RBP-J{kappa}-Dependent Notch Signaling Is Dispensable for Mouse Early Embryonic Development

The Notch signaling pathway is an evolutionarily conserved signalingsystem which has been shown to be essential in cell fate specificationand in numerous aspects of embryonic development in all metazoansthus far studied. We recently demonstrated that several componentsof the Notch signaling pathway, including the four Notch receptorsand their five ligands known in mammals, are expressed in mouseoocytes, in mouse preimplantation embryos, or both. This suggesteda possible implication of the Notch pathway in the first cellfate specification of the dividing mouse embryo, which resultsin the formation of the blastocyst. To address this issue directly,we generated zygotes in which both the maternal and the zygoticexpression of Rbpsuh, a key element of the core Notch signalingpathway, were abrogated. We find that such zygotes give riseto blastocysts which implant and develop normally. Nevertheless,after gastrulation, these embryos die around midgestation, similarlyto Rbpsuh-null mutants. This demonstrates that the RBP-J ${kappa}$ -dependentpathway, otherwise called the canonical Notch pathway, is dispensablefor blastocyst morphogenesis and the establishment of the threegerm layers, ectoderm, endoderm, and mesoderm. These resultsare discussed in the light of recent observations which havechallenged this conclusion.

INTRODUCTION

Top

Introduction

Notch signaling is an evolutionarily conserved system whichcontrols cell fate and has been shown in all metazoans studiedso far, in particular in Drosophila melanogaster, Caenorhabditiselegans, and vertebrates, to be mandatory in various developmentalprocesses, including stem cell homeostasis, cell growth, celldifferentiation, and survival (1, 18, 28). It is therefore nota surprise that mutations perturbing Notch signaling may resultfor humans in various pathologies including cancer (9, 12, 24).

Notch signaling takes place through cell-to-cell contact, andthe core of the pathway is relatively simple. Indeed, upon bindingbetween the Notch receptor on one cell and a ligand on an adjacentcell, the former, a single-path transmembrane protein, undergoesseveral proteolytic steps which result in the release of thesoluble intracellular domain of Notch (NICD). NICD is then translocatedto the nucleus, where it interacts with the transcriptionalregulator CSL [CBF1 in humans/Su(H) in Drosophila/LAG1 in C.elegans], also known as RBP-J ${kappa}$ in mammals. In the absence ofa signal-receptor interaction, RBP-J ${kappa}$ represses transcriptionthrough interactions with a corepressor complex containing ahistone deacetylase. The binding of NICD to RBP-J ${kappa}$ in the nucleusdisplaces the corepressor complex and allows recruitment ofhistone acetylase, a nuclear protein (Mastermind), and othercofactors, resulting in the transcription of various targetgenes (2, 20). This core pathway is further regulated at differentlevels involving many factors (13). Furthermore, interactionswith other signaling pathways have been documented (12, 16).Thus, beyond the relative simplicity of its core, the implementationof the Notch signaling pathway is extremely refined, implicatingthe interplay of many different factors and allowing a veryversatile and cellular context-dependent action (13).

