Yolk sac cell atlas reveals multiorgan functions during human early development.

The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.

Hippo signaling instructs ectopic but not normal organ growth.

The Hippo signaling pathway is widely considered a master regulator of organ growth because of the prominent overgrowth phenotypes caused by experimental manipulation of its activity. Contrary to this model, we show here that removing Hippo transcriptional output did not impair the ability of the mouse liver and Drosophila eyes to grow to their normal size. Moreover, the transcriptional activity of the Hippo pathway effectors Yap/Taz/Yki did not correlate with cell proliferation, and hyperactivation of these effectors induced gene expression programs that did not recapitulate normal development. Concordantly, a functional screen in Drosophila identified several Hippo pathway target genes that were required for ectopic overgrowth but not normal growth. Thus, Hippo signaling does not instruct normal growth, and the Hippo-induced overgrowth phenotypes are caused by the activation of abnormal genetic programs.

Postnatal regulation of B-1a cell development and survival by the CIC-PER2-BHLHE41 axis.

B-1 cell development mainly occurs via fetal and neonatal hematopoiesis and is suppressed in adult bone marrow hematopoiesis. However, little is known about the factors inhibiting B-1 cell development at the adult stage. We report that capicua (CIC) suppresses postnatal B-1a cell development and survival. CIC levels are high in B-1a cells and gradually increase in transitional B-1a (TrB-1a) cells with age. B-cell-specific Cic-null mice exhibit expansion of the B-1a cell population and a gradual increase in TrB-1a cell frequency with age but attenuated B-2 cell development. CIC deficiency enhances B cell receptor (BCR) signaling in transitional B cells and B-1a cell viability. Mechanistically, CIC-deficiency-mediated Per2 derepression upregulates Bhlhe41 levels by inhibiting CRY-mediated transcriptional repression for Bhlhe41, consequently promoting B-1a cell formation in Cic-null mice. Taken together, CIC is a key transcription factor that limits the B-1a cell population at the adult stage and balances B-1 versus B-2 cell formation.

Single-Cell Transcriptome Analysis Reveals Embryonic Endothelial Heterogeneity at Spatiotemporal Level and Multifunctions of MicroRNA-126 in Mice.

BACKGROUND: Endothelial cells (ECs) play a critical role in angiogenesis and vascular remodeling. The heterogeneity of ECs has been reported at adult stages, yet it has not been fully investigated. This study aims to assess the transcriptional heterogeneity of developmental ECs at spatiotemporal level and to reveal the changes of embryonic ECs clustering when endothelium-enriched microRNA-126 (miR-126) was specifically knocked out. METHODS: C57BL/6J mice embryos at day 14.5 were harvested and digested, followed by fluorescence-activated cell sorting to enrich ECs. Then, single-cell RNA sequencing was applied to enriched embryonic ECs. Tie2 (Tek receptor tyrosine kinase)-cre-mediated ECs-specific miR-126 knockout mice were constructed, and ECs from Tie2-cre-mediated ECs-specific miR-126 knockout embryos were subjected to single-cell RNA sequencing. RESULTS: Embryonic ECs were clustered into 11 groups corresponding to anatomic characteristics. The vascular bed (arteries, capillaries, veins, lymphatics) exhibited transcriptomic similarity across the developmental stage. Embryonic ECs had higher proliferative potential than adult ECs. Integrating analysis showed that 3 ECs populations (hepatic, mesenchymal transition, and pulmonary ECs) were apparently disorganized after miR-126 being knocked out. Gene ontology analysis revealed that disrupted ECs were mainly related to hypoxia, glycometabolism, and vascular calcification. Additionally, in vivo experiment showed that Tie2-cre-mediated ECs-specific miR-126 knockout mice exhibited excessive intussusceptive angiogenesis; reductive glucose and pyruvate tolerance; and excessive accumulation of calcium. Agonist miR-126-3p agomir significantly rescued the phenotype of glucose metabolic dysfunction in Tie2-cre-mediated ECs-specific miR-126 knockout mice. CONCLUSIONS: The heterogeneity of ECs is established as early as the embryonic stage. The deficiency of miR-126 disrupts the differentiation and diversification of embryonic ECs, suggesting that miR-126 plays an essential role in the maintenance of ECs heterogeneity.

