Cell, Vol.-65,
927-937,
June
14, 1991, Copyright
0 1991 by Cell Press
Ubiquitous MyoD Transcription at the Midblastula Transition Precedes Induction-Dependent MyoD Expression in Presumptive Mesoderm of X. laevis Ralph A. W. Rupp and Harold Weintraub Department of Genetics and Howard Hughes Medical Institute Fred Hutchinson Cancer Research Center Seattle, Washington 98104
Summary We have used a quantitative reverse transcriptionpolymerase chain reaction assay to detect MyoD mRNA during early embryonic development of Xenopus laevis. We find that during a short period of time following the midblastula transition MyoD becomes transcriptionally activated at a low level ubiquitously throughout the embryo. Restriction of MyoD expression to muscle precursor cells appears as a subsequent event, in which the process of mesoderm induction stabilizes transcription only in the marginal zone of the embryo, the presumptive mesoderm. Introduction MyoD induces the skeletal muscle-specific differentiation program in many different cell types by binding to regulatory DNA sequences of muscle-specific genes. Similar functions have been reported for myogenin (Wright et al., 1989), Myfd (Braun et al., 1989), and MRF4/herculin/ Myf-6 (Rhodes and Konieczny, 1989; Miner and Wold, 1990; Braun et al., 1990). All of these genes share extensive sequence similarity throughout their basic helix-loophelix DNA-binding domains (Murre et al., 1989). Together they constitute the so-called MyoD or myogenic gene family (for recent reviews see Olson, 1990; Lassar and Weintraub, 1991; Weintraub et al., 1991). We know relatively little about how these genes become activated during embryogenesis. In a variety of organisms, which include mouse (Sassoon et al., 1989) quail (de la Brousse and Emerson, 1990), Xenopus (see below), Drosophila (Michelson et al., 1990), and C. elegans (Krause et al., 1990) members of this gene family are found to be expressed at the appropriate time and position within the embryo to suggest their direct involvement in the initial muscle cell commitment event. This suggests that the mechanism for generating muscle cell type is an ancient and conserved process. Recently, several cDNAs have been cloned from X. laevis that appear to be true homologs of MyoD based on the high sequence similarity throughout the entire coding region (Hopwood et al., 1989a; Harvey, 1990; Scales et al., 1990). At least one of these cDNAs is capable of transactivating mouse MyoD and inducing myogenesis in the lOT1/2 cell line (Scales et al., 1990). They represent transcripts from two highly related genes, called MyoDa and MyoDb (Harvey, 1990), which probably arose as the result of a genome duplication event during anuran evolution (Kobel and DuPasquier, 1986). In adult frogs MyoD ex-
pression is restricted to skeletal muscle cells (Hopwood et al., 1989a; Scales et al., 1990) as it is in mammals (Davis et al., 1987). However, early frog embryos contain an unlocalized maternal MyoD RNA pool of unknown function, whose transcripts seem to be derived from the MyoDa gene only (Hopwood et al., 1989a; Harvey, 1990). While zygotic mRNAs of both genes have been detected from early gastrula stages onward, the appearance of RNA precursor molecules of the MyoDa gene at the midblastula transition (MBT) (Harvey, 1990) suggests that the MyoD genes become transcribed immediately at the onset of zygotic gene expression (Newport and Kirschner, 1982). Thus MyoD expression would start early enough to account for the activation of the cardiac actin gene, the first among the known structural muscle genes to become activated during development (Mohun et al., 1986; Hopwood et al., 1989a). Indeed, injection of MyoDb mRNA into 2-cell frog embryos leads to ectopic expression of the cardiac actin gene in ectodermal cells of the animal pole (Hopwood and Gurdon, 1990). In Xenopus, mesoderm is formed in the equatorial region, the marginal zone, of the embryo by induction of nearby animal pole cells with growth factors emitted by vegetal pole cells (reviewed by Gurdon, 1987; Smith, 1989). In explants from the animal pole region (animal cap), which do not differentiate into muscle without induction (Gurdon et al., 1985), the MyoD genes are activated by conjugation with vegetal pole cells (Hopwood et al., 1989a) or by direct incubation with mesoderm-inducing factors (Harvey, 1990). Furthermore, RNA analysis from embryo dissections and in situ hybridizations localized the MyoD transcripts exclusively to the presumptive mesoderm in the marginal zone (Hopwood et al., 1989a; Harvey, 1990). It was therefore concluded that the MyoD genes are activated in a specific region of the embryo as a consequence of mesoderm induction and that their activation marks the commitment of presumptive mesodermal cells to the skeletal muscle cell lineage. We have used a quantitative reverse transcription-polymerase chain reaction (RT/PCR) assay (Veres et al., 1987; Singer-Sam et al., 1990) to detect MyoD mRNAs. Using this highly sensitive assay we report here that the MyoD genes are primarily activated at the MBT in a transient manner throughout the embryo. This transcriptional activation appears to be independent of mesoderm induction. Instead of triggering the activation of these genes, mesoderm induction seems to be a secondary event that stabilizes the transient, ubiquitous expression of the MyoD genes only in a specific subset of embryonic cells. Results Use of a Quantitative RTlPCR Assay for the Expression of XMyoD Genes To gain experimental access to the questions of when and how transcripts of the frog MyoD genes accumulate in a specific subset of embryonal cells, it was necessary to use
Cell 928
A + CEsl3
E
s6
s9
:;r ,-. .
