Regulación de la expresión de tirosina hidroxilasa por transcritos quimera y función de la dopamina en la cardiogénesis

Abstract

A paradigm in molecular biology, lasting for more than two decades, was that one gene gave rise to one protein. Nowadays, it is well-knownthat one gene can give multiple mRNAs by the usage different transcription start sites and different polyadentlation sites, and by alternative splicing. Another unexpected source of variability is the formation of transcription induced chimeres (TIC), mRNAs composed of two gene sequences. In this Thesis we describe for the first time TIC in birds. We have found two TIC in chick and quail embryos which contain tyrosine hydroxylase (TH) and insulin gene sequences. The TIC, named TH-INS1 and TH-INS2 differ in their insulin sequence content. TH-INSI and TH-INSI2 proteins correspond to truncated TH forms that lack the tetramerization domain (TH-INS2) or replace the tetramerization domain with a new amino acid sequence (TH-INS1). Both TH isoforms serve to regulate TH activity, because their enzymatic activity is 6 to 7 times lower than that of canonical TH, and the TIC regulate insulin production since insulin mRNA is interrupter.

TH is the rate limiting enzyme in catecholamines biosynthesis that catalyzes the convertion of L-tyrosine to L-DOPA (catecholamines precursos). We describe in this work the expression pattern of TH during the early chick embryo development. Functional TH is detected since St8, with expression restricted to the heart primordium.

Some authors have proposed a role for catecholamines during heart develpment (Sarasa and Climent, 1991; Zhou et al., 1995; Kobayashi et al., 1995; Thomas et al., 1995).

In this study we show that L-DOPA and dopamine (first catecholamine in catecholamines bisynthesis pathway) added to chick embryos in specifi positions, induced the ectopic expression of cardiac markers such as Tbx5, Nkx2.5, AMHC-1 (atrial myosin), VMHC-1 (ventricular myosin). Moreover, these cells not only express cardiac markers but also are morphologically and ultrastucturally cardiomiocytes-like. In contrast, inhibition of endogenous L-DOPA and dopamine production in chick embryos inhibits AMHC-1 and VMHC-1 expression. Using a genetical manipulation approach, we have found that TH over-expression in chick embryos, induces AMHC-1 and VMHC-1 expression. On the other hand, TH blocking by morphilinos inhibits AMCH-1 expression.

We also demosntrate in this Thesis the implication of L-DOPA and dopamine in mouse embryonic stem cells cardiac differentiation. L-DOPA and dopamine induced the expression of the cardiac markers Nkx2.5, GATA4, Tbx5, MHC and TT2. In parallel, we observed that L-DOPA and dopamine blocked the erytroid lineage differentiation.