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Crim1KST264/KST264 mice display a disruption of the Crim1 gene resulting in perinatal lethality with defects in multiple organ systems

Crim1 is a transmembrane protein, containing six vWF-C type cysteine-rich repeats, that tethers growth factors to the cell surface. A mouse line, KST264, generated in a LacZ insertion mutagenesis gene-trap screen, was examined to elucidate Crim1
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  PATTERNS & PHENOTYPES Crim1 KST264/KST264 Mice Display a Disruption of the Crim1 Gene Resulting in PerinatalLethality With Defects in Multiple OrganSystems David J. Pennisi, 1 Lorine Wilkinson, 1 Gabriel Kolle, 1 Michael L. Sohaskey, 2 Kevin Gillinder, 1 Michael J. Piper, 1,3 John W. McAvoy, 4 Frank J. Lovicu, 5 and Melissa H. Little 1 * Crim1 is a transmembrane protein, containing six vWF-C type cysteine-rich repeats, that tethers growthfactors to the cell surface. A mouse line, KST264, generated in a LacZ insertion mutagenesis gene-trapscreen, was examined to elucidate Crim1 function in development. We showed that  Crim1 KST264/KST264 micewere not null for Crim1 due to the production of a shortened protein isoform. These mice are likely torepresent an effective hypomorph or a dominant-negative for Crim1. Transgene expression recapitulatedknown Crim1 expression in lens, brain, and limb, but also revealed expression in the smooth muscle cells of thedevelopingheartandrenalvasculature,developingcartilage,matureovaryanddetrusorofthebladder.Transgene expression was also observed in glomerular epithelial cells, podocytes, mesangial cells, andurotheliuminthekidney. Crim1 KST264/KST264 micedisplayedperinatallethality,syndactyly,eye,andkidneyabnormalities. The severe and complex phenotype observed in  Crim1 KST264/KST264 mice highlights theimportance of Crim1 in numerous aspects of organogenesis.  Developmental Dynamics 236:502–511, 2007.  ©   2006 Wiley-Liss, Inc.Key words:  Crim1; gene-trap; organogenesis; microphthalmia; syndactyly; skin blebbing  Accepted 13 October 2006 INTRODUCTION There are a growing number of pro-teins described that contain vWF-Ctype cysteine-rich repeats (CRR), in-cluding proteins important in develop-ment, such as chordin, kielin, and am-nionless, as well as the extracellularmatrix component procollagen IIa(Garcia Abreu et al., 2002). One suchprotein, Crim1, was identified as aprotein expressed in a spatially andtemporally restricted manner during organogenesis of the limbs, kidney,lens, pinna, erupting teeth, and testis(Georgas et al., 2000; Kolle et al.,2000; Lovicu et al., 2000). The involve-ment of Crim1 in blood vessel biologywas suggested by the observation thatknockdown of   Crim1  expression in hu-man umbilical vein endothelial cellsdisrupted formation of vascular tubesin culture (Glienke et al., 2002). Inaddition, morpholino-mediated knock-down of Crim1 in the zebrafish em-bryo resulted in aberrant formation of the intersegmental vessels and dorsallongitudinal anastomotic vessel, fur-ther implicating Crim1 in vascular de- velopment (Kinna et al., 2006).The presence of CRR motifs withinCrim1 suggested that Crim1 may actas a modulator of the action of mem-bers of the transforming growth fac- 1 Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia 2 Department of Molecular and Cell Biology, University of California, Berkeley, California 3 Department of Anatomy, University of Cambridge, Cambridge, United Kingdom 4 Save Sight Institute, University of Sydney, NSW, Australia 5 Discipline of Anatomy & Histology, University of Sydney, NSW, AustraliaGrant sponsor: National Health and Medical Research Council of Australia; Grant number: 301056. *Correspondenceto:MelissaH.Little,InstituteforMolecularBioscience,UniversityofQueensland,BrisbaneAustralia,4072.E-mail: