Developmental regulation of gap junction gene expression during mouse embryonic development

DEVELOPMENTAL

BIOLOGY

146,117-130

(1991)

Developmental Regulation of Gap Junction Gene Expression during Mouse Embryonic Development MIYUKINISHI,NALIN

M. KUMAR,ANDNORTON

B. GILULA

The expression of products from three different gap junction genes (ni, & and &) was studied in pre- and postimplantation mouse embryos, during organogenesis, during differentiation of F9 teratocarcinoma cells, and in cultured embryonic stem (ES) cells. In this analysis, the following results were obtained. 1) Pre- and postimplantation mouse embryos. The (yl transcript was the earliest gap junction RNA detected (in the 4 cell stage embryo) and its abundance increased significantly throughout subsequent development. 2) Organogenesis. Evidence was obtained for developmental expression of these three different gap junction genes during early embryogenesis and throughout the late stages of organogenesis. The expression patterns for these genes may be related to differences in gap junctional communication requirements for fetal organ development versus neonatal and adult organ function, or the utilization of different genes by different cell types during organogenesis. 3) During the differentiation of F9 cells in culture, expression of these three genes was modulated. Thus, this is the first evidence for modulation of gap junction gene expression during the differentiation of a single cell type in culture. 4) In an ES cell culture line, (yl was the only gap junction gene product detected. This is consistent with the findings of 1yi expression in the embryonic inner cell mass region and in undifferentiated I(: 1991 Academic Press, Inc. teratocarcinoma Cek INTRODUCTION

Gap junctional communication has been documented between cells in a number of vertebrate embryos (for review, see Guthrie, 1987). Further, these interactions have been proposed to provide a potential regulatory pathway for influencing the orderly process of cell differentiation and patterning that occurs during development and embryogenesis (Guthrie and Gilula, 1989). Recent studies have provided evidence for a multigene family of gap junction genes (for review, see Kumar, 1991). Thus far, five different gap junction genes have been identified in vertebrates (termed (Ye,cyZ,(Y~,& and &) whose protein products are closely related by virtue of sequence and putative topological similarities. At present, there is no direct clarification about the difference in structure/function relationships for the different members of this multigene family. Furthermore, it has been reported that individual cells may coexpress multiple gap junction genes simultaneously (Nicholson

ef al, 1987). In previous studies, it has been shown that oocytes communicate with follicle cells prior to ovulation (Albertini and Anderson, 1974; Gilula et al., 1978; Eppig, 1982). During the final stages of meiotic maturation leading to ovulation, the oocyte becomes uncoupled from surrounding cumulus cells (Gilula et al., 1978; Moor et al., 1980; Dekel et al., 1981; Larsen et al., 1987). Following fertilization gap junction structures are first detected in S-cell stage mouse embryos (Ducibella et al, 1975; Mag-

nuson et al., 1977). This is in agreement with the electrophysiological detection of gap junctional communication between most of the blastomeres of the early 8-cell embryo just prior to compaction (Lo and Gilula, 1979a; Goodall and Johnson, 1982,1984). During mitosis, communication between cells is lost transiently (Goodall and Maro, 1986), being restored in the interphase cells of the morula and blastocyst with no restriction in communication pathways until after implantation (Lo and Gilula, 1979b). Upon progression to the 3%cell stage, the blastomeres segregate into two subpopulations of cells: the presumptive inner cell mass (ICM) and the presumptive trophoblast (Ducibella et al., 1975). All cells of the trophoblast and ICM are connected via functional gap junctions (Lo and Gilula, 1979a,b). In addition, the presence of gap junctions between trophoblast cells and uterine epitheha1 cells (Tachi et al., 1970) indicates that communication pathways are likely to exist between maternal and embryonic cell populations. In the past few years, there have been several studies using perturbation approaches with gap junction specific antibodies that have provided evidence that gap junctional communication can make an important contribution to development and/or differentiation of an organism (Guthrie and Gilula, 1989). In particular, a blockage of communication in the Xe?-lops embryo (Warner et al., 1984) or in the Coelenterate Hydra (Fraser ef al., 1987) leads to defects which are potentially related to patterning processes. In the mouse em117

001%1606/91 $3.00 Copyright All rights

0 1991 by Academic Press, Inc. of reproduction in any form reserved.

DEVELOPMENTAL BIOLOGY

VOLUME 146, 1991

PIG. 1. (a) Comparison of the deduced amino acid sequence of three mouse pap junction cDNAs. Shown are the amino acid sequences for the The asterisks denote identical amino acids among the three mouse al, A, and & gap junction products aligned for maximum similarity. sequences; the dots indicate similar, but not identical, residues that are common to all three sequences. (b) Probes and primers used for PCR analysis and Sl nuclease protection assay of (Y~,PI, and & gap junction transcripts. The probes used for the Sl nuclease assay are indicated by the thin arrows. Shadowed arrows indicate the locations of sense and antisense primers used for PCR analysis, open boxes indicate location of oligonucleotide probes, used for identification of PCR products.

bryo, perturbation of communication in a single cell of a 4-cell stage embryo leads to a decompaction of the progeny of that cell at the compaction stage, resulting in the elimination (decompaction) of these cells from the embryo proper (Lee et ah, 1987). Appropriate strategies for further understanding the potential contribution of gap junctional communication to mouse development will depend critically on understanding the temporal pattern of expression of the different gap junction gene products during mouse embryonic development and cell differentiation. To accomplish this, appropriate gap junction cDNA probes have been isolated and used for analysis of the low abundance transcripts from these genes during early embryonic stages, during organogenesis, and during the differentiation of a teratocarcinoma cell culture line. MATERIAL

Mouse Gap Junction

AND

METHODS

cDNAs

Rat cyl and human & gap junction cDNAs and a synthetic oligonucleotide for 0, were used to isolate homologous mouse sequences from mouse oocyte and ovary cDNA libraries provided by Dr. P. Wasserman, from a mouse blastocyst cDNA library provided by Dr. D. Wang, and from an F9 parietal endoderm cell cDNA library provided by Dr. S. Strickland, using procedures previously described (Sambrook et ab, 1989). A mouse liver cDNA library was generated using poly(A) RNA

obtained from a 4-week-old mouse with a commerical cDNA kit (Invitrogen, San Diego, CA). All isolated cDNA clones that were isolated were sequenced to verify their identity as the mouse homologues to previously isolated gap junction cDNAs (Kumar and Gilula, 1986; Beyer et al., 198’7; Zhang and Nicholson, 1989).

