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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Dev Biol. Author manuscript; available in PMC Oct 15, 2008.
Published in final edited form as:
PMCID: PMC2075093
NIHMSID: NIHMS32357

Cessation of Renal Morphogenesis in Mice

Abstract

The kidney develops by cycles of ureteric bud branching and nephron formation. The cycles begin and are sustained by reciprocal inductive interactions and feedback between ureteric bud tips and the surrounding mesenchyme. Understanding how the cycles end is important because it controls nephron number. During the period when nephrogenesis ends in mice, we examined the morphology, gene expression, and function of the domains that control branching and nephrogenesis. We found that the nephrogenic mesenchyme, which is required for continued branching, was gone by the third postnatal day. This was associated with an accelerated rate of new nephron formation in the absence of apoptosis. At the same time the tips of the ureteric bud branches lost the typical appearance of an ampulla and lost Wnt11 expression, consistent with the absence of the capping mesenchyme. Surprisingly, expression of Wnt9b, a gene necessary for mesenchyme induction, continued. We then tested the postnatal day three bud branch tip and showed that it maintained its ability both to promote survival of metanephric mesenchyme and to induce nephrogenesis in culture. These results suggest that the sequence of events leading to disruption of the cycle of branching morphogenesis and nephrogenesis began with the loss of mesenchyme that resulted from its conversion into nephrons.

Keywords: kidney, renal organogenesis, renal morphogenesis, nephrogenesis, confocal microscopy, postnatal development, mouse, apoptosis, proliferation

Introduction

The kidney has evolved morphologically and physiologically into a highly complex organ in order to meet many metabolic demands of the body. Grobstein described the basic mechanistic framework for understanding the development of the kidney by demonstrating, in cultured explants of mouse tissue, the interdependency and inductive interactions of the ureteric bud and its surrounding mesenchyme (Grobstein, 1955). Studies continue to build on the original framework while filling in the details of molecular control (Shah et al., 2004; Vainio and Lin, 2002; Yu et al., 2004).

Renal morphogenesis depends on a reiterative process in which the ureteric bud grows and repetitively branches in response to mesenchymal signals and in which mesenchymal tissue forms nephrons in response to signals from the tip of each successive branch. At least four morphogenic domains within the cortex of the developing kidney govern the process. The mesenchyme that caps the branch tips of ureteric bud produces GDNF, a member of the transforming growth factor beta superfamily. It is the ligand of Ret and is necessary for normal growth and branching of the ureteric bud tree (Durbec et al., 1996; Pichel et al., 1996; Sanchez et al., 1996; Vega et al., 1996). Both the stromal precursor (Hatini et al., 1996; Mendelsohn et al., 1999) and stromal mesenchyme (Qiao et al., 1999) are also necessary for normal growth and branching as well. The ureteric bud plays multiple roles by participating in a positive feedback loop that maintains branching morphogenesis (Majumdar et al., 2003), by promoting survival of the mesenchyme (Dudley et al., 1999), and by inducing formation of nephrons from mesenchyme (Carroll et al., 2005). The developing nephron itself appears to have a dual role in maintaining normal branching morphogenesis. In the absence of Lim1, nephron development arrests at the renal vesicle stage, yet ureteric bud branching remains normal (Kobayashi et al., 2005). In contrast, absence of Wnt4 not only causes an earlier arrest of nephron development, but also results in severely retarded ureteric bud development. It appears that ureteric bud growth depends on nephron development to at least the renal vesicle stage. In addition, nascent nephrons produce FGF8, which is necessary to prevent apoptosis of the capping mesenchyme (Grieshammer et al., 2005) (Perantoni et al., 2005).

The events leading to the end of this reiterative process have not been explored. Because it controls the total nephron number, the mechanism terminating nephrogenesis has potential broad implications for health and disease (reviewed in (Gross et al., 2005) (Luyckx and Brenner, 2005)) . The mechanism could involve a loss of competency or gradual depletion of the progenitor pool within any one of the domains needed to sustain the reiterative process. Alternatively, it could involve a regulated genetic switch or physiologic sensor, again involving any one of the compartments. We have begun to examine the events leading to the cessation of renal morphogenesis. We found that the nephrogenic mesenchyme converted into nephrons immediately after birth and that the ureteric bud branch tip maintained its capacity to induce new nephrons after nephrogenesis was complete.

