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REFERENCES AND NOTES etching selectivity between the PB and PS domains.

tivity between the PB and PS domains. fer would be patterned with the ordered nanostruc-
___________________________ However, in the case of the ozonated copolymer films ture arrays.
1. H. Masuda and K. Fukuda, Science 268, 1466 (1995); (Fig. 1B), a pattern transfer to silicon nitride can be 25. We thank P. Mansky for helpful discussions. This
R. Gupta, J. J. McClelland, Z. J. Jabbour, R. J. Ce- achieved whether pure CF4 or CF4/O2 is used. project was supported by NSF through the Princeton
lotta, Appl. Phys. Lett. 67, 1378 (1995); M. J. Lercel et 22. We observed that the discreteness of the microdomain Center for Complex Materials under grant DMR
al., J. Vac. Sci. Technol. B 13, 1139 (1995); M. Wen- monolayer thickness was less apparent for SI 68/12 9400362. Parts of the processing were carried out at
del, S. Kuhn, H. Lorenz, J. P. Kotthaus, M. Holland, when compared with SB 36/11. Even at thicknesses the Advanced Technology Center for Photonics and
Appl. Phys. Lett. 65, 1775 (1994); M. A. McCord and below 70 nm, we observed spherical PI microdomains Optoelectronic Materials at Princeton University and
D. D. Awschalom, ibid. 57, 2153 (1990); H. Fang, R. over the entire sample area but with a poorer order. at the Cornell Nanofabrication Center. The silicon
Zeller, P. J. Stiles, ibid. 55, 1433 (1989). 23. C. Harrison et al., in preparation. nitride windows were fabricated at the Cornell Nano-
2. G. M. Whitesides, J. P. Mathias, C. T. Seto, Science 24. We have produced a uniform microdomain monolay- fabrication Center.
254, 1312 (1991). er template by spin-coating over an entire 76-mm
3. D. Hofstadter, Phys. Rev. B 14, 2239 (1976); D. wafer. With a larger ozonator system, the entire wa- 3 February 1997; accepted 16 April 1997
J. Thouless, M. Kohmoto, M. P. Nightingale, M. den
Nijs, Phys. Rev. Lett. 49, 405 (1982).
4. D. Weiss et al., Phys. Rev. Lett. 66, 2790 (1991).
5. W. Kang, H. L. Stormer, L. N. Pfeiffer, K. W. Baldwin,
K. W. West, ibid. 71, 3850 (1993).
6. W. D. Volkmuth and R. H. Austin, Nature 358, 600
Control of Mouse Cardiac Morphogenesis and
(1992); W. D. Volkmuth, T. Duke, M. C. Wu, R. H.
Austin, A. Szabo, Phys. Rev. Lett. 72, 2117 (1994).
Myogenesis by Transcription Factor MEF2C
7. S. Y. Chou, M. S. Wei, P. R. Krauss, P. B. Fischer,
J. Appl. Phys. 76, 6673 (1994). Qing Lin, John Schwarz, Corazon Bucana, Eric N. Olson*
8. T. L. Morkved et al., Science 273, 931 (1996).
9. T. L. Morkved, P. Wiltzius, H. M. Jaeger, D. G. Grier,

