INTRODUCTION

The Drosophila Delta gene is one of the so-called neurogenicgenes required for the proper formation of neural precursor cellsduring neurogenesis of the fly (Lehmann et al., 1981). InDrosophila, neurogenesis is initiated by the separation of neuralprogenitor cells (the neuroblasts) from progenitors of theepidermis (the epidermoblasts). Prior to this separation, neuro-blasts and epidermoblasts are intermingled in the neurogenicectoderm (Campos-Ortega, 1993). Each cell in this region hasthe potential to become either a neuroblast or an epidermoblastand has to choose between these developmental fates (Technauand Campos-Ortega, 1986, 1987). Embryological, genetic andmolecular studies have indicated that cell-to-cell interactions areessential for the proper separation of these two cell lineages, thatthese interactions likely involve direct cell-cell contacts betweenneighboring neuroepidermal cells and that the products of theneurogenic genes participate in this cell communication process(Technau and Campos-Ortega, 1987; Campos-Ortega, 1993).

The products of two of the neurogenic genes, Delta andNotch, are likely mediators of direct cell-cell contacts, and maypass regulatory signals between neighboring neuroectodermalcells. Both genes encode transmembrane proteins containingtandem arrays of epidermal-growth-factor (EGF)-like repeatsin their extracellular domains (Wharton et al., 1985; Vässin etal., 1987; Haenlin et al., 1990). EGF-like repeats are found inmany growth factor and receptor molecules and participate inprotein-protein interactions (Davis, 1990; Muskavitch andHoffmann, 1990). The Delta and Notch proteins can directlybind to each other and specific EGF-like repeats are sufficientand necessary for this binding (Fehon et al., 1990; Rebay etal., 1991; Lieber et al., 1992). Mosaic analysis suggested thatNotch could function as a receptor whereas Delta is more likelyto be a ligand (Technau and Campos-Ortega, 1987; Heitzlerand Simpson, 1993). Delta and Notch function is not restrictedto the developing nervous system. These genes are alsorequired for cell fate decisions in mesodermal cells (Corbin etal., 1991) and are implicated in epithelial-mesenchymal tran-

2407Development 121, 2407-2418 (1995)Printed in Great Britain © The Company of Biologists Limited 1995

The Drosophila Delta (Dl) gene is essential for cell-cell com-munication regulating the determination of various cellfates during development. Dl encodes a transmembraneprotein, which contains tandem arrays of epidermal-growth-factor-like repeats in the extracellular domain anddirectly interacts with Notch, another transmembraneprotein with similar structural features, in a ligand-receptor-like manner. Similarly, cell-cell interactionsinvolving Delta-like and Notch-like proteins are requiredfor cell fate determinations in C. elegans. Notch homo-logues were also isolated from several vertebrate species,suggesting that cell-to-cell signaling mediated by Delta- andNotch-like proteins could also underlie cell fate determina-tion during vertebrate development. However, in verte-brates, no Delta homologues have yet been described.

We have isolated a novel mouse gene, Dll1 (delta-likegene 1), which maps to the mouse t-complex and whosededuced amino acid sequence strongly suggests that Dll1represents a mammalian gene closely related to Drosophila

Delta. Dll1 is transiently expressed during gastrulation andearly organogenesis, and in a tissue-restricted manner inadult animals. Between day 7 and 12.5 of development,expression was detected in the paraxial mesoderm, closelycorrelated with somitogenesis, and in subsets of cells in thenervous system. In adult animals, transcripts were detectedin lung and heart. Dll1 expression in the paraxialmesoderm and nervous system is strikingly similar to theexpression of mouse Notch1 during gastrulation and earlyorganogenesis. The overlapping expression patterns of theDll1 and Notch1 genes suggest that cells in these tissues cancommunicate by interaction of the Dll1 and Notch1proteins. Our results support the idea that Delta- andNotch-like proteins are involved in cell-to-cell communica-tion in mammalian embryos and suggest a role for theseproteins in cellular interactions underlying somitogenesisand development of the nervous system.

Key words: Delta gene, EGF like repeats, somitogenesis, t-complex

SUMMARY

Transient and restricted expression during mouse embryogenesis of Dll1, a

murine gene closely related to Drosophila Delta

Berthold Bettenhausen1, Martin Hrabe de Angelis2, Dominique Simon3, Jean-Louis Guénet3and Achim Gossler1,2,*1Max-Delbrück-Laboratorium in der MPG, Carl-von-Linné-Weg 10, D-50829 Köln, Germany2The Jackson Laboratory, Bar Harbor, Maine 04609, USA3Institut Pasteur, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France

*Author for correspondence

2408

sitions in derivatives of all germ layers (Hartenstein et al.,1992; Tepass and Hartenstein, 1995), suggesting that Delta andNotch are generally involved in choosing between two alter-native fates and in the promotion and maintenance of theepithelial state.

Similarly, Notch and Delta homologues are required in C.elegans for inductive interactions and cell fate decisionsduring nematode development (Greenwald et al., 1983;Austin and Kimble, 1987; Mello et al., 1994; Tax et al.,1994). The existence of Delta- and Notch-like proteins innematodes and flies suggestedthat Delta- and Notch-likeproteins and their interactionsare evolutionary conserved andthat such proteins could also bethe molecular basis for cell-to-cell communication processesduring vertebrate development.Notch homologues were indeedidentified in vertebrates(Coffman et al., 1990; Ellisen etal., 1991; Weinmaster et al.,1991, 1992; del Amo et al.,1992; Reaume et al., 1992;Weinmaster et al., 1992;Bierkamp and Campos-Ortega,1993; Lardelli and Lendahl,1993; Lardelli et al., 1994) andexpression of a dominantversion of the Xenopus Notchgene appeared to redirect cellfates in the Xenopus embryo(Coffman et al., 1993). Arecessive targeted allele of themouse Notch1 gene had noapparent effect on cell fates butresulted in massive and wide-spread cell death in the centraland peripheral nervous system,and led to developmental arrestand embryonic lethality aroundday 11.5 (Swiatek et al., 1994).The lack of a more severe defectof this Notch1 mutation on cellfates has been attributed to thefact that a second mouse Notchgene, Notch2, is expressed inpartly overlapping domains andthus might compensate for theloss of the Notch1 function(Swiatek et al., 1994). Detailedanalysis of a second targetedmutation in Notch1 showed thatNotch1 is required for the coor-dinate segmentation of somitesand suggested that the observedmassive cell death in mutantembryos is a secondary event(Conlon et al., 1995).