We are interested in the first lineage specification of themouse embryo, which results in the formation of the blastocystmade of two different cell lineages: the trophectoderm, whichwill give rise to part of the placenta, and the inner cell mass,which is at the origin of all the tissues of the fetus. Thisprocess implies cell-cell signaling (17, 25), in which Notchsignaling could be implicated. As a first step to address thisquestion, we set out to monitor the expression of various componentsof the Notch signaling core pathway. We found that the genescoding for the four receptors (Notch1, Notch2, Notch3, and Notch4)and the five ligands (Delta-like 1 [Dll1], Dll3, and Dll4 andJagged1 and Jagged2) known in mammals (18) as well as the transcriptionfactor RBP-J ${kappa}$ and the two regulatory proteins Deltex-1 and Deltex-2were transcribed during oogenesis and/or mouse preimplantationdevelopment (4). These observations, which have been subsequentlyconfirmed (34), were consistent with the possibility of Notchsignaling being instrumental in blastocyst formation. On theother hand, it is important to note that disruption of thesegenes by targeted mutagenesis leads either to no obvious phenotype(Notch3 and Notch4) or to embryonic lethality (Notch1, Notch2,Jagged1, Jagged2, Dll1, and Rbpsuh) but not until several daysafter implantation (35), seemingly dismissing the possibilityof Notch signaling being mandatory in preimplantation development.Nevertheless, early embryonic lethality could be rescued forone of the two following reasons. Firstly, there could be functionalredundancy due to the expression of a gene(s) compensating forthe role of the disrupted gene. However, this hypothesis isunlikely because embryos lacking either Rbpsuh, which codesfor RBP-J ${kappa}$ (21), or both PS1 and PS2 presenilin genes (6) implantand develop beyond gastrulation. The second possibility is thatmaternal expression of a given component of the Notch pathwaycould compensate for the zygotic absence of gene function. Toaddress this latter possibility, we decided to disrupt Rbpsuhin such a way that both maternal and zygotic expression wouldbe abrogated. We chose Rbpsuh because it codes for RBP-J ${kappa}$ , whichhas been shown to associate with the NICD of all four Notchreceptors and consequently to be instrumental in the transcriptionalcontrol of target genes mediated by the four receptors (15).RBP-J ${kappa}$ therefore stands as an essential component of the so-calledcanonical Notch signaling (21). Thus, monitoring the fate ofzygotes lacking both maternal and zygotic RBP-J ${kappa}$ should allowus to answer the question of the possible participation of canonicalNotch signaling in the very early steps of mouse embryonic development.

MATERIALS AND METHODS

Top

Materials and Methods

Mice and embryos. We used two lines of transgenic mice: a zona pellucida 3-cre(ZP3-Cre) transgenic mouse line (5) and a mouse line containingan exon 6- and 7-floxed Rbpsuh allele (11). These two lineswere crossed in order to obtain Rbpsuh^f^/; ZP3-Cre^tg^/+ females.Embryos were recovered from mating between Rbpsuh^f^/; ZP3-Cre^tg^/+females and Rbpsuh^f^/ males. The age of the embryos was determinedaccording to the detection of the vaginal plug (day 1).

Mouse genotyping. Genotyping was done by PCR analysis of tail tips of mice orplacental tissue of embryos. The wild-type, floxed, and deletedRbpsuh alleles were detected using the following primers: F1,5'-GTT CTT AAC CTG TTG GTC GGA AAC-3', and R1, 5'-GCT TGA GGCTTG ATG TTC TGT ATT GC-3', for the wild-type allele; F1 andR2, 5'-GGG CTG CTA AAG CGC ATG CT-3', for the floxed allele;F3, 5'-CCT TGG TTT GTT GTT TGG GTT-3', and R3, 5'-GTG GCT CTCAAC TCC CAA TCG T-3', for the deleted allele. The Cre transgenewas detected using the following primers: Cre1, 5'-GGA CAT GTTCAG GGA TCG CCA GGC G-3', and Cre2, 5'-GCA TAA CCA GTG AAA CAGCAT TGC TG-3'.

