogenic transcription factors, as they are all expressed in the pronephros anlage prior to cellular differentiation and their misexpression affects pronephros development. Inhibition of osr1 or osr2 by morpholinos in Xenopus embryos interferes with kidney formation and embryonic overexpression of either of these factors induces ectopic kidney tissue and enlarged pronephros . Overexpression of hnf1b inhibits pronephros formation  and this effect is also seen by using the human HNF1B  implying that the regulatory potential has been conserved during vertebrate evolution. In contrast, lhx1 and pax8 overexpression leads to an enlargement of the pronephros and, if both factors are coexpressed, this effect is increased and even induces ectopic pronephric tubules .
It should be noted that each of these five early nephrogenic transcription factors plays also a crucial role in the development of other organs. The prominent role of these nephrogenic transcription factors is partially also evident in mammalian systems. Whereas null mutation of Osr1 in mice exhibit agenesis of the kidney , Osr2 knock-out has no kidney phenotype , although Osr2 transcripts are expressed in the developing kidney . The kidney-restricted knockout of Hnf1b leads to polycystic kidney disease  and the Lhx1 null mutant even lacks any kidney . In contrast, Pax8 deficient mice exhibit thyroid gland deficiency, but have no pronephric phenotype . Nevertheless, Pax8 plays an essential role in kidney development, as impaired metanephros formation observed in mice deficient for Pax2  is dramatically increased by a lack of any nephric cell lineage, if these embryos lack additionally Pax8 .
To further explore the role of these five nephrogenic transcription factors we have now analyzed the kinetics of their induction in animals caps differentiated into pronephric tissue by activin A and retinoic acid. We then have overexpressed these transcription factors in animal caps and analyzed their potential to induce each other and to stimulate pronephric differentiation in these explants. To allow discrimination between injected mRNAs and endogenous mRNAs we used the human mRNAs that are functionally equivalent, but are not detectable with the Xenopus probes. We use capital letters for these human transcription factors to make a clear distinction. In addition we identified genes induced by HNF1B in these early embryonic cells.
Induction of mRNAs encoding the early nephrogenic transcription factors osr1, osr2, hnf1b, lhx1 and pax8 in animal caps treated with retinoic acid and/or activin A
To clarify which nephrogenic transcription factor transcripts are induced by retinoic acid or activin A alone, we analyzed animal caps treated with either retinoic acid or activin A for three hours (Figure 1C). Treatment with retinoic acid failed to induce the nephrogenic transcription factors, but rather decreased the level of osr1 and pax8 transcripts by 2-fold and 1.6-fold, respectively. In contrast, activin A treatment induced osr2 as well as lhx1, but osr1 in two of four experiments only. Since hnf1b was not induced by activin A alone, we conclude that a synergistic effect of both inducers is needed to induce hnf1b. The lack of induction of pax8 in animal caps treated with activin A or retinoic acid alone is consistent with the no-induction observed in animal caps treated with retinoic acid and activin A for three hours (also compare Figure 1A).
Overexpression of OSR1 and Osr2A leads to enlargement of pronephros and ectopic pronephric tissue
Overexpression of OSR1 alone or in combination with retinoic acid or activin A cannot support pronephros differentiation in animal caps
LHX1 and/or PAX8 or HNF1B are not sufficient to induce pronephros differentiation in animal caps
Since differential addition of retinoic acid and activin A to animal caps has shown that activin A alone induces osr2, lhx1 and frequently also osr1, but never hnf1b that requires retinoic acid in addition (Figure 1C), we wondered whether HNF1B injection might replace retinoic acid to get pronephric induction in animal caps. However, injection of HNF1B mRNA into the animal pole at the two-cell stage, failed to induce pronephric differentiation in the explanted animal caps, even if cultured in the presence of activin A (Figure 5B). In conclusion, LHX1, PAX8 and HNF1B failed to induce pronephros differentiation in animal caps.
Overexpression of HNF1B induces in animal caps genes important for nephrogenesis
Activation of genes by HNF1B in animal caps
7 h (N = 5)
14 h (N = 4)
early nephrogenic TFs
genes involved in nephrogenesis
proximal tubule genes
lhx1 target genes
HNF1B regulates lhx1 transcription by an HNF1 binding site in the lhx1 promoter
To extend these findings to Xenopus embryonic cells we tested some constructs in animal caps that were derived from controls or HNF1B injected eggs. All constructs retaining the HNF1 binding site were transactivated by injected HNF1B, including the minimal construct Ex1(-120/+3), whereas the reporter Ex1(-117/+3) containing the partially deleted HNF1 site was not inducible (Figure 6B). From these results we conclude that the lhx1 promoter carries a functional HNF1 binding site that is active in HEK293 cells as well as in embryonic cells of Xenopus.