The above results and therecent isolation of Jagged, a

mammalian ligand for Notch1 which is similar to theDrosophila Serrate protein (Lindsell et al., 1995), support thenotion that cell-to-cell communication involving Notch-likeproteins is important for vertebrate development. Here wereport on the isolation and expression during embryogenesis ofa novel mouse gene, delta-like gene 1 (Dll1), which is closelyrelated to the Drosophila Delta gene. Dll1 is transientlyexpressed during embryogenesis in groups of cells in thenervous system and in the paraxial mesoderm. The expressiondomains of Dll1 overlap with described patterns of Notch gene

B. Bettenhausen and others

GTCCAGCGGTACCATGGGCCGTCGGAGCGCGCTAGCCCTTGCCGTGGTCTCTGCCCTGCTGTGCCAGGTCTGGAGCTCCGGCGTATTTGAG 91 M G R R S A L A L A V V S A L L C Q V W S S G V F E 26

CTGAAGCTGCAGGAGTTCGTCAACAAGAAGGGGCTGCTGGGGAACCGCAACTGCTGCCGCGGGGGCTCTGGCCCGCCTTGCGCCTGCAGG 181 L K L Q E F V N K K G L L G N R N C C R G G S G P P C A C R 56

ACCTTCTTTCGCGTATGCCTCAAGCACTACCAGGCCAGCGTGTCACCGGAGCCACCCTGCACCTACGGCAGTGCTGTCACGCCAGTGCTG 271 T F F R V C L K H Y Q A S V S P E P P C T Y G S A V T P V L 86

GGTGTCGACTCCTTCAGCCTGCCTGATGGCGCAGGCATCGACCCCGCCTTCAGCAACCCCATCCGATTCCCCTTCGGCTTCACCTGGCCA 361 G V D S F S L P D G A G I D P A F S N P I R F P F G F T W P 116

GGTACCTTCTCTCTGATCATTGAAGCCCTCCATACAGACTCTCCCGATGACCTCGCAACAGAAAACCCAGAAAGACTCATCAGCCGCCTG 451 G T F S L I I E A L H T D S P D D L A T E N P E R L I S R L 146

ACCACACAGAGGCACCTCACTGTGGGAGAAGAATGGTCTCAGGACCTTCACAGTAGCGGCCGCACAGACCTCCGGTACTCTTACCGGTTT 541 T T Q R H L T V G E E W S Q D L H S S G R T D L R Y S Y R F 176

GTGTGTGACGAGCACTACTACGGAGAAGGTTGCTCTGTGTTCTGCCGACCTCGGGATGACGCCTTTGGCCACTTCACCTGCGGGGACAGA 631 V C D E H Y Y G E G C S V F C R P R D D A F G H F T C G D R 206

GGGGAGAAGATGTGCGACCCTGGCTGGAAAGGCCAGTACTGCACTGACCCAATCTGTCTGCCAGGGTGTGATGACCAACATGGATACTGT 721 G E K M C D P G W K G Q Y C T D P I C L P G C D D Q H G Y C 236

GACAAACCAGGGGAGTGCAAGTGCAGAGTTGGCTGGCAGGGCCGCTACTGCGATGAGTGCATCCGATACCCAGGTTGTCTCCATGGCACC 811 D K P G E C K C R V G W Q G R Y C D E C I R Y P G C L H G T 266

TGCCAGCAACCCTGGCAGTGTAACTGCCAGGAAGGCTGGGGGGGCCTTTTCTGCAACCAAGACCTGAACTACTGTACTCACCATAAGCCG 901 C Q Q P W Q C N C Q E G W G G L F C N Q D L N Y C T H H K P 296

TGCAGGAATGGAGCCACCTGCACCAACACGGGCCAGGGGAGCTACACATGTTCCTGCCGACCTGGGTATACAGGTGCCAACTGTGAGCTG 991 C R N G A T C T N T G Q G S Y T C S C R P G Y T G A N C E L 326

GAAGTAGATGAGTGTGCTCCTAGCCCCTGCAAGAACGGAGCGAGCTGCACGGACCTTGAGGACAGCTTCTCTTGCACCTGCCCTCCCGGC 1081 E V D E C A P S P C K N G A S C T D L E D S F S C T C P P G 356

TTCTATGGCAAGGTCTGTGAGCTGAGCGCCATGACCTGTGCAGATGGCCCTTGCTTCAATGGAGGACGATGTTCAGATAACCCTGACGGA 1171 F Y G K V C E L S A M T C A D G P C F N G G R C S D N P D G 386

GGCTACACCTGCCATTGCCCCTTGGGCTTCTCTGGCTTCAACTGTGAGAAGAAGATGGATCTCTGCGGCTCTTCCCCTTGTTCTAACGGT 1261 G Y T C H C P L G F S G F N C E K K M D L C G S S P C S N G 416

GCCAAGTGTGTGGACCTCGGCAACTCTTACCTGTGCCGGTGCCAGGCTGGCTTCTCCGGGAGGTACTGCGAGGACAATGTGGATGACTGT 1351 A K C V D L G N S Y L C R C Q A G F S G R Y C E D N V D D C 446

GCCTCCTCCCCGTGTGCAAATGGGGGCACCTGCCGGGACAGTGTGAACGACTTCTCCTGTACCTGCCCACCTGGCTACACGGGCAAGAAC 1441 A S S P C A N G G T C R D S V N D F S C T C P P G Y T G K N 476

TGCAGCGCCCCTGTCAGCAGGTGTGAGCATGCACCCTGCCATAATGGGGCCACCTGCCACCAGAGGGGCCAGCGCTACATGTGTGAGTGC 1531 C S A P V S R C E H A P C H N G A T C H Q R G Q R Y M C E C 506

GCCCAGGGCTATGGCGGCCCCAACTGCCAGTTTCTGCTCCCTGAGCCACCACCAGGGCCCATGGTGGTGGACCTCAGTGAGAGGCATATG 1621 A Q G Y G G P N C Q F L L P E P P P G P M V V D L S E R H M 536

GAGAGCCAGGGCGGGCCCTTCCCCTGGGTGGCCGTGTGTGCCGGGGTGGTGCTTGTCCTCCTGCTGCTGCTGGGCTGTGCTGCTGTGGTG 1711 E S Q G G P F P W V A V C A G V V L V L L L L L G C A A V V 566

GTCTGCGTCCGGCTGAAGCTACAGAAACACCAGCCTCCACCTGAACCCTGTGGGGGAGAGACAGAAACCATGAACAACCTAGCCAATTGC 1801 V C V R L K L Q K H Q P P P E P C G G E T E T M N N L A N C 596