-XMyoD
a b
i
r
-XMyoD -EF-la
4
5
B
C
s13
s13
: .II
123
RT’a?.e -- E
single
6
7
8
9
Relative
Abundance
4
5
primer multiplex lanes
ratio
13 14 15
of PCR-Products
6
pair
10 1112
7
a
9
PCR
lanes
ratio
mi
E
Figure 1. Quantitation of MyoD Development by RTlPCR
s13
RNA
Levels
during
Early Xenopus
(A) Embryo equivalents of total cellular RNAs from unfertilized eggs (E) or embryos (~6, s9, and ~13) staged according to Nieuwkoop and Faber (1967) were analyzed under standard conditions (see Experimental Procedures) by multiplex RT/PCR (lanes 1-9) or PCRs performed with single primer pairs: XMyoDa, lanes 11 and 14; XMyoDb, lanes 10 and 13; EFla, lanes 12 and 15. Lane 8: parallel sample to lane 7, but 2 cycles less amplification. Lane 9: twice as much of RT sample added to the PCR relative to lane 7. Control reactions performed without reverse transcriptase: no template (C, lane 1); RNA from unfertilized eggs (E, lane 2); RNA from stage 13 embryos (~13, lane 3). The faint diffuse signal seen in RNA of unfertilized eggs (lane 4) at about the appropriate position does not represent authentic MyoDb PCR product, since it does not appear in the PCR performed with the MyoDb gene-specific primer pair alone (lane 10) or any other PCR performed with a single primer pair (lanes 11 and 12). (B and C) The radioactivity of the PCR products was quantitated with a Phosphorlmager System, subtracting as background the signals from appropriate positions of control samples lacking reverse transcriptase (lane 2, background for lanes 4 and 5; lane 3, background for lanes 6-9). (B) shows the relative increase of PCR products during early development as normalized to the earliest signal detected for each gene. Numbers above the table refer to lane numbers of (A). n.d., no signal detectable over background. The bracketed numbers in the column for lane 6 represent the calculated decrease of product amounts, taking into account the amplification efficiency of each primer pair (see Experimental Procedures). (C)compares the product amounts generated by multiplex PCRs with those from PCRs performed with single primer pairs.
an assay system that is both extremely sensitive and able to work with small RNA amounts. We chose for this purpose RTIPCR (Veres et al., 1987; Gilliland et al., 1990; Singer-Sam et al., 1990). In a first step, cDNA is made from total cellular RNA using random hexamer primers. An aliquot of this reaction is then used as template for the PCR, in which gene-specific primers amplify short regions from the respective cDNAs. The PCR mix contains trace amounts of [a-32P]dCTP to allow detection and quantitation of the PCR products. Using published sequences (Hopwood et al., 1989a; Harvey, 1990; Scales et al., lQQO), we designed primer pairs specific for the two Xenopus MyoD genes, which we call MyoDa and MyoDb according to the nomenclature of Harvey (1990). These primers target regions C-terminal of the helix-loop-helix motif to avoid cross-hybridization to cDNAs of other members of the helix-loop-helix protein family(Murre et al., 1989; Benezraet al., 1990). The MyoD primers were also chosen so that they flank expected introns to assure the detection of mRNA and not a nuclear RNA precursor or genomic DNA. Furthermore, we chose a primer pair for the ubiquitously expressed EFla gene (Krieg et al., 1989) to serve as an internal control for the amount of RNA tested (“multiplex PCR”; Chamberlain et al., 1990). The additional controls shown in our figures indicate clearly that the generation of PCR products is strictly dependent on the synthesis of cDNA during the RT reaction and on the presence of both forward and reverse primers during subsequent PCR amplification. It was previously shown that MyoD transcript levels peak in late gastrula embryos (Hopwood et al., 1989a). Based on this, we chose gastrula RNA (stage 13 embryos) to calibrate the maximal RNA input and PCR cycle number that still ensure exponential amplification of the target templates during the PCR (see Experimental Procedures). Like others (Hopwood et al., 1989a; Harvey, lQQO), we detect maternal MyoD transcripts in unfertilized eggs or pre-MBT embryos (Figure 1, lanes 4 and 5). As we show here they are derived exclusively from the MyoDa gene, since our PCR primers differentiate between RNAs of the two highly related MyoD genes (see Figure 7). We detect no transcripts of the MyoDb gene at these stages. From stage 9 onward we observe a strong increase in the product amounts for both MyoD genes and the EFla gene, reflecting the onset of zygotic transcription at the MBT (Figure 1, lanes 6 and 7). The quantitation of the control experiments (see Figure 1 B) reveals that decreasing the cycle number (lane 8 versus lane 7) reduced proportionally the amounts of all PCR products, while doubling the input of RT sample (lane 9 versus lane 7) results in a less than 2-fold increase. This indicates that with the applied cycle number the RNA input of embryo equivalents brings the RT/PCR to the edge of exponential amplification, but is not yet saturating. As can be seen in Figure 1C, the product amounts of the multiplex PCR samples, which contain three primer pairs, match those of PCRs performed with single primer pairs within a factor of 2. In addition, the 37-fold (Figure 1, lane 7) to 54-fold increase (lane 14) of the PCR product representing MyoDa transcripts from unfertilized eggs to stage 13 em-
Primary 929
Activation
t=O -
C +-+++++
of MyoD Genes
in X. laevis
t=12h b 2 ;I.- o ‘; L ol-oa c XQ u
t=o SP ++
VMD + +
+
c T.
1234567 Figure
2. MyoD
89 RNA Levels
in Post-MBT
.._ _,
RT’ase
-
XMyoD
a
“’ -
XMyoD EF-lo
b
10 n 12 Animal
Cap Explants
Total cellular RNA was purified from animal cap explants of stage 9 embryos either immediately after dissection (t = 0, lanes 2 and 3, 812) or after overnight cultivation in 1 x modified Barth’s saline with or without mesoderm-inducing factors (t = 12 hr, lanes 4-7). Final concentrations: XTC cell culture supernatant, I:20 dilution; porcine activin A, 0.5 nglml; basic FGF or TGF-PI (R & D Systems), 40 nglml each (incubations of explants with 1 x modified Barth’s saline supplemented with bFGF/TGF-91 storage solutions did not produce detectable MyoD RNA levels; data not shown). S (lane 9) RNA sample from singly dissected explants, lysed immediately in guanidinium thiocyanate (t = 1 min per explant); P (lane 9) control RNA sample from pool-dissected explants (t = 15 min per 10 explants). RNAs from animal cap subregions (lanes 10-12): V, ventral third; M, midddle region; D, dorsal third of animal caps. Explants were prepared from embryos with strict asymmetrical pigment distribution in respect to dorsallventral blastomeres after the second cleavage. To compensate for the lower RNA amounts of the subdivided explants, two explant equivalents of each fraction were used per RT reaction. Multiplex RT/PCRs were performed under standard conditions.
