Proc. Nat!. Acad. Sci. USA Vol. 88, pp. 9198-9202, October 1991 Developmental Biology
Widespread expression of MyoD genes in Xenopus embryos is amplified in presumptive muscle as a delayed response to mesoderm induction (embryogenesis/helix4oop-helix/gastrulation/myogenesis/RNase protection)
RICHARD P. HARVEY Molecular Biology Unit, The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia
Communicated by G. J. V. Nossal, July 18, 1991 (received for review June 7, 1991)
ABSTRACT The MyoD gene codes for an important regulatory factor in skeletal myogenesis. To explore the relationship between mesoderm induction in Xenopus embryos and expression of MyoD, I have monitored MyoD mRNA levels in normal embryos and cultured explants by RNase protection. Transcription from the two Xenopus MyoD gene copies is activated weakly across the whole embryo at the midblastula transition, and this activation occurs in the absence of mesoderm induction. In response to induction this basal expression is amplified 50- to 100-fold, but in animal-pole explants 6-10 hr elapse before induced mRNAs appear, and this induction requires prior protein synthesis. The promiscuous transcripts disappear from animal explants at a time when induction "competence" is lost, suggesting a link between these events. The data highlight a broad, but transient, permissiveness for MyoD expression in embryos, which is propagated and amplified only in presumptive muscle in response to induction. Moreover, muscle-specific MyoD expression is a relatively late (postgastrulation) event in the mesoderm-induction cascade.
maturation, induces myoblast commitment (2, 3). The consequences of expression in myoblasts are not well documented, but negative regulators, such as Id (17), and cofactors, such as products of the E2A gene (18), influence how MyoD expression is interpreted at different stages of maturation. A separate function of MyoD may be to arrest cell cycling at the onset of myogenic differentiation (19, 20). The Xenopus genome contains two MyoD genes (21-23), referred to in this paper as MyoDa and MyoDb. Maternal transcripts occur at a low level, whereas zygotic expression is muscle-specific from an early stage and a product of mesoderm induction (21, 22). Zygotic expression of the MyoDa gene begins at the midblastula transition (MBT), the time when embryonic transcription is initiated and some 7 hr before appearance of muscle-differentiation products, such as actin. If MyoD expression were muscle-specific at this time, it would imply that the muscle lineage had already been determined and, more generally, that blastomeres become committed to mesodermal lineages during cleavage, perhaps in direct response to the events of mesoderm induction. I show in this paper that MyoD expression at the MBT occurs across the whole embryo and is not amplified in presumptive muscle until early gastrulation. The early lowlevel expression is independent of mesoderm induction, whereas maximal expression requires induction. The broad early permissiveness for MyoD expression may be related to "competence" for mesoderm induction because early expression is down-regulated in explants at a time when competence is lost. MyoD can be classed as an intermediate response gene with respect to induction because in animalpole explants there is a lag of 6-10 hr before induced mRNAs appear and prior protein synthesis is needed. Because MyoD expression is propagated and amplified in muscle after gastrulation begins, muscle determination appears to be a postgastrulation event.
Detailed fate maps have been constructed for organisms, like Xenopus, which are disposed to analysis of developmental problems, but these maps tell us little about how, and when embryonic cells become committed to their respective lineages. These issues are normally studied by cell- or tissuetransplantation experiments, and two progressive states of commitment-specification and determination-have been defined (1). Recent evidence demonstrates that some nuclear proteins containing the structurally conserved helix-loophelix dimerization domain are important regulators of lineage commitment (2-4). Analysis of the function of these proteins and their expression in embryos will help to mark and define the molecular nature of commitment events. A series of in vitro studies, stemming from analysis of the propensity for mouse C3H 1OT1/2 fibroblasts to undergo myogenesis (5), implicate the helix-loop-helix protein MyoD as a key regulator of muscle development (6). Expression of the MyoD gene in primary fibroblasts and various cell lines results in biochemical and morphological conversion of these cells to muscle (7, 8). Three other genes with structural and apparent functional homology to MyoD have now been isolated (myJf, myogenin, and herculin/muscle regulatory factor 4; refs. 9-12) and an additional member of the myogenic pathway, myd, has been inferred from transfection studies (13). The MyoD gene codes for a multifunctional myogenic regulatory factor. It is a DNA-binding protein that transactivates muscle-specific structural genes in differentiating myocytes (14-16). Forced expression of the MyoD gene in cultured cells, under conditions nonpermissive for myogenic
MATERIALS AND METHODS Embryo and Explant Culture. Xenopus laevis eggs were obtained and fertilized as described (24). Embryos were raised in 0.1 x modified Barth solution (MBS) (25) containing Gentamycin at 100 ,ug/ml. Staging was according to Nieuwkoop and Faber (26). Animal and vegetal caps were dissected between stages 8 and 9 and cultured on agarose beds in 0.5x MBS containing Gentamycin at 100 ug/ml and y-globulins (Sigma) at 2 mg/ml, with or without mesoderminducing factors. Basic fibroblast growth factor was from Amersham and used at 20 ng/ml. Mesoderm-inducing factor from the Xenopus XTC cell line (XTC-MIF) (supplied by J. Smith, Medical Research Council, National Institute for Medical Research, Mill Hill, London) was a partially purified
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: MBT, midblastula transition; XTC-MIF, mesoderminducing factor from the Xenopus XTC cell line.
9198
Developmental Biology: Harvey
Proc. Natl. Acad. Sci. USA 88 (1991)
fraction (see ref. 22), dialyzed in phosphate-buffered saline before use. For induction, 4 units/ml were used, where 1 unit/ml is the minimum concentration required to induce muscle-specific actin gene expression. For cycloheximide treatment, stage 8½/2 caps were incubated in 0.5x MBS/ cycloheximide at 5 Ag/ml/y-globulins at 2 mg/ml and Gentamycin at 100 jig/ml for 30 min, followed by a 30-min incubation in the same buffer containing XTC-MIF. Caps were then washed well in 0.5x MBS and incubated in the above buffer without cycloheximide overnight. Serum-free medium conditioned by murine P388D1 macrophages (supplied by D. Hilton, Whitehead Institute, Cambridge, MA) was concentrated 18-fold and used at 1:1 in induction assays. RNA Analysis. For RNase protection analysis, total RNA was extracted from embryos and explants by using the proteinase K/phenol method (24). RNase protection was as described (40). The MyoD probe was an Fsp I/Pst I fragment (nucleotides 526-886) of the MyoDb cDNA (22), cloned into pSP72. Probes were synthesized from EcoRI-linearized plasmid with SP6 polymerase. Hybrids were digested with RNase A at 2 ixg/ml and RNase T1 at 0.1 ,ug/ml at 160C for 30 min. The actin probe was synthesized from the plasmid AC100 (27), linearized with Dde I, and transcribed with SP6 polymerase. Hybrids were digested in RNase A at 2 ;Lg/ml and RNase T1 at 0.1 ,ug/ml for 30 min at 37°C.