CAGCGCGAGAAGGACGTTTCTGTTAGCATCATTGGGGCTACCCAGATCAAGAACACCAACAAGAAGGCGGACTTTCACGGGGACCATGGA 1891 Q R E K D V S V S I I G A T Q I K N T N K K A D F H G D H G 626

GCCAAGAAGAGCAGCTTTAAGGTCCGATACCCCACTGTGGACTATAACCTCGTTCGAGACCTCAAGGGAGATGAAGCCACGGTCAGGGAT 1981 A K K S S F K V R Y P T V D Y N L V R D L K G D E A T V R D 656

ACACACAGCAAACGTGACACCAAGTGCCAGTCACAGAGCTCTGCAGGAGAAGAGAAGATCGCCCCAACACTTAGGGGTGGGGAGATTCCT 2071 T H S K R D T K C Q S Q S S A G E E K I A P T L R G G E I P 686

GACAGAAAAAGGCCAGAGTCTGTCTACTCTACTTCAAAGGACACCAAGTACCAGTCGGTGTATGTTCTGTCTGCAGAAAAGGATGAGTGT 2161 D R K R P E S V Y S T S K D T K Y Q S V Y V L S A E K D E C 716

GTTATAGCGACTGAGGTGTAAGATGGAAGCGATGTGGCAAAATTCCCATTTCTCTCAAATAAAATTCCAAGGATATAGCCCCGATGAATG 2051 V I A T E V * 722

CTGCTGAGAGAGGAAGGGAGAGGAAACCCAGGGACTGCTGCTGAGAACCAGGTTCAGGCGAAGCTGGTTCTCTCAGAGTTAGCAGAGGCG 2341

CCCGACACTGCCAGCCTAGGCTTTGGCTGCCGCTGGACTGCCTGCTGGTTGTTCCCATTGCACTATGGACAGTTGCTTTGAAGAGTATAT 2431

ATTTAAATGGACGAGTGACTTGATTCATATAGGAAGCACGCACTGCCCACACGTCTATCTTGGATTACTATGAGCCAGTCTTTCCTTGAA 2521

ATTTAAATGGACGAGTGACTTGATTCATATAGGAAGCACGCACTGCCCACACGTCTATCTTGGATTACTATGAGCCAGTCTTTCCTTGAA 2611

TTTGGGATTTGTAAAAATATTTTTCATGATATCTGTAAAGCTTGAGTATTTTGTGACGTTCATTTTTTTATAATTTAAATTTTGGTAAAT 2701

ATGTACAAAGGCACTTCGGGTCTATGTGACTATATTTTTTTGTATATAAATGTATTTATGGAATATTGTGCAAATGTTATTTGAGTTTTT 2791

TACTGTTTTGTTAATGAAGAAATTCATTTTAAAAATATTTTTCCAAAATAAATATAATGAACTAC(A)n 2856

Fig. 1. Dll1 nucleotide (EMBL accession number X80903) and deduced amino acid sequence. TheEGF-like repeats are indicated by gray shading, the DSL domain is shaded and framed. The putativemembrane spanning domain is indicated by a black bar. The two polyadenylation signals areunderlined and the ATTTA sequences marked by a line above the sequence.

2409Mouse delta-like gene

expression, suggesting that the protein encoded by Dll1 couldbe involved in cell-cell interactions during nervous systemdevelopment and somitogenesis.

MATERIALS AND METHODS

Cloning and sequencingA 623 bp cDNA fragment, RS15, isolated in a whole-mount in situhybridization screen (Bettenhausen and Gossler, unpublished data), wasused to screen a oligo(dT)-primed day 8.5 embryo cDNA library (giftof Dr B. Hogan). Hybridizations and isolation of clones were done understandard conditions. Phage inserts were subcloned into pBluescriptII SKand sequenced by the dideoxy chain termination method (Sequenase 2.0,US Biochemicals) or automatically with an Applied BiosystemsSequencer A373. Sequence analysis was done with the GCG programBESTFIT (Devereux et al., 1984), database searches were done usingthe BLAST network service (Altschul et al., 1990). The Prosite databasewas screened for protein motifs using MacPattern 3.3.

3′-RACE-PCRTotal day 10.5 embryo RNA was prepared according to (Chom-czynski and Sacchi, 1987), poly(A)+ RNA with oligtex(dT)30latexbeads (Qiagen, Germany) according to the manufacturer’sinstructions. 1 µg poly(A)+ RNA was transcribed into cDNA usingM-MuLV-RT (Stratagene) with oligo(dT)15-primer. RNA, 150 ngprimer and DEPC-H2O were incubated for 10 minutes at 70°C,chilled on ice and shortly centrifuged. 2 µl 10× RT-buffer (Strata-gene), 2.5 µl 10 mM dNTPs, 2 µl 0.1 M DTT and 50 U M-MulV-RT were added to a final volume of 20 µl, incubated for 10 minutesat room temperature and for 60 minutes at 42°C. The reaction wasstopped at 95°C and cooled on ice for 10 minutes. 2 U RNAse H wasadded for a 20 minutes incubation at 37°C. For the PCR reaction 1µl of the cDNA was mixed with 1 pmol of the genespecific primer(5′-GCTTGAGTATTTTGTGACG) and 1 pmol ofthe oligo(dT)15-primer, 1 µl 10 mM dNTP, 5 µl10× PCR-buffer (Boehringer), 2.5 U Taq-Poly-merase (Boehringer) and H2O to a final volume of50 µl. The amplification conditions were 37 cyclesof 30 seconds at 94°C, 1 minutes at 54°C, 2minutes at 72°C followed by 10 minutes at 72°C.The PCR products were cloned into the TA-vector(InVitrogen).

Northern and Southern blot analysisRNA and DNA was separated on by agarose gelelectrophoresis, blotted on Qiagen Nylon Plus(RNA) or on Hybond N (DNA) membranes andbaked at 80°C for 2 hours. Filters were prehy-bridized with 4× SSC, 1% nonfat milk powder, 1%SDS and 500 µg salmon sperm DNA/ml at 65°Cfor 2 hours and hybridized in the presence of 20%dextrane sulfate with radioactivity labeled DNAprobes over night. Filters were washed to the strin-gency of 0.1× SSC, 0.1% SDS at 65°C and exposedto X-ray film at −70°C with an intensifying screen.

