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Original Article |
Kelly{at}brc.ubc.ca
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Key Words: sialomucin • umbilical hernia • podocyte • hematopoiesis • vascular endothelium
Biochemical and sequence analysis have shown that podocalyxin is a 150–165-kD transmembrane protein composed of a mucin domain, a disulfide-bonded globular domain, a transmembrane region, and a highly charged cytoplasmic tail with potential phosphorylation sites for protein kinase C and casein kinase II (Fig. 1). Structurally, it belongs to a large family of highly sulfated cell surface sialomucins of poorly understood functions. The amino acid sequence, protein structure, and genomic organization of podocalyxin suggest it is most closely related to two other molecules, CD34 and endoglycan (Fig. 1; references 5 and 8). CD34 is the prototypic member of this family and has been widely used in human and mouse as marker of hematopoietic progenitor cells and vascular endothelia (for a review, see reference 9). On high endothelial venules (HEVs) CD34 1 and podocalyxin 1 have been shown to act as an adhesive ligand for L-selectin expressed by leukocytes 10. This interaction requires HEV-specific glycosylation of CD34, and similar modifications have not been observed on the majority of vascular endothelial cells (ECs) or hematopoietic cells. Thus, it is remarkable that despite extensive use as a marker (>7,000 CD34-related publications), the functional role of CD34 on hematopoietic cells and most vascular cells remains obscure. Mice lacking CD34 develop normally 1112 although very subtle defects in hematopoietic maturation and function can be detected in in vitro assays or in in vivo assays of allergic responses. Endoglycan is the newest member of this gene family and has a broader distribution as it is expressed by hematopoietic progenitors and some mature blood cells, vascular endothelia, smooth muscle, and a subset of neuronal cells 13. Its function, too, is unknown.
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Podocalyxin (also called podocalyxin-like protein 1 [PCLP-1], Myb-Ets–transformed progenitor (MEP)21, and thrombomucin) is a heavily sialyated and sulfated membrane protein expressed on the apical surface of glomerular epithelial cells or podocytes and on vascular endothelia 12. Although first described as a marker of podocytes and vasculature, podocalyxin expression has also been detected on mesothelial cells lining the coelomic cavity, platelets, and hematopoietic precursors cells 34567. Recent experiments in avians and mice have shown that podocalyxin also marks the earliest detectable hematopoietic progenitors during development 5 as well as bipotent progenitors of blood cells and vascular endothelia called hemangioblasts 7.
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Although mutations in the podocalyxin gene have not yet been linked to nephrotic syndrome and renal failure, several other human and murine gene mutations of podocyte proteins have been identified. These include mutations in nephrin/congenital nephrotic syndrome (NPHS) gene 1, podocin/NPHS gene 2, 
-actinin-4, CD2-associated protein (CD2AP), and 3β1 integrin 2021222324. Although the precise function of the proteins encoded by several of these genes is uncertain they have one common denominator: their mutation leads to the disruption of normal podocyte architecture 252627.
To determine whether podocalyxin plays an essential role in renal, vascular, and hematopoietic function we have disrupted the podocalyxin-encoding gene in mice (podxl–/–). All podxl–/– mice die within the first 24 h of postnatal life from profound defects in kidney and/or gut formation (herniation or omphalocele). Surprisingly, the loss of podocalyxin expression did not result in the massive proteinuria characteristic of leaky podocyte filtration in human nephrotic syndromes. Instead, newborn podxl–/– mice were anuric (no measurable urine in the bladder) and failed to form FPs and SDs. Our data suggest that podocalyxin is indispensable for normal murine development and that its mutation could play a role in podocyte-related renal failure and in omphalocele (see Discussion).
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Targeted Disruption of the Podocalyxin (podxl) Gene.