Recovery of Embryos BALB/c X C57BL/6 Fl mice were used throughout for timed matings. The midnight prior to the appearance of a vaginal plug was defined as 0 days (Hogan et al., 1986). Mice were superovulated with PMS and hCG (Sigma Chemical Co., St. Louis, MO) treatment as described (Hogan et al., 1986). Unfertilized eggs and l-cell stage zygotes were collected 22 hr after the hCG injection. Later stage embryos, up to the blastoeyst stage, were obtained by maintaining the l-cell stage zygotes in culture for an appropriate time. These embryos were maintained in M2 medium (Hogan et ab, 1986) at 37°C in a 5% CO, incubator. Postimplantation embryos were recovered at appropriate times after natural mating to obtain RNA. Embryos and tissues were either processed immediately for RNA or stored in liquid nitrogen prior to use. In order to eliminate associated cumulus cells for some experiments, unfertilized eggs were treated with 300 Kg/y1 hyaluronidase (Sigma) as described (Hogan et al., 1986).

ysis of the ar-fetoprotein (AFP) and laminin Bl mRNA content of the different cell populations. AFP has been shown to be a specific marker for visceral endoderm (Dziadek and Adamson, 19’78) and laminin Bl is enriched in parietal endoderm cells (Strickland et al., 1980).

Embryonal Stem Cells A mouse embryonal stem cell (ES) culture line, CCE (provided by Dr. V. Quaranta), was cultured in DMEM containing 10% FCS and 1000 U/ml LIF (AMRAD Corp.) as previously described (Robertson, 1987).

RNA Isolation

FIG. 2. PCR analysis of gap junction mRNA in preimplantation mouse embryos. RNAs were isolated from different stages of preimplantation mouse embryos, reverse transcribed, and amplified. Samples Lvvereseparated by electrophoresis with a 1.2% agarose gel, transferred to a nylon membrane, and then hybridized with labeled oligonucleotide probes. Adult mouse liver RNA was used as a positive control for p1 and &, and mouse heart RNA was used for CY,.Only (yl transcripts were detected in the different preimplantation stage samples. In the bottom panel, CY,RNA was analyzed from unfertilized eggs btaforc C~1 and after (-I 1 treatment with hyaluronidase.

F.s Cell Culture and Differentiation Parental cells from the mouse F9 teratocarcinoma cell line (provided by Dr. E. Adamson) were cultured in DMEM containing 10% FCS as described (Edwards et nl., 1988). In order to promote differentiation of these cells to visceral endoderm, cells were cultured in untreated petri dishes with DMEM, 10% FCS, and 10 mM retinoic acid (RA; Sigma). For differentiation to parieta1 endoderm, cells were seeded on gelatin-coated plastic dishes in DMEM, 10% FCS, 100 mM RA, 0.2 mM dibutyryl cyclic AMP (dbc AMP), and 0.1 mM isobutyl methylxanthine (IMX, Sigma). Cultures of differentiated visceral endoderm were analyzed after 8 days, and cultures of parietal endoderm after 4 days (Edwards et al., 1988). The differentiation of F9 cells into parietal and visceral endoderm was determined by anal-

RNA was extracted from preimplantation embryos using a CsCl-guanidinium thiocyanate procedure (Rappolee et al., 1989). A Chlamydomonas 32P-/3-tubulin RNA probe was added to the embryo samples to quantify the RNA recovery. The recovery of RNA was approximately 50% for stages between unfertilized egg and blastocyst. Normally 150 embryos from each stage were used for each sample. Total RNA was extracted from preimplantation embryos and embryonic organs by an acid phenol-guanidinium thiocyanate procedure using RNAzol (Cinna/ Biotex). However, it was not possible to isolate intact RNA from placenta or decidua samples using this procedure. Therefore, a CsCl-guanidinium thiocyanate procedure was applied to these tissues (Chirgwin et al., 1979). RNA concentrations were determined by absorbance measurement at 260 nm. The integrity of RNA was analyzed by ethidium bromide staining following electrophoresis on a formaldehyde-agarose gel.

Dye Tmnqfer Determination A solution of 5% Lucifer Yellow (w/v) in PBS was microinjected into cultures of F9 cells using an Eppendorf 5170 (Hamburg, FRG) micromanipulator. Transfer of the dye into adjacent cells was monitored using a Zeiss fluorescent microscope. Micrographs were taken 5 min after microinjection using Kodak TMax film (400 ASA). The number of dye-filled cells per injection was used as an index for the communication capacity of each cell population. Texas Red dextran was microinjected as a control since this compound cannot pass through gap junctions.

PCR ilnalysis The total RNA obtained from equal numbers of preimplantation stage embryos was converted to cDNA using a specific antisense gap junction primer and MMLV reverse transcriptase according to the suppliers’ recommended conditions (BRL, Gaithesburg, MD). Following inactivation of the enzyme by treatment at 94°C for 1 min, Taq polymerase (Promega), sense primer,

120

DEVELOPMENTALBIOLOGY VOLUME146.1991

dNTPs (final concentration 0.2 rnw, and PCR buffer (final concentration 50 mM KCl, 0.1% Triton X-100, 10 mM Tris, pH 8.4,3 mM MgCl,) were added. The amplification reaction was carried out for 35 cycles using a Perkin-Elmer-Cetus thermocycler (2 min at 94°C 3 min at 50°C 3 min at 72°C). The reaction products were analyzed by agarose gel electrophoresis, transferred to nylon membranes (MSI), hybridized with an appropriate oligonucleotide in 6~ SSC, 5~ Denhardt’s, 100 pg/pl yeast RNA at 37”C, and washed three times with 2~ SSC at 42°C. The following primers and probes related to coding regions were used: a1 sense primer, aa 257-263; cyl antisense primer, aa 362-369; q probe, aa 298-306; & sense primer, aa 93-100; & antisense primer, aa 2’74-281; p1 probe, aa 246-252; &sense primer, aa 15-22; & antisense primer, aa 173-185; /J, probe, aa 154-163. The efficiency of amplification by the different PCR primers used in this study was similar for all the different pairs, as judged by the intensity of the hybridization signal using a range of heart or liver RNA concentrations. Sl Nuclease Protection

