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Localization of HstI transcripts to the apical ectodermal ridge in the mouse embryo
DEVELOPMENTAL
BIOLOGY
150,219-222 (19%)
BRIEF NOTE Localization of Hstl Transcripts to the Apical Ectodermal Ridge in the Mouse Embryo HIROAKI R. SUZUKI,* HIROMISAKAM~T~,-~TERUHIKOY~SI-IIDA,~TAKASHI MASAAKITERADA,~ANDMICHAELSOLURSH*
SumnmA,t
*Department of Biology, University of Iowa, Iowa City, Iowa 52242;and tNationa1 Cancer Center Research Institute, l-l, Tsukiji Cchme, Chueku, Tokyo 104,Japan Accepted November 18, 1991 The HstI gene is a transforming gene, coding for a protein of the fibroblast growth factor family (Sakamoto et al, 1986). Previous RNA hybridization studies with the mouse homolog demonstrated the presence of a 3.0-kb transcript in Day 11 and 14 mouse embryos. Here we detect a 3.0-kb transcript in the limb and body of the dissected Day 11 mouse embryo. PCR amplification using HstI-specific primers also showed comparable results. In order to localize the HstI transcripts during development, corresponding HstI cDNA was isolated, and an HstI-specific region was used as a probe for in situ hybridization analysis. Serial sections of embryos from Day 8 (early-somite stages) through Days 9,10,11, and 12 of gestation were examined. With the antisense probe, a signal was detected in the Day 11 and 12 embryo, where it was localized to the apical ectodermal ridge (AER) of the limb bud. This structure is well known for its role in promoting the distal outgrowth of the developing limb bud. Signal was detected in both fore- and hindlimbs during the period of rapid distal growth. This restricted localization suggests a role for HstI in normal embryogenesis, including outgrowth of the limb bud. o lssz Academic press, ~nc.
INTRODUCTION
MATERIALS AND METHODS
The RNA preparation and hybridization conditions Fibroblast growth factors represent a family of mitogenie proteins with related primary structures (Burgess were as described earlier (Yoshida et al, 1988). The and Maciag, 1989; Goldfarb, 1990). In the mammal there primers used in RT-PCR were nucleotides 1029 to 1044 plus 1474 to 1477, and antisense of nucleotides 1562 to are currently seven known family members, including 1543 (Brookes et al, 1989). PCR conditions were as folacidic FGF, basic FGF, INT-2 protein, HSTUK-FGF protein, FGF-5 protein, FGF-6/HST2 protein, and kera- lows: Denaturation, 94”C, 1 min; annealing, 6O”C, 1 min; tinocyte growth factor (KGF), each encoded by a dis- polymerization, 72”C, 1 min; 20 cycles, with Taq DNA tinct gene. FGFs are mitogenic to a large number of polymerase (Promega) in reaction mixture containing 1 ectodermally and mesodermally derived cells and can m&f Mg2+. In order to obtain the HstI cDNA probe, a XgtlO cDNA act as inducers of cell differentiation. While some inforlibrary (Amersham) was made from mouse embryonal mation is available concerning the temporal and spatial distribution of FGF family members during develop- carcinoma F9 cells maintained in the undifferentiated ment (Wilkinson et al, 1988; Joseph-Silverstein et al, state in which they express a high level of HstI mRNA 1989; Haub et ah, 1990; Haub and Goldfarb, 1991; Hebert (Yoshida et aZ., 1988). Screening the library with the et al, 1990, 1991; Fu et ab, 1991), little is known about HstI genomic probe Ml.8 led to the isolation of positive their normal function in situ. Compared to other FGF clones with the sequence identical to those reported by family members, HstI has a particularly restricted pe- others (Brookes et aL, 1989). A 557-bp fragment (seriod of expression, having been detected so far in the quence 1988-2544) encompassing part of the 3’ untranspreimplantation mouse embryo (Hebert et a& 1991) and lated region was subcloned into a pGEM-2 vector (ProDay 11 and 14 mouse embryo but not in the Day 17 em- mega) and used as a probe for in situ hybridization. In situ hybridization was performed on tissue sections bryo or the postnatal mouse by Northern analysis (Yoshida et aL, 1988). The purpose of this study is to de- of Balb/c strain mouse embryos according to the protoscribe the localization of HstI transcripts in the mouse col described previously (Suzuki et aL, 1991). Embryos were fixed in Bouin’s fixative and processed for paraffin embryo. 219
0012-1606/92 $3.00 Copyright All rights
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
220
DEVELOPMENTALBIOLOGYV0~~~~150.1992
-
kb 9.49 7.46
-
4.40
-
2.37 1.35
-
FIG. 1. Northern blot analysis of HstI transcripts in the Il.&day NIH/Swiss mouse embryo. RNA was prepared from the limb (lane l), head (lane 2), remaining body (lane 3), placenta (lane 4), and allantois (lane 5). One microgram each poly-A+ RNA was used with the mouse genomic H&I fragment, Ml.8 as a probe. The RNA preparation and hybridization conditions were as described earlier (Yoshida et al, 1988).
