Novel Frem1-Related Mouse Phenotypes and Evidence of Genetic Interactions with Gata4 and Slit3

The FRAS1-related extracellular matrix 1 (FREM1) gene encodes an extracellular matrix protein that plays a critical role in the development of multiple organ systems. In humans, recessive mutations in FREM1 cause eye defects, congenital diaphragmatic hernia, renal anomalies and anorectal malformations including anteriorly placed anus. A similar constellation of findings–microphthalmia, cryptophthalmos, congenital diaphragmatic hernia, renal agenesis and rectal prolapse–have been described in FREM1-deficient mice. In this paper, we identify a homozygous Frem1 missense mutation (c.1687A>T, p.Ile563Phe) in an N-ethyl-N-nitrosourea (ENU)-derived mouse strain, crf11, with microphthalmia, cryptophthalmos, renal agenesis and rectal prolapse. This mutation affects a highly conserved residue in FREM1’s third CSPG domain. The p.Ile563Phe change is predicted to be deleterious and to cause decreased FREM1 protein stability. The crf11 allele also fails to complement the previously described eyes2 allele of Frem1 (p.Lys826*) providing further evidence that the crf11 phenotype is due to changes affecting Frem1 function. We then use mice bearing the crf11 and eyes2 alleles to identify lung lobulation defects and decreased anogenital distance in males as novel phenotypes associated with FREM1 deficiency in mice. Due to phenotypic overlaps between FREM1-deficient mice and mice that are deficient for the retinoic acid-responsive transcription factor GATA4 and the extracellular matrix protein SLIT3, we also perform experiments to look for in vivo genetic interactions between the genes that encode these proteins. These experiments reveal that Frem1 interacts genetically with Gata4 in the development of lung lobulation defects and with Slit3 in the development of renal agenesis. These results demonstrate that FREM1-deficient mice faithfully recapitulate many of the phenotypes seen in individuals with FREM1 deficiency and that variations in GATA4 and SLIT3 expression modulate some FREM1-related phenotypes in mice.


Introduction
The FRAS1-related extracellular matrix 1 (FREM1) gene encodes an extracellular matrix protein that plays a critical role in the development of multiple organs [1]. During epidermal development, FREM1 is secreted by mesenchymal cells into the basement membrane [2]. In the basement membrane, FREM1 forms a ternary complex with FRAS1 and FREM2, transmembrane proteins that are transported to the plasma membrane of epidermal cells with the help of GRIP1 and are shed from the cell surface by proteolytic processing [2]. In mice, recessive mutations affecting Frem1 or Frem2 lead to diminished expression of FREM1, FRAS1 and FREM2 in the basement membrane, suggesting that these proteins undergo reciprocal stabilization in this location [2]. Loss of the FREM1/FRAS1/FREM2 complex, due to recessive mutations in Frem1, Fras1, Frem2 or Grip1, leads to a loss of epidermal integrity and the development of large fluidfilled blisters over the eyes and/or digits between E10.5 and E12.5. As a result, these mouse strains are often referred to collectively as 'bleb' mutants [3][4][5][6][7][8][9].
Given the known interactions between these proteins, it is not surprising that FREM1-, FRAS1-, FREM2-and GRIP1-deficient mice have overlapping patterns of defects that include cryptophthalmos and syndactyly-which are likely to be secondary effects of blister formation-and renal agenesis. Other features have only been documented in a subset of these mice. For example, abnormal lung lobulation has been documented in mice with mutations in Fras1 and Frem2 but have not been documented in mice with mutations in Frem1 or Grip1 [10,11]. Similarly, recessive mutations in Frem1 have been shown to cause congenital diaphragmatic hernia (CDH) which has not been documented in mice with Fras1, Frem2 or Grip1 mutations [12].
Like FREM1-deficient mice, mice that are deficient for the retinoic acid responsive transcription factor GATA4 have anterior CDH and lung lobulation defects [26][27][28]. Phenotypic similarities-CDH and renal agenesis-also exist between FREM1-deficient mice and mice with recessive mutations in Slit3, which encodes an extracellular matrix protein [29,30]. This prompted us to look for in vivo genetic interactions between these genes. We found that Frem1 and Gata4 interact genetically in the development of lung lobulation defects and that Frem1 and Slit3 interact genetically in the development of renal agenesis.