Methods and Materials

Animals

Embryos were obtained from timed breedings of CD-1 mice. Noon of the day when a vaginal plug was found was counted as embryonic day 0.5 (E0.5). Newborn pups were obtained on the day of birth (P0) through postnatal day 3 (P3). Pairs of kidneys from 5 embryos or newborn pups, each randomly selected from a separate litter, were processed for each age and for each of the confocal microscopy staining conditions.

HoxB7creEGFP (Zhao et al., 2004) mice were bred with Goulding Rosa26 floxed-stop-EGFP mice and the bud tips from the kidneys of P3 pups dissected for co-culture. GFP expression in the Goulding GFP mice can be activated by cre recombination. Breeding with the HoxB7creEGFP mice resulted in GFP expression in ureteric bud derived tissues. Mesenchyme was dissected from Tg(ACTB-EGFP)1Osb (Jackson Laboratory) E11.5embryos for co-culture.

Tissue processing for confocal microscopy

Kidneys were dissected in phosphate buffered saline (PBS). The kidneys or the organ explants were rocked for 1-2 hours in 2% paraformaldehyde in PBS, washed twice with PBS, and then rocked for 1-2 hours in 100% methanol. The tissues were washed twice with cold PBS containing 0.05% Tween-20 (PBT). Kidneys were bisected. Primary antibodies, diluted to 1:250 to 1:400, were added to the tissues in 400μL of PBT containing 2% goat serum and incubated overnight with rocking. Tissues were washed with 5 exchanges of PBT over 8 hours with rocking. The secondary antibodies, diluted to 1:400 in PBT containing 2% goat serum, were added and incubated overnight. The tissues were again washed with 5 exchanges of PBT over 8 hours. Fluorescein-conjugated Dolichos biflorus agglutinin (DBA,Vector), used to mark ureteric bud branches, was diluted 1:60 in PBT and added to the tissues and incubated over night. The tissue was washed for 5-10 minutes and mounted in a depression slide in PBT before they were examined by confocal microscopy. The entire procedure was performed at 4 degree C with pre-cooled reagents.

Antibodies

We used the following primary antibodies: anti-WT1 (c-19, Santa Cruz), anti-Pax2 (Santa Cruz), anti-Uvomorulin (E-cadherin, Sigma), anti-CITED1 (Neomarkers), anti-phospho-Histone H3 (PHH3, Upstate), and anti-cleaved Caspase 3 (Cell Signaling). The secondary antibodies were Alexa 555-conjugated anti-rabbit and Alexa 633-conjugated anti-rat secondary antibodies (Molecular Probes).

Confocal imaging

The tissues were imaged with a Zeiss LSM510 equipped with an Argon (488nm) and two HeNe lasers (543nm and 633nm). We used a multi-track configuration, refractive index correction, and automatic gain control. Approximately 2 micron thick optical sections were obtained every 5 microns to a depth of at least 80 microns. The sections began at the surface of the kidney and were on a plane tangential to it. Two Z-stack series were obtained, one from each of the 2 kidneys of each embryo or pup. The total renal surface area measured from each animal was at least 0.2 mm2. Where indicated, images presented in the figures are merged images of optical sections in order to reduce possible selection bias.

Measurements

All nephrons, from the vesicle stage onward, were identified and counted in each of the Z-stack series and then expressed as the number of nephrons in a given surface area of kidney (density). The diameters of the renal vesicles and glomerular anlage, which could be identified by staining with antibody to WT1, were measured at their greatest diameter and plotted against their depth from the cortical tip of the ureteric bud branch to which it was attached.

In Situ Hybridization

Whole mount in situ hybridization was performed as described before (Patterson et al., 2001). Riboprobes to Wnt11, Wnt7b, and to Foxd1 were used as described previously (Patterson et al., 2001). The Wnt4 riboprobe was contributed by A. McMahon and the Wnt9b by T. Carroll. Riboprobes for Six2, Brn-1, and Aqp2 were derived from Image clones 6504984, 5151907 and 580013, respectively.

Culture

P3 kidneys from HoxB7creEGFP-positive/Goulding GFP-positive pups were dissected and treated with collagenase B (Roche, C. histolyticum) 0.5mg/mL in DMEM with 10%FBS for 40 minutes at 37°C. The GFP-positive bud tips were dissected using a dissecting scope equipped for GFP fluorescence. Kidneys from e11.5 Tg(ACTB-EGFP)1Osb embryos were treated for 15 minutes with collagenase before dissection. The tissues were combined and cultured on polycarbonate filters on the surface of DMEM with 10% FBS for 3-4days.