RT-PCRFor the PCR, 1 µl of each cDNA sample preparedas described above was mixed with 1 pmol each ofthe gene specific primers (5′-GGACTATAAC-CTCGTTCG; 5′-GAAAGACTGGCTCATAGG),1 µl 10 mM dNTPs, 5 µl 10× PCR-buffer(Boehringer), 2.5 U Taq-Polymerase (Boehringer)and H2O to a final volume of 50 µl. The amplifica-tion conditions were 37 cycles of 30 seconds at

94°C, 1 minutes at 50°C, 2 minutes at 72°C followed by 10 minutesat 72°C. The PCR-products were analyzed on an agarose gel, blottedand hybridized with the appropriate cDNA fragment. From eachprepared cDNA a control with β-actin primers was conducted(5′-primer: TGGAATCCTGTGGCATCCATGAAAC; 3′-primer:TAAAACGCAGCTCAGTAACAGTCCG).

Interspecific backcross analysis874 DNA samples from an interspecific backcross involving the lab-oratory strain C57BL/6 and a strain derived from wild specimens ofthe Mus spretus species (EUCIB resource) were typed for variousmarkers as described previously (Breen et al., 1994). Linkage andgenetic distances were computed using the MBx software.

Whole-mount in situ hybridizationCD1 embryos were collected according to Rosen and Beddington(1993). Digoxigenin-labelled sense and antisense riboprobes wereproduced with the Digoxigenin RNA Labeling Kit (BoehringerMannheim) according to the manufacturer’s instructions. Whole-mount in situ hybridizations were performed essentially asdescribed (Wilkinson, 1992), with the following modifications. Allsteps were carried out in Reactivials (Pierce Ltd.). The H2O2bleaching step was omitted and the concentration of the riboprobein the hybridization buffer was doubled. Ten 1-hour post-antibodywashes were done and 2 mg/ml Levamisole was added to the lastwash, which was done overnight at 4°C. After staining, embryoswere washed three times with PBT containing 1 mM EDTA andstored at 4°C. Embryos were cleared overnight in 70% ethanol andphotographed on a Leica Wild stereophotomicroscope with KodakEktachrome 320T film.

HistologyAfter whole-mount in situ hybridization embryos were incubated in20% glucose in PBS/0.1% EDTA overnight at 4°C, embedded inTissue-Tek (Miles Inc.), frozen in liquid nitrogen and sectioned at 20

225 C L . . P G C D D Q H G Y C D K P G E . . . . C K C R V G W Q G R Y C D E

256 C I R Y P G C L . . H G T C Q Q P W Q . . . . C N C Q E G W G G L F C N Q D L N Y

291 C T H H K P C R . N G A T C T N T G Q G S Y T C S C R P G Y T G A N C E L E V D E

331 C A . P S P C K . N G A S C T D L E D S F S . C T C P P G F Y G K V C E L S A M T

369 C A . D G P C F . N G G R C S D N P D G G Y T C H C P L G F S G F N C E K K M D L

408 C G . S S P C S . N G A K C V D L G N S Y L . C R C Q A G F S G R Y C E D N V D D

446 C A . S S P C A . N G G T C R D S V N D F S . C T C P P G Y T G K N C S A P V S R

484 C E . H A P C H . N G A T C H Q R G Q R Y M . C E C A Q G Y G G P N C Q F L L P E P P

100

NH2 COOH

700300200 4000 500 600

A

B

DSL 1 2 3 4 5 6 7 8

Fig. 2. Structure of the Dll1 protein. (A) Hydrophobicity plot of the deduced Dll1protein and schematic drawing showing the positions of the putative leader sequence atthe N terminus (solid black) and the transmembrane spanning region (solid black), theDSL domain (open box labelled DSL) and EGF-like repeats (open boxes 1-8). (B) alignment of the 8 EGF-like repeats. Gaps in the sequence are indicated byperiods, the conserved cysteine and glycine residues are framed.

2410

µm on a Leitz cryostat at −20°C. Sections were heated for 10 minutesat 65°C, washed in PBS, dried overnight and mounted with Mowiol(Harlow and Lane, 1988). Slides were photographed on a ZeissAxiovert using Agfa Ultra 50 film.

RESULTS

Isolation of Dll1Based on the assumption that patterns of gene expression canserve as criteria to define likely candidates for genes with reg-ulatory functions during embryogenesis, we screenedrandomly selected cDNA clones from a mouse day 10.5cDNA library for spatially restricted expression in mouseembryos (Bettenhausen and Gossler, unpublished data).Among novel mouse genes expressed in spatially regulatedpatterns, one clone was isolated, which detected low levels oftranscripts in the nervous system and, more strikingly,abundant mRNA in the tail bud paraxial mesoderm of day 10.5

mouse embryos. Database searches with approximately 200bp of the cDNA sequence suggested that this clone mightrepresent a homologue of the Drosophila Delta (Dl) gene.Additional cDNA clones were isolated by screening a day 8.5mouse embryo cDNA library and by 3′ RACE (see Materialand methods for details). A total of 14 overlapping cDNAclones were analyzed, confirming that these cDNAs representa mouse gene closely related to Drosophila Delta (see below).This mouse gene was therefore called delta-like gene 1 andassigned the gene symbol Dll1.

Sequence analysis of the Dll1 transcriptThe overlapping cDNA clones span 2856 bp and contain onelong open reading frame (ORF) of 2166 bp starting with anATG at position 14. This ORF represents very likely thecomplete coding sequence of Dll1 and encodes a protein of722 amino acids (Fig. 1) which is structurally similar to Dl(Fig. 2A). The N-terminal part begins with a stretch ofhydrophobic amino acids, probably representing a signal

B. Bettenhausen and others

..C...Y....C..FC...........C...G...C..GW.G..C

FVCDEHYYGEGCSVFCRPRDDAFGHFTCGDRGEKMCDPGWKGQYC

VTCARNYFGNRCENFCDAHLAKAARKRCDAMGRLRCDIGWMGPHC

VTCDLNYYGSGCAKFCRPRDDSFGHSTCSETGEIICLTGWQGDYCVQCAVTYYNTTCTTFCRPRDDQFGHYACGSEGQKLCLNGWQGVNC

LAG-2

Dll1

Dl SERRATE

DSL Consensus

Dll1 69Dl 75

Dll1 143Dl 149

Dll1 218Dl 224

Dll1 293Dl 297

Dll1 363Dl 372

Dll1 433Dl 444

Dll1 508Dl 519

Dll1 583Dl 590

Dll1 644Dl 665

Dll1 695Dl 740

Dll1 716Dl 815

Dll1 722Dl 832

AAAAAA

DSL

EGF1

EGF6

EGF2

EGF5

EGF4

EGF7

EGF8

EGF3

A

B

MGRRSALALAVVS-ALLCQVWSSGVFELKLQEFVNKKGLLGNRNCCRG---GSGPPC--ACRTFFRVCLKHYQAS MHWIKCLLTAFICFTVIVQVHSSGSFELRLKYFSNDHGRDNEGRCCSGESDGATGKCLGSCKTRFRVCLKHYQAT