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T. A. Witten, Appl. Phys. Lett. 64, 422 (1994). Members of the myocyte enhancer factor–2 (MEF2) family of MADS (MCM1, agamous,
10. P. Mansky, C. K. Harrison, P. M. Chaikin, R. A. Reg- deficiens, serum response factor)– box transcription factors bind an A-T–rich DNA se-
ister, N. Yao, ibid. 68, 2586 (1996); P. Mansky, P.
Chaikin, E. L. Thomas, J. Mater. Sci. 30, 1987 quence associated with muscle-specific genes. The murine MEF2C gene is expressed
(1995). in heart precursor cells before formation of the linear heart tube. In mice homozygous
iiii
11. Z. Li et al., J. Am. Chem. Soc. 118, 10,892 (1996). for a null mutation of MEF2C, the heart tube did not undergo looping morphogenesis,
12. F. S. Bates, Science 251, 898 (1991); and G.
H. Fredrickson, Annu. Rev. Phys. Chem. 41, 525
the future right ventricle did not form, and a subset of cardiac muscle genes was not
(1990). expressed. The absence of the right ventricular region of the mutant heart correlated with
13. The underlying silicon nitride layer is grown by a down-regulation of the dHAND gene, which encodes a basic helix-loop-helix transcrip-
plasma-enhanced chemical vapor deposition tech-
nique. See S. M. Sze, VLSI Technology (McGraw-
tion factor required for cardiac morphogenesis. Thus, MEF2C is an essential regulator
Hill, New York, ed. 2, 1988), pp. 233–271. of cardiac myogenesis and right ventricular development.
14. M. Park et al., in Symposium Proceedings, vol. 461,
Morphological Control in Multiphase Polymer Mix-
tures, Materials Research Society (MRS), Boston, 2
to 6 December 1996 (MRS, Pittsburgh, PA, 1997),
pp. 179 –184. The mechanisms that regulate heart forma- co-expressed in the precardiogenic meso-
15. Because of the discrete microdomain size produced tion during embryogenesis are only begin- derm beginning at embryonic day 7.75
by self-assembly, spin-coated copolymer films on
hard substrates rearrange into discrete film thick- ning to be elucidated (1). Members of the (E7.75), and MEF2A and MEF2D are ex-
nesses upon annealing, which typically results in a MEF2 family of transcription factors bind a pressed about 12 hours later (3, 8). MEF2
nonuniform topography [see G. Coulon, D. Ausserre, conserved A-T–rich DNA sequence associ- gene expression is detected in skeletal
T. P. Russell, J. Phys. France 51, 777 (1990)]. How-
ever, by spin-coating initially at these discrete thick-
ated with most cardiac muscle structural muscle precursors in the somites and in
nesses, a uniform film can be produced even after genes (2) and are expressed in cardiogenic smooth muscle cells beginning at about
annealing. precursor cells and differentiated cardiomyo- E9.0. Loss-of-function mutations in a sin-
16. S. M. Sze, VLSI Technology (McGraw-Hill, New
York, ed. 2, 1988), pp. 184 –232.
cytes during embryogenesis (3). MEF2 fac- gle MEF2 gene in Drosophila, D-mef2, pre-
17. The RIE parameters used here are CF4 (100%), 2 tors are also expressed in skeletal and smooth vent differentiation of cardiac, skeletal,
mtorr, 10 standard cubic centimeters per minute muscle cell lineages (3, 4), and MEF2 bind- and visceral muscle cells (9), but the func-
(SCCM), 24 W, and ;290 VDC [DC self-bias voltage ing sites are essential for expression of muscle tions of the vertebrate MEF2 genes in the
of cathode (sample holder) with respect to plasma]
and also CF4/O2 (90/10%), 25 mtorr, 20 SCCM, 20 genes in all three muscle cell types (5). embryo have not been determined.
W, and ;170 VDC. There are four MEF2 genes in vertebrate To investigate MEF2C function during
18. We obtained the TEM micrographs by spin-coating species, designated MEF2A, -B, -C, and -D, mouse embryogenesis, we inactivated this
the copolymer films on thin (;75 nm) silicon nitride
windows (50 mm by 50 mm) (Fig. 2). In this way, one share homology in an NH2-terminal gene with a targeting vector (10) that
can easily image the microdomain morphologies MADS-box and an adjacent motif known deleted the second protein-coding exon,
with the substrate intact before RIE and later directly as the MEF2 domain (5). These protein which encodes amino acids 18 to 86 (Fig.
view the patterned silicon nitride after RIE. The high-
resolution TEM assists in characterizing and tuning domains mediate DNA binding, homo- and 1). The MADS and MEF2 domains are
the processing steps. Silicon nitride windows have heterodimerization, and interaction with contained in residues 1 to 56 and 57 to 86,
been previously used in TEM micrography [see (8)]. basic helix-loop-helix (bHLH) transcrip- respectively, and the residues deleted by
Having a resolution and contrast of the image but not
significantly at the 10-nm length scale.
tion factors (6–8). the mutation are essential for DNA bind-
19. The spherical microdomain monolayer shown in Fig. In the mouse, MEF2B and MEF2C are ing and dimerization (7). The vector was
2A is produced from SB 36/11, which exhibits a introduced into embryonic stem (ES) cells
cylindrical morphology in bulk. A change from a cy-
lindrical to a spherical morphology in thin films with a
Q. Lin and E. N. Olson, Department of Molecular Biology by electroporation, clones were isolated
few nanometers of variation in film thickness has
and Oncology, University of Texas Southwestern Medical after positive-negative selection (11), and
Center, 5323 Harry Hines Boulevard, Dallas, TX 75235 –
been observed and discussed previously (14).
9148, USA.
genomic DNA was analyzed by Southern
20. The depth (or height) of the patterned structures is blot analysis for gene replacement at the
J. Schwarz, Division of Cardiology, Department of Inter-
estimated from the etching times and rates (Figs. 2
and 3). In the case of the patterned silicon nitride
nal Medicine, University of Texas Medical School, Hous- MEF2C locus (12). The frequency of ES
ton, TX 77030, USA.
layer on a silicon substrate in Fig. 3, the estimated
C. Bucana, Department of Cell Biology, University of Tex-
cell clones bearing a targeted MEF2C al-
number was confirmed by SEM on the cross section lele was 1: 7. Three targeted ES cell clones
as M. D. Anderson Cancer Center, Houston, TX 77030,
of a cracked sample.
21. In the case of the osmium-stained copolymer films (Fig.
USA. were injected into blastocysts isolated
1C), the addition of O2 to CF4 is necessary to obtain an * To whom correspondence should be addressed. from C57BL/6J mice to generate chimeras,

