Hoxa11 and Hoxd11 regulate branching morphogenesis of the ureteric bud in the developing kidney

Larry T. Patterson, Martina Pembaur and S. Steven Potter*

Division of Nephrology and Hypertension and Division of Developmental Biology, The Children’s Hospital Research Foundation, Cincinnati, OH 45229, USA

*Author for correspondence (e-mail: steve.potter{at}chmcc.org)

Accepted March 23, 2001


    SUMMARY
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hoxa11 and Hoxd11 are functionally redundant during kidney development. Mice with homozygous null mutation of either gene have normal kidneys, but double mutants have rudimentary, or in extreme cases, absent kidneys. We have examined the mechanism for renal growth failure in this mouse model and find defects in ureteric bud branching morphogenesis. The ureteric buds are either unbranched or have an atypical pattern characterized by lack of terminal branches in the midventral renal cortex. The mutant embryos show that Hoxa11 and Hoxd11 control development of a dorsoventral renal axis. By immunohistochemical analysis, Hoxa11 expression is restricted to the early metanephric mesenchyme, which induces ureteric bud formation and branching. It is not found in the ureteric bud. This suggests that the branching defect had been caused by failure of mesenchyme to epithelium signaling. In situ hybridizations with Wnt7b, a marker of the metanephric kidney, show that the branching defect was not simply the result of homeotic transformation of metanephros to mesonephros. Absent Bf2 and Gdnf expression in the midventral mesenchyme, findings that could by themselves account for branching defects, shows that Hoxa11 and Hoxd11 are necessary for normal gene expression in the ventral mesenchyme. Attenuation of normal gene expression along with the absence of a detectable proliferative or apoptotic change in the mutants show that one function of Hoxa11 and Hoxd11 in the developing renal mesenchyme is to regulate differentiation necessary for mesenchymal-epithelial reciprocal inductive interactions.

Key words: Organogenesis, Kidney, Renal, Branching, Development, Hox, Metanephros, Mouse


    INTRODUCTION
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Renal organogenesis has served as a model system for the study of multiple developmental mechanisms such as branching morphogenesis and mesenchyme-to-epithelial conversion. These processes depend upon the successful integration of inductive interactions between tissues. Inductive events in the kidney are functionally well described (Saxen, 1987) and remain of great interest at the molecular level. Ureteric bud induction from the Wolffian duct and its subsequent growth and branching to form the collecting system occurs in response to signaling from nephrogenic and stromal progenitor mesenchyme. The mesenchyme secreted factors, GDNF (Pichel et al., 1996) and amphiregulin (Lee et al., 1999) are two of probably several soluble morphogens that regulate ureteric bud development. In addition, mutation of an enzyme involved in proteoglycan synthesis (Bullock et al., 1998) and inhibition of extracellular matrix sulfation (Davies et al., 1995) within the nephrogenic mesenchyme demonstrate that normal ureteric bud branching morphogenesis requires extracellular matrix as well. The signaling mechanism for ureteric bud growth and branching does not require mesenchymal-epithelial cell contact, as demonstrated by ureteric bud growth in the absence of mesenchyme (Qiao et al., 1999). The mechanism does require the presence of more than one mesenchymal cell type. With loss of the winged helix Bf2 transcription factor (Foxd1 – Mouse Genome Informatics) from the stromal mesenchyme progenitors, normal ureteric bud growth and branching is impaired (Hatini et al., 1996). The loss of {alpha}8 integrin from the mesenchyme immediately adjacent to the bud also impairs ureteric bud branching morphogenesis (Muller et al., 1997). One distinguishing feature in renal development is the complex process of mesenchymal morphogenesis. After induction of the bud by the mesenchyme, the bud provides signals that promote mesenchyme survival and induce nephronogenesis. The induced mesenchyme condenses, becomes polarized epithelium and forms vesicles. The vesicles then elongate and differentiate to form the mature glomeruli and tubules of the metanephric kidney. In the absence of ureteric bud derived signals, the mesenchyme becomes apoptotic. The processes of branching morphogenesis and nephronogenesis must conform to a set pattern of development.

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.