sectioning. %-labeled sense and antisense RNA probes were synthesized upon the linearized template. Stringent washes were carried out in 2~ SSC, 50% formamide at 6O”C, followed by 0.1X SSC at 50°C. RESULTS AND DISCUSSION
In order to detect the HstI message in different regions of the Day 11.5 mouse embryo, RNA blot hybridization was carried out on poly-A+ RNA isolated from limb buds, heads, remaining bodies, placentae, and allantois, utilizing the mouse HstI genomic fragment, Ml.8 as a probe. This probe encompasses exons 2 and 3 but not exon 1 (or GC-rich intron 1) which tends to cross-hybridize to the other FGF family member, HstB/FGF 6, even under highly stringent conditions (data not shown). As shown in Fig. 1 mouse HstI expression was detected in the limb bud as a 3.0-kb band and in the body as a 3.0-kb band and a lighter additional band of 3.5 kb. No signal was detected in the other samples. By RTPCR technique using H&I-specific primers (specific priming data not shown), no band was detected by ethidium bromide staining after 20-cycle amplification. But when the same RT-PCR products were analyzed by Southern blot hybridization with the Ml.8 probe, we could detect HstI transcripts in samples from limb, head, and body, but not in samples of placenta or allantois (Fig. 2). The signal in the body sample was slightly
stronger than that in the limb sample. In contrast the signal in the head sample was very weak. By RT-PCR after an additional 20-cycle amplification (re-PCR) we could detect the comparable bands using ethidium bromide staining (data not shown). The 3.0-kb transcript observed in the limb and body samples is consistent with our previous report (Yoshida et aL, 1988) and may represent mature HstI mRNA. In contrast, we could detect an additional lighter band of 3.5 kb in the body sample. This larger product may represent pre-mRNA. The size difference is consistent with the possible retention of either the 399-bp intron 1 or the 429-bp intron 2. Since the primers used in the RT-PCR analysis flank the second intron and rule out its retention (Fig. 2), the second transcript may be due to the retained first intron. To address this issue, the cloning of the corresponding cDNA is underway, since the GCrich nature of intron 1 poses problems in specificity of priming PCR as well as Northern hybridization. In order to localize the HstI transcripts during development, corresponding HstI cDNA was isolated and the 3’ untranslated region was used as a probe for in situ hybridization. In cross and sagittal sections of Day 8 (early somite) as well as mid-sagittal sections of Day 9, 10, 11, and 12 mouse embryos, we were unable to detect any particular localization of the HstI message. In contrast, in cross sections through the body of Day 11 and 12 embryos localization of message was detected in the apical ectodermal ridge (AER) of the limb bud (Figs. 3 and 4). Transcripts of HstI were detected in all regions (anterior, distal, and posterior) of the AER in both the fore and hind limb buds. As far as the sensitivity per-
FIG.2. RT-PCR analysis of HstI transcripts in 11.5-day p.c. embryo. (A) Ethidium bromide-stained reverse transcriptase-polymerase chain reaction (RT-PCR) products in 11.5-day p.c. embryo of NIH/ Swiss mouse. Lanes 1 and 6, limb; lanes 2 and 7, head; lanes 3 and 8, body; lanes 4 and 9, placenta; and lanes 5 and 10, allantois. Oligonucleotides specific to the mouse HstI were used as primers in lanes 1 to 5. Oligonucleotides specific to @a&in were used as primers in lanes 6 to 10. (B) Southern blot hybridization of RT-PCR products in A probed with the mouse genomic HstI fragment, M1.8. Lanes 1 and 6, limb; lanes 2 and 7, head; lanes 3 and 8, body; lanes 4 and 9, placenta; and lanes 5 and 10, allantois. The positions of molecular weight markers are indicated.
