Tissue-specific expression of the mouse Q10 H-2 class-I gene during embryogenesis

145

Gene, 61 (1987) 145-154 Elsevier GEN 02229

Tissue-specific (Recombin~t cells)

expression of the mouse QlO II-2 class-1 gene during embryogenesis DNA; major histocompatibility complex; secreted antigen; development; hematopoiesis; stem

Brigitte David-Watine, Catherine Transy, Gabriel Gachelin and Philippe Kouritsky Unit& de Siologie ~o~~culaire du G&e, U. 277 ICIER

- U.A.C. I IS CNRS, lnstitut Pasteur,

75724Paris Ckdex I5 (France)

Received 12 August 1987 Revised 29 September 1987 Accepted 1 October 1987

SUMMARY

We have studied the pattern of expression of the Ql0 gene, a H-2 class-I gene located in the major histocompatibility complex which encodes a soluble class-1 molecule, in the mid-gestation mouse embryo, and compared it to those of two other class-1 genes, namely Kd and 37, the latter gene located in the thymus leukemia region We found that the steady-state amount of these different mRNAs gradually increased from day 13 to day 18. By comparison with the level of expression of these genes in adult liver, the increase during gestation was fairly more marked for QlO mRNA than for the others. F~he~ore, we found that the QlO gene is transiently expressed in the endoderm layer of the visceral yolk sac and in the fetal heart. Expression in the latter tissue decreases abruptly while increasing in the liver. It has been proposed that the QlO protein is involved in immune tolerance. However, the time course of expression of QlO mRNA and its tissue distribution during embryogenesis suggest that the 010 protein could play a role in the differentiation of hematopoietic stem cells.

INTRODUCTION

The H-2 class-1 multigene family comprises 30 to 40 closely related genes located in the MHC on chromosome 17 of the mouse. Most of these genes code for cell-surface 45kDa glycoproteins, noncovalently associated to 82-microglobulin (Steinmetz et al., 1982; Weiss et al., 1984). The classical Correspondence to: Dr. B. David-Watine, Unite de Biologie Moleculaire du Gene, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris CCdex 15 (France) Tel. (1)45.68.85.45.

Abbreviations: AC, acetate; AFP, a-feto protein; bp, base pair(s); H-2, ‘histocompatibility (gene) 2’; hybridization buffers,

highly polymorphic H-2 antigens, encoded by a few (two to six) genes located in the H-2D/L and H-2K regions, serve as r~o~ition elements for both allospecific and virus-specific or H-2-restricted cytotoxic T cells. Thus, like class-II MHC molecules, they may present fragmented rather than nominal antigens (Townsend et al., 1986; Kourilsky et al., 1987). They are expressed at the surface of most see MATERIALS AND METHODS, section c; kb, kilobase or 1000 bp; MHC, major histocompatibility complex; nt, nucleotide{s); PBS, phosphate buffered saline; PolIk, Klenow {large) fragment of E. coli DNA polymerase I; SDS, sodium dodecyl sulfate; TL, thymus leukemia (Iocus); TLa, thymus leukemia antigen; VYS, visceral yolk sac.

037X-l119/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)

146

adult somatic cells. All other class-I genes have been assigned to the TL region of the MHC (Winoto et al., 1983; Weiss et al., 1984; Stephan et al., 1986). These include the genes encoding the serologically defined Qa-Tla cell-surface antigens, the expression of which is restricted to certain cell lineages, or even to certain differentiation stages (Flaherty, 1980; Rabinowitz et al., 1986). Their biological roles are as yet unknown, and it may be that at least some of them have a non-immunological function. The QIO class-I gene is located in the Tla region. It displays several original features. First, it has been reported as being expressed only in liver cells of the adult (Cosman et al., 1982). Second, the QJO gene sequence encodes a polypeptide with a modified transmembrane domain and is devoid of an intracytoplasmic domain. Accordingly, a QlO molecule has been detected as a soluble protein in the serum of most mouse strains (Devlin et al., 1985; Kress et al., 1983). Two mRNA products from the unique QlO gene have so far been detected: one obeys the conventional splicing rules for class-I antigens, whereas the other lacks the third exon which encodes the second domain of class-I antigens (Lalanne et al., 1985). Finally, the synthesis of QlO mRNAs has also been investigated during embryogenesis by Northern blot analysis using a QlO specific probe, and compared with that of all others H-2 class-I genes in RNAs using a non-specific class-1 gene probe. QlO RNA is detectable in the emb~onic liver since day 12 and in the visceral yolk sac (VYS) of day 16 embryos of the outbred mouse strain CD1 (Stein et al., 1986). We report here comparative studies on the expression of the QIO gene during early embryogenesis, in a genetically well defined inbred strain of mouse, namely the DBA/2 (H-2d) strain, From the embryological view point, the liver can be considered as being predominantly of endodermal origin. In addition, from day 11 of embryonic development up to two weeks after birth, the liver is an important haematopoieti~ organ, as it is colonized by haematopoietic stem cells of mesodermal origin. The VYS also acts as a primary then accessory erythropoietic organ until day 13 of gestation. VYS is composed of endoderma and extra embryonic mesoderm layers that can be separated following mild enzymatic treatment (Hogan et al, 1986). The very similar functions supported by the VYS and the fetal liver in hematopoiesis (Meehan et al., 1984) led

