ANÁLISIS DE LA REGULACIÓN DE LA HUELGA EN EL PERÚ
3.2. Huelga y constitución en el Perú
3.2.2. Análisis del derecho de huelga en la Constitución de
septation
Null mutants for caspase-8, F ADD and FLIP develop cardiac defects and die during the period of septation (Varfolomeev et a l, 1998; Yeh et a l, 1998; Yeh et a l
2000). These molecules are all involved in the death receptor-mediated apoptotic pathway and are reportedly expressed in the embryonic heart (Varfolomeev et a l, 1998; Yeh et a l, 1998; Yeh et a l, 2000). All three mutants develop poorly formed trabeculae and thinned ventricular walls, display signs of cardiac failure, such as pericardial
oedema, and die at E13.5, E l 1.5 and E10.5 respectively. Caspase-8 and F ADD both transduce the signal from activated death receptors causing apoptosis. FLIP, in contrast, is believed to inhibit apoptosis, although in some circumstances, may enhance it
(Section 1.1.3.2). In the FLIP and caspase-8 mutants, the cardiac defects are potentially secondary to some other defect, such as the hyperaemia observed in caspase-8 null embryos (Varfolomeev et a l, 1998; Yeh et a l, 2000). In the case of the FADD mutant, chimeric studies showed that embryos in which the heart was mainly made up of non mutant cells were less likely to be abnormal at E l 1.5 than those in which the heart mainly consisted of FADD null cells, suggesting that the heart defect is probably primary (Yeh et a l, 1998).
Interestingly, TUNEL analysis of the FADD and FLIP knockouts showed no obvious differences in PCD between the hearts of null and wild type embryos, leading
the authors of these papers to suggest that these molecules had some as yet undescribed downstream function, other than apoptosis (Yeh et a l, 1998; Yeh et a l, 2000), although notably these analyses were not described quantitatively. Despite the finding that components of the death receptor-mediated pathway are important in cardiac development, no death receptor or death ligand null mice show any cardiac defects. This might suggest that during cardiogenesis there is some, as yet undiscovered, death receptor/ligand interaction, or that more than one death receptor/ligand interaction occurs causing redundancy.
In contrast to the effects of perturbations in death receptor-mediated apoptotic pathways, null mutants for components of the stress-mediated apoptotic pathway, such as the Apaf-1 and Caspase-9, do not have an obvious cardiac phenotype (Kuida et a l,
1998; Yoshida et a l, 1998). This suggests that the stress-mediated pathways are not vital for cardiac development.
The neurofibromin-1 (NF-1) null mouse has poorly fused outflow tract cushions, and develops a double outlet right ventricle. It also appears to have reduced cell death, but increased proliferation, within both the outflow tract and atrioventricular cushions, and dies showing signs of cardiac failure at E l2.5 (Lakkis and Epstein, 1998).
Clinically, mutations in the NF-1 gene are involved in the development of Von Recklinghausen’s neurofibromatosis, and the gene is thought to act as a tumour
suppressor (Lakkis and Epstein, 1998). Although the exact mechanism is unclear, NF-1
appears to act on the ras pathway (Section 1.1.2), potentially implicating NF-1 in causing the cell death seen in the endocardial cushions. This is complicated by the fact that if, as the paper suggests, the effect was solely through ras and therefore its action on p53, one may expect to see a defect in cardiac apoptosis in p53 null mice, which has not been reported (Norimura et a l, 1996).
Another group of genes that may be important in endocardial cushion cell death are those encoding the retinoic acid receptors (RARs). Mice null for R A R ^ and retinoid X receptor a{RX Rd) do not have properly fused outflow tract cushions (Ghyselinck et a l, 1998). In contrast to the NF-1 null mice, they appear to have increased apoptosis within their outflow tract cushions at E12.5 (Ghyselinck et a l, 1998). Again, the mechanisms through which the retinoid receptors may be functioning to affect PCD in the outflow tract cushions remain unclear. Taken together, the data on the NF-1 and
RARj3/RXRanu\\ mice suggest that appropriate outflow tract apoptosis may be important in proximal outflow tract septation. Furthermore, these data suggest that failure of proximal outflow tract septation does not directly affect apoptosis, or else the apoptotic phenotype in the cardiac outflow tract cushions of NF-1 and RAR^/RXRanull
embryos would be expected to be similar.
c-Jun is known to have pro- and anti-apoptotic effects (Jochum et a l, 2001). Furthermore, the c-Jun knockout mouse is reported to have a common arterial trunk and neural crest defects (Fferl et a l, 1999). Although the extent of apoptosis in the outflow tract cushions of c-Jun null embryos has not yet been examined, such analysis may prove insightful given that c-Jun is a downstream target of death receptor activation.
BMP-4 has been shown to be important for cell death in the interdigital regions of the limb (Zou and Niswander, 1996) although the exact mechanism by which it does so is unknown, it is believed to act through the muscle segmental homeobox-2 {Msx-2)
gene (Marazzi et a l, 1997). Zhao and Rivkees (2000) showed that cultured mouse endocardial cushions, at F I 1.5, can be stimulated by BMP-4 to undergo increased PCD. This was taken as evidence that BMP-4 signalling may play a role in endocardial
cushion PCD. Interestingly, Abdelwahid et a l (2001) have recently shown that the PCD in the endocardial cushions occurs in complementary regions to BMP-4 and Msx-
2.
provides further support for a mechanism in which BMP-4, via Msx-2, could stimulate endocardial cushion apoptosis.
Together, these data suggest that apoptosis in the heart is not regulated in a simple manner, but is influenced by a variety of factors. Furthermore, there appears to be a particularly important role for the death receptor-mediated pathway in heart development, although perhaps not necessarily by influencing levels o f apoptosis in the heart.
Pexieder (1975) used two approaches to alter cell death in the chick. First, he altered blood flow through the heart by clamping the 6^^ aortic arches at E4, mimicking pulmonary stenosis. This resulted in a reduced level of PCD in the atrioventricular cushions and also delayed their fusion. Secondly, he used chemical teratogens, specifically cyclophosphamide, which he found increased PCD in the outflow tract cushions, and dexamethasone, which reduced PCD in the outflow tract cushions. In some cases, ventricular septal defects, and outflow tract alignment defects were observed, resulting in either transposition of the great arteries, or overriding of the interventricular septum by the aorta. This evidence further suggests that normal PCD is important in cardiac development, although again, exactly why remains uncertain.
1.4 Aims
This first aim of this thesis is to provide a detailed qualitative and quantitative spatio-temporal map of PCD during cardiac development. This is the basis for the rest of the thesis, and would also help to assess the effects on heart development of
perturbations in cardiac apoptosis. The second aim is to investigate how cell death may be involved in outflow tract development. This alludes to the questions of whether PCD is involved in outflow tract shortening and myocardialisation, and whether neural crest cells are involved in apoptosis in the outflow tract cushions o f the heart. Thereafter, the role of death receptors in developmental cardiac apoptosis is investigated. Finally, the possibility that PCD is required for normal interventricular septal development is addressed by use of an inhibitor of apoptosis in whole embryo culture.