Germ cell apoptosis is critical to maintain Caenorhabditis elegans offspring viability in stressful environments

Maintaining reproduction in highly variable, often stressful, environments is an essential challenge for all organisms. Even transient exposure to mild environmental stress may directly damage germ cells or simply tax the physiology of an individual, making it difficult to produce quality gametes. In Caenorhabditis elegans, a large fraction of germ cells acts as nurse cells, supporting developing oocytes before eventually undergoing so-called physiological germ cell apoptosis. Although C. elegans apoptosis has been extensively studied, little is known about how germline apoptosis is influenced by ecologically relevant environmental stress. Moreover, it remains unclear to what extent germline apoptosis contributes to maintaining oocyte quality, and thus offspring viability, in such conditions. Here we show that exposure to diverse environmental stressors, likely occurring in the natural C. elegans habitat (starvation, ethanol, acid, and mild oxidative stress), increases germline apoptosis, consistent with previous reports on stress-induced apoptosis. Using loss-of-function mutant alleles of ced-3 and ced-4, we demonstrate that eliminating the core apoptotic machinery strongly reduces embryonic survival when mothers are exposed to such environmental stressors during early adult life. In contrast, mutations in ced-9 and egl-1 that primarily block apoptosis in the soma but not in the germline, did not exhibit such reduced embryonic survival under environmental stress. Therefore, C. elegans germ cell apoptosis plays an essential role in maintaining offspring fitness in adverse environments. Finally, we show that ced-3 and ced-4 mutants exhibit concomitant decreases in embryo size and changes in embryo shape when mothers are exposed to environmental stress. These observations may indicate inadequate oocyte provisioning due to the absence of germ cell apoptosis. Taken together, our results show that the central genes of the apoptosis pathway play a key role in maintaining gamete quality, and thus offspring fitness, under ecologically relevant environmental conditions.

A gene desert required for regulatory control of pleiotropic Shox2 expression and embryonic survival

ABSTRACTThe Shox2 homeodomain transcriptional regulator is known for its critical functions during mouse embryogenesis, enabling accurate development of limbs, craniofacial structures, neural populations and the cardiac conduction system. At the genomic level, the Shox2 gene is flanked by an extensive gene desert, a continuous non-coding genomic region spanning over 500 kilobases that contains a multitude of evolutionarily conserved elements with predicted cis-regulatory activities. However, the transcriptional enhancer potential of the vast majority of these elements in combination with the biological necessity of the gene desert have not yet been explored. Using transgenic reporter assays in mouse embryos to validate an extensive set of stringent epigenomic enhancer predictions, we identify several novel gene desert enhancers with distinct tissue-specific activities in Shox2 expressing tissues. 4C-seq chromatin conformation capture further uncovers a repertoire of gene desert enhancers with overlapping activities in the proximal limb, in a compartment essential for Shox2-mediated stylopod formation. Leveraging CRISPR/Cas9 to delete the gene desert region contained in the Shox2 topologically associated domain (TAD), we demonstrate that this complex cis-regulatory platform is essential for embryonic survival and required for control of region-specific Shox2 expression in multiple developing tissues. While transcription of Shox2 in the embryonic limb is only moderately affected by gene desert loss, Shox2 expression in craniofacial and cardiac domains is nearly abolished. In particular, Shox2 transcripts in the sinus venosus (SV) encompassing the sinoatrial node (SAN) were depleted in embryos lacking the gene desert, likely accounting for the embryonic lethality due to Shox2-dependency of the SAN pacemaker. Finally, we discover a 1.5kb SV enhancer within the deleted gene desert region, which may act as a genomic module controlling the development of the cardiac conduction system. In summary, our results identify a gene desert indispensable for pleiotropic patterning and highlight the importance of these extensive regulatory landscapes for embryonic development and viability.

Placental thrombo-inflammation impairs embryonic survival by reducing placental thrombomodulin expression

Excess platelet-activation by extracellular vesicles (EVs) results in trophoblast inflammasome activation, IL-1β activation, preeclampsia (PE) and partial embryonic lethality. Furthermore, embryonic thrombomodulin (TM) deficiency, which causes embryonic lethality hallmarked by impaired trophoblast proliferation, has been linked with maternal platelet-activation. Hence, we hypothesized that placental TM loss, platelet-activation, and embryonic lethality are mechanistically linked to trophoblast inflammasome activation. Here, we uncover a unidirectional interaction of placental inflammasome activation and reduced placental TM-expression: while inflammasome inhibition did not rescue TM-null embryos from lethality, the inflammasome-dependent cytokine IL-1β reduced trophoblast TM expression and impaired pregnancy outcome. Likewise, EVs known to induce placental inflammasome activation, reduced trophoblast TM expression and proliferation. Trophoblast TM expression correlated negatively with IL-1β expression and positively with platelet numbers and trophoblast proliferation in human PE placentae, implying translational relevance. Soluble TM treatment or placental TM restoration ameliorated the EV-induced PE-like phenotype in mice, preventing placental thrombo-inflammation and embryonic death. Collectively, the lethality of TM-null embryos is not a consequence of placental NLRP3 inflammasome activation. Conversely, EV-induced placental inflammasome activation reduces placental TM expression, which promotes placental and embryonic demise. These data identify a new function of placental TM in PE and suggest that soluble TM limits thrombo-inflammatory pregnancy complications.