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Am J Physiol Regul Integr Comp Physiol 292: R1768-R1774, 2007. First published February 1, 2007; doi:10.1152/ajpregu.00442.2006
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Sex Differences in Renal and Cardiovascular Function: Physiology and Pathophysiology

Effects of dietary protein restriction on nephron number in the mouse

Chantal C. Hoppe,1 Roger G. Evans,2 John F. Bertram,1 and Karen M. Moritz1

Departments of 1Anatomy and Cell Biology and 2Physiology, Monash University, Melbourne, Victoria, Australia

Submitted 28 June 2006 ; accepted in final form 21 January 2007


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In rats, maternal protein restriction reduces nephron endowment and often leads to adult hypertension. Sex differences in these responses have been identified. The molecular and genetic bases of these phenomena can best be identified in a mouse model, but effects of maternal protein restriction on kidney development have not been examined in mice. Therefore, we determined how combined prenatal and postnatal protein restriction in mice affects organ weight, glomerular number and dimensions, and renal expression of angiotensin receptor mRNA, in both male and female offspring. C57/BL6/129sv mice received either a normal (20% wt/wt; NP) or low (9% wt/wt; LP) protein diet during gestation and postnatal life. Offspring were examined at postnatal day 30. Protein restriction retarded growth of the kidney, liver, spleen, heart, and brain. All organs except the brain weighed less in female than male offspring. Protein restriction increased normalized (to body weight) brain weight, with females having relatively heavier brains than males. The effects of protein restriction were not sex dependent, except that normalized liver weight was reduced in males but increased in females. Glomerular volume, but not number, was greater in female than in male mice. Maternal protein restriction reduced nephron endowment similarly in male and female mice. Renal expression of AT1A receptor mRNA was approximately sixfold greater in female than male NP mice, but similar in male LP and female LP mice. We conclude that maternal protein restriction reduces nephron endowment in mice. This effect provides a basis for future studies of developmental programming in the mouse.

stereology; nephron deficit; sex; mice; kidney

THE HYPOTHESIS THAT ADULT disease can have developmental origins has been a major focus of experimental and human studies in the last decade (36). There is now considerable evidence that the in utero and early postnatal environment has a major impact on adult behavior, physiology, and risk of adult disease (2, 7, 16, 29, 30, 36, 40, 41). There is also considerable evidence that the effects of the perinatal environment on offspring development and adult renal and cardiovascular function can be sex specific (23, 30, 36, 41, 42). The most widely studied model of developmental programming is maternal protein restriction in rats. Rat dams fed a low-protein (LP; 8–9% wt/wt) diet during pregnancy give rise to offspring with a 10–30% deficit in nephrons, a finding consistent across a range of studies (14, 17, 39, 41, 43). This is often associated with hypertension in adulthood (41). Nephron number data in female offspring following maternal protein restriction, however, are somewhat conflicting. Some studies have shown females from protein-restricted mothers have a nephron deficit (17, 24, 39, 44). In contrast, Woods et al. (42) recently found that female rats subjected to maternal protein restriction did not have a reduced nephron endowment. Importantly, studies that have investigated both sexes following protein restriction have either used a relatively severe form of protein restriction (39), maceration techniques (24), or pooled male and female data, making it difficult to discern sex differences (17). Importantly, we are aware of no previous studies that have specifically investigated whether the effects of modest protein restriction on nephron endowment are sex dependent in the mouse.

The increasing evidence that interactions between genotype, sex, and the developmental environment dominate adult cardiovascular health, calls for intense study of the molecular and genetic bases of developmental programming. Currently, such studies can best be performed in mice. A most powerful approach to assessing the pathophysiological role of potential genes involved in developmental programming is to examine the consequences of gene disruption using genetically modified animals. To date, the mouse is the only mammalian species in which complex investigations of molecular mechanisms can be easily performed. Before such studies can be performed, we must define the effects of maternal protein restriction on nephron endowment in the mouse. However, there are no previously published reports of the effects of maternal protein restriction on renal development in mice. Therefore, in the present study we tested the primary hypothesis that in mice, combined prenatal and postnatal protein restriction reduces nephron endowment, alters glomerular morphology, and retards the growth of major organs. There is strong evidence from studies in rats that changes in the renin/angiotensin system contribute to reduced nephron endowment associated with maternal protein restriction (41). Therefore, we also measured renal expression of angiotensin receptors. Our secondary hypothesis was that the effects of combined prenatal and postnatal protein restriction are sex dependent, so we studied both male and female offspring. Our findings suggest that maternal protein restriction reduces nephron endowment in mice and that the effects of protein restriction on the development of the kidney and other organs are largely independent of sex. Thus, our current study characterizes a new model of maternal protein restriction in mice.


