The focus on assessing embryo-specific defects in lethal knockout (KO) mouse lines has resulted in an underrepresentation of documented placental abnormalities. Presented herein is a uniform analysis of 22 distinct KO mouse lines that exhibit homozygous lethality in a narrow mid-gestation window. All genes altered by each KO have human orthologs, most are implicated in human disease, yet almost all are understudied. To unravel the role each plays in mammalian development, null embryonic and placental phenotype analysis was performed, and wild type (WT) expression of each KO gene was assessed. While the null phenotype of each KO line falls into two broad embryonic categories, the placental phenotypes are diverse and coincide with the onset of gross embryonic defects. The co-occurrence of null embryonic and placental defect onset coupled with WT gene expression highlights that many could be essential in either the embryo or placenta. This analysis serves to guide mid-gestation placental analysis, underscores the importance of routine analysis of the entire conceptus during mid-gestation lethality, and provides functional annotation for each understudied human ortholog.

Caspase-6 (casp-6), is a member of the apoptotic family of caspases (cysteine aspartic proteases) and has been shown to be involved in several neurodegenerative diseases, including Alzheimer's disease. Drug discovery efforts have focused on specific inhibition of casp-6, and it is important that lead compounds demonstrate engagement and inhibition of the protease in vivo. A casp-6 overexpressing mouse model was reported, but is no longer available to the scientific community. We developed alternative means of activating casp-6 in commercially available mice as a platform to test potential casp-6 inhibitors. Lipopolysaccharides (LPS) from E. coli was used to stimulate an immunogenic response in WT C57BL/6NJ mice, which has been shown to activate the NLRP1 inflammasome, which activates casp-1 and subsequently, casp-6. Key tissues (brain, colon, and thymus) were sampled and evaluated for casp-6 activation by immunoblotting for both active casp-6 and a cleaved lamin A, a casp-6 substrate, and by directly measuring VEIDase activity in tissue lysates. Using in-house bred WT C57BL/6NJ mice, which had never been subjected to shipping, with a single intraperitoneal dose of LPS (5 mg/kg) and a 4-hour incubation, we observed robust casp-6 activation in the thymus. We also established VEIDase activity assays as a more reliable marker of casp-6 function than monitoring levels of casp-6 or cleaved substrates, and established the importance of using a non-immunogenic vehicle. We observed casp-6 activation in aged mice as a second useful model, which also further implicates casp-6 in age-related neurodegenerative pathways. These are new means to test the efficacy of casp-6 inhibitors using a simple in vivo assay that can robustly activate casp-6 in an easily accessible laboratory mouse strain. We foresee that these developments will drive drug discovery efforts aimed at identifying casp-6 inhibitors in various disease contexts, including neurodegeneration.

Proper junction organization within the trophectoderm is essential for blastocyst integrity and implantation. Epithelial junctions are well characterized in many somatic cell types with a typical apical-basal arrangement of junction type: tight junctions, adherens junctions, and then desmosomes. Here, we reveal that the early mural trophoblast of both mouse and human blastocysts exhibit a distinct junction arrangement in which desmosomes are positioned medially within adherens junctions, leaving a basal E-cadherin “tail”, which colocalizes with ATP1A1, a marker for Na+/K+-ATPase pumps. As blastocysts develop and expand approaching hatching and implantation, the organization of junctions in mural trophoblast shifts to the canonical arrangement, and we show that this reorganization requires the ATPase pump function. Together, these data uncover a dynamic remodeling of trophoblast junctions, suggesting a novel connection between junction organization, blastocyst maturation, hatching, and successful implantation.

Uncontrolled inflammation is the driver of numerous lung diseases. Current treatments, including corticosteroids and bronchodilators, can be effective. However, they often come with notable side effects. siRNA is a promising therapeutic modality for immune regulation. However, effective delivery of siRNA is challenged by issues related to cellular uptake and localization within tissues. This study investigates a series of guanidinium-functionalized polymers (Cn-Guan) designed to explore the effects of amphiphilicity on siRNA complexation and efficiency in vitro and in vivo. Nine polymers with varying side chain lengths (C3, C5, C7) and molecular weights (17 kDa, 30 kDa, 65 kDa) were synthesized, forming polyplexes with siRNA. Characterization revealed that C7-Guan/si_scr polymers exhibited the smallest polyplex sizes and the tightest complexation with siRNA. In vitro studies showed that 65 kDa polymers had the highest gene knockdown efficiency, with C3 and C5-Guan/si_TNF-α achieving ∼70 % knockdown, while C7-Guan/si_TNF-α achieved ∼30 %. In vivo, C7-Guan/Cy5-siRNA demonstrated the highest lung accumulation, and all polymers showed ∼70 % TNF-α knockdown with a low siRNA dosage (0.14 mg/kg) in a murine lung inflammation model. C7-Guan polymers, despite lower in vitro efficiency, were quite effective in vivo, potentially due to enhanced serum stability. These findings demonstrate that Cn-Guan/siRNA polyplexes are effective and safe for attenuating pulmonary inflammation and provide important insights for the development of future siRNA delivery vectors for lung disease treatment.