Assay

The RNA from cell cultures, embryonic regions, and tissues was analyzed by an Sl nuclease protection assay using antisense single strand DNA synthesized in the presence of r2P]dCTP as described (Davis et al., 1986). A mixture of the &, &and o1 probes (over 25,000 cpm individually) was hybridized to 5 pg of total RNA for each sample in 10 ~1hybridization buffer (76% formamide, 20 mMTris-HCl (pH 7.4), 0.4 MNaCl, 10 mMEDTA, 0.1% SDS) at 50°C overnight. A layer of mineral oil was placed on top of the reaction mixture to prevent evaporation. Following hybridization, the reaction mixture was diluted with 90 ~1of Sl nuclease mixture (0.3 MNaCl, 60 mM Na acetate (pH 4.5), 3.3 mM ZnSO,, 20 mg/ml denatured salmon sperm DNA, 2000 U/ml Sl nuclease) and digested at 28°C. The temperatures used for hybridization and digestion were optimal for sensitivity and specificity for the gap junction genes studied. It was verified that the probe was in excess by using different RNA/ DNA ratios. The resulting protected fragments were ethanol precipitated and then analyzed on a 6% polyacrylamide gel containing 7 Murea. The bound radioactivity was determined by autoradiographic exposure at -70°C to Kodak XAR-5 film using an intensifying screen. Embryos were manually freed from the zona pellucida after incubation in acid Tyrode’s solution (pH 2.5) for several minutes as described (Nicholson et ab, 1975). Following fixation for several hours in Lavdowsky’s fixative at room temperature (ethanol:formalin:acetic

FIG. 3 (a) Immunohistochemical localization of the q gap junction antigen in cell surface membrane regions of preimplantation embryos. Embryos were treated with preimmune rabbit IgG at the 8-ccl1 stage (A) and blastocyst stage (B), or treated with (yl gap junction prptidr antibodies at the early 8-cell stage (C), late g-cell stage (D), 8- to 16.cell stage (E), and hlastocyst stage (F). Phase-contrast images appear on the left, with indirect immunofluorescence of the same specimens on the right. Bar, 10 pm. (b) Immunohistochemical localization of CY,gap junction antigen in the inner cell mass region of postimplantation embryos. Fertilized eggs were collected and maintained in culture. The cultured embryos were treated with either preimmune rabbit IgG(A) or LYEgap junction peptide antibodies (8 and C).
121

122

DEVELOPMENTALBIOLOGY

V0~~~~146,1991

b

FIG. 4 (a) Sl nuclease protection assay for gap junction transcripts in mouse embryonic stem (ES) cells. Five micrograms of total RNA was analyzed for each sample. Mouse liver RNA was used as a positive control for fir and &, while heart RNA was used as a positive control for (pi. (?I was the only detectable gap junction transcript in the ES cell RNA. (b) Immunohistochemical localization of (Yegap junction antigen in regions of cell-cell contact between ES cells. ES cells were treated with 01~gap junction peptide antibodies (A) or with preimmune rabbit IgG (B). Phase contrast images appear on the left, with indirect immunofluorescence of the same specimens on the right. Bar, 10 Frn.

acidwater, in proportions of 50:10:4:40), embryos were rinsed for 45 min in four changes of 0.1 M phosphate buffer (pH 7.4) containing 0.05 M lysine, followed by a brief rinse in PBS. The embryos were then incubated for 1 hr at 3’7°C with PBS containing 3% BSA and 3% normal goat serum (Vector Laboratory). After rinsing in PBS, the embryos were incubated with either preimmune IgGs or gap junction antibodies for 1 hr at room temperature, followed by incubation with fluorescein conjugated goat anti-rabbit IgG (Southern Biotechnology) diluted 1:50 in PBS. After several rinses with PBS, the embryos were mounted in PBS containing 0.1% N,NJ’,N’-tetraethyl-p-phenylenediamine in 90% glycerol. The embryos were viewed with a Zeiss Axiophot microscope with epifluorescence, and photographs were taken with Kodak T-MAX 400 film. Affinity-purified antibodies prepared against synthetic peptides corresponding to a carboxy-terminal domain of & (262-280 aa) and to a cytoplasmic domain of & (112-125 aa) were used for these studies, and the procedures for their production and use have been described previously (Milks et al., 1988; Risek et al., 1990). Similarly, a synthetic peptide corresponding to a postu-

lated carboxy-terminal domain of a1 (3’70-381 aa) was used to generate a rabbit polyclonal antibody that has similar characteristics to an al-antibody reported previously from this laboratory (Risek et ah, 1990). RESULTS

Cloning and Characterixatim cDNAs

of Mouse Gap Junction

To study the temporal expression of the gap junction genes during mouse embryogenesis by Sl nuclease analysis, it was necessary to isolate cDNAs for mouse gap junction mRNAs. For this purpose, human & and rat a1 gap junction cDNAs (Kumar and Gilula, 1986; Risek et al., 1990) and a synthetic oligonucleotide derived from the partial amino acid sequence for & (Nicholson et al., 1987) were used as probes to screen several mouse cDNA libraries. Clones for 01~gap junction cDNA were obtained from mouse cDNA libraries generated using mouse RNAs from ovary, blastocyst, and F9 parietal endoderm (PE) cells. & and & cDNAs were not detected in these libraries. However, the mouse & and & gap junction cDNAs were isolated from a mouse liver cDNA library.

123

+

p2 b

FIG. 5 (a~ Sl nuclease protection assay of gap junction transcripts during differentiation of the F9 cell culture line. Five micrograms of total RNA was analyzed by this technique using RNA isolated from cultures of undifferentiated cells (Undif.), visceral endoderm-like (V.E.) cells, and parietal endoderm-like (P.E.) cells. Although CY~and & RNAs were detected in all three populations, & RNA was only detected in thr visceral endoderm-like cells. (b) Analysis of gap junctional communication (dye transfer) in F9 cell cultures. Lucifer Yellow was injected into the center cell and fluorescence images were recorded 5 min later. The images on top represent the phase-contrast images of the cells that were analyzed by dye injection below. (A) IJndifferentiated F9 cells, (B) visceral endoderm-like cells, (C) parietal endoderm-like cells. Bar, 10 pm.