   METHODS
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 METHODS
RESULTS
 DISCUSSION
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Animals and Their Diets

Experiments were conducted in accordance with the National Health and Medical Research Council of Australia Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (7th ed., 2004) and were approved by the Monash University, Biochemistry, Anatomy and Microbiology Animal Ethics Committee. Previously unmated female C57/BL6 mice were fed either a normal-protein (NP; 20% wt/wt; Growth Diet AIN93G, Specialty Feeds, WA, Australia) or near-isocaloric LP (9% wt/wt; 9% Protein Modified AIN93G, Specialty Feeds, WA, Australia) diet from 2 wk before mating with 129SV males. The LP diet was rendered near-isocaloric by the addition of sucrose and a small amount of starch, making it a high-sucrose diet. After compensation of low protein by sucrose-starch, the LP diet was nearly isocaloric with energy content being only 3.7% less than the NP diet. The compositions of the diets are given in Table 1. Female breeders were maintained on their given diet throughout pregnancy and lactation. Only first litters were studied. Protein restriction continued after birth to ensure that offspring were protein restricted throughout the entire period of nephrogenesis, which in mice, extends into the postnatal period. Offspring were also maintained on their mother's designated diet after weaning at 21 days, to avoid the potentially confounding effects of altering dietary protein intake in the week before animals were killed for post mortem measurements. We have previously used this approach in rats (14), so our current observations allow a direct comparison across the two species. Male and female offspring were used in all of the experiments. To minimize handling stress, animals were not weighed at birth but were weighed every 5 days of age, from postnatal day 5.


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  		Table 1. Composition of the diets

 
Morphometric Measurements and Stereological Estimation of Kidney Volume, Glomerular Number, and Glomerular Volume

At postnatal day 30, mice were humanely killed via cervical dislocation and the kidneys, liver, spleen, heart, and brain were removed and weighed. Left kidneys were placed in 10% formalin for stereological estimation of total nephron number, and right kidneys were snap frozen and stored at –70°C for analysis of gene expression. Total body weight and the weights of internal organs were determined in a total of 12–16 animals from each diet and sex group, obtained from 4–6 different litters.

Total glomerular number was estimated in 4–7 male and female animals per dietary group. Left kidneys were decapsulated and sliced at 1 mm, and every slice was processed for embedding in glycolmethacrylate (Technovit 7100; Kulzer, Friedrichosdorf, Germany). Tissue blocks were exhaustively sectioned at 20 µm, and every 10th and 11th section (section pair) was collected and stained with Periodic-Acid Schiff's stain.

Total glomerular number (Nglom) and mean glomerular (Vglom) and mean renal corpuscle (Vcorp) volumes were determined as previously described using unbiased stereological techniques (4). In brief, the areas of all sampled kidney sections were estimated using stereological point counting with the aid of a microfiche reader using a magnification of x19.2. Grid points overlying all sections were designated Ps, and those overlying complete sections were designated Pf. The total number of glomeruli in a kidney was determined using physical disectors in a known fraction of the kidney, the so-called physical disector/fractionator combination. Two light microscopes modified for projection were employed, and each pair of sections was mounted on the microscopes. Corresponding fields on the two sections were found, and glomeruli sampled by an unbiased counting frame in one section that were not present in the section pair were counted (Q–). Nglom was estimated using Nglom = 10 x (Ps/Pf) x [1/(2fa)] x Q–, where 10 is the inverse of the section sampling fraction, and Ps/Pf and 1/(2fa) give the fraction of the total section area used to count glomeruli.

Kidney volume (Vkid) was estimated using the Cavalieri principle (4) through Vkid = 10 x t x a(p) x Ps, where t was section thickness and a(p) was the area of the stereological grid associated with each grid point after adjustment for magnification.

