The Drosophila melanogaster l(2)gl gene encodes a protein homologous to the cadherin cell-adhesion molecule family

DEVELOPMENTAL

BIOLOGY

133,425-436

(1989)

The Drosophila meknogaster /(Z)gl Gene Encodes a Protein Homologous to the Cadherin Cell-Adhesion Molecule Family CHRISTIAN

KLAMBT,' STEPHAN MULLER,REINHARD RITA

Institut

fiir

Biologic

III,

Universittit

ROSSA,

FRANK

Freiburg,

TOTZKE,

Schiinxlestrasse

Accepted

December

AND

OTTO

1, D-7800

L~~TZELSCHWAB,' SCHMIDT

Freiburg,

Federal

Republic

of Germany

19, 1988

Mutations in the recessive Drosophila tumor gene 1(2))81 affect growth and structural properties of neural tissues and imaginal discs during larval development. We have analyzed the cellular localization of transcripts and a 130-kDa protein encoded by the l(Z))91 gene, using ill situ hybridization and immunofluorescence techniques. Transcripts of maternal origin are detected in freshly laid eggs and are homogeneously incorporated into blastoderm cells. The protein is found at low levels in all embryonic tissues after blastoderm formation. In later stages differential expression of the protein is observed, particularly in cells of the nervous system. The protein is located at the cell surface of dissociated embryonic cells. Anti-l(Z)gl sera show cross-reaction to a mouse protein that is localized at cell-cell contact sites in tissue culture cells. Moreover, amino acid sequence homology to deduced amino acid sequences of members of the vertebrate cadherin cell-adhesion molecule family suggests that the l(2)gl gene product may have properties of a cell-adhesion molecule. (0 1989 Academic Press, I~C. INTRODUCTION

Recessive mutations at the l(2)gl gene locus affect the developmental transition of embryonic cells toward a differentiated state (Hadorn, 1955; Gateff and Schneiderman, 1974; Gateff, 1978). In homozygous mutant Z(2)gZ tissues no obvious morphological anomalies are observed during embryogenesis and most of the larval development. Phenotypic effects of the mutation are visible at the end of third instar, when mutant larvae invariably fail to metamorphose and some imaginal primordia, such as larval brain hemispheres and imaginal discs, continue to grow in a disorganized, unstructured fashion. Within the mutant brain, neuroblasts of the presumptive optic centers fail to differentiate into neurons. Instead, both neuroblasts and ganglion mother cells accumulate, causing enlargement of the brain hemispheres (Gateff and Schneiderman, 1974). The l(2)gl gene has recently been cloned (Mechler et al., 1985). Two transcripts, 5.5 and 4.3 kb in length, are expressed during embryonic development and in thirdinstar larvae (Mechler et al., 1985). Both transcripts are absent in homozygous mutant Z(Z)glDV275 larvae (Ltitzelschwab et al., 1986). Sequence analysis of an almostfull-length wild-type cDNA clone representing the 5.5-

1 Present address: Institut fur Entwicklungsphysiologie, sitat KBln, Gyrhofstrasse 1’7, D-5000 Cologne 41, FRG. a Present address: Max-Planck Institut fur Biochemie, ferspitz 18a, D-8033 Martinsried, FRG.

UniverAm Klop-

425

kb transcript revealed an open reading frame of 3479 bp. A deduced protein product of 1161 or 1114 amino acids, depending on the initiation signal chosen, amounts to a molecular weight of about 130 kDa (Ltitzelschwab et a,l., 1987). In addition, a 78-kDa protein has been postulated from a cDNA fragment which corresponds to a differentially spliced transcript (Jacob et al., 1987). Western blot experiments using Z(2)gZ-specific antibodies directed against fusion proteins revealed a 130-kDa protein (Klambt and Schmidt, 1986; LiitzelSchwab et al., 1987). Here we present evidence that the l(2)gl protein is located on the surface of embryonic cells that have ceased to divide or are in the process of stopping cell division. Moreover, direct amino acid sequence homology to the extracellular domain of members of the family of cadherin cell-adhesion molecules suggests that the l(2)gl protein has properties of a cell-adhesion molecule. RESULTS

Distribution

of Transcripts

in the Embryo

Transcripts are detected in freshly laid eggs by in situ hybridization on embryo sections, using a radioactive labeled cDNA fragment as a probe (Fig. 1). During nuclear migration most of the label is accumulated at the cortex of the egg, where it is incorporated into the blastoderm cells (Fig. Id). Transcripts are homogeneously distributed in all tissues. The amount of transcript is increased until mid-embryogenesis due to zygotic ex0012-1606189$3.00 Copyright All rights

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

426

FIG. 1. Localization of l(Z)gl transcripts in early embryos. Bright-field (left) and corresponding after autoradiography. Orientation of embryos is anterior right. (a) Stage 1 embryo (O-O.25 hr); embryo (1.05-1.20 hr); (d) stage 4 embryo (1.20-2.10 hr).

pression of the l(Z)gl gene. At this stage differential amounts of transcripts are detected in parts of the nervous system (not shown). The l(2)gl Gene Encodes a B’O-kDa

Protein

To raise antibodies against the amino acid sequence, which can be predicted from the pE7-9 cDNA sequence (Liitzelschwab et al., 1987), we expressed subfragments containing parts of the open reading frame in bacteria and used the corresponding fusion proteins for immunization (see Materials and Methods). Three antisera from different fusion proteins were tested on Western blots with protein extracts from wild-type and homozygous mutant l(.)gl brains. These antisera recognize a 130-kDa wild-type protein that is absent in protein extracts from homozygous l(2)gl tissues (Fig. 2). The l(2)gl Protein