VSPEPPCTYGSAVTPVLGVDSFSLPDGAGI-DPAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLI IDTTSQCTYGDVITPILGENSVNLTDAQRFQNKGFTNPIQFPFSFSWPGTFSLIVEAWH-DTNNSGNARTNKLLI

SRLTTQRHLTVGEEWSQDLHSSGRTDLRYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGDRGEKMCDPGWKGQ

YCTDPICLPGCDDQHGYCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTH YCHIPKCAKGCE--HGHCDKPNQCVCQLGWKGALCNECVLEPNCIHGTCNKPWTCICNEGWGGLYCNQDLNYCTN

HKPCRNGATCTNTGQGSYTCSCRPGYTGANCELEVDECAP--SPCKNGASCTD---LEDSFSCTCPPGFYGKVCE HRPCKNGGTCFNTGEGLYTCKCAPGYSGDDCENEIYSCDADVNPCQNGGTCIDEPHTKTGYKCHCANGWSGKMCE

LK--GDEATVRDTHSKRDTKCQSQSSAG----EEKIAPTLRGGEIPDRKRPESVYSTSKDTKYQSVYVLS----- QKQLNTDPTLMHRGSPAGSSAKGASGGGPGAAEGKRISVLGEGSYCSQRWPSLAAAGVAGACSSQLMAAASAAGT

----------------IKNTNKKADFHGDHG------------------------AKKSSFKVRYPTVDYNLVRDTGSNSGLTFDGGNPNIIKNTWDKSVNNICASAAAAAAAAAAADECLMYGGYVASVADNNNANSDFCVAPLQRAKS

PFPWVAVCAGVVLVLLLLLGCAAVVVCVRLKLQKHQPPPEPCGGETETMNNLANCQREKDVSVSIIGATQ----- TNAQVVLIAVFSVAMPLVAVIAACVVFCMKRKRKRAQEKDDAEARKQNEQNAVATMHHNGSGVGVALASASLGGK

GYTGKNCSAPVSRCEHAPCHNGATCHQRGQRYMCECAQGYGGPNCQFLLPEPPPGPMVVDLSE----RHMESQGG

------EKKMDLCGSSPCSNGAKCVDLGNSYLCRCQAGFSGRYCEDNVDDCASSPCANGGTCRDSVNDFSCTCPP

LSAMTCADGPCFNGGRCSDNPDG------GYTCHCPLGFSGFNC----------------------------- EKVLTCSDKPC-HQGICRNVRPGLGSKGQGYQCECPIGYSGPNCDLQLDNCSPNPCINGGSCQPSGKCICPAG

FSGTRCETNIDDCLGHQCENGGTCIDMVNQYRCQCVPGFHGTHCSSKVDLCLIRPCANGGTCLNLNNDYQCTCRA

GFTGKDCSVDIDECSSGPCHNGGTCMNRVNSFECVCANGFRGKQCD----EESYDSVTFDAHQYGATTQARADGL

---AEKDECVI--ATEVDGTAQQQRSVVCGTPHM

VTCDDHYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECJagged

QRLLVQQVLEVSSEWKTNKSESQYTSLEYDFRVTCDLNYYGSGCAKFCRPRDDSFGHSTCSETGEIICLTGWQGD

Fig. 3. Sequence alignment of theDll1 protein. (A) Sequence alignmentof the Dll1 and Drosophila Dlproteins. Identical amino acids areboxed and shaded, gaps that wereintroduced for the alignment areindicated by lines. The DSL domainsand EGF-like repeats shared by bothproteins are marked by a black barabove the alignment. Thehydrophobic stretches representingthe putative transmembrane domainsare indicated by a hatched bar. (B) Sequence alignment of the DSLdomains of Dll1, rat Jagged,Drosophila Delta and Serrate, and C.elegans lag-2. Identical amino acidsare indicated in bold, the consensussequence is given below thealignment.

2411Mouse delta-like gene

peptide. A second highly hydrophobic region characteristicfor membrane spanning regions is present in the C-terminalthird, suggesting that Dll1 is a transmembrane protein withan extracellular domain of approximately 545 amino acidsand a cytoplasmic portion of approximately 150 amino acids.Starting at residue 225 the extracellular domain containseight EGF-like repeats (shaded regions in Fig. 1). Thededuced Dll1 protein shares about 40% overall identity withDl and up to 70% identity in individual EGF-like repeats (Fig.3A). Additional regions of high homology are in the N-terminal region upstream the EGF-like repeats (Fig. 3B) andinclude the so-called DSL (Delta-Serrate-lag2) domain (Taxet al., 1994), which is required for binding of Delta andSerrate to Notch (Muskavitch, 1994), and which is alsopresent in Jagged, a rat ligand for Notch1 (Lindsell et al.,1995). The deduced Dll1 protein lacks one EGF-like repeatcompared to Drosophila Dl and is 110 amino acids shorter,which is largely accounted for by the shorter cytoplasmaticportion of the Dll1 protein.

The 3′ untranslated region of the Dll1 mRNA comprises 677bp and contains two polyadenylation signals, one 39 bp afterthe coding region, the other at the 3′ end followed 13 bp down-stream by a stretch of adenosine residues. These adenosineresidues probably correspond to the beginning of the poly(A)tail, suggesting that the 3′ end of the gene is included in thecDNA clones. The 3′ untranslated region contains severalcopies of a A(T)3A or A(T)>3A sequence, which has beenimplicated in the regulation of specific mRNA degradation(Shaw and Kamen, 1986; Wickens, 1990; Huarte et al., 1992),and is also present in the 3′ untranslated region of DrosophilaDl (Vässin et al., 1987).

Probes from Dll1 detected a single transcript of approxi-mately 3.8 kb in northern blothybridizations of poly(A)+

RNA from day 10.5 mouseembryos (Fig. 4A), suggest-ing that about 900 bp repre-senting the 5′ untranslatedregion and the poly(A) tail ofthe mature Dll1 message aremissing from the completemRNA sequence.