scribed during oogenesis, resulting in a stable maternal mRNA, and in embryos beginning at the MBT when the embryonic genome first becomes transcriptionally active (22). To analyze expression from the MyoDb gene, an RNase protection probe was constructed from a region of the MyoDb cDNA that is highly conserved between MyoDb and MyoDa. With limited RNase digestion, this probe detects MyoDb mRNA as a full-length species and MyoDa mRNA as a partially digested species (Fig. 1). The identity of the bands was confirmed using synthetic MyoDa or MyoDb mRNAs transcribed from corresponding cDNA clones (22). Fig. 1 shows an RNase protection analysis ofRNA samples taken at hourly intervals from 5- to 15-hr-old embryos. This RNA series, which has been characterized previously for MyoDa and other markers, spans the MBT, which occurs between 6 and 7 hr (22). Three points about initiation of MyoD expression are noteworthy. (i) Only the MyoDa gene contributes to the maternally inherited mRNA-i.e., bands seen in pre-MBT samples. To validate this conclusion RNA was analyzed from eggs of eight independent females, and all samples contained only MyoDa mRNA (data not shown). (ii) MyoDb expression, like that of MyoDa, initiates at the MBT. Both MyoD genes are therefore activated in the zygote as soon as transcription first begins. (iii) Zygotic MyoD transcripts accumulate slowly and only reach levels significantly above that of the maternal RNA after gastrulation. With this probe it has also been shown that both genes are activated, although to different extents, in animal-pole explants (animal caps) induced to form mesoderm by contact with vegetal-pole cells or by culture with mesoderm-inducing factors (Fig. 2). Weak induction is seen with basic fibroblast growth factor, consistent with its poor ability to induce dorsal mesoderm (30). Strong induction is seen in animal/vegetal recombinants and in animal caps treated with P388D1 macrophage-conditioned medium or XTC-MIF, which contain factors similar or identical to activin A (31, 32). With all inducing regimens, as well as in normal embryos, the MyoDb
RESULTS Only the MyoDa Gene Is Expressed During Oogenesis. The Xenopus genome contains two MyoD genes that are thought to be functionally equivalent (22, 23). Many genes have been duplicated during the evolution of polyploidy in anurans (28), but the two Xenopus c-myc genes are regulated differently (29), one being expressed only maternally. It seemed important, therefore, to analyze expression from both MyoD genes, in case regulatory or functional differences had evolved. It has previously been shown that the MyoDa gene is tran0 )
0
L
-* 406_ 360-*
z
z
>- a E -O-E
r=
H- r,-
1-~-
u
F-
>a
~A5
'6B TI 5
6
7
umU.
&
~Li
_m a--
K
9AS-wL..^ -
__OA_,* .. .
22 9-* 2 1 7-_-
9199
.
.
M oDa
N
_w
oM . v
.ow
FIG. 1. Kinetics of activation of MyoD genes in normal development. The RNase protection probe used in this analysis protects both MyoDa and MyoDb transcripts. Protected bands are identified in control assays using synthetic mRNAs transcribed from cDNA clones with T7 polymerase (T7-MyoDa and T7-MyoDb RNAs). For reasons not clear, synthetic RNAs always protect a doublet in these assays, whereas embryo RNA protects single bands for each mRNA. RNA samples were extracted from embryos at hourly intervals from 5-15 hr postfertilization (5-15 hpf). This same RNA series has been previously characterized for MyoDa, a-cardiac actin, N-cell-adhesion molecule, Xhox-1A, GS17, and cytoskeletal actin RNAs (22). The position of the MBT was determined by the onset of transcription of the GSI7 gene (24).
9200
Developmental Biology: Harvey
mRNA reaches levels -5-fold above that of MyoDa. Presumably, the MyoDb gene promoter is more responsive to induction mechanisms. MyoD Expression Is a Delayed Response to Mesoderm Induction. The early activation of MyoD genes and their induction by mesoderm inducers raise two questions about the formation of mesoderm in Xenopus (22). (i) Is MyoD expression a primary transcriptional response to mesoderm induction? (ii) Is MyoD expression at the MBT specific to presumptive muscle, as would be expected were blastomeres already assigned to the muscle lineage at this point? The first question has been addressed. A true immediate-early response to mesoderm induction, like that from the homeobox gene Mix.], is characterized by rapid appearance of transcripts in response to induction, even in the absence of protein synthesis (33). Before induction with XTC-MIF, animal caps were incubated in cycloheximide under conditions that inhibit 90%o ofprotein synthesis for at least 4 hr (33). This treatment almost totally inhibited induction ofthe MyoD genes and expression of the muscle-specific actin gene (Fig. 2), as well as elongation movements characteristic of induced caps. On the other hand, cytoskeletal actin expression, a marker of tissue mass and viability, was minimally affected. The kinetics of MyoD gene activation in XTC-MIFinduced explants was analyzed to determine how soon MyoD transcripts appear (Mix.1 mRNA appears within 30 min). In U-)
z
C') Li
z 0 L
W LL
Li
C
L+
LF-
co n
Q cL
e
x
L-
LiJE
s
U')
X
L
r) IC z
-
U LLI
Proc. Natl. Acad. Sci. USA 88 (1991)
0
z
U] -C
4k--
LLI
'v
FIG. 2. Activation of MyoDa and MyoDb genes in animal-pole explants cultured overnight in basic fibroblast growth factor (bFGF), XTC-MIF, medium conditioned with murine P388D1 macrophages (P388D1-CM) or animal explants combined with vegetal-pole cells (A/V recombinants). In one sample (XTC-MIF + CHX), explants were treated with cycloheximide before addition of XTC-MIF. Induction of muscle in explants was monitored by RNase protection with a muscle-specific a-cardiac actin probe (MS-Actin). This probe also detects cytoskeletal actins (CY-Actin), which serve as a measure of explant viability and tissue mass. For MyoD protections, 12 animal caps were used; for actin protections, 3 caps were used. Control protection (tRNA) contains 20 jig of yeast tRNA.