Vglom was estimated using Vglom = VVglom,kid / NVglom,kid, where VVglom,kid was estimated by dividing the number of grid points overlying glomerular profiles by the number of points overlying kidney sections. A similar point counting approach was used to estimate Vcorp. NVglom,kid was obtained by dividing Nglom by Vkid. To calculate total glomerular and total renal corpuscle volumes, mean volumes were multiplied by Nglom.

Gene Expression Studies

Total RNA was extracted from half of the right kidney using a commercially available kit (Qiagen, Valencia, CA). Samples were DNase treated to eliminate any residual genomic DNA. Samples were reverse transcribed to form cDNA and an ABI Prism 7700 Sequence Detector System (PE Biosystems, Foster City, CA) was used to perform real-time PCR (26). This assay was used to determine relative levels of mRNA expression for the AT1A, AT1B, and AT2 receptors. All primers and TaqMan probes were designed using Primer Express TM Version 1.0 (PE Biosystems) (see Table 2).


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  		Table 2. Primer and probe sequences

 
A comparative CT (cycle of threshold fluorescence) method was utilized with 18S used as an endogenous reference. To calculate the relative expression levels in each sample, the CT value for 18S was subtracted from the CT value of the gene of interest to give a {Delta}CT value. The {Delta}CT value of the calibrator was then subtracted from each individual sample to give a {Delta}{Delta}CT value. The calibrator used was the mean {Delta}CT of the normal protein male group. This number was then inserted into the formula 2–{Delta}{Delta}CT to give the expression level relative to the calibrator.

Statistics

Data are reported as means ± SE. Statistical analyses were performed using the software packages SYSTAT (Version 11.0, SPSS, Chicago, IL) and SPSS (Version 14.0, SPSS). Mean and SE of data from individual mice were weighted by litter, via least squares regression. Data were analyzed by univariate ANOVA, the factors comprising "protein intake" and "sex" but weighted for "litter" as recommended by Festing (9). The main effect of protein intake (PProtein) tested for effects of protein restriction independent of sex. The main effect of "sex" (PSex) tested for sex differences independent of protein intake. The interaction term (PSex·Protein) tested whether the effects of protein restriction differed in male and female mice. Analysis of changes in body weight over the first 30 days of postnatal life was performed by ANCOVA, and individual regression lines were determined by the least-squares method (19). P ≤ 0.05 was considered statistically significant (GenBank accession no. NM_007429).


   RESULTS
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DISCUSSION
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Body and Organ Weight, Litter Size

Effects of protein restriction. Litter size was similar for LP (7.6 ± 1.8 pups) and NP (6.6 ± 2.5 pups) dams (P = 0.25, unpaired t-test), and therefore, no adjustment of litter size was performed. There were no observed adverse effects of protein restriction on survival of pups to 30 days of age. At postnatal day 30, body weight and the absolute weights of the kidneys, liver, spleen, heart, and brain were less in LP than in NP offspring (Table 3, Absolute Values). The smaller weights of all organs in LP offspring, except the brain, were proportional to the deficit in body weight, as evidenced by the fact that protein restriction did not significantly alter the ratios of organ weight to body weight (Table 3, Corrected Values). In contrast, the ratio of brain weight to body weight was significantly increased by protein restriction.


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  		Table 3. Effects of protein restriction and sex on body and organ weights at postnatal day 30

 
Sex differences. Body weight and the weight of all organs except the brain were less in females than males (Table 3, Absolute Values). Sex differences in the weights of the liver, spleen, and heart were in proportion to differences in body weight, as evidenced by the fact that sex did not significantly affect the normalized weights of these organs. In contrast, normalized kidney weight was significantly less in female mice than in male mice. Normalized brain weight, on the other hand, was significantly greater in female than in male mice.

The effects of protein restriction on body weight and the weights of internal organs were similar in male and female mice, with the exception of the liver. Protein restriction reduced liver weight by 28% in male mice, but only by 11% in female mice. Consequently, protein restriction reduced normalized liver weight in males (by 9%) but increased it in females (by 12%).

Covariant analysis of body weight up to postnatal day 30. There was a highly significant positive relationship between postnatal age and body weight (r2 = 0.90–0.93 in the four groups of mice; PAge < 0.001). The rate of growth was greater in male than in female mice (PAge·Sex = 0.008). Protein restriction significantly reduced the slope of the growth curve (PAge·Protein < 0.001), but the magnitude of this effect was similar in male and female mice (PAge·Protein·Sex = 0.511) (Fig. 1).


