Division of Nephrology and Hypertension and Division of Developmental Biology, The Childrens Hospital Research Foundation, Cincinnati, OH 45229, USA
*Author for correspondence (e-mail: steve.potter{at}chmcc.org)
Accepted March 23, 2001
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SUMMARY |
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Key words: Organogenesis, Kidney, Renal, Branching, Development, Hox, Metanephros, Mouse
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INTRODUCTION |
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Hox genes are a well described family of clustered genes that encode homeodomain-containing transcription factors. Relatively little is known about the role of Hox genes during organogenesis. We have described morphological changes within the reproductive tract that suggest the Hox genes regulate positional information during organogenesis (Hsieh-Li et al., 1995; Gendron et al., 1997). During neurogenesis, Hox genes have been shown to control both dorsoventral and anteroposterior patterning of the hindbrain (Davenne et al., 1999). In the developing gut, it has been demonstrated that Hoxd13 regulates regionally restricted inductive signaling (Roberts et al., 1998). Over 15 Hox genes are expressed in the developing kidney (Davies and Brandli, 1997 The Kidney Development Database http//mbisg2.sbc.man.ac.uk/kidbase/kidhome.html and http://golgi.ana.ed.ac.uk/kidhome.html.). These genes may control cell proliferation, inductive signaling pathways and/or anteroposterior-dorsoventral patterning of the kidney or its multiple components. We have shown that two members of the family, Hoxa11 and Hoxd11, exhibit functional redundancy in formation of the kidney, as well as the forelimb and vertebra (Davis et al., 1995). Homozygous mutant mice for either Hoxa11 or Hoxd11 have normal kidneys; however, the kidneys of the double homozygous mutants are absent or rudimentary.
To better understand the function of Hoxa11 and Hoxd11 in kidney development, we defined their expression domains and examined early renal developmental anomalies of Hoxa11/Hoxd11 double mutant mice. Although Hoxa11 and Hoxd11 expression was limited to the mesenchyme in the early developing kidney, the most striking mutant defect was in the pattern of ureteric bud branching morphogenesis. This establishes a role for Hoxa11/Hoxd11 in mesenchyme for patterning ureteric bud branching morphogenesis. Expression analysis of the mutant renal mesenchyme demonstrated an altered mesenchymal character. There was loss of expression of genes that are crucial for ureteric bud morphogenesis, as well as diminished expression of genes that are normally induced in the mesenchyme by the ureteric bud. These findings support the hypothesis that Hox genes control pattern formation in kidney development by promoting mesenchyme-epithelial reciprocal inductive signaling.
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MATERIALS AND METHODS |
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Immunostaining
Embryonic day 10.5, 11.5 and 13.5 embryos from CD-1 and mutant mice were snap frozen in embedding media and sectioned on a cryostat. Serial sections were stained with antibodies to Hoxa11, Pax2, cytokeratin (Sigma) and phosphorylated histone H3 (Upstate Biotechnology 06-570), following the procedure of Dressler and Douglass, 1992 (Dressler and Douglass, 1992). The antibody to Hoxa11 has previously been described (Gendron et al., 1997). Polyclonal rabbit antibody from immune and preimmune serum was purified from a protein A column. Sections (6 µm) were fixed for 5 minutes with 3% paraformaldehyde in PBS. The sections were then permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 10 minutes and washed in PBS 0.05% Tween (PBST). The primary antibodies were diluted 1:100 to 1:200 in 2% goat serum PBST and incubated with the sections for 1 hour. The slides were washed in PBST and incubated with rhodamine- or fluorescein-conjugated secondary antibody diluted in goat serum PBST.
Dissected embryonic urogenital blocks from double heterozygous Hoxa11/Hoxd11 matings were processed for whole-mount antibody detection of cytokeratin. The tissue was fixed in methanol and incubated 4-5 hours in 2% goat serum PBST with 1:100 dilution of anti-pan cytokeratin (Sigma C2562). After washing, the tissue was incubated with FITC-conjugated anti-mouse IgG secondary antibody (Sigma F2012) in PBST (2% goat serum). Photographic slides were taken with an Olympus BHS model microscope with a reflected light fluorescence attachment. The slides were scanned into Adobe Photoshop.