bryos (lanes 4 and 7) correlates very well with the 40-fold increase measured via RNAase protection assays by Hopwood et al. (1989a). The RT/PCR assay used here is therefore capable of measuring accurately and simultaneously the relative transcript levels of several genes, allowing the use of the EFla primer pair as a true internal control. For the following experiments, the RNA input was set so not to exceed the PCR product amounts generated with the concentration of stage 13 embryo RNA used in Figure 1, thereby guaranteeing quantitative performance of the RTI PCR assay. The Two MyoD Genes Are Ubiquitously Transcribed in Xenopus Embryos in a Transient Manner Preceding Lineage-Specific Restriction The activation of the MyoD genes has been shown to be the earliest known muscle-specific response to mesoderm induction in embryos and isolated animal cap explants. In situ hybridization has localized the position of the zygotically expressed MyoD RNA to the marginal zone of early gastrulaembryos, which includes presumptive muscle tissue (Hopwood et al., 1989a; Harvey, 1990). To our surprise, however, we found both MyoD genes expressed in uninduced animal cap explants from earlier blastula embryos (stage 9; Figure 2, lane 3), when RNA was prepared immediately after dissection. Subdividing these explants into ventral, middle, and dorsal parts, the MyoD transcripts are almost equally distributed (Figure 2, lanes
10-12). To date, in more than ten independent experiments, we have not obtained a single RNA sample derived from animal caps of about stage 8X-10 embryos that lacked zygotic MyoD transcripts (data not shown). To prove that the explants were not induced we undertook a number of control experiments. Reducing the time span from the start of dissection to the completion of cell lysis to 1 min did not affect the abundance of the MyoD PCR products (Figure 2, lanes 8 and 9), which are derived from spliced mRNAs (see above; Figure 7). If the MyoD genes were induced by experimental manipulation, then RNA induction, processing, and steady-state mRNA accumulation would have to be completed within 1 min. This time frame is inconsistent with known estimates for these processes (Lewin, 1980). When cultivated overnight in neutral saline, animal cap explants had formed undifferentiated round balls (see Figure 3) and had lost all MyoD mRNAs (Figure 2, lane 4). The control primers present in the multiplex PCR generated a strong EFl a signal, indicating that these explants were indeed viable. In contrast, incubation of the animal cap explants with mesoderminducing factors (Figure 2, lanes 5-7) counteracted the loss of MyoD mRNA and resulted in high level expression of both MyoD genes. In a further experiment, we dissected embryos from preMBT to early gastrula stages into animal caps, marginal zones, and vegetal poles and immediately purified their RNAs (Figure 3). When cultured separately, these parts of the embryos are known to form ectodermal derivatives including epidermis and nerve, embryoid bodies containing notochord and muscle, and poorly differentiated endodermal cells, respectively (Gurdon et al., 1985). Analyzed by RT/PCR, cells of all three regions of stage 9-10 embryos expressed roughly equivalent levels of MyoD mRNAs, irrespective of their future developmental fate. Using a primer pair for GS17 to identify the MBT (Krieg and Melton, 1985), MyoD transcripts, particularly clearly for the MyoDb gene, were detectable as soon as zygotic transcription had started (Figure 3, compare lanes 2 and 3). Again, animal caps and vegetal pole cells from early gastrulae, which have been cultivated overnight (Figure 3, lane 6), are completely lacking or show only trace amounts of MyoD PCR products. In contrast, RNA from marginal zone samples, cultivated overnight, contained greatly increased MyoD RNA levels as a consequence of mesoderm induction. Morphologically this was reflected by the establishment of an anterior-posterior axis by gastrulation movements (Figure 3; Symes and Smith, 1987). These observations argue strongly against the possibility that the MyoD signals result from contamination of both pole explants with prospective mesoderm of the marginal zone. There appears to be a temporal sequence concerning both accumulation and disappearance of MyoD transcripts. Compared with respective levels of MyoDa and EFl a PCR products, animal caps from stage 9 embryos showed clearly more MyoDb PCR product than vegetal pole explants. Later on in stage 10% embryos, almost all MyoDb transcripts had disappeared from cells of the animal pole, while the vegetal pole explants were reaching
Cell 930
AC t=o NF: t post
fertil.
(h)
on.
7
8
9
10
lO@#
4.5
5.3
7.5
10
11.6
ra +14
VP GS17
(AC)
AC
Mz
VP
-
XMyoD
a -
XMyoD
b -
EF-la
-
XMyoD
a -
XMyoD
b -
EF-la
-
XMyoD
a -
XMyoD
b -
EF-la
t=o
on.
..:
.
-
123456 Figure
3. Both MyoD
Genes
Are Transcribed
throughout
the Xenopus
Embryo
during
a Short
Period
of Time after the MBT
Embryos were dissected into animal caps (AC), marginal zones (MZ), and vegetal poles (VP) during early blastula to early gastrula stages, and RNA was purified immediately from the explants (t = 0). From stage 10% embryos, sibling explants were incubated in 1 x modified Barth’s saline overnight, before RNA was harvested (t = 14 hr, lane 6). RT/PCRs were performed under standard conditions. To determine the onset of zygotic transcription, RT samples of animal cap explants were subjected to 28 PCR cycles using a primer pair specific for the gene GSI 7 (Krieg and Melton, 1985). Freshly dissected (t = 0; stage 9 embryos) and overnight cultivated explants (on.) are shown to the right. Note the morphological differences between overnight incubated animal caplvegetal pole explants and marginal zone explants. Sibling embryos of the overnight explants had reached stage 18-I 9 (Nieuwkoop and Faber, 1967).