Tissue-specific responses that constrain glucose oxidation and increase lactate production with the severity of hypoxemia in fetal sheep.

Fetal hypoxemia decreases insulin and increases cortisol and norepinephrine concentrations and may restrict growth by decreasing glucose utilization and altering substrate oxidation. Specifically, we hypothesized that hypoxemia would decrease fetal glucose oxidation and increase lactate and pyruvate production. We tested this by measuring whole body glucose oxidation and lactate production, and molecular pathways in liver, muscle, adipose, and pancreas tissues of fetuses exposed to maternal hypoxemia for 9 days (HOX) compared with control fetal sheep (CON) in late gestation. Fetuses with more severe hypoxemia had lower whole body glucose oxidation rates, and HOX fetuses had increased lactate production from glucose. In muscle and adipose tissue, expression of the glucose transporter GLUT4 was decreased. In muscle, pyruvate kinase (PKM) and lactate dehydrogenase B (LDHB) expression was decreased. In adipose tissue, LDHA and lactate transporter (MCT1) expression was increased. In liver, there was decreased gene expression of PKLR and MPC2 and phosphorylation of PDH, and increased LDHA gene and LDH protein abundance. LDH activity, however, was decreased only in HOX skeletal muscle. There were no differences in basal insulin signaling across tissues, nor differences in pancreatic tissue insulin content, ß-cell area, or genes regulating ß-cell function. Collectively, these results demonstrate coordinated metabolic responses across tissues in the hypoxemic fetus that limit glucose oxidation and increase lactate and pyruvate production. These responses may be mediated by hypoxemia-induced endocrine responses including increased norepinephrine and cortisol, which inhibit pancreatic insulin secretion resulting in lower insulin concentrations and decreased stimulation of glucose utilization.NEW & NOTEWORTHY Hypoxemia lowered fetal glucose oxidation rates, based on severity of hypoxemia, and increased lactate production. This was supported by tissue-specific metabolic responses that may result from increased norepinephrine and cortisol concentrations, which decrease pancreatic insulin secretion and insulin concentrations and decrease glucose utilization. This highlights the vulnerability of metabolic pathways in the fetus and demonstrates that constrained glucose oxidation may represent an early event in response to sustained hypoxemia and fetal growth restriction.

EpoR stimulates rapid cycling and larger red cells during mouse and human erythropoiesis.

The erythroid terminal differentiation program couples sequential cell divisions with progressive reductions in cell size. The erythropoietin receptor (EpoR) is essential for erythroblast survival, but its other functions are not well characterized. Here we use Epor-/- mouse erythroblasts endowed with survival signaling to identify novel non-redundant EpoR functions. We find that, paradoxically, EpoR signaling increases red cell size while also increasing the number and speed of erythroblast cell cycles. EpoR-regulation of cell size is independent of established red cell size regulation by iron. High erythropoietin (Epo) increases red cell size in wild-type mice and in human volunteers. The increase in mean corpuscular volume (MCV) outlasts the duration of Epo treatment and is not the result of increased reticulocyte number. Our work shows that EpoR signaling alters the relationship between cycling and cell size. Further, diagnostic interpretations of increased MCV should now include high Epo levels and hypoxic stress.

Sequence logic at enhancers governs a dual mechanism of endodermal organ fate induction by FOXA pioneer factors.

FOXA pioneer transcription factors (TFs) associate with primed enhancers in endodermal organ precursors. Using a human stem cell model of pancreas differentiation, we here discover that only a subset of pancreatic enhancers is FOXA-primed, whereas the majority is unprimed and engages FOXA upon lineage induction. Primed enhancers are enriched for signal-dependent TF motifs and harbor abundant and strong FOXA motifs. Unprimed enhancers harbor fewer, more degenerate FOXA motifs, and FOXA recruitment to unprimed but not primed enhancers requires pancreatic TFs. Strengthening FOXA motifs at an unprimed enhancer near NKX6.1 renders FOXA recruitment pancreatic TF-independent, induces priming, and broadens the NKX6.1 expression domain. We make analogous observations about FOXA binding during hepatic and lung development. Our findings suggest a dual role for FOXA in endodermal organ development: first, FOXA facilitates signal-dependent lineage initiation via enhancer priming, and second, FOXA enforces organ cell type-specific gene expression via indirect recruitment by lineage-specific TFs.