Animal caps are a suitable system to analyse nephrogenesis in vitro, because pronephros differentiation can be induced by treatment with activin A and retinoic acid . Activin A simulates as a TGF-β family member the vegetalizing factor 1 (Vg1) . This factor whose maternal mRNA is localized to the vegetal pole of Xenopus eggs [49, 50] has mesoderm-inducting activity and is an essential regulator of embryonic patterning . On the other hand retinoic acid regulates major embryonic growth and patterning decisions and its availability is regulated by synthesizing and metabolizing enzymes . In Xenopus retinal dehydrogenase (RALDH2) creates a critical retinoic acid concentration gradient along the anteroposterior axis  and it was shown that retinoic acid treatment of embryos leads to larger pronephros , whereas defective retinoic acid signalling impairs pronephros development .
Although the animal cap assay represents a powerful system to analyse key molecular events in nephrogenesis, it has some limitations, since pronephros differentiation does not occur in all animal caps. In our hands treated animal caps revealed pronephric induction rate of about 60-85% comparable to about 80% described previously [15, 18]. Significantly, animal caps often died during the four day incubation and this lethality was increased upon mRNA injection, but a clear activation of pronephros differentiation was seen in surviving activin A and retinoic acid treated animal caps. The inhomogeneous response of individual animal caps seen by antibody staining was also observed when comparing the induction of specific transcripts between different experiments (Figure 1 and 4). This experimental variation was also evident in transactivation of promoter luciferase reporter constructs (Figure 6B), a limitation reported previously [42, 56]. In these experiments the variable outcome is possibly further increased, since reporter constructs and mRNAs cannot be introduced at exactly the same level into each animal cap. In spite of these technical difficulties, we successfully used the animal caps to identify several transcriptional regulatory pathways in this differentiating system.
Our analysis of animal caps treated with activin A and retinoic acid revealed the known induction of lhx1 (Taira et al., 1992) and pax8 , but also induced expression of osr1, osr2 and hnf1b (Figure 1A). Interestingly, in animal caps the kinetics of induction reflects the expression in vivo, an observation made for other induced RNAs previously [16, 57]. Thus, the expression of osr1, osr2, lhx1 and hnf1b after 1.5 hours treatment corresponds with their embryonic expression at early gastrula [19–21] and pax8 expressed later in animal caps agrees with its expression in late gastrula .
In animal caps activin A can induce osr2 and lhx1 alone reflecting its strong inducing activity in animal caps . In contrast retinoic acid fails to induce any of the five factors (Figure 1C) correlating the fact that retinoic acid does not induce pronephros [13, 59, 60]. Previous experiments showed a low lhx1 induction in retinoic acid treated animal caps [21, 61] that possibly reflect subtle differences in the induction protocol or the animals used.
The fast induction of osr2, hnf1b and lhx1 reflects most likely a direct activation by activin A and retinoic acid, since the induction of these transcripts was not inhibited by cycloheximide treatment (Figure 1B), a finding that has been previously reported for activin A induced lhx1 transcripts [21, 62]. Consistent with such a direct activation an actvin A respose element (ARE) has been identified in the lhx1 promoter [42, 43].
Previous experiments showed that it is possible to induce tissue differentiation by overexpressing transcription factors in animal caps. For instance overexpressed Xbra leads to mesoderm differentiation with muscle, mesothelium and mesenchyme [63, 64], whereas Sox1  or Zic3  induce neural tissues. In contrast, overexpression of the five nephrogenic transcription factors failed to trigger pronephros differentiation in animal caps (Figure 4 and 5). However, as we used only the common pronephric markers 4A6 and 3G8, we cannot exclude that some other pronephric differentiation products are induced.
In other experiments the failure of injected transcription factor to induce differentiation in animal caps could be overcome by adding growth factors. For example the induction of neural crest differentiation by Pax3 and Zic1 requires Wnt signaling  or Neptune induces erythropoiesis only together with GATA1 and bFGF . Concerning pronephros differentiation the ectodermal character of the animal pole cells has possibly first to be changed to a mesodermal fate by adding mesoderm inducing factors. Hence, we added activin A to OSR1 treated animal caps, but without enhancing pronephros differentiation of animal caps (Figure 4C). Since Osr1 has essential functions at the beginning of kidney development [26, 69] and the ability to induce the three early nephrogenic transcription factors hnf1b, lhx1 and pax8 in vivo , its inability to induce pronephros differentiation is striking. However, murine cells expressing Osr1 although multipotent and necessary to build the metanephric precursors require signals from the surrounding tissues for kidney development . Similarly in zebrafish osr1 is required to limit endoderm differentiation to allow kidney development . Since OSR1 did not improve the differentiation potential of activin A and retinoic acid treated animal caps (Figure 4C), other signalling molecules are missing. In this context it may be relevant that ectopic kidney tissues in OSR1 (Figure 3B), osr2 (Tena et al., 2007) or lhx1 and pax8 [23, 25] overexpressing embryos were found exclusively close to the pronephros. This suggests that signals in the region of pronephros anlage are needed. Most recently Wnt11b has been proposed as such a signal .