us to investigate the QlO gene expression in these two organs during the second half of mouse embryogenesis, as well as in relevant organs taken from embryos at, or greater than, day 12 of gestation. It is known that the expression of class-I mRNAs and/or class I antigens is very low before this time (Ozato et al., 1985; Morello et al., 1985). We have also examined several embryonic organs (heart, head of day-14 embryo) and embryonic membranes which are not of endodermal origin. As controls for the expression of the QlO gene, we have studied the expression of two other class-I genes: the H-2Kd gene and its two 5’ splicing products (Lalanne et al., 1983; 1985; Transy et al., 1984), and the 37 gene, a non-polymorphic gene of the TL region which might encode a so far undescribed class-I molecule, found transcribed in all adult tissues so far studied (Lalanne et al., 1985; Transy et al., 1987).

MATERIALS

AND METHODS

(a) Mouse embryos

Embryos from day 12.5 to day 18 were obtained from DBA/2 x DBA/2 matings. The presence of a vaginal plug was taken as day 0 of pregnancy. The embryos, once harvested from the pregnant mice, were immediately dissected in PBS (per liter: 0.257 g NaH,PO, +H,O; 2.250 g Na,HPO,* H,O, 8.767 g NaCl; final pH 7.4; without calcium and magnesium); all organs, placenta and yolk sac were washed in PBS and frozen in liquid nitrogen. Visceral yolk sacs of day-13-14 embryos were separated by enzymatic digestion into its endodermal and extra-mesodermal layers (Hogan et al., 1986). (b) Preparation of RNA

Total RNA from tissues was prepared using the procedure of Cathala et al. (1983) by homogeneization in 5 M guanidine monothiocyanate and precipitation in 4 M LiCl. Preparations from embryonic tissue were carried out on pooled organs of 5-10 embryos. Total RNA from cells cultured in vitro were prepared using the hot phenol procedure (Sherrer, 1969).

(c) Sl nuclease mapping

Sl nuclease was purchased from BoehringerMannheim. The probes used were: (i) a MstII-Hi&I 556bp fragment of the J? probe ~H-2~-24 labeled by with [ y-32P]ATP and T4 kinase (Maxam and Gilbert, 1980) at the MstII site for the analysis of Kd mRNAs (Transy et al., 1984). This probe is protected by the E;“imRNA over 122 bp and by another mRNA of unknown origin over 289 bp. (ii) A 1200-bp Bali-EcoRI fragment of ~H-2~-18 labeled at the Ball: site for the analysis of QltI mRNA (Lalanne et al., 1985). This probe protects two bands at 330 and 450 bp which corresponds to the two mRNAs of QlO. Ten pg of plasmid DNA was used to produce each probe. Aliquots (about 20 000 cpm) of each probe were co-precipitated with 20 to 50 pg of total RNA, then resuspended in 30 yl of hybridization buffer (0.4 M NaCl, 40 mM Pipes, pH 6.4, 1 mM EDTA and 80 % formamide). After heating 10 min at 85”C, the samples were incubated 16 h at 60’ C for hybridization. S 1 nuclease digestion was performed for 2 h at 37’ C with 100 units of enzyme in 300 ~1 of buffer containing 0.2 M NaCl, 30 mM NaAc, pH 4.5 and 3 mM ZnAc (Favolaro et al., 1980). After phenol extraction and ethanol precipitation, the samples were subjected to electrophoresis in a 6% polyacrylamide-8 M urea gel, together with HpaII digested pBR322 as markers, labeled with [a-3zP]dCTP using PolIk. The gels were usually run during 4 h at 1400 V, then fixed in acetic acid and methanol (10 : 10% v/v) and dried at 80°C for 2 h.