RT-PCR on ovulated oocytes. Females were superovulated by injection of 5 units of pregnantmare serum gonadotrophin (Calbiochem) followed by injectionof 5 units of human chorionic gonadotrophin (Intervet) 48 hlater. Sixteen hours after human chorionic gonadotrophin injection,oocytes were collected and the cumulus cells were removed bya hyaluronidase treatment (0.5 mg/ml). Poly(A)⁺ RNAs were isolatedfrom 50 to 100 oocytes using Dynabeads (mRNA DIRECT kit; DYNAL).Poly(A)⁺ RNAs were reverse transcribed during 60 min at 42°Cusing 200 units of SuperScript II (Invitrogen). An equivalentof five oocytes was then used for nested reverse transcription-PCR(RT-PCR). Conditions for RT-PCR were 96°C for 5 min andthen 30 cycles of 96°C for 1 min, 60°C for 1 min, and72°C for 30 s, followed by 10 min at 72°C. A secondround of PCR was performed under similar conditions using 1µl of the first PCR mixture. Two nested primer pairs wereused: RBP1, 5'-GGC ACT CCC AAG ATT GAT A-3', and RBP2, 5'-GGTCCG CCA GCC AGT CCA G-3', and RBP3, 5'-CAG ACA AGG CCG AGT ACAC-3', and RBP4, 5'-GTT TCG GCT TCT ACA TCC C-3'. Hypoxanthinephosphoribosyltransferase primers (5'-GTT CTT TGC TGA CCT GCTGGA TTA C-3' and 5'-GTC AAG GGC ATA TCC AAC AAC AAA C-3') wereused to check the amount and integrity of cDNAs.

RESULTS

Top

Results

Rbpsuh disruption in oocytes. In order to deplete the Rbpsuh maternal store, we adopted aconditional mutagenesis approach. To do this, we used two differentlines of transgenic mice: ZP3-Cre transgenic (ZP3-Cre^tg/tg)mice, which express Cre specifically during early oogenesis(5, 19), and Rbpsuh^f/f mice, which carry Rbpsuh floxed allelesin which loxP sites flank exons 6 and 7 coding for the DNA bindingdomains of RBP-J ${kappa}$ such that, upon Cre action, these exons aredeleted, resulting in a null allele, Rbpsuh (31). Thus, as depictedin Fig. 1A, ZP3-Cre^tg/tg females were crossed with Rbpsuh^f/fmales, giving rise to Rbpsuh^f^/+;ZP3-Cre^tg^/+ progeny. Femalesborn from this cross, which produce oocytes with one Rbpsuh-disruptedallele, due to Cre expression in oogenesis (Fig. 1A), were thenbackcrossed to an Rbpsuh^f/f male. The progeny of this crossshowed a balanced distribution of Rbpsuh^f^/ and Rbpsuh^f^/+ animals(25 of the former and 21 of the latter, respectively) (Fig.1B and 1C), therefore demonstrating consistent expression ofCre in oocytes, with very efficient deletion of the Rbpsuh^fallele.

View larger version (29K):
[in this window]
[in a new window]
FIG. 1. Disruption of Rbpsuh in the oocyte. (A) Successive crosses to obtain zygotes deprived of maternal and zygotic Rbpsuh products. (B) Schematic representation of the wild type (+), floxed (f), and deleted ( ${Delta}$ ) Rbpsuh alleles. The PCR primers used for genotyping mice are indicated by arrowheads. Black boxes represent exons. (C) PCR of genomic DNA of Rbpsuh^f^/ and Rbpsuh^f^/+ mice. +, wild-type Rbpsuh allele; f, floxed Rbpsuh allele; ${Delta}$ , deleted Rbpsuh allele; M, molecular weight markers (Smartladder, small fragment; Eurogentec).

Rbpsuh^f^/; ZP3-Cre^tg^/+ females produce oocytes deprived of maternal Rbpsuh functional transcripts. Rbpsuh^f^/; ZP3-Cre^tg^/+ females obtained in the latter cross wererecovered. To demonstrate that these females produce oocytesdeprived of a functional Rbpsuh allele, the deletion of codingexons 6 and 7 of Rbpsuh was monitored in ovulated oocytes, usingnested RT-PCR. The first step was designed to amplify the 3'region of the mRNA encompassing exons 7 to 11. RT-PCR shouldefficiently amplify transcripts produced by wild-type Rbpsuh(Rbpsuh⁺) and Rbpsuh^f alleles but not the nonfunctional transcriptproduced by the Rbpsuh allele, as it lacks exons 6 and 7. Ascan be seen in Fig. 2, the presence of Rbpsuh transcripts wasreadily detected in oocytes from Rbpsuh^f/f; ZP3-Cre^+/+ females,by the presence of a band of the predicted size (181 bp). Incontrast, the same primers did not reveal any transcript allowingthe amplification of a 181-bp fragment in oocytes from Rbpsuh^f^/;ZP3-Cre^tg^/+females. This demonstrated the absence of transcripts containingthe region coding for the DNA binding region and therefore theabsence of a functional Rbpsuh maternal contribution.