BRIEF NOTE
221
FIG. 3. Bright-field and dark-field micrographs of a cross section of a Day 11.5 mouse embryo cut at the level of the hindlimb bud through the posterior distal region and probed with an antisense riboprobe to H&I. The signal is in the apical ectodermal ridge (AER) at the tip of the stage 6 limb bud (short arrows), equivalent to the stage 24 chick wing bud. Weak signal can also be found in a group of cells (arrows), probably within the myotome of the somite (see text for explanation). The apparently high signal in the left coelom and some other regions is a reflection of darkly stained cells, such as red blood cells, otherwise the background level (very fine grains) is very low. This is clear in Fig. 4. Bar, 500 pm.
mitted, signal in the AER was detected in the forelimbs of stage 5 and 6, and in the hindlimbs of stage 4 and 5 of the mouse limb staging system (Wanek et al, 1989), equivalent to stage 24 to 25 and stage 22 to 25, respectively, of the chick wing bud (Hamburger and Hamilton, 1951). The observed level of HstI hybridization was relatively low and thus the signal was detectable only under the most stringent washing conditions. Signal was not detected in limbs younger than these stages or in the stage 12 (Day 16) forelimb bud, equivalent to the stage 35 chick wing. Nonridge ectoderm and limb mesoderm were negative at all stages examined. Sense probe was used at each stage examined and did not show any localization (not shown).
In the mouse, development of the forelimb precedes considerably that of the hindlimb, but the signal was detected in both limb buds at comparable developmental stages. Since a fully developed AER is not observed histologically until stage 4 and the AER becomes reduced after stage 7 (Wanek et a& 1989), correlation between HstI expression and the developmental maturation of the AER is clear. It is noteworthy that while we detected signal in all regions of the AER, the signal was clearer in the posterior half of the AER. So far, two of the FGF family members have been found in the early limb bud; basic FGF antigen was detected in the cytoplasm of skeletal muscle myoblasts (Joseph-Silverstein et aL, 1989) and FGF-5 transcripts were localized in a
222
DEVELOPMENTAL BIOLOGY
VOLUME 150.1992
by NIH Grants HD05505 and DE05837 and by a grant-in-aid from the Ministry of Health and Welfare for a Comprehensive lo-year Strategy for Cancer Control, Japan.
REFERENCES
FIG. 4. An enlargement of a region similar to Fig. 3 showing signal in the AER. Nonridge ectoderm and limb mesoderm are negative. Silver grains can be seen in both bright-field and dark-field micrographs while some darkly stained cells show a superficial reflection in the dark field micrograph. Bar, 100 pm.