same way starting from the 250-bp PstI fragment corresponding to the second domain of ~H-2~-37 (Transy et al., 1987). The plasmids were digested by l%dIII, phenolchloroform-extracted, ethanol-precipitated and adjusted to 1 mg/ml. Preparation of [cr-32P]UTPlabeled probe was done according to the procedure of Melton et al. (1984). Aliquots of probe (2.4 x lo5 cpm) were co-precipitated with total RNA as for S 1 mapping. The samples were resuspended in 30 ~1 of hybridization buffer. After heating 10 min at 85”C, the samples were incubated 16 h at 45°C for hybridization. RNase digestion was performed for 90 min at 37 oC with RNase A (4 ~g/ml) and RNase Tl (2 @g/ml) in 300 ~1 of buffer (0.3 M NaCl, 10 mM Tris * HCl, pH 7.5, 5 mM EDTA). RNase digestion was terminated by the addition of 20 ~1 10% SDS and 50 pg of proteinase K and an additional incubation at 37°C for 15 min. After extraction with phenolchloroform, and precipitation with 10 pg tRNA and ethanol, the precipitate was analysed by denaturing 6% acrylamide-8 M urea gel electrophoresis, under conditions essentially the same as for Sl mapping experiments. RNase A and TI were purchased from Sigma.

RESULTS

(a) Expression of the Q-l0 gene during mid-embryonic development (1) Expression in embryonic liver cells

(d) RNAse mapping

The QlO-riboprobe, Ribo-QlO-250, was obtained by subcloning the 250-bp PstI fragment containing a sequence specific for the QlO gene (Cosman et al., 1982) isolated from the 3’noncoding region of the ~H-2~-19 cDNA (Lalanne et al., 1985) into the PSP65 (Melton et al., 1984) vector at the PstI site; the orientation was determined to be an anti-sense mRNA alter polymerization with the SP6 polymerase. Ribo-QlO-110 was obtained by deleting a poly(A)+ stretch and a stretch of GC from the const~ction of the cDNA from the 250-bp PstI fragment of the ~H-2~-19 cDNA and cloning the fragment (as a 242-bp doublet) into PSP65. The 37-riboprobe Ribo-37.2 was prepared in the

Embryos of DBAj2 mice were taken at various times of pregnancy and dissected. The analysis of QlO gene expression was performed on total RNA isolated from day 12, 13, 14, 18 DBA/2 fetal livers, and from the livers of 6-week-old DBAj2 mice. We performed an RNase mapping analysis (Melton et al., 1984) using the ribo-QlO 250 probe which is unique for the QlO gene product. Results in Fig. 1 show protection of a major 145-bp fragment as expected for the protection of the QlO probe by the homologous RNA. The protection pattern is identical at all stages, indicating the expression of the QlO gene, although variable, as early as day 12 (Fig. lA, lanes e to h). Dilutions of total RNA from adult liver were made in order to allow a semi-quantitative comparison

148

4 probe

160t

*I

147,

a

bcdefg

h

-QlO i 1’

i

122,

-% abcdefghi

C Fig 1. Expression

of QZO mRNA

adult and the embryo, tion mapping.

an analysis

in different performed

Total RNAs were hybridized

Ribo-QlO-250

probe

(panels

probe (panel C) overnight

tissues

from the

by RNase

protec-

with aliquots

of the

A and B) or the Ribo-QlO-110

at 45°C in 30 ~1 ofhybridization

buffer

1 mM EDTA and 80%

(0.4 M NaCl, 40 mM PIPES,

pH 6.4,

formamide),

10 min at 85°C for denaturation.

