Maternally-preset program of apoptosis and caspases involved in execution of the apoptosis at midblastula transition (MBT) but not before in <i>Xenopus laevis</i> embryogenesis

Abstract

To study gene control mechanisms in <i>Xenopus</i> embryos, we analyzed polyamines, cloned SAMDC (S-adenosylmethionine decarboxylase), a key enzyme of polyamine metabolism, and microinjected its mRNA into <i>Xenopus</i> fertilized eggs. The microinjection induced a large increase in SAMDC activity, exhaustion of the substrate SAM (S-adenosylmethionine), and execution of apoptosis at the stage called midblastula transition (MBT). By tracing GFP (green fluorescence protein)-marked apoptotic cells, we reached a conclusion that the apoptosis provides pre-blastula embryos with a fail-safe mechanism of early development. We analyzed caspase mRNAs and found that caspase-9 and -3 mRNAs are maternal mRNA and activation of caspase-9 is one of the key steps for the execution of the apoptosis. We also found that over- expression of caspase-8, and in addition p53, a tumor suppressor protein, also induces apoptosis at MBT, just like the overexpression of SAMDC and caspase-9 does. The apoptosis induced by p53 was suppressed by Xdm-2, a negative regulator of p53, and by a peptide inhibitor and a dominant-negative type mutant of caspase-9, but not by those of caspase-8. By contrast, apoptosis induced by SAMDC was suppressed by peptide inhibitors and dominant-negative mutants of both caspase-9 and caspase-8, but not by Xdm-2. Unlike caspase-9 mRNA, caspase-8 mRNA was not a maternal mRNA, but newly expressed during cleavage stage (pre-MBT stage) only in embryos overexpressed with SAMDC. In SAMDC-induced apoptotic embryos activities to process procaspase-8 and procaspase-9 appeared, whereas in p53-induced apoptotic embryos only activity to process procaspase-9 appeared. Thus, <i>Xenopus</i> embryos have at least two pathways to execute the maternal program of apoptosis: One induced by SAMDC overexpression through activation of caspase-9 and <i>do novo</i> expression of caspase-8 gene, and the other induced by p53 overexpression through activation of caspase-9 but not caspase-8. In <i>Xenopus</i> embryos, it has long been believed that zygotic genes are silent until MBT, but results obtained with caspase-8 may provide a novel example of gene expression before MBT

Commitment to nutritional endoderm in Eleutherodactylus coqui involves altered nodal signaling and global transcriptional repression

“…Before MBT in X. laevis , early embryonic cells are transcriptionally silent, and survive using maternally supplied stockpiles of proteins and RNAs (Newport and Kirshner, ; Kisielewska and Blow, ). Although MBT is a complex mechanism, onset of zygotic/embryonic gene expression is considered as one strong parameter in identifying it (Gerhart, ; Heasman, ; Skirkanich et al, ; Shiokawa, ). In X. laevis , MBT occurs at NF8.…”

RNA species whose transcription is totally silent in pre-MBT stage are not mRNA but rRNA and possible involvement of weak bases (ammonium salts and/or amines) in the transcriptional silence of rRNA genes during the pre-MBT stage in <i>Xenopus</i> early embryos

“…In the cellular activities to be checked here are included the level of the methyl donor S-adenosylmethionine (SAM), DNA structure, DNA replication, DNA methylation, RNA transcription, and translation, as shown in the experiments which utilized SAMDC mRNA [33][34][35], γ-ray [36][37][38], cycloheximide [36][37][38][39], and 5-azadeoxycytidine (5-Aza-CdR) [40] as stimuli for execution of the apoptosis. From the experiments to trace cell lineage, we reached the conclusion that this apoptosis is to check and eliminate damaged cells shortly after MBT, and constitutes a surveillance or a "fail-safe" mechanism to save the rest of the embryo for normal development [33,35,41,42]. Since…”

“…However, if the number of apoptotic cells was small, such cells are confined within the blastocoel and disappear due to apoptosis, permitting the rest of the embryo continues on development. From Shiokawa [42].…”