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*
INSERM U 489 and University of Paris VI, AP-HP, Paris, France
The University of Texas, M.D. Anderson Cancer Center, Houston,
Texas.
Correspondence to Dr. Jerome A. Rossert, INSERM U489 and Department of Nephrology, Tenon Hospital, 4 rue de la Chine, 75020 Paris, France. Phone: 33-1-56-01-60-29; Fax: 33-1-56-01-69-99; E-mail: jerome.rossert{at}tnn.aphop-paris.fr
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As for many other structures, the combinatorial action of different cell-specific transcription factors is very likely to play a critical role in the development of the Wolffian duct and of the ureteric bud. Different transcription factors such as Emx2, Pax-2, Lim-1, and GATA-2 are expressed in the ureteric bud and are involved in kidney development (reviewed in reference 2). Emx2 is expressed predominantly in the ureteric bud, and in mice that lack Emx2, the ureteric bud invades the metanephric mesenchyme but does not branch and never induces mesenchymal cells to condense (3,4). Pax-2 is expressed both in the ureteric bud and in the metanephric mesenchyme; mice that lack Pax-2 do not have a ureteric bud, whereas hemizygous mice have hypoplastic kidneys (5). Lim-1 is expressed in the ureteric bud and its derivatives and in the developing nephrons (6). The very few Lim-1 null mice that survive until birth lack kidneys (7). GATA-2 is expressed in different tissues, including the ureteric bud, and mice that do not express GATA-2 selectively in the developing kidney display an abnormal junction between the ureter and the bladder (8). A few other genes that encode transcription factors such as GATA-3 or Hoxb-7 are also expressed in the developing ureteric bud (9,10), but their role in kidney development is still unknown.
Zinc finger proteins have emerged as a major class of eukaryotic transcription factors. They are characterized by their DNA-binding domain containing cysteine or histidine residues that bind zinc atoms, and they can be divided into different subgroups depending on the amino acid residues that are important for zinc binding and on the spacing between these amino acid residues. For example, factors that belong to the subfamily of Krüppel-like transcription factors have two cysteine and two histidine residues that bind the zinc ion, and the consensus sequence of their zinc finger motif is Cys-X2-4-Cys-X12-His-X3-4-His (reviewed in reference 11). This subfamily, which is itself part of the TFIIIA subclass (reviewed in reference 12), includes ubiquitously expressed transcription factors but also transcription factors that have a restricted pattern of expression and that can play important roles during organ differentiation, such as EKLF, LKLF, or GKLF/EZF (13,14,15,16,17,18).
We report here the characterization of a mouse cDNA encoding a zinc finger protein, called Klf6, which belongs to the Krüppel-like family of transcription factors. Northern blot analyses and in situ hybridization experiments showed that the corresponding gene was expressed early during embryonic development and had a restricted pattern of expression. In particular, during kidney development, the expression of Klf6 was restricted mostly to the collecting duct system, which suggests that this transcription factor may play a role during the development of the renal excretory system.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 
gt10 library from 16.5-d p.c. mouse embryos (Clontech, Cambridge,
UK) was screened using the cDNA obtained by PCR as a probe. Among positive
clones, two were cloned in pBluescript KS and entirely sequenced. A 290-bp
fragment derived from the 5' end of one of these clones was then used to
screen again the same library. Three positive clones were isolated and
sequenced.
Cell-Free Transcription and Translation Experiments
In vitro transcription and translation of cDNA cloned into
pBluescript KS was performed using a reticulocyte lysate system (Promega,
Madison, WI) according to the manufacturer's instructions, in the presence of
[35S]methionine. The radiolabeled peptides were resolved by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized
by fluorography.
