Failure of ventral closure and axial rotation in …...regulatory step in the control of the Delta-Notch pathway responsible for cell-fate selection is proteolysis of the Notch receptor

4863Development 125, 4863-4876 (1998)Printed in Great Britain © The Company of Biologists Limited 1998DEV4090

Failure of ventral closure and axial rotation in embryos lacking the proprotein

convertase Furin

Anton J. M. Roebroek 1,*, Lieve Umans 2, Ilse G. L. Pauli 1, Elizabeth J. Robertson 3, Fred van Leuven 2, Wim J. M. Van de Ven 1 and Daniel B. Constam 3,*1Laboratory for Molecular Oncology, 2Experimental Genetics Group, Center for Human Genetics, University of Leuven andFlanders Interuniversity Institute for Biotechnology (VIB), Herestraat 49, B-3000 Leuven, Belgium3Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Ave, Cambridge, MA 02138, USA*Authors for correspondence (e-mail: [email protected] and [email protected])

Accepted 2 October; published on WWW 12 November 1998

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We have examined the role of Furin in postimplantation-stage mouse embryos by analyzing both the expressionpattern of fur mRNA and the developmental consequencesof a loss-of-function mutation at the fur locus. At earlystages (day 7.5), furmRNA is abundant in extraembryonicendoderm and mesoderm, anterior visceral endoderm, andin precardiac mesoderm. 1 day later fur is expressedthroughout the heart tube and in the lateral platemesoderm, notochordal plate and definitive gut endoderm.Embryos lacking Furin die between days 10.5 and 11.5,presumably due to hemodynamic insufficiency associatedwith severe ventral closure defects and the failure of theheart tube to fuse and undergo looping morphogenesis.Morphogenesis of the yolk sac vasculature is also

abnormal, although blood islands and endothelialprecursors form. Analysis of cardiac and endodermalmarker genes shows that while both myocardial precursorsand definitive endoderm cells are specified, their numbersand migratory properties are compromised. Notably,mutant embryos fail to undergo axial rotation, even thoughNodal and eHand, two molecular markers of left-rightasymmetry, are appropriately expressed. Overall, thepresent data identify Furin as an important activator ofsignals responsible for ventral closure and embryonicturning.

Key words: Development, Turning, Heart morphogenesis, EndoderTGFβ, Processing, Mouse

SUMMARY

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INTRODUCTION

Specific proteolytic modifications are a powerful means which to regulate the biological activities of proteins. Alsduring embryonic development, proteases control tactivation and turnover of many important key regulatomolecules. For example, in Drosophila the syncytiblastoderm is patterned along the dorsoventral axis bygradient of mature Spätzle protein, which is established by ventrally localized proteolytic activity of Easter (Chasan anAnderson, 1989; Stein and Nusslein-Volhard, 1992). In tventral blastoderm, Spätzle signaling leads to repressionseveral target genes, includingtolloid (Kirov et al., 1993).Thus, localized expression of tolloid in the dorsal blastodermlocally enhances the dorsalizing activity of DPP, thereallowing specification of dorsalmost cell fates (Shimell et a1991). Mechanistic insights into the function of thmetalloproteases encoded by tolloid and its Xenopushomologue xolloid have come from studies demonstrating ththey selectively degrade the DPP/BMP antagonists SOG Chordin, respectively (Marqués et al., 1997; Piccolo et a1997). Similarly, in both arthropods and vertebrates, a kregulatory step in the control of the Delta-Notch pathw

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responsible for cell-fate selection is proteolysis of the Notcreceptor (reviewed in Greenwald, 1998). Recent data show tthe cytoplasmic domain of Notch is cleaved and, upon ligabinding of the extracellular domain, translocates to the nucle(Kopan et al., 1996; Schroeter et al., 1998). Genetic abiochemical evidence have implicated the metalloproteaencoded by kuzas a potential candidate for Notch processinand turnover (reviewed in Greenwald, 1998); however, in vitNotch can also be cleaved by Furin or related proteases (Loget al., 1998).

Furin is the mammalian prototype of a family of serinproteases that have catalytic domains resembling the bacteprotease subtilisin (Roebroek et al., 1986; for review see Vde Ven et al., 1993). Three members of this protease gefamily, including PC1/3, PC2 and PC4, are expresseexclusively in endocrine tissues or in testis, respectively. Loof function mutations have shown that PC1/3 and PC2 are brequired for generating physiological amounts of mature insu(Furuta et al., 1997; Jackson et al., 1997). The lack of PC2 aimpairs the production of mature glucagon (Furuta et al., 199By contrast, PC4 is uniquely required for normal sperm fertilit(Mbikay et al., 1997). These findings demonstrate that the moubiquitous, closely related activities of Furin, PACE4, PC5/6

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PC7 cannot effectively compensate for the loss of PC1/3, Por PC4. The physiological roles of Furin, PACE4, PC5/6 aPC7 remain poorly understood since their normal in vivsubstrates are unknown, as is the extent to which their subsspecificities overlap under physiological conditionCoexpression experiments and in vitro studies have implicaFurin as a major player in the processing of proproteins thatspecifically cleaved after R-X-X-R sequences (Molloy et a1992), such as insulin-like growth factor I (Duguay et al., 199insulin receptor (Bravo et al., 1994), hepatocyte growth facreceptor (Komada et al., 1993), α-integrins (Lehmann et al.,1996) and matrix metalloproteinases (Pei and Weiss, 199Similarly, Furin has been shown to efficiently process tprecursor forms of TGFβ-related molecules, including TGFβ1(Dubois et al., 1995), activin A (Roebroek et al., 1993Mullerian inhibiting substance (Nachtigal and Ingraham, 199BMP4 (Cui et al., 1998; D. B. C. and E. J. R., unpublished) aNodal (D. B. C. and E. J. R., unpublished). Under physiologicconditions, the partially overlapping activities of PACE4, PC5and PC7 may also contribute to the processing of at least sof these precursor proteins. In keeping with this suggestionprevious analysis of their expression patterns during embryodevelopment led us to propose that the overlapping activitiesFurin, PACE4, PC5(6) and PC7 may locally control thavailability of mature BMP ligands (Constam et al., 1996Unlike PACE4 and PC5/6, Furin mRNA has not beedocumented to be locally restricted to specific tissues in mouse embryo. In rat embryos, however, furmRNA isexpressed specifically in the extraembryonic endoderm amesoderm during early primitive streak stages (Zheng et 1994). During late somite stages, localized expression of fur isalso seen in the cardiovascular system (Zheng et al., 19raising the possibility that Furin is specifically required in thetissues during early embryonic development.