View larger version (20K):
[in this window]
[in a new window]
FIG. 2. Deletion of floxed Rbpsuh allele in oocytes produced by Rbpsuh^f^/; ZP3-Cre^tg^/+ females. (A) Primers used to detect Rbpsuh transcripts by nested RT-PCR. (B) cDNA equivalent to that in five eggs was prepared from Rbpsuh^f/f (f/f) or from Rbpsuh^/ ( ${Delta}$ / ${Delta}$ ) oocytes obtained from Rbpsuh^f/f and Rbpsuh^f; ZP3-Cre^tg/+ females, respectively. It was subjected to a nested RT-PCR using the two pairs of primers (RBP1-RBP2 and RBP3-RBP4 indicated in panel A). The experiment was performed twice, using oocytes from different females in each case. As a control for cDNA synthesis and amount, RT-PCR was performed using hypoxanthine phosphoribosyltransferase (HPRT) primers.

Rbpsuh^/ zygotes deprived of maternal transcripts implant and develop beyond gastrulation. Having obtained females producing oocytes lacking maternal Rbpsuhcontribution, we then asked whether zygotes lacking both maternaland zygotic Rbpsuh contribution could survive and develop normallybeyond implantation and gastrulation despite the complete absenceof RBP-J ${kappa}$ and therefore the disruption of the RBP-J ${kappa}$ -dependentcanonical Notch signaling. To do this, Rbpsuh^f^/;ZP3-Cre^tg^/+females were crossed with Rbpsuh^/+ males and embryos were recoveredat different times during development. An examination of embryosat 4 days postcoitum (dpc) revealed that the blastocysts appearedmorphologically normal (data not shown). We then examined embryosat 9 dpc to 11 dpc. All together, 85 embryos were collected,carefully examined, and genotyped. Among them, 45 were homozygous(Rbpsuh^/) mutant and 40 were heterozygous (Rbpsuh^/+), furtherconfirming the highly efficient activity of the Cre recombinasein oocytes. At 9 dpc, none of eight mutant embryos examinedexhibited obvious morphological anomalies. From 10 dpc, Rbpsuh^/embryos showed severe growth retardation, and they exhibiteda reduction in size compared to Rbpsuh^/+ embryos with associateddefects (Fig. 3A and B). These defects became more pronounced1 day later. Somites were poorly formed in Rbpsuh^/ embryos,and they were disorganized (Fig. 3C and D). The neural tubeof Rbpsuh^/ embryos appeared twisted in shape (Fig. 3C and D),and the anterior neuropore was not closed at 10 dpc whereasclosure was evident between 9 and 10 dpc in wild-type embryos(data not shown). Mutant embryos had also a distended pericardium(Fig. 3B and F) and a compact unfused allantois (Fig. 3E). Takentogether, these results show that Rbpsuh^/+ and Rbpsuh^/ zygotesare produced at Mendelian ratios and that the latter, whichlack both maternal and zygotic RBP-J ${kappa}$ , develop normally throughthe blastocyst stage, implantation, and gastrulation. Laterin development, starting at 9.5 dpc, Rbpsuh^/ embryos exhibitgross developmental anomalies, as originally described by Okaet al. for homozygous-null Rbpsuh embryos (21).