A partial mouse podocalyxin cDNA clone (534 bp) was produced by reverse transcription PCR of mouse kidney RNA using primers to the 3'-coding sequence of chicken podocalyxin: 5'-GAATTCGGCTTCTCGTGGAACTGCTGTGTCACT-3' and 5'-GAATTCGGCTTCTCCTCATCTAGGTCATCCTTGG-3'. This probe was used to screen a 129/SvJ phage library in 
FIXII (Stratagene), and 3 independent mouse podxl genomic clones were identified and purified. A mutant allele of the podxl genomic locus was generated by inserting a neomycin resistance cassette in the antisense orientation between the XbaI site in exon 5 and an engineered XhoI site in exon 8 of the m-podxl gene, thereby deleting the majority of exons 5, 6, 7, and 8. The 5.5-kb XbaI fragment containing part of the 5th exon was subcloned into the XbaI site of the pPNT vector 28 to create the 5' arm of homology in the knockout (KO) vector (29, 30). The 3' arm of homology was created by PCR of an 892-bp fragment from the 3' region of the MEP21 coding sequence using primers 5'-GCGGCCGCTTACTAGGCATGGTTTTATG-3' and 5'-CTCGAGACACCTCTGATCTGTCTGCTGGTC-3', and this was cloned into the NotI and XhoI sites of pPNT. The resulting vector was electroporated into E14 embryonic stem (ES) cells and these were selected for resistance to G418 and against sensitivity to ganciclovir. Of 672 resistant ES cell clones, 384 were screened by Southern blot analysis for presence of a 5-kb HindIII fragment using the strategy depicted in Fig. 2. Blastocyst injection of these ES cells and production of germline transmitting chimeras was performed by RCC Ltd., using standard protocols. Genomic DNA from mouse tail cuts or tissue samples was isolated using the DNAeasy Tissue Kit (QIAGEN). Genotypes were determined by PCR using wild-type and KO specific primers (5'-AGTGAGAGACACATTGGGTAACT-3' wild-type allele specific, 5'-TATCGCCTTCTTGACGAGTTCTT-3' KO allele specific, and 5'-GAGGATTTGTGCACTCTACATGTG-3' common primer; GIBCO BRL). The wild-type allele resolves as a 760-bp band, while the KO allele resolves as a 550-bp band.
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40 µg per sample) were resolved on 0.7% agarose gels, denatured with NaOH, and transferred to nylon membranes. Membranes were hybridized with radiolabeled probes corresponding to the 2,200-bp ApaI fragment of the podocalyxin 3' untranslated region (see Fig. 2). Hybridization of radiolabeled probes and removal of unbound probe was performed in NaHPO4/SDS buffer as described by Church and Gilbert 33. In this assay, the wild-type podxl locus resolves as a 4-kb HindIII fragment while the homologously recombined locus resolves as a diagnostic 5-kb fragment. For Northern blot analysis, total RNA was prepared by lysis and fractionation in guanidinium/acetate/phenol/chloroform as described by Chomczynski and Sacchi 34. Approximately 10 µg of each RNA was resolved on a 1% agarose-formaldehyde gel and blotted onto nylon membranes (GeneScreen; Dupont). [32P]-labeled probes were generated from the 490-bp SmaI-HindIII fragment encoding the mucin domain of the mouse podocalyxin cDNA or from a 1,000-bp PstI fragment of the glyceraldehyde-3-phosphate dehydrogenase cDNA as a control for RNA loading 35.
Histological Analyses and Immunohistochemistry.