Here we have performed a detailed analysis of texpression pattern of fur in the early mouse embryo usingmRNA whole-mount in situ hybridization. To explore itspotential function during embryogenesis, we have algenerated a loss-of-function mutation at the fur locus. Furin-deficient embryos die between 10.5 and 11.5 dpc, probadue to hemodynamic insufficiency resulting from seveventral closure defects. In mutant embryos, the cardiogemesoderm either fails to fuse at the ventral midline, resultiin cardia bifida, or forms a single linear tube that fails undergo looping morphogenesis. Interestingly, Furideficient embryos also fail to turn, and their allantois does nattach to the chorion. To further characterize the turning aventral closure defects, we analyzed the expression onodallacZ reporter allele, and a panel of cardiac- anendoderm-specific molecular markers. Based on our findinwe propose that Furin activities promote the migration andproliferation of definitive endoderm, and also contribute the activation of cardiogenic signals.

MATERIALS AND METHODS

Whole-mount in situ hybridization and histological analyisWhole-mount in situ hybridization using digoxygenin-labelled RNprobes was performed as described (Wilkinson, 1992). The riboproused were as described: shh (Echelard et al., 1993), HNF3β (Sasaki

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and Hogan, 1993), MLC2V (Lyons et al., 1995a) and -2A (Kubalak al., 1994), eHand (Biben and Harvey, 1997), GATA4 (Arceci et a1993) and Flk-1 (Yamaguchi et al., 1993). In addition, a 130 bfragment of the α-cardiac actin gene (provided by R. Harvey) watranscribed using T3 RNA polymerase to synthesize an antisense Rprobe. The Furin riboprobe comprised nucleotides 2014-277corresponding to the 3′ end of the Furin coding sequence (gb:L26489The Nkx2.5 probe comprised nucleotides 820-1488 of murinNkx2.5/Csx (gb:X75415; kindly provided by S. Izumo). Followingphotography, embryos were dehydrated in a series of graded ethand in xylene, embedded in paraffin wax and sectioned at 10 µm. Forhistological analysis, embryos were embedded as described, sectioat 7 µm, and stained with Haematoxylin and Eosin.

Derivation of mutant miceA cosmid containing the 5′ region of the mouse fur gene was isolatedfrom a 129/Sv genomic cosmid library by colony hybridization usinthe mouse fur cDNA (Creemers et al., 1992) as a molecular probe6 kb NotI fragment, comprising exon 1A to intron 6 sequences, wsubcloned into pGEM-5Zf(+). A 1.8 kb BglII fragment encodingthe hygromycin B phosphotransferase gene, fused to tphosphoglycerate kinase promoter, was cloned into a KpnI site in exon4 via KpnI-BglII adaptors. The adaptor sequence GTACTAGCTTTC-GTTGCCGGATC contained a stop codon (underlined) in-frame withe fur coding sequence in exon 4, and disrupts the KpnI site presentin exon 4. The insert of the resulting plasmid was excised with Nand electroporated into E14 embryonic stem cells (Doetschman et1987). Hygromycin B-resistant colonies were analyzed by Southeblotting using KpnI-digested genomic DNA. Probes were obtained PCR amplification of nucleotides 298-763 (probe L), 764-1231 (proR1) and 970-1231 (probe R2) of the fur cDNA, corresponding to ex2 to exon 4, exon 5 to exon 8, or exon 7 to exon 8 sequencrespectively. An additional probe used to confirm correct targeticomprised a 529 bp fragment of the hygromycin B phosphotransfergene amplified by PCR using primers 5′-CAGCGAGAGCCTGACC-TATTGC-3′ and 5′-CGATCCTGCAAGCTCCGGATG-3′ (probe H).

Correctly targeted ES cell clones were injected into C57Bl/6 usistandard procedures. The resulting chimeras were mated to C57mice, and transmission of the mutant fur allele was confirmed bySouthern blotting and PCR analysis of tail DNA samples derived froF1 progeny (Fig. 2). Heterozygous offspring carrying the mutant allewere backcrossed to C57Bl/6 mice for three generations to redethe pathogen-free progeny used for the subsequent analysis. Pgenotyping was carried out using the hygromycin-specific primedescribed above to detect the mutant allele. Primers 5′-CGGTGAC-TATTACCACTTCTGGCACAGAGC-3′ and 5′-AAACAGAAGAA-GCCAGGGTGAGCCTCATCC-3′were used to amplify a wild-type-specific 373 bp fragment extending from exon 3 into the fourintron of the fur gene. No product was amplified from the targeteallele using the second primer pair, presumably because hygromycin cassette interfered with the amplification under thconditions used.

To analyze fur mRNA expression in mutant tissues, total RNA frommouse liver was isolated using TRIzol Reagent (Life Technologieand analyzed by northern blot hybridization as described previou(Roebroek et al., 1993) using probes L1, R2 or H, respectively. Eqloading of RNA in each lane was confirmed by rehybridizing thmembranes with a hamster α-actin cDNA probe. To analyze furexpression in mutant embryos, total RNA isolated from 9.5 dpembryos was subjected to RT-PCR using the Perkin Elmer GeneaRNA PCR kit. The first strand cDNA synthesis was primed with aoligonucleotide corresponding in sequence to positions 1212-1231the fur cDNA complementary to exon 8. In the subsequent PCreaction, a fragment comprising exon 3 to exon 6 sequences amplified using primers 5′-CGGTGACTATTACCACTTCTGGCA-CAGAGC-3′ and 5′-TGTCATTCATCTGTGTGTACCGAGGCTG-TG-3′.

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alleleThe nodallacZ allele was maintained on a 129/Sv background, ananimals were genotyped as described (Collignon et al., 1996). Fanalysis of β-galactosidase expression in a fur null background,heterozygous fur+/− males were mated to nodallacZ/+ females. Theresulting doubly heterozygous offspring were crossed to fur+/−

partners. Embryos were dissected between 7.5 and 8.5 dpc genotyped using yolk sac DNA. Staining of β-galactosidase activitywas as described (Hogan et al., 1994).