Figure 1
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  		Fig. 1. Growth curves for male and female mice. Male and female mice were exposed to normal or low-protein diet both in utero and postnatally until postnatal day 30. Data are presented as within-litter averages in a scatterplot and fitted to lines of best fit by the method of least squares using individual animals as the experimental unit. Univariate ANCOVA was performed on these data, weighted by litter. See text for statistical analysis.

 
Nephron Number

Total glomerular number, and therefore nephron number, was similar in male and female NP mice (male = 10,755 ± 937, female = 10,695 ± 864). Glomerular number was significantly less in protein-restricted male offspring (by 22%, 8,364 ± 468) and female offspring (by 16%, 8,977 ± 494) (Fig. 2A). Similarly, kidney volume was significantly less in LP than in NP mice (PProtein < 0.001). Mean and total glomerular and renal corpuscle volumes were greater in female than in male mice. There were no apparent interactions between the respective effects of sex and protein intake (Fig. 2).


Figure 2
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  		Fig. 2. Nephron number and glomerular dimensions. Male and female mice were exposed to normal or low-protein diets both in utero and postnatally until postnatal day 30. Unbiased stereological techniques were used to determine total glomerular (nephron) number (A); mean glomerular volume (Vglom) (B); total glomerular volume (TGV) (C); mean renal corpuscle volume (Vcorp) (D); and total renal corpuscle volume (TCV) (E). For mice exposed to a normal protein diet, n = 4–6 animals from n = 3 or 4 litters. For mice exposed to a low protein diet, n = 6 or 7 animals from n = 4 litters. Values are means ± SE of the mean. P values are the outcomes of univariate analysis of variance weighted for litter, testing the main effects of "protein diet" and "sex," and for interactions between these factors.

 
Renal Gene Expression

In male mice, relative expression of AT1A receptor mRNA was similar in LP and NP animals. AT1A receptor mRNA was approximately sixfold greater in NP female mice than in corresponding male mice, but similar in LP female mice compared with the corresponding male mice (Fig. 3). There were no significant effects of protein or sex on AT1B receptor expression (Fig. 3). Low levels of AT2 receptor mRNA were detected in all mice, but the level of expression was highly variable between animals. There were no apparent effects of sex or protein intake on AT2 receptor expression (data not shown). A positive control from E17.5 kidney tissue revealed a significantly greater expression of AT2 receptor mRNA compared with mice from postnatal day 30.


Figure 3
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  		Fig. 3. Relative expression of AT1A and AT1B receptor mRNA. Male and female mice were exposed to a normal or low-protein diet both in utero and postnatally until postnatal day 30. Real-time PCR was performed using tissue from the left kidneys of these animals. Data are presented as means ± SE of the mean, analyzed statistically as for Fig. 2.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The most important finding of the present study was that combined pre- and postnatal protein restriction led to a reduction in nephron endowment, of similar magnitude in male and female mice. Furthermore, growth retardation in this model of protein restriction, both in terms of total body weight and the weights of individual organs, was with the exception of the liver and was of a similar magnitude in male and female offspring. However, we did identify underlying sex differences in gross body and renal morphology. Interestingly, both mean glomerular volume and total glomerular volume were greater in female mice than male mice. Furthermore, renal AT1A receptor gene expression was greater in female than in male NP mice, although similar in female and male LP mice. The results of this study highlight the importance of analyzing offspring of both sexes in experimental models of maternal protein restriction.