Mouse Studies
All experiments using mouse models were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The associated protocols were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine (Animal Welfare Assurance #A3832-01).
All efforts were made to minimize suffering. Euthanasia was carried out using methods consistent with the recommendations of the Panel of Euthanasia of the American Veterinary Medical Association and included carbon dioxide (CO 2 ) inhalation or an overdose of an inhaled anesthetic, such as isoflurane, in an appropriate enclosure.

Generation of crf11 Mice by N-ethyl-N-nitrosourea (ENU) Mutagenesis
ENU mutagenesis was carried out using 8-to 12-week-old male C57BL/6J mice given 300 mg/kg of N-ethyl-N-nitrosourea. ENU was administered in three 100 mg/kg intraperitoneal injections at 1-week intervals, as previously described [31]. These mice were then bred and intercrossed to screen for viable recessive phenotypes. The craniofacial 11 (crf11) strain (MGI: 2671571) was identified based on the presence of unilateral and bilateral microphthalmia and/or cryptophthalmos and variable craniofacial defects [32].

Mapping and Cloning of the crf11 Allele
Mice from the crf11 strain were backcrossed to 129S6/SvEvTac mice. The progeny of these crosses were intercrossed and mice carrying the crf11 allele were identified based on their eye phenotypes. After several generations of backcrossing, crf11 mice were genotyped using single nucleotide polymorphism (SNP) markers that discriminate between C57BL/6J and 129S6/ SvEvTac strains. Linkage analysis was performed as previously described and the crf11 allele was found to be linked to markers on mouse chromosome 4 [33]. Additional backcrosses were carried out and crf11 mice were genotyped by amplifying and sequencing regions of the genome which harbored SNPs known to vary between the C57BL/6J and 129S6/SvEvTac strains.
Candidate genes from the crf11 interval-including Frem1-were selected for further mutation screening. The coding region and associated intron/exon junctions of Frem1 were amplified by PCR and the resulting amplification products were sequenced. Primer sequences are available on request. Sequence traces were analyzed using Sequencher 4.7 software (Gene Codes Corporation).

Phenotypic Analysis of Mice Bearing the crf11 and eyes2 Alleles
Necropsies were performed on Frem1 crf11/crf11 , Frem1 crf11/eyes2 , and Frem1 eyes2/eyes2 mice to determine the spectrum and penetrance of Frem1-associated phenotypes on a mixed B6J/129S6 background. Similar studies were performed on a congenic Frem1 eyes2/eyes2 mouse line that had been backcrossed to C57BL6/J mice for at least 8 generations [12].

In situ Hybridization and Immunohistochemistry
In situ hybridization probes were created to target Frem1, Fras1 and Frem2 transcripts. The probe targeting Frem1 was designed using Sequencher 4.7 software (Gene Codes Corporation, Ann Arbor, MI, USA), while the probes targeting Fras1 and Frem2 were based on in situ probe designs from the Eurexpress database (http://www.eurexpress.org/ee/; Frem2 template ID T38972; Fras1 template ID T36392), as previously described [12]. In situ studies were performed by the IDDRC RNA In Situ Hybridization core at Baylor College of Medicine, on sagittal sections of E14.5, C57BL6/J embryos as previously described [12].
Immunohistochemistry was performed on paraffin sections of E14.5 wild-type C57BL6/J embryos using a mouse polyclonal primary antibody targeted against GATA4 (Sc-25310, Santa Cruz Biotechnology, Santa Cruz, CA) as previously described [27].