their peak level. This is not a general phenomenon, since the acccumulation of GS17 transcripts was indistinguishable among the different explants within the resolution of this experiment (data not shown). The MyoDb Transcripts in Animal Cap Cells Represent Correctly Initiated Sense-Strand mRNAs We have shown that both MyoD PCR fragments are generated from spliced transcripts (see first section of Results;
Figure 7). Since the RT reaction is random primed, however, our assay does not indicate whether the template RNA is sense or antisense, nor do we know whether transcription initiates at the authentic MyoD promoters. To clarify these points we designed two RT/PCR experiments for transcripts of the MyoDb gene, whose analysis is, unlike those of the MyoDa gene, unaffected by maternal RNA. We reverse transcribed RNA from late blastula animal cap explants using oligonucleotide primers that specifi-
Primary 931
Activation
of MyoD
Genes
in X. laevis
;;-i%-
XM;.Db-gene
R2 -
/
PCR-primer
’ -FR-
RT-primer RT’ase
RNA RT’ase
7-N && + +
8 - +
12
Figure
--PCR-
RTIPCR-
34
4. Characterization
5678
of the Transient
RT-PCR E-s17 ac-s9 -+ + - + + R2F2 F3 P2 F2 F3 F3
1 2
MyoDb
Transcripts
from Post-MET
3 4 56 Animal
-FcR-
genomic
DNA template
7 8 9 1011
Cap Explants
(A) RT reactions with RNA from animal cap explants of stage 9 embryos were either primed with random hexanucleotide primers (lanes 1 and 2) or with primers that hybridize specifically to antisense (RTl, lane 3) or sense RNA (RT2, lane 4) of the MyoDb locus. Excess RTI and RT2 primers were removed afterward from specifically primed RT reactions via the PrimeErase protocol (Stratagene). Samples were subjected to standard PCR amplification. Control PCRs, which were performed with IO fg of Hindlll-linearized pXMyoDb and various primer combinations under otherwise standard conditions, but a 55OC primer annealing temperature, demonstrate comparable amplification efficiencies of RTl and RT2. The schematic diagram at the top shows relative primer target positions and is not drawn to scale. (6) RNA from stage 17 neurulae (lanes 1 and 2) or from animal cap explants (stage 9 embryos; lanes 3-6) was analyzed by RT/PCR under otherwise standard conditions, but with a 58% PCR primer annealing step. The weak signals produced by the F3-R2 primer combination are not dependent on cDNA synthesis (compare lanes 5 and 6) and are most likely due to DNA contamination of the RNA sample (see also lanes 2 and 3). Control PCRs (lanes 7-l l), which were performed with 100 ng of genomic DNA as template under the same conditions as RT/PCRs, demonstrate comparable amplification efficiencies of F3 and F2 primers in combination with the R2 primer. The diagram shows the XMyoDb gene with its putative transcriptional start site as well as the relative positions of the PCR primers used in this experiment (not drawn to scale). The authenticity of all PCR products was further confirmed by diagnostic restriction enzyme digests (BamHI in [A]; Ddel in [B]; data not shown).
tally hybridize to antisense (RTl ; Figure 4A) or sense RNA (RT2) of the MyoDb locus. Subsequent PCR analysis with the primer pair used so far in our assays (F/R) produced the authentic DNA fragment (Figure 4A, compare lane 4 with lanes 2 and 5) exclusively from RTBprimed cDNA (compare lanes 3 and 4). Therefore all the MyoDb transcripts, which we have found transiently expressed in animal cap cells, represent sense mRNA containing the MyoD open reading frame.
-
XMyoD a XMyoD b
-
EF-la
-
_
1
2 3
Figure
4 5
6
7
8 9 10 1112l3l415
5. MyoD RNA Levels
in Late Embryonic
16 and Adult Frog Tissues
Approximately 400 ng of total cellular RNA from tissues of either postmetamorphic (lanes 2-l 1) or late embryonic animals (stage 50; lanes 12-15) was analyzed for MyoD and EFla RNA by multiplex RTlPCR under standard conditions. Lane 16 shows the products of the RTIPCR performed with RNA of one animal cap equivalent (stage 9 embryo).
To determine the transcriptional start site of these MyoDb transcripts (Figure 4B), we designed two forward primers located immediately upstream (F3; R. A. W. R., unpublished data; see Experimental Procedures) or downstream (F2) of the S’end of our cDNA, as well as a reverse primer (R2) roughly400 bp downstream of these. The analysis of RNA from animal cap explants of stage 9 embryos revealed that only the F2-R2 primer combination detected MyoD cDNAs (Figure 46, lanes 3 and 4). The same result isfound with RNAfromstage 17embryos(Figure4B, lanes 1 and 2). We thus have indirectly mapped the transcriptional start site for these MyoDb transcripts to the 75 bp long region between the two forward primers. This suggests that the transient expression of the MyoDb gene in animal cap cells is accomplished by use of the same promoter as in somitic cells of neurula embryos (Hopwood et al., 1989a). MyoD RNA Levels Are Tightly Regulated during Development and in Adult Frogs Northern blot analysis provided evidence for a strictly muscle-specific expression of MyoD in adult frogs (Hopwood et al., 1989a; Scales et al., 1990). We therefore tested RNA from late embryonic or adult tissues in the much more sensitive RTlPCR assay to see whether MyoD transcription is shut off completely, or whether low levels of ectopic expression could also be detected later in development (Figure 5). As expected, RNA from adult leg muscle shows high level expression of both MyoD genes compared with the signals found in late blastula animal cap
Cell 932
B
A RNA pcDNA PCR-primer
RNA el. primer sffic. (%)
ac (s9) t=O on. -- + - +
Figure 6. Comparing Transient Transcription of MyoD with the Expression Pattern of other Genes in Post-MBT Animal Cap Explants
RT/PCR assays were performed under standard conditions except for a lowered primer annealing temperature (56OC) during PCR. (A) and (8) represent independent experiments. 100 MyoDb MyoDb (A) Comparing the amplification efficiencies of PCR primer pairs. RNA from stage 6 embryos XMRF4 30 XMRF4 was reverse transcribed and subjected to separate PCR amplifications with primer pairs specard.act. 140 cific for the above listed genes. Lanes 1 and 4, card.act. both primers present; lane 2, forward primer 270 Xtwi only; lane 3, reverse primer only. Lanes 2-4,5 Xtwi fg of EcoRI-linearized plasmid DNA containing XGATA 140 respective cDNA inserts was added to the RT XGATA reactions (plasmid size differences
