An experimental system was devised to study the mechanisms by which cells become committed to the cardiac myocyte lineage during avian development. Chick tissues from outside the fate map of the heart (in the posterior primitive streak (PPS) of a Hamburger & Hamilton stage 4 embryo) were combined with potential inducing tissues from quail embryos and cultured in vitro. Species-specific RT-PCR was employed to detect the appearance of the cardiac muscle markers chick Nkx-2.5 (cNkx-2.5), cardiac troponin C and ventricular myosin heavy chain in the chick responder tissues. Using this procedure, we found that stage 4–5 anterior lateral (AL) endoderm and anterior central (AC) mesendoderm, but not AL mesoderm or posterior lateral mesendoderm, induced cells of the PPS to differentiate as cardiac myocytes. Induction of cardiogenesis was accompanied by a marked decrease in the expression of rho-globin, implying that PPS cells were being induced by anterior endoderm to become cardiac myocytes instead of blood-forming tissue. These results suggest that anterior endoderm contains signaling molecules that can induce cardiac myocyte specification of early primitive streak cells. One of the cardiac muscle markers induced by anterior endoderm, cNkx-2.5, is here described for the first time. cNkx-2.5 is a chick homeobox-containing gene that shares extensive sequence similarity with the Drosophila gene tinman, which is required for Drosophila heart formation. The mesodermal component of cNkx-2.5 expression from stage 5 onward, as determined by in situ hybridization, is strikingly in accord with the fate map of the avian heart. By the time the myocardium and endocardium form distinct layers, cNkx-2.5 is found only in the myocardium. cNkx-2.5 thus appears to be the earliest described marker of avian mesoderm fated to give rise to cardiac muscle.

Reference

Antin
P. B.
,
Taylor
R. G.
,
Yatskievych
T.
(
1994
)
Precardiac mesoderm is specified during gastrulation in quail.
Dev. Dynam
200
,
144
154
Bacon
R. L.
(
1945
)
Self-differentiation and induction in the heart of Amblystoma.
J. Exp. Zool
98
,
87
125
Biehl
J.
,
Holtzer
S.
,
Bennett
G.
,
Sun
T.
,
Holtzer
H.
(
1985
)
Cultured chick blastodisc cells diverge into lineages with different IF isoforms.
Ann. N. Y. Acad. Sci
455
,
158
66
Bisaha
J. G.
,
Bader
D.
(
1991
)
Identification and characterization of a ventricular-specific avian myosin heavy chain, VMHC1: expression in differentiating cardiac and skeletal muscle.
Dev. Biol
148
,
355
364
Bodmer
R.
(
1993
)
The gene tinman is required for specification of the heart and visceral muscles in Drosophila.
Development
118
,
719
729
Bodmer
R.
,
Jan
L. Y.
,
Jan
Y. N.
(
1990
)
A new homeobox-containing gene, msh-2, is transiently expressed early during mesoderm formation in Drosophila.
Development
110
,
661
669
Dodgson
J. B.
,
Stadt
S. J.
,
Choi
O. R.
,
Dolan
M.
,
Fischer
H. D.
,
Engel
J. D.
(
1983
)
The nucleotide sequence of the embryonic chicken beta-type globin genes.
J. Biol. Chem
258
,
12685
12692
Fujisawa-Sehara
A.
,
Nabeshima
Y.
,
Hosoda
Y.
,
Obibata
T.
,
Nabeshima
Y.
(
1990
)
Myogenin contains two domains conserved among myogenic factors.
J. Biol. Chem
265
,
15219
23
Gannon
M.
,
Bader
D.
(
1995
)
Initiation of cardiac differentiation occurs in the absence of anterior endoderm.
Development
121
,
2439
2450
Garcia-Martinez
V.
,
Schoenwolf
G. C.
(
1993
)
Primitive streak origin of the cardiovascular system in avian embryos.
Dev. Biol
159
,
706
719
Gonzalez-Sanchez
A.
,
Bader
D.
(
1990
)
In vitro analysis of cardiac progenitor cell differentiation.
Dev. Biol
139
,
197
209
Hamburger
V.
,
Hamilton
H. L.
(
1951
)
A series of normal stages in the development of the chick embryo.
J. Morph
88
,
49
92
Han
Y.
,
Dennis
J. E.
,
Cohen-Gould
L.
,
Bader
D. M.
,
Fischman
D. A.
(
1992
)
Expression of sarcomeric myosin in the presumptive myocardium of chicken embryos occurs within six hours of myocyte commitment.
Dev. Dynam
193
,
257
265
Inagaki
T.
,
Garcia-Martinez
V.
,
Schoenwolf
G. C.
(
1993
)
Regulative ability of the prospective cardiogenic and vasculogenic areas of the primitive streak during avian gastrulation.