Immunoperoxidase staining of snap frozen embryo tissues was performed using anti-PCLP-1 antibody 7, anti-CD34-biotin (BD PharMingen), and anti-CD31 (platelet endothelial cell adhesion molecule [PECAM]-1; BD PharMingen), followed by Vector Elite developing reagents (Vector Laboratories) and methyl green counterstain, as described previously 5. For paraffin sections, embryonic day 19 kidneys were fixed in 10% formalin, embedded in paraffin, and sectioned. After deparafinization and hydration the tissue sections were treated with target unmasking fluid (Signet Labs) for 16 h at 90°C 34 and stained with monoclonal antibodies to glomerular epithelial protein (GLEPP)1, nephrin, or control rat IgG. Antibody binding was revealed by staining with biotinylated goat anti–rat antibodies, followed by streptavidin-peroxidase, and 3,3'-diaminobenzidine–developing reagent using ABC reagents and protocols from Vector Laboratories. Sections were counterstained with periodic acid-Schiff's reagent using standard protocols 3. Immunofluorescence staining was performed essentially as described previously 36. Cryostat sections of mouse E19 kidneys were fixed in acetone and incubated with blocking solution (10% goat or donkey serum in PBS). Primary antibodies used were chicken anti–mouse GLEPP1 (courtesy of Dr. Roger Wiggins, University of Michigan, Ann Arbor, MI); rabbit anti–mouse nephrin (courtesy of Dr. Lawrence Holzman, University of Michigan); rabbit anti–mouse collagen 4(IV) and laminin 5 (courtesy of Dr. Jeff Miner, Washington University School of Medicine, St. Louis, MO); mouse monoclonal anti-synaptopodin (courtesy of Peter Mundel, Albert Einstein College of Medicine, Bronx, NY); rabbit anti–human Wilm's tumor protein 1 (Santa Cruz Biotechnology, Inc.); rat anti–mouse PECAM-1 (BD PharMingen); and rat anti–mouse laminin, laminin β1 and β2 (Chemicon International). For collagen 4(IV) staining, sections were pretreated with 6 M urea, 0.1 M glycine for 1 h at 4°C 37. Secondary antibodies were FITC-conjugated goat anti–rat (Southern Biotechnology Associates, Inc.), Cy3-conjugated goat anti–rabbit (Jackson ImmunoResearch Laboratories), FITC-conjugated goat anti–mouse (Cappel), and FITC-conjugated donkey anti–chicken (Lampire Biological Laboratories). All incubations were carried out in blocking solution. A Nikon Diaphot microscope and a Hamamatsu digital camera were used for acquisition of immunofluorescence images.
Transmission Electron Micrographs.
For transmission electron micrographs (TEMs), newborn kidneys were fixed in cold glutaraldehyde/cacodylate buffer. After plastic embedding, one micron sections were stained with toluidine blue. Selected samples containing glomeruli were thinly sectioned, stained with uranyl acetate, and examined by TEM as described previously 38. The most mature glomeruli on the sections with open capillary loops and urinary spaces were examined.
Flow Cytometric Analysis.
For single color, indirect immunofluorescence analysis, 106 cells from fetal liver, spleen, or bone marrow were preincubated with Fc block (anti-mCD16/32. clone 2.4G2; reference 39) in PBS containing 10% FCS and 0.05% azide. These cells were then stained with either biotin-, FITC-, or phycoerythrin-conjugated monoclonal antibodies to CD34 40, Sca-1 41, c-kit 42, CD41, Mac1 43, B220 44, CD3 45, CD4 46, CD8 47, or with unlabeled antibodies to Ter119 48, Gr-1 49, or PCLP-1 7 followed by goat anti–rat-FITC-coupled or biotin-coupled secondary antibodies (BD PharMingen) as described previously 50. All flow cytometric analyses were performed using a FACScanTM (Becton Dickinson).
Blood, Urine, and Amniotic Fluid Analysis.
Newborn mice were left for 30 min under a heating lamp and then killed by decapitation. Blood was collected from the aorta in 50-µl heparinized capillary tubes, and urine was collected in 5-µl capillary tubes after gentle massage of the bladder. The volume of urine produced from each mouse was measured and a 2-µl sample was analyzed for protein content by 10% SDS-PAGE under nonreducing conditions. After electrophoresis, proteins were visualized by silver staining. Blood samples were clotted and serum was analyzed for their creatinine/urea content using a modified creatinine kit (Sigma-Aldrich). Amniotic fluid was collected at 15 d after coitum and 1 µl of unconcentrated amniotic fluid was analyzed by 10% SDS-PAGE. As an indirect measure of blood pressure, day 18 embryos were rapidly dissected and placed in 1 ml of PBS with 1 mM EDTA. The umbilical cord was severed and embryonic blood was allowed to flow freely for 60 s. The cord was then clamped, the embryo and placenta were removed, and the number of RBCs released into the PBS was counted.
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To confirm that podocalyxin expression had been ablated in podxl–/– mice, Northern blot analysis was performed with RNA isolated from 16-d fetal lungs of podxl+/+, podxl+/–, and podxl–/– embryos, using the 5' region encoding the podocalyxin extracellular domain (lying outside the region of recombination) as a probe (Fig. 2 C). This analysis revealed the presence of the expected 5-kb podocalyxin transcript in wild-type embryos, reduced expression in heterozygote, and a complete lack of hybridizing transcripts in podxl–/– embryos suggesting a loss of expression from the targeted allele. This was confirmed at the protein level by cell surface immunofluorescence analysis of 15-d fetal liver cells using a monoclonal antibody to mouse podocalyxin (Fig. 2 D). Hematopoietic cells from podxl+/+ and podxl+/– mice expressed high levels of podocalyxin on their surface, while podxl–/– showed no reactivity. From these analyses we conclude that the engineered rearrangement in the podocalyxin locus has resulted in a complete loss of podocalyxin expression in podxl–/– mice.