m.little@imb.uq.edu.auDOI 10.1002/dvdy.21015 Published online 14 November 2006 in Wiley InterScience (www.interscience.wiley.com). DEVELOPMENTAL DYNAMICS 236:502–511, 2007  ©   2006 Wiley-Liss, Inc.  tor-beta (TGF-  ) superfamily, as doeschordin (Larrain et al., 2000). We have demonstrated that Crim1 canbind to bone morphogenetic protein(BMP) -2, -4 and -7, but only withinthe context of the cell and not in solu-tion, suggesting a cell-autonomousmode of action (Wilkinson et al.,2003). Crim1 forms a complex withthe preprotein forms of such ligands,tethering the preproteins to the cellsurface, thus retarding their secretionas mature active dimers (Wilkinson etal., 2003). While possibly antagonistic,Crim1-ligand binding may act to pro-long ligand activity by allowing slowrelease of the mature protein or byrestricting the distance over which li-gand can act.In this study, we have investigatedthe role of Crim1 in mouse develop-ment by characterizing the KST264mouse line. This line was generated aspart of a previously reported gene-trap screen designed to identify novelproteins containing signal sequences(secreted and transmembrane pro-teins; Skarnes et al., 1995; Leighton etal., 2001). KST264 mice have a   -ga-lactosidase/neomycin res (  -Geo) cas-sette inserted into intron 1 of the Crim1  gene, which results in a fusionof the coding region of exon 1 of the Crim1  gene and   -Geo. We refer tohomozygotes for this insertion as Crim1 KST264/KST264 mice. This inser-tion results in perinatal lethality inhomozygous animals with defects in a variety of organ systems, including the limbs, eyes, and kidneys. We showthat the  Crim1 KST264/KST264 mouseline is not a “null” mutant but retainsexpression of a minor, alternativelyspliced isoform of   Crim1 . Analysis of transgene expression in  Crim1   / KST264  mice has revealed additionalsites of   Crim1  expression, particularlyin the smooth muscle cells of specificdeveloping arterial vascular beds. RESULTS Crim1 KST264/KST264 MiceDisplay a Disruption of  Crim1  Expression, but AreNot Null The present study used a mouse linemutant for  Crim1 , KST264, generatedin a secretory gene-trap screen (Leigh-ton et al., 2001; Fig. 1A). We haveused this mouse line to further ourunderstanding of   Crim1  biology. Webegan by confirming that the mouseline represented a bona fide  Crim1 mutation and analyzing   Crim1  tran-script levels. Reverse transcriptase-polymerase chain reaction (RT-PCR)was performed on kidney mRNA from Fig. 1.  The KST264 line is a  Crim1  mutant.  A:  Ideogram of the  Crim1  locus and a schematic representation of the transgene, insertion site of thegene-trap and  Crim1  mRNA splice variants (discussed below) (SA, splice acceptor; TM, transmembrane domain; PLAP, placental alkaline phospha-tase;   -Geo, a fusion between   -galactosidase and neomycin phosphotransferase; IRES, internal ribosome entry site; pA, polyadenylation site).  B: Reverse transcriptase-polymerase chain reaction (RT-PCR) of wild-type (    /    ) and heterozygous  Crim1   /KST264 (    /KST264) 15.5 days post coitum(dpc) kidney mRNA, showing amplification of a 1,046-bp amplicon using a  Crim1  exon 1 primer and a   -Geo primer in the    /KST264 lane. C: RT-PCR analysis of wild-type (    /    ) and homozygous  Crim1 KST264/KST264 (KST264/KST264) 15.5 dpc kidney mRNA. An mRNA splice variant lackingexon 2 (261 bp) is weakly expressed in wild-type samples but appears more prominently in  Crim1 KST264/KST264 homozygous kidneys (KST264/KST264)(X1-X3). Transcripts containing exon 2 were not detected in  Crim1 KST264/KST264 kidneys (X2). RT-PCR using primers to the 3  portion (exons 12 and 16)of  Crim1  also indicate a down-regulation of  Crim1  in  Crim1 KST264/KST264 kidneys (X12-X16). Primers to glyceraldehyde-3-phosphate dehydrogenase(GAPDH) were used as a control.  