patch of cells near the base of the limb (Haub and Goldfarb, 1991). Thus HstI localization in the AER is unique. Besides the limb bud, we also detected transcripts in the body samples on Northern blots. By using in situ hybridization, we saw a very low level of signal in the somites, probably in a subset of cells in the myotome. The signal was found in a very limited sample of the Day 11.5 embryo all along the body axis (forelimb, flank, and hindlimb levels). However, further study must be done to elucidate HstI expression in this region. The apical ectodermal ridge has been the focus of considerable interest since Saunders (1948) demonstrated that its presence is required for the progressive elongation of the developing limb bud along its proximal-distal axis. The basis for the activity of the AER is not known, however, it has been shown to have mitogenic activity in vitro (Reiter and Solursh, 1982). The detection of HstI mRNA in the AER raises the interesting possibility that HstI might be involved in AER function and play a role in limb outgrowth. The limb bud has been well studied as a model system for the identification of mechanisms of morphogenesis and pattern formation because it is relatively accessible for experimental studies. It may be possible to take advantage of such features of the system now for the analysis of the role and function of HstI expression during limb outgrowth. The authors thank Li Huang for her help in preparing tissue samples used in the in situ hybridization studies. The work was supported
Brookes, S., Smith, R., Thurlow, J., Dickson, C., and Peters, G. (1989). The mouse homologue of HstI k-FGF: sequence, genome organization and location relative to int-2. Nucleic Acids Res. 17,4037-4045. Burgess, W. H., and Maciag, T. (1989). The heparin-binding (fibroblast) growth factor family of proteins. Annu. Rev. Biochem 58, 575-606. Fu, Y.-M., Spirito, P., Yu, Z.-X., Biro, S., Sasse, J., Lei, J., Ferrans, V. J., Epstein, S. E., and Casscells, W. (1991). Acidic fibroblast growth factor in the developing rat embryo. J. CeUBiol 114,1261-1273. Goldfarb, M. (1990). The fibroblast growth factor family. Cell Growth wer. 1,439-445. Hamburger, V., and Hamilton, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol 88,49-92. Haub, O., Drucker, B., and Goldfarb, M. (1990). Expression of the murine fibroblast growth factor 5 gene in the adult central nervous system. Proc Nat1 Ad Sci USA 87,8022-8026. Haub, O., and Goldfarb, M. (1991). Expression of the fibroblast growth factor-5 gene in the mouse embryo. Development 112,397-406. Hebert, J. M., Basilica, C., Goldfarb, M., Haub, 0.. and Martin, G. R. (1990). Isolation of cDNAs encoding four mouse FGF family members and characterization of their expression patterns during embryogenesis. Dev. Bid 138,454-463. Hebert, J. M., Boyle, M., and Martin, G. R. (1991). mRNA localization studies suggest that murine FGF-5 plays a role in gastrulation. Development 112,407-415. Joseph-Silverstein, J., Consigli, S. A., Lyser, K. M., and Ver Pault, C. (1989). Basic fibroblast growth factor in the chick embryo: Immunolocalization to striated muscle cells and their precursors. J. CeU Biol. 108,2459-2466. Reiter, R. S., and Solursh, M. (1982). Mitogenic property of the apical ectodermal ridge. Dev. Biol. 93,28-35. Sakamoto, H., Mori, M., Taira, M., Yoshida, T., Matsukawa, S., Shimizu, K., Sekiguchi, M., Terada, M., and Sugimura, T. (1986). Transforming gene from human stomach cancers and a noncancerous portion of stomach mucosa. Proc Natl. Acad Sci USA 83, 39974001. Saunders, J. W., Jr. (1948). The proximo-distal sequence of origin of the parts of the chick wing and the role of the ectoderm. J. Exp. Zod 108,363-403. Suzuki, H. R., Padanilam, B. J., Vitale, E., Ramirez, F., and Solursh, M. (1991). Repeating developmental expression of G-Hox 7, a novel homeobox-containing gene in the chicken. Dev. Biol 148,375-388. Wanek, N., Muneoka, K., Holler-Dinsmore, G., Burton, R., and Bryant, S. V. (1989). A staging system for mouse limb development. J. Exp. zoo1 249,41-49. Wilkinson, D. G., Peters, G., Dickson, C., and McMahon, A. P. (1988). Expression of the FGF-related proto-oncogene int-2 during gastrulation and neurulation in the mouse. EMBO J. 7,691-695. Yoshida, T., Tsutsumi, M., Sakamoto, H., Miyagawa, K., Teshima, S., Sugimura, T., and Terada, M. (1988). Expression of the Hstl oncogene in human germ cell tumors. Biockm~ Biophys Res. Commun 155,1324-1329.