Perinatal Lethality, Omphalocele, and Edema in podxl–/– Mice.
PCR and Southern blot analyses of 6-wk-old progeny from heterozygous crosses between podxl+/– mice revealed the complete absence of any podxl–/– offspring suggesting embryonic or perinatal lethality in mice lacking podocalyxin. To more accurately pinpoint the time of disappearance, embryos were harvested at various stages of development and genotyped by PCR. Although we observed the expected Mendelian frequency of podxl+/+, podxl+/–, and podxl–/– offspring throughout embryonic development, all podxl–/– mice died within the first 24 h of postpartum life (Table ). No statistically significant differences were observed in the birth weight of podxl+/+ newborns and their podxl–/– littermates. Likewise, no significant differences were noted in the organ weight or macroscopic appearance of the lungs, liver, heart, gut, and kidneys of the majority of the podxl–/– mice (although two notable defects were observed in a subset of the podxl–/– mice, see below). Perinatal lethality persisted when podxl–/– mice were delivered by Caesarean section and placed with foster mothers. Thus, the data suggest that the loss of podocalyxin results in perinatal lethality due to defects intrinsic to podxl–/– mice.
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25% of the podxl–/– embryos (3/12 at embryonic day 18 and 4/15 at embryonic day 15) exhibited mild to severe edema. This usually appeared as subdermal swelling as early as 15 d after coitum (Fig. 3) and a mildly turgid trunk and appendages at day 18 (data not shown). More strikingly, 30% of all podxl–/– mice were born with an "omphalocele" or herniation of the gut into the umbilical cord (Fig. 4A and Fig. B). No direct correlation was observed between embryonic edema and omphalocele; podxl–/– embryos at day 18 were equally likely to have one, the other, or both defects. However, neither defect was ever observed in any day 18 podxl+/+ or podxl+/–mice suggesting a strict correlation with podocalyxin loss.
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Podocalyxin Deficiency Leads to Symptoms Consistent with Neonatal Anuric Renal Failure.
The observation that only a subset of newborn podxl–/– have overt defects, and yet 100% of these mice die perinatally prompted us to perform more detailed analyses of organ function in these mice. Since podocalyxin is most prominently expressed in kidneys, we aspirated bladder contents to analyzed urine from newborns. Strikingly, 100% of the podxl–/– newborns (12/12) exhibited a lack of urine in the bladder suggestive of severely impaired kidney function (Fig. 5 A). Consistent with anuria (and not proteinuria) SDS-PAGE analysis of blood serum and amniotic fluid proteins showed no significant differences in protein constituents from wild-type and KO mice. Anuria is one of several clinical features of acute renal failure and can lead to intravascular volume expansion and hypertension. Other symptoms include increased blood pressure and elevated levels of serum creatinine and urea. Although direct measurement of blood pressure was not possible in newborn mice, we attempted to measure this indirectly by assessing cardiac output. Day 18 embryos were collected by Caesarean section, the umbilical cord of each embryo was severed and the RBCs were collected in a solution of PBS/EDTA for 60 s. These were then counted as a rough measure of cardiac output/blood pressure (Fig. 5 B). While no significant differences were observed between podxl+/+ and podxl+/– embryos, podxl–/– embryos exhibited an 15-fold increase in released RBCs over the 60-s interval (most were released within the first 15 s). Consistent with this observation, podxl–/– embryos frequently appeared pale when the umbilical cords were severed in solutions that prevented clotting. This did not reflect an inability to clot as no differences in pallor were observed when the umbilical cords were severed in the absence of aqueous anti-coagulants, and we have not observed any differences in the frequency or function of platelets in podxl–/– mice (see below). An increase in blood pressure may offer an explanation for the edema observed in podxl–/– embryos: excessive pressure could drive fluid into the extravascular spaces. Although there were no significant differences in serum creatinine/urea levels between wild-type and podxl-deficient mice, it is likely that such changes would only appear after 1 or 2 d of life postpartum (during embryogenesis these would be removed maternally). Newborn mice with anuric renal failure have been described to consistently die in the first day of life 52535455, and thus it is likely that podxl–/– mice die due to kidney failure.