1404 SCIENCE z VOL. 276 z 30 MAY 1997 z www.sciencemag.org


REPORTS
two of which transmitted the mutation the linear heart tube stage (E8.0–8.25), cause of the apparent absence of the future
through the germ line. Mice heterozygous and contractions were initiated in the right ventricle, the remaining portion of the
for the targeted allele showed no discern- heart tubes of mutants, although they were ventricular chamber was displaced slightly to
ible phenotype and were intercrossed to slower and less rhythmic than normal. At the left. The atrioventricular (AV) demar-
obtain MEF2C homozygous null offspring. E9.0, the heart rate of wild-type and cation was apparent in the mutant, but the
The genotypes of offspring from heterozy- MEF2C heterozygous embryos was 58 6 3 AV canal did not elongate. Also, the sinus
gous intercrosses were determined within beats per minute, compared with 26 6 4 venosus, which normally forms from the pos-
1 to 3 weeks after birth by Southern blot beats per minute for mutants (14). In wild- terior portion of the atrial chamber, did not
analysis of DNA obtained from tail biop- type embryos, the heart tube initially ex- form in the mutant.
sies. No neonates homozygous for the hibits a peristaltic beat that becomes se- As the ventricular chambers develop,
MEF2C mutation were found among 189 quential after looping, with strong atrial the trabeculae form as fingerlike projec-
offspring and no neonatal lethality was contraction preceding ventricular con- tions along the inner myocardial wall,
observed, indicating that the homozygous traction. In mutants, the atrial chamber which becomes clearly separated from the
mutation resulted in embryonic lethality. exhibited weak contractions, and the hy- endocardial layer by a loose mesenchyme
We determined the genotypes of em- poplastic ventricular chamber appeared to called the cardiac jelly. Histological sec-
bryos between E6.5 and E12.5 by Southern vibrate only in response to atrial contrac- tions of wild-type hearts at E9.0 revealed
blot or polymerase chain reaction (PCR) tions; there was no evidence of indepen- well-defined atrial and ventricular cham-
analysis (12) of DNA isolated from yolk dent ventricular contractions. Mutant em- bers, with trabeculae projecting from the
sacs. Homozygous mutants were detected bryos also exhibited severe pericardial ef- inner ventricular walls (Fig. 3, A, C, and