In our experiments we overexpressed either the human or murine transcription factors in order to distinguish the activity of the endogenous gene from the injected RNA, because previous experiments have shown equivalence between Xenopus and human hnf1b. This we confirmed in principle for OSR1 and Osr2A (Figure 3) as well as for coinjected LHX1 and PAX8 (see text). However, in contrast to the Xenopus factors, human LHX1 coinjecetd with PAX8 cannot induce ectopic pronephric tissue and such subtle differences may limit the use of mammalian factors in Xenopus embryos.
Investigating the influence of HNF1B on 26 potential hnf1b target genes we could show the activation of nine genes in injected animal caps (Table 1). The induced genes include the transcription factor lhx1, hnf1a, hnf4a and tfe3 suggesting the activation of various transcriptional cascades. This assumption is supported by the observation that the lhx1 target genes cer1  and chrd , both with important roles in nephrogenesis, were also induced. Since other lhx1 target genes were not activated (Table 1), we assume that some lhx1 target genes are inhibited by HNF1B expression. It is striking that cer1 is induced at very high levels and peaked off within seven hours indicating a transient activation of cer1 by lhx1 which itself is only transiently induced by HNF1B (Table 1). The activation of hnf1a was expected, as HNF1B expression in the Xenopus embryos activates hnf1a  and the hnf1a promoter contains a functional HNF1 site . Since HNF1B induces the expression of osr1 and osr2 we deduce a positive feedback loop, as osr1 and osr2 are able to induce hnf1b . Furthermore, an increased expression of the signalling molecules wnt11b  and gdnf  were found, both of which play a role in nephrogenesis. Similarly, the downregulation of the fibroblast growth factor receptor (fgfr4c) may be relevant, as fgfr4c down-regulation is needed to allow pronephros development . The delayed induction of two other important molecules in nephrogenesis, pax2  and esd , implied an indirect activation. The fact that several genes crucial for pronephros development and expressed later in more differentiated pronephric tissues were not activated in animal caps by HNF1B, may either reflect that hnf1b acts differentially in Xenopus compared to mammals or more likely other signals are missing in the undifferentiated animal cap. Albeit our data show that HNF1B regulates in embryonic cells many genes potentially essential for pronephros development.
A function of Hnf1b in early nephrogenesis has most recently been reported also in mice by tetraploid and diploid embryo complementation to overcome early embryonic lethality of homozygous knock-out mice . Significantly, in these mice Lhx1 was shown to be regulated by Hnf1b and to contain an HNF1 binding site in the far upstream promoter. Thus, the regulation of lhx1 by hnf1b seems to be evolutionary conserved, as a 10-fold increase in the expression level of lhx1 in HNF1B differentiated animal caps is seen (Table 1) and this induction is mediated by an HNF1 binding site in the Xenopus lhx1 promoter region (Figure 6).
The myc-Rc/CMVHNF1B expression vector has been described previously . The mouse Osr2A plasmid was kindly provided by S. Kawai (Osaka University Graduate School of Dentistry, Osaka, Japan). The full-length open reading frame of the human gene OSR1 (IRATp970D0444D6; RZPD, Gemany) was cloned into pCS2+MT  to generate N-terminal myc-tag fusion proteins using 5'-GCTCTAGAGATGGGCAGCAAAACCTTGCC-3' and 5'-GCTCTAGATTAGCATTTGATCTTGGAGGTTTT-3' as forward and reverse primers, respectively. The XbaI restriction sites for cloning are underlined and the construct was verified by sequencing. The full-length open reading frames of the human PAX8 (RC200651) and LHX1 (RC210977) in pCMV6 were obtained from OriGene Technologies.
The pRL-Con renilla luciferase construct  and the HNF-4a P2-285 pGL3-Basic reporter plasmid  have been described. The lhx1 gene luciferase fusion constructs Ex-1:A, Ex-2:C, Ex-2:D and Ex-5:B  were kindly provided by M.L. Rebbert (NICHD, USA). The lhx1 reporter construct harbouring the HNF1 binding site in the promoter region (Ex-2:C) was used for additional constructs. XhoI/HindIII DNA fragments containing the intact or mutated HNF1 binding site in the promoter region to the transcription start generated by PCR using the primers Ex1(-120/+3): 5'-CCGCTCGAGGCTTAATGGTT-3' (forward), Ex1(-117/+3): 5'-CCGCTCGAGGGTTTACCAG-3' (forward) and 5'-CCCAAGCTTTCCCTTTGGTTAT-3' (reverse) were inserted into the XhoI and HindIII digested pGL3-Basic vector (Promega). The restriction sites for cloning are underlined. Both constructs were verified by sequencing.