D:  Quantitative RT-PCR using real-time PCR was conducted to determine the change in  Crim1  transcript levelsbetween wild-type,  Crim1   /KST264 and  Crim1 KST264/KST264 15.5 dpc kidneys. Primer pairs specific for exon 1, exon 2, exon 3, exons 1–3, exon 11, andexon 17 were used and transcript levels expressed as a proportion of wild-type levels. Solid bars, wild-type; striped bars,  Crim1   /KST264 ; open bars, Crim1 KST264/KST264 . Error bars represent the standard deviation of the mean (SDM).  E:  RT-PCR reveals the existence of an exon 2-minus splice variantof  Crim1  in wild-type 8.5 dpc embryos and wild-type 15.5 dpc lung, eye, rib, and heart. This transcript is also present in 8.5 dpc and 10.5 dpc Crim1 KST264/KST264 embryos, 13.5 dpc  Crim1 KST264/KST264 hindlimb, and 15.5 dpc  Crim1 KST264/KST264 lung, brain, eye, rib, and heart.  F:  Crim1 proteinin the membrane fraction of 17.5 dpc wild-type,  Crim1   /KST264 , and  Crim1 KST264/KST264 whole kidney extracts. Note that the predominant band ofalmost 150 kDa in wild-type sample diminishes in  Crim1   /KST264 sample. Conversely, an immunoreactive protein of just over 25 kDa in size is abundantin the  Crim1 KST264/KST264 sample, detectable in the  Crim1   /KST264 sample, and undetectable in the wild-type sample. Crim1 KST264/KST264 FUNCTION IN MOUSE DEVELOPMENT 503  wild-type, heterozygous  Crim1   /KST264 ,and homozygous  Crim1 KST264/KST264 15.5 days post coitum (dpc) embryos(Fig. 1B). PCR using a  Crim1  exon 1primeranda  -Geoprimeronheterozy-gous  Crim1   /KST264 15.5 dpc kidneycDNA yielded an amplicon of 1,046 bp,but none was apparent from wild-typecDNA. Subcloning and sequencing of the RT-PCR amplicon confirmed thatthetranscriptwaschimericforexon1of  Crim1  and the transgene (data notshown), suggesting that the gene-trapcassette was inserted into intron 1.RT-PCR on 15.5 dpc embryonic kid-ney mRNA with primers designed toexon 1 and exon 3 produced an ampli-con (435 bp) of the expected size forthe reported transcript in wild-typesamples. No such amplicon was de-tected in  Crim1 KST264/KST264 samples(Fig. 1C). However, a shorter ampli-con (261 bp) was apparent in bothwild-type and  Crim1 KST264/KST264 samples. Subcloning and sequencing of this RT-PCR amplicon from Crim1 KST264/KST264 homozygotesdemonstrated that an in-frame Crim1  transcript lacking exon 2 wasproduced (data not shown). Further-more, RT-PCR with exon 2-specificprimers failed to detect any evidenceof a transcript containing exon 2 in Crim1 KST264/KST264 homozygotes. Analysis of the 3   portion of the Crim1  transcript with RT-PCR using primerstoexons12and16producedanampliconoftheexpectedsize(797bp)inwild-type and  Crim1 KST264/KST264 sam-ples, in addition to a shorter, minorPCR product (Fig. 1C). The level of transcript in  Crim1 KST264/KST264 ho-mozygotes appeared reduced relativeto the wild-type. This finding was con-firmed by quantitative real-time PCRon whole kidney cDNA from 15.5 dpcwild-type,  Crim1   /KST264 , and Crim1 KST264/KST264 embryos (Fig. 1D).Relative to wild-type, we found similarmRNA levels for  Crim1   /KST264 tran-scriptswiththeexceptionofexon2-con-taining transcripts, that were some-what reduced. Transcript levels for Crim1 KST264/KST264 , however, weregreatly diminished, with exon 2-con-tainingtranscriptsremainingundetect-able.We also tested for the presence of anexon 2-minus  Crim1  transcript inother tissues. We found that exon2-minus splice variants were presentin wild-type 8.5 dpc embryos and 15.5dpc lung, eye, rib, and heart (Fig. 1E).Moreover, RT-PCR revealed exon2-minus  Crim1  transcripts were de-tectable in 8.5 dpc and 10.5 dpc Crim1 KST264/KST264 embryos, 13.5 dpc Crim1 KST264/KST264 hindlimb and 15.5dpc  Crim1 KST264/KST264 lung, brain,eye, ribs, and heart (Fig. 1E). Takentogether, the data demonstrate