RESULTS

Expression of fur mRNA between embryonic days7.5 and 8.5Using whole-mount in situ hybridization, we examined thexpression of fur in mouse embryos between 7.5 and 8.5 dpThe probe was derived from coding sequences 3′ to thecatalytic domain to exclude regions that are most conservamong related proprotein convertases. At the earliest staexamined (7.5 dpc), fur transcripts are detected inextraembryonic tissues, and in the proximal region of thembryo (Fig. 1A). By late gastrulation, a diffuse signal iassociated with the node (Fig. 1A). In the extraembryontissues, furmRNA is localized to both the mesoderm andprimitive endoderm, but is excluded from the most proximaprimitive endoderm contacting the extraembryonic ectoder(Fig. 1B). At more distal levels, fur mRNA is stronglyexpressed in the most anterior visceral endoderm, and in underlying precardiac mesoderm, as well as in ectodermal cein the antero-proximal region of the epiblast (Fig. 1C). Bheadfold stage, fur is strongly expressed in the allantois, anin the anterior intestinal portal (AIP) both within mesodermaand endodermal cells subjacent to the headfolds (Fig. 1D).the node region, high levels of fur mRNA are apparent in theperiphery of the ventral notochordal plate (Fig. 1E).

From the 4-6 somite stage onward (8.5 dpc), furis stronglyexpressed in the primitive heart, with particularly high leveapparent in the inflow tract and in the AIP, where ventrclosure is proceeding in a caudal direction. High expressilevels persist in the allantois (Fig. 1F). Furin mRNA is alsdetected throughout the foregut endoderm (Fig. 1G,H), andthe lateral plate mesoderm of the trunk (Fig. 1I). In the tailbu

Fig. 1. Expression of furmRNA in 7.5-8.5 dpc mouse embryosanalyzed by whole-mount in situ hybridization. (A) Whole-mountview of a 7.5 dpc embryo. (B) Transverse sections through theextraembryonic and (C) embryonic regions of a similar 7.5 dpcembryo. Dashed lines in A indicate the level of sectioning in B andC, respectively. (D) Frontal-lateral and posterior (E) whole-mountview of a headfold stage embryo (8.0 dpc). (F) Whole-mount viewand transverse sections through (G) the heart, (H) the AIP, (I) thetrunk, and (J,K) through the tailbud of an 8.5 dpc embryo which hformed 3-4 pairs of somites. aip, anterior intestinal portal; al,allantois; ave, anterior visceral endoderm; cf, chorionic fold; cip,caudal intestinal portal; cp, notochordal plate; cv, cardinal veins; eand ee, embryonic and extraembryonic region; em, extraembryonmesoderm; en, endocardium; ep, epiblast; fg, foregut; h, heart; hghindgut; lp, lateral plate; m, mesoderm; mc, prospectivemyocardium; n, node; nf, neural fold; np, neural plate; so, somite;vys, visceral yolk sac. In A-D and F, anterior is to the left; in G-K,the left side of the embryo is to the right.

4865Role of Furin in ventral morphogenesis

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fur is most strongly expressed in the notochordal plate (Fi1J), and more caudally in the hindgut and caudal intestinportal (cip; Fig. 1K).

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Targeted inactivation of fur by homologousrecombinationThe targeting construct used to inactivate the furlocusconsisted of a 6 kb NotI fragment comprising part of exon 1Athrough exon 6. A PGKhygro cassette was inserted into e4 together with adapter sequences which abolish a residKpnI site and provide an in-frame stop codon to disrupt thefurcoding sequence (Fig. 2A). Following electroporation into EES cells, individual drug-resistant colonies were analyzedSouthern blotting using a cDNA probe corresponding to exo7-8 (probe R2). Of 44 drug-resistant colonies analyzed, secorrectly targeted ES cell lines were recovered. Two out

Fig. 2. Targeted inactivation of the furgene by homologous recombinlocus and targeting vector. Due to the insertion of a hygromycin recodon, the KpnI restriction site in exon 4 is abolished in the mutant of the probes used to screen for homologous recombination evenEdigested genomic DNA isolated from parental (E14) or targeted (EPCR genotyping analysis of embryos collected at 9.5 dpc from fur+/− int(D) Northern blot analysis of total liver RNA from wild-type (+/+) anan α-actin-specific probe (lower panel). The upper panels show th(middle panel). The arrow indicates the presence of a transcript dexpression in 9.5 dpc embryos. The upper panel shows PCR prodproducts were digested with KpnI. Resistance to KpnI is indicative of crwere as indicated.

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seven correctly targeted ES cell lines (ROE-1 and -2, Fig. 2Bgave rise to germline chimeras. Heterozygous (C57Bl/6 ×129)F1 offspring derived from line ROE-1 were intercrossed, andviable offspring were genotyped. Of 231 progeny, 78 (34%were wild type, 153 (66%) were heterozygous, but nohomozygous mutants were recovered. Similarly, of 19newborn F2 progeny derived from ROE-2 intercrosses, nonewere homozygous mutant. To determine the timing odevelopmental arrest, embryos from heterozygous intercromatings were genotyped by PCR. No homozyogous mutaembryos were recovered from 11.5 dpc onwards, although this stage, 17 (27%) out of 63 decidua contained remnants

ation in ES cells. (A) Diagram showing the relevant regions of the fursistance gene (hygB) and adaptor sequences providing an in-frame stopallele. Solid black and shaded bars (bottom) indicate the relative positionsts. E, coRI; H, HindIII; K, KpnI; N, NotI. (B) Southern blot analysis of KpnI-14-Roe-1 and E14-Roe-2) ES cell lines, respectively. (C) Representative

ercross matings. Appropriate PCR controls were included as indicated.d heterozygous (+/−) mice using the fur cDNA probe L (upper panels) or

e same hybridization exposed either for 3 days (upper panel) or for 14 dayserived specifically from the targeted allele. (E) RT-PCR analysis of furmRNAucts comprising exon 3 to exon 6 sequences, whereas in the lower panel, theseyptic splice events (see text for details). Genotypes and control PCRs


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resorbed tissues. In contrast at 10.5 dpc, 39 (24%) of 1embryos analyzed were homozygous mutant (Fig. 2suggesting that fur−/− embryos die between 10.5 and 11.5 dp

To confirm that the targeted fur allele is non-functional, totalliver RNA was isolated from wild-type and heterozygousiblings and analyzed by northern blotting. In all sampltested, the fur cDNA probe L hybridized to a 4.3 kb transcrcorresponding to wild-type fur mRNA. The abundance of thtranscript was reduced by approximately 50% in RNA froheterozygotes compared to wild type (Fig. 2D). Iheterozygotes, an additional 6.0 kb transcript was detected also hybridizes with probes R2 and H (data not showsuggesting that the targeted allele is transcribed at a redulevel and gives rise to a mRNA where the Furin coding regiis disrupted by insertion of the PGKhygro cassette.