Cap Methyltransferase 1 (CMTR1) facilitates the addition of a 5' methyl cap on eukaryotic mRNA molecules. Using a knock-out (KO) allele, we demonstrate that CMTR1plays an essential role during gastrulation. In the absence of CMTR1, mutant embryos undergo early lethality, arresting prior to organogenesis with severe developmental delay apparent at E7.5. Multiple molecular approaches indicate significant disruptions in the ability of the CMTR1-KO embryo to form the three primary germ layers - likely driving the observed gastrulation failure. Our analysis of CMTR1 has revealed an unexpected sexually dimorphic phenotype. Female CMTR1 null embryos are more severely delayed and have increased differentially expressed genes compared to male mutants; presumably causing a variety of downstream consequences and a more severe developmental phenotype. Importantly, we do not observe defects in X-inactivation, suggesting that there are unidentified sexually dimorphic mechanisms active during early embryonic stages, prior to the onset of known differences between XX and XY embryos. In sum, we illustrate the necessity of CMTR1 during embryonic development and reveal novel insights into differences in gene regulation pathways between sexes prior to organogenesis.

Memory CD8 T cells provide long-lasting immunity, but their developmental origins remain incompletely defined. Growing evidence suggests that functional heterogeneity exists within the naïve T cell pool, shaping lineage potential before antigen stimulation. Here, we identify a subpopulation of naïve CD8 T cells expressing death-associated protein-like 1 (Dapl1) that contains preprogrammed precursors biased toward memory differentiation. The differentiation of these precursors is independent of Dapl1 but relies on the transcription factor B-cell lymphoma/leukaemia 11b (Bcl11b), resulting in the generation of Dapl1+ central memory-like CD8 T cells after infection and stem-like memory cells in cancer. Dapl1+ naïve T cells originate among mature thymocytes and gradually appear in the periphery postnatally. Peripheral Dapl1+ and Dapl1- populations show limited plasticity, supporting a thymic-imprinting model. These findings reveal a developmentally imprinted subset of naïve CD8 T cells committed to memory fate, uncovering an alternative pathway for memory T cell generation offering new avenues for therapeutic application.

Memory CD8 T cells play a vital role in providing lasting immune protection, yet their origins remain incompletely understood. Contrary to classical models, emerging evidence suggests that heterogeneity within the naive T cell pool may influence fate decisions prior to antigen encounter. However, the markers of naive T cell heterogeneity have not yet been clearly defined. Here, we describe intraclonal heterogeneity within the naive T cell population marked by the protein Dapl1. Using novel monoclonal antibodies and a reporter-knockout mouse model, we found that Dapl1-positive naive CD8 T cells exhibit distinct phenotypes compared to their Dapl1-negative counterparts. Furthermore, this population includes a subset of pre-programmed precursors biased toward memory lineage fate. The differentiation of these precursors is independent of Dapl1 but relies on the transcription factor Bcl11b, resulting in the generation of Dapl1-positive central memory-like CD8 T cells in response to infection, and stem-like memory cells in response to cancer. Notably, naive Dapl1-positive T cells originate in the thymus among mature thymocytes and gradually appear in the periphery within several days after birth. Our findings suggest that committed memory precursors in the Dapl1-positive population may represent an alternative pathway for memory CD8 T cell generation, offering new avenues for therapeutic application. ### Competing Interest Statement The authors have declared no competing interest.