ChromosomallocalizationTo address whether Dll1might qualify as a candidategene for a known mousemutation affecting embryonicdevelopment, we determinedits chromosomal localization.The results of the analysisconducted with the DNAsamples of the EUCIBresource placed Dll1 at mapposition 8.5 on mouse Chr 17.Dll1 thus maps to the mouset-complex, which harborsvarious embryonic lethalmutations (Bennett, 1975;Artzt et al., 1982; Artzt,

1984). One of these, tw18, prevents somite formation and leadsto embryonic death around day 9 of development (Bennett andDunn, 1960; Moser and Gluecksohn-Waelsch, 1967), and isassociated with a deletion in the distal inversion of the t-complex (Búcan et al., 1987). Because Dll1 expression isclosely correlated with somitogenesis (see below), we analyzedby Southern blot hybridizations whether there are detectablealterations of Dll1 in DNA samples prepared from +/tw18 mice.Dll1 was present in tw18 DNA and, on this level of analysis,indistinguishable from other t-haplotype DNA (Fig. 4D). Thisindicates that Dll1 does not map into the tw18 deletion andexcludes Dll1 as a candidate gene for the tw18-associatedmutation. Further mapping will be required to qualify orexclude Dll1 as a candidate for any of the other t-associatedembryonic mutations.

Expression of Dll1The temporal expression profile of Dll1 during embryogenesisand the distribution of transcripts in adult tissues were analyzedby RT-PCR (Fig. 4B). Dll1 mRNA is present at least as earlyas on day 8.5 of development and persists at least until day12.5. Transcripts were no longer detected in day 15.5 and 18.5fetuses. In the adult tissues examined, high levels ofDll1expression were detected in lung, and low levels were foundin the heart. Thus, Dll1 is transiently expressed during embryo-genesis and expression is reactivated after birth in a tissue-specific manner.

The spatial pattern of Dll1 expression during gastrulationand early organogenesis was determined by whole-mount insitu hybridization of embryos between day 6 and 12.5 of devel-opment and by examination of sectionend whole-mountstained embryos. Dll1 transcripts were first detected in

Fig. 4. Expression analysis of Dll1 during embryogenesis and in adult tissues, and deletion mapping oftw18 DNA. (A) Northern blot with poly(A)+ RNA from day 10. 5 p. c. embryos hybridized with a probecovering 1.1 kb of the Dll1 coding sequence. (B) RT-PCR analysis of poly(A)+ RNA from adult tissuesand from day 8.5, 10.5, 12.5, 15.5 and 18.5 mouse embryos. (C) Control RT-PCR with β-actin. (D)Southern blot analysis of homozygous t-haplotype (tw5g/tw5g), heterozygous tw18 (T qk tf / tw18) and wild-type (T qk tf / + +) genomic DNA restricted with BglII and TaqI, respectively. tw18 DNA shows therestriction fragments indicative of wild-type and t-haplotype DNA, demonstrating that Dll1 is not deletedor altered in tw18.

2412 B. Bettenhausen and others

2413Mouse delta-like gene

midstreak embryos in the embryonic mesoderm and primitivestreak (Fig. 5A). In late streak embryos, expression wasconfined to the posterior mesoderm and extended to the distaltip of the egg cylinder (Fig. 5A). During subsequent develop-mental stages, Dll1 expression remained restricted to theposterior mesoderm. The anterior expression boundary

sharpened and was consistently found just anterior to the node(Fig. 5B,C). Transcripts accumulated just posterior to thisborder resulting in characteristic stripes of Dll1-expressingcells perpendicular to the anterior-posterior axis on both sidesof the node. Lower levels of transcripts were detected posteriorto these stripes. No expression was detected in the node itself

Fig. 6. Dll1 expression in day 9 and 9.5 embryosas detected by whole-mount in situ hybridization.(A,B) Lateral, (C,D) frontal views of a day 9(A,C) and 9.5 (B,D) embryo showing details ofDll1 expression in the nervous system. In day 9embryos, Dll1 is expressed in the presumptiveforebrain (small arrow in A), midbrain (largearrow in A and C) and ectodermal placodes(arrowheads in A and B) and cervical spinal cord.Note that not all cells of these regions expressDll1. In day 9.5 embryos, Dll1 is expressedthroughout the central nervous system, the opticand otic vesicles, the ectodermal cells of thelateral nasal process (large arrow in B), and incells of the ectodermal placodes overlying thedeveloping cranial ganglia VIII-X (arrowheads inB). OT, otic vesicle. Bars, 80 µm (A,B,D); 50 µm(C).

Fig. 5. Dll1 expression between day 7 and 12.5 as detected bywhole-mount in situ hybridization. (A) Early-, mid- and late-streakembryos, lateral view. Expression is first detected in mid-streakembryos in the embryonic mesoderm (arrow) and in late-streakembryos confined to the posterior mesoderm. (B) Early neural foldstage embryo, posterior view. Expression is in the posteriormesoderm and primitive streak. Note absence of Dll1 transcriptsfrom the node (arrowhead). (C) Late neural fold stage embryo,ventral view. Dll1 expression in the mesoderm displays a sharpborder of expression just anterior to the node. (D) 5-somiteembryo, dorsal view. Dll1 expression is in the presomiticmesoderm, caudal halves of the condensed somites and in theneuroepithelium of the presumptive midbrain region.(E) 10-somite

embryo, dorsal view. Dll1 expression is in the presomiticmesoderm, in caudal halves of the condensed somites and in groupsof cells in the neuroepithelium of the presumptive midbrain region.(F) Day 9 embryo, lateral view. Expression is as in E withadditional strong expression in the forebrain, ectodermal placodesand spinal cord. (G) Day 9.5 embryo, lateral view. Dll1 isexpressed throughout the central nervous system, in the presomiticmesoderm, and in the posterior halves of somites. (H) Day 10.5embryo, lateral view. Dll1 expression in the presomitc mesodermin the tail tip, brain vesicles and spinal cord. (H). Day 12.5 embryo,lateral view. Dll1 expression is confined to the tail tip of theembryo. Bars, 30 µm (A,B); 50 µm (C); 80 µm (E,D); 100 µm(F,G); 150 µm (H); 200 µm (I).