thatthere is at least one minor splice vari-antof  Crim1 ,whichsplicesaroundthegene-trap cassette that lacks exon 2,and continues to be expressed in the Crim1 KST264/KST264 homozygotes.This variant would be expected tohave a deletion of 57 amino acids in acysteine-rich region N-terminal to thefirst CRR repeat.To determine whether there was achange in Crim1 protein levels in themutant mice, we performed immuno-blotting on the membrane fraction of 17.5 dpc wild-type,  Crim1   /KST264 ,and  Crim1 KST264/KST264 whole kidneyextracts. Using an antibody that de-tects the C-terminal, cytoplasmic por-tion of Crim1 (Wilkinson et al., 2003),we detected a predominant band of almost 150 kDa in wild-type samples Fig. 2.  Expression of   -galactosidase in  Crim1   /KST264 heterozygotes reveal novel sites of Crim1expression.  A:  An X-Gal stained  Crim1   /KST264 adult kidney shown in hemi-mount reveals Crim1-  -gal expression in an afferent artery and arterioles (arrow) and glomeruli (arrowheads).  B:  A cross-section of an adult  Crim1   /KST264 kidney reveals Crim1-  -gal expression in proximal tubules(arrow), mesangial cells (double arrowhead), glomerular epithelial cells (open arrowhead), andpodocytes (arrowhead).  C:  A section of an adult  Crim1   /KST264 kidney comparable to that in B aftercryosectioning and X-Gal staining was performed directly on the section. Note that a pattern ofexpression was observed similar to the adult  Crim1   /KST264 kidney in B, with Crim1-  -gal expres-sion in vascular smooth muscle (open arrowhead) and structures of the glomerulus (arrow).  D:  A cross-section of an adult  Crim1   /KST264 kidney reveals Crim1-  -gal expression in distal tubules(arrow).  E:  A section of an adult  Crim1   /KST264 kidney comparable to that in D after cryosectioningand X-Gal staining was performed directly on the section. Again, a pattern of expression wasobserved similar to the adult  Crim1   /KST264 kidney in D, with Crim1-  -gal expression in distaltubules (arrow).  F:  Posterior view of an 8.5 days post coitum (dpc)  Crim1   /KST264 embryo showingCrim1-  -gal expression in the yolk sac (arrow). Note the localized expression in the blood islandsof the yolk sac (open arrowhead).  G:  A 10.5 dpc  Crim1   /KST264 embryo showing Crim1-  -galexpression in the heart (double arrowhead), eye, branchial arches (arrowhead), and restricted sitesof the brain (open arrowhead) and spinal chord (arrow).  H:  A 12.5 dpc  Crim1   /KST264 embryoshowing Crim1-  -gal expression in the eye, the primordium of the pinna (arrow), and spinal cord(open arrowhead).  I:  A 15.5 dpc  Crim1   /KST264 embryo showing Crim1-  -gal expression in the eye,cartilage within the pinna (arrow), and vibrissae (open arrowhead). J:  A cross-section of the embryoin G showing Crim1-  -gal expression in the endothelium of the dorsal aorta (open arrowhead) andthe motor neurons (arrow).  K:  A cross-section of the embryo in G showing Crim1-  -gal expressionin the pericardium (arrow) and epicardial cells adjacent to the myocardium (open arrowheads).  L:  A forelimb of a 12.5 dpc  Crim1   /KST264 embryo showing Crim1-  -gal expression associated withthe developing digits.  M:  Crim1-  -gal expression persists in the forelimb of a 15.5 dpc Crim1   /KST264 embryo.  N:  A cross-section of a 16.5 dpc  Crim1   /KST264 forelimb showing Crim1-  -gal expression in the basal layer of the epidermis (arrow) and cartilage condensates (open arrow-head).  O:  The heart of a 17.5 dpc  Crim1   /KST264 embryo after X-gal staining. Note the expressionof Crim1-  -gal in the large coronary artery (arrow) and adventitia of the outflow vessels (openarrowhead), as well as the epicardial covering of the ventricles and atria. P–R: Cross-sections of theheart in (O) detailing Crim1-  -gal expression in epicardial cells (arrows, P). Q: Crim1-  -gal isexpressed in the smooth muscle cells (arrow) and the endothelium (open arrowhead) of a coronaryartery (lumen denoted by an asterisk). R: Crim1-  -gal expression in endocardial endothelial cells ofa trabeculated portion of the left ventricle (open arrowheads).  