927-937,
June
14, 1991, Copyright
0 1991 by Cell Press
Ubiquitous MyoD Transcription at the Midblastula Transition Precedes Induction-Dependent MyoD Expression in Presumptive Mesoderm of X. laevis Ralph A. W. Rupp and Harold Weintraub Department of Genetics and Howard Hughes Medical Institute Fred Hutchinson Cancer Research Center Seattle, Washington 98104
Summary We have used a quantitative reverse transcriptionpolymerase chain reaction assay to detect MyoD mRNA during early embryonic development of Xenopus laevis. We find that during a short period of time following the midblastula transition MyoD becomes transcriptionally activated at a low level ubiquitously throughout the embryo. Restriction of MyoD expression to muscle precursor cells appears as a subsequent event, in which the process of mesoderm induction stabilizes transcription only in the marginal zone of the embryo, the presumptive mesoderm. Introduction MyoD induces the skeletal muscle-specific differentiation program in many different cell types by binding to regulatory DNA sequences of muscle-specific genes. Similar functions have been reported for myogenin (Wright et al., 1989), Myfd (Braun et al., 1989), and MRF4/herculin/ Myf-6 (Rhodes and Konieczny, 1989; Miner and Wold, 1990; Braun et al., 1990). All of these genes share extensive sequence similarity throughout their basic helix-loophelix DNA-binding domains (Murre et al., 1989). Together they constitute the so-called MyoD or myogenic gene family (for recent reviews see Olson, 1990; Lassar and Weintraub, 1991; Weintraub et al., 1991). We know relatively little about how these genes become activated during embryogenesis. In a variety of organisms, which include mouse (Sassoon et al., 1989) quail (de la Brousse and Emerson, 1990), Xenopus (see below), Drosophila (Michelson et al., 1990), and C. elegans (Krause et al., 1990) members of this gene family are found to be expressed at the appropriate time and position within the embryo to suggest their direct involvement in the initial muscle cell commitment event. This suggests that the mechanism for generating muscle cell type is an ancient and conserved process. Recently, several cDNAs have been cloned from X. laevis that appear to be true homologs of MyoD based on the high sequence similarity throughout the entire coding region (Hopwood et al., 1989a; Harvey, 1990; Scales et al., 1990). At least one of these cDNAs is capable of transactivating mouse MyoD and inducing myogenesis in the lOT1/2 cell line (Scales et al., 1990). They represent transcripts from two highly related genes, called MyoDa and MyoDb (Harvey, 1990), which probably arose as the result of a genome duplication event during anuran evolution (Kobel and DuPasquier, 1986). In adult frogs MyoD ex-
pression is restricted to skeletal muscle cells (Hopwood et al., 1989a; Scales et al., 1990) as it is in mammals (Davis et al., 1987). However, early frog embryos contain an unlocalized maternal MyoD RNA pool of unknown function, whose transcripts seem to be derived from the MyoDa gene only (Hopwood et al., 1989a; Harvey, 1990). While zygotic mRNAs of both genes have been detected from early gastrula stages onward, the appearance of RNA precursor molecules of the MyoDa gene at the midblastula transition (MBT) (Harvey, 1990) suggests that the MyoD genes become transcribed immediately at the onset of zygotic gene expression (Newport and Kirschner, 1982). Thus MyoD expression would start early enough to account for the activation of the cardiac actin gene, the first among the known structural muscle genes to become activated during development (Mohun et al., 1986; Hopwood et al., 1989a). Indeed, injection of MyoDb mRNA into 2-cell frog embryos leads to ectopic expression of the cardiac actin gene in ectodermal cells of the animal pole (Hopwood and Gurdon, 1990). In Xenopus, mesoderm is formed in the equatorial region, the marginal zone, of the embryo by induction of nearby animal pole cells with growth factors emitted by vegetal pole cells (reviewed by Gurdon, 1987; Smith, 1989). In explants from the animal pole region (animal cap), which do not differentiate into muscle without induction (Gurdon et al., 1985), the MyoD genes are activated by conjugation with vegetal pole cells (Hopwood et al., 1989a) or by direct incubation with mesoderm-inducing factors (Harvey, 1990). Furthermore, RNA analysis from embryo dissections and in situ hybridizations localized the MyoD transcripts exclusively to the presumptive mesoderm in the marginal zone (Hopwood et al., 1989a; Harvey, 1990). It was therefore concluded that the MyoD genes are activated in a specific region of the embryo as a consequence of mesoderm induction and that their activation marks the commitment of presumptive mesodermal cells to the skeletal muscle cell lineage. We have used a quantitative reverse transcription-polymerase chain reaction (RT/PCR) assay (Veres et al., 1987; Singer-Sam et al., 1990) to detect MyoD mRNAs. Using this highly sensitive assay we report here that the MyoD genes are primarily activated at the MBT in a transient manner throughout the embryo. This transcriptional activation appears to be independent of mesoderm induction. Instead of triggering the activation of these genes, mesoderm induction seems to be a secondary event that stabilizes the transient, ubiquitous expression of the MyoD genes only in a specific subset of embryonic cells. Results Use of a Quantitative RTlPCR Assay for the Expression of XMyoD Genes To gain experimental access to the questions of when and how transcripts of the frog MyoD genes accumulate in a specific subset of embryonal cells, it was necessary to use
Cell 928
A + CEsl3
E
s6
s9
:;r ,-. .