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4(IV), and laminins 5, β1, and β2) and podocyte markers (Wilm's tumor gene product 58, GLEPP1 56, nephrin [20, 57], and synaptopodin 59). We found no evidence of loss of expression of any GBM proteins in wild-type and podxl–/– kidneys (Fig. 6, and data not shown). The mature glomeruli of the newborn podxl+/+ and podxl–/– mice showed similar expression levels of collagen and laminin isoforms in mature GBM (Fig. 6B and Fig. C, and data not shown). Normally, podocyte FP and GBM proteins appear to have an overlapping staining pattern at the light microscopy level. This reflects the fact that the thin FPs of podocytes are in very close proximity to the GBM rather than true "colocalization". Interestingly we noted a consistent reduction in the degree of "side-by-side" podocyte and GBM-marker staining in podxl–/– kidneys. For example, while dual immunofluorescence-labeling showed the normal close proximity of the podocyte markers nephrin and GLEPP1 with the GBM in wild-type mice (note the red/green proximity in Fig. 6B and Fig. C), there was a marked decrease in the degree of overlap in the podxl–/– glomeruli. This indicates either a greater-than-normal distance between the podocyte apical membranes and the GBM (cell thickening) or incomplete coverage of the basement membranes by podocyte FPs.
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Role of Podocalyxin in Glomerular Structure and Function.
In kidneys, the final stage of glomerular filtrate production occurs when the ultrafiltrate passes through the filtration slits between neighboring podocyte FPs. The specialized cell–cell junctions between podocyte FPs forming the slit diaphragm provides the last barrier for filtrate production (Fig. 8). The permeability of the glomerular filter is highly dependent on the filtration slit surface area and the filtration properties of the slit diaphragm 6061. The podocytes of podxl–/– mice do not form FPs or SDs and instead form impermeable TJs. The absence of SDs and the reduction of filtration slit area due to the lack of interdigitating FPs in the podxl–/– mice probably leads to glomerular filters with reduced permeability that result in anuria at birth.
It has been speculated that the charge of podocalyxin is required for maintaining the spacing between FPs 15 since podocalyxin is the major constituent of the glycocalyx at their apical surface. This is supported by the fact that the inducible, ectopic expression of podocalyxin in Chinese hamster ovary cells and Madin-Darby canine kidney epithelial cells results in the inhibition of cell aggregation and in an altered organization of junctional proteins in adherent cell monolayers 62. Moreover, ectopic expression was shown to inhibit the formation of electrical-resistant monolayers of epithelial cells, indicating that podocalyxin can block the formation of TJs. Our data clearly support and extend this finding; in the absence of podocalyxin, JCs persist between podocytes and they fail to migrate down the lateral surface towards the basement membrane where they are normally modified to form SDs. The failure of the podocytes in podxl–/– mice to form permeable cell–cell junctions (SDs) is consistent with a role for podocalyxin as an antiadhesin on the surface of the podocytes and as a potential regulator of cell junctions.
It has been suggested that one way podocalyxin regulates cell–cell junctions is by interacting with the actin cytoskeleton, possibly via the actin-associated protein ezrin. The COOH-terminal tail of podocalyxin (D-T-H-L) is highly homologous to that of endoglycan and CD34 (D-T-H/E-L; Fig. 1 A). All three molecules contain the consensus sequence (X-S/T-X-V/I/L) recognized by proteins with PDZ protein interaction domains 63. PDZ-containing proteins can act as scaffolds to link transmembrane proteins in multiprotein complexes that may include the actin cytoskeleton 64. Indeed, one of us (unpublished data) recently found that the COOH terminus of podocalyxin interacts with NHERF-2, a PDZ domain-containing protein that can link transmembrane proteins to the actin cytoskeleton via ezrin 65. In light of the present studies we feel it is likely that this linkage is important in the modification of JCs in mature podocytes.