Lectin staining
Mutant and normal urogenital blocks from E13.5 embryos were stained with fluorescein-conjugated Dolichos biflorus agglutinin (DBA, Vector). The tissue was dissected and fixed for 10 minutes in 2% paraformaldehyde in PBS, treated with 3% bovine serum albumin (BSA) in PBS 0.05% Triton X-100 for 1 hour at 37°C, and incubated with DBA 1:40 in PBS Triton for 1 hour. After washing, the tissue was transferred to a slide for photography. So as not to distort the structures, care was taken not to compress the tissue with a coverslip.
Whole-mount in situ hybridization
Mutant and normal urogenital blocks were dissected and hybridized with riboprobes as described previously (Wilkinson, 1992; Hogan, 1994). Wnt11 probe was obtained from a 782 bp PvuII fragment of EST 349486 (Research Genetics). The pCMV Pax2 construct, kindly provided by Gregory Dressler, was used to obtained a 575 bp XbaI/BamHI Pax2 riboprobe (Dressler et al., 1993). A 399 bp cDNA fragment was amplified using tgtccagtgtggagaactttactg and ctctacacctcaaaaagggcttag primers, cloned and used for a Bf2 riboprobe. The Wnt7b EST clone 334147 contained a 530 bp fragment that was used as a riboprobe. The Wt1 and Gdnf riboprobes were kind gifts from Andreas Schedl (from Buckler et. al., 1991) and Frank Costantini (Srinivas et. al., 1999), respectively. A 504 bp EcoRI/SmaI fragment was subcloned from pSHlox Hoxd11 and used for Hoxd11 riboprobe (a gift from M. Todd Valerius). Clones for Hoxd10 and Hoxd12 riboprobes were generously provided by Denis Duboule (Dolle et al., 1989), and those for Hoxc10 and Hoxc11 by Alexander Awgulewitsch (Peterson, 1994). Whole-mount tissues were post-fixed in paraformaldehyde, embedded in paraffin, sectioned and counterstained with nuclear Fast Red (Vector Laboratories).
Embryonic proliferation and apoptosis
Day 13.5 urogenital blocks were snap frozen and parasagital sections stained. At least four sections were stained for proliferation or for apoptosis. Antibody to phosphorylated histone H3 was used to detect mitotic cells (Correia and Conlon, 2000; Wei et al., 1999). Tissue was also stained with antibody to cytokeratin and with DAPI to identify the epithelium of the branching ureteric bud and all nucleated cells, respectively. Digital images were obtained on an Olympus BX60 microscope for each marker and were merged using Photoshop before counting nuclei. Between 1800 and 3000 nucleated mesenchymal cells per section were counted and the number of mitotic mesenchymal cells expressed as a fraction of the total number of cells. The proportion of proliferating cells on the dorsal and ventral halves of the kidney was also determined. Sections for apoptosis were first stained with antibody to cytokeratin as above, then post-fixed in 4% PFA for 15 minutes. Following the in situ cell death detection kit protocol (Roche), the tissue was permeabilized for 2 minutes at 4°C with 0.1% Triton and 0.1% sodium citrate. After washing, apoptotic cells were fluorescein labeled using terminal transferase for one hour at 37°C. The slides were then stained with DAPI. Pyknotic cells, ureteric bud epithelium and all nucleated cells could then be detected and counted by fluorescence microscopy. Similar to mitotic cells, the proportion of apoptotic cells was determined.
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RESULTS |
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To better understand the origin of the double mutant phenotype, we examined ureteric bud formation and branching morphogenesis in mutant kidneys during early organogenesis. In total, 66 double homozygous mutants were examined. At E11.5, mutants, like control littermates, showed a single outgrowth from the caudal segment of the Wolffian duct (Fig. 1A,B). At E13.5, however, all double mutants exhibited defects in branching morphogenesis. The severity was variable, with less affected mutants (Fig. 2B,C) showing long intervals between the initial and subsequent branches. In the severely affected mutants, the outgrowths remained unbranched though they were more elongated than the initial E11.5 bud and they still maintained a rounded tip. The mesenchyme surrounding these outgrowths of the nephric duct was not condensed around the tip and was easily fragmented (Fig. 2D). Thus, the earliest identified renal phenotype was impaired branching morphogenesis of the ureteric bud while ureteric bud growth or elongation remained intact.
