Lnx2-mediated signaling and heterogeneity of ribosomal proteins during early embryogenesis in mice

Abstract

PART I Impaired blastocyst formation in Lnx2-knockdown mouse embryos Ligand of Numb-protein X 2 (LNX2) is an E3 ubiquitin ligase that is known to regulate Notch signaling by participating in NUMB protein degradation. Notch signaling is important for differentiation and proliferation in mammals, and plays a significant role in blastocyst formation during early embryonic development. In this study, I investigated Lnx2 in mouse preimplantation embryos. Expression analysis showed that Lnx2 is expressed in oocytes and preimplantation embryos. Lnx2- knockdown embryos normally progress to the morula stage, but the majority of them do not develop into normal blastocysts. Transcript analysis revealed that the expression levels of genes critical for cell lineage specification, including octamer-binding transcription factor 4 (Oct4), are increased in Lnx2 knockdown embryos. Furthermore, the expression levels of Notch and Hippo signaling-related genes are also increased by Lnx2 knockdown. Collectively, my results show that Lnx2 is important for blastocyst formation in mice, suggest that this may act via lineage specification of inner cell mass, and further show that Lnx2 may be involved in transcriptionally regulating various genes implicated in early embryonic development. Keywords Lnx2; Notch signaling; preimplantation embryo; blastocyst; inner cell mass|PART II Functional characterization and heterogeneity analysis of ribosomal proteins in mouse preimplantation embryos Translational control is important during the mammalian preimplantation phase, when maternal RNAs and proteins are degraded and de novo synthesis of RNAs and proteins increases. Proteins are synthesized in ribosomes, which are assembled from ~82 ribosomal proteins (RPs). The function of ribosomes varies depending on the resident RPs, suggesting that ribosome heterogeneity can lead to functional specialization. Only a few studies have investigated the function of RPs during preimplantation embryonic development. Here, I performed functional analyses on six RP-encoding genes—Rpl4, Rps9, Rps11, Rpl13a, Rpl19, and Rpl39—in mouse preimplantation embryos. Knockdown (KD) of each of these RP genes, except Rpl39, affected morula-to-blastocyst transition, producing phenotypes that varied somewhat in their details. Rpl4-, Rpl13a-, and Rpl19-KD embryos showed fragmentation and strong arrest of cell proliferation, whereas Rps9-and Rps11-KD embryos showed severe fragmentation with relatively weak arrest of cell proliferation. In the case of Rpl39, single-KD embryos developed normally, but double-KD embryos with its paralog Rpl39-like (Rpl39l) inhibited normal blastocyst development. Protein misfolding signals were also activated in Rpl39-KD and Rpl39l + Rpl39 double-KD embryos, confirming a previous finding that RPL39 and RPL39L are associated with ribosome exit tunnels. My results suggest the presence of different groups of proteins that require an RPL39-containing ribosome or RPL39/RPL39L-containing ribosome for correct folding in early embryos. Taken together, the results of the present study demonstrate that ribosomal proteins are fundamentally important for normal blastocyst formation and development, but not all ribosomal proteins contribute equally to embryonic development, providing a novel example of ribosome heterogeneity in preimplantation embryos. Keywords De novo protein synthesis; morula-to-blastocyst transition; mouse preimplantation embryo; protein folding; ribosomal protein; ribosome heterogeneity