Results

Mesenchyme

We used confocal microscopy to examine the mesenchyme of intact kidneys during the postnatal period. Confocal microscopy is useful for examination of later stages of renal development because it provides rapid interpretation of complex structures. It has the advantages of being able (1) to use immunostaining to mark and discriminate between multiple individual structures, (2) to obtain a series of sections without loss of sections and without sectioning artifact or distortion, and (3) to rapidly collate the sections and construct a 3-dimensional image from the series. We took an unconventional approach by obtaining optical slices of the kidney in planes parallel to the tangent of the kidney surface rather than perpendicular to it. This approach allowed us to quickly examine multiple planes, each representing a single developmental stage. With this strategy nephrogenesis could be examined in superficial slices while maturing nephrons could be examined in deeper slices.

During renal development condensed mesenchymal tissue surrounds the tips of the branching ureteric bud. This tissue is induced to become nephron and as cells are lost to the new nephron, additional cells must be generated to replenish them, otherwise nephrogenesis will cease. We found that by postnatal day 3 that there was complete loss of the capping mesenchyme, as identified by CITED1 immunostaining and by Six2 in situ hybridization (Fig.1). The loss of the mesechymal substrate that contributes to new nephrons is earlier than what has been typically reported for the end of nephrogenesis (Larsson et al., 1980). The difference can easily be attributed to the length of time needed to progress from a renal vesicle to a fully mature nephron with a glomerular capillary tuft. Thus immature nephrons are present when nephrogenesis, the birth of new nephrons, is complete.

Fig.1
Capping nephrogenic mesenchyme rapidly converted to nephrons in the immediate postnatal period

There are three potential fates of the capping mesenchyme that could account for its sudden loss. Proliferation could slow or cease, the cells could undergo apoptosis, or the cells could differentiate either into nephrons or perhaps into a different mesenchymal lineage, such a stromal mesenchyme. We tested each of these possibilities. Proliferating cells within the capping mesenchyme were easily identified in the postnatal period and appeared in a random pattern similar to what was seen with the antibody to pHH3 in the prenatal kidney (Fig. 1). Fragmenting nuclei of apoptotic cells, identified by immunohistochemistry using an antibody to activated Caspase 3, were rarely seen within the nephrogenic region in the postnatal period, again similar to what we found in the prenatal period (Fig. 1). On the other hand, apoptotic cells were common immediately interior to the nephrogenic zone both in the prenatal and postnatal period at a depth of approximately 40 microns from the surface. At later stages, when nascent nephrons were very superficial, apoptotic cells were also closer to the surface. Although the cells appeared to be mesenchyme cells, it was not clear whether they were derived from the nephrogenic mesenchyme and had failed to become part of the nephron or were derived from stromal precursor cells and were part of a remodeling process. Lineage studies would be required to distinguish between these two possibilities. Nevertheless, there was there was no appreciable change in apoptosis that might explain the loss of metanephric mesenchyme.

We examined the mesenchyme by in situ hybridization with a riboprobe to Foxd1 (formerly Bf2) to determine whether there was evidence for conversion to stromal mesenchyme (Fig. 1) and did not find expansion of the stromal precursor population. In fact the staining intensity in the nephrogenic region with this probe decreased during the first 3 postnatal days. Therefore, it was unlikely that capping mesenchyme had differentiated into another mesenchymal lineage. Finally, we used an antibody to WT1 and to Pax2 to determine whether mesenchyme converted to nephrons, and found that renal vesicles surrounded the tips and replaced the mesenchyme in the first three postnatal days (Fig.1). Therefore, the loss of nephrogenic mesenchyme occurred because of conversion to nephrons without replenishment from the progenitor pool and not by cell death. It appeared that the signaling necessary for mesenchyme survival and differentiation did not diminish at the end of nephrogenesis.

The data was consistent with an intact inductive potential of the tips of the ureteric bud branches in the postnatal period. One would predict from these studies, based on the rapid disappearance of the capping mesenchyme, that normal signaling to the ureteric bud would be disrupted. The tip would detect the change in signaling which in turn would lead to altered gene expression and cessation of branching morphogenesis.

Ureteric bud branch tips

We next examined the UB tips in the postnatal period to identify changes predicted by the changes in the mesenchyme. We could not quantify the number of tips in the postnatal period to determine when branching ceased, because the changes in and the complexity of the tip did not allow us to discern where one tip ended and another started. A change in tip morphology however suggested that branching ceased in the interval between birth and postnatal day 3. Prior to birth the typical pattern or relationship of branch tip to nephron is approximately 1:1 and the tips are characteristic smoothly contoured ampullae. After birth the ampulla, the site of dichotomous branching, thinned and became scalloped with each concavity being the location of an attaching newly formed nephron (Fig. 2). The loss of the ampulla would suggest the end of branching morphogenesis.