---
A c- t i r,
Acr;~,,i.r ---Act
FIG. 3. Kinetics of expression of MyoD genes in animal-pole explants treated with XTC-MIF. Stage 8/2-9 explants were cultured in XTC-MIF and sampled at the times indicated (1-22 hr) before RNA extraction and RNase protection with probes for MyoD (12 caps) and actin (3 caps). Induced 0-hr caps were extracted immediately after dissection and were not incubated in XTC-MIF. The uninduced 22-hr sample was incubated overnight without induction. Note that cytoskeletal actins are also induced in caps, as they are in normal development, but this is not a response to induction.
Developmental Biology: Harvey
Proc. Natl. Acad. Sci. USA 88 (1991)
diate response, as it occurs between induction of immediateearly genes (such as Mix. 1) and differentiation products, such as cardiac actin. The induction-dependent influences that result in activation of the MyoD genes are at present unknown (see Discussion). Induction-Independent MyoD Expression Occurs Across the Whole Embryo. In normal development animal caps do not form muscle but contribute mainly to epidermis and brain. To further investigate the expression of MyoD RNAs in uninduced caps, animal and vegetal caps were cultured for various periods without inducers, and extracted RNA was analyzed for MyoD transcripts (Fig. 4). MyoD genes were transcribed in both types of explant, although with different kinetics. Because caps are dissected shortly after the MBT, this early phase must account for expression initiated at the MBT in normal embryos (see Fig. 1). It is unlikely that animal caps are induced in these experiments-for example, during dissection-because the strong induction of MyoD or actin transcription characteristic of induced caps (Figs. 2 and 3) has never been seen in several experiments. The data in Fig. 3 suggest that induction may have a weak positive effect on the early expression of MyoD genes. In a separate experiment the level of early transcripts in uninduced animal caps after 5 hr in culture was compared with levels in caps induced for the same period with basic fibroblast growth factor, XTC-MIF, P388D1 macrophageconditioned medium, or vegetal-pole cells. In each case the levels of both MyoDa and -b transcripts were unaltered (data not shown). Similarly, treatment of caps with cycloheximide before induction did not change the level of early transcripts. Therefore, products produced in the early phase of mesoderm induction do not amplify or sustain the basal level of MyoD transcription occurring across the embryo. The kinetics of expression of early transcripts differs between animal and vegetal caps. Interestingly, transcripts in animal caps are degraded rapidly at a time (6-11 hr in culture; Fig. 4) corresponding to stage 101/2-12 in normal development. This is also when the capacity ("competence"; ref. 34)
9201
for these cells to be induced to mesodermal lineages is lost (ref. 35; see Discussion). In vegetal-pole cells, transcripts persist much longer, still being detectable after 11 hr and weakly after 22 hr of culture. It is an interesting possibility that vegetal cells, which are the source of mesoderm inducers in normal development (36), can respond weakly to autoinduction. The Mix.] gene, which gives an immediate-early response to induction in animal caps, is also expressed in the vegetal-pole cells, as well as presumptive mesoderm in normal blastulae (33).
DISCUSSION The two Xenopus MyoD gene copies are regulated differently during oogenesis and are induced to different extents during embryogenesis. The significance of this to development is unknown, as is the relevance of the maternal RNA per se. Whether it reflects real functional differences between the gene products or is simply a consequence of reduced selection pressure on duplicated genes is still an open question and will require further analysis. In an RNase protection assay, both MyoDa and MyoDb genes were found to be transcribed at low levels across the whole embryo, beginning at the MBT, and this early transcription is independent of mesoderm induction. This result has also recently been reported by Rupp and Weintraub (37) using a quantitative PCR assay. The number of early transcripts from each MyoD gene was estimated to be 105 per animal cap (data not shown), compared with _107 for later embryos (21, 22). What is the significance of these early transcripts? It is possible that the early transcription phase is necessary for induction-dependent MyoD expression. Autoactivation (38), triggered by the events of induction, would seem a plausible mechanism to connect the two phases. Alternatively, there may be a nonmyogenic function for MyoD in early blastomeres. However, because the low level of early transcripts is probably too low to produce a significant level of tran-
0
ANIMAL CAPS
LL
O
L
0
L
LU
0
-
L
v ~ I ro
L -
VEGETAL CAPS L
L.
CN
.- N
0
L
-_
r_
-
rn
Lfl
Iq~
LC -~ ko- (N
4-
Myo D b
4- MyoDa
I.
19r a
W, -Am- W, .w
10
*-MS -Act in +4-CY-Actin
FIG. 4. Time course of expression of MyoD genes in cultured animal and vegetal-pole explants without induction. Explants were cut at stage
81/2-9 and cultured without induction for the times indicated (0-22 hr). RNA extracted at each time point was analyzed by RNase protection for MyoD transcripts (12 explants) and actin transcripts (3 explants). The egg sample was taken from eggs that were fertilized to produce the embryos used in this experiment; only the MyoDa gene contributes to the maternal RNA pool.