S,T:  The 15.5 dpc  Crim1   /KST264 embryonic kidney sections reveal Crim1-  -gal expression in the vascular smooth muscle (arrow, S)andtheurothelium(openarrowhead,S),inadditiontothedevelopingtubules(arrows,T),mesangialcells (double arrowhead, T), glomerular epithelial cells (open arrowhead, T), and podocytes (arrow-head, T).  U:  Expression of Crim1-  -gal in the suprarenal ganglion of the adrenal gland (arrow) andcells of the adrenal gland proper (open arrowheads) of a 15.5 dpc  Crim1   /KST264 embryo.  V: Expression of Crim1-  -gal in trophoblasts of the placenta of a 17.5 dpc  Crim1   /KST264 embryo.  W: Expression of Crim1-  -gal in the epithelium of the 17.5 dpc  Crim1   /KST264 lung.  X: Expression in thebasal layer of the epidermis (arrow) and epithelium of a developing hair follicle (open arrow) in theskin of a 17.5 dpc  Crim1   /KST264 embryo.  Y:  Expression in the transitional epithelium (arrowhead),lamina propria (open arrowhead), and muscular layer (arrows) of the adult  Crim1   /KST264 bladder. Z: Expression in the vascular smooth muscle of an artery in the adult  Crim1   /KST264 bladder.  AA: Expression in Sertoli cells (arrow), smooth muscle cells (arrowhead), and Leydig cells (openarrowhead)ofanadult Crim1   /KST264 mousetestis. BB: ExpressionofCrim1-  -galinthegranulosacellsof an ovarian follicle of a 17.5 dpc  Crim1   /KST264 embryo.  CC:  Expression of Crim1-  -gal and in theatresing follicle of an adult  Crim1   /KST264 ovary. Expression is also seen in the ovarian surface epithe-lium(BB,CC).da,dorsalaorta;ep,epicardium;lp,laminapropria;mu,muscularis;my,myocardium;nt,neural tube; pc, pericardium; te, transitional epithelium; tr, trabeculated myocardium. 504 PENNISI ET AL.  Fig. 2. Crim1 KST264/KST264 FUNCTION IN MOUSE DEVELOPMENT 505  (Fig. 1F), comparable to that expectedfor full-length protein (Wilkinson etal., 2003). This immunoreactive pro-tein was slightly diminished in Crim1   /KST264 samplesandfaintlyde-tected in  Crim1 KST264/KST264 samples.Conversely, an immunoreactive proteinofjustover25kDawasabundantinthe Crim1 KST264/KST264 sample, detectableinthe Crim1   /KST264 sample,andunde-tectable in the wild-type sample. Crim1 KST264/KST264 homozygous miceretain isoforms of   Crim1  mRNA andresidual protein and, therefore, can-not be regarded as null. Therefore, weuse the terminology  Crim1   /KST264 and  Crim1 KST264/KST264 for mice het-erozygous and homozygous for thismutation, respectively. Expression of the TransgeneReveals Additional Sites of Crim1 Expression In  Crim1   /KST264 adult kidneys,Crim1-  -Geo was expressed stronglyin the entire arterial plexus, including the afferent arterioles, but was not de-tected in the venous plexus (Fig. 2A).Section data of adult  Crim1   /KST264 kidneys revealed X-Gal staining in theGECs, podocytes, mesangial cells,proximal tubules (Fig. 2B), and distaltubules (Fig. 2D). To verify that noartifacts were introduced due to themethod chosen for X-Gal staining, Crim1   /KST264 adult kidneys werecryosectioned then incubated with X-Gal directly on the tissue section. Thestaining pattern observed for thesetissues (Fig. 2C,E) was consistentwith those stained in whole-mountand sectioned after paraffin embed-ding.Whole-mount X-Gal staining re- vealed an onset of Crim1-  -Geo ex-pression around 8.5 dpc. Staining wasevident in the yolk sac and around theectoplacental cone (Fig. 2F). High lev-els of staining were concentrated inregions of the yolk sac adjacent to theposterior part of the embryo properand in the nascent blood