The expression vectors encoding OSR1, Osr2A, HNF1B, LHX1 and PAX8 proteins and the GFP encoding expression vector (pCSGFP2) were linearized and in vitro transcribed with RNA polymerases (Nielsen and Shaprio, 1986). The restriction enzymes and RNA polymerases used are given in Additional file 1. Capped mRNA encoding the different proteins together with 100 pg of capped green fluorescent protein (GFP) mRNA as internal control were injected into one blastomere of the two-cell stage and after two days, the injected side was scored under a stereofluorescence microscope for the presence of GFP. In case of animal explants, the capped mRNA together with GFP is injected into the animal region of Xenopus embryos in each blastomere of the two-cell stage.
Animal cap assays
Xenopus late blastulae, stage 9 , were de-jellied by treatment with 2% cysteine hydrochloride in water. The presumptive ectoderm (animal cap) was isolated with loops of 20 μm platinum wire heated to about 450°C for a few microseconds using the Gastromaster (Xenotek Engineering, Belleville, USA). The explants were incubated for three hours in Steinberg's solution (58 mM NaCl, 0.67 mM KCl, 0.34 mM Ca(NO3)2, 0.83 mM MgSO4, 3 mM HEPES, pH 7.8) containing recombinant human activin A (10 ng/ml; Sigma, A4941) and all-trans retinoic acid (10-4M; Sigma, R2625) or only in Steinberg's solution for controls and for explants from mRNA injected embryos. After three-times washing with Steinberg's solution the explants were cultured in Steinberg's solution at 20°C until they were equivalent to stage 40-42 in normal embryos (four days) and used for whole-mount immunostaining.
RNA from pools of 30 animal caps was isolated with peqGold RNAPure (PeqLab) followed by phenol/chloroform extraction. For cDNA synthesis the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) was used. SYBR-Green real-time PCR was performed on a 7900HT Sequence Detection System (Applied Biosystems) using Power-SYBRGreen Mix (Applied Biosystems). Templates were determined in duplicate and for primers used see Additional file 2. Results are normalized to ornithin decarboxylase (odc) expression levels. In all cases water only and reverse transcriptase negative controls failed to produce specific products. The fold induction of the early transcription factors was obtained by comparison of treated and untreated animal caps.
Whole-mount immunostaining of four day cultured animal caps or animals at the swimming larval stage were done as described . The difference between the injected and the non-injected sides of embryos was evaluated by measuring the whole area using the lateral view with the widest diameter from the dorsal to the ventral side of the immunostained pronephros including the pronephric tubules and the anterior part of the pronephric duct. The measurements were made using AxioVision 4.6 software (Carl Zeiss Imaging Solutions), and the non-injected side was used as a reference for each animal. The values representing kidney size obtained from each mRNA injected embryo were compared to values obtained from GFP control-injected embryos (data adapted from ). Significant differences were scored using the Student's t-test to calculate p-values.
Cell culture and transient transfection assays
The HEK293 (HNF1B) cell line  contains a tetracycline-inducible HNF1B transgene. In a 96-well plate (17,500 cells/well) 30 ng of the promoter constructs were cotransfected using FuGeneHD (Roche) with 0.05 ng of renilla luciferase plasmid pRL-Con for normalization of transfection efficiencies. Four hours after transfection HNF1B expression was induced by the addition of 1 μg/ml doxycycline. Twenty-four hours after transfection firefly and renilla luciferase activities were measured in triplicate with the Dual-Luciferase Reporter Assay (Promega).
Luciferase reporter assays in animal caps
50 pg reporter constructs and 150 pg HNF1B mRNA were coinjected into the animal region of Xenopus embryos at the two-cell stage together with renilla luciferase as internal control. Animal caps were cut, cultured in Steinberg's solution for four hours and pools of four caps were assayed by the Dual-Luciferase Reporter Assay (Promega). As the absolute levels of luciferase activity varied between pools of animal caps (data not shown, also described previously ), the Mann-Whitney-test was used to score significant differences.
We thank Christoph Waldner for critical reading of the manuscript, Elizabeth A. Jones for the antibodies 3G8 and 4A6, Martha L. Rebbert for the lhx1 luciferase reporter constructs and S. Kawai for the Osr2A plasmid. We also thank Ludger Klein-Hitpass for instruction on quantitative RT-PCR analysis and Tanja Boes for statistical advice.
Parts of this work were supported by the Deutsche Forschungsgemeinschaft [Graduiertenkolleg 1431/1].
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