Is Located on the Cell Surface

The localization of the l(2)gl protein in tissue sections suggests that it is associated with the cellular membranes or intercellular space (KlZmbt and Schmidt,

dark-field (right) (b) stage 2 embryo

micrographs were taken (0.25-1.05 hr); (c) stage 3

1986). To test the extracellular localization of the l(2)gl protein experimentally, we performed immunostaining of dissociated viable embryonic cells, using purified antiserum H (see Materials and Methods). This experiment shows that the l(2)gl protein is localized on the surface of cells (Fig. 3~). Staining of single cells reveals a patchy pattern, as observed for many membranebound proteins (Bourguignon and Bourguignon, 1984). Attempts to remove the protein from isolated membranes by salt washes were not successful (Kltimbt, unpublished results), suggesting that the l(2)gl protein is fixed to the cell surface. If the dissociated cells are kept at high density (3 X 106/ml Kc medium under vigorous agitation for 1 hr), they begin to aggregate and eventually form clumps that increase in size as aggregation continues. During the formation of such cell aggregates the fluorescence signal changes from a patchy distribution to an intense signal at the contact sites of cells (Fig. 3e). Thus the l(2)gl protein seems to be readily mobile in the plane of the membrane. The patchy appearance of the l(2)gl protein on single cells was reduced when experiments were performed at 4°C instead of 20°C probably due to a reduced membrane fluidity at 4°C. Similar

KL;~MBT W lgl

wt

H lgl

ET AL.

D. melanogasfer

P

wt

lgl

wt

-

180

-

116

-

84

-

58

-

48.5

-

36.5

-

26.5

c

FIG. 2. Western blot of Drosophila proteins treated with anti-l(Z)ol sera. Protein extracts (25 pg) of wild-type larval brain (lane wt) and homozygous mutant l(2)gl brain tissue (lane lgl) were separated on SDS-PAGE 10% gel, transferred onto nitrocellulose, and incubated with purified antisera J, W, and H (see Fig. 5). Bound antibodies were visualized by alkaline phosphatase reaction. Molecular weight markers are /3-galactosidase (116 kDa), fructose-6-phosphate kinase (84 kDa), fumarase (48.5 kDa), and lactate dehydrogenase (36.5 kDa).

results were obtained using purified l(.)gl-specific antisera from other fusion proteins (data not shown). This confirms suggestive evidence from the deduced amino acid sequence (Liitzelschwab et al., 1987) that the l(??)gl protein is located at the surface of embryonic cells, where it is fixed either to the cellular membrane or to the extracellular matrix in a still unknown way. The 13%kDa Protein Nerve Cells

Is Localized

on Embryonic

Until gastrulation the 130-kDa protein is distributed in low amounts in almost every tissue of the Drosophila embryo (Klambt and Schmidt, 1986). Toward mid-embryogenesis, different amounts of protein are detected in some tissues, particularly in pole cells, which appear to express more protein compared to somatic cells (data not shown). At later stages, relatively high amounts of l(.)gl protein are detected in neuroblasts of the presumptive optic lobes within the developing supraesophageal ganglion and in the embryonic neuropil (Klambt and Schmidt, 1986). The occurrence of the protein at axoplasmic membranes of the neuropil during embry-

l(2)gl

427

Gene

onic development is restricted to axogenesis. Since much less label can be observed in larval or adult neuropil, it is of particular interest to investigate the localization of the l(2)gl protein at a cellular level during embryonic development, stages 12-13 of Drosophila during which axogenesis begins, extending until stage and Harten17 (Thomas et al., 1984; Campos-Ortega stein, 1985). Figure 4 shows sections of a stage 13 embryo, immunostained with antiserum H. Axon bundles of the peripheral nervous system (PNS) are known to be located at the segmental boundaries, extending into neighboring tissues (Thomas et al., 1984; Campos-Ortega and Hartenstein, 1985). The pattern of labeled cells at this stage coincides with the distribution of axonforming cells, suggesting that l(2)gl protein is expressed in relatively high amounts in differentiating nerve cells. Thus labeling of neuropil structures, as observed in older embryos (Klambt and Schmidt, 1986), reflects increased l(2)gl protein expression in nerve cells undergoing axogenesis, rather than accumulation of axoplasmic membranes. As l(2)gl protein is present in high amounts at nerve cells during axogenesis, we asked whether we could observe any defect or irregularities in the PNS in homozygous mutant embryos. Whole mounts of stage 14 to 16 embryos derived from a heterozygous l(Z)glDV275 strain were examined, using immunofluorescence with the monoclonal antibody 22ClO that specifically stains the developing PNS (Zipursky et al., 1984). Although 25% of these embryos are homozygous mutants, all embryos examined (about 50) showed a normal PNS organization (data not shown). This suggests that either the l(2)gl protein is not essential for correct development of these cells or that enough functional protein is provided by maternally contributed gene products. Structural Hom,ologies Adhesion Molecules

to Cadherin

Cell-

Analysis of predicted l(2)gl protein sequences revealed several small repeated units (Ltitzelschwab et al., 1987). On the basis of internal structural similarities, the deduced l(Z)91 protein sequence can be subdivided into four regions termed A, B, C, and D (Fig. 5). These regions also show sequence homology to the deduced sequences of members of the family of vertebrate cadherin adhesion molecules (chicken L-CAM: Gallin et al., 1987; mouse uvomorulin: Ringwald et al., 1987; mouse P-cadherin: Nose et al., 1987). In Fig. 5 only L-CAM is depicted for structural comparison. Region A shows a characteristic spacing of three N-glycosylation sites which, together with additional amino acid homologies, define a repeated sequence about 60 amino acids long. It is found in three copies (“glycosylation site repeats”)

428

FIG. 3. Localization immunofluorescence shown on the right. localization of label.

of the l@)gl protein on embryonic cell surfaces. The using purified serum H. Immunofluorescence pictures are (a, b) Controls, with only secondary antibodies applied. (e, f) Reaggregated cells. The l(Z)gl protein accumulates at

1(2)g/ protein is detected on dissociated Drosophila cells by on the left; the corresponding bright-field photographs are (c. d) Single embryonic cell stained, showing slight patchy cell-cell contact sites. Bar represents 10 pm.