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Wnt7b expression in Hoxa11/Hoxd11 mutant kidneys
Hoxa11 and Hoxd11 were expressed in the metanephric mesenchyme of the developing kidney, while the primary observed morphological defect in Hoxa11/Hoxd11 double mutants was in the branching of the ureteric bud. This suggested that the mutant mesenchyme did not properly signal the ureteric bud. Three different models could explain this altered metanephric mesenchyme-ureteric bud interaction. First, as observed in the adjacent paraxial segmented mesoderm, these Hox genes may pattern the anterior-posterior axis of the intermediate mesoderm. In this case, loss of Hoxa11 and Hoxd11 would cause a homeotic transformation of segment identity resulting in anteriorization or conversion of the metanephros into mesonephros. Second, Hoxa11 and Hoxd11 may simply regulate proliferation of the mesodermal segment that gives rise to the primordium of the metanephric mesenchyme. Reduced mass of mesenchymal tissue would then perhaps result in reduced signaling. Finally, these Hox genes may simply be viewed as upstream regulators of mesenchyme differentiation or of specific morphogens that are required for normal bud growth and branching.
The metanephros and mesonephros are morphologically distinct. Features that distinguish the normal anterior primitive mesonephros and the posterior mature metanephros are the number of outgrowths from the Wolffian duct, their length and the subsequent branching of those outgrowths. The mesonephros contains multiple short outgrowths from the duct that do not branch, while the metanephros has a single bud that arborizes. As shown earlier, there was a single outgrowth from the Wolffian duct in the mutant mice that sometimes branched. The absence of branching or rudimentary branching in the mutant was consistent with a mesonephric-type outgrowth from the caudal end of the Wolffian duct. Therefore, we characterized this outgrowth using a molecular marker specific for the metanephric bud.
Wnt7b is expressed in the caudal segment of the Wolffian duct and the ureteric bud of the metanephros, but is not detectable in the mesonephros. In normal E13.5 mice, Wnt7b expression was detected in the ureter leading to the metanephric kidney and in the branches within the developing kidney (Fig. 4A). In situ hybridizations of double homozygous mutant littermates showed Wnt7b expression in the caudal outgrowth from the Wolffian duct, suggesting that the duct maintained metanephric identity (Fig. 4B). The ureteric bud of the mutant again elongated without branching and appeared more tortuous than normal. This molecular characterization argues that mutation of the Hoxa11 and Hoxd11 genes did not result in homeotic transformation of the metanephros into mesonephros.
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Mesenchyme analysis
We further studied the nature of the mutant mesenchyme using molecular markers for stromal precursor mesenchyme (Bf2; Hatini et al., 1996), induced mesenchyme (Pax2; Dressler and Douglass, 1992), early undifferentiated mesenchyme (Wt1; Armstrong et al., 1993) and a mesenchyme signaling factor (Gdnf; Pichel, 1996). The early metanephric mesenchyme gives rise to two distinct cell lineages, the nephrogenic lineage, which forms the nephrons, and the stromal lineage, which gives rise to the renal interstitial mesenchyme. In situ hybridization with Bf2 probe was used to determine if the ventral cells differentiated into stromal precursor cells. Surprisingly, there was no Bf2 expression on the mid-ventral surface of the mutant kidney (Figs 6F, 7B). Therefore, not only is the nephrogenic mesenchyme not replaced by stromal precursor mesenchyme, but Hoxa11/Hoxd11 function is required for normal stromal cell differentiation on the ventral surface of the developing kidney. Whether this is due to absence of bud tips or directly to loss of Hoxa11 and Hoxd11 is not known.
Pax2 is normally expressed in the ureteric bud, its branches and within the induced mesenchyme. Normally, cortical mesenchyme Pax2 expression obscures underlying Pax2-positive ureteric bud branches after whole-mount in situ hybridization. Hybridizations with Pax2-specific probe showed limited induction in mutant metanephric mesenchyme. The E13.5 mutant kidney ventral surface shown in Fig. 6I, for example, contained only a single area of induced mesenchyme. This in situ further demonstrated the abnormal branching pattern of the ureteric bud in mutants. By E13.5 the ureter should insert into the kidney medially; however, a lateral insertion was found in the mutant kidneys. In addition, the primary branches failed to form the normal initial series of dichotomous branches. It should also be noted that areas of induced mesenchyme were greatly reduced even on the dorsal surface of the mutant kidney, despite evidence of ureteric bud branching (Fig. 6H). Thus, in the absence of Hoxa11/Hoxd11, reciprocal inductive interactions are reduced. The mesenchyme to bud signaling was deficient, resulting in defective bud branching and the mesenchyme induction in response to bud signaling was poor, as measured by Pax2 expression. These results provide evidence that the Hoxa11/Hoxd11 genes control more than just proliferation of the mesenchyme during kidney development. The expression of Wt1 was globally reduced in the mutant kidney, on both dorsal and ventral surfaces (Fig. 6K,L), further illustrating the altered nature of the mutant metanephric mesenchyme. Not unexpectedly, decreased Gdnf expression was seen on the ventral surface of the mutant kidney and could explain the branching defect (Fig. 6J).
