Developmental cell death in invertebrates and vertebrates is often controlled by systemic signals, which provide the trigger for cell and tissue destruction (Jacobson et al., 1997
; Baehrecke, 2002
). However, it is not well understood why these signals induce death only at a particular time and only in some cells and tissues, but not in others. The experimental data presented in this study support a model that explains how a specific tissue of the fruit fly D. melanogaster
is singled out for destruction in response to the steroid hormone 20E (). It explains why the larval salivary glands are destroyed in response to a particular 20E pulse, the prepupal pulse, and why they survive earlier pulses of the same hormone. It thus provides a framework for our understanding of how tissue-specific developmental cell death is precisely timed.
In more general terms, our data suggest that a key event in acquiring competence for a cell death response to a systemic signal is the loss of a tissue-specific survival factor. This loss occurs in response to a temporal signal that precedes the death-inducing signal. In the salivary glands, the tissue-specific survival factor is Fkh, and the signal that leads to the loss of Fkh is provided by the late-larval 20E pulse. Our data show that loss of Fkh is required for the death response to the prepupal 20E pulse (). They further suggest that the salivary glands survive all previous hormone pulses because they are protected by the presence of Fkh (). Thus, Fkh not only plays a key role in determining the tissue selectivity of salivary gland death, but also in the proper timing of this event. Elimination of Fkh is mediated by the 20E-induced transcription factor BR-C
(Renault et al., 2001
). We find that fkh
is strongly expressed at the normal time of salivary gland death in the rbp5
mutant of BR-C
, which demonstrates that BR-C
is required for the continued repression of fkh
beyond the larval–prepupal transition (). The derepression of fkh
is sufficient to explain why the salivary glands do not die in the mutant.
Loss of the survival factor renders critical death regulators responsive to the death-inducing signal. In the salivary glands, these death regulators are the IAP antagonists hid
, which together are required for salivary gland death (Yin and Thummel, 2004
). In the absence of Fkh, the two genes are inducible by hormone, as shown by the premature induction of hid
after RNAi knockdown of fkh
(). Importantly, loss of fkh
by itself is not sufficient to activate hid
, or to kill the salivary glands within the ~36 h between fkh
knockdown and the late-larval 20E pulse. Strong activation of hid
and death only occur in response to the hormonal signal. After elimination of Fkh, the hormone induces expression of hid
at a level that is sufficient to kill (). This observation supports the conclusion that there are no other repressors present in prepupal salivary glands that are sufficient to prevent hormonal induction of cell death. All that seems to be needed to induce death is the hormone 20E and one or more 20E-induced transcriptional activators.
Previous work has shown that E74A
play the role of hormone-induced activators of hid
in late-prepupal salivary glands (Jiang et al., 2000
). Both E74A
are required for the induction of hid
, which has the characteristics of a secondary-response gene. Intriguingly, the activation of hid
after premature loss of fkh
shows the same secondary-response characteristics, suggesting that the same 20E-induced transcription factors are responsible for the activation of hid
by the late-larval 20E pulse (). Full induction of rpr
depends not only on BR-C
but also on direct binding of the hormone receptor EcR/Usp to the gene (Jiang et al., 2000
). Thus, rpr
has characteristics of both a primary- and secondary-response gene, leading to an earlier induction of the gene in response to the prepupal 20E pulse. Again, premature activation in response to the late-larval pulse shows the same temporal characteristics (). This suggests that E74A
are responsible for the premature activation of hid
after knockdown of fkh
, and that such an activation is normally prevented by the presence of Fkh. Our immunostaining data support this conclusion by showing that Fkh protein is still present in the larval salivary glands at the time when the two genes are active. It only disappears from the tissue 2–4 h APF (). These data explain why E74A
mediate a death response exclusively to the prepupal 20E pulse, despite a very similar induction pattern of the two genes in response to the preceding late-larval pulse.
Our results exclude that repression of hid and rpr is mediated by the fkh target sens. Repression may thus be mediated by another downstream target of fkh or by direct binding of Fkh to transcriptional control regions of hid and rpr. In support of the latter possibility, we found that the first intron of hid contains a cluster of 13 Fkh binding sites. One of these sites exhibits strong binding of Fkh in in vitro DNA-binding assays, whereas the other sites have weak to moderate binding affinity (unpublished data; de Banzie, J., personal communication). Although this region may function as a silencer of hid expression in vivo, lacZ reporter gene assays in transgenic flies did not reveal that it has an enhancer function. We were not able to identify a similar binding site cluster in rpr.
Our microarray data identify other apoptosis-related genes that are down- or up-regulated by Fkh. Therefore, it is likely that Fkh protects cells from death by interfering with the cell death program at multiple levels. Regulation of genes such as Ark
, or PDK1
, is likely to mediate a general function of fkh
as a survival factor. This function appears to be required for the survival of the developing salivary glands during embryogenesis (Myat and Andrew, 2000
). However, it is not essential for the survival of postembryonic salivary glands, as demonstrated by the failure of the glands to die in the absence of Fkh during prepupal development. Our data confirm this conclusion by showing that the salivary glands fail to undergo PCD within the ~36 h between the premature knockdown of fkh
and the steroid induction of death. They separate a general protective function of Fkh from a specific function that Fkh has in the control of steroid-induced developmental PCD.
Tissue-specific developmental cell death controlled by steroid hormone plays an important role not only in insects but also in humans and other vertebrates. Glucocorticoids, for instance, control the development of the immune system by killing specific types of thymocytes (Ashwell et al., 2000
). Many genes regulated by glucocorticoids are coregulated by the vertebrate FOXA counterparts of Fkh (Friedman and Kaestner, 2006
). It will be interesting to see whether FOXAs have evolutionarily conserved functions in glucocorticoid-induced death and in other types of developmental cell death.