    MATERIALS AND METHODS
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Breeding and genotyping Hoxa11/Hoxd11 mutant mice
Hoxa11 and Hoxd11 mutant mice have been previously described (Small and Potter, 1993; Davis et al., 1995). The homozygous mice are sterile and heterozygotes have significantly reduced fertility. The colony has been maintained on a mixed genetic background of three strains of mice (C57, C3H and CF1). For this study, 66 mutants were generated by matings between compound heterozygous parental mice. The day when the vaginal plug was observed was considered to be embryonic day (E) 0.5. Embryos with at least one wild-type allele for each gene were used as control normal littermates. The genotypes of all embryos were determined by PCR as before for Hoxa11 (Small and Potter, 1993). PCR genotyping for wild type and mutant Hoxd11 alleles was performed using the oligonucleotide primers: cgctgtccctacaccaagtaccagatccgc, tccagtgaaatattgcagacggtccctgtt and gtttcagcagtgttggctgtattttcccac.

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.


    RESULTS
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Early defects of the Hoxa11/Hoxd11 double mutant kidney
We previously reported the newborn kidney phenotype of mice homozygous mutant for Hoxa11, Hoxd11, or both. The single mutant kidneys appeared morphologically normal, whereas double mutant kidneys were severely reduced in size. These mutant kidneys contained both glomeruli and tubules (Davis et al., 1995).

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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Fig. 1. Normal initiation of ureteric bud in Hoxa11/Hoxd11 mutant embryos. Dissected E11.5 urogenital blocks were stained with antibody to cytokeratin to identify the Wolffian duct and its outgrowths. Single outgrowths (arrows) from the Wolffian duct (wd) were identified in the normal littermate (A) and in the double mutant embryo (B). Scale bar, 0.2 mm.

 


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Fig. 2. Disrupted ureteric bud branching and growth in the Hoxa11/Hoxd11 mutant embryos. Dissected E 13.5 urogenital blocks from mutant embryos (B-D) and normal littermates (A) were stained with cytokeratin (A-C) or DBA lectin (D) to identify the ureteric bud (u) and its branches. An abnormal branching pattern with elongated primary branches is seen in mutants (B,C) compared with the normal pattern (A). A severely affected mutant kidney (D) showed absence of branching in a bud (arrows) dissected free of the nephric duct. Scale bar, 0.2 mm.

 
Hoxa11 and Hoxd11 expression in the developing kidney
To better understand the apparent function of the Hoxa11 and Hoxd11 genes in promoting ureteric bud branching we defined their expression patterns in the early developing kidney. We found that expression of these genes was restricted early in wild type development to the caudal end of the intermediate mesoderm. At E10.5, Hoxa11 was present in the primordial metanephric blastema, adjacent and dorsomedial to the Wolffian duct expansion and at the level of the hindlimb buds (Fig. 3A). On the adjacent section, Pax2 expression was strong in the Wolffian duct and was initiating in the metanephric mesenchyme undergoing induction (Fig. 3B). Also, whereas Pax2 was expressed in the mesonephros, Hoxa11 was absent (data not shown). Later, at E11.5, Hoxa11 was expressed within the metanephric mesenchyme surrounding the branching ureteric bud (Fig. 3C) and at E13.5 it persisted in the nephrogenic mesenchyme around the tips of the bud and to a lesser extent in the stromal mesenchyme. Consistent with Hox gene transcriptional function, Hoxa11 immunostaining was predominately nuclear. Thus, Hoxa11 was a specific early marker of the progenitors of the metanephric mesenchyme prior to ureteric bud outgrowth, with expression restricted to the caudal segment of the intermediate mesoderm.



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Fig. 3. Early renal expression patterns of Hoxa11 and Hoxd11. E10.5 (A,B) and E11.5 (C) embryos were sectioned transversely and stained with polyclonal antibody to Hoxa11 (A,C) or Pax2 (B). Hoxa11 is expressed in the limb bud (lb) and in the posterior intermediate mesoderm (arrowhead), but not in the Wolffian duct (arrow), which stains for Pax2. On E11.5 (C), Hoxa11 can only be detected in the mesenchyme surrounding the ureteric bud (ub) and not in the bud itself. E10.5 (D), E11.5 (E) and E13.5 (F) embryos were stained by whole-mount in situ hybridization with a riboprobe to Hoxd11. At E10.5, staining was found in the posterior hindlimb bud (lb) field and in the intermediate mesoderm (im). The dorsal view of the tissue was taken after removal of the overlying neural tube. An oblique view of an E11.5 (E) whole-mount urogenital block showed expression posteriorly in the metanephros (arrow) and the very posterior tip of the developing gonad (go). At E13.5 (F), expression in the metanephros (m) was consistent with mesenchymal expression that overlaps with Hoxa11 expression. Sections of the Hoxd11 stained kidneys show expression in the mesenchyme (G) around a branch of the ureteric bud (arrow). ad, adrenal; nt, neural tube. Scale bar, 0.1 mm