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at roughly the predicted Mendelian fre- fusion (Fig. 2, A and B), indicative of E). The endocardium was also evident in
quency between E6.5 and E9.5. Wild-type hemodynamic insufficiency. wild-type embryos at this stage. In mutant
and mutant embryos were similar in size up At E8.5, the heart tube normally initiates hearts, the trabeculae were poorly devel-
to the 14-somite stage (E9.0), and the first rightward looping, a process that is necessary
branchial arch appeared to form normally to orient the atrial and ventricular chambers
in the mutant. However, the mutants de- and to align the outflow tract with the vas- Wild type MEF2C (-/-)
veloped no more than 20 somites and culature. Looping morphogenesis is accom-
showed retarded growth after E9.0. No panied by formation of the conotruncus in
viable mutants were observed by E10.5, the anteriormost region and later by forma-
indicating that the lethality of the tion of the common ventricular and atrial
MEF2C mutation was fully penetrant by chambers. Further maturation of the looped
that stage. cardiac tube yields the future right and left
The most obvious morphologic defects ventricles and the common atrial chamber.
in mutant embryos were within the heart. In mutant embryos, the heart tube did not
The heart develops from bilaterally sym- undergo rightward looping (Fig. 2, C to F),
metric cardiogenic primordia that coalesce and there was no morphologic evidence of
at the midline to form a primitive heart the future right ventricle. Rather, a single
tube (13). Mutant and wild-type embryos hypoplastic ventricular chamber was fused
were microscopically indistinguishable at directly to an enlarged atrial chamber. Be-

Fig. 1. Targeting strategy for


the MEF2C locus. Diagram
of MEF2C, which contains
465 amino acids, is shown
at the top, above a diagram
of the mouse MEF2C locus
around coding exon 2,
which encodes residues 18
to 86. The first intron in the
coding region is .10 kb. In
the targeting vector (10), the
neomycin-resistance gene
(neo) was transcribed in the
same direction as MEF2C.
Homologous recombination
resulted in deletion of coding
exon 2. The structure of the
targeted allele is shown at Fig. 2. Cardiac defects in MEF2C null embryos.
the bottom. Insertion of neo (A) Wild-type and (B) mutant embryos at E9.0. The
introduced an Eco RI site lower portion of the wild-type embryo was re-
that could be used to distin- moved for a clear view of the heart. Note severe
guish the wild-type and mu- pericardial effusion in the mutant. Hearts of the
tant alleles. Position of the 39 wild-type (C) and mutant (D) embryos are shown.
probe used for Southern Scanning electron micrographs of wild-type (E)
blot analysis of yolk sac DNA and mutant (F) embryos at E9.0. Abbreviations: a,
is indicated. Hybridization of Atrium; bc, bulbus cordis; lv, future left ventricle;
Eco RI– digested DNA with ps, pericardial sac; and v, ventricular chamber.
the 39 probe yielded fragments of 11.5 and 3.2 kb from the wild-type and mutant loci, respectively. Bars 5 2 mm.