Measurement of Fecal Diameter and Anogenital Distance
To minimize variation, measurements of fecal diameter and anogenital distance were measured on P28-P30 littermates on a C57BL/6J background by a single individual. Feces was collected from mice of different genotypes and imaged using a Nikon SM21500 microscope (Nikon Instruments, Melville, NY, USA) with an attached Zeiss Axiocam MRc camera (Carl Zeiss AG, Oberkochen, Germany). Fecal diameter was then measured using the AxioVision Release 4.6.1.0 software (Carl Zeiss AG). For measurements of anogenital distance, the anogenital regions of euthanized mice were pressed against a clear plastic surface and measured from the other side. In female mice, the anogenital distance was measured from the center of the anal opening to the center of the vaginal opening. In male mice, the anogenital distance was measured from the center of the anus to the base of the genital papilla.

Statistics
Chi-square analyses were performed using the Opus12 chi-square calculator (http://www.opus12.org/Chi-Square_Calculator.html) to assess genotype-and strain-dependent differences in the penetrance of individual phenotypes. A Fisher Exact test was used for comparisons in which expected frequencies were less than five. For anogenital distance and fecal diameter studies, results were analyzed using one-way analysis of variance performed using the IBM SPSS Statistics software (IBM, Armonk, NY, USA).
After several generations of backcrossing to 129S6/SvEvTac mice, the crf11 phenotype was linked to a region of mouse chromosome 4. This region contained Frem1 which was considered a promising candidate gene based on published reports of other FREM1-deficient mice with similar phenotypes (Table 1) [1,2,4,7,12].
Sequencing of the Frem1 coding region and intron/exon boundaries revealed a homozygous c.1687A.T change in DNA samples from crf11 mice which was not found in DNA from C57BL/6J and 129S6/SvEvTac control mice ( Fig. 2A). This change causes an isoleucine to phenylalanine change (p.Ile563Phe) at a highly conserved amino acid residue in FREM1's third CSPG domain (Figs. 2B & 2C). CSPG domains are cadherin repeat-like repetitive units which are a hallmark of the FRAS-FREM protein family [1]. The p.Ile563Phe change was predicted to be ''Non Tolerated'' by SIFT (http://sift.jcvi.org/) and damaging by PANTHER PSEC (http://www.pantherdb.org/tools/ csnpScoreForm.jsp), two online programs that predict the effects of amino acid substitutions on protein function [34,35].

The crf11 Allele Fails to Complement the eyes2 Allele of Frem1
To confirm that the phenotype of the crf11 strain was due to changes affecting Frem1 function, we crossed crf11 mice with eyes2 mice which are homozygous for a previously described nonsense Frem1 Phenotypes, Interactions with Gata4 & Slit3 mutation in Frem1 (p.Lys826*) [12]. The phenotypes seen in the resulting compound heterozygous progeny (Frem1 crf11/eyes2 ) are summarized in Table 2 and include unilateral and bilateral cryptophthalmos and/or microphthalmia, CDH and renal agenesis.
This failure of the crf11 allele to complement the eyes2 allele of Frem1 suggests that an ENU-induced defect in the Frem1 gene is  the cause of the crf11 phenotype. Since the crf11 and eyes2 mouse lines were created in the same mutagenesis screen, and have been maintained on similar B6J/129S6 backgrounds, it is less likely that failure to complement is due, instead, to strain specific variants linked to the mutant locus [12,32,42]. However, these studies cannot rule out the possibility that the c.1687A.T change is not the cause of the crf11 phenotype but is, instead, linked to a causative mutation which was not identified in our sequencing analysis.
Expression of Frem1, Fras1, Frem2 in the Developing Lung Lung lobulation defects have been reported previously in FRAS1-deficient and FREM2-deficient mice [10,11]. In the case of FRAS1-deficient mice, branching morphogenesis-the process by which reciprocal interactions between the epithelium and its underlying mesenchyme produce the individual branches of the bronchial tree-was found to be normal [10,43,44]. Further evaluations performed on embryos at E14.5, suggested that defects in the basement membrane of the visceral pleura surrounding the developing lung were the underlying cause of the lung lobe fusions seen in FRAS1-deficient mice [10].
If similar defects underlie the lung lobulation defects in FREM1 and FREM2-deficient mice, we would expect that Frem1 and Frem2 would be expressed along with Fras1 in cells adjacent to the basement membrane of the visceral pleura. To determine the expression patterns of these genes in the developing lung, we performed in situ hybridization studies on wild-type embryos at E14.5. At this time point, Frem1 transcripts were detected in the underlying mesenchymal cells of the developing lung but not in the mesothelial cells of visceral pleura (Fig. 4A-C). In contrast, Fras1 and Frem2 transcripts were detected in the mesothelial cells of visceral pleura but were not detected in the underlying mesenchymal cells (Fig. 4D-I). This pattern is identical to the expression pattern of these genes in the developing diaphragm [12]. A similar expression pattern is also seen in the developing skin, where FREM1 is secreted by mesenchymal cells into the basement membrane and FRAS1 and FREM2 are released into the basement membrane from epidermal cells [2].