9202
Developmental Biology: Harvey
scription factor, this expression probably more likely reflects a general capacity for MyoD transcription, which is then propagated and amplified specifically in presumptive muscle by the events of mesoderm induction. In animal caps this capacity for transcription is of limited duration because early transcripts are lost after several hours. It is probably significant that the time of this loss corresponds to when the competence of animal caps to be induced to mesoderm is also lost. The window of competence during early development may, therefore, represent, in part, a permissiveness for critical lineage regulatory genes, such as MyoD, to respond to the early events of induction. Rupp and Weintraub (37) have also noted, using PCR, that some other potential regulatory genes (but not all) show a broad and transient expression phase. Loss of competence may involve a general repression of these loci. It is not known whether the early mRNAs are translated into MyoD protein. However, their deadenylylated state in blastulae suggests that these early RNAs may not be loaded onto polysomes (22). Full induction-dependent MyoD expression does not occur until many hours after the addition ofinducers to animal caps. Furthermore, it is totally dependent on the prior synthesis of new proteins in response to induction. For these reasons the MyoD genes can be regarded as intermediate-response genes to induction because they are expressed after immediateearly genes like Mix.I but before lineage differentiation products, such as cardiac actins. The specific amplification of MyoD gene expression in a muscle-specific manner occurs only after the beginning of gastrulation. Because MyoD is believed to be a critical myogenic regulator and is part of an auto-activating and cross-regulating myogenic regulatory signal (2, 3), definitive commitment to the muscle lineage would appear to be a postgastrulation event. This idea fits well with estimates of commitment in other germ layers from a singlecell transplantation assay (39). Which gene products interact with the MyoD gene promoter to ensure muscle-specific activation is now an important question. I thank Ralph Rupp and Hal Weintraub for releasing data before publication, Ora Bernard for basic fibroblast growth factor, Jim Smith for XTC-MIF, Doug Hilton for P388D1-conditioned medium, Donna West and colleagues for animal care, and Jerry Adams and Jane Visvader for comments on the manuscript. This work was supported by a Queen Elizabeth II Fellowship and a grant from the National Institutes of Health (HD26024-02). 1. Slack, J. M. W. (1983) in From Egg to Embryo: Determinative Events in Early Development, eds. Barlow, P. W., Green, P. B. & Wylie, C. C. (Cambridge Univ. Press, Cambridge, U.K.), pp. 18-20. 2. Olsen, E. N. (1990) Genes Dev. 4, 1451-1461. 3. Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., Benezra, R., Blackwell, T. K., Turner, D., Rupp, R., Hollenberg, S., Zhuang, Y. & Lassar, A. (1991) Science 251, 761-766. 4. Campos-Ortega, J. A. & Knust, E. (1990) Eur. J. Biochem. 190, 1-10. 5. Taylor, S. M. & Jones, P. A. (1979) Cell 17, 771-779. 6. Davis, R. L., Weintraub, H. & Lassar, A. B. (1987) Cell 51, 987-1000.
Proc. Natl. Acad Sci. USA 88 (1991) 7. Weintraub, H., Tapscott, S. J., Davis, R. L., Thayer, M. J., Adams, M. A., Lassar, A. B. & Miller, A. D. (1989) Proc.
Natl. Acad. Sci. USA 86, 5434-5438. 8. Lin, A.-Y., Dechesne, C. A., Eldridge, J. & Paterson, B. M. (1989) Genes Dev. 3, 986-996. 9. Braun, T., Buschhausen-Denker, G., Bober, E., Tannich, E. & Arnold, H. H. (1989) EMBO J. 8, 701-709. 10. Wright, W. E., Sassoon, D. A. & Lin, V. K. (1989) Cell 56, 607-617. 11. Rhodes, S. J. & Konieczny, S. F. (1989) Genes Dev. 3, 20502061. 12. Mueller, P. R. & Wold, B. (1989) Science 246, 780-786. 13. Pinney, D. F., Pearson-White, S. H., Konieczny, S. F., Latham, K. E. & Emerson, C. P., Jr. (1988) Cell 53, 781-793. 14. Murre, C., McCaw, P. S. & Baltimore, D. (1989) Cell 56, 777-783. 15. Lassar, A. B., Busken, J. N., Lockshon, D., Davis, R. L., Apone, S., Hauschka, S. D. & Weintraub, H. (1989) Cell 58, 823-831. 16. Weintraub, H., Davis, R., Lockshon, D. & Lassar, A. (1990) Proc. Natl. Acad. Sci. USA 87, 5623-5627. 17. Benezra, R., Davis, R. L., Lockshon, D., Turner, D. L. & Weintraub, H. (1990) Cell 61, 49-59. 18. Murre, C., McCaw, P. S., Vaessin, H., Caudy, M., Jan, Y. N., Cabrera, C. V., Buskin, J. N., Hauschka, S. D., Lassar, A. B., Weintraub, H. & Baltimore, D. (1989) Cell 58, 537-544. 19. Sorrentino, V., Pepperkok, R., Davis, R. L., Ansorge, W. & Phillipson, L. (1990) Nature (London) 345, 813-815. 20. Crescenzi, M., Fleming, T. P., Lassar, A. B., Weintraub, H. & Aaronson, S. A. (1990) Proc. Natl. Acad. Sci. USA 87, 84428446. 21. Hopwood, N. D., Pluck, A. & Gurdon, J. B. (1989) EMBO J. 8, 3409-3417. 22. Harvey, R. P. (1990) Development 108, 669-680. 23. Scales, J. B., Olsen, E. N. & Perry, M. (1990) Mol. Cell. Biol. 10, 1516-1524. 24. Krieg, P. A. & Melton, D. A. (1985) EMBO J. 4, 3463-3471. 25. Gurdon, J. B. (1977) Methods Cell Biol. 16, 125-138. 26. Nieuwkoop, P. D. & Faber, J. (1956) Normal Table ofXenopus laevis (Daudin) (North-Holland, Amsterdam). 27. Dworkin-Rastl, E., Kelly, D. B. & Dworkin, M. B. (1986) J. Embryol. Exp. Morphol. 91, 153-168. 28. Fritz, A. F., Cho, K. W. Y., Wright, C. V. E., Jegalian, B. G. & De Robertis, E. M. (1989) Dev. Biol. 131, 584-588. 29. Vriz, S., Taylor, M. & Mechali, M. (1989) EMBO J. 8, 4091-4097. 30. Slack, J. M. W., Darlington, B. G., Heath, J. K. & Godsave, S. F. (1987) Nature (London) 326, 197-200. 31. Smith, J. C., Price, B. M., Van Nimmen, K. & Hylebroeck, D. (1990) Nature (London) 345, 729-731. 32. Thomsen, G., Woolf, T., Whitman, M., Sokol, S., Vaughan, J., Vale, W. & Melton, D. A. (1990) Cell 63, 485-493. 33. Rosa, F. (1989) Cell 57, 965-974. 34. Waddington, C. H. (1932) Philos. Trans. R. Soc. London B 221, 179-230. 35. Jones, E. A. & Woodland, H. R. (1987) Development 101, 557-563. 36. Nieuwkoop, P. D. (1973) Adv. Morphog. 10, 1-39. 37. Rupp, R. A. & Weintraub, H. (1991) Cell 65, 927-937. 38. Tapscott, S. J., Davis, R. L., Thayer, M. J., Cheng, P.-F., Weintraub, H. & Lassar, A. B. (1988) Science 242, 405-411. 39. Heasman, J., Snape, A., Smith, J. C. & Wylie, C. C. (1986) J. Embryol. Exp. Morphol. 97, Suppl., 65-73. 40. Krieg, P. A. & Melton, D. A. (1988) Methods Enzymol. 155, 397-415.