The vacuoles observed in the podocytes of podxl–/– mice are similar to the podocyte vacuoles seen in human or rodent models of renal disease 66676869. These vacuoles can occur in the setting of minimal change disease or in models where there is extensive podocyte injury (puromycin aminonucleoside–induced nephrosis). They have been hypothesized to result from the abnormal passage of fluid from the basolateral to apical surface of the podocyte in situations where there is extensive FP effacement or disruption of normal slit diaphrams 7071. Considering the lack of permeable cell–cell junctions and FPs in the podxl–/– mice, these podocyte vacuoles may result from the lack of a paracellular pathway for filtrate production and this "salvage pathway" may contribute to the bulk of fluid seen in the urinary space of the podxl–/– mice.
Podocalyxin Deficiency: Relation to other Podocyte-specific Mutations.
Several proteins proposed to be involved in the formation of JCs between podocytes have been identified recently as crucial regulators of podocyte function. These include nephrin/NPHS1, podocin/NPHS2, CD2AP, P-cadherin, β
catenin, -actinin-4, and ZO-1 162021222372. All of these proteins are expressed at the SDs between processes and have been speculated to participate in the formation of modified AJs 1626. Nephrin and P-cadherin are thought to interact by homotypic recognition and to be responsible for maintenance of the filtration slits 162026. CD2AP has been shown to interact with the intracellular domain of nephrin 23 whereas β catenins bind the intracellular domain of P-cadherin. Human mutations in NPHS2 or actinin 4 result in proteinuria and FP effacement 2122. Ablation of CD2AP and nephrin in mice leads to nephrotic syndrome with massive proteinuria and effacement of the FPs. A striking difference between podxl–/– mice and mice lacking nephrin or CD2AP is that the latter mice exhibit massive proteinuria and are still able to develop podocyte FPs 2023 whereas the podxl–/– mice lack FPs and produce no urine. These results support the contention that podocalyxin has a fundamentally distinct function in podocyte cell junctions (increased permeability) from nephrin/CD2AP (increased barrier function).
Aside from the proteins found in JCs, only a small number of other membrane proteins located at the apical surfaces of podocytes have been defined. They include podoplanin, a protein that is also expressed on nonpolarized cells 73, and GLEPP1, a transmembrane protein tyrosine phosphatase 38. GLEPP1 appears to regulate podocyte FP structure since deletion of this gene (ptpro) leads to toe-like podocyte FPs that are shorter and broader than the FPs of normal mice. Although ptpro–/– mice are viable and develop normally, they have a reduced glomerular filtration rate and after partial nephrectomy display a predisposition to hypertension. Thus, although the defects in ptpro–/– mice are much milder than those of podxl–/– mice, they also display a reduced glomerular filtration rate due to abnormalities in FP formation.
The fact that podxl–/– podocytes express the normal repertoire of both apical and junctional-complex podocyte markers suggests that maturation of these cells is relatively normal and that the observed defects in urine production are not due to defects in downstream protein expression. Rather, our results argue that podocalyxin loss leads directly to structural malformations of FPs. Similar to the neonatal lethality observed in podxl–/– mice, there are several other reports of anuric mice that die in the first day of life 52535455. However, it is noteworthy that most of these mutations result in much more severe kidney defects (agenesis) or in pleiotropic defects in other vital organs. Thus, podocalyxin loss represents the most selective, lethal anuria described to date.
Role of Podocalyxin in Resolution of Physiologic Omphalocele.
In normal mouse development, the gut herniates into the umbilical space through the umbilical ring beginning at embryonic day 12 and retracts back into the peritoneal cavity by the 16th day of embryonic development 58. This "physiologic omphalocele" is resolved by the expansion of the peritoneal cavity and retraction of the gut from the umbilical cord. While only a subset of podocalyxin-null mice displayed omphalocele at birth, all null mice showed delay in omphalocele resolution in utero. Surprisingly, some heterozygotes showed a similar delay suggesting a dosage effect of this mucin on the resolution of omphalocele. Omphalocele is a relatively common congenital birth defect in man, affecting 1:6,000 children, but the molecular details of its etiology are poorly understood. In many cases omphalocele has been linked to syndromes with abdominal organomegaly or defects in the development of the anterior abdominal wall leading to failure of umbilical ring closure 74. However, examination of podxl–/– mice revealed no abdominal organomegaly. Because of the dosage effect and because podocalyxin is expressed by the mesothelial cells lining the peritoneal membrane, we speculate that mesothelial function of this molecule is to provide an antiadhesive surface and facilitate retraction of the gut through the umbilical ring (see below).