FIG. 4. Expression of l(Z)gl protein on nerve cells. Localization of the l(.)gl protein on growing nerves in stage 13 embryos using purified serum H. (a) Immunofluorescence on a l&pm tissue section showing an unidentified nerve cell at axogenesis. (b, c and d, e) show labeling of neurons belonging to the peripheral nervous system in two serial 16-rrn tissue sections. The fixation conditions chosen only allow detection of cells containing relatively large amounts of protein.

KLAMBT

ET AL

D. melanogaster

l(2)gl

429

Gene

L-CAM

PTH6

P

PTB5

H

PTH2

W

PTBal

J

FIG. 5. l(2)gZ and L-CAM protein structure. Schematic drawing of the structural organization of similar protein regions in the L-CAM as an example for a cadherin molecule and the l(2)gl protein. The L-CAM protein (Gallin et aZ., 1987) spans the membrane (TM domain) and has a 16-kDa intercellular domain (i). The l(2)gl protein exhibits no transmembrane domain (Ltitzelschwab et al., 1987) and is attached to the membrane and/or the extracellular matrix by an unknown mechanism. Four regions conserved between L-CAM and l(2)gl amino acid sequences are indicated, A and B are repeats containing putative N-glycosylation sites. B is a modified A-type region. C and D represent smaller regions containing negatively and positively charged amino acids, respectively. The amino acid homology between L-CAM and 1(2)gl sequences is shown in Fig. 6. P, H, W, and J denote the antisera raised against the protein regions indicated. Abbreviations: o, intercellular space; TM, transmembrane domain.

within the l(2)gl sequence (Fig. 6A). Repeats lglA2 and lglA3 are contiguous and located at the C-terminal end of the protein, whereas lglA1 is located at a more interior position. The l(2)gl repeat lglA3 shows sequence homology to the L-CAM sequence, with 17 of 60 amino acids identical and 6 amino acids representing homologous exchanges. This amounts to an overall homology of 38%. Two putative N-glycosylation sites are located at conserved positions in the L-CAM sequence and one each in uvomorulin and P-cadherin, which have fewer glycosylation sites (heavily lined boxes in Fig. 6). Region B of the l(2)gl protein sequence can be aligned over 140 amino acids with cadherin sequences and shows an overall homology of 39% when compared to L-CAM (Fig. 6B, 31 identical amino acids and 24 homologous exchanges). Two cysteine residues are conserved, and all putative N-glycosylation sites are located at conserved positions (heavily lined boxes in Fig. 6B). Additional repeated amino acid sequences are observed in the middle of the l(2)gl protein, termed regions Cl and C2 in Fig. 5. Both are direct repeats of 70 amino acids and show a high content of negatively charged amino acids (about 22%). Of 29 negatively charged amino acids, 22 are located at conserved positions in both repeats. A homologous region found in the L-CAM sequence has an overall homology of about 33% (12 identical and 11 homologous positions in 70 amino acids) when L-CAM sequencesare compared to the l(2)gl repeat C2 (Fig. 6C). Similar homology exists to uvomorulin and P-cadherin. Two regions marked a and b in

Fig. 6C might be similar to sequences that have been suggested for Ca”’ binding (Ringwald et al., 1987). Two regions, Dl and D2, are enriched in positively charged amino acids. A region homologous to Dl is also found in the amino acid sequences of the cadherin adhesion molecule family. The overall homology of Dl to L-CAM is 35%, with 7 identical and 9 homologous amino acids out of 45 (Fig. 6D). In summary, four distinct regions of the l(2)gl protein contain sequence homology to more than half of the extracellular domains of the members of the cadherin adhesion molecule family. This homology supports the hypothesis that the two types of molecules have similar biological functions. Anti-l(2)gl Antibodies Cross-React with Vertebrate Proteins l(2)gl gene sequences are detected in other Drosophila species by Southern blot analysis. However, we could not observe significant cross hybridization to mouse or human genomic DNA even under low-stringency conditions (G. Scherer, D. Eik, and 0. Schmidt, data not shown). To determine conserved regions within the l(2)gl protein, we analyzed Western blots containing protein extracts derived from several Drosophila species (D. virilis, D. hydeii, D. pseudoobscura, D. pinicola, D. meycatoria, and D. novamexicana). Antisera directed against different parts of the protein were tested and the degree of cross-reaction toward a 130-kDa-like pro-

430

DEVELOPMENTAL

A

BIOLOGY

VOLUME

133,1989

B

EARPVERQIKAP

lgll

lgl

Y

1g12

L-CAN

DEV-

"vol.

FTR

AEH..

P-cad.

.NA

G In.

A--uV.E.R--.T.....Hu

lgll

CLL

FAKT

lg12

-QQ

VN-G

1913

TNKA

L-CAM

IIE-

-I

V. P-cad.

.E.

f

YIP T-

.A. . A .

VVQ-

I -IF -v.

1gl

GSPS-

L-an

-SFEI

-E.

““Drn

-NHQF

.

P-cad.

.I.".r";.lV

-NYQF El

-I

P Q '4I.m.lTl.l.l.

.IS

u-u-

I

lgll lgl2

1g1

lg13

-[SIC

-1 SLAVSRGD

L-CAM

L-CAM

uvom.

""ml.

P-cad.

.d

P-cad

[.lN

S SI.mQl.

T

$.

EAW

C

1g1

a

L-an uvom .

lg11

c

SVY

lg12

T

FKT

L-CAM

H

DPW

P-cad.

.

P-cad.

TH.lS$.

+uQ

D TUD

L itwS

YSNISYPITC EIL 1g12

DAAADN

L-CAM

--PVFI

HTV

""ox".

--.E.T

.K.

P-cad.

- - . K . T

. Q .

Ll

LFD

1PESF -VQI

b -N.

FIG. 6. Amino acid homology between Z(2)gl and cadherins. sequences are indicated with L-CAM, uvomorulin with uvom.

L

The l(Z)gl sequences are drawn in the upper line(s) and marked lgl. L-CAM , and P-cadherin with P-cad. In uvomorulin and P-cadherin sequences, amino

KLAMBT

ET AL,.