islands. By10.5 dpc, Crim1-  -Geo expression wasdetected in the embryo proper, with X-Gal staining in the eye, forebrain,and branchial arches (Fig. 2G), consis-tent with previously reported sites of  Crim1  expression (Georgas et al.,2000; Kolle et al., 2000; Lovicu et al.,2000). At 12.5 dpc, strong staining was observed in the eye, ear, spinalcord, limbs, and peridermal cells (Fig.2H). At 15.5 dpc, Crim1-  -Geo expres-sion remained strong in the eye, ear,limbs, and the chordal regions of thespinal cord, and was also detected inthe nascent vibrissae follicles (Fig. 2I).Section data at 10.5 dpc revealedCrim1-  -Geo expression in the dorsalaorta, motor neurons of the spinalcord (Fig. 2J), the pericardium, anddeveloping epicardium (Fig. 2K).Crim1-  -Geo expression was presentin the developing limbs (Fig. 2L,M)and cartilage of the developing digits(Fig. 2N). Crim1-  -Geo expression re-mained prominent during heart devel-opment, with X-Gal staining in theepicardium, the coronary arteries, andsome endocardial cells at 17.5 dpc(Fig. 2O–R).In contrast to other organs exam-ined, the X-Gal staining observed inthe kidney did not completely repli-cate previously described  Crim1  ex-pression.  Crim1  mRNA expressionwas reported in renal vesicles, comma-and S-shaped bodies of the developing nephrons of the kidney (Georgas et al.,2000). Whereas  Crim1  mRNA expres- TABLE 1. Frequency of Phenotypes Among Crim1 KST264/KST264 Embryos a Stage(dpc)Number of Crim1 KST264/KST264 embryosPhenotypeEye andlens sizeLimbsyndactylyKidneysizeFacialhematomaSkinblebbing 10.5 5 0 (0%) 0 (0%) n.d. 0 (0%) 0 (0%)11.5 7 0 (0%) 0 (0%) n.d. 0 (0%) 2 (29%)12.5 8 4 (50%) 4 (50%) n.d. 0 (0%) 8 (100%)13.5 3 3 (100%) 3 (100%) 0 (0%) 0 (0%) 3 (100%)14.5 4 4 (100%) 4 (100%) 0 (0%) 0 (0%) 0 (0%)15.5 21 21 (100%) 21 (100%) 21 (100%) 4 (19%) 1 (5%)16.5 7 7 (100%) 7 (100%) 7 (100%) 2 (29%) 0 (0%)17.5 12 12 (100%) 12 (100%) 12 (100%) 1 (8%) 0 (0%) a DPC, days post coitum; n.d.; no data. Fig. 3.  Homozygosity for a gene-trap insertioninto  Crim1  results in cerebral edema; eye, rib,kidney, and skull defects; and syndactyly.  A: Frontal views of the heads of 14.5 days postcoitum (dpc) wild-type and  Crim1 KST264/KST264 embryos reveal hematoma on the forehead ofthe homozygote (arrow, right).  B:  Whole-mount(left panels) and representative hematoxylin andeosin-stained, mid-sagittal sections throughthe center of the optic cups (right panels) ofeyes from 16.5 dpc  Crim1   /KST264 (top panels)and  Crim1 KST264/KST264 (lower panels) show arestriction of the aperture of the anterior eyechamber (bars), a reduction in lens size and anaccumulation of cells in the posterior eyechamber (closed arrowhead) in the Crim1 KST264/KST264 mice.  C:  An X-Gal stained12.5 dpc  Crim1 KST264/KST264 embryo showingblebbing of the skin (arrows).  D:  Analysis of limbdevelopmentat12.5dpc,15.5dpc,andpostnatal(P) 0 in  Crim1   /KST264 (top panels) and Crim1 KST264/KST264 (lower panels) animals revealssyndactyly in homozygotes. At 12.5 dpc, there isanindicationoffusionofdigits3and4inhomozy-gous limbs. At 15.5 dpc, there is clear fusion ofdigits 3 and 4 in homozygous limbs. Analysis oftheperinatalforelimbsandhindlimbsusingAlcianblue and Alizarin red staining reveals evidence forsoft and hard tissue syndactyly in homozygousanimals.  E–G:  15.5 dpc embryonic kidneysshown in whole-mount from wild-type (E), Crim1   /KST264 (F), and  Crim1 KST264/KST264 (G) lit-termates.  H:  The relative length of fresh, unfixed15.5 dpc embryonic kidneys collected from wild-type,  Crim1   /KST264 , and  Crim1 KST264/KST264 em-bryos are graphically represented. Error bars rep-resent the standard deviation of the mean (SDM).Kidneys from homozygotes were smaller thanwild-type littermates (  P    0.05). 506 PENNISI ET AL.

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