www.sciencemag.org z SCIENCE z VOL. 276 z 30 MAY 1997 1405


oped and the lumen of the ventricular was decreased significantly but was still of the cardiac muscle genes in MEF2C
chamber was extremely narrow (Fig. 3, B detectable at a low level in the hearts of mutant embryos was a specific response to
and F). The cardiomyocytes within the the mutants (Fig. 4, A to D) (15). In the absence of MEF2C rather than a sec-
ventricular wall and the endocardial cells contrast, other cardiac muscle genes, in- ondary result of embryonic demise, be-
also appeared to be disorganized. In the cluding MLC2V (Fig. 4, E and F) and cause it preceded growth retardation of
atrial chamber of the mutant, the myocar- MLC2A (15), were expressed at normal the embryo and was specific to a certain
dial wall was thin and the cardiac jelly was levels in the mutants. Expression of subset of genes.
absent (Fig. 3D). The AV canal was MLC2V is normally localized to the ven- The bHLH genes dHAND and eHAND
present in the mutant; however, the en- tricular region of the developing heart by are expressed in complementary patterns
docardial cushions did not form. Red E9.0 (16). In the mutants, MLC2V expres- that demarcate distinct regions of the devel-
blood cells were seen in the mutant em- sion was localized to the hypoplastic ven- oping mouse heart (18–20). dHAND is nor-
bryos, but very few were present in the tricular chamber, indicating that the AV mally expressed throughout the heart tube
heart, suggesting that circulation was im- chambers were specified correctly. before expression becomes localized predom-
paired. Schematic diagrams showing the Quantitative reverse transcriptase– inantly to the right side of the looping heart
morphologies of wild-type and mutant PCR (RT-PCR) (17) confirmed the re- tube (19, 20). In contrast, eHAND is ex-
hearts at E9.0 are shown in Fig. 3, G and sults of whole mount in situ hybridization pressed in the conotruncus and future left
H, respectively. and showed that several muscle-specific ventricle, but expression is excluded from
Whole mount in situ hybridization of transcripts were down-regulated four- to the region of the heart tube that gives rise to
embryos at E9.0, before the mutants began fivefold in the mutants. Down-regulation the right ventricle (18–20). In the MEF2C

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to show retarded growth, demonstrated a
reduction in expression of several markers
Wild type MEF2C (-/-) Wild type MEF2C (-/-)
of cardiac differentiation. Transcripts for
atrial natriuretic factor (ANF), cardiac
a-actin, and a-myosin heavy chain were
down-regulated to background levels, and
myosin light chain (MLC)–1A expression
ANF dHAND

Wild type MEF2C (-/-)


A B

MLC1A eHAND

C D

E F MLC2V Nkx2.5

Fig. 4. Analysis of cardiac gene expression by whole mount in situ hybrid-


G H ization. Wild-type (A, C, E, G, I, and K) and mutant (B, D, F, H, J, and L)
embryos were stained by in situ hybridization for the indicated transcripts
(3, 28). For a clearer view of the heart, heads and tails were dissected away
hf from the embryos in (I), (J), and (L). Note that dHAND expression is restrict-
hf ed to the right ventricular region of the heart in the wild-type embryo and is
B not expressed in the heart of the mutant. eHAND is expressed in the
A conotruncus and left ventricular region in the wild-type heart; in the mutant,
C D there is no gap in expression that would represent the right ventricle. Tails
Left were dissected away from embryos in (C), (D), and (K). (F), (I), (J), and (L) are
ventricle Atrium frontal views; other embryos are viewed from the side. Abbreviations: a,
Atrium; ct, conotruncus; h, heart; lm, lateral mesoderm; lv, left ventricle; rv,
Fig. 3. Hematoxylin and eosin staining of thin right ventricle; and v, ventricular chamber. Bars 5 5 mm. (M) Quantitative
sections of hearts of wild-type and mutant embry- RT-PCR analysis of gene expression in wild-type (wt) and MEF2C mutant
os. Wild-type (A, C, and E) and mutant (B, D, and (mu) embryos. PCR amplifications were performed with cDNA synthesized
F) embryos at E9.0 were fixed, sectioned, and from RNA isolated from E9.5 embryonic heart or whole embryos at E8.5
stained with hematoxylin and eosin (27 ). (G and H) and E9.0 and gene-specific primers for the indicated transcripts. L7 was
Diagrams of planes of section. Abbreviations: a, used as a loading control. Transcripts for dHAND were down-regulated in
Atrium; bc, bulbus cordis; hf, headfold; lv, left ven- the heart by E9.0, as determined by in situ hybridization. The expression of dHAND transcripts in the E8.5
tricle; tb, trabeculae; and v, ventricular chamber. and E9.0 RNA samples used for RT-PCR reflects its expression in the lateral mesoderm and not in the
Bars 5 1 mm. heart. RNA concentrations were quantitated by PhosphorImager analysis.