D. melanogaster

tein in other Drosophila species was used as an indication for the degree of conserved amino acid sequences. The amount of cross-reaction observed was high in serum H and reduced in the order W, P, J (see Materials and Methods and Fig. 5). To look for cross-reactive proteins in vertebrates, we performed immunofluorescence on murine embryonic tissue culture cells using purified serum H. No immunofluorescence signal was observed in either undifferentiated or differentiated mouse F9 embryonic carcinoma cells (Bernstine et al., 1973). In contrast, the mouse embryonic carcinoma cell line PCC4 (Nicolas et al., 1975) showed immunofluorescence at cell-cell contact sites (Fig. 7). Only purified serum H and not the preserum showed a signal. The label at the cell-cell contact sites resembles the immunofluorescence signal found on aggregates forming from dissociated embryonic Drosophila cells (Figs. 3c, e) and suggests that l(2)gl homologous proteins may exist in vertebrates as well. DISCUSSION

In situ hybridization on sections of early embryos revealed a maternal contribution of l(2)gl transcripts. Its homogeneous distribution in blastoderm cells could account for the low level of l(2)gl protein present in most of the tissues during early embryogenesis. After gastrulation, differing amounts of the protein are detected in embryonic tissues, presumably reflecting differential expression of the l(2)gl gene. In a nonepithelial overgrowth mutant lethal(l)dislarge-l (Stewart et al., 1972), a maternal effect has been observed in embryos lacking both maternal and zygotic activity of the l(l)dIQ-1 gene (Perrimon, 1988). The embryonic phenotype can be rescued to some extent by introduction of a wild-type copy of the gene through the sperm or by the presence of maternal gene products (Perrimon, 1988). At the gastrulation stage presumptive larval tissues and pole cells are intensively labeled, whereas most of the embryonic CNS is labeled only weakly, except for two cell types: neuroblasts of the presumptive optic lobes within the brain primordia and nerve cells undergoing axogenesis. Although cell and tissues, expressing the protein, are histologically rather divers, the pattern

l(2)gl

Gene

431

of protein distribution agrees with the notion that a common feature of cells expressing the protein is the absence or slowing down of cell division. This is seen in tissues and cells that undergo terminal differentiation, including larval primordia and nerve cells undergoing axogenesis. Other cells expressing the protein during embryogenesis are known to remain rather inactive until larval stages, when they resume cell division, as reported for pole cells and neuroblasts of presumptive optic lobes (Campos-Ortega and Hartenstein, 1985). Thus the l(2)gl protein appears to be expressed in cells that have ceased to divide or are preparing to stop cell division in order to differentiate. A similar coincidence is observed at the time of metamorphosis, when the l(2)gl gene is expressed a second time in development. At the end of third instar the l(2)gl protein is detected in larval brain and imaginal disc cells (Klambt and Schmidt, 1986). In mutant larvae, in the absence of the protein, these cells continue to grow in a disperse fashion and fail to differentiate into adult structures (Gateff, 1987a, b). It is therefore likely that the l(2)gl protein is required in a process concomitant with proliferation arrest. The absence of ecdysone secretion in mutant larvae could be the result of phenotypically affected ring glands (Hadorn, 1955; Aggarwal and King, 1969; Gateff, 1978). Mutant larvae can be induced to pupate by the implanting of wild-type ring glands (Hadorn, 1955) or by the injection of ecdysone (Karlson and Hauser, 1952). Under these conditions cells stop growing, at least temporarily, but fail to differentiate into adult structures. Similarly, mutant imaginal discs implanted into wild-type larvae stop growing at metamorphosis of the host, but form no cuticle structures (Gloor, 1943; Gateff and Schneiderman, 1974; Gateff, 1978). An alternative explanation for the continuing cell division observed in mutant larvae could be the absence of termination of cell proliferation, an intrinsic property of particular tissues as suggested for imaginal discs (Bryant, 1987; Bryant and Simpson, 1984). Analysis of mutations from other gene loci that affect growth control of imaginal primordia at the larval-pupal transition, such as lethal (2)giant discs (Bryant and Schubiger, 1971) and lethal(l)dislarge-1 (Stewart et al., 1972), indicates that pupariation is probably delayed as a result

acids identical to those of L-CAM are indicated by dots. Homologous putative N-linked glycosylation sites (N X S/T) are boxed with heavy lines; within these glycosylation sites nonhomologous amino acids are included. Identical and homologous amino acids (L, I, V, A; T, S; D, E; R, H, K) are lighter boxes. (A) Sequence A is found at three locations within Z(Z)gZ protein. Repeats A2 and A3 form a tandem array. The corresponding sequences in the cadherin molecules are given below. (B) Sequence B found once in the Z(.)gZ protein and in each of the cadherin proteins extends about 140 amino acids in length. Putative glycosylation sites and cysteine residues are boxed with a heavy line. (C) Sequences C are found in a tandem organization in the Z@)gZ protein and once in each of the cadherins. The C repeats contain a high degree of negatively charged amino sequences (Ringwald et al, 1987). (D) Sequence D is found acids. The black bars denoted a and b mark regions that are putative Ca2+ -binding twice in the Z(Z)gZ protein and once in each of the cadherins; it is enriched in positively charged amino acids. The alignments are drawn with small gaps (-) and insertions to optimize homology.

432

DEVELOPMENTALBIOLOGY

VOLUME 133,1989

F ‘IG. 7. l(Z)gl antibodies detect a mouse protein. Cross-reaction of purified anti-l(2)gl and incubated with purified serum H. Immunofluorescence fixe *d with cold methanol lot: ilized at cell-cell contact sites. Preserum gave no signal. Bar represents 10 gm.