Widespread expression of MyoD genes in Xenopus embryos is amplified in presumptive muscle as a delayed response to mesoderm induction (embryogenesis/helix4oop-helix/gastrulation/myogenesis/RNase protection)
RICHARD P. HARVEY Molecular Biology Unit, The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia
Communicated by G. J. V. Nossal, July 18, 1991 (received for review June 7, 1991)
ABSTRACT The MyoD gene codes for an important regulatory factor in skeletal myogenesis. To explore the relationship between mesoderm induction in Xenopus embryos and expression of MyoD, I have monitored MyoD mRNA levels in normal embryos and cultured explants by RNase protection. Transcription from the two Xenopus MyoD gene copies is activated weakly across the whole embryo at the midblastula transition, and this activation occurs in the absence of mesoderm induction. In response to induction this basal expression is amplified 50- to 100-fold, but in animal-pole explants 6-10 hr elapse before induced mRNAs appear, and this induction requires prior protein synthesis. The promiscuous transcripts disappear from animal explants at a time when induction "competence" is lost, suggesting a link between these events. The data highlight a broad, but transient, permissiveness for MyoD expression in embryos, which is propagated and amplified only in presumptive muscle in response to induction. Moreover, muscle-specific MyoD expression is a relatively late (postgastrulation) event in the mesoderm-induction cascade.
maturation, induces myoblast commitment (2, 3). The consequences of expression in myoblasts are not well documented, but negative regulators, such as Id (17), and cofactors, such as products of the E2A gene (18), influence how MyoD expression is interpreted at different stages of maturation. A separate function of MyoD may be to arrest cell cycling at the onset of myogenic differentiation (19, 20). The Xenopus genome contains two MyoD genes (21-23), referred to in this paper as MyoDa and MyoDb. Maternal transcripts occur at a low level, whereas zygotic expression is muscle-specific from an early stage and a product of mesoderm induction (21, 22). Zygotic expression of the MyoDa gene begins at the midblastula transition (MBT), the time when embryonic transcription is initiated and some 7 hr before appearance of muscle-differentiation products, such as actin. If MyoD expression were muscle-specific at this time, it would imply that the muscle lineage had already been determined and, more generally, that blastomeres become committed to mesodermal lineages during cleavage, perhaps in direct response to the events of mesoderm induction. I show in this paper that MyoD expression at the MBT occurs across the whole embryo and is not amplified in presumptive muscle until early gastrulation. The early lowlevel expression is independent of mesoderm induction, whereas maximal expression requires induction. The broad early permissiveness for MyoD expression may be related to "competence" for mesoderm induction because early expression is down-regulated in explants at a time when competence is lost. MyoD can be classed as an intermediate response gene with respect to induction because in animalpole explants there is a lag of 6-10 hr before induced mRNAs appear and prior protein synthesis is needed. Because MyoD expression is propagated and amplified in muscle after gastrulation begins, muscle determination appears to be a postgastrulation event.
Detailed fate maps have been constructed for organisms, like Xenopus, which are disposed to analysis of developmental problems, but these maps tell us little about how, and when embryonic cells become committed to their respective lineages. These issues are normally studied by cell- or tissuetransplantation experiments, and two progressive states of commitment-specification and determination-have been defined (1). Recent evidence demonstrates that some nuclear proteins containing the structurally conserved helix-loophelix dimerization domain are important regulators of lineage commitment (2-4). Analysis of the function of these proteins and their expression in embryos will help to mark and define the molecular nature of commitment events. A series of in vitro studies, stemming from analysis of the propensity for mouse C3H 1OT1/2 fibroblasts to undergo myogenesis (5), implicate the helix-loop-helix protein MyoD as a key regulator of muscle development (6). Expression of the MyoD gene in primary fibroblasts and various cell lines results in biochemical and morphological conversion of these cells to muscle (7, 8). Three other genes with structural and apparent functional homology to MyoD have now been isolated (myJf, myogenin, and herculin/muscle regulatory factor 4; refs. 9-12) and an additional member of the myogenic pathway, myd, has been inferred from transfection studies (13). The MyoD gene codes for a multifunctional myogenic regulatory factor. It is a DNA-binding protein that transactivates muscle-specific structural genes in differentiating myocytes (14-16). Forced expression of the MyoD gene in cultured cells, under conditions nonpermissive for myogenic
MATERIALS AND METHODS Embryo and Explant Culture. Xenopus laevis eggs were obtained and fertilized as described (24). Embryos were raised in 0.1 x modified Barth solution (MBS) (25) containing Gentamycin at 100 ,ug/ml. Staging was according to Nieuwkoop and Faber (26). Animal and vegetal caps were dissected between stages 8 and 9 and cultured on agarose beds in 0.5x MBS containing Gentamycin at 100 ug/ml and y-globulins (Sigma) at 2 mg/ml, with or without mesoderminducing factors. Basic fibroblast growth factor was from Amersham and used at 20 ng/ml. Mesoderm-inducing factor from the Xenopus XTC cell line (XTC-MIF) (supplied by J. Smith, Medical Research Council, National Institute for Medical Research, Mill Hill, London) was a partially purified
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: MBT, midblastula transition; XTC-MIF, mesoderminducing factor from the Xenopus XTC cell line.
9198
Developmental Biology: Harvey
Proc. Natl. Acad. Sci. USA 88 (1991)
fraction (see ref. 22), dialyzed in phosphate-buffered saline before use. For induction, 4 units/ml were used, where 1 unit/ml is the minimum concentration required to induce muscle-specific actin gene expression. For cycloheximide treatment, stage 8½/2 caps were incubated in 0.5x MBS/ cycloheximide at 5 Ag/ml/y-globulins at 2 mg/ml and Gentamycin at 100 jig/ml for 30 min, followed by a 30-min incubation in the same buffer containing XTC-MIF. Caps were then washed well in 0.5x MBS and incubated in the above buffer without cycloheximide overnight. Serum-free medium conditioned by murine P388D1 macrophages (supplied by D. Hilton, Whitehead Institute, Cambridge, MA) was concentrated 18-fold and used at 1:1 in induction assays. RNA Analysis. For RNase protection analysis, total RNA was extracted from embryos and explants by using the proteinase K/phenol method (24). RNase protection was as described (40). The MyoD probe was an Fsp I/Pst I fragment (nucleotides 526-886) of the MyoDb cDNA (22), cloned into pSP72. Probes were synthesized from EcoRI-linearized plasmid with SP6 polymerase. Hybrids were digested with RNase A at 2 ixg/ml and RNase T1 at 0.1 ,ug/ml at 160C for 30 min. The actin probe was synthesized from the plasmid AC100 (27), linearized with Dde I, and transcribed with SP6 polymerase. Hybrids were digested in RNase A at 2 ;Lg/ml and RNase T1 at 0.1 ,ug/ml for 30 min at 37°C.