Dev. Dynam
197
,
57
68
Jacobson
A. G.
(
1960
)
Influences of ectoderm and endoderm on heart differentiation in the newt.
Dev. Biol
2
,
138
154
Jacobson
A. G.
(
1961
)
Heart determination in the newt.
J. Exp. Zool
146
,
139
152
Jacobson
A. G.
,
Duncan
J. T.
(
1968
)
Heart induction in salamanders.
J. Exp. Zool
167
,
79
103
Jacobson
A. G.
,
Sater
A. K.
(
1988
)
Features of embryonic induction.
Development
104
,
341
359
Komuro
I.
,
Izumo
S.
(
1993
)
Csx: A murine homeobox-containing genespecifically expressed in the developing heart.
Proc. Natl. Acad. Sci. USA
90
,
8145
8149
Lash
J.
,
Holtzer
S.
,
Holtzer
H.
(
1957
)
An experimental analysis of the development of the spinal column.
Exp. Cell Res
13
,
292
303
Lin
Z. Y.
,
Dechesne
C. A.
,
Eldridge
J.
,
Patterson
B. M.
(
1989
)
An avian muscle factor related to MyoD1 activates muscle-specific promoters in nonmuscle cells of different germ-layer origin and in BrdU-treated myoblasts.
Genes Dev
3
,
986
996
Lints
T. J.
,
Parsons
L. M.
,
Hartley
L.
,
Lyons
I.
,
Harvey
R. P.
(
1993
).
Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants.
Development
119
,
419
431
Maisonpierre
P. C.
,
Hastings
K. E.
,
Emerson
C. P.
(
1987
)
The cloning and the codon and amino acid sequence of the quail slow/cardiac troponin C cDNA.
Methods Enzymol
139
,
326
37
Montgomery
M. O.
,
Litvin
J.
,
Gonzalez-Sanchez
A.
,
Bader
D.
(
1994
)
Staging of commitment and differentiation of avian cardiac myocytes.
Dev. Biol
164
,
63
71
Nascone
N.
,
Mercola
M.
(
1995
)
An inductive role for the endoderm in Xenopus cardiogenesis.
Development
121
,
515
523
Nieto
M. A.
,
Sargent
M. G.
,
Wilkinson
D. G.
,
Cooke
J.
(
1994
)
Control of cell behavior during vertebrate development by Slug, a zinc finger protein.
Science
264
,
835
839
Panabieres
F.
,
Piechaczyk
M.
,
Rainer
B.
,
Dani
C.
,
Fort
P.
,
Riaad
S.
,
Marty
L.
,
Imbach
J. L.
,
Jeanteur
P.
,
Blanchard
J.-M. M.
(
1984
)
Complete nucleotide sequence of the messenger RNA coding for chicken muscle glyceraldehyde-3-phosphate dehydrogenase.
Biochem. Biophys. Res. Commun
118
,
767
783
Pownall
M. E.
,
Emerson
C. P.
(
1992
)
Sequential activation of three myogenic regulatory genes during somite morphogenesis in quail embryos.
Dev. Biol
151
,
67
79
Sassoon
D. A.
,
Lyons
G.
,
Wright
W. E.
,
Lin
V.
,
Lassar
A. B.
,
Weintraub
H.
,
Buckingham
M.
(
1989
)
Expression of two myogenic regulatory factors, myogenin and MyoD1 during mouse embryogenesis.
Nature
341
,
303
307
Sater
A. K.
,
Jacobson
A. G.
(
1989
)
The specification of heart mesoderm occurs during gastrulation in Xenopus laevis.
Development
105
,
821
830
Sugi
Y.
,
Lough
J.
(
1994
)
Anterior endoderm is a specific effector of terminal cardiac myocyte differentiation of cells from the embryonic heart forming region.
Dev. Dynam
200
,
155
162
Tonissen
K. F.
,
Drysdale
T. A.
,
Lints
T. J.
,
Harvey
R. P.
,
Krieg
P. A.
(
1994
).
XNkx-2.5, a Xenopus gene related to Nkx-2.5 and tinman: Evidence for a conserved role in cardiac development.
Dev. Biol
162
,
325
328
Toyota
N.
,
Shimada
Y.
,
Bader
D.
(
1989
)
Molecular cloning and expression of chicken cardiac troponin C.
Circ. Res
65
,
1241
6
Weiskirchen
R.
,
Siemeister
G.
,
Hartl
M.
,
Bister
K.
(
1993
)
Sequence and expression of a glyceraldehyde-3-phosphate dehydrogenase gene from quail embryo fibroblasts.
Gene
128
,
269
272
Wilens
S.
(
1955
)
The migration of heart mesoderm and associated areas in Amblystoma punctatum.
J. Exp. Zool
129
,
579
605
This content is only available via PDF.