-XMyoD
a b
i
r
-XMyoD -EF-la
4
5
B
C
s13
s13
: .II
123
RT’a?.e -- E
single
6
7
8
9
Relative
Abundance
4
5
primer multiplex lanes
ratio
13 14 15
of PCR-Products
6
pair
10 1112
7
a
9
PCR
lanes
ratio
mi
E
Figure 1. Quantitation of MyoD Development by RTlPCR
s13
RNA
Levels
during
Early Xenopus
(A) Embryo equivalents of total cellular RNAs from unfertilized eggs (E) or embryos (~6, s9, and ~13) staged according to Nieuwkoop and Faber (1967) were analyzed under standard conditions (see Experimental Procedures) by multiplex RT/PCR (lanes 1-9) or PCRs performed with single primer pairs: XMyoDa, lanes 11 and 14; XMyoDb, lanes 10 and 13; EFla, lanes 12 and 15. Lane 8: parallel sample to lane 7, but 2 cycles less amplification. Lane 9: twice as much of RT sample added to the PCR relative to lane 7. Control reactions performed without reverse transcriptase: no template (C, lane 1); RNA from unfertilized eggs (E, lane 2); RNA from stage 13 embryos (~13, lane 3). The faint diffuse signal seen in RNA of unfertilized eggs (lane 4) at about the appropriate position does not represent authentic MyoDb PCR product, since it does not appear in the PCR performed with the MyoDb gene-specific primer pair alone (lane 10) or any other PCR performed with a single primer pair (lanes 11 and 12). (B and C) The radioactivity of the PCR products was quantitated with a Phosphorlmager System, subtracting as background the signals from appropriate positions of control samples lacking reverse transcriptase (lane 2, background for lanes 4 and 5; lane 3, background for lanes 6-9). (B) shows the relative increase of PCR products during early development as normalized to the earliest signal detected for each gene. Numbers above the table refer to lane numbers of (A). n.d., no signal detectable over background. The bracketed numbers in the column for lane 6 represent the calculated decrease of product amounts, taking into account the amplification efficiency of each primer pair (see Experimental Procedures). (C)compares the product amounts generated by multiplex PCRs with those from PCRs performed with single primer pairs.
an assay system that is both extremely sensitive and able to work with small RNA amounts. We chose for this purpose RTIPCR (Veres et al., 1987; Gilliland et al., 1990; Singer-Sam et al., 1990). In a first step, cDNA is made from total cellular RNA using random hexamer primers. An aliquot of this reaction is then used as template for the PCR, in which gene-specific primers amplify short regions from the respective cDNAs. The PCR mix contains trace amounts of [a-32P]dCTP to allow detection and quantitation of the PCR products. Using published sequences (Hopwood et al., 1989a; Harvey, 1990; Scales et al., lQQO), we designed primer pairs specific for the two Xenopus MyoD genes, which we call MyoDa and MyoDb according to the nomenclature of Harvey (1990). These primers target regions C-terminal of the helix-loop-helix motif to avoid cross-hybridization to cDNAs of other members of the helix-loop-helix protein family(Murre et al., 1989; Benezraet al., 1990). The MyoD primers were also chosen so that they flank expected introns to assure the detection of mRNA and not a nuclear RNA precursor or genomic DNA. Furthermore, we chose a primer pair for the ubiquitously expressed EFla gene (Krieg et al., 1989) to serve as an internal control for the amount of RNA tested (“multiplex PCR”; Chamberlain et al., 1990). The additional controls shown in our figures indicate clearly that the generation of PCR products is strictly dependent on the synthesis of cDNA during the RT reaction and on the presence of both forward and reverse primers during subsequent PCR amplification. It was previously shown that MyoD transcript levels peak in late gastrula embryos (Hopwood et al., 1989a). Based on this, we chose gastrula RNA (stage 13 embryos) to calibrate the maximal RNA input and PCR cycle number that still ensure exponential amplification of the target templates during the PCR (see Experimental Procedures). Like others (Hopwood et al., 1989a; Harvey, lQQO), we detect maternal MyoD transcripts in unfertilized eggs or pre-MBT embryos (Figure 1, lanes 4 and 5). As we show here they are derived exclusively from the MyoDa gene, since our PCR primers differentiate between RNAs of the two highly related MyoD genes (see Figure 7). We detect no transcripts of the MyoDb gene at these stages. From stage 9 onward we observe a strong increase in the product amounts for both MyoD genes and the EFla gene, reflecting the onset of zygotic transcription at the MBT (Figure 1, lanes 6 and 7). The quantitation of the control experiments (see Figure 1 B) reveals that decreasing the cycle number (lane 8 versus lane 7) reduced proportionally the amounts of all PCR products, while doubling the input of RT sample (lane 9 versus lane 7) results in a less than 2-fold increase. This indicates that with the applied cycle number the RNA input of embryo equivalents brings the RT/PCR to the edge of exponential amplification, but is not yet saturating. As can be seen in Figure 1C, the product amounts of the multiplex PCR samples, which contain three primer pairs, match those of PCRs performed with single primer pairs within a factor of 2. In addition, the 37-fold (Figure 1, lane 7) to 54-fold increase (lane 14) of the PCR product representing MyoDa transcripts from unfertilized eggs to stage 13 em-
Primary 929
Activation
t=O -
C +-+++++
of MyoD Genes
in X. laevis
t=12h b 2 ;I.- o ‘; L ol-oa c XQ u
t=o SP ++
VMD + +
+
c T.
1234567 Figure
2. MyoD
89 RNA Levels
in Post-MBT
.._ _,
RT’ase
-
XMyoD
a
“’ -
XMyoD EF-lo
b
10 n 12 Animal
Cap Explants
Total cellular RNA was purified from animal cap explants of stage 9 embryos either immediately after dissection (t = 0, lanes 2 and 3, 812) or after overnight cultivation in 1 x modified Barth’s saline with or without mesoderm-inducing factors (t = 12 hr, lanes 4-7). Final concentrations: XTC cell culture supernatant, I:20 dilution; porcine activin A, 0.5 nglml; basic FGF or TGF-PI (R & D Systems), 40 nglml each (incubations of explants with 1 x modified Barth’s saline supplemented with bFGF/TGF-91 storage solutions did not produce detectable MyoD RNA levels; data not shown). S (lane 9) RNA sample from singly dissected explants, lysed immediately in guanidinium thiocyanate (t = 1 min per explant); P (lane 9) control RNA sample from pool-dissected explants (t = 15 min per 10 explants). RNAs from animal cap subregions (lanes 10-12): V, ventral third; M, midddle region; D, dorsal third of animal caps. Explants were prepared from embryos with strict asymmetrical pigment distribution in respect to dorsallventral blastomeres after the second cleavage. To compensate for the lower RNA amounts of the subdivided explants, two explant equivalents of each fraction were used per RT reaction. Multiplex RT/PCRs were performed under standard conditions.