To further analyze transcripts arising from the mutant allea cDNA fragment comprising exon 3 to exon 6 sequences wamplified by RT-PCR using RNA from 9.5 dpc embryos. both wild-type and heterozygous embryos we detected a bp product together with a significantly less abundant 300fragment (Fig. 2E). Unexpectedly, small amounts of similproducts were also detected in homozygous mutant embrTo test whether any of these fragments are derived from levels of functional mRNA produced from the targeted furallele, they were digested with KpnI. As shown in Fig. 1E(lower panel) the majority of the RT-PCR product obtainefrom wild-type or heterozygous embryos was cleaved by KpnI,giving rise to two closely migrating fragments of 184 and 2bp, respectively. However, both the 300 bp and a minor fractof the 391 bp fragment were resistant to KpnI treatment,were the products amplified from homozygous mutaembryos, suggesting that these PCR products likely reprelow levels of aberrant splicing events. To confirm this, thKpnI-resistant fragments obtained from fur−/− embryos werefurther amplified in a second round of PCR. Direct sequencof the larger fragment revealed that a cryptic splice donpresent within the rudimentary KpnI site (GGTAC) of exon 4had been used to splice around the PGKhygro casseresulting in a transcript where exon 4 is truncated by sevnucleotides. This truncation causes a frame shift and disruthe open reading frame of Furin after amino acid 12Moreover, sequence analysis of the KpnI-resistant fragmentsshowed that the same cryptic splice site is also used in wtype embryos. Finally, sequencing of the smaller RT-PCfragment revealed that it was derived from transcripts devof exon 4 sequences. Thus, exon 3 is spliced in-frame to e5. An analogous splice event has been observed in a muallele of the closely related PC2 gene (Furuta et al., 199However, as in the case of PC2, a fur transcript devoid of exon4 can only give rise to a protein which lacks the multibasmotif required for autoactivation of the zymogenic pro-FuriTherefore, it is highly unlikely that any functional protein cabe generated from the targeted furallele.

Multiple tissue abnormalities in Furin-deficientembryosWhile homozygous mutant embryos survive until 10.5 dpthey display several overt abnormalities. Most noticeabmorphogenesis of the yolk sac vasculature is disrupted, andembryos fail to undergo axial rotation. As shown in Fig. 3,well organized vascular network consisting of both capillari

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and vitelline vessels has developed throughout the yolk sacnormal 10.5 dpc embryos (Fig. 3A,E), whereas in mutalittermates, the capillaries are poorly organized and vitellinvessels do not form (Fig. 3B,F). Fetal red blood cells aclearly recognizable, but are pooled in a single patch on oside of the yolk sacs (Fig. 3B). Occasionally, the endodermsurface of mutant yolk sacs is ruffled in appearancpresumably due to failure of the vasculature to expand (F3F). Blood islands can also clearly be seen in mutants 1 dearlier (9.5 dpc, Fig. 3I), but their number is reduced (Fi3J,K) compared to control littermates (Fig. 3G). However, thcellular architecture of both visceral endoderm and mesodeappear to be morphologically normal (Fig. 3I). The presenof Flk-1 transcripts further suggests that extraembryonmesoderm is competent to give rise to endothelial precurs(Fig. 3C,D; Yamaguchi et al., 1993).

During early somite stages (4-6 somites), normal embryinitiate the process of axial rotation. While wild-type oheterozygous embryos have completed this process withinhours (Fig. 3G), mutant littermates consistently fail to tur(Fig. 3J,K), and by 9.5 dpc they are approximately 30% smalthan controls. Noteably, the gut endoderm and the body whave failed to properly fuse at the ventral midline (Fig. 3KM,O). This ventral fusion defect is frequently associated wicardia bifida (77%; n=26), and as development proceeds, wiincreasingly severe disorganization of all ventral tissues (F3O). In less severe cases (Figs 3K, 5I,J) a single heart toccasionally forms, but these consistently fail to underglooping morphogenesis. Only in rare instances (9%; n=45)were these rudimentary hearts found to be beating at the tof dissection (9.5-10.5 dpc). However, this is not due to tabsence of myocytes, since both the epimyocardium andendothelial layer lining the inside of the heart tube are readdistinguishable (Fig. 3L).

The midgut region is also highly abnormal in furmutants.The ventralmost aspect of the embryo normally occupied the gut tube comprises the cardinal veins, which asignificantly enlarged and engorged with nucleated red blocells (Fig. 3K,M). Presumably, cardiac insufficiency limits thblood flow between the yolk sac and the embryonvasculature, and this is likely to be responsible for thdevelopmental arrest and death occurring between 10.5 to 1dpc. Additional defects seen in mutant embryos at 9.5 dinclude exencephaly and kinking of the neural plate and neutube (Fig. 3K). The anteroposterior body axis is frequentshortened in the trunk and tail region, and the somites abnormally compact and small (Fig. 3J). Interestingly, in spof continued growth, the allantois fails to fuse with the chorioand instead becomes highly vacuolated by 9.5 dpc, anddisplaced laterally as opposed to projecting towards tchorion (Figs 3J,N, 5M, 7E).

nodal lacZ expression is unaffected by the lack ofFurinDuring normal development, axial rotation is preceded by tasymmetric expression of the TGFβfamily members Nodal,Lefty-1 and Lefty-2 (Collignon et al., 1996; Lowe et al.1996; Meno et al., 1996, 1997), consistent with the idea ththe direction of heart looping and embryonic turning is likelto be controlled by the overlapping activities of severTGFβ-related signals. To test whether the left-right signalin

4868 A. J. M. Roebroek and others

Fig. 3. Histologicalexamination of fur−/− embryosbetween 9.5 and 10.5 dpc.(A) Control and (B-D)homozygous mutant yolk sacs.(C) Whole-mount view and (D)section through a mutant yolksac (9.5 dpc) stained for Flk-1mRNA. (E) Section throughcontrol 10.5 dpc yolk sac,compared to (F) yolk sac of amutant litter mate.(G) Transverse section of a 9.5dpc control embryo and (H), athigher magnification, of itsyolk sac. (J) Parasagittal and(K,N) transverse sectionsthrough mutant littermates ofthe embryo shown in G, and (I)higher magnification of a bloodisland in the mutant yolk sac ofthe embryo shown in K. Notethat the mutant embryos havenot turned (J,K), and theirallantoides failed to fuse withthe chorion (J,N). In themutants, the neural tube iskinked and fails to close (K),and the fusion of the heart tubeand of the gut epithelium at theventral midline is highlyabnormal (L,M), leading to theformation of a heart that failsto loop (J-L), or of twoseparate heart tubes, which by10.5 dpc are highlydisorganized (O). al, allantois;am, amnion; bc, blood cell;bi, blood island; bw, body wall;ca, common atrial chamber;ch, chorion; cv, cardinal veins;da, dorsal aorta; dm, dorsalmesocardium; ec, endothelialcell; em, extraembryonicmesoderm; en, endocardium;ep, epithelial layer; fg, foregut;hg, hindgut; ht, heart tube;lht/rht, left and right heart tube;luv, left umbilical vein; mc,myocardium; np, neural plate;nt, neural tube; sh, right sinushorn; so, somite; ve, visceralendoderm; vv, vitelline vessel;vys, visceral yolk sac.