of overgrowth of imaginal discs, suggesting a feedback signaling of termination of growth and onset of metamorphosis (Bryant, 1987). What is the molecular role of the l(2)gl protein? The l(2)gl protein is located on the cell surface of dissociated living embryonic cells, as shown by immunofluorescence labeling using anti-l(2)gl antibodies. The observed patchy localization (Fig. 3) suggests that the Z(2)gZprotein is easily mobile in the plane of the membrane and appears to accumulate at cell-cell contact regions of cell aggregates. Relatively high levels of l(2)gl protein can be detected on single nerve cells beginning to extend axons (Fig. 4). This indicates a cell-specific expression of the l(2)gl protein and could imply a function of the l(.)gl protein in cell-cell interaction. Cell adhesion and migration are important features of vertebrate as well as invertebrate development (Takeichi, 19’77;Edelman, 1983, 1986; Gauger et ah, 1985; Yamada, 1985; Palka, 1987; Noll, 1985). The ability to form fascicles via axonaxon contact is mediated by adhesion-like molecules (Rathjien et ah, 1987; Chang et ab, 1987). Specific nerve

antiserum with proteins of mouse PCC4 cells. Cells w pere labeling of cells. Anti-l(2)g)gl antibodies bind to a pro1 ;ein

outgrowth is probably mediated by a different set of adhesion molecules that guide axons to their target to form a remarkably constant pattern in the nervous system (Goodman et aZ.,1984; Bastiani et al., 1987; Pate1 et al., 1987). The l(Z)gl protein probably does not provide specific axon guiding or pathfinding functions, since it is present at the cell surface of virtually all differentiating nerve cells. Therefore, we propose that the biological function of the Z(.)gZ protein is to facilitate cellcell contact in general.

Homologous

Sequences in Vertebrates

A short repeated sequence motif within the l(2)gl protein spanning a putative glycosylation site was found in a number of extracellular proteins, suggesting to us that the l(2)gl protein is potentially involved in cell-cell interaction (Ltitzelschwab et ah, 1987). The possible cell-adhesion function of the l(2)gl protein reported in this paper is supported by sequence homologies to the cadherin cell-adhesion molecule family. Moreover, the aggregation of dissociated embryonic

KL;IMBT

ET AL.

D. melarwgaster

cells is changed in the presence of fusion protein, indicating that the l(2)gl protein is involved in the connection of cells (Klambt, unpubl. results). Antisera directed against the most conserved part of the l(2)gl protein (antiserum H, Fig. 5) bind to a mouse protein in PCC4 cells that is accumulated at cell-cell contact sites. This suggests the existence of mouse proteins with epitopes in common with the l(Z)gl protein. The crossreacting protein is not necessarily uvomorolin. In fact, anti-uvomorolin antibodies stain the entire cell membrane of PCC4 cells (R. Kemler, personal communication). Thus, anti-l(2)gl antibodies probably recognize a related protein in mouse cells, which is not identical to uvomorulin. Cadherins are a family of Ca’+-dependent adhesion molecules. E-cadherin (Nagafuchi et al, 1987) and uvomorulin (Ringwald et al., 1987) in the mouse are corresponding proteins. Further distinct members of this family are P-cadherin (Nose and Takeichi, 1986; Nose et ab, 1987) and N-cadherin (Hatta et al., 1985). The biological properties and sequence homologies reported in the present paper suggest that the l(2)gl protein exerts functions during development similar to those described for the family of cadherin molecules. Sequence homology is found in four regions of the l(2)gl protein, which might be associated with different functions. For example, since cadherin adhesion molecules exert their function in a Ca’+-dependent fashion (Takeichi, 1977; Gallin et al, 1983). l(2)gl sequences may also be involved in Ca’+-dependent adhesion processes. The C regions in the l(2)gl protein and the corresponding region of the cadherin molecules exhibit a conserved spacing of negatively charged amino acids which might bind Ca’+. The consensus sequence deduced from the alignment of all three C-type regions reveals no direct homology to previously described Ca’+-binding regions (Lawler and Hynes, 1986; Vyas et al., 1987), but within l(2)gl regions C weak similarities to two putative Ca’+-binding sequences are found (Fig. 6c), as discussed by Ringwald et al. (1987). Despite many homologies between the l(2)gl protein and the cadherin cell-adhesion molecule family, it should be noted that structural differences exist between the proteins. The deduced l(2)gl protein sequence lacks a transmembrane region; nevertheless it appears to be bound to the cellular surface. Its mode of binding to cellular membrane and/or extracellular matrix remains to be clarified. In contrast, cadherins in vertebrates are integral molecules of cellular membranes containing an intracellular domain. Moreover, those regions with homology to cadherins are repeated more than once in the l(2)gl protein sequence. Given the molecular properties of the protein, it is conceivable that the protein is involved in the adhesion

l(2)gl

433

Gene

of cells, which cease to divide. The phenotypic effects of the l(2)gl mutation suggest that this cell contact may be a prerequisite for the differentiation of cells. MATERIALS

AND

METHODS

In Situ Hybridization The in situ hybridization on sections was performed as described (Akam, 1983; Hafen et al., 1983) with the following modifications. The pretreatment of the sections was in 0.1 N HCl and the subsequent Pronase incubation was omitted. Tritiated probes were synthesized using the random priming method (Feinberg and Vogelstein, 1983). Denatured pE7-9.A cDNA fragment (75 ng) was incubated for 14 hr at lS”C, yielding labeled DNA fragments up to 300-500 bp in length. Incorporation of radioactivity was l-5 X lo6 cpm/pg DNA. After hybridization the slides were coated with Kodak NTB-3 emulsion and stored for lo-14 days at 4°C for autoradiography. Slides were developed with Kodak D-19 and fixed with Tetenal fixer. Sections were stained with Giesma for 2 min, mounted in DePex (Serva, Heidelberg), and examined with a Leitz Laborlux microscope using light- and dark-field illumination. Expression of Fusion Proteins Subfragments from the open reading frame of the cDNA clone pE7-9 were subcloned in the expression vector PATH, allowing a fusion to the 3’-end of the bacterial trpE gene (the PATH vector system was kindly provided by T. J. Korner). The plasmids were transformed in C600 (F-, thi-1, thr-1, ZeuB6, ZacYl, tonA21, supE44, h-) to express the fusion proteins. The bacteria were grown to midlog (ODsg5: 0.4) in MS/glycerol, and the expression of the fusion protein was induced at 37°C by the addition of 5pg/ml /3-indole-acrylic acid (Sigma, Munich). The bacteria were harvested 4 hr after induction by pelleting, resuspended in 10 mM Tris-HCl, pH 7.5, and incubated with 0.1 mg/ml lysozyme for 1 hr on ice. After several freeze-thaw cycles the bacteria were sonified 3X 2 min and incubated for 10 min at 37°C with 0.1 yg/ml DNase I. The extract was centrifuged through a sucrose cushion, according to Frasch et al. (1987). For the cell aggregation experiments the pellet containing the fusion proteins was resuspended in distilled water. For immunization the pellet was resuspended in PBS and the proteins were analyzed on SDS-polyacrylamide gels. The pellet contains up to 80% fusion protein. Proteins were separated on a preparative 10% SDS-PAGE gel and stained with an aqueous Coomassie blue solution (White and Wilcox, 1984). The gel band containing the fusion protein band was cut out of the gel after destaining in water and ground to a fine powder under liquid NZ.