after being heated

RNase digestion

was performed

A (4 pg/ml) and RNase

for 90 min at 37°C with RNase

Tl (2 pg/ml) in 300 ~1 of buffer (0.3 M

NaCl, 10 mM Tris . HCl, pH 7.5,5 mM EDTA). RNase digestion was terminated

by the addition

proteinase

K and additional

extraction

with phenol-chloroform

tRNA and ethanol, blue]

is dissolved

MATERIALS

with 10 pg

in 4 ~1 of loading

0.1% (w/v) xylene-cyanol,

(see

sections b and d). Autoradiogram electrophoresed

at 37°C for 15 min. After

and precipitation

the precipitate

buffer [80% formamide, bromophenol

of 20 /.II 10% SDS and 50 pg

incubation

AND

0.1 y0 (w/v) METHODS,

ofthe RNase resistant

on a 6% polyacrylamide-8

material

M urea denaturing

gel are shown. The M, markers

were s2P-end-labelled

the HpaII restriction

of pBR322: (A) lanes a and n; (B)

fragments

lane a; (C) lane d. Size numbers nt. (A) Comparison

with different

indicated

DNA from

in the margin

amounts

are in

of total RNA from

adult liver. (B) Adult liver and fetal liver and heart. The fragment specific

for the QlO mRNA

non-digested

abc

d

is about

probe Ribo-QlO-250

total RNA is analysed

143-145

is 240-242

nt long. The

nt long. 10 pg of

in each assay and yeast tRNA

is added

149

between the embryonic and adult levels of QlO gene expression using identical amounts of total RNA (Fig. IA, lanes j-m). The level of QIO mRNA in adult liver is about five times the expression observed at day 18 (Fig. IB, lanes c, d). It is much lower and variable in day 12-14 liver cells (Fig. lB, lanes e-g). However, it should be noted that the intensity of the signal does not appear to be proportional to the amount of mRNA added (Fig. IA, lanes j, k, 1, m). The preparations of RNAs from a given embryonic stage are independent from each other: it should be pointed out that in a given progeny, not all of the embryos have matched the very same stage. This is probably the reason why the signal obtained with the RNA from day-12 embryonic liver (Fig. lA, lane g) is stronger than the signal obtained with liver RNA of the same stage of development on Fig. 1B (lane g). The relative incertainty concerning the age of the embryos holds true also for older embryos. Because two alternative mRNA products of the QIO gene were detected in the adult liver (Lalanne et al., 1985), we carried out Sl mapping analysis using a DNA probe derived from ~H-2~-18 (Lalanne et al., 1985) hybridized with mRNAs isolated from the liver of adults and embryos. As shown in Fig. 2A (lanes d-h, lanes b and c showing the size of the probes and lane i being a control for digestion), we detected the same relative amounts of the two Q10 mRNA products, just as in the adult liver: a double

to dilutions undigested

of liver RNA

up to 10 pg. The position

probe and the protected

mRNA arc indicated of the different indicated

by arrows

RNAs (organ,

fragment

on the right margins. stage of development

The origin in days) is

above each lane. (A) Lanes: b, c, d, VYS RNA of day

14, 13, 12; e, f, g, fetal liver RNA ofdays RNA;

of the

specific for the QlO

i, adult kidney

14, 13, 12; h, adult liver

RNA; j, k, 1, m, dilutions

of adult liver

RNA, 100 ng, 200 ng, 1 ug, 10 pg. (B) Lanes: b, undigested

probe;

c, adult liver RNA (5 pg); d, fetal liver RNA (25 pg); e, f, fetal liver RNA of days

14 and 13 respectively

(25 p(p); g, fetal liver

RNA of day 12 (12 pg); h, i, fetal liver RNA of days 14 and 13 (12 pg). (C) Expression in its separated

layers

a). The protected

section

(95 nt) is indicated digested

fragment

by an arrow

RNA

Ribo-QlO-110

from

VYS;

layer

of VYS; c, total

endodermal

layer

of VYS; d, @aI1

digested

on the right margin

are in nt.

indicated

total

RNA RNA

The nonis 240 nt

section d). Lanes: a,

mesodermal numbers

METHODS,

on the left margin.