View larger version (87K):
[in this window]
[in a new window]
FIG. 3. Rbpsuh^/ zygotes lacking both maternal and zygotic Rbpsuh products develop until beyond gastrulation, when they exhibit gross developmental abnormalities. (A and B) Rbpsuh^/ (right) and Rbpsuh^f^/ (left) embryos were isolated from crosses between Rbpsuh^f^/; ZP3-Cre^tg^/+ females and Rbpsuh^f^/ males at 10 dpc (A) and 11 dpc (B). (C) Defective somitogenesis (white arrowhead) and neurogenesis (black arrowhead) in mutant embryos at 10 dpc. (D) Enlarged view of the boxed part of the embryo in panel C showing abnormal somites (white arrowhead) and neural tube (black arrowhead). (E) Unfused allantois in Rbpsuh^/ embryos. (B and F) Distended pericardium in two different Rbpsuh^/ embryos (white arrowheads).

DISCUSSION

Top

Discussion

In this study, we addressed the possibility of a role for Notchsignaling in early development in the mouse. This was basedon our recent observation that several essential actors of theNotch pathway are expressed in oocytes and/or preimplantationembryos from the fertilized oocyte to the blastocyst stage,where the first cell fate specifications of the mouse embryohave taken place (4). To address this question, we generatedzygotes lacking both maternal and zygotic expression of Rbpsuh,a gene which has been shown to be an essential component ofthe core of Notch signaling (21) but also a bottleneck in theNotch cascade, i.e., being implicated in the signaling throughall four Notch receptors (15). We showed not only that suchzygotes give rise to normal blastocysts but that the latterimplant and develop through gastrulation and finally die aroundday 10 to 11 dpc, exhibiting the same phenotypic anomalies asreported earlier for embryos homozygous for a null mutationin the Rbpsuh gene (21). This strongly suggests that the canonicalRBP-J ${kappa}$ -dependent Notch signaling system is dispensable for theearly development of the mouse embryo, including gastrulationand therefore the formation of the three embryonic germ layers.In light of the described expression of the Notch signalingpathway (4, 34), this finding is quite unexpected. It shouldbe noted that a similar conclusion was reached in a very recentstudy by Shi et al. (29). These authors used a strategy similarto the one used in the present study but targeted Pofut1, agene which encodes the protein O-fucosyltransferase 1 (O-FucT-1)and has been shown, both in Drosophila (22, 27) and in mouse(30), to act upstream in the canonical Notch signaling pathwayand to be an essential component of this pathway. The authorsgenerated embryos lacking both maternal and zygotic Pofut1 geneproduct and reported that these embryos develop beyond the blastocyststage until after gastrulation (29). Thus, our study providesfurther evidence and substantiates the unexpected notion ofthe dispensability of the canonical RBP-J ${kappa}$ -dependent Notch pathwayfor mouse early embryonic development.

Recent studies of Drosophila have given further insight intothe molecular mechanisms underlying the function of O-fucosyltransferase1 in Notch signaling. Firstly, it was shown to be required forthe activity of the full-length Notch receptor and essentialfor the physical interaction of Notch with its ligands Deltaand Serrate (23, 27). Secondly, it was demonstrated that, besidesits well-established activity of fucosylating serine and threoninein EGF repeats of the Notch extracellular domain (reviewed inreference 10), O-fucosyltransferase 1 has a chaperone activityrequired for Notch egression from the endoplasmic reticulumand subsequent transport to the membrane. Thus, it would beessential for the proper localization of Notch at the cell surface.To date, these results have not been extended to mammals. However,should it be the case, one would assume that in embryos lackingboth maternal and zygotic O-FucT-1, like those generated byShi et al. (29), both O-fucosylation and chaperone activityof O-FucT-1 could not take place, resulting in defective ligand-receptorbinding and therefore the absence of NICD production. Thus,in such embryos, both RBP-J ${kappa}$ -dependent and RBP-J ${kappa}$ -independentmodes of Notch signaling would be precluded, reinforcing thenotion that Notch signaling is dispensable for mammalian earlyembryonic development.

