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INS1 chimeric mRNA also generates a small amount of insulin (8). Besides this
unusual post-‐transcriptional regulation, the th gene displays an unexpected
expressionpattern.
We found that the expression of th mRNA in the chick embryo antecedes
development of thenervous system. thtranscriptsweredetected fromgastrulation
onwards, and they were enriched in the cardiogenic region. Before focusing this
overview in the heart THduring cardiogenesis, it is worth however to emphasize
the remarkably similar features of a human embryo and a chick embryo during
that period (Figure 3). In vertebrates, the heart is initially formed by the fusion of
the two bilateral endocardial tubes arising from the splanchnic lateral plate
mesoderm. The resultant primitiveheart tube, locatedat theventralmidlineof the
organism, undergoes a complex series ofmovements and tissue remodelingevents
that leads to the formationof thematurechamberedorgan(12).
3. USEOF THECHICKEMBRYOASAMODEL FORSTUDYINGTHEROLEOF TH IN
CARDIOGENESIS
As very clearly stated by Brand (in a kind editorial comment of our work,
13), “despite its evolutionarydistance tomammals, the chick embryo is a valuable
model toworkout themechanismof cardiac specification. Theembryo is largeand
accessible and, therefore, manipulations at the time of cardiac specification and
early heart formation are easily performed”. Research in the last two decades
utilizing this model organism has identified several signaling molecules that are
important for cardiac induction(14).
The fact that the chick embryo can be accessed and manipulated without
disrupting early development, allows to perform experiments of gain of function
and loss of function starting during gastrulation, and to analyze the effects on
embryonic organogenesis (Figure 4). Factors or antibodies may be added and, if
theaimis to look for effects over thenext several days, theembryo canbe reached
throughawindowin the shell and incubation can continue. To look for short-‐term
effects, as in the series of studies reviewed here, the addition of molecules or
plasmid DNA was carried out maintaining the embryo in culture under specific
tension conditions (15). The chick embryos were treated at stages 3-‐5 (12-‐22
hours of development, according to Hamburger and Hamilton (16) and were
studied at stages 11-‐12 (less than 50 hours of development). The addition of
dopamineor enzymatic inhibitorsof THactivitycouldbe carriedout usingheparin
or resinmicrobeads, implanted in theembryounderneath theepiblast (Figure4B).
The uptake by the embryo tissues of plasmid DNA containing TH, GFP (Green
Fluorescence Protein) or TH antisense-‐morpholino DNA was facilitated by
electroporation(Figure4C).