Fig. 4. The lack of Furin does not perturb the asymmetric expressionof nodal. (A) Control and (B-D) fur−/− embryos normally express anodallacZ reporter allele in the left lateral plate mesoderm at 8.5 dpc(B), and in the node between 7.5 (D) and 8.5 dpc (C). Note that evenin a fur−/− background, a single copy of functional nodal is sufficientfor normal development at least until 8.5 dpc. h, heart; hf, headfold;lp, lateral plate; n, node; so, somite; anterior is to the left.

Fig. 5. Defective ventral closure in fur−/− homozygotes. (A) Incontrol 9.5 dpc embryos, expression of shh marks midline structures,including the floorplate and the notochord (nc), as well as theprimitive gut (g). (B) Homozygous mutant exhibiting cardia bifida.No gut tube has formed, but shh-positive cells are situated at theventral surface on each side of the embryo. (C,D) Frontal (C) andposterior view (D) of a normal embryo at 8.5 dpc stained for HNF3βmRNA. (E) Ventrolateral view of a stage-matched mutant littermateof the embryo shown in C,D. No HNF3β-positive cells are detectedin the AIP, and the hindgut has not yet formed. (F) α-cardiac actinexpression throughout the heart tube of an 8.5 dpc control embryothat is about to turn. (G) In mutant littermates, no heart tube hasformed. (H-K) Expression of α-cardiac actin is reduced in therudimentary hearts of fur−/− embryos (I-K) compared to controllittermates (H). Note the absence of a normally looped heart tube. (L) Normal embryo (9.5 dpc) expressing myosin light chain 2Vthroughout the heart tube (h). (M) Bifid heart of a mutant embryo inwhich MLC2V expression marks two separate heart tubes. A,B,D-F,H,J,K: lateral view; C,G,I,M: frontal view. aip, anterior intestinalportal; al, allantois; g, gut; h, heart; hg, hindgut; n, node; nc,notochord; np, neural plate.

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pathway responsible for the sidedness of Nodal expressiodisturbed in Furin-deficient embryos, a nodallacZ allele wasintroduced into the fur−/− background. Since insertion of aβgeo cassette in this allele disrupts nodalfunction (Collignonet al., 1996), this experiment also allowed us to investigwhether a single functional copy of nodal becomeshaploinsufficient in a fur null background. Males doublyheterozygous for fur and nodallacZ were mated to fur+/−

females. Embryos were recovered at 8.5 dpc, genotyped stained to examine nodallacZ expression. Of 99 embryosanalyzed, 49 were nodallacZ heterozygotes and expressed β-galactosidase, and 26 were homozygous mutant for fur. Ofthese, 14 (54%) were heterozygous for the nodallacZ allele butappeared morphologically normal, suggesting that even ifur null background, a single copy of functional nodal issufficient for normal development at least up to this stagMoreover, β-galactosidase expression was unperturbed eitin the node or the left lateral plate mesoderm of Furideficient embryos (Fig. 4B,C) compared to stage-matchwild-type litter mates (Fig. 4A). NodallacZ was also found tobe normally expressed in the node of mutant embryos at dpc (n=2; Fig. 4D). Together, these results suggest that failure to undergo axial rotation is not associated wideregulation of nodal expression.

Ventral closure and cardiac defectsVentral morphogenesis is initiated as a rostral to caudal alateral to ventral displacement of embryonic mesodesubjacent to the headfolds, which is fated to fuse at the venmidline and to form a linear heart tube (Kaufman, 1992Concomitant with these cell movements, the most anterdefinitive endoderm involutes to form the foregut pockewhich then becomes extended caudally during midgformation. While the molecular basis of endoderspecification and its subsequent migration, growth apatterning remain largely unknown, limited insight into thregulation of ventral closure has come from functional studof the transcription factor GATA4. Loss of GATA4 results ifailure of precardiac mesoderm to fuse at the ventral midlinConsequently, two separate heart tubes develop in the majoof mutant embryos (Kuo et al., 1997; Molkentin et al., 1997

To characterize the ventral closure defects of fur mutants inmore detail, we analyzed the expression of a number of magenes by whole-mount mRNA in situ hybridizationSpecifically, we assessed expression of sonic hedgehog (and HNF3β,which identify midline structures and definitiveendoderm. At 9.5 dpc, shh is expressed in the notochord in the overlying floorplate of the neural tube both in contrand mutant embryos (Fig. 5A,B), indicating that thesstructures develop normally. By contrast, in the midgut regiof mutant embryos, shh-positive endoderm cells are ectopicsituated at the ventrally exposed surface of the primitive ve(Figs 5B, 6B) and are undetectable at more caudal levels (6C), with the exception of the CIP (Fig. 6D) and hindgdiverticulum (Fig. 6E). Similar to shh, HNF3β staining did notreveal any defects in midline structures of embryos analyzat 8.5 dpc (compare the mutant in Fig. 5E to the similarly sizcontrol embryo in Fig. 5C,D). By contrast, in all of the mutaembryos analyzed (n=5), HNF3β-positive cells aresignificantly reduced in number both in the AIP and CIP regicompared to control littermates, and a hindgut has not

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To characterize the basis of the observed heart defectsFurin-deficient embryos, we analyzed the expression of a paof cardiac markers. In normal embryos, α-cardiac actin isdetected at 8.5 dpc in the future myocardial layer throughothe heart tube (Fig. 5F; Sassoon et al., 1988). α-Cardiac actinis also expressed in mutant embryos, indicating the preseof myocardial precursors (Fig. 5G). However, the field of α-cardiac actin positive cells is reduced in size in the mutan(n=3) compared to control littermates, and it has not fusedthe ventral midline. Even 1 day later (9.5 dpc; n=4),cardiogenic mesenchyme cells expressing α-cardiac actinappeared to be reduced in numbers in mutants comparedcontrols, and they failed to form a looping heart tube (Fig. 5HK). Next, we examined expression of myosin light chai(MLC)-2V, which by 9.5 dpc is normally restricted to theventricular and proximal outflow tract of the primitive hear(Figs 5L, 6F; O’Brien et al., 1993). High levels of MLC-2VmRNA were also detected in mutant embryos (n=4), althoughtheir heart tubes were abnormally short and bifid (Figs 5M6G). Similar results were obtained using a probe for MLC-2Awhich is expressed throughout the heart prior to 11.5 d(Kubalak et al., 1994; data not shown). Together, these resconfirm that cardiac myocytes are specified in mutant embrybut that a severe ventral closure defect has a major impacheart tube morphogenesis.