Fig. 2
Ureteric bud branch tip morphology and gene expression pattern changed in the early postnatal period

As predicted by the loss of capping mesenchyme, Wnt11, a marker which is responsive to GDNF/Ret signaling in the ureteric bud branch tip, and therefore sensitive to mesenchyme loss, was significantly down-regulated in the postnatal period (Fig. 2). We were unable to detect any change in cell death or proliferation that might contribute to the changes in the tips. As found in the mesenchyme, apoptotic cells were infrequent in the ureteric bud branch tips in the nephrogenic region (Fig. 1). Unlike Wnt11, other gene markers within the branches of the ureteric bud continued to show strong expression (Fig.2). One of the genes, Wnt9b, is necessary for mesenchyme induction by the ureteric bud. This marker profile was consistent with reduced mesenchyme to tip signaling, but continued tip to mesenchyme signaling in the postnatal period.

Next, in order to examine the function of the ureteric bud branch tips in the postnatal period we tested the inductive activity of the postnatal tips in co-culture experiments with early nephrogenic mesenchyme. We used Hoxb7creGFP transgenic mice in order to visualize and micro-dissect tips from postnatal day 3 kidneys. While the ureteric bud branch tips of these transgenic mice express GFP, the nephrons do not. The tips were combined with embryonic day 11.5 mesenchyme dissected from Tg(ACTB-EGFP)1Osb transgenic mice which allowed us to determine tissue lineage. GFP expression in these mice is strong and ubiquitous in the developing kidney. Using this strategy the capping mesenchyme and any glomerular structure that is induced in culture and also derived from the e11.5 mesenchyme will express GFP, whereas any contaminating nephron carried over from the dissection of the ureteric bud of the postnatal kidney will not express it. In 6 of 7 cultures, nephrons were induced in the GFP positive mesenchyme derived from embryos by postnatal tips (Fig. 3). In addition, these nephrons were connected to the ureteric bud tips dissected from the Hoxb7creGFP pups. This demonstrates not only that the tips were capable of inducing mesenchyme but also that they were still subject to invasion by developing nephrons. Finally, we found clusters of GFP-positive capping mesenchyme that also stained with anti-WT1 antibody around the tips dissected from postnatal kidneys (Fig 3). This indicated that the postnatal tip was still able to prevent apoptosis of the mesenchyme. These characteristics of the postnatal ureteric bud branch tip demonstrate that the tip remained competent despite the arrest of both branching and nephrogenesis by this stage.

Fig. 3
The postnatal day 3 ureteric bud branch tip still had the potential to induce nephrons and promote survival of metanephric mesenchyme

Control experiments showed that the cultures were not contaminated by ureteric bud tips adherent to the E11.5 mesenchyme. Compared to ureteric buds derived from the Tg(ACTB-EGFP)1Osb mice, the tip from the Hoxb7creGFPmice expressed GFP only very weakly in culture and was difficult to detect when the confocal laser power was adjusted so that the EGFP expression controlled by ACTB did not over-saturate the image. Thus, we were able to distinguish potential contamination of the cultures by tips from the Tg(ACTB-EGFP)1Osb transgenic mice because of an intense GFP signal. We did not find ureteric buds from Tg(ACTB-EGFP)1Osb mouse embryos in any of the cultures. In addition, nephrons did not develop in isolated mesenchyme that was cultured alone in the absence of ureteric bud tips. The control mesenchyme tissue died in culture (data not shown).

Stromal precursor

When the stromal precursor mesenchyme is abnormal, as found in the Foxd1 (Hatini et al., 1996) and Pbx1 (Schnabel et al., 2003) mutant mice, there is expansion of the capping mesenchyme rather than a loss of mesenchyme that we saw in the postnatal period. We would therefore not expect that the inciting event that led to cessation of nephrogenesis to be caused by loss of or diminished signaling from stromal precursor mesenchyme. As noted above, when we evaluated the stromal precursor mesenchyme by in situ hybridization with a probe for Foxd1, we found that the signal had decreased by the postnatal day 3, following the change in capping mesenchyme (Fig. 1). Again, as found in both the capping mesenchyme and ureteric bud tree, apoptosis was rare within the stromal precursor population in the nephrogenic region (Fig. 1). Therefore cell death did not explain the loss of expression.