Dorsoventral renal patterning
In an attempt to explain the dorsoventral pattern, we examined the normal mesenchymal expression patterns of several other Hox genes in the kidney at E13.5. We found approximately equal ventral and dorsal expression of Hoxc10, Hoxc11, Hoxd10 and Hoxd12 (Fig. 8). Thus, we were unable to explain the apparent dorsal compensation for loss of both Hoxa11 and Hoxd11.
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DISCUSSION |
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Defective branching morphogenesis of the ureteric bud in mutant kidneys
Hoxa11 and Hoxd11 showed similar patterns of expression, present in the early metanephric mesenchyme, later surrounding the invading and branching bud, and down-regulating during mesenchyme-to-epithelia conversion. The most striking developmental alteration in the double mutant kidney, however, was defective branching morphogenesis of the ureteric bud, which did not express Hoxa11/Hoxd11. This suggested defective signaling from the mesenchyme to the ureteric bud.
The function of Hox genes in vertebrate development has been a controversial subject. In Drosophila, however, it is generally agreed that Hom-C (Drosophila Hox) genes have an important patterning function. Null mutations in Drosophila Hox genes often result in anteriorizations of segment identity, while misexpression mutations often convert structures to a more posterior identity (for a review see Manak and Scott, 1994). The roles of Hom-C genes during Drosophila organogenesis have also been examined. For example, they pattern the gut along the anterior-posterior axis. Spatially restricted expression of Hom-C genes within the gut mesoderm is required for mesoderm-endoderm interactions and the formation of constrictions between the four chambers of the midgut (Reuter et al., 1990; Immergluck et al., 1990; Capovilla et al., 1994). The Hom-C genes of the mesoderm appear to control the expression of the transforming growth factor ß family member dpp, which then serves to induce labial expression in the endoderm.
In vertebrates, however, it has been suggested that the Hox genes function in a manner that is surprisingly different from that observed in flies. Duboule has proposed that all mammalian Hox genes regulate cell proliferation and not cell identity (Douboule, 1995). In its extreme form this model states that all mammalian Hox genes control identical or functionally equivalent downstream targets that are involved in the regulation of cell proliferation. This model is consistent with the observations that Hox proteins have similar in vitro DNA target binding specificities, single Hox gene knockout mice have relatively mild phenotypes, and double knockout mice sometimes have reduced or absent structures (Davis et al., 1995). It is also consistent with the results of a number of studies that suggest at least some role for Hox genes in controlling cell proliferation rates (Goff and Tabin, 1997).
To better understand the nature of the morphogenic defects in the Hoxa11/Hoxd11 mutant kidneys additional gene expression studies were performed. Even when unbranched, the ureteric buds of mutant embryos did not lose metanephric identity and continued to express Wnt7b, a marker of the metanephros. We were thus unable to detect evidence of a homeotic transformation of metanephros to mesonephros, one potential result of loss of Hox gene function.
Not only was there a defect in branching, but in situ hybridization with ureteric bud tip specific riboprobes demonstrated that there was a distinct patterning defect. In the most severely affected kidneys the bud completely failed to branch. In the less severely affected kidneys, however, there was a relatively normal distribution of bud tips on the dorsal surface and at the anterior and posterior poles, but an absence of bud tips in the mid-ventral region of the kidney. This demonstrates a biologically important difference between cells along the dorsoventral axis of the kidney. In the ventral domain, which because of rotation was formerly the lateral domain, Hoxa11/Hoxd11 expression is rigidly required for branching to occur. In contrast, in the dorsal domain, formerly the medial domain, near normal branching can often take place, even in the absence of Hoxa11/Hoxd11 function. To our knowledge, this is the first reported observation of a dorsoventral axis in the kidney. Review of the literature indicates that branching morphogenesis and tubulogenesis are normally synchronous in all quadrants of the kidney. It is therefore unlikely that our observations represent an accentuation of a lag in development of the ventral aspect of the kidney. A functional difference also has been reported in the cells along the anteroposterior axis of the kidney, as defined by differing responses to bone morphogenetic protein 4 in organ culture (Raatikainen-Ahokas et al., 2000). With over 15 Hox genes expressed in the developing kidney, it is possible that dorsoventral or anteroposterior Hox gene gradients could control development of the renal axes and compensate for the absence of Hoxa11/Hoxd11 in the dorsal domain.