BIOLOGY
150,219-222 (19%)
BRIEF NOTE Localization of Hstl Transcripts to the Apical Ectodermal Ridge in the Mouse Embryo HIROAKI R. SUZUKI,* HIROMISAKAM~T~,-~TERUHIKOY~SI-IIDA,~TAKASHI MASAAKITERADA,~ANDMICHAELSOLURSH*
SumnmA,t
*Department of Biology, University of Iowa, Iowa City, Iowa 52242;and tNationa1 Cancer Center Research Institute, l-l, Tsukiji Cchme, Chueku, Tokyo 104,Japan Accepted November 18, 1991 The HstI gene is a transforming gene, coding for a protein of the fibroblast growth factor family (Sakamoto et al, 1986). Previous RNA hybridization studies with the mouse homolog demonstrated the presence of a 3.0-kb transcript in Day 11 and 14 mouse embryos. Here we detect a 3.0-kb transcript in the limb and body of the dissected Day 11 mouse embryo. PCR amplification using HstI-specific primers also showed comparable results. In order to localize the HstI transcripts during development, corresponding HstI cDNA was isolated, and an HstI-specific region was used as a probe for in situ hybridization analysis. Serial sections of embryos from Day 8 (early-somite stages) through Days 9,10,11, and 12 of gestation were examined. With the antisense probe, a signal was detected in the Day 11 and 12 embryo, where it was localized to the apical ectodermal ridge (AER) of the limb bud. This structure is well known for its role in promoting the distal outgrowth of the developing limb bud. Signal was detected in both fore- and hindlimbs during the period of rapid distal growth. This restricted localization suggests a role for HstI in normal embryogenesis, including outgrowth of the limb bud. o lssz Academic press, ~nc.
INTRODUCTION
MATERIALS AND METHODS
The RNA preparation and hybridization conditions Fibroblast growth factors represent a family of mitogenie proteins with related primary structures (Burgess were as described earlier (Yoshida et al, 1988). The and Maciag, 1989; Goldfarb, 1990). In the mammal there primers used in RT-PCR were nucleotides 1029 to 1044 plus 1474 to 1477, and antisense of nucleotides 1562 to are currently seven known family members, including 1543 (Brookes et al, 1989). PCR conditions were as folacidic FGF, basic FGF, INT-2 protein, HSTUK-FGF protein, FGF-5 protein, FGF-6/HST2 protein, and kera- lows: Denaturation, 94”C, 1 min; annealing, 6O”C, 1 min; tinocyte growth factor (KGF), each encoded by a dis- polymerization, 72”C, 1 min; 20 cycles, with Taq DNA tinct gene. FGFs are mitogenic to a large number of polymerase (Promega) in reaction mixture containing 1 ectodermally and mesodermally derived cells and can m&f Mg2+. In order to obtain the HstI cDNA probe, a XgtlO cDNA act as inducers of cell differentiation. While some inforlibrary (Amersham) was made from mouse embryonal mation is available concerning the temporal and spatial distribution of FGF family members during develop- carcinoma F9 cells maintained in the undifferentiated ment (Wilkinson et al, 1988; Joseph-Silverstein et al, state in which they express a high level of HstI mRNA 1989; Haub et ah, 1990; Haub and Goldfarb, 1991; Hebert (Yoshida et aZ., 1988). Screening the library with the et al, 1990, 1991; Fu et ab, 1991), little is known about HstI genomic probe Ml.8 led to the isolation of positive their normal function in situ. Compared to other FGF clones with the sequence identical to those reported by family members, HstI has a particularly restricted pe- others (Brookes et aL, 1989). A 557-bp fragment (seriod of expression, having been detected so far in the quence 1988-2544) encompassing part of the 3’ untranspreimplantation mouse embryo (Hebert et a& 1991) and lated region was subcloned into a pGEM-2 vector (ProDay 11 and 14 mouse embryo but not in the Day 17 em- mega) and used as a probe for in situ hybridization. In situ hybridization was performed on tissue sections bryo or the postnatal mouse by Northern analysis (Yoshida et aL, 1988). The purpose of this study is to de- of Balb/c strain mouse embryos according to the protoscribe the localization of HstI transcripts in the mouse col described previously (Suzuki et aL, 1991). Embryos were fixed in Bouin’s fixative and processed for paraffin embryo. 219
0012-1606/92 $3.00 Copyright All rights
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
220
DEVELOPMENTALBIOLOGYV0~~~~150.1992
-
kb 9.49 7.46
-
4.40
-
2.37 1.35
-
FIG. 1. Northern blot analysis of HstI transcripts in the Il.&day NIH/Swiss mouse embryo. RNA was prepared from the limb (lane l), head (lane 2), remaining body (lane 3), placenta (lane 4), and allantois (lane 5). One microgram each poly-A+ RNA was used with the mouse genomic H&I fragment, Ml.8 as a probe. The RNA preparation and hybridization conditions were as described earlier (Yoshida et al, 1988).