1406 SCIENCE z VOL. 276 z 30 MAY 1997 z www.sciencemag.org


REPORTS
mutant, dHAND was expressed at normal structural genes for cardiac muscle was not crotiter plates, and genomic DNA was analyzed on
Southern blots.
levels in the heart tube before looping (Fig. up-regulated, whereas others were expressed 12. Homologous recombination at the MEF2C locus
4M) (15). However, dHAND transcripts normally. Some of the cardiac genes that were was detected by Southern blot or PCR analysis of
were down-regulated at the time of looping, independent of MEF2C, such as MLC2V, genomic DNA isolated from ES cells, yolk sacs, or tail
concomitant with failure of the right ven- contain essential MEF2-binding sites in their biopsies. For Southern blots, genomic DNA was di-
tricular region to form (Fig. 4, G, H, and M). control regions (24), suggesting that down- gested with Eco RI and hybridized to a probe from a
region immediately 39 of the region of homology
eHAND was expressed in the mutant, but stream genes in the pathway for cardiomyo- used for targeting. For PCR, the following oligonu-
expression was contiguous throughout the cyte differentiation can discriminate between cleotide primers were used: neo primer, 59-GGCAT-
heart tube without the gap in expression that different members of the MEF2 family and GCTGGGGATGCGGTGGGCTC-39; MEF2C MADS
box primer, 59-AGTACAACGAGCCGCACGAGAG-
normally characterizes the future right ven- that another member of the MEF2 family CCG-39; and MEF2C short-arm primer, 59-GTCAC-
tricle (Fig. 4, I and J). This distortion in supports activation of those cardiac genes that CT TAAGACATAAAGCACCCTCC-39.
eHAND expression and the lack of dHAND are expressed in the MEF2C mutant. MEF2B 13. R. L. DeHaan, in Organogenesis, R. L. DeHaan and
H. Ursprung, Eds. (Holt Rinehart Winston, New York,
expression in the heart tube of the mutant is coexpressed with MEF2C throughout the 1964), pp. 377– 419.
are consistent with absence of the future early stages of cardiogenesis (3, 8), and it was 14. Heart rates were determined in E9.0 embryos obtained
right ventricle. The cardiac homeobox gene up-regulated in the MEF2C mutant, consis- from timed matings of MEF2C heterozygotes. Pregnant
Nkx-2.5 (21) was expressed throughout the tent with the possibility that it may partially females were killed by cervical dislocation; embryos
were removed and placed in phosphate-buffered saline
developing heart in wild-type and mutant substitute for MEF2C. That cardiac develop- at 37°C. Fourteen wild-type and six mutant embryos
embryos (Fig. 4, K and L). MEF2B, which is ment occurs normally in MEF2B-null mice were analyzed. The average values were statistically
normally coexpressed with MEF2C during (25) also suggests that MEF2C shares func- significant, as assessed by a standard t test.
15. Q. Lin and E. N. Olson, unpublished data.

