Some mice lacking intrinsic, as well as death receptor induced apoptosis and necroptosis, can survive to adulthood

Programmed cell death, in particular the intrinsic apoptotic pathway, has been shown to play a critical role in the shaping of tissues during embryonic development. The multi-BCL-2 Homology (BH) domain effectors of apoptosis, BAX, BAK, and BOK, are essential for cell killing in the intrinsic apoptotic pathway. It was therefore surprising that we found earlier that a few mice lacking all effectors of apoptosis (Bax;Bak;Bok triple knockout), albeit many fewer than expected based on Mendelian ratios, could reach weaning or even adulthood. This indicated that death receptor induced apoptosis or necroptosis, a lytic form of programmed cell death, may also have roles in embryogenesis alongside the intrinsic apoptotic pathway. To explore this, we generated Bax;Bak;Bok;caspase-8;Mlkl quintuple knockout mice, which lack not only intrinsic apoptosis but also death receptor induced apoptosis (loss of caspase-8) and necroptosis (loss of MLKL). These foetuses exhibited similar defects to the Bax;Bak;Bok triple knockout mice and, intriguingly, a small number of Bax;Bak;Bok;caspase-8;Mlkl quintuple knockout mice could reach weaning or even adulthood. These findings identify the contributions of these three programmed cell death pathways to embryonic development and show that despite the absence of all of them, development to adulthood is possible, albeit very rare.


INTRODUCTION
Programmed cell death plays a critical role in embryonic development by removing cells that are no longer needed, damaged, or infected [1]. There are several distinct pathways to programmed cell death, including apoptosis, which can be activated through the intrinsic (aka mitochondrial or BCL-2 regulated) or the death receptor induced (aka extrinsic) pathway [2], necroptosis, which can be triggered through activation of death receptors when caspase-8 that is essential for extrinsic apoptosis is absent, or pyroptosis, which can be activated by signals from diverse pathogens and requires caspases-1 or -11 and gasdermin D [3]. The intrinsic apoptotic pathway is regulated by the BCL-2 protein family [2]. In healthy cells, prosurvival BCL-2 family members, such as BCL-2, BCL-XL, and MCL-1, restrain the effectors of cell death, BAK and BAK. Stress conditions, such as nutrient deprivation or anoikis (cell detachment) enhance transcription or cause a post-transcriptional increase of the pro-apoptotic BH3-only proteins (e.g., BIM, PUMA). These critical initiators of apoptosis bind with high affinity to and inhibit the pro-survival BCL-2 proteins, thereby unleashing BAX and BAK to cause mitochondrial outer membrane permeabilisation (MOMP), the point-of-no return in apoptosis signalling. MOMP allows the release of apoptogenic factors (e.g., cytochrome c, SMAC/DIABLO) from the intermitochondrial membrane space into the cytoplasm and this leads to the activation of the cascade of caspases that cause the ordered dismantling of the cell [1,2]. BOK structurally resembles BAX and BAK and can also cause MOMP and apoptosis, but it is neither restrained by the pro-survival BCL-2 proteins nor activated by BH3-only proteins [4,5].
The intrinsic apoptotic pathway has long been thought to be the major process of programmed cell death that is critical for embryogenesis [6]. Morphological and histological analyses of Bax −/− Bak −/− and Bax −/− Bak −/− Bok −/− embryos and mice identified those developmental processes that require apoptosis to occur normally. Common abnormalities seen in E18.5 Bax −/− Bak −/− and Bax −/− Bak −/− Bok −/− foetuses include cleft palate/cleft face, aortic arch defects, omphalocele and curled fingers, toes, and tail [5]. Surprisingly, however, many tissues that were thought to depend on apoptosis for development appeared normal in E18.5 Bax −/− Bak −/− and Bax −/− Bak −/− Bok −/− foetuses and some of these animals reached the age of weaning or even early adulthood [5,6]. This raised the possibility that additional programmed cell death pathways, in particular death receptor induced apoptosis and/or necroptosis, might play a role in embryogenesis alongside intrinsic apoptosis. In so-called type 2 cells, death receptor induced apoptosis requires BAX and BAK and would thus be blocked in cells from Bax −/− Bak −/− Bok −/− embryos [7]. However, in so called type 1 cells death receptor induced activation of caspase-8 with consequent activation of the effector caspases suffices for cell killing with no need for engagement of the intrinsic apoptotic pathway by caspase-8 mediated activation of the pro-apoptotic BH3-only protein BID [8]. Thus, death receptor induced apoptosis would be possible in type 1 cells in Bax −/− Bak −/− Bok −/− embryos and can only be abrogated by the loss of caspase-8. Necroptosis, a lytic form of programmed cell death that is executed by the activation of the pore-forming protein MLKL, is induced when death receptors are stimulated and caspase-8 is absent or inhibited [9]. We examined the impact of the combined absence of both apoptotic pathways and necroptosis on embryonic development.