A comparison of the deduced amino acid sequences for the three mouse gap junction cDNAs is shown in Fig. la. The nucleotide sequence data reported in this paper have been submitted to the EMBL, GenBank, and DDBJ Nucleotide Sequence Databases, and assigned the accession numbers. The mouse ail, p, and & gap junction sequences were about 95% homologous in the coding region to their rat counterparts at both the amino acid and the nucleotide level. Overall, the sequences were less conserved in the 3’-nontranslating region. From analysis of these sequences, it is possible to infer that the members of the gap junction multigene family have a number of common features (Milks et al, 1988), namely: (a) the presence of four transmembrane domains with one domain, M3, contributing to the channel space; (b) a short NH,-terminal region exposed on the cytoplasmic surface with a variable length carboxyl terminus also on the cytoplasmic surface; and (c) the presence of two extracellular loops that contain a characteristic spacing of cysteines. Analysis of Gal, Junction Transcripts Preimplantation Embryos by PCR

in

On the basis of the information obtained from the cDNA clones, primers and probes were synthesized for

PCR amplification of RNA, and plasmids were constructed for use in an Sl nuclease protection assay. The probes used in the Sl assay were designed to produce unique size products when hybridized to different gap junction RNAs. While PCR is a much more sensitive analytical technique than the Sl nuclease assay, it is subject to complications, such as signals arising from DNA contamination. However, the signal from contaminating DNA could be eliminated by pretreating the RNA with DNAase. Since limited amounts of RNA were obtained from early embryos, PCR was used for the analysis of preimplantation embryos, and the Sl nuclease assay was used for postimplantation embryos. To determine the temporal pattern of gap junction gene expression during embryogenesis, total RNA was prepared from embryos at different stages of development. One hundred fifty embryos were collected at each stage for extraction of RNA. Since each mouse embryo at early stages contains only -300 pg of RNA (Piko and Clegg, 1982), Escherichia coli rRNA was added as a carrier to improve the recovery. In order to quantify the recovery of RNA, synthetic radioactive Chlamydomonas @-tubulin RNA was added (the @-tubulin gene has no homology to gap junction genes). Analysis of the data indicates that the RNA recovery was approximately

124

DEVELOPMENTALBIOLOGY V0~~~~146.1991 Placenta

Embryo

FIG. 6. Sl nuclease protection assay of gap junction mRNA in decidua, placenta, and embryo samples. Decidua samples also contain the embryo. Five micrograms of total RNA was analyzed from each sample. The three gap junction transcripts were analyzed simultaneously for the decidua and placenta samples. For analysis of the embryo RNA, a, was hybridized separately from & and &. Developmentally, u1 was expressed earliest, followed by & and finally /3r.

50% at all stages. The RNA from each stage was divided into three aliquots. Following DNase treatment, the RNA was reverse-transcribed using specific gap junction primers and then amplified by the PCR technique. The primers and probes used are shown in Fig. lb. Each pair of primers used was equally efficient in amplifying control RNA. RNA from mouse heart and RNA from liver, which have been shown previously to contain cyl, &, and &, respectively, were used as controls to demonstrate that the primers successfully amplified RNA from these sources (Fig. 2). With this procedure no gap junction transcripts were detected until the 4-cell stage; at this stage, LY~gap junction transcripts were detected, and this signal increased in intensity at subsequent stages (Fig. 2). Transcripts for & and ,&could not be detected at any of these early stages. Further, this result provides additional evidence for the absence of contaminating DNA in the RNA samples since the primers used can also amplify DNA. In order to determine if the detectable 01~transcripts in the unfertilized eggs were due to contaminating granulosa cells from the cumulus, RNA was isolated from unfertilized eggs before and after treatment with hyaluronidase. In the nonhyaluronidase treated eggs, a strong (Yesignal was present, and this

signal was absent from the eggs that received the enzyme treatment (Fig. 2). These results were confirmed also by Sl nuclease analysis (data not shown). Therefore, these results suggest that none of the gap junction transcripts analyzed were present in the unfertilized egg, but some cyl was present in the cumulus cells that were removed by the hyaluronidase treatment. Immunolocalixation of Gap Junction and Postimplantation Embryos

Antigen

in Pre-

Following the RNA analysis, efforts were made to detect and localize the three gap junction proteins at the 8to 32-cell stage by indirect immunofluorescence. After fixation and permeabilization, the embryos were incubated with preimmune IgGs or antibodies specific for the CQ,&, and pZantigens, followed by treatment with an FITC-conjugated goat anti-rabbit IgG. Figure 3a contains results obtained with embryos at different stages. A punctate fluorescence was observed in the cell surface membrane region at stages beyond the late 8 cell stage embryo, with no detectable staining in the early 8 cell stage embryo. This localization was observed only with a1 antibodies and it was consistent with regions involved in cell-cell contact. No distinct and reproducible

fluorescence signals were observed with preimmune IgGs or peptide antibodies specific for & and 0, proteins. The embryos were cultured until free from the zona pellucida and similarly analyzed for gap junction proteins at postimplantation stages by indirect immunofluorescence. While the fully expanded blastocyst contains about 64 cells, of which about 20 form the ICM (Hogan, 1986), it is difficult to distinguish morphologically between the two cell types in the early blastocyst stage. However, as shown in Fig. 3b, the 01~gap junction antibodies clearly distinguished between the two cell types, with the o1 product distinctively localized to the cells of the ICM. No & or &gap junction antigens were detected at these early postimplantation stages (data not shown). Analysis of Gap Junction mRNA, Protein, Coupling in ES an,d F9 Cells