bryos (lanes 4 and 7) correlates very well with the 40-fold increase measured via RNAase protection assays by Hopwood et al. (1989a). The RT/PCR assay used here is therefore capable of measuring accurately and simultaneously the relative transcript levels of several genes, allowing the use of the EFla primer pair as a true internal control. For the following experiments, the RNA input was set so not to exceed the PCR product amounts generated with the concentration of stage 13 embryo RNA used in Figure 1, thereby guaranteeing quantitative performance of the RTI PCR assay. The Two MyoD Genes Are Ubiquitously Transcribed in Xenopus Embryos in a Transient Manner Preceding Lineage-Specific Restriction The activation of the MyoD genes has been shown to be the earliest known muscle-specific response to mesoderm induction in embryos and isolated animal cap explants. In situ hybridization has localized the position of the zygotically expressed MyoD RNA to the marginal zone of early gastrulaembryos, which includes presumptive muscle tissue (Hopwood et al., 1989a; Harvey, 1990). To our surprise, however, we found both MyoD genes expressed in uninduced animal cap explants from earlier blastula embryos (stage 9; Figure 2, lane 3), when RNA was prepared immediately after dissection. Subdividing these explants into ventral, middle, and dorsal parts, the MyoD transcripts are almost equally distributed (Figure 2, lanes
10-12). To date, in more than ten independent experiments, we have not obtained a single RNA sample derived from animal caps of about stage 8X-10 embryos that lacked zygotic MyoD transcripts (data not shown). To prove that the explants were not induced we undertook a number of control experiments. Reducing the time span from the start of dissection to the completion of cell lysis to 1 min did not affect the abundance of the MyoD PCR products (Figure 2, lanes 8 and 9), which are derived from spliced mRNAs (see above; Figure 7). If the MyoD genes were induced by experimental manipulation, then RNA induction, processing, and steady-state mRNA accumulation would have to be completed within 1 min. This time frame is inconsistent with known estimates for these processes (Lewin, 1980). When cultivated overnight in neutral saline, animal cap explants had formed undifferentiated round balls (see Figure 3) and had lost all MyoD mRNAs (Figure 2, lane 4). The control primers present in the multiplex PCR generated a strong EFl a signal, indicating that these explants were indeed viable. In contrast, incubation of the animal cap explants with mesoderminducing factors (Figure 2, lanes 5-7) counteracted the loss of MyoD mRNA and resulted in high level expression of both MyoD genes. In a further experiment, we dissected embryos from preMBT to early gastrula stages into animal caps, marginal zones, and vegetal poles and immediately purified their RNAs (Figure 3). When cultured separately, these parts of the embryos are known to form ectodermal derivatives including epidermis and nerve, embryoid bodies containing notochord and muscle, and poorly differentiated endodermal cells, respectively (Gurdon et al., 1985). Analyzed by RT/PCR, cells of all three regions of stage 9-10 embryos expressed roughly equivalent levels of MyoD mRNAs, irrespective of their future developmental fate. Using a primer pair for GS17 to identify the MBT (Krieg and Melton, 1985), MyoD transcripts, particularly clearly for the MyoDb gene, were detectable as soon as zygotic transcription had started (Figure 3, compare lanes 2 and 3). Again, animal caps and vegetal pole cells from early gastrulae, which have been cultivated overnight (Figure 3, lane 6), are completely lacking or show only trace amounts of MyoD PCR products. In contrast, RNA from marginal zone samples, cultivated overnight, contained greatly increased MyoD RNA levels as a consequence of mesoderm induction. Morphologically this was reflected by the establishment of an anterior-posterior axis by gastrulation movements (Figure 3; Symes and Smith, 1987). These observations argue strongly against the possibility that the MyoD signals result from contamination of both pole explants with prospective mesoderm of the marginal zone. There appears to be a temporal sequence concerning both accumulation and disappearance of MyoD transcripts. Compared with respective levels of MyoDa and EFl a PCR products, animal caps from stage 9 embryos showed clearly more MyoDb PCR product than vegetal pole explants. Later on in stage 10% embryos, almost all MyoDb transcripts had disappeared from cells of the animal pole, while the vegetal pole explants were reaching
Cell 930
AC t=o NF: t post
fertil.
(h)
on.
7
8
9
10
lO@#
4.5
5.3
7.5
10
11.6
ra +14
VP GS17
(AC)
AC
Mz
VP
-
XMyoD
a -
XMyoD
b -
EF-la
-
XMyoD
a -
XMyoD
b -
EF-la
-
XMyoD
a -
XMyoD
b -
EF-la
t=o
on.
..:
.
-
123456 Figure
3. Both MyoD
Genes
Are Transcribed
throughout
the Xenopus
Embryo
during
a Short
Period
of Time after the MBT
Embryos were dissected into animal caps (AC), marginal zones (MZ), and vegetal poles (VP) during early blastula to early gastrula stages, and RNA was purified immediately from the explants (t = 0). From stage 10% embryos, sibling explants were incubated in 1 x modified Barth’s saline overnight, before RNA was harvested (t = 14 hr, lane 6). RT/PCRs were performed under standard conditions. To determine the onset of zygotic transcription, RT samples of animal cap explants were subjected to 28 PCR cycles using a primer pair specific for the gene GSI 7 (Krieg and Melton, 1985). Freshly dissected (t = 0; stage 9 embryos) and overnight cultivated explants (on.) are shown to the right. Note the morphological differences between overnight incubated animal caplvegetal pole explants and marginal zone explants. Sibling embryos of the overnight explants had reached stage 18-I 9 (Nieuwkoop and Faber, 1967).