Function and Redundancy of CD34-related Molecules in Development.
Despite the widespread expression of sialomucins in many tissues their function is still poorly understood and, until now, ablation of sialomucins have not resulted in a lethal phenotype. This suggests that most sialomucins are either dispensable for normal development or that there are mechanisms that compensate for their loss such as redundancy. The podxl–/– mice provide the first example of a sialomucin that is critically required for the normal development and postnatal survival of mice. Since podocalyxin deficiency causes defects in tissues that selectively express podocalyxin but not CD34 (gut mesothelial cells and podocytes), while coexpressing tissues are spared (vascular endothelia and hematopoietic precursors), it is tempting to speculate that CD34 and podocalyxin can, indeed, cross-compensate. Our observation that CD34 expression is upregulated in podxl–/– mice is consistent with this idea and it will now be interesting to determine whether podxl–/–/cd34–/– double KO mice exhibit hematopoietic and vascular defects (unpublished data).
Two opposing hypotheses have been proposed for the functions of the CD34 family of sialomucins: adhesion and antiadhesion. In favor of the adhesion model both podocalyxin and CD34, when expressed by HEVs, can act as adhesive tethers for activated leukocytes migrating into lymph nodes 810. Lymphocytes use L-selectin (a C-type animal lectin) to bind to specific glycoforms of CD34 and podocalyxin expressed on the surface of HEVs and this is the initiating step in lymphocyte extravasation from blood into the lymph nodes. A caveat, however, is that L-selectin binding to these molecules is glycosylation specific and the appropriate modifications have usually only been observed on HEVs. Therefore, it is likely that in most tissues, podocalyxin and CD34 have alternate functions other than adhesion. Based on a number of studies, other researchers have speculated that podocalyxin (and in some circumstances CD34; reference 75) can act as an antiadhesion molecules or molecular "TeflonTM" by virtue of its negatively charged mucin domain (1, 2, 15, 62, and see above).
One exciting hypothesis is that, in fact, these molecules can serve both adhesive and antiadhesive functions. Under the majority of circumstances, these molecules provide a barrier to adhesion, increase monolayer permeability, and aid in modifying JCs. In the special case of the HEVs, however, these molecules provide dual functions. In the first step they provide tethers for leukocytes expressing L-selectin binding. Subsequently, however, podocalyxin and CD34 move to the junctions between ECs where they act to "spread" the endothelia and facilitate leukocyte transmigration. This is consistent with previous reports showing movement of CD34 to junctions in response to cell activation 75. Analysis of mice lacking multiple members of the CD34 family offers an opportunity to test this model and should clarify this issue.
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Acknowledgments |
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5 and collagen 4(IV), Roger Wiggins for antibody to GLEPP1, Lawrence Holzman for antibody to mouse nephrin, Peter Mundel for antibody to synaptopodin, and Atsushi Miyajima and Takahiko Hara for antibody to mouse podocalyxin. Helpful suggestions and critical evaluation of this manuscript were provided by Drs. Peter Mundel, Jeffrey Miner, Hermann Ziltener, John Schrader, Ian Clark-Lewis, Fabio Rossi, Julie Nielsen, and Erin Drew. Special thanks to Maj Britt Hansen and MBH studies for advice, graphics, and prints. C. Duhme was supported by the Boehringer Ingelheim Fonds. This work was funded by Canadian Medical Research Council grant MT-15477 (to K.M. McNagny) and National Institutes of Health grant DK02264-01A1 (to D.B. Kershaw) and an American Heart Association grant-in-aid (to D.B. Kershaw). K.M. McNagny is a Canadian Medical Research Council Scholar.
Submitted: 16 November 2000
Revised: 23 April 2001
Accepted: 15 May 2001
R. Doyonnas and D.B. Kershaw contributed equally to this manuscript.
C. Duhme's present address is Green College Residence, University of British Columbia, Vancouver, BC, Canada.
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