Northern Blot Analysis
Total RNA was isolated from various mouse tissues using an RNeasy kit
(Qiagen, Valencia, CA) and following the manufacturer's instructions. Fifteen
µg of total RNA were then electrophoresed through formaldehyde-agarose
gels, transferred to nylon membranes (Hybond-N, Amersham Pharmacia Biotech,
Piscataway, NJ), and hybridized with a 32P-radiolabeled probe
containing 192 bp of the coding sequence and 308 bp of the 3'
untranslated region, following standard procedures. After 18 to 20 h of
hybridization, high-stringency washes were performed, the last wash being done
at 65°C for 10 min in 0.1 x SSC (1 x SSC is 0.15 M NaCl plus
0.015 M citrate Na), 0.1% SDS. To control for loading of RNA samples, each
membrane was then stripped and reprobed using a 32P-radiolabeled
probe corresponding to the mouse glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) gene. Labeling of the probes with [
-32P]dCTP was
performed using a random priming labeling kit (Roche Diagnostics, Basel,
Switzerland) and following the manufacturer's instructions. In some cases, the
intensity of the signal was quantified using a Storm 860 PhosphorImager
(Molecular Dynamics, Sunnyvale, CA).
In Situ Hybridization
The in situ hybridization study was performed using whole mouse
embryos that ranged from 11.5 to 15.5 d p.c. and using different organs
obtained from newborn mice and from 4-wk-old mice. Embryos and organs were
collected under sterile conditions, fixed in freshly made 4% paraformaldehyde
at 4°C for 4 to 10 h, and embedded in paraffin. Five to 7-µ sections
were made. 35S-radiolabeled Klf6 sense and antisense
riboprobes were synthesized by in vitro transcription of a linearized
pBluescript KS plasmid containing the 500-bp sequence that was used as a probe
in Northern blot experiments. Transcripts were synthesized using the
maxiscript T3/T7 kit (Ambion, Austin, TX) and [35S]UTP, following
the manufacturer's instructions. Before use, the RNA probes were incubated
with 1 U of DNase I for 20 min at 37°C and purified using G50 columns
(Amersham Pharmacia Biotech). Hybridization was performed as described by
Sibony et al. (19).
Briefly, slides were deparaffinized, microwaved for 12 min, refixed in 4%
paraformaldehyde, digested with proteinase K (20 µg/ml), postfixed in 4%
paraformaldehyde, dehydrated through increasing concentrations of ethanol, air
dried, and incubated with the probe (5 x 104 to 5 x
105 cpm/section) overnight at 50°C in a solution containing 2
x SSC, 10% (wt/vol) dextran sulfate, 1 mg/ml denatured salmon sperm DNA
(Roche Diagnostics), 70 mM DTT, and 50% formamide. Washes were then performed
under high-stringency conditions, first in 5 x SSC supplemented with 10
mM DTT, at 50°C, second in 2 x SSC supplemented with 50% formamide
and 10 mM DTT, at 55°C, and then in TNE (10 mM Tris HCl [pH 7.5], 0.5 M
NaCl, 5 mM EDTA [pH 8.0]) at 37°C. The third wash performed in TNE was
done at 37°C for 20 min in a solution that was supplemented with 20
µg/ml RNase A to minimize the background. After the washing steps, the
slides were dehydrated with graded ethanol solutions, coated with NTB2 film
emulsion (Kodak, New Haven, CT), and stored under desiccant at 4°C for 3
to 5 wk. After being photographically developed and fixed, sections were
counterstained with hematoxylin (0.5%) and eosin (1%), observed, and
photographed on an Axioplan 2 microscope (Zeiss,
Göttingen, Germany).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gt10 mouse library for clones containing
the entire open reading frame. Sequencing of positive clones enabled us to
identify a 1.5-kb cDNA with a single open reading frame, which encoded a
protein containing three Cys2-His2 zinc fingers
(Figure 1). This open reading
frame contained a stop codon 4 bp downstream of the third zinc finger, but it
extended up to the 5' end of the cDNA. To identify clones extending
further upstream, we rescreened the same library using a 290-bp probe located
at the 5' end of the cDNA. Three additional clones were isolated and
sequenced, but they did not extend further upstream. A computer-based search
of the dbest database also identified three mouse clones, but they extended at
most 40 bp upstream of the previous cDNA.