 
Hoxd11 exhibited a pattern of expression similar to Hoxa11. At E10.5 Hoxd11 was expressed in the intermediate mesoderm adjacent to the hindlimb buds (Fig. 3D). At E11.5 it was expressed in the mesenchyme surrounding the ureteric bud (Fig. 3E) and at E13.5, Hoxd11 expression was maintained in the mesenchyme surrounding the termini of the branching ureteric bud (Fig. 3F,G). We observed no dorsoventral asymmetry in expression of either Hoxa11 or Hoxd11 in the developing kidneys on days E11.5 and E13.5.

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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Fig. 4. The outgrowth from the Wolffian duct of mutant embryos maintained Wnt7b expression that is characteristic of the metanephros. By in situ hybridization, Wnt7b was not detectable in the mesonephros (data not shown). (A) Wnt7b was expressed in the posterior Wolffian duct, in the ureter (arrow), the branches and not the tips of the bud in the normal E13.5 metanephros (m). (B) In the E13.5 mutant kidney, Wnt7b was expressed in the unbranched outgrowth (arrows) of the Wolffian duct. Scale bars, 0.2 mm.

 
Proliferation and apoptosis
Altered mesenchymal growth could explain the mutant phenotype. Immunostaining for phosphorylated histone H3, a marker of mitotic cells, was examined in mutant and normal E13.5 kidneys. We found normal numbers of mitotic cells in mutant kidneys (Fig. 5, Table 1). Additionally, we found the mitotic cells were evenly distributed between the dorsal and ventral halves of the kidneys. Likewise, normal numbers of apoptotic cells were identified by TUNEL assay in mutant kidneys (Fig. 5, Table 1). The apoptotic cells were also evenly distributed between the dorsal and ventral halves. It remains possible that very subtle changes in proliferation or apoptosis, not detected in these studies, contributed to reduction in renal size. The diminished branching of the ureteric bud, however, was probably the main cause of attenuated renal growth during this early stage before obvious nephron development. Further reduced renal size at subsequent stages of development would be expected because of the reduced number of ureteric bud tips needed for nephron induction and later growth of the kidney. Because we did not detect a change in proliferation or apoptosis, we argue that a change in early mesenchymal growth alone cannot account for the defect in branching morphogenesis.



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Fig. 5. TUNEL assay (B,D) and immunostaining for phosphorylated histone H3 (A,C) showed similar rates of apoptosis and mitosis, respectively, in Hoxa11/Hoxd11 mutant (A,B) and normal (C,D) kidneys. Digital photographs for phosphorylated histone H3 (red), cytokeratin (a marker of ureteric bud epithelium, green), and DAPI (a marker for all nuclei, blue) were merged (A,C). Likewise, photographs of the TUNEL assay (green), cytokeratin (red) and DAPI (blue) were merged (B,D). Scale bar, 0.1 mm.

 

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Table 1.
 
Abnormal pattern of ureteric bud branching and metanephric growth in Hoxa11/Hoxd11 double mutants
We further examined the pattern of ureteric bud branching and the distribution of branch termini in Hoxa11/Hoxd11 double mutants by in situ hybridizations with riboprobes for Wnt11, Ret and Emx2. In the normal kidney, Wnt11 and Ret expression is restricted to branch tips, and Emx2 is expressed in a slightly broader domain that extends from the most distal bifurcation to the branch tips. In the most severely affected Hoxa11/Hoxd11 double mutant kidneys, with no branching of the ureteric bud, riboprobes for Wnt11, Emx2 and Ret did not hybridize with the unbranched tip (data not shown). In the less severely affected Hoxa11/Hoxd11 mutant kidneys, however, the branch tips were reduced in number, but still hybridized to Wnt11, Emx2 and Ret. Surprisingly, the mid-ventral E13.5 mutant kidney was devoid of branch termini (Fig. 6C), while the poles and dorsum (Fig. 6B) appeared more normal. Sections confirmed the absence of organized epithelial structures of ureteric bud branches within this field of the kidney (Fig. 7A). This was a reproducible pattern in the less severely affected kidneys, with the ventral surface showing the most dramatic branching defect.