Connexin Expression Is Altered in Liver Development of Yotari (dab1 -/-) Mice.

Disabled-1 (Dab1) protein is an intracellular adaptor of reelin signaling required for prenatal neuronal migration, as well as postnatal neurotransmission, memory formation and synaptic plasticity. Yotari, an autosomal recessive mutant of the mouse Dab1 gene is recognizable by its premature death, unstable gait and tremor. Previous findings are mostly based on neuronal abnormalities caused by Dab1 deficiency, but the role of the reelin signaling pathway in nonneuronal tissues and organs has not been studied until recently. Hepatocytes, the most abundant cells in the liver, communicate via gap junctions (GJ) are composed of connexins. Cell communication disruption in yotari mice was examined by analyzing the expression of connexins (Cxs): Cx26, Cx32, Cx37, Cx40, Cx43 and Cx45 during liver development at 13.5 and 15.5 gestation days (E13.5 and E15.5). Analyses were performed using immunohistochemistry and fluorescent microscopy, followed by quantification of area percentage covered by positive signal. Data are expressed as a mean ± SD and analyzed by one-way ANOVA. All Cxs examined displayed a significant decrease in yotari compared to wild type (wt) individuals at E13.5. Looking at E15.5 we have similar results with exception of Cx37 showing negligible expression in wt. Channels formation triggered by pathological stimuli, as well as propensity to apoptosis, was studied by measuring the expression of Pannexin1 (Panx1) and Apoptosis-inducing factor (AIF) through developmental stages mentioned above. An increase in Panx1 expression of E15.5 yotari mice, as well as a strong jump of AIF in both phases suggesting that yotari mice are more prone to apoptosis. Our results emphasize the importance of gap junction intercellular communication (GJIC) during liver development and their possible involvement in liver pathology and diagnostics where they can serve as potential biomarkers and drug targets.

Effect of Maternal Marginal Zinc Deficiency on Development, Redox Status, and Gene Expression Related to Oxidation and Apoptosis in an Avian Embryo Model.

Maternal severe zinc (Zn) deficiency resulted in growth retardation and high mortality during embryonic development in human. Therefore, this study is aimed at evaluating the effect of maternal marginal Zn deficiency on the development and redox status to avoid severe Zn deficiency using an avian model. A total of 324 laying duck breeders at 214 days old were randomly allotted into 3 dietary Zn levels with 6 replicates of 18 ducks per replicate. The birds were fed experimental diets including 3 dietary supplemental Zn levels of 0 mg/kg (maternal Zn-deficient group, 29.2 mg Zn/kg diet), 60 mg/kg (maternal Zn-adequate group), and 120 mg/kg (maternal Zn-high group) for 6 weeks. Dietary Zn levels had on effect on egg production and fertility (P > 0.05), whereas dietary Zn deficiency decreased breeder plasma Zn concentration and erythrocytic alkaline phosphatase activity at week 6 and inhibited erythrocytic 5'-nucleotidase (5'-NT) activity at weeks 2, 4, and 6 (P < 0.05), indicating that marginal Zn-deficient status occurred after Zn depletion. Maternal marginal Zn deficiency increased embryonic mortality and contents of superoxide anion radical, MDA, and PPC and reduced MT content and CuZnSOD activity in duck embryonic livers on E29. The MDA content was positively correlated with embryonic mortality. Maternal marginal Zn deficiency increased BCL2-associated X protein and Caspase-9 mRNA expressions as well as decreased B-cell lymphoma-2 and MT1 mRNA and signal AKT1 and ERK1 protein expressions (P < 0.05). Breeder plasma Zn concentration and erythrocytic 5'-NT activities at week 6 were positively correlated with GSH-Px activity and GPx, MT1, and BCL2 mRNA expressions in embryonic livers on E29. In conclusion, erythrocytic 5'-NT activity could be more rapid and reliable to monitor marginal Zn-deficient status. Marginal Zn deficiency impaired hatchability and antioxidant defense system and then induced oxidative damage and apoptosis in the embryonic liver, contributing to the greater loss of duck embryonic death.

Liver and brain differential expression of one-carbon metabolism genes during ontogenesis.