Nephron

We examined developing nephrons for changes during the early postnatal period by quantifying the change in nephron density and the size and location of renal vesicles and glomerular anlagen. The density of nephrons/mm2 continued to increase in the postnatal period and the rate of increase appeared to accelerate between days 0 and 2 (Fig. 4). The density increased from 225 nephrons/mm2 to 378 nephrons/mm2 in the 3 days between e16.5 and birth and then from 378 nephrons/mm2 to 580 nephrons/mm2 in the 2 days between birth and P2. The density then reached a plateau with a small, insignificant decline by P3. The change in density can also be visualized in the early postnatal period with a marker for pretubular aggregates, Wnt4 (Fig. 5). Early in the postnatal period the expression of Wnt4 was clustered around ureteric bud tips; later, by P3 it was distributed more evenly and by P5 was no longer detectable. Wide unstained spaces were found between the clusters at birth. These spaces were filled in by P2, consistent with increased density of early nephrons and the finding of accelerated nephrogenesis noted earlier. The acceleration could have occurred because of a sudden change in signaling which in turn created an imbalance between maintenance and consumption of a progenitor pool. Cessation did not occur because of a more gradual senescence and consumption of progenitors that would be predicted to cause slowing of the rate of nephrogenesis. The staining also demonstrates the rapid, orderly progression of nephron maturation during the post-natal period.

Fig. 4
Nephron density increased and mature nephrons were located more superficially in the postnatal period
Fig. 5
The increased density of newly induced nephrons can be seen using a marker for pretubular aggregates

In addition to density, the location of maturing nephrons changed during the early postnatal period. As expected, before birth we found a strong correlation between the diameter of the vesicle or more mature glomerular anlage and the depth of the structure from the surface. This is a simple reflection of the fact that interior nephrons were induced earlier and had more time to grow and mature than newly induced nephrons at the surface (Fig.4). A strong correlation, with an R2 = 0.66, was found at birth as well, whereas by P2 and P3 when large, maturing vesicles were abundant near the surface next to the ureteric bud tips rather than subjacent to the tips, the correlation was weak, with the R2 falling to 0.21.

Interestingly, a change in polarity could be seen in these superficial nephrons that develop postnatally. The polarity of WT-1 staining within the early vesicle changed so that intense staining was found still in opposition to the ureteric bud, except it was lateral rather than interior to the bud (Fig.1). While the proximal-distal axis of the early nephron is perpendicular to the surface of the kidney prior to birth, the axis of early nephrons on postnatal day 3 appeared to rotate so that at least initially it lies parallel to the tangent of the kidney surface.

Nephron development in the postnatal period changed in other ways as well. Up until birth, nephrons attached singly to the tip of a ureteric bud branch where, with few exceptions, they remained attached until birth. There were no arcades. After birth we found progressively more nephrons attached to the stalk rather than just to the tips and could identify neighboring nephrons that jointly attached to the ureteric bud tree by a common connecting segment (not shown). By PND3 multiple newly induced nephrons could be seen associating with each ureteric bud branch tip.

We also examined kidneys of postnatal day 7 mice. We could not identify any early stage nephrons such as vesicles (data not shown). The glomeruli of all nephrons reached at least stage 3 of the staging system used by Kazimierczak (Kazimierczak, 1980). As it takes a few days to reach this stage after induction, these results indicate that new nephron formation ceased well before PND7, although maturation continues beyond this time.

Discussion

The kidney develops by repeated cycles of ureteric bud branching and nephron formation. Once the developmental process ends, the program to create new nephrons cannot be reactivated. After renal injury, existing nephrons may be repaired, but no new nephrons will form. Therefore, the events that control termination of the cycles of branching and nephrogenesis also control the final nephron number. The mechanism therefore has direct clinical relevance as evidence mounts showing that a deficit in nephron number is associated with renal disease and hypertension (Gross et al., 2005; Luyckx and Brenner, 2005). Here we provide initial insight into the mechanism by examining morphologic and molecular changes, as well as functional activity at the end of renal development in mice.

Maintenance of nephrogenesis and branching rely on signaling by four regions in the cortex of the developing kidney. One region, the capping mesenchyme, is consumed in the process of nephron formation. In order for development to continue the tissue must theoretically double with each new generation of ureteric bud branch bifurcation to maintain balance. Doubling of this compartment must occur in the face of mesenchyme consumption that leads to the development of new nephrons. The balance appears to be upset in Foxd1 (formerly Bf-2) (Hatini et al., 1996) and Pbx1 (Schnabel et al., 2003) mutant mice in which the condensed mesenchyme accumulates as a result of diminished consumption or ineffective progression to epithelium, and is also upset in Six2 mutant mice in which the mesenchyme is lost in early development partly because of dysregulated mesenchyme induction and premature mesenchyme consumption (Self et al., 2006).