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the earliest stages of cardiogenesis (3, 8), was tions with MEF2B. 16. T. X. O’Brien, K. J. Lee, K. R. Chien, Proc. Natl.
up-regulated more than sevenfold in the In addition to their expression in the Acad. Sci. U.S.A. 90, 5157 (1993).
hearts of mutants at E9.5 (Fig. 4M), whereas developing heart, members of the MEF2 17. For RT-PCR, total RNA was prepared from isolated
MEF2A and MEF2D transcripts were ex- family are expressed later in development in hearts at E9.5 and from whole embryos at E8.5 and
pressed at comparable levels in wild-type and E9.0, treated with ribonuclease-free deoxyribonu-
skeletal and smooth muscle cells (3, 4, 8), clease I, and resuspended in 20 ml of water. First
mutant hearts. in the nervous system (26), and eventually strand cDNA synthesis was performed with 10 ml
The phenotype of MEF2C mutant em- in a wide range of cell types. MEF2C may of RNA with Moloney murine leukemia virus reverse
bryos demonstrates that MEF2C is required have additional functions in these other transcriptase (BRL) and random primers. PCR am-
plification was performed with 0.5 ml of the cDNA,
for looping of the cardiac tube, develop- cell types. 0.1 mCi of [32P]deoxycytidine triphosphate (dCTP),
ment of the right ventricle, and expression and gene-specific primers under conditions of lin-
of a subset of cardiac muscle genes. The REFERENCES AND NOTES earity for each individual primer set. Most oligonucle-
___________________________ otide primers spanned introns. To confirm that sam-
cardiogenic defects seen in MEF2C mutant 1. E. N. Olson and D. Srivastava, Science 272, 671 ples were not contaminated with genomic DNA, we
embryos are distinct from those seen in (1996); M. C. Fischman and D. Y. R. Stanier, Circ. also performed duplicate PCRs without RT. All PCR
other mouse mutants (22) and reveal a Res. 74, 757 (1994). products corresponded to the size predicted for the
2. L. A. Gossett, D. J. Kelvin, E. A. Sternberg, E. N. corresponding transcript. PCR cycles were as fol-
cardiogenic regulatory program for right lows: 99°C for 2 min then 18 to 25 cycles of 96°C for
Olson, Mol. Cell. Biol. 9, 5022 (1989).
ventricular development. 3. D. G. Edmondson, G. E. Lyons, J. F. Martin, E. N. 30 s, 58°C for 30 s, and 72°C for 45 s. PCR products
MEF2C is expressed uniformly Olson, Development 120, 1251 (1994). were separated by 6% polyacrylamide gel electro-
4. A. E. Chambers et al., Genes Dev. 8, 1324 (1994); M. phoresis and quantitated by analysis on a phosphor-
throughout the heart tube. Therefore, the Imager (Molecular Dynamics).
W. Wong, M. Pisegna, M. F. Lu, D. Leibham, M.
selective ablation in the MEF2C mutant Perry, Dev. Biol. 166, 683 (1994); B. Nadal-Ginard 18. D. Srivastava, P. Cserjesi, E. N. Olson, Science 270,
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rise to the right ventricle suggests that A. B. Firulli et al., Circ. Res. 78, 196 (1996). N. Olson, Dev. Biol. 170, 664 (1995).
5. E. N. Olson, M. Perry, R. A. Schulz, Dev. Biol. 172, 19. D. Srivastava, T. Thomas, Q. Lin, D. Brown, E. N.
MEF2C regulates expression or activity of 280 (1995). Olson, Nature Genetics, in press.
a regionally restricted regulatory factor re- 6. R. Pollock and R. Treisman, Genes Dev. 5, 2327 20. C. Biben and R. P. Harvey, personal communication.
quired for development of this region of (1991); J. F. Martin et al., Mol. Cell. Biol. 14, 1647 21. T. Lints et al., Development 119, 419 (1993).
(1994); J. F. Martin, J. J. Schwarz, E. N. Olson, Proc.