and Dye

An established mouse ES culture line, CCE (Robertson et ul., 19861, was analyzed for gap junction expression since these cells represent: (a) the properties of the undifferentiated stem cells of the embryonic inner cell mass region; and (b) appropriate vehicles for creating transgenic animals (via chimeras). With an Sl assay, only cyl mRNA was detected in the ES cells (Fig. 4a), and a1 antigen was the sole junction protein observed by immunohistochemistry (Fig. 4b). Further, the a1 antigen was principally localized to regions of cell-cell contact. The F9 teratocarcinoma cell line is derived from mouse embryonal carcinoma cells. This cell line has a very low frequency of spontaneous differentiation. However, exposure to retinoic acid promotes rapidly dividing F9 stem cells to differentiate into cells resembling either parietal endoderm (in the presence of CAMP) or visceral endoderm (see Materials and Methods). Analysis of RNA isolated from these three populations of cells by the Sl nuclease assays indicated that all the different cell types expressed cyl mRNA at very high levels (Fig. 5a). p2 RNA was also detected in the three different cell populations, but at different levels. & was most abundant in the visceral endoderm population, less abundant in the undifferentiated cells, and barely detectable in the parietal endoderm cells. 0, was detected only in the visceral endoderm population. In addition, junctional communication pathways (i.e., Lucifer Yellow dye coupling) were present in all three populations of F9 cells (undifferentiated, visceral, and parietal endodermal cells) (Fig. 5b). Gap Junction mRNA Expression in Postimplantation Embryos, Decidua, und Placenta The analysis of embryonic gap junction RNA was extended developmentally by using RNA isolated from nat-

urally mated mice at different days post coitum (p.c.). Five micrograms of total RNA was analyzed by the Sl nuclease protection assay with a mixture of the three gap junction probes. The probes used are shown in Fig. lb. Since all three probes contain vector sequences that do not hybridize to gap junction RNA, the protected bands are slightly shorter than the original probes. The vector sequences that are attached to the probe are essential for demonstrating the complete digestion of RNA by the Sl nuclease. Sometimes multiple Sl nuclease resistant fragments were observed. These may have resulted from multiple sites of transcriptional initiation, alternative splicing, or from limited “nibbling” of the ends by Sl nuclease. In this assay genomic DNA was not protected by any of these probes under the hybridization conditions used, demonstrating the specificity of this assay for RNA (Fig. 6, lane 2). In a decidua sample that also contains the embryo at 5.5 days p.c., both o(~and & gap junction RNAs were detected (Fig. 6, decidua). Although p2 RNA levels declined at 6.5 and 7.5 days p.c., they subsequently increased at 9.5 days p.c. No 8, signal was observed at these stages. At stages up to 9.5 days p.c., the results of gap junction RNA analysis are complicated by the fact that it was not possible to separate the embryo completely from uterine tissues. Nevertheless, it can be concluded that B, was not present in substantial amounts in the embryo at that time since no signal was observed at any of these stages. At 9.5 days p.c. and beyond, it was possible to separate the embryo from placental tissues and, thus, analyze each separately (Fig. 6). In RNA samples isolated from the placenta, both (yl and flZ gap junction RNA were detected (Fig. 6, placenta), with &far more abundant than No.The expression pattern of & did not appear to change from 11.5 to 17.5 days p.c., unlike (Yewhich gradually decreased in this tissue during development. Analysis of RNA from the 9.5 days p.c. embryo also indicated expression of both a!1and &. However, in contrast to the placental tissue, 01~was more abundant (Fig. 6, embryo). Furthermore, analysis of whole embryo RNA indicated that a1 expression was relatively constant until birth, whereas ,B, gradually increased during development. 8, was first detected in whole embryos at 15.5 days p.c., and this is about the same time that fil expression was detected in liver (see Fig. 7a). These results suggest the following temporal expression pattern for these three genes in the mouse embryo: No, followed by & and then &. Gap Junction

mRNA Expression

during OygarLogerLesis

To determine the temporal expression of gap junction mRNA during organogenesis, RNA was isolated from several organs separately and analyzed by the Sl assay.

126

DEVELOPMENTALBIOLOGY VOLUME146,1991

Heart

a

b

Lung

Liver

Stomach

Spleen

FIG. 7 (a) Sl nuclease protection assay of gap junction mRNA during organogenesis of the heart, liver, and kidney. Five micrograms of total RNA (except for the l- and 4-week liver samples, 2.5 pg) was hybridized to a mixture of three antisense single-stranded DNA probes for each sample, as described in under Materials and Methods. (b) Sl nuclease protection assay of gap junction mRNA during the development of lung, stomach, thymus, and spleen. Five micrograms of total RNA was analyzed for each sample. (c) Sl nuclease protection assay of gap junction mRNA during the development of the head (embryo) and brain (after birth). Five micrograms of total RNA was analyzed for each sample. Embryonic RNA was extracted from the entire head at each indicated developmental stage. After birth, RNA was extracted from brain tissue, not including the spinal cord.

NISHI,

C

KUMAR,

AND GILLILA

Brain

Head

FIG. 7-('orttir~rrd

In mesodermally derived organs, such as the heart and spleen, only a1 transcripts were detected (Figs. 7a and 7b). The heart began to beat at 9 days p.c., and RNA from the 13.5 days p.c. embryo contained high levels of cu, RNA similar to the adult organ (Fig. 7a). The abundant expression of CY~RNA in the heart did not change during development. Similarly, only LYEwas detected in the spleen (Fig. 7b). The faint bands that were observed in the Sl analysis of heart RNA (Fig. 7a) were due to contamination by another fragment of the 01~cDNA. Since the size of this contaminating band was different from any of the other gap junction probes, it did not create complications for analyzing the results. The differential modulation of gap junction expression was most evident in the analysis of kidney organogenesis. In the kidney, all three transcripts were detected (Fig. 7a). The cyl transcript was expressed most strongly at 15.5 days p.c., but decreased gradually, with only a weak signal detectable in ii-week-old mice. p1 and & RNAs gradually increased in abundance during development of the kidney, with 0, detected first at 15.5 days p.c., followed by /3, at 17.5 days p.c. In adult mouse (4 weeks) kidney, pZ expression was the strongest, followed by &, and finally No. This expression pattern is completely opposite to that found in the 15.5 days p.c. embryonic kidney.