scribed during oogenesis, resulting in a stable maternal mRNA, and in embryos beginning at the MBT when the embryonic genome first becomes transcriptionally active (22). To analyze expression from the MyoDb gene, an RNase protection probe was constructed from a region of the MyoDb cDNA that is highly conserved between MyoDb and MyoDa. With limited RNase digestion, this probe detects MyoDb mRNA as a full-length species and MyoDa mRNA as a partially digested species (Fig. 1). The identity of the bands was confirmed using synthetic MyoDa or MyoDb mRNAs transcribed from corresponding cDNA clones (22). Fig. 1 shows an RNase protection analysis ofRNA samples taken at hourly intervals from 5- to 15-hr-old embryos. This RNA series, which has been characterized previously for MyoDa and other markers, spans the MBT, which occurs between 6 and 7 hr (22). Three points about initiation of MyoD expression are noteworthy. (i) Only the MyoDa gene contributes to the maternally inherited mRNA-i.e., bands seen in pre-MBT samples. To validate this conclusion RNA was analyzed from eggs of eight independent females, and all samples contained only MyoDa mRNA (data not shown). (ii) MyoDb expression, like that of MyoDa, initiates at the MBT. Both MyoD genes are therefore activated in the zygote as soon as transcription first begins. (iii) Zygotic MyoD transcripts accumulate slowly and only reach levels significantly above that of the maternal RNA after gastrulation. With this probe it has also been shown that both genes are activated, although to different extents, in animal-pole explants (animal caps) induced to form mesoderm by contact with vegetal-pole cells or by culture with mesoderm-inducing factors (Fig. 2). Weak induction is seen with basic fibroblast growth factor, consistent with its poor ability to induce dorsal mesoderm (30). Strong induction is seen in animal/vegetal recombinants and in animal caps treated with P388D1 macrophage-conditioned medium or XTC-MIF, which contain factors similar or identical to activin A (31, 32). With all inducing regimens, as well as in normal embryos, the MyoDb
RESULTS Only the MyoDa Gene Is Expressed During Oogenesis. The Xenopus genome contains two MyoD genes that are thought to be functionally equivalent (22, 23). Many genes have been duplicated during the evolution of polyploidy in anurans (28), but the two Xenopus c-myc genes are regulated differently (29), one being expressed only maternally. It seemed important, therefore, to analyze expression from both MyoD genes, in case regulatory or functional differences had evolved. It has previously been shown that the MyoDa gene is tran0 )
0
L
-* 406_ 360-*
z
z
>- a E -O-E
r=
H- r,-
1-~-
u
F-
>a
~A5
'6B TI 5
6
7
umU.
&
~Li
_m a--
K
9AS-wL..^ -
__OA_,* .. .
22 9-* 2 1 7-_-
9199
.
.
M oDa
N
_w
oM . v
.ow
FIG. 1. Kinetics of activation of MyoD genes in normal development. The RNase protection probe used in this analysis protects both MyoDa and MyoDb transcripts. Protected bands are identified in control assays using synthetic mRNAs transcribed from cDNA clones with T7 polymerase (T7-MyoDa and T7-MyoDb RNAs). For reasons not clear, synthetic RNAs always protect a doublet in these assays, whereas embryo RNA protects single bands for each mRNA. RNA samples were extracted from embryos at hourly intervals from 5-15 hr postfertilization (5-15 hpf). This same RNA series has been previously characterized for MyoDa, a-cardiac actin, N-cell-adhesion molecule, Xhox-1A, GS17, and cytoskeletal actin RNAs (22). The position of the MBT was determined by the onset of transcription of the GSI7 gene (24).
9200
Developmental Biology: Harvey
mRNA reaches levels -5-fold above that of MyoDa. Presumably, the MyoDb gene promoter is more responsive to induction mechanisms. MyoD Expression Is a Delayed Response to Mesoderm Induction. The early activation of MyoD genes and their induction by mesoderm inducers raise two questions about the formation of mesoderm in Xenopus (22). (i) Is MyoD expression a primary transcriptional response to mesoderm induction? (ii) Is MyoD expression at the MBT specific to presumptive muscle, as would be expected were blastomeres already assigned to the muscle lineage at this point? The first question has been addressed. A true immediate-early response to mesoderm induction, like that from the homeobox gene Mix.], is characterized by rapid appearance of transcripts in response to induction, even in the absence of protein synthesis (33). Before induction with XTC-MIF, animal caps were incubated in cycloheximide under conditions that inhibit 90%o ofprotein synthesis for at least 4 hr (33). This treatment almost totally inhibited induction ofthe MyoD genes and expression of the muscle-specific actin gene (Fig. 2), as well as elongation movements characteristic of induced caps. On the other hand, cytoskeletal actin expression, a marker of tissue mass and viability, was minimally affected. The kinetics of MyoD gene activation in XTC-MIFinduced explants was analyzed to determine how soon MyoD transcripts appear (Mix.1 mRNA appears within 30 min). In U-)
z
C') Li
z 0 L
W LL
Li
C
L+
LF-
co n
Q cL
e
x
L-
LiJE
s
U')
X
L
r) IC z
-
U LLI
Proc. Natl. Acad. Sci. USA 88 (1991)
0
z
U] -C
4k--
LLI
'v
FIG. 2. Activation of MyoDa and MyoDb genes in animal-pole explants cultured overnight in basic fibroblast growth factor (bFGF), XTC-MIF, medium conditioned with murine P388D1 macrophages (P388D1-CM) or animal explants combined with vegetal-pole cells (A/V recombinants). In one sample (XTC-MIF + CHX), explants were treated with cycloheximide before addition of XTC-MIF. Induction of muscle in explants was monitored by RNase protection with a muscle-specific a-cardiac actin probe (MS-Actin). This probe also detects cytoskeletal actins (CY-Actin), which serve as a measure of explant viability and tissue mass. For MyoD protections, 12 animal caps were used; for actin protections, 3 caps were used. Control protection (tRNA) contains 20 jig of yeast tRNA.