434

DEVELOPMENTALBIOLOGY

To raise antibodies against the complete protein sequence predicted by the open reading frame (ORF) found in pE7-9, we expressed subfragments containing open reading frame sequences in bacterial expression vectors. To obtain a fusion protein that encodes the N-terminal portion of the ORF, the 5’-untranslated leader sequence was removed by exonuclease Ba131 treatment. The shortened pE7-9 insert was subsequently cloned into the SmaI site of pUC13 to yield pE7-9.A. Sequence analysis revealed that one of the two potential initiation codons had been removed, leaving the second ATG at position 540 as a potential initiation start signal (Kozak, 1984). Four restriction fragments of pE7-9.A (Liitzelschwab et al., 1987) were subcloned into the appropriate expression vectors and the fusion proteins were expressed in bacteria. All fusion proteins obtained in bacterial extracts were in a size range corresponding to the coding capacity of the cloned cDNA fragment. Fusion proteins were recovered from preparative PAGE gels and used for immunization. Isolation. of Polyclonal Antisera

For immunization of rabbits the gel powder was resuspended in 1 ml extraction buffer (according to White and Wilcox, 1984: 50 mM Tris-HCl, 0.1 mM EDTA, 100 mM NaCl, 1 mM DTT, pH 7.9). New Zealand white rabbits, 2-3 months old, were immunized with 100-200 pg fusion protection well mixed with 1 ml of Freund’s complete adjuvant (Sigma) for the first boost and with Freund’s incomplete adjuvant (Sigma) for the following boosts, given at 4- to 5-week intervals. Bleeding was performed 10 days after the boost. The protein for antiserum J is from pPTBa1, containing an EcoRI-Hind111 fragment coding for the amino end of the 130-kDa protein. Antiserum W is from pPTH2, containing an internal 700-bp Hind111 fragment. Antiserum H is from pPTB5, containing an internal 500-bp BamHI fragment. Antiserum P is from pPTH6, containing a Hind111 fragment for the carboxyl end. The antisera were purified by adsorbing out on TrpE protein and whole bacterial extract coupled to CNBr-activated Sepharose 4B (Pharmacia) and subsequently tested on Western blots containing protein extracts from Drosophila wild-type and homozygous mutant l(2)gl brains. In wild-type protein extracts from larval brain, all four antisera recognize a 130-kDa protein that is absent in protein extracts from homozygous l(2)gl tissue. Protein blots were performed as described (Kllmbt and Schmidt, 1986). Immun$?uorescence on Tissue Sections and Fixed Cells

Staged embryos were grown at 20°C and dechorionated with 3% Na-hypochloride. Sections were prepared

Vot~~~133,1989

according to Mitchison and Sedat (1983) as described earlier (Klambt and Schmidt, 1986). The dechorionated embryos were fixed in a 4% paraformaldehyde in PBS/n-heptane mixture for 5 min. The fixed embryos were transferred into a precooled methanol/n-heptane mixture at -70°C and shaken vigorously for 10 min. After the mixture was rapidly warmed to room temperature, the devitellinized embryos sank to the bottom. The supernatant was removed and the embryos were washed extensively four times with 10 ml methanol and successively rehydrated with PBS. The embryos were mounted in Tissue Tek O.T.C. (Miles Scientific). Sections of 8-16 pm were cut on a cryotome and positioned on gelatin-coated slides. PCC4 cells were grown on HzSO,-treated coverslips. The cells were fixed with methanol at -20°C for 10 min. After three washings with PBS for 5 min, the coverslips and the slides were incubated with purified serum H (diluted 1:lOO) in PBS/3% BSA for 25 min, washed three times for 5 min, and incubated for 25 min with the second FITC-conjugated anti-rabbit IgG antibody (Sigma, diluted 1:lOO in PBS/3% BSA). After a 15-min washing, the sections were mounted in 60% glycerol and inspected under indirect uv light with a standard Zeiss microscope using a fluorescein filter set (450-490). Photographs were taken using Ilford HP5 1600 ASA and forced development with Ilford Microphen developer. Immunojluorescence of Dissociated Embryonic Cells

Dissociated cells were prepared essentially as described by Seecof and Unanue (1968) and Chan and Gehring (1971). Rapidly laying females were kept in large population cages on 3% agar (in 30% apple juice, 1% ethanol, 1% acetic acid, and 1% yeast extract) and egg laying was allowed for 2 hr. The embryos were collected on a nylon mesh (pore size 60 pm) and kept at 20°C for another hour. After the embryos were washed with a balanced saline solution (BS; Chan and Gehring, 1971), the chorion was removed with 3% Na-hypochlorite. The embryos were washed extensively with BS and transferred into sterile BS. Dispersion of the embryos was carried out in a 2-ml glass homogenizer by 7 gentle strokes. The suspension was filtered through a nylon mesh (pore size 50 pm) to remove the vitelline membrane and larger cell clumps. The cells were pelleted in a clinical centrifuge at 100~ and resuspended in 30 ml BS. After two more washings, the cells were adjusted to 1 X lo6 cells/ml BS. For immunofluorescence labeling, anti-l(2)gl antiserum was added (dilution 1:80) and incubated under agitation for 20 min at 20°C. After three washings, anti-rabbit FITC-conjugated secondary antibody (Sigma) was added (dilution 1:lOO) and incubated

KLAMBT

ET AL

D.

for 20 min in the dark. After the cells were washed three times, they were inspected under a standard Zeiss microscope. We thank Dorothea Gltick for help with the antibodies and Western blots, Rolf Kemler for providing laboratory space to perform initial experiments to look for cross-reactive proteins in the mouse, and K. F. Fischbach for passing on an aliquot of the 22ClO antibody. We thank J. Campos-Ortega, K. F. Fischbach, D. Kllmbt, K. Sander, M. Brand, and M. Haenlein for critical reading of the manuscript and valuable suggestions. C. K. was supported by a fellowship of the Land BadenWtirttemberg. This work was supported by the Deutsche Forschungsgemeinschaft.