2414

or in the axial mesoderm anterior to the node (Fig. 5B,C).When the first somites were formed, the stripe of strong Dll1expression was always in the anterior presomitic mesodermand appeared to mark the nascent somites (Fig. 5E, and datanot shown). This apparent correlation was maintained untilapproximately day 9.5. Thereafter, all cells in the presomiticmesoderm expressed Dll1 at similar levels (Fig. 6 and data notshown). In the condensed somites, Dll1 expression was down-regulated and lower levels of Dll1 transcripts were restrictedto the posterior halves (Figs 5D,E, 6A, 7A-C, 8A). Initially, all condensed somitesexpressed Dll1 in the caudal halves.However, in embryos with more than 12somites, Dll1 expression was detected onlyin the 12-14 posteriormost somites (Figs5G,H, 6). During subsequent development,successively fewer of the condensedsomites concomitantly expressed Dll1 intheir caudal halves and, around day 12.5,Dll1 transcripts were found exclusively inthe very tip of the tail (Fig. 5I). In slightlyolder embryos, Dll1 expression was nolonger detected (data not shown).

Expression in mesodermal cells was notexclusively in the paraxial mesoderm. Inday 9.5 embryos with approximately 12-14somites, Dll1 expression was detected in themesonephric mesoderm and, in day 10.5embryos, in the mesonephric tubules (Fig.8C and data not shown). In day 10 embryos,low levels of Dll1 transcripts were detectedalso in myotome cells of all somites anteriorto the hindlimb buds (Fig. 8B).

In the central nervous system (CNS),Dll1 expression was first detected inembryos on day 8 of development. Dll1expression levels in the CNS increased untilday 9.5 of development and thereafterdecreased again. Dll1 transcripts were nolonger detected in the nervous system ofday 12.5 embryos. In day 8 embryos with 6to 8 somites, Dll1 was expressed in the neu-roepithelium of the presumptive midbrainand forebrain (Fig. 5D,E, and data notshown). On day 9, additional expressionwas found in the neuroectoderm of thehindbrain and in scattered cells of thesurface ectoderm of the head. On day 9.5,strong expression of Dll1 was foundthroughout the entire neural tube withexception of the posterior neuropore (Figs5G, 6B,D, 7A,C). Strong expression wasalso detected in the optic and otic vesicles,in scattered cells of the nasal placode and inthe ectodermal placodes overlaying thedeveloping craniofacial ganglia VIII-X(Fig. 6B). In day 10.5 embryos, Dll1 wasexpressed in a very similar pattern, althoughat lower levels. Additional Dll1-expressingcells were found in the neural crest anddorsal root ganglia (Figs 7D, 8C, and data

not shown). Cells in the posterior part of the neural tube didnot express detectable levels of Dll1 (data not shown).

Expression in the neuroectoderm consisted of a fine-grainedpattern of Dll1 expressing and non-expressing cells (Figs7C,D, 8). Cells expressing Dll1 at high levels were concen-trated in the dorsal region of the spinal cord and were foundmore frequently in the outer than in the ependymal layer (Fig.8, and data not shown). The distribution of Dll1 expressingcells differed along the anterior-posterior axis. In the brain

B. Bettenhausen and others

Fig. 7. Dll1 expression in day 9.5 and 10.5 embryos as detected by whole-mount in situhybridization. (A,B) Dorsal views of a day 9.5 embryo; (C,D) dorsolateral view of a day10.5 embryo, showing Dll1 expression in the paraxial mesoderm and nervous system.Dll1 expression in the condensed somites is confined to the caudal compartments(arrowheads in A-C) and in day 10.5 embryos detected only in the posteriormost 5somites (B). Expression in the nervous system is not homogenous but restricted toindividual cells or groups of cells (arrows in B and C, large arrow in D) and also found inthe dorsal root ganglia (small arrow in D). Arrowhead on D indicates Dll1 expression inthe myotome (compare also Fig. 8). Bars, 100 µm (A,D); 80 µm (B,C).

2415Mouse delta-like gene

vesicles and the cervical portion of the spinal cord, Dll1-expressing cells were restricted to the dorsal region of the neu-roectoderm whereas in the trunk Dll1-expressing cells werealso present in the ventral part (data not shown).

DISCUSSION

We have isolated a mouse gene, Dll1, whose deduced aminoacid sequence shows significant homology to the DrosophilaDelta protein (Kopczynski et al., 1988; Haenlin et al., 1990).The homologous regions include the EGF-like repeats and theso-called DSL (Delta-Serrate-lag-2) domain, a variant of theEGF repeat that is required for binding of Delta and Serrate toNotch (Muskavitch, 1994). Since this domain is missing inother mammalian genes with sequence homology toDrosophila Delta (Laborda et al., 1993; Smas and Sul, 1993),Dll1 presently represents the mammalian gene most closelyrelated to Dl. In Dll1, the overall structure and all regionsknown to be of importance for Dl function during fly devel-opment are well conserved, suggesting that the Dll1 protein isa potential ligand for mammalian Notch protein(s) and that itmight be involved in mediating cell-to-cell communicationduring development, similar to Jagged, which is closely relatedto Drosophila Serrate (Lindsell et al., 1995).

Dll1 is transiently expressed during gastrulation and earlyorganogenesis in a spatially and tissue-restricted manner con-sistent with functions of Dll1 in mesoderm development, insomitogenesis and in the nervous system. Dll1 expression wasnot found in the embryonic ectoderm or neuroectoderm ofearly and mid-streak embryos and was first detected exclu-sively in the embryonicmesoderm and primitivestreak. The absence of Dll1transcripts from ectodermalcells at these stages arguesagainst a role of Dll1 in thespecification of neuroectoder-mal cells, which is in contrastto the pivotal function of theDrosophila Delta gene forneurogenesis (Campos-Ortega, 1993). Expression ofDll1 in the neuroectodermwas first detected in head-fold-stage embryos in the pre-sumptive midbrain region andhigh levels of Dll1 transcriptsdid accumulate in the CNSonly after day 9 p.c. Individ-ual cells or small cell clustersexpressing Dll1 were initiallyfound in the dorsal and ventralregions of the neural tube, butwere later virtually absentfrom the ventral neural tube inits anterior region. Duringneural tube development,signals from the notochord,the ventral midline and thedorsal region of the neural

tube regulate the differentiation of distinct cell types along thedorsoventral axis (Placzek et al., 1991; Yamada et al., 1991,1993; Basler et al., 1993). Dll1 expression appears to reflectthe dorsoventral patterning of the neural tube, suggesting thatit might be implicated in this early regional specification andthat its expression might become restricted to the dorsal regionas a consequence of repression in ventral cells or specific acti-vation in dorsal cells in response to either ventralizing or dor-salizing signals.