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Analysis of the cDNA showed that it contained only one open reading frame,
with three ATG codons located at its 5' end
(Figure 1). The most upstream
ATG and the most down-stream ATG were not in a favorable context to serve as
initiation codons (20). By
contrast, the second ATG codon, which was located 99 bp downstream of the
first one and 12 bp upstream of the third one, was in a favorable context for
translation initiation according to Kozak
(20). It is therefore likely
that the major translation product initiates at this second ATG codon. This
codon is followed by an open reading frame of 846 nucleotides capable of
encoding a 282-amino acid peptide containing three zinc fingers of the
krüppel-like type
(Cys-X2-4-Cys-X3-Phe-X5-Leu-X2-His-X-Arg/Lys-X-His);
only one mismatch (Phe
Tyr) was present in the first zinc finger
(Figure 1) (11). These zinc fingers are
separated by spacers of seven amino acid residues
(Figure 1). The first spacer
corresponds to the consensus sequence Thr/Ser-Gly-Glu-Arg/Lys-Pro-Phe/Tyr-X,
which has been described for Krüppel-like
proteins (11), and the second
one adheres to this consensus sequence with only one mismatch (GluAla).
A comparison of the zinc finger region with other available DNA sequences
confirmed the existence of strong homologies with other zinc finger proteins
of the Krüppel-like class
(Figure 2A). Besides the three
zinc fingers, the protein contains an N-terminal domain (amino acids 29 to
118) that is rich in acidic residues (23.3% of glutamic acid and aspartic
acid) and thus could be a transcription activation domain
(Figure 2B). The central part
of the protein also contains both a serine- and a proline-rich domain (42.2%
of serine and proline residues between amino acids 82 and 152) and a
glutamine-rich domain (30.3% of glutamine residues between amino acids 164 and
196) that could also act as transcription activation domains
(Figure 2B). Just upstream of
the first zinc finger motif, a sequence corresponds to a consensus core
nuclear localization signal (Arg-Arg-Arg-Val-His-Arg). This protein also
contains two potential protein kinase C phosphorylation sites (Thr-Thr-Lys at
position 122 to 124 and Ser-Gly-Lys at position 180 to 182)
(21), suggesting that it may
undergo posttranslational modifications.
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Since we cloned this mouse cDNA, partial human and rat cDNA that code for proteins that are, respectively, 95% and 98% identical to our protein have been isolated (Genbank accession numbers U44975, AB017493, AF001417) (22,23,24). The rat protein has been called Zf9 (24) and the human one CPBP/GBF (22,23), but the Human Gene Nomenclature Committee has recently suggested renaming the latter protein KLF6. Accordingly, we decided to call our protein Klf6, because it is the mouse ortholog of KLF6.
In Vitro Transcription—Translation
To confirm that the predicted protein can be synthesized in an eukaryotic
system, we transcribed in vitro a cDNA containing the entire open
reading frame corresponding to Klf6 and cloned it into pBluescript KS and then
translated it using a reticulocyte lysate. By doing so, we isolated a protein
with the predicted size of approximately 32 kD
(Figure 2C), which is in good
agreement with the predicted molecular mass for Klf6 (31.9 kD).
Expression Pattern of Klf6 in Embryos and Adult Mice
Northern blot analyses using total RNA isolated from mouse embryos ranging
from 11.5 to 15.5 d p.c. and from newborn mice showed that Klf6 was
expressed as soon as 11.5 d p.c. and that its expression persisted all through
embryonic development (Figure
3A). At all stages of development, a single band of approximately
4.5 kb could be detected.
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As a first approach to determine which organs express Klf6, we performed Northern blot analyses of different tissues in newborn and adult mice. In both cases, Klf6 was expressed at high levels in lung and intestine and at lower levels in brain, heart, and kidney (Figure 3B, and data not shown). By contrast, no expression could be detected in liver or spleen (Figure 3B, and data not shown).