Drosophila.

129,171-200.

EMBO

of Ultrabithorax

transcripts

in

J. 2, 20’75-2084.

BASTIANI, M. J., HARRELSON, A. L., SNOW, P. M., and GOODMAN, C. S. (1987). Expression of fasciculin I and II glycoproteins on subsets of axon pathways during neuronal development in the grasshopper. Cell 48, 745-755.

BERNSTINE, E. G., HOOPER, M. L., GRANDCHAMP, S., and EPHRUSSI, B. (1973). Alkaline phosphatase activity in mouse teratoma. Proc. Natl. Acad Sci. USA 70,3899-3903. BOURGUIGNON,L. Y., and BOURGUIGNON,G. J. (1984). Capping and the cytoskeleton. Int. Rev. Cytol. 187, 195-224. BRYANT, P. J. (1987). Experimental and genetic analysis of growth and cell proliferation in Drosophila imaginal discs. In “Genetic Regulation of Development” (W. F. Loomis, Ed.). A. R. Liss, New York. BRYANT, P. J., and SCHUBIGER, G. (1971). Giant and duplicated imaginal discs in a new lethal mutant of Drosophila melanogaster. Dew. Biol.

24,233-263.

BRYANT, P. J., and SIMPSON, P. (1984). Intrinsic and extrinsic control of growth in developing organs. Q. Rev. Biol. 59,387-415. CAMPOS-ORTEGA, J. A., and HARTENSTEIN, V. (1985). “The Embryonic Development of Drosophila melanogaster.” Springer Verlag, Berlin/Heidelberg. CHAN, L.-N., and GEHRING, W. (1971). Determination of blastoderm cells in Drosophila melanogaster. Proc. Natl. Acad. Sci. 1JSA 68, 2217-2221. CHANG, S., RATHJEN, F. G., and RAPER, J. A. (1987). Extension of neurites on axons is impaired by antibodies against specific neural cell surface glycoproteins. J. Cell Biol 104,355-362. EDELMAN, G. M. (1983). Cell adhesion molecules. Science 219,450-457. EDELMAN, G. M. (1986). Cell adhesion molecules in the regulation of animal form and tissue pattern. Rev. Cell Biol. 2, 81-116. FEINBERG, A. P., and VOGELSTEIN, B. (1983). A technique for labeling DNA restriction endonuclease fragments to high specific activity. Anal.

B&hem.

435

GATEFF, E., and SCHNEIDERMAN, H. A. (1969). l(2)gP: Lethal giant larvae”. &OS. Inj Service 44,47-48. GATEFF, E. (1978a). Malignant neoplasms of genetic origin in Drosophila. Science 200,1448-1459. GATEFF, E. (197813). Malignant and benign neoplasms of Drosophila melanogaster. In (M. Ashburner and R. T. F. Wright, Eds.), “The Genetics and Biology of Drosophila,” Vol. 2b, pp. 181-261. Academic Press, New York. GATEFF, E., and SCHNEIDERMAN, H. A. (1974). Developmental capacities of benign and malignant neoplasms in Drosophila. RouxlArch. Dev. BioL

176,23-65.

GAUGER, A., FEHON, R. C., and SCHUBIGER, G. (1985). Preferential binding of imaginal disc cells to embryonic segments of Drosophila. Nature

(London)

Rev. Suisse

AGGARWAL, S. K., and KING, R. C. (1969). A comparative study of the ring gland from wildtype and l(2)gl mutant Drosophila melanogaster. J. Morph01

l(2)gl Gene

313,395-397.

GLOOR, H. (1943). Entwicklungsphysiologische Untersuchungen an den Gonaden einer Lethalrasse (lgl) von Drosophila melanogaster.

REFERENCES

AKAM, M. E. (1983). The localization

melanogaster

132,6-13.

FRASCH, M., HOEY, T., RUSHLOW, C., DOYLE, H., and LEVINE, M. (1987). Characterization and localization of the even-skipped protein of Drosophila. EMBO J. 6, 749-759. GALLIN, W. J., EDELMAN, G. M., and CUNNINGHAM, B. A. (1983). Characterization of L-CAM, a major cell adhesion molecule from embryonic liver cells. Proc. NatL Acad Sci. USA 80, 1038-1042. GALLIN, W. J., SORKIN, B. C., EDELMAN, G. M., and CUNNINGHAM, B. A. (1987). Sequence analysis of a cDNA clone encoding the liver cell adhesion molecule L-CAM. Proc. Natl. Acad. Sci. USA 84,2808-2812.

ZooL 50,339-393.

GOODMAN, C. S., BASTIANI, M. J., DOE, C. Q., DULAC, S., HELFAND, S. L., KUWADA, J.-Y., and THOMAS, J. B. (1984). Cell recognition during neuronal development. Science 225,1281-1279. HADORN, E. (1955). “Letalfaktoren.” Thieme, Stuttgart. HAFEN, E., LEVINE, M., GARBER, R. L., and GEHRING, W. J. (1983). An improved in situ hybridization method for the detection of cellular RNAs in Drosophila tissue sections and its application for localizing transcripts of the homeotic antennapedia gene complex. EMBOJ. 2, 617-626. HATTA, K., OKADA, T. S., and TAKEICHI, M. (1985). A monoclonal antibody disrupting calcium dependent cell-cell adhesion of brain tissue: Possible involvement of its target antigen in animal pattern formation. Proc. Natl. Acad. Sci. USA 82,2789-2793. JACOB, L., OPPER, M., METZROTH, B., PHANNAVONG, B., and MECHLER, B. M. (1987). Structure of the l(2)gl gene of Drosophila and delimitation of its tumor suppressor domain. Cell 50,215-225. KARLSON, P., and HAUSER, G. (1952). Uber die Wirkung des Puparisierungshormons bei den Wildformen und der Mutante l(2)gl von Drosophila

melanogaster.