AND METHODS, b,

in the yolk sac and AND

specific for the QlO mRNA

probe used in the experiment,

long (see MATERIALS total

of the QlO mRNA (see MATERIALS

from from

the

separate

the separate pBR322;

size

intense band at 450 bp and a single fainter band at 330 bp. As a control (lanes c-h) we use /I-actin mRNA which is expressed evenly during embryogenesis, even in the liver, as a result of the high rate of cell division of this organ. /?-Actin is no longer a good control in the adult tissue where the rate of division of liver cells is decreasing. (2) Visceral yolk sac (VYS) During late embryogenesis it has been found that fetal liver and VYS synthesize similar sets of serum proteins (Meehan et al., 1984). In a similar way, we have found that the QIO gene is expressed by VYS cells on days 12-13. Stein et al. (1986) have shown that Q10 mRNA decreases very quickly after day 15. By contrast, neither 12-14-day total placental cells, nor cells of the PYS-2 cell line (which simulates parietal yolk sac cells) expressed the QlO mRNAs (Fig. 2B, lanes g-i). Because the genuine VYS is composed of two distinct layers, in addition to hematopoietic cells, we addressed the question of the specific expression of the QIO gene in the endodermal layer of VY S, after it was separated from the extra-embryonic mesodermal layer (Hogan et al., 1986) (Fig. lC, lanes a-c). The QlO mRNAs are detected only in endodermal cells. The endodermal or extra-embryonic mesodermal origin of the two mRNA preparations was controlled for by hybridization with an AFP probe, since the expression of the AFP gene is known to be restricted to the endodermal layer of the VYS (Dziadek et al., 1983) (not shown). The VYS endoderm derives from cells of the primitive endoderrn that do not colonize endodermal tissues of the fetus (Gardner, 1983), whereas the primitive ectoderm lineage gives rise to the ectodermal, mesodermal and endodermal tissues of the fetus, to germ cells and to the mesodermal components of the extra-embryonic membranes and of placenta (Gardner and Rossant, 1979). However, the visceral endoderm of VYS shares some differentiation properties with the endoderm-derived cells of the fetal large intestine and fetal liver (Meehan et al., 1984). We have thus examined for the expression of QlO mRNAs extracted from different organs of mainly mesodermal and ectodermal origin in fetal and adult mouse. We could detect a signal in mRNAs prepared from fetal heart at day 13 but not at day 14 as analysed by

150

A

Qt9 probe) 622w 527, Ractin

c I,

UR mm

*

-450010

4 probe 622 L527 -450

a10

4 330 a10

a

bC(Iefghi

m

abcdefghi

j

j

Fig. 2. Expression of the two mRNA products of the QlO gene analysed with the S 1 mapping technique. Total RNAs are co-precipitated with aliquots (about 20000 cpm) of the Ball-EcoRI probe (see MATERIALS AND METHODS, sections band c) and the /I-actin probe (a 430-bp BglII-HinfI fragment from the pAL41 plasmid containing a p-actin cDNA (Alonso et al., 1986; s2P-end-labeled at the BglII site). The samples are heated at 85 “C for 10 min and then incubated for 16 h at 60°C for hybridization in 300 ~1 of hybridization buffer (see Fig. 1). Sl nuclease digestion is performed for 2 h at 37°C with 100 units of enzyme in 300 ~1 of buffer containing 0.2 M NaCl, 30 mM NaAc, pH 4.5 and 3 mM ZnAc. After phenol extraction and precipitation, the samples are resuspended in 2 ~1 of loading buffer (80% formamide, 0.1% (w/v)xylene-cyanol, 0.1% (w/v) bromophenol blue). Autoradiograms ofthe Sl-resistant material electrophoresed on a 6 % polyacrylamide-8 M urea denaturing gel are shown. (A) In the liver at different stages of development with S-actin mRNA used as control. Lanes: a, j, ffpaI1 digested pBR322 DNA. The position of undigested probes is indicated by arrows on the left (probe). The position ofthe fragments specific for each QlO mRNA is indicated by arrows on the right margin (330-450 nt) as are the protected fragments of /I-actin (180-200 nt). Lanes: b, undigested QZOprobe, 1200 bp Bali-EcoRI fragment 32P-end-labeled at the EalI site (see MATERIALS AND METHODS, section c); c, undigested actin probe, 430-bp EgZII-HintI fragment, 32P-end-labeled DNA at the BglII site; d, adult liver RNA; e, f, g, h, fetal liver RNA from days 18, 14, 13 and 12, respectively; i, control of digestion with yeast tRNA hybridized with the two probes and treated the same as all the other samples. (B) Expression of the two mRNA products of the QZO gene in different organs from the adult and the embryo. Lanes: a, HpaII digested pBR322 DNA. The position of undigested probe and the fragments specific for the two QZOmRNA (330-450 nt) are indicated by arrows on the right margin; b, control of digestion, yeast tRNA; c, adult liver RNA; d, adult kidney RNA; e, adult heart RNA; f, fetal heart RNA of day 13; g, RNA from the PYS-2 cell line; h, VYS RNA; i, RNA from placenta of day 12; j, adult thymus RNA.