Northern blot experiments were also performed using a PhosphorImager to analyze the levels of expression of Klf6 during metanephros development and afterward. They showed that Klf6 was expressed at similar levels in kidneys obtained from 15.5-d p.c. mouse embryos, newborn mice, 10-d-old mice, and 4-mo-old mice (Figure 3C).
The pattern of expression of Klf6 was assessed more precisely by performing in situ hybridization experiments with 11.5-, 12.5-, 13.5-, and 15.5-d p.c. mouse embryos and with tissues from newborn mice (Figures 4,5,6). The cDNA used to synthesize the 35S-radiolabeled sense and antisense riboprobes was identical to the one used to synthesize the probe for Northern blot experiments. In all of the cases, slides that were hybridized with the sense riboprobe did not show any staining (data not shown). A labeling of the lung buds was observed as soon as day 12.5 p.c. (Figure 4B), and from this stage until birth, Klf6 was expressed in the lung buds, in the bronchi, and in the layer of mesothelial cells that covers the lung buds and will form the pleura (Figure 4, B through D, and data not shown). The expression of Klf6 in the digestive tract was very weak at 11.5 d p.c., but it became stronger 1 d later and persisted until birth (Figure 4, and data not shown). It was restricted to epithelial cells lining the lumen of the developing intestine (data not shown). In the nervous system, Klf6 was expressed in some discrete areas of the brain, but it was also strongly expressed in ganglia such as root ganglia or the trigeminal ganglion (Figure 4). Besides these organs, Klf6 was expressed in the peritoneum and in the pericardium (Figure 4). By contrast, no labeling could be detected in spleen, myocardium, muscles, or skeleton (Figure 4). The liver was almost entirely negative, with the exception of a few cells scattered in the organ (data not shown).
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Expression Pattern of Klf6 during Kidney Development
Because of sequence homologies between Klf6 and WT-1, the pattern of
expression of Klf6 during kidney development was analyzed in detail.
At 11.5 and 12.5 d p.c., the Wolffian duct penetrated the mesonephric
mesenchyme, and this mesenchyme was differentiated into epithelial structures
that correspond to primitive nephrons. At these stages, Klf6 was
expressed in the Wolffian duct (Figure 5, A
through F), but it was not expressed in the mesonephric mesenchyme
(Figure 5, A through F). In
particular, mesonephric tubules were negative
(Figure 5F).
At 12.5 d p.c., the ureteric bud sprouted from the distal portion of the Wolffian duct and penetrated into the metanephric blastema, but only one or very few branchings occurred. At this stage, Klf6 was expressed in the ureteric bud not in the metanephric mesenchyme (data not shown).
At 13.5 d p.c., the ureteric bud branched within the metanephric mesenchyme, and this mesenchyme gave rise to comma-shaped and S-shaped bodies. At this stage, Klf6 was expressed in the ureter and in the ureteric bud (Figure 5, G and H, and data not shown). By contrast, it was not expressed in the metanephric mesenchyme (Figure 5, G and H).
At 15.5 d p.c., nephrons at all developmental stages can be observed, the most mature ones being located in the juxtamedullary area. Klf6 was expressed in the ureteric bud, in the developing collecting ducts (Figure 5, I and J), but also in fully differentiated glomeruli (data not shown). By contrast, it was not expressed in tubular structures derived from the metanephric mesenchyme or in noncapillarized glomeruli (Figure 5, I and J). Analysis of glomeruli showed that the labeling was restricted to cells located within the mesangial area, which correspond to mesangial cells and possibly to endothelial cells (data not shown). Klf6 was also expressed in the urogenital sinus, which will form the bladder, and in the urethra (Figure 4D).
At birth, the pattern of expression was similar to the one observed at 15.5 d p.c. Klf6 was detected in mature glomeruli and in the collecting ducts but not in other tubular structures or in the interstitium (Figure 6, A through C, and data not shown).