We next analyzed expression of eHand, a transcription facexpressed in the heart, pericardium, and in vitelline aumbilical vessels (Biben and Harvey, 1997; Firulli et al., 1998As expected within the heart tube, eHand was expressed infuture left ventricle both in wild-type and fur+/− embryos (Fig6H). Interestingly, eHand is also asymmetrically expressedthe rudimentary heart tube(s) of fur−/− embryos (Fig. 6I,J).Thus, both in bifid hearts as well as in cases where a sinheart tube forms, eHand is expressed preferentially on the side (n=5; Fig. 6I,J shows an intermediate situation whereheart tube has partly fused). Caudal to the heart, strong bilatexpression of eHand is observed in tissues fated to form ventral body wall and the vitelline and umbilical vesselrespectively. However, the lateral mesodermal tissues failfuse at the ventral midline, and they are severely disorganizmaking it difficult to distinguish the vitelline and umbilicalvessels in transverse sections (Fig. 6K).

The cardia bifida defects that develop in fur−/− mutants arestrikingly similar to those seen in embryos lacking thtranscription factor GATA4 (Kuo et al., 1997; Molkentin et al.1997). On the other hand, fur−/− embryos forming a single hearttube more closely resemble embryos lacking the homeobtranscription factor Nkx2.5, where the rudimentary heart tufails to loop and instead bends in a ventral as opposed to latdirection (Lyons et al., 1995a). In light of these similarities, was of interest to examine whether abnormalities in thexpression patterns of either of these transcription factocontribute to the heart defects in fur mutants. At early primitivestreak stages, GATA4 is widely expressed in visceral endodeand nascent mesoderm. As the definitive endoderm migratedisplace the primitive visceral endoderm, GATA4 mRNAbecomes confined to the most proximal domain abutting textraembryonic tissue (Fig. 7A-C). At the advanced primitivstreak stage, GATA4 mRNA is expressed in a crescent


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mesoderm subjacent to the headfold (Fig. 7A, right-membryo). By 8.5 dpc, GATA4 transcripts are highly abundathroughout the AIP and the heart tube, as well as in the allan(Fig. 7D,E). In all 28 embryos collected from intercrosmatings and examined at 7.5 dpc, the expression patterGATA4 appeared normal, indicating that the loss of Furinunlikely to interfere with the expression of GATA4. LikewiseGATA4 expression appeared to be normal in the AIP of mut8.5 dpc embryos (n=4, Fig. 7D). Only in the most anteraspect of bifid mutant hearts were GATA4 expression levreduced, probably due to a delay in the migration of precardmesoderm (Fig. 7D). Interestingly, GATA4 expression wconsistently absent in the allantois of mutant embryos (F7E), even though twist mRNA, a marker for mesodermal cein the allantois, was normally expressed (Fuchtbauer, 19data not shown).

In control embryos (8.0-8.5 dpc), high levels of Nkx2transcripts are detected in cardiogenic mesoderm subjacethe headfold, and throughout the newly formed heart tube (F7F,G). In comparison, Nkx2.5 mRNA expression was absor dramatically reduced in early headfold stage embrylacking Furin (n=9, Fig. 7F), and detectable only at reduclevels during subsequent stages (n=4; Fig. 7H), indicating thateither the numbers of cardiomyocytes or Nkx2.5 transcripts cell are compromised.

DISCUSSION

In the early post-implantation rat embryo, fur mRNA has beenshown to be expressed in both embryonic and extraembrylineages (Zheng et al., 1994). Consistent with these data,here report that in the mouse embryo fur mRNA is expressedin the extraembryonic endoderm and mesoderm durprimitive streak stages. We have also documented strongexpression in the anterior visceral endoderm and underlying cardiogenic mesoderm. 1 day later (8.5 dpc), furmRNA is abundantly expressed in the myocardial aendocardial layers of the heart, in definitive gut endodelateral plate mesoderm, the allantois, and in the ventral laof the node. While the physiological substrates of Furin these tissues have not yet been identified, we note a stridegree of overlap in the distribution of fur transcripts with thatof candidate substrates belonging to the TGFβfamily. Forexample during gastrulation, both BMP2 and BMP7 aexpressed in anterior visceral endoderm (Lyons et al., 199Winnier et al., 1995; Zhang and Bradley, 1996). At the timeventral closure, BMP2 as well as TGFβ2 transcripts are foundwithin cardiogenic mesoderm (Dickson et al., 1993; Winnet al., 1995; Zhang and Bradley, 1996), and TGFβ2 mRNA alsohas been detected in the adjacent pharyngeal endod(Dickson et al., 1993). At slightly later stages (8.5 dpTGFβ2, BMP4 and BMP7 mRNAs are abundant in thmyocardium throughout the heart tube (Jones et al., 19Dickson et al., 1993; Lyons et al., 1995b), whereas endocardial layer expresses TGFβ1 (Dickson et al., 1993).Interestingly, Nodal and BMP7 are both expressed in the nand notochordal plate, whereas BMP4, -5, -7, Nodal and Le2 transcripts are present in lateral plate mesoderm (Colliget al., 1996; Lowe et al., 1996; Meno et al., 1996, 1997; Dudand Robertson, 1997; M. Solloway and E. J. R., unpublishe

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Finally, we note that multiple BMPs including BMP2, -4, -5-6 and -7 are all coexpressed with Furin in the allantois (Jonet al., 1991; Lyons et al., 1995a; Winnier et al., 1995; MSolloway and E. J. R., unpublished). Together witbiochemical data showing that Furin and related proteasenhance cleavage of TGFβ-related precursor proteins such asTGFβ1 (Dubois et al., 1995), activin A (Roebroek et al., 1993Mullerian inhibiting substance (Nachtigal and Ingraham1996), BMP4 (Cui et al., 1998; D. B. C. and E. J. Runpublished), and Nodal (D. B. C. and E. J. R., unpublishedthese overlapping expression patterns indicate that Furin mcontribute to the proteolytic activation of at least a sub-set TGFβ-related growth factors.