---
A c- t i r,
Acr;~,,i.r ---Act
FIG. 3. Kinetics of expression of MyoD genes in animal-pole explants treated with XTC-MIF. Stage 8/2-9 explants were cultured in XTC-MIF and sampled at the times indicated (1-22 hr) before RNA extraction and RNase protection with probes for MyoD (12 caps) and actin (3 caps). Induced 0-hr caps were extracted immediately after dissection and were not incubated in XTC-MIF. The uninduced 22-hr sample was incubated overnight without induction. Note that cytoskeletal actins are also induced in caps, as they are in normal development, but this is not a response to induction.
Developmental Biology: Harvey
Proc. Natl. Acad. Sci. USA 88 (1991)
diate response, as it occurs between induction of immediateearly genes (such as Mix. 1) and differentiation products, such as cardiac actin. The induction-dependent influences that result in activation of the MyoD genes are at present unknown (see Discussion). Induction-Independent MyoD Expression Occurs Across the Whole Embryo. In normal development animal caps do not form muscle but contribute mainly to epidermis and brain. To further investigate the expression of MyoD RNAs in uninduced caps, animal and vegetal caps were cultured for various periods without inducers, and extracted RNA was analyzed for MyoD transcripts (Fig. 4). MyoD genes were transcribed in both types of explant, although with different kinetics. Because caps are dissected shortly after the MBT, this early phase must account for expression initiated at the MBT in normal embryos (see Fig. 1). It is unlikely that animal caps are induced in these experiments-for example, during dissection-because the strong induction of MyoD or actin transcription characteristic of induced caps (Figs. 2 and 3) has never been seen in several experiments. The data in Fig. 3 suggest that induction may have a weak positive effect on the early expression of MyoD genes. In a separate experiment the level of early transcripts in uninduced animal caps after 5 hr in culture was compared with levels in caps induced for the same period with basic fibroblast growth factor, XTC-MIF, P388D1 macrophageconditioned medium, or vegetal-pole cells. In each case the levels of both MyoDa and -b transcripts were unaltered (data not shown). Similarly, treatment of caps with cycloheximide before induction did not change the level of early transcripts. Therefore, products produced in the early phase of mesoderm induction do not amplify or sustain the basal level of MyoD transcription occurring across the embryo. The kinetics of expression of early transcripts differs between animal and vegetal caps. Interestingly, transcripts in animal caps are degraded rapidly at a time (6-11 hr in culture; Fig. 4) corresponding to stage 101/2-12 in normal development. This is also when the capacity ("competence"; ref. 34)
9201
for these cells to be induced to mesodermal lineages is lost (ref. 35; see Discussion). In vegetal-pole cells, transcripts persist much longer, still being detectable after 11 hr and weakly after 22 hr of culture. It is an interesting possibility that vegetal cells, which are the source of mesoderm inducers in normal development (36), can respond weakly to autoinduction. The Mix.] gene, which gives an immediate-early response to induction in animal caps, is also expressed in the vegetal-pole cells, as well as presumptive mesoderm in normal blastulae (33).
DISCUSSION The two Xenopus MyoD gene copies are regulated differently during oogenesis and are induced to different extents during embryogenesis. The significance of this to development is unknown, as is the relevance of the maternal RNA per se. Whether it reflects real functional differences between the gene products or is simply a consequence of reduced selection pressure on duplicated genes is still an open question and will require further analysis. In an RNase protection assay, both MyoDa and MyoDb genes were found to be transcribed at low levels across the whole embryo, beginning at the MBT, and this early transcription is independent of mesoderm induction. This result has also recently been reported by Rupp and Weintraub (37) using a quantitative PCR assay. The number of early transcripts from each MyoD gene was estimated to be 105 per animal cap (data not shown), compared with _107 for later embryos (21, 22). What is the significance of these early transcripts? It is possible that the early transcription phase is necessary for induction-dependent MyoD expression. Autoactivation (38), triggered by the events of induction, would seem a plausible mechanism to connect the two phases. Alternatively, there may be a nonmyogenic function for MyoD in early blastomeres. However, because the low level of early transcripts is probably too low to produce a significant level of tran-
0
ANIMAL CAPS
LL
O
L
0
L
LU
0
-
L
v ~ I ro
L -
VEGETAL CAPS L
L.
CN
.- N
0
L
-_
r_
-
rn
Lfl
Iq~
LC -~ ko- (N
4-
Myo D b
4- MyoDa
I.
19r a
W, -Am- W, .w
10
*-MS -Act in +4-CY-Actin
FIG. 4. Time course of expression of MyoD genes in cultured animal and vegetal-pole explants without induction. Explants were cut at stage
81/2-9 and cultured without induction for the times indicated (0-22 hr). RNA extracted at each time point was analyzed by RNase protection for MyoD transcripts (12 explants) and actin transcripts (3 explants). The egg sample was taken from eggs that were fertilized to produce the embryos used in this experiment; only the MyoDa gene contributes to the maternal RNA pool.
