MOLECULAR AND CELLULAR BIOLOGY, JUlY 1990, p. 3801-3805 0270-7306/90/073801-05$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 10, No. 7
Developmental Expression of Myeloid Leukemia Inhibitory Factor Gene in Preimplantation Blastocysts and in Extraembryonic Tissue of Mouse Embryos FRANCOIS CONQUET AND PHILIPPE BRULET* Unite de Genetique Cellulaire du College de France et de l'Institut Pasteur, 28, rue du Dr Roux, 75724 Paris Cedex 15, France Received 13 November 1989/Accepted 9 April 1990
Murine leukemia inhibitory factor (LIF) protein is a growth factor which has the ability to maintain the developmental potential of pluripotent embryonic stem cells through a specific receptor. We have examined the expression pattern of the LIF gene from the preimplantation stage (3.5 days post coitum) to the midgestation stage (12.5 days post coitum) of the mouse embryo. LIF transcripts were detected at the preimplantation blastocyst stage, whereas no transcripts were detectable in embryonic stem cells. LIF gene transcription continued in the extraembryonic tissue of the 7.5-day and in the placenta of 9.5-, 10.5-, and 12.5-day post coitum embryos. No transcripts were detected in the embryo proper of the corresponding stages. Our results suggest that this growth factor is synthesized in the extraembryonic part of the embryo and acts on the embryonic tissues during early mouse development.
extraembryonic ectoderm layer of the 7.5-day p.c. embryo (Fig. 1C and E). Because maternal cells are known to contaminate extraembryonic tissues, it was important to assess the signal strength in decidua. As shown in Fig. 1D, no signal was detectable in maternal tissues, nor was a signal found in the embryo proper of the same stage (Fig. 1B). At 10.5 days p.c., a weak signal was detectable in the placenta only (data not shown). In order to confirm the data obtained by in situ hybridization, we used a very sensitive technique of transcription detection which consists of a specific reverse transcription (RT) step followed by amplification by PCR (RT-PCR) (13, 17) (Fig. 2). This method allowed detection of transcripts of low copy number in early embryos, obviating the need to obtain the large amounts of embryonic tissue required for standard RNA analysis. A total of 250 blastocysts were recovered at the stage 3.5 days p.c. and pooled in 5 p.l of phosphate-buffered saline (PBS). Ten 7.5-day p.c. embryos were harvested and detached from maternal tissue before being transversally separated along the constriction separating the embryo proper from the extraembryonic portion (Fig. 1A, arrowheads) (22), and the two parts were separately pooled in 5 p.1 of PBS prior to RT-PCR analysis (Fig. 2B). It is noteworthy that the ectoplacental cone is potentially contaminated by maternal cells. For this reason, a control RT-PCR was performed on decidual cells (Fig. 2B). Bisection of the 7.5-day p.c. embryo allowed clear separation of extraembryonic tissue from the embryo proper (Fig. 1); however, further distinction between various extraembryonic layers present at this stage of development, such as parietal (distal) yolk sac, visceral (proximal) yolk sac, and allantois, was not possible. For blastocysts and 7.5-day p.c. embryos, no RNA purification was performed, and RT-PCR was done directly on
Murine leukemia inhibitory factor (LIF) protein is known to have distinct in vitro biological activities. It induces macrophage differentiation in the Ml murine myeloid leukemia cells and suppresses their proliferation (10, 12), whereas it supports the proliferation of murine interleukin-3-dependent leukemic DA-la cells (15). In addition, it was recently demonstrated that LIF is identical to the cholinergic neuronal differentiation factor from heart cells (24). Furthermore, embryonic stem (ES) cells cultured in the presence of purified recombinant LIF retain their potential to form chimeric mice when injected into a blastocyst and allowed to colonize the germ line (21, 23). ES cells, which most closely resemble inner cell mass (ICM) cells (2), have a membrane receptor specific to LIF protein (21, 23). Taking into account these data, it was reasonable to think that LIF might play an important role during mouse embryogenesis. The LIF gene expression pattern during early mouse development was studied by two complementary techniques. It was first examined by in situ hybridization on 7.5and 10.5-day post coitum (p.c.) embryos (Fig. 1). The LIF probe used was an antisense 35S-labeled riboprobe synthesized from a fragment of the mouse LIF cDNA cloned by the polymerase chain reaction (PCR) (Fig. 2A) with the Bluescript T3-T7 transcription system (Stratagene). Sephadex G50-purified antisense probe was applied to a 7-p.m paraffin section (106 cpm per slide) (19). Autoradiographic exposure was for 3 weeks. A sense probe from the same fragment was used as a negative control on adjacent sections (Fig. 1F and G). A signal was detectable (about threefold over background) in the extraembryonic portion and particularly in the
*
Corresponding author. 3801
3802
NOTES
_'es4*
MOL. CELL. BIOL.