Male FREM1-deficient Mice have Decreased Anogenital Distances
Individuals with BNAR syndrome have a spectrum of anorectal malformations that includes anal stenosis and anteriorly placed anus [19]. To determine if the development of rectal prolapse in FREM1-deficient mice was due, in part, to anal stenosis or anteriorly placed anus, we compared the fecal diameters and anogenital distances of P28-P30 Frem1 eyes2/eyes2 mice and their wild-type littermates on a C57BL/6J background. No significant differences in fecal diameter were found between mice of these genotypes (Fig. 5A & 5B). While no difference was seen in the anogenital distance of female Frem1 eyes2/eyes2 mice and their female wild-type littermates (Fig. 5C), a significant difference in average anogenital distance was found between male Frem1 eyes2/eyes2 mice and their male wild-type littermates (0.89 cm vs. 1.05 cm, p = 0.026) (Fig. 5D). This difference was not attributable to a difference in overall size since the average weight and length of P28-P30 wild-type and Frem1 eyes2/eyes2 mice on this background are not significantly different (14.4 g vs. 15.5 g, p = 0.58; 14.8 cm vs. 14.9 cm, p = 0.6).

Frem1 and Gata4 Interact Genetically in the Development of Lung Lobulation Defects
Gata4 +/Dex2 mice, which harbor a deletion in exon 2 of Gata4, have anterior congenital diaphragmatic hernias that are similar to those seen in FREM1-deficient mice [12,26]. Defects in Gata4 also cause lung lobulation defects. Specifically, embryos that are homozygous for the V238G missense mutation in Gata4 (Gata4 ki/ ki ), which die between E11.5 and E13.5, have abnormal lung lobulation with failure of accessory lobe development being evident in cultured lungs harvested at E11.5 and E12.5 [28]. Loss of the accessory lobe in Gata4 ki/ki mice is attributable to defects in branching morphogenesis.
To determine if Gata4 and Frem1 are expressed in the same cells of the lung, we performed immunohistochemical analyses of GATA4 expression at E14.5. These studies revealed GATA4 nuclear staining in the mesothelial cells of the visceral pleura, where Fras1 and Frem2 are expressed (Figs. 4J & 4K), but little evidence of GATA4 nuclear staining in the mesenchymal cells of the lung where Frem1 is expressed. This is consistent with previous published reports in which GATA4 expression was found mainly in the mesothelial cells of the developing lungs at E11.5, a time point after secondary lung branches have been established [28].
Renal agenesis was not seen in Frem1 eyes2/+ mice (0/110) or in Frem1 eyes2/+ ;Slit3 +/2 mice (0/126). However, a statistically significant increase in the prevalence of renal agenesis was seen between Frem1 eyes2/eyes2 and Frem1 eyes2/eyes2 ;Slit3 +/2 mice (2.7% vs. 13.6%, p,0.02). These results suggest that Frem1 and Slit3 interact genetically in the development of renal agenesis, since this increase in prevalence is more than can be attributed to the additive effects of homozygosity for the eyes2 allele (2.7%) and heterozygosity for the disrupted Slit3 allele (0%) alone. Figure 5. Male Frem1 eyes2/eyes2 mice on a C57BL6 background have decreased anogenital distance. Comparisons of fecal diameter and anogenital distance were made between female and male P28-P30 Frem1 eyes2/eyes2 mice and their sex-matched wild-type littermates on a C57BL6 background. A-B) No significant difference was seen in the fecal diameters of female or male Frem1 eyes2/eyes2 mice compared to their wild-type littermates. C) No significant difference was seen in the anogenital distances of female Frem1 eyes2/eyes2 mice compared to their wild-type littermates. D) The anogenital distances of male Frem1 eyes2/eyes2 mice was significantly smaller than that of their wild-type littermates (p = 0.026) despite having similar weights and lengths. Error bars show standard error of the mean. doi:10.1371/journal.pone.0058830.g005 Table 3. Effects of Gata4 haploinsufficiency on the prevalence of Frem1-associated phenotypes.