the heart. A candidate for such a factor is Natl. Acad. Sci. U.S.A. 90, 5282 (1993); Y. T. Yu et
22. I. Lyons et al., Genes Dev. 9, 1654 (1995); J. Ros-
sant, Circ. Res. 78, 349 (1996).
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dHAND, which is expressed throughout Development 118, 1095 (1993); D. Leifer et al., Proc. Cell 83, 1125 (1995); S. Kaushal, J. W. Schneider, B.
the linear heart tube and becomes restrict- Natl. Acad. Sci. U.S.A. 90, 1546 (1993); J. C. Mc- Nadal-Ginard, V. Mahdavi, Science 266, 1236
Dermott et al., Mol. Cell. Biol. 13, 2564 (1993). (1994).
ed to the future right ventricle as looping 7. J. D. Molkentin et al., Mol. Cell. Biol. 16, 2627 (1996); 24. J. D. Molkentin and B. E. Markham, J. Biol. Chem.
is initiated. Mice homozygous for a null Y.-T. Yu, J. Biol. Chem. 271, 24675 (1996). 268, 19512 (1993); S. Navankasattusas, H. Zhu, A.
mutation in dHAND exhibit a cardiac 8. J. Molkentin et al., Mol. Cell. Biol. 16, 3814 (1996). V. Garcia, S. M. Evans, K. R. Chien, Mol. Cell. Biol.
9. B. Lilly et al., Science 267, 688 (1995); B. A. Bour et 12, 1469.
phenotype similar to that of the MEF2C al., Genes Dev. 9, 730 (1995); G. Ranganayakulu et 25. J. Molkentin, Q. Lin, E. N. Olson, unpublished data.
mutant (19), suggesting that MEF2C reg- al., Dev. Biol. 171, 169 (1995).
26. G. E. Lyons, B. K. Micales, J. Schwarz, J. F. Martin,
ulates dHAND expression in the future 10. The MEF2C targeting vector was constructed as shown
E. N. Olson, J. Neurosci. 15, 5727 (1995).
in Fig. 1, with the neomycin resistance gene under con-
right ventricular region of the heart tube trol of the phosphoglycerol kinase promoter in the same 27. J. F. Martin, A. Bradley, E. N. Olson, Genes Dev. 9,
or that these two transcription factors co- transcriptional orientation as MEF2C. A thymidine ki- 1237 (1995).
28. Sources of probes were as follows: ANF [R. Zeller et
operate to control right ventricular devel- nase gene under control of the herpes simplex virus
al., Genes Dev. 1, 693 (1987); MLC1A [(P. Barton et
promoter pMC1-HSV tk [S. L. Mansour, K. R. Thomas,
opment, as has been described for MEF2 M. R. Capecchi, Nature 336, 348 (1988)] was cloned al., J. Biol. Chem. 263, 12669 (1988)]; MLC2V (16);
and myogenic bHLH proteins in the skel- into an Xho I site immediately 59 of the 59 arm of genomic eHAND and dHAND (18); and Nkx-2.5 (21).
etal muscle lineage (23). MEF2C and homology. All the cloning junctions in the targeting vec- 29. We thank J. Martin for isolating the MEF2C genomic
tor were confirmed by DNA sequencing. The targeting clone, A. Tizenor for assistance with graphics, and D.
dHAND may control right ventricular de- vector was linearized by digestion with Not I before Srivastava and members of the Olson laboratory for
velopment by specifying a subpopulation electroporation. helpful discussions. Supported by grants from NIH,
of cardiogenic cells within the cardiac 11. The MEF2C targeting vector was introduced by elec- the Muscular Dystrophy Association, the American
troporation into 129 ES cells [A. P. McMahon and A. Heart Association, and the Human Frontiers Scienc-
tube or by regulating the expansion of es Program to E.N.O.
Bradley, Cell 62, 1073 (1990)]. After positive-nega-
such a population. tive selection, surviving clones were isolated and
In the absence of MEF2C, a subset of replica-plated onto SN76/7 fibroblasts in 96-well mi- 5 December 1996; accepted 2 April 1997

www.sciencemag.org z SCIENCE z VOL. 276 z 30 MAY 1997 1407

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