A second potential effect of Hoxa11/Hoxd11 loss of function is a diminished proliferative capacity of the mesenchyme. Because of the dorsoventral defect, we were not only able to compare mutant with wild-type kidneys, but also severely affected ventral mutant mesenchyme to the more normal dorsal mesenchyme. Even with this advantage, we were unable to detect a change in proliferation. Similarly, we were unable to find a change in apoptosis. Absence of change in proliferation or apoptosis and the observed qualitative difference in the ventral domain of the mutant kidney argues against a proliferation restricted function for Hoxa11/Hoxd11. The developing mutant mesenchyme was not simply reduced in size. Some regions of the mutant mesenchyme remained able to induce budding, while others did not. These results contrast with other reports such as the description of the Myc mutant embryos kidneys, where the kidneys were hypoplastic, had reduced proliferation, but maintained normal structure (Moens et al., 1993; Bates et al., 2000). In this case, Myc was thought to play a role in proliferation without disturbing pattern.
Additional defects in mutant metanephric mesenchyme
Perturbation of mutant mesenchyme differentiation was detected by in situ hybridizations. First, the expression level of Wt1 was severely reduced in all regions of mutant mesenchyme, further indicating altered character and not just reduced mass. Along with the reduced Wt1 expression, we also found decreased Gdnf expression in the ventral mesenchyme. Hoxa11/d11 appear to be upstream of very early events in intermediate mesoderm differentiation. Pax2 expression in mesenchyme was also reduced, even in regions where relatively normal bud branching was observed. In the early developing wild-type kidney, Pax2 expression is found initially in the ureteric bud, which then induces Pax-2 expression in the metanephric mesenchyme. This reduced mesenchymal expression of Pax-2 in mutants therefore suggests a defective response to ureteric bud induction.
We also examined the expression of the winged-helix transcription factor Bf2, a stromal lineage marker, in the mutant metanephric mesenchyme. There was no Bf2 expression in the ventral domain of the mutant metanephric mesenchyme, indicating that the failure of this mesenchyme to induce branching morphogenesis of the bud was not simply the result of a decision en masse to form stroma instead of nephrogenic mesenchyme. Similarly, the nephrogenic mesenchyme did not simply fail to proliferate, resulting in its replacement by stromal mesenchyme. The reduced Bf2 expression in the mutant mesenchyme is also of interest because of the similarities between the Bf2 and Hoxa11/Hoxd11 mutant phenotypes. In each case there is a striking defect in branching morphogenesis (Hatini et al., 1996). These observations suggest that Bf2 lies downstream of Hoxa11/Hoxd11 in kidney development, and that, in addition to altered Gdnf expression, the altered Bf2 expression in Hoxa11/Hoxd11 mutants at least partly accounts for the observed kidney phenotype.
Finally, based on expression analysis, we found no candidate Hox gene that could account for compensation on the dorsal surface of the mutant kidney. In situ hybridization showed roughly equivalent dorsoventral levels of expression for each of the four Hox genes tested. Despite ventral expression of several Hox genes, total compensation for loss of Hoxa11 and Hoxd11 in the mutant did not occur. The overlapping expression of these and perhaps other Hox genes raises a provocative question: what is the function of each individual Hox gene in renal development? As in C. elegans, the individual Hox genes may serve multiple functions, including proliferation and differentiation (Salser and Kenyon, 1996). In aggregate, the Hox genes may control proliferation within the kidney thus accounting for the apparent preservation of mesenchymal proliferation in the mutant ventral mesenchyme. Hoxa11 and Hoxd11 appear to have attained a separate and distinct function during renal mesenchyme differentiation. As a result, the reciprocal inductive interactions between the metanephric mesenchyme and ureteric bud are greatly attenuated in the kidneys of mutant embryos. Elucidation of downstream effectors of these and other Hox genes will greatly aid in our understanding of their function. Additionally, detailed examination of kidneys in double knockouts of other paralogous Hox genes will enhance our understanding.
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ACKNOWLEDGMENTS |
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