sectioning. %-labeled sense and antisense RNA probes were synthesized upon the linearized template. Stringent washes were carried out in 2~ SSC, 50% formamide at 6O”C, followed by 0.1X SSC at 50°C. RESULTS AND DISCUSSION
In order to detect the HstI message in different regions of the Day 11.5 mouse embryo, RNA blot hybridization was carried out on poly-A+ RNA isolated from limb buds, heads, remaining bodies, placentae, and allantois, utilizing the mouse HstI genomic fragment, Ml.8 as a probe. This probe encompasses exons 2 and 3 but not exon 1 (or GC-rich intron 1) which tends to cross-hybridize to the other FGF family member, HstB/FGF 6, even under highly stringent conditions (data not shown). As shown in Fig. 1 mouse HstI expression was detected in the limb bud as a 3.0-kb band and in the body as a 3.0-kb band and a lighter additional band of 3.5 kb. No signal was detected in the other samples. By RTPCR technique using H&I-specific primers (specific priming data not shown), no band was detected by ethidium bromide staining after 20-cycle amplification. But when the same RT-PCR products were analyzed by Southern blot hybridization with the Ml.8 probe, we could detect HstI transcripts in samples from limb, head, and body, but not in samples of placenta or allantois (Fig. 2). The signal in the body sample was slightly
stronger than that in the limb sample. In contrast the signal in the head sample was very weak. By RT-PCR after an additional 20-cycle amplification (re-PCR) we could detect the comparable bands using ethidium bromide staining (data not shown). The 3.0-kb transcript observed in the limb and body samples is consistent with our previous report (Yoshida et aL, 1988) and may represent mature HstI mRNA. In contrast, we could detect an additional lighter band of 3.5 kb in the body sample. This larger product may represent pre-mRNA. The size difference is consistent with the possible retention of either the 399-bp intron 1 or the 429-bp intron 2. Since the primers used in the RT-PCR analysis flank the second intron and rule out its retention (Fig. 2), the second transcript may be due to the retained first intron. To address this issue, the cloning of the corresponding cDNA is underway, since the GCrich nature of intron 1 poses problems in specificity of priming PCR as well as Northern hybridization. In order to localize the HstI transcripts during development, corresponding HstI cDNA was isolated and the 3’ untranslated region was used as a probe for in situ hybridization. In cross and sagittal sections of Day 8 (early somite) as well as mid-sagittal sections of Day 9, 10, 11, and 12 mouse embryos, we were unable to detect any particular localization of the HstI message. In contrast, in cross sections through the body of Day 11 and 12 embryos localization of message was detected in the apical ectodermal ridge (AER) of the limb bud (Figs. 3 and 4). Transcripts of HstI were detected in all regions (anterior, distal, and posterior) of the AER in both the fore and hind limb buds. As far as the sensitivity per-
FIG.2. RT-PCR analysis of HstI transcripts in 11.5-day p.c. embryo. (A) Ethidium bromide-stained reverse transcriptase-polymerase chain reaction (RT-PCR) products in 11.5-day p.c. embryo of NIH/ Swiss mouse. Lanes 1 and 6, limb; lanes 2 and 7, head; lanes 3 and 8, body; lanes 4 and 9, placenta; and lanes 5 and 10, allantois. Oligonucleotides specific to the mouse HstI were used as primers in lanes 1 to 5. Oligonucleotides specific to @a&in were used as primers in lanes 6 to 10. (B) Southern blot hybridization of RT-PCR products in A probed with the mouse genomic HstI fragment, M1.8. Lanes 1 and 6, limb; lanes 2 and 7, head; lanes 3 and 8, body; lanes 4 and 9, placenta; and lanes 5 and 10, allantois. The positions of molecular weight markers are indicated.