We found that the capping mesenchyme, identified by staining with antibody to CITED1 and by in situ hybridization for Six2, was absent by postnatal day 3. Loss of the mesenchyme could have occurred by one or a combination of several mechanisms: cell death, decreased production, increased rate of differention to create new nephrons, or possibly conversion to an alternate cell type such as stromal mesenchyme. In evaluating these possibilities we found that cell death was uncommon in the nephrogenic region, even during the postnatal period in question. Because signaling by the ureteric bud and by the early nephron is necessary to prevent apoptosis, presumably these signaling pathways remained intact. Proliferation did not abate but continued in the cortical region, initially in the mesenchyme and later in nascent nephrons that replaced the mesenchyme. We were unable to directly examine proliferation of progenitor cells because their molecular characteristics and exact location have not been well defined. Proliferation however could be seen in the capping mesenchyme in the postnatal period. We also did not find evidence for conversion to and expansion of the stromal progenitor population as detected by in situ hybridization for Foxd1.

Six2 expression continued in the capping mesenchyme until P2 in the same way that CITED1 was expressed and was only lost when there was no longer any detectable capping mesenchyme. The timing of the loss of Six2 expression did not anticipate the loss of the mesenchyme. This suggests that the loss of expression of Six2, an essential gene for progenitor cell self-renewal, is not an early initiating event in the termination of nephrogenesis.

We found evidence for the final possibility: increased differentiation into nephrons. It appeared that nephron density had increased at a greater rate in the immediate postnatal period than in the prenatal period. This was quantified by confocal microscopy and can be seen by comparing in situ hybridization for Wnt4 at P0 and P2. Thus, not only did the signaling pathway to prevent apoptosis appear intact, but also did the signaling pathway for mesenchyme induction. We also found in vitro evidence that signaling by ureteric bud tips remained intact. The P3 bud branch tips, which continued to express Wnt9b during the postnatal period, were still able to induce new nephrons when co-cultured with nephrogenic mesenchyme. At this stage there appears to be a shift in the balance between the signals that determine the fate of the progenitor population. The shift favors induction and could be caused by increased inductive signaling, increased sensitivity to induction, or to decreased sensitivity to a growth factor or to an inhibitor of differentiation. Any of the changes could lead to loss of the capping mesenchyme and a cascade of events terminating renal development.

During the same period when the capping mesenchyme was lost, the stromal precursor compartment was lost as indicated by down-regulation of Foxd1 expression. As with the capping mesenchyme, proliferation continued in this compartment postnatally and apoptosis was rare. Loss of the stromal precursors by maturation could initiate termination, however, if this was the initiating event, then we might expect to find accumulation of capping mesenchyme in the postnatal kidney as was found in the Foxd1 mutant mouse in which there is failure of stromal mesenchyme signaling. Over the same developmental period when we identified the mesenchymal changes, we also found morphologic and molecular changes in the terminus of each branch of the ureteric bud that suggested the end of branching.

In conclusion, nephrogenesis and branching morphogenesis end relatively abruptly in the postnatal period in mice with a burst of nephrogenesis. It appears to be a regulated process in which the character of nephron formation changes as nephrogenesis accelerates and all remaining nephrogenic mesenchyme is synchronously converted to nephrons. The cap mesenchyme cells are not replaced in the process. Mesenchyme markers continue to be expressed normally and the ureteric bud retains in inductive potential. The events suggest that there is sudden unopposed mesenchyme induction after birth, a sudden shift in the signaling that determines mesenchyme fate. The changes occur so quickly after birth that a physiologic trigger at or after parturition may be entertained as a possible cause, just as it has been for lung development (Chuang and McMahon, 2003). The mechanism for the completion of renal morphogenesis in mice would then need to be reconciled with the mechanism in man where nephrogenesis ends well before parturition.

Acknowledgments

We thank Carl Bates for the HoxB7creGFP transgenic mice and Martyn Goulding for the Goulding Rosa26 floxed-stop-EGFP transgenic mice. We would also like to thank S. Potter and E. Brunskil for their thoughtful review of the manuscript. Portions of this work were supported by NIH grants DK02702 and DK61916

Footnotes

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