In kidneys that were isolated from 4-wk-old mice, Klf6 was still strongly expressed in glomeruli and in the cortical portion of the collecting ducts (Figure 6D, and data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1(I) collagen gene
(22,24). Few data are available regarding the expression pattern of Klf6, and in particular its expression has not been studied during embryonic development. In human, Northern blot analyses have shown that CPBP/GBF is expressed at various levels in different malignant cell lines and that it is expressed at high levels in placenta, lung, and arguably pancreas (22,23). In rat, RNase protection assays using adult tissues have shown that Zf9 is expressed at high levels in lung and intestine, as well as in different cell lines, including stellate cells (24). Our experiments showed that Klf6 was already expressed in 11.5-d p.c. mouse embryos and that Klf6 had a restricted pattern of expression. It was expressed at high levels in organs such as lung, intestine, and brain, whereas it was not expressed in other organs such as spleen or skeleton and was very faintly expressed in liver.
In situ hybridization experiments during kidney development showed that Klf6 was expressed during the development of the mesonephros and of the metanephros and that its expression was mostly restricted to the excretory system: it was expressed in the Wolffian duct, in the ureteric bud, and in the collecting ducts, whereas it was not expressed in the mesonephric mesenchyme or in the metanephric mesenchyme or in their derivatives. In addition to the excretory system, Klf6 was expressed only in the mesangial area of glomeruli, this labeling appearing at the capillary loop stage and persisting thereafter. The restricted pattern of expression of Klf6 suggests that it may play a role during kidney development, in particular in the differentiation of the ureteric bud system. A search for promoters that contain a GGNGNGGGN consensus sequence and thus likely to bind Krüppel-like factors (11) showed that this sequence is present in the promoters of genes that are selectively expressed in collecting duct cells, such as the arginine vasopressin type 2 receptor gene, the aquaporin 2 gene, and the aquaporin 3 gene, reinforcing the idea that Klf6 may play a role in the differentiation of collecting duct cells.
Most of the genes that are expressed in the ureteric bud during kidney development, such as Emx2, Lim-1, or Pax-2, have a pattern of expression that is quite different from the one of Klf6; in particular, they are also expressed in tubular structures that are derived from the metanephric mesenchyme (3,5,6). By contrast, the pattern of expression of Klf6 in the kidney is strikingly similar to the one of GATA-3, which encodes a zinc finger protein of the Cys4 class (9). During mesonephros development, GATA-3 is expressed in the Wolffian duct and in its derivatives but not in the mesonephric blastema (9). At the metanephric stage, GATA-3 is expressed in the excretory system and in mesangial cells but not in other structures derived from the metanephric mesenchyme. The similarities between the patterns of expression of GATA-3 and of Klf6 in the developing kidney are reminiscent of previous observations made for two other genes of the same families: GATA-1 and EKLF. Both genes are expressed in erythroid cells, and both proteins cooperate to induce a normal differentiation of red blood cells, probably through direct protein—protein interactions (13,26,27). It is possible that Klf6 and GATA-3 also directly interact to induce a normal differentiation of the collecting duct system. One of the specificities of GATA-3 is that its tissue-specific expression is controlled by a modular arrangement of different cis-acting elements, some of them being located far from the coding sequence (28). In particular, the cis-acting element that is responsible for the expression of GATA-3 in the developing kidney is located approximately 100 kb upstream of the transcription start site (28). As shown by an analysis of sequence tag sites (sequence tag site WI-12084), the human KLF6 gene is located on chromosome 10, less than 10 cm away from GATA-3. Thus, it is tempting to hypothesize that the same cis-acting element controls the renal expression of both genes, as suggested for other genes (29,30).
In conclusion, we have isolated a mouse cDNA that encodes a protein that belongs to the Krüppel-like class of zinc finger transcription factors and that we named Klf6. During kidney development, Klf6 mRNA was expressed selectively in the Wolffian duct, the ureteric bud, the collecting ducts, and the mesangium. This pattern of expression suggests that Klf6 may play a role in the development of the kidney, in particular of the renal collecting duct system. Furthermore, strong similarities between the patterns of expression of Klf6 and GATA-3 during kidney development suggest that these two proteins could interact to regulate renal development.
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