Mammalian preimplantation development culminates in the formation of a blastocyst that undergoes extensive gene expression regulation to successfully implant into the maternal endometrium. Zinc-finger HIT domain-containing (ZNHIT) 1 and 2 are members of a highly conserved family, yet they have been identified as subunits of distinct complexes. Here, we report that knockout of either Znhit1 or Znhit2 results in embryonic lethality during peri-implantation stages. Znhit1 and Znhit2 mutant embryos have overlapping phenotypes, including reduced proportion of SOX2-positive inner cell mass cells, a lack of Fgf4 expression, and aberrant expression of NANOG and SOX17. Furthermore, we find that the similar phenotypes are caused by distinct mechanisms. Specifically, embryos lacking ZNHIT1 likely fail to incorporate sufficient H2A.Z at the promoter region of Fgf4 and other genes involved in cell projection organization resulting in impaired invasion of trophoblast cells during implantation. In contrast, Znhit2 mutant embryos display a complete lack of nuclear EFTUD2, a key component of U5 spliceosome, indicating a global splicing deficiency. Our findings unveil the indispensable yet distinct roles of ZNHIT1 and ZNHIT2 in early mammalian embryonic development.

e-Lysine acetylation is a prominent histone mark found at transcriptionally active loci. Among many lysine acetyl transferases, nonspecific lethal complex (NSL) members are known to mediate the modification of histone H4. In addition to histone modifications, the KAT8 regulatory complex subunit 3 gene (Kansl3), a core member of NSL complex, has been shown to be involved in several other cellular processes such as mitosis and mitochondrial activity. Although functional studies have been performed on NSL complex members, none of the four core proteins, including Kansl3, have been studied during early mouse development. Here we show that homozygous knockout Kansl3 embryos are lethal at peri-implantation stages, failing to hatch out of the zona pellucida. When the zona pellucida is removed in vitro, Kansl3 null embryos form an abnormal outgrowth with significantly disrupted inner cell mass (ICM) morphology. We document lineage-specific defects at the blastocyst stage with significantly reduced ICM cell number but no difference in trophectoderm cell numbers. Both epiblast and primitive endoderm lineages are altered with reduced cell numbers in null mutants. These results show that Kansl3 is indispensable during early mouse embryonic development and with defects in both ICM and trophectoderm lineages.

Self-assembly of siRNA with a block copolymer featuring guanidinium and zwitterion functionalized blocks generates core-shell-like nanovectors that provide cytosolic access to siRNA and efficiently evade phagocytic clearance. The guanidinium-functionalized inner block complexes siRNA in the nanovector interior and enables cytosolic delivery. The zwitterionic outer block provides a non-interacting shell on the nanovectors that reduces macrophage uptake in vitro and phagocytic clearance and enhances tumor localization in vivo. These nanovectors were used to treat a 4T1 (murine) model of triple-negative breast cancer (TNBC). The nanovectors deliver siRNA efficiently to 4T1 triple-negative breast cancer cells in vitro, with high selectivity relative to macrophages. This efficiency and selectivity translate into in vivo efficacy: diblock nanovectors evaded phagocytic clearance and efficiently localized in an aggressive murine 4T1 orthotopic model, with a ~3-fold increase of vector residing in the tumor compared to the homopolymer nanovectors. This increased localization efficiently knocked down STAT3 (~80%) and provided tumorostasis (100% growth inhibition) at a low dose of 0.14 mg/kg. The in vitro and in vivo efficacy of these nanovectors demonstrate the potential of engineered polymer architectures to generate effective self-assembled siRNA therapeutics that avoid phagocytic clearance for the treatment of diseases requiring systemic administration.

Early embryonic development is a finely orchestrated process that requires precise regulation of gene expression coordinated with morphogenetic events. TATA-box binding protein-associated factors (TAFs), integral components of transcription initiation coactivators like TFIID and SAGA, play a crucial role in this intricate process. Here we show that disruptions in TAF5, TAF12 and TAF13 individually lead to embryonic lethality in the mouse, resulting in overlapping yet distinct phenotypes. Taf5 and Taf12 mutant embryos exhibited a failure to implant post-blastocyst formation, and Taf5 mutants have aberrant lineage specification within the inner cell mass. In contrast, Taf13 mutant embryos successfully implant and form egg-cylinder stages but fail to initiate gastrulation. Strikingly, we observed a depletion of pluripotency factors in TAF13-deficient embryos, including OCT4, NANOG and SOX2, highlighting an indispensable role of TAF13 in maintaining pluripotency. Transcriptomic analysis revealed distinct gene targets affected by the loss of TAF5, TAF12 and TAF13. Thus, we propose that TAF5, TAF12 and TAF13 convey locus specificity to the TFIID complex throughout the mouse genome.