their peak level. This is not a general phenomenon, since the acccumulation of GS17 transcripts was indistinguishable among the different explants within the resolution of this experiment (data not shown). The MyoDb Transcripts in Animal Cap Cells Represent Correctly Initiated Sense-Strand mRNAs We have shown that both MyoD PCR fragments are generated from spliced transcripts (see first section of Results;
Figure 7). Since the RT reaction is random primed, however, our assay does not indicate whether the template RNA is sense or antisense, nor do we know whether transcription initiates at the authentic MyoD promoters. To clarify these points we designed two RT/PCR experiments for transcripts of the MyoDb gene, whose analysis is, unlike those of the MyoDa gene, unaffected by maternal RNA. We reverse transcribed RNA from late blastula animal cap explants using oligonucleotide primers that specifi-
Primary 931
Activation
of MyoD
Genes
in X. laevis
;;-i%-
XM;.Db-gene
R2 -
/
PCR-primer
’ -FR-
RT-primer RT’ase
RNA RT’ase
7-N && + +
8 - +
12
Figure
--PCR-
RTIPCR-
34
4. Characterization
5678
of the Transient
RT-PCR E-s17 ac-s9 -+ + - + + R2F2 F3 P2 F2 F3 F3
1 2
MyoDb
Transcripts
from Post-MET
3 4 56 Animal
-FcR-
genomic
DNA template
7 8 9 1011
Cap Explants
(A) RT reactions with RNA from animal cap explants of stage 9 embryos were either primed with random hexanucleotide primers (lanes 1 and 2) or with primers that hybridize specifically to antisense (RTl, lane 3) or sense RNA (RT2, lane 4) of the MyoDb locus. Excess RTI and RT2 primers were removed afterward from specifically primed RT reactions via the PrimeErase protocol (Stratagene). Samples were subjected to standard PCR amplification. Control PCRs, which were performed with IO fg of Hindlll-linearized pXMyoDb and various primer combinations under otherwise standard conditions, but a 55OC primer annealing temperature, demonstrate comparable amplification efficiencies of RTl and RT2. The schematic diagram at the top shows relative primer target positions and is not drawn to scale. (6) RNA from stage 17 neurulae (lanes 1 and 2) or from animal cap explants (stage 9 embryos; lanes 3-6) was analyzed by RT/PCR under otherwise standard conditions, but with a 58% PCR primer annealing step. The weak signals produced by the F3-R2 primer combination are not dependent on cDNA synthesis (compare lanes 5 and 6) and are most likely due to DNA contamination of the RNA sample (see also lanes 2 and 3). Control PCRs (lanes 7-l l), which were performed with 100 ng of genomic DNA as template under the same conditions as RT/PCRs, demonstrate comparable amplification efficiencies of F3 and F2 primers in combination with the R2 primer. The diagram shows the XMyoDb gene with its putative transcriptional start site as well as the relative positions of the PCR primers used in this experiment (not drawn to scale). The authenticity of all PCR products was further confirmed by diagnostic restriction enzyme digests (BamHI in [A]; Ddel in [B]; data not shown).
tally hybridize to antisense (RTl ; Figure 4A) or sense RNA (RT2) of the MyoDb locus. Subsequent PCR analysis with the primer pair used so far in our assays (F/R) produced the authentic DNA fragment (Figure 4A, compare lane 4 with lanes 2 and 5) exclusively from RTBprimed cDNA (compare lanes 3 and 4). Therefore all the MyoDb transcripts, which we have found transiently expressed in animal cap cells, represent sense mRNA containing the MyoD open reading frame.
-
XMyoD a XMyoD b
-
EF-la
-
_
1
2 3
Figure
4 5
6
7
8 9 10 1112l3l415
5. MyoD RNA Levels
in Late Embryonic
16 and Adult Frog Tissues
Approximately 400 ng of total cellular RNA from tissues of either postmetamorphic (lanes 2-l 1) or late embryonic animals (stage 50; lanes 12-15) was analyzed for MyoD and EFla RNA by multiplex RTlPCR under standard conditions. Lane 16 shows the products of the RTIPCR performed with RNA of one animal cap equivalent (stage 9 embryo).
To determine the transcriptional start site of these MyoDb transcripts (Figure 4B), we designed two forward primers located immediately upstream (F3; R. A. W. R., unpublished data; see Experimental Procedures) or downstream (F2) of the S’end of our cDNA, as well as a reverse primer (R2) roughly400 bp downstream of these. The analysis of RNA from animal cap explants of stage 9 embryos revealed that only the F2-R2 primer combination detected MyoD cDNAs (Figure 46, lanes 3 and 4). The same result isfound with RNAfromstage 17embryos(Figure4B, lanes 1 and 2). We thus have indirectly mapped the transcriptional start site for these MyoDb transcripts to the 75 bp long region between the two forward primers. This suggests that the transient expression of the MyoDb gene in animal cap cells is accomplished by use of the same promoter as in somitic cells of neurula embryos (Hopwood et al., 1989a). MyoD RNA Levels Are Tightly Regulated during Development and in Adult Frogs Northern blot analysis provided evidence for a strictly muscle-specific expression of MyoD in adult frogs (Hopwood et al., 1989a; Scales et al., 1990). We therefore tested RNA from late embryonic or adult tissues in the much more sensitive RTlPCR assay to see whether MyoD transcription is shut off completely, or whether low levels of ectopic expression could also be detected later in development (Figure 5). As expected, RNA from adult leg muscle shows high level expression of both MyoD genes compared with the signals found in late blastula animal cap
Cell 932
B
A RNA pcDNA PCR-primer
RNA el. primer sffic. (%)
ac (s9) t=O on. -- + - +
Figure 6. Comparing Transient Transcription of MyoD with the Expression Pattern of other Genes in Post-MBT Animal Cap Explants
RT/PCR assays were performed under standard conditions except for a lowered primer annealing temperature (56OC) during PCR. (A) and (8) represent independent experiments. 100 MyoDb MyoDb (A) Comparing the amplification efficiencies of PCR primer pairs. RNA from stage 6 embryos XMRF4 30 XMRF4 was reverse transcribed and subjected to separate PCR amplifications with primer pairs specard.act. 140 cific for the above listed genes. Lanes 1 and 4, card.act. both primers present; lane 2, forward primer 270 Xtwi only; lane 3, reverse primer only. Lanes 2-4,5 Xtwi fg of EcoRI-linearized plasmid DNA containing XGATA 140 respective cDNA inserts was added to the RT XGATA reactions (plasmid size differences