Discussion
Deleterious Mutations in Frem1 cause the crf11 Phenotype The ENU-derived mouse line crf11 displays several phenotypes which mimic previously described models of Frem1 deficiency ( Table 1). Sequencing of the Frem1 gene revealed a homozygous missense mutation (c.1687A.T, p.Ile563Phe) that affects a highly conserved residue in FREM1's third CSPG domain. The CSPG domains of chondroitin sulfate proteoglycan 4 (CSPG4) have been shown to interact with various growth factors and collagen molecules [47][48][49][50]. It is possible that the CSPG domains of FREM1 play a similar role.
Since the c.1687A.T, p.Ile563Phe mutation was predicted to be deleterious and to result in decreased protein stability, and the crf11 allele failed to complement the eyes2 allele of Frem1 (p.Lys826*), we conclude that homozygosity for this change is the cause of the crf11 phenotype [12,32].

Recessive Mutations in Frem1 Cause Lung Lobulation Defects
Lung lobulation defects have not been described previously in FREM1-deficient mice. However, we found complete and partial fusions between the cranial, middle and caudal lobes of the right lung in Frem1 crf11/crf11 , Frem1 crf11/eyes2 and Frem1 eyes2/eyes2 mice on a mixed B6Brd/129S6 background. This demonstrates that FREM1 plays a critical role in normal lung lobulation.
The lung lobulation defects seen in FREM1-deficient mice mirror those found in FRAS1 and FREM2-deficient mice. In FRAS1-deficient mice, defects in the basement membrane of the visceral pleura surrounding the developing lung have been implicated in the development of lung lobe fusions [10]. Since FREM1, FRAS1 and FREM2 form a ternary structure that is required for maintenance of epidermal integrity, it is possible that they also work together in the basement membrane of the visceral pleura to modulate lung lobe development. In support of this hypothesis, we have shown that Frem1 is expressed in the mesenchymal cells that underlie the basement membrane of the visceral pleura and that Fras1 and Frem2 are expressed in the mesothelial cells of the visceral pleura at E14.5. We note that FREM1 and FRAS1 proteins have also been detected previously in the visceral pleura at E14.5 [51]. Taken together, these data suggest that FREM1 protein from mesenchymal cells and FRAS1 protein from mesothelial cells may be secreted into the basement membrane of the visceral pleura in a manner analogous to that documented in the mesenchymal and epidermal cells of the developing skin [2]. Although the presence of FREM2 protein in the basement membrane of the visceral pleura has not been demonstrated, the expression pattern of Frem2 suggests that it may also be secreted from mesothelial cells into the basement membrane.
Lung lobulation defects have yet to be documented in individuals with FREM1 deficiency. However, in one large literature review, abnormal lung lobulation was documented in 5/117 (4.3%) of cases of Fraser syndrome, which can be caused by recessive mutations in FRAS1, FREM2 and GRIP1 [8,13,52]. Since lung lobulation defects are typically asymptomatic-and their diagnosis is often made incidentally-it is possible that such defects are also present in a subset of individuals with FREM1 deficiency but have gone undetected.