Exosome Complex Components 1 and 2 (EXOSC1 and 2) are two proteins in the RNA Exosome complex whose main function is 5' → 3' RNA degradation and processing. The RNA exosome complex is comprised of nine subunits that form two separate components: the S1/KH cap and the PH-core. EXOSC1 and 2 are both part of the S1/KH cap and are involved in binding nascent RNA. As part of a systemic characterization of early lethal alleles produced by the Knockout Mouse Project, we have examined Exosc1 and Exosc2 homozygous null (mutant) embryos to determine developmental and molecular phenotypes of embryos lacking their functions. Our studies reveal that Exosc1 null embryos implant and form an egg cylinder but are developmentally delayed and fail to initiate gastrulation by embryonic day 7.5. In contrast, Exosc2 null embryos are lethal during peri-implantation stages, and while they do form a morphologically normal blastocyst at E3.5, they cannot be recovered at post-implantation stages. We show the absence of stage-specific developmental and altered lineage-specification in both Exosc1 and Exosc2 mutant embryos and conclude that these genes are essential for the successful progression through early mammalian development.

The success of the CD8 T cell-mediated immune response against infections and tumors depends on the formation of a long-lived memory pool, and the protection of effector cells from exhaustion. The advent of checkpoint blockade therapy has significantly improved anti-tumor therapeutic outcomes by reversing CD8 T cell exhaustion, but fails to generate effector cells with memory potential. Here, using in vivo mouse models, we show that let-7 miRNAs determine CD8 T cell fate, where maintenance of let-7 expression during early cell activation results in memory CD8 T cell formation and tumor clearance. Conversely, let-7-deficiency promotes the generation of a terminal effector population that becomes vulnerable to exhaustion and cell death in immunosuppressive environments and fails to reject tumors. Mechanistically, let-7 restrains metabolic changes that occur during T cell activation through the inhibition of the PI3K/AKT/mTOR signaling pathway and production of reactive oxygen species, potent drivers of terminal differentiation and exhaustion. Thus, our results reveal a role for let-7 in the time-sensitive support of memory formation and the protection of effector cells from exhaustion. Overall, our data suggest a strategy in developing next-generation immunotherapies by preserving the multipotency of effector cells rather than enhancing the efficacy of differentiation.

Uncontrolled inflammation is responsible for acute and chronic diseases in the lung. Regulating expression of pro-inflammatory genes in pulmonary tissue using small interfering RNA (siRNA) is a promising approach to combatting respiratory diseases. However, siRNA therapeutics are generally hindered at the cellular level by endosomal entrapment of delivered cargo and at the organismal level by inefficient localization in pulmonary tissue. Here we report efficient anti-inflammatory activity in vitro and in vivo using polyplexes of siRNA and an engineered cationic polymer (PONI-Guan). PONI-Guan/siRNA polyplexes efficiently deliver siRNA cargo to the cytosol for highly efficient gene knockdown. Significantly, these polyplexes exhibit inherent targeting to inflamed lung tissue following intravenous administration in vivo. This strategy achieved effective (>70%) knockdown of gene expression in vitro and efficient (>80%) silencing of TNF-α expression in lipopolysaccharide (LPS)-challenged mice using a low (0.28 mg/kg) siRNA dosage.

In female mice, the gene dosage from X chromosomes is adjusted by a process called X chromosome inactivation (XCI) that occurs in two steps. An imprinted form of XCI (iXCI) that silences the paternally inherited X chromosome (Xp) is initiated at the 2- to 4-cell stages. As extraembryonic cells including trophoblasts keep the Xp silenced, epiblast cells that give rise to the embryo proper reactivate the Xp and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI require the lncRNA Xist, which is expressed from the X to be inactivated. The X-linked E3 ubiquitin ligase Rlim (Rnf12) in conjunction with its target protein Rex1 (Zfp42), a critical repressor of Xist, have emerged as major regulators of iXCI. However, their roles in rXCI remain controversial. Investigating early mouse development, we show that the Rlim-Rex1 axis is active in pre-implantation embryos. Upon implantation Rex1 levels are downregulated independently of Rlim specifically in epiblast cells. These results provide a conceptual framework of how the functional dynamics between Rlim and Rex1 ensures regulation of iXCI but not rXCI in female mice.