The liver is the first endodermal-derived organ analyzed where & mRNA could be detected as early as 11.5 days p.c. (Fig. 7a). This is earlier than in RNA prepared from the whole embryo where fil was initially detected at 15.5 days p.c. &was also detected in the 11.5 days p.c. sample upon longer exposure of the autoradiogram. Subsequently, both 0, and 0, RNAs progressively increased in the liver beyond 17.5 days p.c., although the ratio of & to & mRNA appeared to remain constant, similar to the value found in adult mouse liver. Different amounts of RNA were used in the analysis of RNA for the l- and 4-week liver samples shown in Fig. 7a. In other endodermally derived organs such as lung, stomach, and intestine, & was detected at later stages (Fig. 7b). & was detected in the stomach of newborn mice and in the lung of ll-week-old mice. The developmental expression of these different gap junction genes was found in several organs. For example, in the lung CY~ was detected at 15.5 days p.c., followed by & in the newborn mouse, and /J1 in the ll-week-old mouse (Fig. 7b). In the stomach & was detected earlier at 17.5 days p.c., with the level of&in stomach remaining relatively constant during neonatal development (Fig. 7b). a1 and & were detected in the stomach of newborn mice, and their levels gradually increased in abundance during development of the stomach. In the thymus, cyl was detectable at 15.5 days p.c. and it remained relatively constant (Fig. 7b), although there appeared to be an elevated level at birth which subsequently decreased to preterm levels. However, &was not detected in the thymus until birth. These results suggest that, although 8, is found in predominately endodermal-derived organs, not every organ derived from the endoderm, such as the thymus, expresses &. Expression of the three different gap junction genes was also examined in the ectodermally derived brain. From 11.5 to 17.5 days p.c., RNA was extracted from the entire head region, since it was difficult to isolate the brain at these early stages. As shown in Fig, 7c, a1 and & were already expressed at 11.5 days p.c., and both remained almost at the same level throughout subsequent embryonic and postnatal development, whereas & was virtually absent in the embryonic head. To analyze the gap junction expression pattern more precisely, brain RNA from newborn mice and subsequent stages was examined. The expression of a1 and & was relatively constant in the brain during this time period (Fig. 7c), whereas 0, transcripts were not detected in the brain until 4 weeks after birth. The 0, levels then appeared to remain constant during subsequent development. DIS(:USSION

In this study the distribution and temporal expression of three different gap junction genes have been an-

128

DEVELOPMENTALBIOLOGY V0~u~~146,1991

alyzed in the developing mouse embryo. The results have provided evidence that the a1 gap junction gene, and not the fll and & genes, was exclusively expressed in preimplantation embryos, starting from the 4-cell stage onward. Immunostaining indicated that the late E-cell stage embryo contained detectable gap junctions, whereas they were absent in early E-cell stage embryos. It is possible that gap junction antigen was not detected by immunofluorescence at earlier stages when RNA was detected because of a greater relative sensitivity of the PCR method for RNA, compared to immunofluorescence for protein. However, an alternative explanation could be that the gap junctions are not assembled until the late E-cell stage. This would be in agreement with earlier electrophysiological studies which indicate that gap junctional communication is first detectable in the early E-cell embryo just prior to compaction (Lo and Gilula, 1979a; Goodall and Johnson, 1982, 1984). The presence of a, RNA and protein at the 4-cell stage would be consistent also with experiments using metabolic inhibitors on mouse embryos (McLachlin et ah, 1983; Kidder et al., 1985). These studies indicate that adding inhibitors at the 4-cell stage did not prevent the appearance of gap junctions at the E-cell stage as measured by ionic coupling. In addition, the synthesis of 01~RNA at the 4-cell stage resembles the situation with the ZO-1 component of tight junctions which is also synthesized to a limited extent at the late 4-cell stage, and the mRNA for encoding it is inherited by the E-cell stage embryo for translation (Fleming et ub, 1989). In the present study, no &, &, or 01~gap junction RNA was detected in zygotes prior to the 4-cell stage. This finding is in contrast to a previous study which reported & protein, but not RNA, in zygotes through late morula (Barron et al., 1989). That study concluded that & protein is inherited as a oogenetic product. Since no RNA for &, & or a1 was detected in unfertilized oocytes stripped of cumulus cells in the present study, it is highly unlikely that the hypothesis from the previous study (Barron et al., 1989) will be valid. The first stage at which gap junction RNA was detected is the 4-cell stage, and this would be consistent with activation of the mouse embryonic genome which occurs at the 2-cell stage (Flach et al., 1982; Piko and Clegg, 1982). Once the QI~gene is activated, it appears to remain active since the a1 RNA was detected at all stages up to adult. In contrast, p1 and & gap junction RNAs were not detectable in any stages prior to implantation. However, while the complete absence of any particular gap junction transcripts at a given stage cannot be ruled out, the results do suggest that 01~gap junctions are the first to be detected. In a previous study, it was found that injection of gap junction antibodies into mouse embryos can block com-

munication and subsequently lead to decompaction and the failure of cavitation (Lee et al., 1987). The polyclonal antibodies used in that study were prepared against & gap junction protein isolated from rat liver. However, it is known that those antibodies can bind to a number of different gap junction gene products (Zimmer et al., 1986; Warner et al., 1984; Fraser et al., 1987), including potentially (Ye.Therefore, the previous results (Lee et al., 1987) are consistent with the present study. Since & and a1 gap junction RNAs were detected in the 5.5 days p.c. decidua/embryo sample, these gene products may be involved in the communication between embryonic tissue and uterus. The & RNA was modulated in this sample since it was diminished in the 6.5 and 7.5 days p.c. samples, while it increased again at 9.5 days p.c. The reason for this modulation is not known; however, the increase may be related to placental development. The kidney represents an organ, like the uterus (Risek et al,, 1990), where the expression of a1 is modulated up and down at different stages of kidney development. Interestingly, the a1 expression pattern in kidney, like placenta, decreased during development. One possible explanation for the high level of expression of LYE in fetal kidney is that the developing kidney is highly vascularized, and this vascularization (which utilizes cyl) decreases with development. It is interesting to note that between 11.5 and 15.5 days p.c. the expression of & in the liver was relatively constant at a low value. Subsequently, p1 increased dramatically at 17.5 days p.c. and was maintained at this increased level throughout later stages. This is intriguing because AFP synthesis peaks at 15.5 days p.c., decreasing at later stages (Janzen et al., 1982). It has been suggested recently that AFP synthesis is related to a loss of junctional communication in the liver (Gleiberman et al., 1989). It remains to be determined if there is a causal relationship between the low levels of & and & at stages up to 15.5 days p.c. and AFP synthesis at these stages. The observed modulation of gap junction genes during organogenesis may be related to the differences in communication requirements that exist for fetal organ development and adult organ function. Alternatively, the modulation may simply reflect the use of different genes by different cells that contribute to organogenesis at different stages of development. This latter possibility can be clarified in the future by in situ hybridization analysis. Analysis of F9 cells indicated that & was only detected in visceral endoderm-like cells. Since the visceral endoderm appears to mimic the fetal liver, the finding of P1and ,& predominately in these cells is in agreement with the presence of these two products in mouse liver (Nicholson et al., 1987). Thus, it may be concluded that in