RNAse mapping with the Ribo-QlO-250 probe (Fig. lB, lanes h, i). We could also detect it in the adult kidney but not in adult heart as shown on Fig. 2B (lanes d, e, f), in an Sl mapping experiment, using day- 13 fetal heart mRNAs as comparison, nor in spleen or lymph nodes, all organs of nonendodermal origin (not shown). mRNAs from the fetal head (day 14), adult brain and thymus, all organs which are predominantly of ectodermal

origin, did not give any signal, even upon exposure (Fig. 2B, lane j as an example).

over-

(b) Analysis of the expression of the Kd gene and the 37 gene in fetal liver The expression of two other H-2 class-I genes, Kd and 37 was analysed separately with most of the mRNAs studied above, as controls for the analysis

151

557,

:r 2

527

4

-

I

-

II

299

of the expression of the QlO gene. It was interesting to analyse the expression of the recently discovered 37 gene (Lalanne et al., 1985) which may encode a poorly polymorphic, ubiquitous class-I antigen (Transy et al., 1987). The Kd gene encodes several transcripts produced by alternative splicing both in the 5’ and 3’ end of the gene; the Kd probe isolated from ~H-2~-24 which we used to perform a Sl mapping analysis of the liver mRNA allowed us to discriminate between the two transcripts alternatively spliced in the 5’ end of the gene (Transy et al., 1984). The normally spliced ZY-2KdmRNA protected a 122-bp band present but very faint from day 12 to day 14; since the alternative spliced mRNA gives a ten to 20 times less intense protected band of 289 bp in the adult liver (Fig. 3, lane b), this transcript cannot be detected in day-12-14 embryos (Fig. 3, lanes c-f).

4309

-

1,

Ir

122*

_

bcdefghi

Fig. 3. Expression organs.

different

of the two mRNA

products

The Sl mapping

556-bp

MATERIALS

242 t 238 -

I

a

MstII-Hi&

147

()

II

different

4

hagment

AND

amounts

assay

from ~H-2~-24

METHODS,

of the Kd gene in

217~

(see Fig. 2) and the are described

section c. The origin

in and

Fig. 4. Expression

from day 18 (12 pg); d, e, f, fetal liver RNA from days 14,13 and

RNase mapping

12, respectively

are described

h, placental

(25 pg); g, fetal heart RNA from day 14 (25 pg);

RNA (25 pg). The arrow

cates the position ofthe undigested

at 557 (left margin)

probe. The fragments

for Kd (122 bp) and for the other mRNA (289 bp) are also indicated pBR322.

Numbers

by arrows.

on the margins

product

of the K* gene

Lanes a, i, HpaII

are in nt.

indi-

specific digested

bcdefgh

a

of total RNAs used in each assay are indicated

above each lane. Lanes: b, adult liver RNA (12 pg); c, fetal liver

of the gene 37 mRNA in different tissues. The assay (see Fig. 1) and the Ribo-37.2

in MATERIAL

probe used

section d. The

AND METHODS,

origin of the RNAs used in each assay is indicated

above each

lane. Lanes: b, adult liver RNA; c, d, e, fetal liver of days 18, 14 and 13, respectively

(of embryonic

development)

heart RNA from day 14; g, placental specific fragment indicated

protected

on the right margin. are in nt.

f, fetal of the

by the Ribo-37.2

long. Lanes a, h, HpaII-digested left margin

RNAs;

RNA. The position

The undigested pBR322;

probe at 250 nt is probe is 305 bp

size numbers

on the

152

The RNase mapping analysis of the 37 gene in embryonic liver, using the Ribo-37.2 probe (Transy et al., 1987) is shown in Fig. 4 (lanes b, c, d, e). Gene 37 and gene Kd are expressed in the fetal heart (day 14) and in the placenta whereas Q1CJis not (Fig. 2B, lane i; Fig. 3, lanes h, i; Fig. 4, lanes f, g). The level of expression of these three gene products gradually increases in the liver from day 12-14 to day 18. The amount of the Q10 mRNA in the expressing organs (as analysed with two probes mapping in two different regions of the mRNAs) appears to be proportionally higher than those of Kd and 37 mRNAs, when compared to the adult.