9202
Developmental Biology: Harvey
scription factor, this expression probably more likely reflects a general capacity for MyoD transcription, which is then propagated and amplified specifically in presumptive muscle by the events of mesoderm induction. In animal caps this capacity for transcription is of limited duration because early transcripts are lost after several hours. It is probably significant that the time of this loss corresponds to when the competence of animal caps to be induced to mesoderm is also lost. The window of competence during early development may, therefore, represent, in part, a permissiveness for critical lineage regulatory genes, such as MyoD, to respond to the early events of induction. Rupp and Weintraub (37) have also noted, using PCR, that some other potential regulatory genes (but not all) show a broad and transient expression phase. Loss of competence may involve a general repression of these loci. It is not known whether the early mRNAs are translated into MyoD protein. However, their deadenylylated state in blastulae suggests that these early RNAs may not be loaded onto polysomes (22). Full induction-dependent MyoD expression does not occur until many hours after the addition ofinducers to animal caps. Furthermore, it is totally dependent on the prior synthesis of new proteins in response to induction. For these reasons the MyoD genes can be regarded as intermediate-response genes to induction because they are expressed after immediateearly genes like Mix.I but before lineage differentiation products, such as cardiac actins. The specific amplification of MyoD gene expression in a muscle-specific manner occurs only after the beginning of gastrulation. Because MyoD is believed to be a critical myogenic regulator and is part of an auto-activating and cross-regulating myogenic regulatory signal (2, 3), definitive commitment to the muscle lineage would appear to be a postgastrulation event. This idea fits well with estimates of commitment in other germ layers from a singlecell transplantation assay (39). Which gene products interact with the MyoD gene promoter to ensure muscle-specific activation is now an important question. I thank Ralph Rupp and Hal Weintraub for releasing data before publication, Ora Bernard for basic fibroblast growth factor, Jim Smith for XTC-MIF, Doug Hilton for P388D1-conditioned medium, Donna West and colleagues for animal care, and Jerry Adams and Jane Visvader for comments on the manuscript. This work was supported by a Queen Elizabeth II Fellowship and a grant from the National Institutes of Health (HD26024-02). 1. Slack, J. M. W. (1983) in From Egg to Embryo: Determinative Events in Early Development, eds. Barlow, P. W., Green, P. B. & Wylie, C. C. (Cambridge Univ. Press, Cambridge, U.K.), pp. 18-20. 2. Olsen, E. N. (1990) Genes Dev. 4, 1451-1461. 3. Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., Benezra, R., Blackwell, T. K., Turner, D., Rupp, R., Hollenberg, S., Zhuang, Y. & Lassar, A. (1991) Science 251, 761-766. 4. Campos-Ortega, J. A. & Knust, E. (1990) Eur. J. Biochem. 190, 1-10. 5. Taylor, S. M. & Jones, P. A. (1979) Cell 17, 771-779. 6. Davis, R. L., Weintraub, H. & Lassar, A. B. (1987) Cell 51, 987-1000.
Proc. Natl. Acad Sci. USA 88 (1991) 7. Weintraub, H., Tapscott, S. J., Davis, R. L., Thayer, M. J., Adams, M. A., Lassar, A. B. & Miller, A. D. (1989) Proc.
Natl. Acad. Sci. USA 86, 5434-5438. 8. Lin, A.-Y., Dechesne, C. A., Eldridge, J. & Paterson, B. M. (1989) Genes Dev. 3, 986-996. 9. Braun, T., Buschhausen-Denker, G., Bober, E., Tannich, E. & Arnold, H. H. (1989) EMBO J. 8, 701-709. 10. Wright, W. E., Sassoon, D. A. & Lin, V. K. (1989) Cell 56, 607-617. 11. Rhodes, S. J. & Konieczny, S. F. (1989) Genes Dev. 3, 20502061. 12. Mueller, P. R. & Wold, B. (1989) Science 246, 780-786. 13. Pinney, D. F., Pearson-White, S. H., Konieczny, S. F., Latham, K. E. & Emerson, C. P., Jr. (1988) Cell 53, 781-793. 14. Murre, C., McCaw, P. S. & Baltimore, D. (1989) Cell 56, 777-783. 15. Lassar, A. B., Busken, J. N., Lockshon, D., Davis, R. L., Apone, S., Hauschka, S. D. & Weintraub, H. (1989) Cell 58, 823-831. 16. Weintraub, H., Davis, R., Lockshon, D. & Lassar, A. (1990) Proc. Natl. Acad. Sci. USA 87, 5623-5627. 17. Benezra, R., Davis, R. L., Lockshon, D., Turner, D. L. & Weintraub, H. (1990) Cell 61, 49-59. 18. Murre, C., McCaw, P. S., Vaessin, H., Caudy, M., Jan, Y. N., Cabrera, C. V., Buskin, J. N., Hauschka, S. D., Lassar, A. B., Weintraub, H. & Baltimore, D. (1989) Cell 58, 537-544. 19. Sorrentino, V., Pepperkok, R., Davis, R. L., Ansorge, W. & Phillipson, L. (1990) Nature (London) 345, 813-815. 20. Crescenzi, M., Fleming, T. P., Lassar, A. B., Weintraub, H. & Aaronson, S. A. (1990) Proc. Natl. Acad. Sci. USA 87, 84428446. 21. Hopwood, N. D., Pluck, A. & Gurdon, J. B. (1989) EMBO J. 8, 3409-3417. 22. Harvey, R. P. (1990) Development 108, 669-680. 23. Scales, J. B., Olsen, E. N. & Perry, M. (1990) Mol. Cell. Biol. 10, 1516-1524. 24. Krieg, P. A. & Melton, D. A. (1985) EMBO J. 4, 3463-3471. 25. Gurdon, J. B. (1977) Methods Cell Biol. 16, 125-138. 26. Nieuwkoop, P. D. & Faber, J. (1956) Normal Table ofXenopus laevis (Daudin) (North-Holland, Amsterdam). 27. Dworkin-Rastl, E., Kelly, D. B. & Dworkin, M. B. (1986) J. Embryol. Exp. Morphol. 91, 153-168. 28. Fritz, A. F., Cho, K. W. Y., Wright, C. V. E., Jegalian, B. G. & De Robertis, E. M. (1989) Dev. Biol. 131, 584-588. 29. Vriz, S., Taylor, M. & Mechali, M. (1989) EMBO J. 8, 4091-4097. 30. Slack, J. M. W., Darlington, B. G., Heath, J. K. & Godsave, S. F. (1987) Nature (London) 326, 197-200. 31. Smith, J. C., Price, B. M., Van Nimmen, K. & Hylebroeck, D. (1990) Nature (London) 345, 729-731. 32. Thomsen, G., Woolf, T., Whitman, M., Sokol, S., Vaughan, J., Vale, W. & Melton, D. A. (1990) Cell 63, 485-493. 33. Rosa, F. (1989) Cell 57, 965-974. 34. Waddington, C. H. (1932) Philos. Trans. R. Soc. London B 221, 179-230. 35. Jones, E. A. & Woodland, H. R. (1987) Development 101, 557-563. 36. Nieuwkoop, P. D. (1973) Adv. Morphog. 10, 1-39. 37. Rupp, R. A. & Weintraub, H. (1991) Cell 65, 927-937. 38. Tapscott, S. J., Davis, R. L., Thayer, M. J., Cheng, P.-F., Weintraub, H. & Lassar, A. B. (1988) Science 242, 405-411. 39. Heasman, J., Snape, A., Smith, J. C. & Wylie, C. C. (1986) J. Embryol. Exp. Morphol. 97, Suppl., 65-73. 40. Krieg, P. A. & Melton, D. A. (1988) Methods Enzymol. 155, 397-415.