F9 cells the gap junction transcripts analyzed in this study are modulated during differentiation in a specific manner that is in agreement with the analysis of fully differentiated tissues. The finding of multiple gap junction transcripts in F9 cells and their derivatives is the first demonstration that gap junctional gene expression can be modulated during the differentiation of a single cell type in culture. At later stages when RNA from whole embryos was analyzed, it is clear that there was an overall temporal pattern of expression for the gap junction genes with w1 being expressed first, followed by &, and then & gap junction RNA. This pattern resembles the situation in Xenopus where a temporal expression of gap junction genes has also been observed (Gimlich et ul., 1990). In whole embryos a specific transcript could go undetected because of dilution by other species of RNA. This could be avoided by enriching for the transcripts of interest by analyzing specific organs. Thus, in liver, & gap junction RNA was detected as early as 11.5 days p.c., in contrast to the situation with total embryo RNA. In the liver, & and & transcript levels remained relatively constant throughout development, indicating a close coordinate regulation of these two genes in this organ. Although it is not known if such regulation occurs in the same cell, it has been observed in the adult liver that both fll and & gap junction proteins exist in the same cell (Nicholson et al., 1987). It was also observed that many of the endoderm derived organs such as stomach, lung and liver expressed & and & RNA, whereas mesoderm derived organs such as heart, spleen, and muscle (data not shown), exclusively expressed a1 gap junction RNA. However, the distribution of a1 gap junction transcripts appeared to be more widespread, since it was present in many endodermal derived organs as well. In addition, the absence of & in the thymus, an endodermally derived organ, suggests that not every organ derived from the endoderm expresses &. Finally, the three gap junction transcripts analyzed were expressed at different periods of development for each organ. Thus, it can be concluded that each of the gap junction genes (a,, &, and &) is differentially regulated during development, but some coordinated regulation may occur within an organ. Each organ consists of an intricate mixture of many cell types in a similar environment. Possibly by using a unique expression pattern for different gap junctional products, the cells within the diverse population can retain their individuality. The authors gratefully acknowledge Dr. S. Strickland, L)r. D. Wang, and Dr. 1’. Wasserman for their generous access to the mouse cDNA libraries, Dr. E. Adamson and Dr. L. Gudas for their advice on the F9 tcratocarcinoma cells, Dr. J. Price for assistance in obtaining the mouse embryos, Dr. M. Mesnil for the dye-transfer studies, and Theresa Byrd-Tallcy, Rchecca Cochran, and Laura Goe for their secretarial

assistance. This work was supported by NIH Grants GM 37904,(N. B. Gilula), GM 37907 (N. 8. Gilula and N. M. Kumar) and EYO6884(N. M. Kumar).

REFERENCES ALBERTINI, D. F., and ANDERSON,E. (1974). The appearance and structure of intercellular connections during ontogeny of the rabbit ovarian follicle with particular references to gap junctions. .I &ll Biol. 63,234-250.

BARRON,D. J., VALDIMARSSON,G., PAUL, D. L., and KIDDER, G. M. (1989). Connexin 32, a gap junction protein is a persistent oogenetic product through preimplantation development of the mouse. %I,. Gem?. 10 318-72‘3. e . BEYER. E. i., PAUL, D. L., and GOODENOUGH, D. A. (1987). Connexin 43: A protein from rat heart homologous to a gap junction protein from liver. ,I. (B/I Rio/. 10.5,2621-2629. CHIRGWIN,J. M., PRZBYLA, il. E., MACDONALD, R. J., and RUTTER, W. J. (1979). Isolation of hiolo~ically active rihonucleic acid from sources enriched in rihonuclease. Biochwnistry 18, 5294-5299. DAVIS, L. G.. DIBNER. M. D., and BATLERY,J. F. (1986). “Methods in Molecular Riolog)-.” Elsevier, New York. DEKEL, N., LAWRENCE,T. S.. GILULA, N. B., and BEERS,W. H. (1981). Modulation of cell communication in the mammalian cumulus-oocyte complex. L)PP. Hid. 86, X6-362. DUCIBELLA, T., ALBERTINI, T., ANDERSON,E., and BIGGERS,J. D. (7975). The preimplantation mammalian embryo: Characterization of intercellular junctions and their appearance during development. Drc Bird. 45, 231-250. DZIADEK, M., and ADAMSON,E. (1978). Localisation and synthesis of
FLACH, G., JOHNSON,M. H., BRAUDE, P. R., TAYLOR, R. A. S., and BOLTON,\‘. N. (198%).The transition from maternal to embryonic control in the 2 cc~llmouse embryo. h’Ml3O.J. 1, 681-686. FLEMING,T. I’., MCCONNELL,J., JOHNSON,M. H., and STEVENSON,B. R. (1989). Development of tight junctions de novo in the mouse earls cmhryo: (‘ontrol of assembly of the tight junction-specific protein ZO-1. .I. (“VII Kid. 108, 1407-1418. FRASER,S. E.. GREEN, C. R., BODE, H. R.. and GILULA, N. B. (1987). Selective disruption of gap junctional communication interferes with a patterning process in Hydra. Scic>r/rc237, 49-55. GILULA, N. B., EPSTEIN,M. L., and BEERS,W. H. (1978). Cell communication and ovulation. ,1 (‘fd/ Rid. 78, 58-75. GIMLICH, R. I,., KUMAR, N. M., and GILULA, N. B. (1990). Differential regulation of the levels of three different gap junction mRNAs in X~r/o[~/t.sembryos. .J. (‘cl! Bid. 110, 597-605. GLEIBERMAN,A. S.. SHAROVSKAYA,Y. Y., and CHAILAKHJAN, L. M. (19x9). “Contact inhihition” of tu-fetoprotein synthesis and junctional communication in adult mouse hepatocgte culture. Ex/I. Co// nei 184, 22%‘V4. -t GOODALL,H., and JOHNSON,M. H. (1982). IJse of carhoxgfluorescrin diacrtate to studs- formation of permeable channels between mouse hlastomews. IVU/UR 295, 524-526. GOODALLH., and JOHNSON,M. H. (1984). The nature of intercellular coupling lvithin the preimplantation mouse embryo. ,J. Fwhrqrd E.rji.