FREM1 Deficiency Causes Reduced Anogenital Distance in Mice
Anal stenosis and anteriorly placed anus are among the anorectal anomalies seen in individuals with FREM1 deficiency [19][20][21]. While we found no evidence for anal stenosis in studies of fecal diameter, we show that male Frem1 eyes2/eyes2 mice on a C57BL6 background have decreased anogenital distances. Anteriorly placed anus is commonly associated with chronic constipation which may, in turn, predispose to rectal prolapse through excessive straining during defecation [53]. This suggests that reduced anogenital distance may contribute to the development of the rectal prolapse in FREM1-deficient mice. However, it is unlikely that abnormal anal position is the only factor that leads to a propensity to develop rectal prolapse in FREM1-deficient mice, since this propensity is seen in both males and females and no differences were seen in the anogenital distances of female Frem1 eyes2/eyes2 mice compared to their wildtype littermate controls.
Anogenital distance is a sexually dimorphic trait in rodents and humans with males having an anogenital distance that is 2 to 2.5 times larger than in females [54]. In rodents, male anogenital distance has been shown to be dependent on exposure and response to androgens during a ''masculinization programming window'' which has not been well defined in mice but is thought to occur between E13.5 and E17.5 based on observations in rats [55][56][57]. The dependence of male perineal development on the ability to produce and respond to androgens-particularly testosteronemay explain why male eyes2 mice have reduced anogenital distances compared to their wild-type littermates while the anogenital distances of female eyes2 and wild-type mice are indistinguishable.
A complete inability to respond to androgens-as seen in complete androgen insensitivity syndrome (OMIM #300068)-can cause male fetuses to develop female external genitalia [58]. Since eyes2 male mice have male external genitalia and are fertile, we can assume that they are able to produce and respond to androgens in utero. However, further studies will be needed to determine if male eyes2 embryos and mice have lower levels of androgen production or attenuated responses to androgens when compared to their sexmatched wild-type littermates.
Although decreased anogenital distance has not been documented in FRAS1, FREM2, or GRIP1-deficient mice, one large literature review showed that anteriorly placed or displaced anus was documented in 7/117 (6%) of individuals with Fraser syndrome, which can be caused by the genes that encode these proteins [15]. The spectrum of anorectal anomalies-which can also include anal stenosis, rectal atresia, and imperforate anus-is also similar between individuals with FREM1 deficiency and Fraser syndrome [15,[19][20][21][22][23][24][25]. This suggests that FREM1, FRAS1, FREM2 and GRIP1 may function together to ensure normal anorectal development.
Gata4 as a Modifier of Lung Lobulation Defects in FREM1-deficient Mice We have previously reported a statistically significant difference between the prevalence of CDH in Frem1 eyes2/eyes2 mice on an inbred mixed B6Brd/129S6 background and on a C57BL/6J background [12]. In this report we have shown that similar differences exist in the prevalence of lung lobulation defects in Frem1 eyes2/eyes2 mice on these backgrounds. These findings provide evidence of the existence of genetic factors that influence the penetrance of CDH and lung lobulation defects caused by FREM1 deficiency [12].
Since GATA4-deficient mice have CDH and lung lobulation defects, we looked for evidence of a genetic interaction between Frem1 and Gata4 [12,26]. Although no evidence of a genetic interaction was seen in the development of CDH, the prevalence of lung lobulation defects increased significantly in FREM1 eyes2/ eyes2 mice with the addition of a Gata4-null allele, from 19.7% to 55.2% (p,0.001). This increase was more than could be attributed to the additive effect of the GATA4-null allele alone, which resulted in only a 4.3% rate of lung lobulation defects in Frem1 eyes2/+ mice. This suggests that Frem1 and Gata4 interact genetically in the development of lung lobulation defects and that Gata4 expression can modulate the penetrance of this FREM1related phenotype in mice.
Lungs from Gata4 ki/ki embryos fail to form accessory lobes in culture due to an abnormality in branching morphogenesis [28]. In contrast, the lung lobe fusion defects seen in Frem1 eyes2/ + ;Gata4 +/2 and Frem1 eyes2/eyes2 ;Gata4 +/2 mice did not affect the accessory lobe and are similar to those seen in FRAS1-deficient mice, which have been attributed to defects in the basement membrane of the visceral pleura surrounding the developing lung [10]. At E14.5, GATA4 is expressed in the nuclei of mesothelial cells surrounding the developing lungs while Frem1 is expressed in the mesenchymal cells that underlie the basement membrane of the visceral pleura. This makes it unlikely that GATA4 directly regulates the expression of Frem1. However, GATA4 may regulate the expression of Fras1 and/or Frem2 in a cell-autonomous fashion since these genes are expressed in mesothelial cells.