Two sets of observations based on the generation of knockoutmice might, however, challenge this contention. In fact, disruptionof either Brainiac1 or mouse Notchless (mNle) genes resultsin a dramatic early phenotype: the embryos homozygous for anull mutation die either before implantation as regards to Brainiac1(32) or shortly after with a degeneration of the inner cellmass in the case of mNle (3). Importantly, both Brainiac1 andNotchless have been shown in Drosophila to interact geneticallywith members of the Notch signaling pathway (7, 8, 26). Brainiacwas initially characterized as a neurogenic gene, with mutantflies exhibiting neural hyperplasia and epidermal hypoplasia(7), and recently shown to code for a glycosyltranferase, involvedin the glycosphingolipid biosynthetic pathway (33). In addition,it has been implicated in the proper differentiation of theepithelial cells surrounding the oocyte (7). Both phenotypesin fly as well as the results of a very recent study of theC. elegans Brainiac gene (bre-5) uncovering its functional implicationupstream of the lin12 (Notch) signaling system (14) point toa role for Brainiac in Notch signaling which could be conservedin mammals. It is therefore tempting to speculate that defectivepreimplantation development of mouse embryos lacking Brainiacmight reflect some misfunctioning of the Notch pathway. However,other explanations related to the fact that Brainiac is a glycosyltransferasecannot be excluded whereby Brainiac-dependent glycosphingolipidsynthesis might be important in other ways. Other findings suggesta role for Notch in mouse preimplantation development relatedto the observation that embryos lacking mNle die very shortlyafter implantation (3). Notchless was initially discovered inDrosophila in a genetic screen for modifiers of Notch activity:loss-of-function dominantly suppressed the wing notching inducedby certain Notch alleles (26). It was also shown that dependingon the context, Notchless regulates either negatively or positivelythe Notch pathway. Furthermore, using a biochemical approach,it was demonstrated that Notchless binds to NICD (26). Similarlyand importantly, we recently demonstrated that murine Nle proteinhas conserved its ability to modulate Notch activity (3). Therefore,the phenotypic consequences of disrupting mNle, the mouse orthologof Notchless, namely, death of embryos shortly after implantation,were consistent with the notion that Notch signaling is implicatedin very early development, in apparent contradiction to thedelayed phenotypic consequences of Pofut1 or Rbpsuh disruption.One possible explanation to resolve this contradiction wouldbe to assume that mNle acts to repress Notch signaling or maintainit below a certain level and that its abrogation would resultin enhanced canonical Notch signaling with detrimental consequencesfor the development of the embryo.

In conclusion, the phenotypic consequences of the abrogationof zygotic and maternal Rbpsuh contribution (this study) onthe one hand and Pofut1 (29) on the other hand strongly suggestthat Notch signaling is dispensable in preimplantation mousedevelopment. However and intriguingly, both the expression ofvarious components of the Notch pathway and the phenotypic analysisof embryos mutant for either Brainiac (32) or mNle (3), twogenes recognized as modulators of the Notch pathway, are consistentwith an opposite conclusion. Presently, it remains unclear howto reconcile these different studies, until the precise molecularmechanisms underlying the phenotypes observed are elucidated.

ACKNOWLEDGMENTS

We thank T. Honjo (Kyoto, Japan) for the RBP-J ${kappa}$ ^f/f mice and B.Knowles (Bar Harbor, Maine) and D. Solter (Freiburg, Germany)for the ZP3-Cre^tg/tg transgenic line. We thank S. Tajbakhshfor critical reading of the manuscript.

This work was supported by the Centre National de la RechercheScientifique (CNRS), the Association pour la Recherche contrele Cancer (ARC), the Institut Pasteur GPH07 on stem cells, andthe Agence Nationale pour la Recherche (contract number 05-JC05-41835).S.C. received grants from the Pasteur-Negri-Weizmann Counciland the ARC. C.S. received grants from CNRS (Bourse de Doctoratpour les Ingénieurs).

FOOTNOTES

* Corresponding author. Mailing address: Unité Biologie du Développement, CNRS URA 2578, Institut Pasteur, Paris, France. Phone: 33 1 45 68 85 54. Fax: 33 1 45 68 86 34. E-mail: chbabi{at}pasteur.fr.

REFERENCES

Top