Treatment of wound biofilm infections faces challenges from both pathogens and uncontrolled host immune response. Treating both issues through a single vector would provide enhanced wound healing. Here, we report the use of a potent cationic antimicrobial polymer to generate siRNA polyplexes for dual-mode treatment of wound biofilms in vivo. These polyplexes act both as an antibiofilm agent and a delivery vehicle for siRNA for the knockdown of biofilm-associated pro-inflammatory MMP9 in host macrophages. The resulting polyplexes were effective in vitro, eradicating MRSA biofilms and efficiently delivering siRNA to macrophages in vitro with concomitant knockdown of MMP9. These polyplexes were likewise effective in an in vivo murine wound biofilm model, significantly reducing bacterial load in the wound (∼99% bacterial clearance) and reducing MMP9 expression by 80% (qRT-PCR). This combination therapeutic strategy dramatically reduced wound purulence and significantly expedited wound healing. Taken together, these polyplexes provide an effective and translatable strategy for managing biofilm-infected wounds.

Heterogeneous nuclear ribonucleoprotein L (hnRNPL) is a conserved RNA binding protein (RBP) that plays an important role in the alternative splicing of gene transcripts, and thus in the generation of specific protein isoforms. Global deficiency in hnRNPL in mice results in preimplantation embryonic lethality at embryonic day (E) 3.5. To begin to understand the contribution of hnRNPL-regulated pathways in the normal development of the embryo and placenta, we determined hnRNPL expression profile and subcellular localization throughout development. Proteome and Western blot analyses were employed to determine hnRNPL abundance between E3.5 and E17.5. Histological analyses supported that the embryo and implantation site display distinct hnRNPL localization patterns. In the fully developed mouse placenta, nuclear hnRNPL was observed broadly in trophoblasts, whereas within the implantation site a discrete subset of cells showed hnRNPL outside the nucleus. In the first-trimester human placenta, hnRNPL was detected in the undifferentiated cytotrophoblasts, suggesting a role for this factor in trophoblast progenitors. Parallel in vitro studies utilizing Htr8 and Jeg3 cell lines confirmed expression of hnRNPL in cellular models of human trophoblasts. These studies [support] coordinated regulation of hnRNPL during the normal developmental program in the mammalian embryo and placenta.

As a highly conserved DNA polymerase (Pol), Pol δ plays crucial roles in chromosomal DNA synthesis and various DNA repair pathways. However, the function of POLD2, the second small subunit of DNA Pol δ (p50 subunit), has not been characterized in vivo during mammalian development. Here, we report for the first time, the essential role of subunit POLD2 during early murine embryogenesis. Although Pold2 mutant mouse embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at gastrulation stages. Outgrowth assays reveal that mutant blastocysts cannot hatch from the zona pellucida, indicating impaired blastocyst function. Notably, these phenotypes can be recapitulated by small interfering RNA (siRNA)-mediated knockdown, which also exhibit slowed cellular proliferation together with skewed primitive endoderm and epiblast allocation during the second cell lineage specification. In summary, our study demonstrates that POLD2 is essential for the earliest steps of mammalian development, and the retarded proliferation and embryogenesis may also alter the following cell lineage specifications in the mouse blastocyst embryos.

Retinoic acid (RA), a metabolite of vitamin A, is a small molecule and morphogen that is required for embryonic development. While normal RA signals are required for hepatic development in a variety of vertebrates, a role for RA during mammalian hepatic specification has yet to be defined. To examine the requirement for RA in murine liver induction, we performed whole embryo culture with the small molecule RA inhibitor, BMS493, to attenuate RA signaling immediately prior to hepatic induction and through liver bud formation. BMS493 treated embryos demonstrated a significant loss of hepatic specification that was confined to the prospective dorsal anterior liver bud. Examination of RA attenuated embryos demonstrates that while the liver bud displays normal expression of foregut endoderm markers and the hepato-pancreatobiliary domain marker, PROX1, the dorsal/anterior liver bud excludes the critical hepatic marker, HNF4α, indicating that RA signals are required for dorsal/anterior hepatic induction. These results were confirmed and extended by careful examination of Rdh10trex/trex embryos, which carry a genetic perturbation in RA synthesis. At E9.5 Rdh10trex/trex embryos display a similar yet more significant loss of the anterior/dorsal liver bud. Notably the anterior/dorsal liver bud loss correlates with the known dorsal-ventral gradient of the RA synthesis enzyme, Aldh1a2. In addition to altered hepatic specification, the mesoderm surrounding the liver bud is disorganized in RA abrogated embryos. Analysis of E10.5 Rdh10trex/trex embryos reveals small livers that appear to lack the dorsal/caudal lobes. Finally, addition of exogenous RA prior to hepatic induction results in a liver bud that has failed to thicken and is largely unspecified. Taken together our ex vivo and in vivo evidence demonstrate that the generation of normal RA gradients is required for hepatic patterning, specification, and growth.