Slit3 as a Modifier of Renal Agenesis in FREM1-deficient Mice
Although Slit3-null mice have central diaphragmatic herniaswhich are anatomically different than the retrosternal anterior hernias seen in Frem1 crf11/eyes2 and Frem1 eyes2/eyes2 mice-a similar pattern of decreased cell proliferation in the central tendon region of the diaphragm has been documented in both Slit3-null mice and Frem1 eyes2/eyes2 mice [12,29,30]. Slit3-null mice also have unilateral and bilateral renal agenesis that is similar to that seen in FREM1deficient mice [29,30]. This prompted us to look for evidence of a genetic interaction between Frem1 and Slit3.
No increase in CDH prevalence was seen in Frem1 eyes2/eyes2 mice with the addition of a disrupted Slit3 allele. In contrast, the prevalence of renal agenesis increased significantly from 2.7% in Frem1 eyes2/eyes2 mice to 13.6% in Frem1 eyes2/eyes2 ;Slit3 +/2 mice (p,0.02). This increase was more that could be attributed to the additive effect of the disrupted Slit3 allele, since renal agenesis was not seen in Frem1 eyes2/+ ;Slit3 +/2 mice (0/126). This suggests that Frem1 and Slit3 interact genetically in the development of renal agenesis and that Slit3 expression can modulate the penetrance of this FREM1-related phenotype in mice.
During normal development of the metanephric kidney, the ureteric bud extends out from the mesonephric duct and penetrates the metanephric mesenchyme. FRAS1, FREM1 and FREM2 are expressed around the ureteric bud at E11 and appear to undergo reciprocal stabilization as previously demonstrated in the skin [2,59]. In FRAS1-deficient mice, lack of ureteric bud initiation from the mesonephric duct, or failure of the ureteric bud to penetrate the metanephric mesenchyme, causes the metanephric mesenchyme to undergo fulminant apoptosis, resulting in renal agenesis [59,60]. Apoptosis of cells in the metanephric mesenchyme, leading to renal agenesis, is also seen in mice that lack the cytosolic adapter protein GRIP1, which plays a critical role in trafficking FRAS1 and FREM2 to the plasma membrane [2,61]. It is possible that a similar mechanism underlies the development of renal agenesis in FREM1-deficient mice.
In situ hybridization studies performed at E12.5 demonstrate Frem1 expression in the metanephric mesenchyme surrounding the growing uretic bud [1]. It is likely that the FREM1 protein detected around the ureteric bud during kidney development is secreted from these cells. Whole mount in situ hybridization studies of kidney explant cultures from E11.5 embryos do not detect Slit3 transcripts in the invading ureteric tree or in the metanephric mesenchyme. However, Slit3 transcripts were found at high levels in the intermediate mesoderm surrounding the metanephric mesenchyme after one day in culture (E12.5) [62]. Hence, Frem1 and Slit3 appear to be expressed in adjacent cell populations in the developing kidney. Further studies will be needed to determine the molecular mechanism by which decreased SLIT3 expression causes renal agenesis in Slit3-null mice and modulates the prevalence of renal agenesis caused by FREM1 deficiency.