BRIEF NOTE
221
FIG. 3. Bright-field and dark-field micrographs of a cross section of a Day 11.5 mouse embryo cut at the level of the hindlimb bud through the posterior distal region and probed with an antisense riboprobe to H&I. The signal is in the apical ectodermal ridge (AER) at the tip of the stage 6 limb bud (short arrows), equivalent to the stage 24 chick wing bud. Weak signal can also be found in a group of cells (arrows), probably within the myotome of the somite (see text for explanation). The apparently high signal in the left coelom and some other regions is a reflection of darkly stained cells, such as red blood cells, otherwise the background level (very fine grains) is very low. This is clear in Fig. 4. Bar, 500 pm.
mitted, signal in the AER was detected in the forelimbs of stage 5 and 6, and in the hindlimbs of stage 4 and 5 of the mouse limb staging system (Wanek et al, 1989), equivalent to stage 24 to 25 and stage 22 to 25, respectively, of the chick wing bud (Hamburger and Hamilton, 1951). The observed level of HstI hybridization was relatively low and thus the signal was detectable only under the most stringent washing conditions. Signal was not detected in limbs younger than these stages or in the stage 12 (Day 16) forelimb bud, equivalent to the stage 35 chick wing. Nonridge ectoderm and limb mesoderm were negative at all stages examined. Sense probe was used at each stage examined and did not show any localization (not shown).
In the mouse, development of the forelimb precedes considerably that of the hindlimb, but the signal was detected in both limb buds at comparable developmental stages. Since a fully developed AER is not observed histologically until stage 4 and the AER becomes reduced after stage 7 (Wanek et a& 1989), correlation between HstI expression and the developmental maturation of the AER is clear. It is noteworthy that while we detected signal in all regions of the AER, the signal was clearer in the posterior half of the AER. So far, two of the FGF family members have been found in the early limb bud; basic FGF antigen was detected in the cytoplasm of skeletal muscle myoblasts (Joseph-Silverstein et aL, 1989) and FGF-5 transcripts were localized in a
222
DEVELOPMENTAL BIOLOGY
VOLUME 150.1992
by NIH Grants HD05505 and DE05837 and by a grant-in-aid from the Ministry of Health and Welfare for a Comprehensive lo-year Strategy for Cancer Control, Japan.
REFERENCES
FIG. 4. An enlargement of a region similar to Fig. 3 showing signal in the AER. Nonridge ectoderm and limb mesoderm are negative. Silver grains can be seen in both bright-field and dark-field micrographs while some darkly stained cells show a superficial reflection in the dark field micrograph. Bar, 100 pm.
patch of cells near the base of the limb (Haub and Goldfarb, 1991). Thus HstI localization in the AER is unique. Besides the limb bud, we also detected transcripts in the body samples on Northern blots. By using in situ hybridization, we saw a very low level of signal in the somites, probably in a subset of cells in the myotome. The signal was found in a very limited sample of the Day 11.5 embryo all along the body axis (forelimb, flank, and hindlimb levels). However, further study must be done to elucidate HstI expression in this region. The apical ectodermal ridge has been the focus of considerable interest since Saunders (1948) demonstrated that its presence is required for the progressive elongation of the developing limb bud along its proximal-distal axis. The basis for the activity of the AER is not known, however, it has been shown to have mitogenic activity in vitro (Reiter and Solursh, 1982). The detection of HstI mRNA in the AER raises the interesting possibility that HstI might be involved in AER function and play a role in limb outgrowth. The limb bud has been well studied as a model system for the identification of mechanisms of morphogenesis and pattern formation because it is relatively accessible for experimental studies. It may be possible to take advantage of such features of the system now for the analysis of the role and function of HstI expression during limb outgrowth. The authors thank Li Huang for her help in preparing tissue samples used in the in situ hybridization studies. The work was supported
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