To our knowledge, this is the first report of reduced nephron endowment in a mouse model of maternal protein restriction. We used a relatively mild degree of protein restriction, similar to that we have used previously in rats (14). However, it is important to note that the LP diet contains ~3.7% less energy than the NP diet, and contains a higher sucrose content and a different starch: sucrose ratio. Nevertheless, in the present study in the mouse, the LP diet did not reduce litter size or offspring survival. Our current observations do not address the impact of combined pre- and postnatal protein restriction on adult cardiovascular health. However, our characterization of this new mouse model of maternal protein restriction provides a basis for future studies to examine both adult cardiovascular outcomes and the molecular mechanisms that mediate renal developmental programming in the mouse. A nephron deficit, as seen in our study in both male and female LP mice, has been implicated in the development of adult diseases, via Brenner's hypothesis (5). For example, maternal protein restriction in rats can reduce offspring nephron endowment and lead to adult hypertension (17, 41). The nephron deficit seen in protein-restricted rats is characterized by a 10–30% reduction in glomerular number (14, 17, 41, 43). Thus, at least in this respect, our findings in mice replicate previous observations in the rat. Interestingly, estimates of female offspring nephron number following maternal protein restriction, have produced somewhat conflicting data (17, 39, 42, 43). For example, Zimanyi et al. (44), using unbiased techniques, found that female protein-restricted rats had a nephron deficit compared with controls (44). In contrast, Woods et al. (42) found that female offspring of moderately protein-restricted rat dams had normal nephron endowment (42). Strain and diet variation may account for these disparate findings in rats. Of particular importance to the study of nephron number is the use of unbiased stereology. Alternatives to this "gold-standard" technique are subject to considerable bias, leading to inaccurate results. In our present study using unbiased stereology, we have shown that maternal protein restriction can lead to a nephron deficit in both male and female mice. We were unable to detect a sex-dependent difference in the magnitude of this effect.

However, we were able to detect sex-dependent differences in underlying renal morphology in mice, in that female mice were found to have larger glomeruli and renal corpuscles than males, yet have smaller kidneys. Total glomerular volume and total renal corpuscle volume was also found to be greater in female than in male mice. Two previous studies investigated sex differences in glomerular morphology in mice at a similar age yet failed to detect sex differences in renal corpuscle diameter or glomerular area (18, 28), although neither study employed unbiased stereological techniques. Okada et al. (28) found no sex differences in cortical corpuscle diameter in DBA/2CrSlc mice at 30 days of age, although corpuscle diameter was greater in males from 90 days of age. Lovegrove et al. (18) found no sex differences in glomerular area in wild-type mice at 70 days of age. Differences between our current observations and those of these previous studies might be explained by strain differences or by differences in the stereological techniques used. Our findings merit further investigation of adult mice of both sexes to determine whether the greater glomerular size in females confers a greater or decreased survival advantage under certain physiological conditions.

Total body weight and the weight of all organs studied were less in LP than in NP mice. These observations are consistent with the outcomes of previous studies of combined prenatal and postnatal protein restriction in rats (6, 14). In our study, retarded growth, of all organs except the brain, was in proportion to the retardation in total body weight. This contrasts with the effects of combined prenatal and postnatal protein restriction in the rat, where the weights of the spleen and the liver were found to be reduced proportionally more than was total body weight (14). "Brain sparing" following protein restriction occurred in both sexes in mice, consistent with previous findings in the rat (3, 6). Similarly, brain sparing has been shown in rabbits and sheep subjected to intrauterine growth restriction (12, 29) and occurs in human growth-restricted infants (10, 11, 20). Brain sparing may confer an immediate survival advantage to the offspring, while growth of other organs is compromised.

The effects of protein restriction on gross organ morphology were not sex dependent, except in the case of the liver. Protein restriction retarded growth of the liver far more in male mice than in female mice. Sex-dependent effects of maternal protein restriction on hepatic glycogen stores have been identified in rats (1). The significance of these sex-dependent effects of protein restriction on liver development remains to be determined. They may reflect the potential for sex-dependent developmental programming of metabolic disorders.

Female mice in our study had a significantly lower kidney and body weight than males, consistent with observations from previous studies (18, 28). Brain weight, as a proportion of total body weight, was greater in female than male mice. This has not previously been demonstrated in mice, although our observations are consistent with the results of previous studies in rats (21, 31).