RESULTS
The observations that many tissues in which apoptosis was proposed to play a role appear surprisingly normal in Bax −/− ;Bak −/− ;Bok −/− embryos suggested that additional programmed cell death pathways might operate alongside the intrinsic apoptotic pathway to allow the shaping of tissues during embryonic and foetal development. To explore this hypothesis, we generated mice that lacked not only the multi-BH (BCL-2 homology) domain effectors of apoptosis, BAX, BAK and BOK, but additionally were also deficient in caspase-8, which is essential for death receptor induced apoptosis [10], and MLKL, which is needed for necroptosis [11]. Note that loss of caspase-8 causes embryonic lethality~E11.5 due to aberrant necroptosis that can be prevented by concomitant absence of RIPK3 [12,13] or MLKL [14], which are both essential for necroptosis. For this we crossed  (Table 1; p = 0.5 and p = 1, respectively; Fisher's exact test). This demonstrates that it is possible to obtain adult mice that lack the intrinsic as well as the death receptor apoptotic pathways and necroptosis. Importantly, the loss of the death receptor apoptotic pathway and necroptosis does not appear to affect the frequency of mice reaching weaning or even adulthood that was previously observed in Bax −/− ;Bak −/− ;Bok −/− TKO mice [5].

DISCUSSION
Since in embryos deficient for the intrinsic apoptotic pathway (Bax −/− ;Bak −/− ;Bok −/− ) many tissues that were long thought to require apoptosis for development appeared surprisingly normal and since some of these mice could even reach early adulthood [5], we explored whether foetuses deficient in both intrinsic as well as death receptor induced apoptosis and necroptosis (Bax −/− ; Bak −/− ;Bok −/− ;Casp8 −/− ;Mlkl −/− ) would have more severe abnormalities. Our findings presented here demonstrate that, while the penetrance of the observed defects varied between these two genotypes, overall, the additional absence of death receptor induced apoptosis and necroptosis, due to the loss of caspase-8 and MLKL, had no effect on the survival rate and little effect on the developmental abnormalities caused by the absence of the intrinsic apoptotic pathway (loss of BAX, BAK, and BOK). Nevertheless, the three defects that were more common in Bax −/− ;Bak −/− ;Bok −/− ;Casp8 −/− ;Mlkl −/− than in Bax −/− ;Bak −/− ; Bok −/− pups, aortic arch defects, extra finger skin tissue, and curly tails, could indicate some role of the extrinsic apoptotic pathway or necroptosis in restricted tissues. Loss of necroptosis alone [11] or combined absence of death receptor induced apoptosis plus necroptosis do not cause any developmental abnormalities [13,14]. However, inhibition of the intrinsic apoptotic pathway synergises with defects in the death receptor apoptotic pathway in causing lymphadenopathy [15,16]. We, therefore, assume that, if any additional defects in embryonic development exist at all, it is the loss of death receptor induced apoptosis rather than the absence of necroptosis that causes an increase in some developmental abnormalities in Bax embryogenesis but cannot be removed in this manner due to the absence of BAX, BAK, and BOK, could be induced to undergo death receptor induced apoptosis instead, with the ligand(s) for the relevant death receptor(s) presumably provided by neighbouring cells. This would mean that these cells must be type 1 cells, since type 2 cells require the BH3-only protein BID as well as BAX and/or BAK for death receptor induced killing (i.e. in type 2 cells the loss of BAX and BAK would already suffice to block death receptor induced apoptosis) [7].

W T B a k -/ -B a x -/ -B o k -/ -B a x -/ -B a k -/ -B o k -
In conclusion, our work shows that, surprisingly, a small number of mice can reach weaning and even adulthood despite the complete absence of the intrinsic as well as the death receptor induced apoptotic pathways and also necroptosis. Perhaps additional cell death pathways, either known ones, such as cell death associated with autophagy [17], or yet to be discovered cell death pathways also play roles alongside apoptosis in embryonic and foetal development.

MATERIALS AND METHODS Mice
All experiments with mice were performed with the approval of the Walter and Eliza Hall Institute Animal Ethics Committee. Animals were handled according to the Australian code of practice for the care and use of animals for scientific purposes.

Microscopy
For detailed phenotypic examination, E18.5 foetuses were euthanised by cooling. Dissections were performed using the Stemi 2000-C dissecting microscope. Pups were photographed with a digital camera (AxioCam HR, Carl Zeiss).  Table 1 or in the figure legends.