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Fig. 6. Altered dorsoventral pattern and gene expression in the kidneys of Hoxa11/Hoxd11 mutant mice. E13.5 kidneys from Hoxa11/Hoxd11 double mutants (B,C,E,F,H,I,K,L,N,O) and normal littermates (A,D,G,J,M) were hybridized with riboprobes for Wnt11 (A-C), Bf2 (D-F), Pax2 (G-I), Wt1 (J-L) and Gdnf (M-O). The dorsal (not shown) and ventral regions of the normal kidneys showed an even distribution of ureteric bud termini (Wnt11), stromal mesenchyme (Bf2), induced mesenchyme (Pax2), uninduced mesenchyme (Wt1) and Gdnf expression. The dorsal region of the mutant kidneys showed near normal distribution of bud termini (B) and stromal mesenchyme (E). In contrast, the dorsum showed patchy Pax2 (H) and reduced Wt1 (K) expression. The mid-ventral region was more severely affected in the mutants with loss of ureteric bud branch termini (C, arrow) and stromal mesenchyme (F, arrow). Areas of induced mesenchyme (I) and expression of Wt1 (L) were diminished in the mutant ventral metanephric mesenchyme. A patch of induced mesenchyme (white arrow) was found on the surface of the left kidney (I). Also of note were the lateral entry and elongated primary branches (black arrow) of the ureteric bud in the mutant. The expression of Pax2 in the branches of the bud in the interior of the kidney stands out because of the lack of Pax2 expression in the overlying mesenchyme (I). Gdnf expression was significantly decreased on the ventral surface of the mutant kidney (O). ad, adrenal; go, gonad; gu, gut; m, metanephros. Scale bars, 0.2 mm.

 


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Fig. 7. Sections of Hoxa11/Hoxd11 mutant kidneys showed the mid-ventral region consists of mesenchyme and contains no organized epithelium of ureteric buds. Longitudinal sections through E13.5 kidney hybridized with the Wnt11 probe (A) showed bud tips (arrow) and central mesenchyme (arrowhead). Transverse section through E13.5 kidney hybridized with Bf2 (B) showed Bf2 expression on the dorsal (d) but not on the ventral (v) surface.

 
We also observed an apparent defect in position of the mutant kidneys. The kidneys of the double mutants were located more caudal and medial than normal, and had close approximation of the inferior poles. The less severely affected E13.5 kidneys often showed a somewhat dumb-bell shape, consistent with the reduced bud growth and branching, and nephron induction in the mid-ventral region (Fig. 6B,C). These results showed that the Hoxa11/Hoxd11 mutations did influence overall renal growth that again can partly be explained by the diminished number of ureteric bud branches.

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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Fig. 8. Anteroposterior or dorsoventral gradients of expression were not found in the kidney for other Hox genes. Wild-type E13.5 kidneys were hybridized with riboprobes to Hoxc10 (A), Hoxc11 (B), Hoxd10 (C) and Hoxd12 (D). The intensity of staining for these genes on the ventral surface (shown) was equivalent to the dorsal surface and varies with distance from the termini of the branches. Note also that the expression of Hoxc11 is globally very weak. Scale bar, 0.1 mm.

 

    DISCUSSION
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we examined the expression patterns of Hoxa11 and Hoxd11 in the developing kidney, and defined the kidney mutant phenotype in Hoxa11/Hoxd11 double mutants. The major conclusions are that Hoxa11/Hoxd11 regulate metanephric mesenchyme-ureteric bud inductive interactions during patterning of the kidney. The mechanism for control of these interactions appears to be independent of metanephric mesenchyme growth.

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.


    ACKNOWLEDGMENTS
 
Many thanks to M. Todd Valerius for many insightful discussions and to D. Witte for assistance with the TUNEL assay. We gratefully acknowledge G. Dressler for the Pax2 riboprobe, A. Schedl for the Wt1 riboprobe, F. Costantini for the Gdnf riboprobe, D. Duboule for the Hoxd10 and Hoxd12 riboprobes, and A. Awgulewitsch for the Hoxc10 and Hoxc11 riboprobes. This work was supported by NIH grant HD32061 to S. S. P.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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