In the current study, we measured renal expression of AT1A, AT1B, and AT2 receptor mRNA in 30-day-old mice. AT1A-receptor mRNA was detected in all kidneys, as was AT1B- and AT2-receptor mRNA expression. Compared with our positive control of fetal kidney tissue, where the AT2 receptor is constitutively highly expressed, the levels of AT2 receptor in postnatal day 30 tissues was considerably lower and highly variable. This is perhaps explained by the fact that after birth, levels of AT2 receptor expression are reduced markedly (35), and the timing of this reduction to very low levels may vary between individual mice. In the mouse kidney, the AT1 receptor is expressed in developing glomeruli and S-shaped bodies, as well as in proximal and distal tubules, where it is thought to mediate cellular differentiation and proliferation during nephrogenesis (15). Expression is greatest at the end of gestation and decreases after birth. We found considerably greater renal AT1A receptor mRNA levels in female NP mice compared with male NP mice at postnatal day 30. This observation is consistent with the results of a previous study in rats (23), although other studies have failed to replicate this observation (24). The greater levels of renal AT1A receptor expression in NP female than NP male mice might reflect sex differences in the time-course of renal development, in that they are "leftover" from the higher levels of AT1A receptor expression present during nephrogenesis in these relatively young (adolescent) mice. This phenomenon could possibly contribute to the sex differences in glomerular morphology observed in the present study. We might also therefore expect to find an eventual decrease in AT1A receptor expression in female mice beyond 30 days of age. Studies are currently under way to investigate these possibilities.

There is now considerable evidence, from a number of models of developmental programming, for roles of abnormalities in the renal renin/angiotensin system (32). For example, maternal protein restriction in rats reduced renal renin mRNA levels and renal tissue ANG II levels in newborn male pups and resulted in adult hypertension (41). Moreover, AT1 receptor density was increased, while AT2 receptor density was reduced, in protein-restricted male rats at 4 wk of age (33). We found a clear effect of maternal protein restriction on renal AT1A receptor expression but only in female offspring. Thus, renal AT1A receptor expression was considerably less in LP compared with NP female mice, but at a similar level to both LP and NP male mice. Previous studies of the effects of maternal protein restriction on AT1A receptor expression have produced conflicting results (22–24, 33, 34). Our current data raise the possibility that the sex-dependent effects of maternal protein restriction on renal AT1A receptor expression might lead to sex differences in the impact of maternal protein restriction on adult disease. This hypothesis merits further investigation.

One important caveat must be applied to our current findings. The genetic background of the male breeder mice (129sv) differed from that of the female breeders (C57/BL6). We took this approach to ensure hybrid vigor in the offspring, since we are currently undertaking intensive studies of cardiovascular and renal physiology in these animals in adulthood. The advantages of a hybrid strain include offspring vigor, uniformity, and litter sizes that are often larger than those of pure isogenic strains (9). It does, however, mean that the single X chromosomes of the male offspring are exclusively of the C57/BL6, whereas the females have both the 129sv and C57/BL6 X chromosome. However, we believe this is unlikely to have greatly confounded our current observations since, in mice, X inactivation is imprinted with preferential silencing of the paternal X chromosome (37). This means that the C57/BL6 X chromosome should dominate in both male and female mice. There is a small possibility that some X-linked genes are still expressed from the inactive X chromosome. However, this phenomenon is less frequent in mice than humans (13).

Perspectives

Sex has important influences on adult risk of disease (38). It might therefore be expected that sex could greatly influence adult cardiovascular and renal function. Indeed, there is now evidence that this is the case in the model of maternal protein restriction in rats (42), while other studies have identified important differences between the sexes in basal renal hemodynamics (25, 27) and renal function (8). Our present study is, to our knowledge, the first to examine the effects of maternal protein restriction in mice on offspring renal morphology and incorporates measurements in both male and female offspring. Our results indicate that maternal protein restriction reduces nephron endowment in the offspring of mice, as is the case in rats (14). This effect, and many of the other effects of maternal protein restriction, was similar in male and female offspring. However, there were sex-dependent effects of protein restriction on renal AT1A receptor expression and liver weight, the significance of which requires further study. We also identified sex differences in underlying glomerular morphology that have not previously been described. The mouse model that we describe here should facilitate future study of the molecular bases of developmental programming and its sex dependence.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Health and Medical Research Council of Australia (NH&MRC) Grants 384207, 384101, and 384279 and by an Australian Postgraduate Award (to C. C. Hoppe) through the Commonwealth Government of Australia. R. G. Evans is also supported by a NH&MRC Senior Research Fellowship, and K. M. Moritz is supported by a NH&MRC Career Development Award.


   ACKNOWLEDGMENTS
 
The authors acknowledge the expert technical assistance of Sue Connell and Debbie Arena.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Hoppe, Dept. of Anatomy and Cell Biology, School of Biomedical Sciences, Monash Univ., Victoria, 3800, Australia (e-mail: Chantal.Hoppe{at}med.monash.edu.au)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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