Xo~~j~hol. 79, S-76.

130

DEVELOPMENTALBIOLOGY

GOODALL,H., and MARO, B. (1986). Major loss of junctional coupling during mitosis in early mouse embryos. J. Gel2Biol. 102, 568575. GUTHRIE, S. C. (1987). Cell-to-cell communication. In “Intercellular Communication in Embryos.” W. C. DeMello, Ed., pp. 223-244. Plenum, New York. GUTHRIE,S. C., and GILULA, N. B. (1989). Gap junctional communication and development. TINS 12,12-16. HOGAN, B., COSTANTINI,F., and LACY, E. (1986). “Manipulating the Mouse Embryo, A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. JANZEN,R. G., ANDREWS,G. K., and TAMAOKI, T. (1982). Synthesis of secretory proteins in developing mouse yolk sac. Dev. BioL 90,18-23. KIDDER, G. M., GREEN,A. F., and MCLACHLIN,J. R. (1985). On the use of cu-amanitin as a transcriptional blocking agent in mouse embryos: A cautionary note. J. Exp. Zoo1 233,155-159. KUMAR, N. M. (1991). Gap junctions. A multigene family. Adv. Struct. Bid., in press. KUMAR, N. M., and GILULA, N. B. (1986). Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein. J. Cell Bid. 103, 767-776. LARSEN, W. J., WERT, S. E., and BRUNNER,G. D. (1987). Differential modulation of follicle cell gap junction populations at ovulation. Den Biol. 122, 61-71. LEE, S., GILULA, N. B., and WARNER, A. E. (1987). Gap junctional communication and compaction during preimplantation stages of mouse development. Cell 51,851-860. LO, C. W., and GILULA, N. B. (1979a). Gap junctional communication in the preimplantation mouse embryo. Cell l&399-409. Lo, C. W., and GILULA, N. B. (197913).Gap junctional communication in the postimplantation mouse embryo. Cc/l 18,411-422. MAGNUSON,T., DEMSEY,A., and STACKPOLE,C. W. (1977). Characterization of intercellular junctions in the preimplantation mouse embryo by freeze-fracture and thin-section electron microscopy. Deu. Biol. 61, 252-261. MCLACHLIN,J. R., CAVENEY,S., and KIDDER, G. M. (1983). Control of gap junction formation in early mouse embryos. Dev. Biol. 98,155164. MILKS, L. C., KUMAR, N. M., HOUGHTEN,R., UNWIN, N., and GILULA, N. B. (1988). Topology of the 32.kD liver gap junction protein determined by site-directed antibody localizations. EMBO J. 7, 2967 2975.

MOOR,R. M., SMITH, M. W., and DAWSON,R. M. C. (1980). Measure-

VOLUME146.1991

ment of intercellular coupling between oocytes and cumulus cells using intracellular markers. Exn. Cell Res. 126, 15-29. NICHOLSON,B., DERMIETZEL,R., TEPLOW,D.,TRAuB, O., WILLECKE,K., and REVEL, J. P. (1987). Hepatic gap junctions are comprised of two homologous proteins of MW 28,000and 21,000.Nature 329,732-734. NICHOLSON,G. A., YANAGIMACHI, R., and YANAGIMACHI, H. (1975). Ultrastructural localization of lectin binding sites in the zona pellucida and plasma membrane of mammalian eggs. J. Cell BioL 66, 263-275.

PIKO, L., and CLEGG,K. B. (1982). Quantitative changes in total RNA, total poly(A), and ribosomes in early mouse embryos. Den. BioL 89, 362-378.

RAPPOLEE,D. A., WANG, A., MARK, D., and WERB Z. (1989). Novel method for studying mRNA phenotypes in single or small numbers of cells. J. Cell Biochem. 39, 1-11. RISEK, B., GUTHRIE,S., KUMAR, N., and GILULA, N. B. (1990). Modulation of gap junction transcript and protein expression during pregnancy in the rat. J. Cell BioL 110,269-282. ROBERTSON,E. J. (1987). “Teratocarcinomas and Embryonic Stem Cells, A Practical Approach.” IRL Press, Oxford. ROBERTSON,E., BRADLEY, A., KUEHN, M., and EVANS, M. (1986). Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature 323, 445-448. SAMBROOK,J., FRITSCH,E. F., and MANIATIS, T. (1989). “Molecular Cloning, A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. STRICKLAND,S., SMITH, K. K., and MAROTTI, K. R. (1980). The hormonal induction of differentiation in teratocarcinoma stem cells: Generation of parietal endoderm by retinoic acid and dibutyryl cyclic AMP. Cell 21, 347-355. TACHI, S., TACHI, C., and LINDNER, H. R. (1970). Ultrastructural features of blastocyst attachment and trophoblastic invasion in the rat. % Reprod. Fertil. 21, 37-56. WARNER,A. E., GUTHRIE, S. C., and GILULA, N. B. (1984). Antibodies to gap junctional protein selectively disrupt junctional communication in the early amphibian embryo. Nature 311,127-131. ZHANG,J., and NICHOLSON,B. J. (1989). Sequence and tissue distribution of a second protein of hepatic gap junctions, Cx26, as deduced from its cDNA. J.Cell BioL 109, 3391-3401. ZIMMER,D. B., GREEN,C. R., EVANS, W. H., and GILULA, N. B. (1986). Topological analysis of the major protein in isolated intact rat liver gap junctions and gap junction-derived single membrane structures. J. BioL Chem. 262,7751-7763.