The most conspicuous expression of Dll1 was observed inmesodermal cells and was closely correlated to somitogenesis.This subdivision of the paraxial mesoderm into a metamericseries of epithelial blocks of cells along the anterior-posterioraxis begins during gastrulation and progresses in ananterior-posterior sequence, while concomitantly newmesoderm cells are being generated caudally from theprimitive streak and later from the tail bud (Hogan et al., 1985;Rugh, 1990). Dll1 transcripts were first detected in mid-streakembryos in the whole embryonic mesoderm, but when themesoderm extended more anteriorly and laterally, Dll1expression remained restricted to the posterior (presumptivepresomitic) mesoderm with a sharp expression border justanterior to the node. Subsequently, bilateral bands of cellsapparently corresponding to the nascent somites at the anteriorend of the presomitic mesoderm accumulated high levels ofDll1 transcripts. This region of strong Dll1 expression at theanterior end of the presomitic mesoderm just posterior to themost recently formed somite was maintained as the successivesomites were formed. The association of Dll1 expression withthe nascent somites suggests that Dll1 is implicated in cellularinteractions that lead to the condensation of the epithelial

Fig. 8. Dll1 expression in day 10.5 embryos as detected in sections after whole-mount in situhybridization. (A) Dll1 expression in the caudal halves of somites and in individual cells in the neuraltube. (B) Dll1 expression in myotome cells (arrowheads) and spinal cord. (C) Dll1 expression in themesonephric tubules (arrowheads), spinal cord and dorsal root ganglia (arrows). Note absence of Dll1-expressing cells from the ventral region of the spinal cord. Bar, 8 µm.

2416

somites from the mesenchymal presomitic mesoderm. Inanalogy to the requirement of the Drosophila Delta gene forepithelial-mesenchymal transitions (Hartenstein et al., 1992;Tepass and Hartenstein, 1995), the role of Dll1 during somiteformation could be to promote the epithelial state of somitecells, potentially by changing their adhesive properties. In thecondensed somites Dll1 expression was down regulated, andlower levels of mRNA were detected only in the caudal halves.Dll1 expression thus appears to reflect the compartmentaliza-tion of somites into the functionally different cranial andcaudal halves (Keynes and Stern, 1984; Stern and Keynes,1986; Davies et al., 1990; Goldstein et al., 1990), suggestingthat Dll1 might play a role in the specification of the caudalsomite compartment and in establishing different properties insomitic cells during subsequent differentiation.

In analogy to Drosophila, the Dll1 protein is a likelycandidate for a ligand for mammalian Notch protein(s). Directinteraction between membrane-bound Dll1 and Notch proteinsrequires concurrent expression of both proteins in the same orin neighboring cells. At present, the distribution of anymammalian Notch or of the Dll1 protein is not known.However, the expression patterns of Dll1 and Notch1, the onlymammalian Notch gene whose expression in early embryos hasbeen reported in detail thus far (del Amo et al., 1992; Reaumeet al., 1992), are strikingly similar during gastrulation and earlyorganogenesis, strongly supporting the possibility that the Dll1and Notch1 proteins can directly interact. The similarities ofthe temporal and spatial expression patterns of Dll1 and Notch1is particularly striking in the paraxial mesoderm. Expression ofboth genes was detected first in mid streak mouse embryos inthe embryonic mesoderm and primitive streak. Subsequently,both genes are expressed in the posterior mesoderm and a sharpborder of expression is established just anterior to the nodefollowed by a band of cells accumulating transcripts of bothgenes. During early somitogenesis, high levels of transcripts ofboth genes are accumulating in the condensing somites andlater in the whole presomitic mesoderm. Similarly, Dll1 andNotch1 are coexpressed in the nervous system, ectodermalplacodes and the mesonephric kidney. Assuming that cellsexpressing these genes also contain the functional proteins,direct interaction of the Dll1 and Notch1 proteins couldmediate cell-to-cell communication in these tissues. The available expression data suggest that many if not all cellsin the embryonic and presomitic mesoderm express both genes.This appears to be different in the condensed and differentiat-ing somites and in the nervous system. Notch1 expression inthe neural tube was reported in the ventricular zone (Wein-master et al., 1991; del Amo et al., 1992; Reaume et al., 1992),whereas our data show that most but not all Dll1-expressingcells are in the outer layer (Fig. 8). In the differentiatingsomites, Notch1 transcripts were found in the sclerotome butnot in the dermatome and myotome (del Amo et al., 1993)whereas Dll1 transcripts were detected in the myotome butwere absent from the other somite-derived cell types. Furtheranalysis will be required to determine unambiguously whichcells coexpress both genes and when and where cells exclu-sively expressing either gene are in contact. In addition, theexpression of the other Notch genes during early embryogen-esis has to be analyzed in more detail to determine whether theDll1 protein could also interact with their gene products. Thevarious mouse Notch genes are expressed from at least as early

as day 8.5 onwards throughout embryogenesis and in manyadult tissues (Weinmaster et al., 1991; del Amo et al., 1992;Reaume et al., 1992; Lardelli and Lendahl, 1993). In contrast,Dll1 is transiently expressed during embryogenesis and wasonly detected in adult lung and heart (Fig 4B). This suggeststhat, whereas Notch function appears to be required through-out development and in many adult tissues, Dll1 has specificfunctions during gastrulation and early organogenesis, and ina subset of Notch-expressing adult tissues, and implies thatthere are other ligands that interact with the Notch proteins atlater stages of development and in most tissues of the adultorganism. Good candidates for such ligands, besides theJagged gene (Lindsell et al., 1995), are a second mouse Deltaand a Serrate homologue, which have been isolated (DomingosHenrique, personal communication).

The existence of several Delta and Notch genes in mouseraises the possibility of combinatorial interactions among theDelta and Notch proteins during mammalian development.Further biochemical and genetic analysis will be required toprove that mammalian Delta and Notch proteins directlyinteract, to clarify whether the different Delta and Notch proteinsare functionally interchangeable, to determine whether interac-tion between particular Delta and Notch proteins leads tospecific responses in the communicating cells and to elucidatethe biological functions of these proteins during embryogenesis.

We thank Dr Domingos Henrique for communicating unpublishedresults, Dr Jörg Sprengel for his help with the sequence analysis, DrBrigid Hogan for the gift of the day 8.5 cDNA library and Dr KarenArtzt for the various t-haplotype DNAs and Dr José Campos-Ortegafor helpful discussions and critical comments. This work wassupported by the Minister of Research and Technology (BMFT),Germany, and The Jackson Laboratory program award 95-09. M.HdA. is supported by a fellowship of the Deutsche Forschungsge-meinschaft. This work was facilitated by the microchemistry serviceof The Jackson Laboratory (NIH core grant CA 34196).

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(Accepted 8 May 1995)

B. Bettenhausen and others