Z. Naturforsch.

7b, 80-83.

KLBBT, C., and SCHMIDT, 0. (1986). Developmental expression and tissue distribution of the lethal (2) giant larvae protein in Drosophila melanogaster. EMBO J. 5, 2955-2961. KOZAK, M. (1984). Compilation and analysis of sequences upstream from the start site in eukaryotic mRNAs. Nucleic Acids Res. 12, 857-872. LAWLER, J., and HYNES, R. 0. (1986). The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins. J. Cell Biol. 103,1635-1648. L~TZELSCHWAB, R., MOLLER, G., WILDER, B., SCHMIDT, O., FURBASS, R., and MECHLER, B. (1986). Insertion mutation inactivates the expression of the recessive oncogene lethal (2) giant larvae, a recessive Drosophila tumor gene. Mol. Gen. Genet. 204,58-64. L~~TZELSCHWAB,R., KL;IMBT, C., ROSSA, R., and SCHMIDT, 0. (1987). Protein product of a recessive Drosophila tumor gene l(2)gl has potential cell adhesion properties. EMBO J. 6,1791-1797. MECHLER, B. M., MCGINNIS, W., and GEHRING, W. J. (1985). Molecular cloning of lethal (2) giant larvae, a recessive oncogene of Drosophila melanogaster. EMBO J. 4,1551-1557. MITCHISON, T. J., and SEDAT, J. (1983). Localization of antigenic determinants in whole Drosophila embryos. Den BioL 99,261-264. NAGAFUCHI, A., SHIRAYOSHI, Y., OKAZAKI, K., YASUDA, K., and TAKEICHI, M. (1987). Transformation of cell adhesion properties by exogeneously introduced E-cadherin cDNA. Nature (London) 329, 341-343.

NICOLAS, J. F., DUBOIS, P., JACOB, H., GAILLARD, J., and JACOB, F. (1975). Teratocarcinome de la souris: Differentiation en culture d’

436

DEVELOPMENTAL

BIOLOGY

un lignee de cellules primitives a potentialites multiple. Ann. Microbiol. (Innstitut Pasteur) 126 A, 3-22. NOSE, A., and TAKEICHI, M. (1986). A novel cadherin cell adhesion molecule: Its expression patterns associated with implantation and organogenesis of mouse embryos. J. Cell Biol. 103,2649-2658. NOSE, A., NACAFUCHI, A., and TAKAEICHI, M. (1987). Isolation of placental cadherin cDNA: Identification of a novel gene family of cell-cell adhesion molecules. EMBO J 6, 3655-3661. OGOU, S., YOSHIDA-NORO, C., and TAKEICHI, M. (1983). Calcium-dependent cell-cell adhesion molecules common to hepatocytes and tertocarcinoma stem cell. J. Cell Biol. 97, 944-948. PALKA, J. (1987). 99,307-309.

Axon

guidance

in the insect

perifery.

Development

PATEL, N. P., SNOW, P., and GOODMAN, C. S. (1987). Characterization and cloning of fasciclin III: A glycoprotein expressed on a subset neurons and axon pathways in Drosophila. Cell 48,975-988. PERRIMON, recessive 392-407.

N. (1988). oncogene

The maternal of Drosophila

effect

of lethal(l)dislarge-l: Dew.

melanogaster.

Biol.

of A 127,

RATHJEN, F. G., WOLFF, J. M., FRANK, R., BONHOEFFER, F., and RUTISHAUSER, U. (1987). Membrane glycoproteins involved in neurite fasciculation. J. Cell Biol. 104, 343-353. RINGWALD,

M.,

SCHUH,

R., VESTWEBER,

D., EISTETTER,

H., LOTT-

VOLUME

133, 1989

SPHCH, F., ENGEL, J., DBLz, R., JWHNI~;, F., EPPLEN, J., MAYER, S., MOLLER, C., and KEMLER, R. (1987). The structure of cell adhesion molecule uvomorulin: Insight into the molecular mechanism of Caz+-dependent cell adhesion. EMBO J 6,3647-3653. SEECOF, R. L., and UNANUE, R. L. (1968). Differentiation of embryonic Drosophila cells in vitro. &‘q. Cell Res. 50, 654-660. STEWART, M., MURPHY, C., and FRISTROM, J. (1972). The recovery and preliminary characterization of X-chromosome mutants affecting imaginal discs of Drosophila melanogaster. Dew. Biol. 93, 240-256. TAKEICHI, M. (1977). Functional correlation between cell adhesive properties and some cell surface proteins. J. Cell Biol. 75,464-474. THOMAS, J. B., BASTIANI, M. J., BATE, M., and GOODMAN, C. S. (1984). From grasshopper to Drosophila: A common plan for neuronal development. Nature (London) 310,203-207. VYAS, N. K., VYAS, M. N., and QUIOCHO, F. A. (1987). A novel binding site in the galactose-binding protein of bacterial transport and chemotaxis. Nature (London) 327,635-638. WHITE, R. A. H., and WILCOX, M. (1984). Protein products of the bithorax complex in Drosophila. Cell 39, 163-171. YAMADA, K. M. (1985). Cell surface interactions with extracellular materials. Rev. Biochem. 52, 761-799. ZIPURSKY, S. L., VENKATESH, T. R., TEPLOW, D. B., and BENZER, S. (1984). Neuronal development in the Drosophila retina: Monoclonal antibodies as molecular probes. Cell 36,15-26.