To test whether Furin is required for normal development the mouse embryo, we generated a loss of function mutatiat the fur locus using homologous recombination.Homozygous mutant embryos survive until 10.5 dpc, but dshortly thereafter, presumably due to hemodynaminsufficiency. The yolk sacs of mutant embryos fail to develolarge blood vessels, although patches of blood islancontaining embryonic nucleated red blood cells form, anendothelial precursors expressing the molecular marker Flkare present. A similar defect has been observed in embrydeficient in either the transcription factor SCL/tal-1 or TGFβ1(Dickson et al., 1995; Robb et al., 1995; Visvader et al., 1998raising the possibility that Furin may be similarly required fothe terminal differentiation of endothelial cells. We also founthat the allantois, a tissue that expresses abundant levels offurmRNA, fails to fuse with the chorion and instead becomehighly vacuolated by 9.5 dpc in mutant embryos. Interestinglchorioallantoic fusion is also abolished in embryos lackinboth BMP5 and BMP7 (M. Solloway and E. J. R.unpublished), consistent with the idea that a decrease in BMsignaling may contribute to this defect in Furin-deficienembryos. Chorioallantoic fusion also is inhibited in embryolacking either VCAM-1 or α4-integrin (Gurtner et al., 1995;Kwee et al., 1995; Yang et al., 1995), two cell-surface proteiexpressed within the allantois or chorion, respectively, whicinteract with each other to mediate cell-cell adhesion (Elicesal., 1990). Possibly, Furin activities are required to maintathe expression of VCAM-1. Alternatively, Furin may play arole in the proliferation or migration of primitive streak-derivedmesodermal cells which form the allantois. However, mutaallantoides are not overtly reduced in size compared to wtype, and they appropriately express twist mRNA, whichidentifies a population of mesodermal cells (Fuchtbauer, 199indicating that proliferation and migration of these cells arrelatively normal. In marked contrast, GATA4 expression iabsent in mutant allantoides, suggesting perhaps that cewhich would normally express GATA4 fail to adopt theirnormal fate. Alternatively, in this cell population GATA4 maybe a downstream target of factors activated by Furin.

The most profound defect in fur−/− embryos is their failureto undergo ventral closure, i.e. to form a looping heart tube aa coherent primitive gut. While definitive endoderm, identifieby the expression of HNF3β or shh mRNA, is formed, fusionof this sheet of cells at the ventral midline is delayed and fato proceed in the midgut region. As a result, only the foregand hindgut diverticula develop in mutant embryos, whereas tmidgut is missing. Furin activities therefore seem to bintimately involved in either the proliferation or migration of

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Fig. 6. Transverse tissue sections obtained from embryos stained by whole-mount in situ hybridization. (A-E) Sections of the shh-stainedembryos shown in Fig. 5A,B. At the entrance to the foregut, shh-positive cells have failed to involute in the mutant (B, open arrow), but not inthe normal embryo (A). (C) In the trunk region of the same mutant embryo shown in B, shh mRNA highlights the absence of gut endoderm. (D) At the level of the caudal intestinal portal and of (E) the hindgut, shh expression marks the presence of both the notochord and definitiveendoderm. (F,G) MLC2V mRNA staining in sections through normal (F) or mutant (G) hearts, respectively (9.5 dpc). (H-K) Expression ofeHand in normal (H) or mutant (I-K) 9.5 dpc embryos in cells of the rudimentary left ventricle (H-J, open arrows), and in the trunk and tailregion. Note that in the mutant embryo, the body wall has not fused at the ventral midline (K). a, common atrial chamber; bc, bulbus cordis; bw,body wall; cip, caudal intestinal portal; da, dorsal aorta; en, endocardium; fg, foregut; fp, floorplate; g, gut; hg, hindgut; ht, heart tube; lp, lateralplate; lv, left ventricle; mc, epimyocardium; mg, midgut; nc, notochord; nt, neural tube; rv, right ventricle; so, somite; uv, umbilical vein; v,common ventricular chamber; va, vitelline artery.

definitive endoderm cells. In keeping with this hypothesis, hilevels of fur mRNA expression are seen in the node anotochordal plate, and in the definitive endoderm, particulain the regions of the AIP and CIP where ventral fusion norma

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occurs. Within tissues fated to form the heart, cardiomyocyteidentified by the expression of α-cardiac actin, MLC-2V orMLC-2A are eventually specified, although their numberappear to be reduced, and they fail to fuse at the ventral midli


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Fig. 7. Whole-mount in situ hybridization of GATA4 and Nkx2.5expression in embryos derived from fur+/− intercross matings.(A) GATA4 mRNA expression in four representative 7.5 dpcembryos. (B,C) Transverse serial sections from the proximalembryonic region of a 7.5 dpc embryo stained for GATA4 mRNA.The level of sectioning in B is next to the extraembryonic region,whereas the section in C is derived from a slightly more distal leve(D) Frontal view of embryos stained for GATA4 at 8.5 dpc. Note thbifid hearts in the mutants. (E) Ventral view of embryos (8.5 dpc)showing GATA4 expression in the allantois (al) of control embryosbut not in that of fur−/− mutants. (F) Embryos (8.0 dpc) stained forNkx2.5 mRNA. Nkx2.5 expression is reduced in mutant embryos(n=9) compared to control litter mates. (G,H) During early somitestages, Nkx2.5 mRNA expression is also reduced in fur−/− mutants(n=4, H) compared to control littermates (G). aip, anterior intestinportal; al, allantois; en, visceral endoderm; ep, epiblast; ht, hearttube; m, embryonic mesoderm. In A-C and E, anterior is to the lef