I
*
Vol. 10, No. 7
Developmental Expression of Myeloid Leukemia Inhibitory Factor Gene in Preimplantation Blastocysts and in Extraembryonic Tissue of Mouse Embryos FRANCOIS CONQUET AND PHILIPPE BRULET* Unite de Genetique Cellulaire du College de France et de l'Institut Pasteur, 28, rue du Dr Roux, 75724 Paris Cedex 15, France Received 13 November 1989/Accepted 9 April 1990
Murine leukemia inhibitory factor (LIF) protein is a growth factor which has the ability to maintain the developmental potential of pluripotent embryonic stem cells through a specific receptor. We have examined the expression pattern of the LIF gene from the preimplantation stage (3.5 days post coitum) to the midgestation stage (12.5 days post coitum) of the mouse embryo. LIF transcripts were detected at the preimplantation blastocyst stage, whereas no transcripts were detectable in embryonic stem cells. LIF gene transcription continued in the extraembryonic tissue of the 7.5-day and in the placenta of 9.5-, 10.5-, and 12.5-day post coitum embryos. No transcripts were detected in the embryo proper of the corresponding stages. Our results suggest that this growth factor is synthesized in the extraembryonic part of the embryo and acts on the embryonic tissues during early mouse development.
extraembryonic ectoderm layer of the 7.5-day p.c. embryo (Fig. 1C and E). Because maternal cells are known to contaminate extraembryonic tissues, it was important to assess the signal strength in decidua. As shown in Fig. 1D, no signal was detectable in maternal tissues, nor was a signal found in the embryo proper of the same stage (Fig. 1B). At 10.5 days p.c., a weak signal was detectable in the placenta only (data not shown). In order to confirm the data obtained by in situ hybridization, we used a very sensitive technique of transcription detection which consists of a specific reverse transcription (RT) step followed by amplification by PCR (RT-PCR) (13, 17) (Fig. 2). This method allowed detection of transcripts of low copy number in early embryos, obviating the need to obtain the large amounts of embryonic tissue required for standard RNA analysis. A total of 250 blastocysts were recovered at the stage 3.5 days p.c. and pooled in 5 p.l of phosphate-buffered saline (PBS). Ten 7.5-day p.c. embryos were harvested and detached from maternal tissue before being transversally separated along the constriction separating the embryo proper from the extraembryonic portion (Fig. 1A, arrowheads) (22), and the two parts were separately pooled in 5 p.1 of PBS prior to RT-PCR analysis (Fig. 2B). It is noteworthy that the ectoplacental cone is potentially contaminated by maternal cells. For this reason, a control RT-PCR was performed on decidual cells (Fig. 2B). Bisection of the 7.5-day p.c. embryo allowed clear separation of extraembryonic tissue from the embryo proper (Fig. 1); however, further distinction between various extraembryonic layers present at this stage of development, such as parietal (distal) yolk sac, visceral (proximal) yolk sac, and allantois, was not possible. For blastocysts and 7.5-day p.c. embryos, no RNA purification was performed, and RT-PCR was done directly on
Murine leukemia inhibitory factor (LIF) protein is known to have distinct in vitro biological activities. It induces macrophage differentiation in the Ml murine myeloid leukemia cells and suppresses their proliferation (10, 12), whereas it supports the proliferation of murine interleukin-3-dependent leukemic DA-la cells (15). In addition, it was recently demonstrated that LIF is identical to the cholinergic neuronal differentiation factor from heart cells (24). Furthermore, embryonic stem (ES) cells cultured in the presence of purified recombinant LIF retain their potential to form chimeric mice when injected into a blastocyst and allowed to colonize the germ line (21, 23). ES cells, which most closely resemble inner cell mass (ICM) cells (2), have a membrane receptor specific to LIF protein (21, 23). Taking into account these data, it was reasonable to think that LIF might play an important role during mouse embryogenesis. The LIF gene expression pattern during early mouse development was studied by two complementary techniques. It was first examined by in situ hybridization on 7.5and 10.5-day post coitum (p.c.) embryos (Fig. 1). The LIF probe used was an antisense 35S-labeled riboprobe synthesized from a fragment of the mouse LIF cDNA cloned by the polymerase chain reaction (PCR) (Fig. 2A) with the Bluescript T3-T7 transcription system (Stratagene). Sephadex G50-purified antisense probe was applied to a 7-p.m paraffin section (106 cpm per slide) (19). Autoradiographic exposure was for 3 weeks. A sense probe from the same fragment was used as a negative control on adjacent sections (Fig. 1F and G). A signal was detectable (about threefold over background) in the extraembryonic portion and particularly in the
*
Corresponding author. 3801
3802
NOTES
_'es4*
MOL. CELL. BIOL.
I
*