DISCUSSION

Earlier studies on adult animals have suggested that the QlO gene is expressed only in adult liver cells and that the QlO molecule is secreted into the serum (Kress et al., 1983; Devlin et al., 1985). The QlO gene is also expressed during embryonic life. We confirm here, using DBA/2 inbred mice, the results reached by Stein et al. (1986) on embryos of the CD1 outbred mouse strain, namely that the QlO gene is expressed by the endodermal layer of the VYS up to day 14, as well as the fetal liver. In addition, we detected the expression of Q10 mRNA in other organs, such as the fetal heart (or more precisely the cardiac area until day 13) and the adult kidney, which are both mesodermal derivatives (preliminary results of in situ hybridization suggest the presence of QlO mRNA in the vicinity of the tubules). From these results we conclude that the expression of the QlO gene is not strictly associated with endodermal cells or tissues of endodermal origin as it may have been concluded from the results reported previously. It is worth pointing out that embryonic liver is a complex organ. The hepatic parenchyme is not only made up of hepatocytes but also of Kupffer cells, endothelial cells, etc.. . Hematopoietic stem cells are also present. In situ hybridization on histological sections of different organs from the adult and the embryo are in progress and should help in defining more accurately which cell types express QlO mRNA during development. Also, we were struck by the time course of the appearance and disappearance of QlO mRNA in these embryonic organs (VYS,

liver, heart) which correlates with difTerent steps of hematopoiesis in the developing embryo. The first erythropoietic stem cells differentiate in the VYS from day 7 of gestation until day 13-14: QlO gene is expressed in these tissues to this very date. This primitive erythropoiesis is taken over by hematopoietic differentiation which occurs in diffuse intraembryonic foci, which some cells leave to colonize the definitive hematopoietic organ rudiments, such as liver and spleen, during fetal life. These diffuse foci, described essentially in the chick embryo, surround the aortic area (Dieterlin-Lievre et al., 1981). If they also exist in the mouse embryo, these diffuse foci might account for the QlO positivity of the embryonic cardiac area. The QlO gene product has been implicated in a wide variety of possible functions, on the sole basis of its temporal and tissue-specific patterns of expression. It has been hypothetically involved in self tolerance (Cosman et al., 1982) or maternal tolerance to the fetus (Stein et al., 1986). In addition, whatever function the QlO molecule has in the adult, this function alone cannot be vital since the QlO protein is absent from the serum of H-2f adult animals (Lew et al., 1986). Its presence or absence in H-2’embryos has not been looked at so far. The function of QlO in the embryo is unknown as well, but might be different from that in the adult. On the basis of the time course of its expression in various embryonic tissues, we propose the hypothesis that QlO expression might be associated with the differentiation of embryonic hemopoietic stem cells, either as some sort of locally acting growth factor, or through cellto-cell contacts. In this respect, it should be noted that Q10 molecules might associate with the cell membrane through an anchoring lipidic domain, as described for other cell-surface antigens (Low et al., 1985; Stroynowski et al., 1987). Also, compartmentalization of the QlO molecule might exist, as was shown, for example, in the case of the CSF-1 molecule (Gordon et al., 1987). The participation of cellular and molecular components of the immune system to the differentiation of haematopoietic cells has long been discussed. Negative and positive regulation by T cells through the release of soluble factors (colony-stimulating factors, interferon y, other factors) is well documented, although this regulation appears to take place at later stages of differentiation (Marchal et al., 1986). The QlO

153

molecule

might be one of the components

complex

interactions.

of these

Gordon,

H.Y.,

Riley,

G.P.,

Compartmentalization (GM-CSF)

Nature

B., Costantini,

layers separated the Mouse

ACKNOWLEDGEMENTS

Harbor

This

work

Institut

was

National

supported

by grants

de la Sante

Medicale,

the Institut

Pasteur

ale contre

le Cancer.

B.D.-W.

fellowship

from the

et de la Recherche

awarded by the Minis&e

by a basement

marrow

E.: Separation

membrane.

A Laboratory

Cold Spring Harbor,

P., Chaouat,

G., Rabourdin-Combe,

principles

of tissue

In Manipulating

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