during the process of ventral closure, leading to cardia bifidathe majority of mutant embryos. Interestingly, a very similaphenotype develops in embryos lacking GATA4 (Kuo et a1997; Molkentin et al., 1997). Since both Furin and GATA4 aexpressed in the anterior regions of visceral endoderm andthe underlying cardiogenic mesoderm, one possibility is ththey act in a common pathway. However, with the exception the allantois, GATA4 mRNA expression is largely normal iFurin-deficient embryos, suggesting that it acts independenfrom Furin to regulate ventral fusion of precardiac mesoderMoreover, chimera analysis has shown that embryos largcomposed of GATA4−/− cells develop normally in conjunctionwith wild-type visceral endoderm (Narita et al., 1997)suggesting that GATA4 activities are required exclusively in thprimitive visceral endoderm lineage to direct ventramorphogenesis. Most recently, chimera analysis has also bhighly informative in delineating distinct functions for thetranscription factor HNF3β in the extraembryonic visceralendoderm and in embryonic lineages (Dufort et al., 1998). ThHNF3β is required during gastrulation in the visceral endoderfor normal elongation of the primitive streak, whereas at a lastage it is essential in the embryo proper for the specificatiof node and notochord cell fates. Interestingly, in conjunctiowith wild-type visceral endoderm, chimeric embryos largelcomposed of HNF3β−/− cells fail to form definitive fore- andmidgut endoderm, and they fail to undergo heart loopinmorphogenesis and axial rotation. We are currently examiniwhether Furin activities are essential only in the viscerendoderm, or whether this protease is required in boextraembryonic and embryonic lineages, as is the case HNF3β.

In a small subset of Furin-deficient embryos, a single hetube occasionally develops, but subsequently fails to undelooping morphogenesis. A similar heart defect is seen embryos deficient for either Nkx2.5 or MEF2C (Lyons et al1995a; Lin et al., 1997). Interestingly, Nkx2.5 mRNA, markincardiomyocyte lineages, is expressed at significantly reduclevels in fur mutants, accounting perhaps in part for the failurof heart looping. This reduction in the expression levels Nkx2.5 is consistent with the idea that Furin activities macontribute to the activation of BMPs in the heart field, sincstudies in both chick and Drosophila embryos have implicatTGFβ/BMP-related signals in the specification of cardialineages (Frasch, 1995; Schultheiss et al., 1997; Andree et1998). Thus, the cardiac markers GATA4 and Nkx2.5 aectopically induced in anterior paraxial mesoderm treated wrecombinant BMP2 or BMP4 (Schultheiss et al., 1997; Andreet al., 1998), whereas cardiac myogenesis assayed by expression of Nkx2.5 is inhibited in tissue explants treated wthe BMP antagonist Noggin, strongly arguing for a role oendogenous BMP signaling in the specification of cardiamyocytes (Schultheiss et al., 1997). Tissue recombinatiexperiments in chick and frog embryos suggest that in additito BMP activities, inductive signals derived from definitiveendoderm are also required for normal heart developme(Nascone and Mercola, 1995; Schultheiss et al., 1995). Tdelay in the migration of definitive endoderm observed in fumutants is therefore likely to contribute to the heart defects.support of this conclusion, similar heart defects occur HNF3βchimeras that lack fore- and midgut endoderm (Dufoet al., 1998).

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In addition to defects in ventral closure, Furin-deficieembryos consistently fail to undergo axial rotation, a procethat is normally preceded by the rightward looping of the hetube. The inability of furmutants to turn is unlikely to resultsolely from abnormal heart looping morphogenesis, sinsimilar heart defects do not interfere with axial rotation embryos lacking Nkx2.5 or MEF2C (Lyons et al., 1995a; Let al., 1997). Likewise, impaired yolk sac vasculogenesisunlikely to account for the failure of axial rotation, sincembryos with similar vascular defects owing to the loss TGFβ1 or the transcription factor SCL/tal-1 turn normall(Dickson et al., 1995; Robb et al., 1995; Visvader et al., 199Possibly, the inability to turn results from mechanicconstraints associated with the endoderm defects. Howeapart from a delay in hindgut formation, fur mutants show noovert indications of mechanical obstruction at early somstages when embryonic turning is normally initiated, and thmorphology and size are strikingly normal up to this stagMoreover, the left-right axis is established since two molecumarkers of left-right asymmetry, Nodal and eHand, aexpressed appropriately in fur−/− embryos. Given theobservation that fur is coexpressed with both nodal and lefty2 in the left lateral plate mesoderm, it is possible that Furinrequired to generate physiological amounts of these ligandssupport of this hypothesis, overexpression of Furin leads tdramatic increase in the efficiency of Nodal precursor cleavain COS cells (D. B. C. and E. J. R., unpublished). Howevthe turning defect is unlikely solely to reflect inefficienprocessing of Nodal or Lefty, since in iv/iv embryos theabsence of asymmetric nodaland leftyexpression does notabolish, but rather randomizes the direction of axial rotati(Lowe et al., 1996; Meno et al., 1996). Thus, Furin seemsbe required for the activation of additional precursor proteinpotentially including BMP4, -5 and -7, all of which arestrongly expressed in the lateral plate (Dudley and Roberts1997; M. Solloway and E. J. R., unpublished).

While biochemical evidence and the striking overlap expression patterns support the notion that Furin may responsible for activating TGFβ-related growth factors, loss ofFurin has less severe consequences than mutationscomponents of these signaling pathways, including nodal(Zhou et al., 1993; Conlon et al., 1994), bmp4(Winnier et al.,1995), or receptors for TGFβfamily members (Mishina et al.,1995; Gu et al., 1998) and their downstream targets (Sirardal., 1998; Waldrip et al., 1998). This clearly suggests that activation of TGFβ/BMP signaling pathways involvesconvertase activities other than Furin. However, the phenotof fur mutants may in part reflect impaired function of TGFβ-related signals in tissues that strongly express Furin, and ikeeping with our previous hypothesis that the overlappiactivities of several related convertases including FurPACE4, PC5(6) and/or PC7 may promote the productionmature TGFβ-related ligands to control their localconcentration (Constam et al., 1996). In future studies, it wbe important to examine the processing efficiency of these other candidate Furin substrates in cells derived from fur−/−

embryos that specifically lack Furin activities.

This work was supported by grants from the Fonds voWetenschappelijk Onderzoek-Vlaanderen (FWO), by a grant of Interuniversity-network for Fundamental Research (IUAP) of th

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Belgian government, by the action-program for Biotechnology of thFlemish government (VLAB/COT-008), and the National Institutes oHealth. L. U. received a post-doctoral research fellowship from the U. Leuven research fund. D. B. C. is the recipient of fellowship 823A46642 from the Swiss National Science Foundation.

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Failure of ventral closure and axial rotation in …...regulatory step in the control of the Delta-Notch pathway responsible for cell-fate selection is proteolysis of the Notch receptor