One-carbon metabolism (1C metabolism) is of paramount importance for cell metabolism and mammalian development. It is involved in the synthesis or modification of a wide variety of compounds such as proteins, lipids, purines, nucleic acids and neurotransmitters. We describe here the evolution of expression of genes related to 1C metabolism during liver and brain ontogeny in mouse. The level of expression of 30 genes involved in 1C metabolism was quantified by RT-qPCR in liver and brain tissues of OF1 mice at E9, E11, E13, E15, E17, P0, P3, P5, P10, P15 developmental stages and in adults. In the liver, hierarchical clustering of the gene expression patterns revealed five distinct clades of genes with a first bifurcating hierarchy distinguishing two main developmental stages before and after E15. In the brain most of the 1C metabolism genes are expressed but at a lower levels. The gene expression of enzymes involved in 1C metabolism show dramatic changes during development that are tissue specific. mRNA expression patterns of all major genes involved in 1C metabolism in liver and brain provide clues about the methylation demand and methylation pathways during embryonic development.

Hepatic Stellate Cell: A Double-Edged Sword in the Liver.

Hepatic stellate cells (HSCs) are located in the space of Disse, between liver sinusoidal endothelia cells (LSECs) and hepatocytes. They have surprised and excited hepatologists for their biological characteristics. Under physiological quiescent conditions, HSCs are the major vitamin A-storing cells of the liver, playing crucial roles in the liver development, regeneration, and tissue homeostasis. Upon injury-induced activation, HSCs convert to a pro-fibrotic state, producing the excessive extracellular matrix (ECM) and promoting angiogenesis in the liver fibrogenesis. Activated HSCs significantly contribute to liver fibrosis progression and inactivated HSCs are key to liver fibrosis regression. In this review, we summarize the comprehensive understanding of HSCs features, including their roles in normal liver and liver fibrosis in hopes of advancing the development of emerging diagnosis and treatment for hepatic fibrosis.

Early Embryo Exposure to Assisted Reproductive Manipulation Induced Subtle Changes in Liver Epigenetics with No Apparent Negative Health Consequences in Rabbit.

Embryo manipulation is a requisite step in assisted reproductive technology (ART). Therefore, it is of great necessity to appraise the safety of ART and investigate the long-term effect, including lipid metabolism, on ART-conceived offspring. Augmenting our ART rabbit model to investigate lipid metabolic outcomes in offspring longitudinally, we detected variations in hepatic DNA methylation ART offspring in the F3 generation for embryonic exposure (multiple ovulation, vitrification and embryo transfer). Through adult liver metabolomics and proteomics, we identified changes mainly related to lipid metabolism (e.g., polyunsaturated fatty acids, steroids, steroid hormone). We also found that DNA methylation analysis was linked to changes in lipid metabolism and apoptosis genes. Nevertheless, these differences did not apparently alter the general health status. Thus, our findings suggest that ART is likely to be a player in embryo epigenetic events related to hepatic homeostasis alteration in adulthood.

Role of mitochondrial dysfunction and PINK1/Parkin-mediated mitophagy in Cd-induced hepatic lipid accumulation in chicken embryos.

The present study was performed to investigate the effects of Cd exposure on lipid metabolism and mitochondrial dysfunction and to explore the role of mitophagy in Cd-induced dysregulation of lipid metabolism in chicken embryo liver tissues and hepatocytes. To this end, seven-day-old chicken embryos were exposed to different concentrations of Cd for 7 days, and primary chicken embryo hepatocytes were treated with Cd at four different concentrations for 6 h. Furthermore, the mitophagy inhibitor cyclosporine A (CsA) was used to investigate the role of mitophagy in Cd-induced disruption of lipid metabolism. Lipid accumulation, the expression levels of genes involved in lipid metabolism, mitochondrial dysfunction, and mitophagy were measured. The results demonstrated that Cd exposure increases hepatic triglyceride (TG) accumulation and the expression levels of lipogenic genes while decreasing those of lipolytic genes. Furthermore, Cd exposure was observed to alter mitochondrial morphology in terms of reduced size, excessive mitochondrial damage, and the formation of mitophagosomes. The co-localization of lysosome-associated membrane glycoprotein 2 and LC3 puncta was significantly increased in primary chicken embryo hepatocytes after Cd exposure. Moreover, Cd exposure increased LC3, PINK1, and Parkin protein expression levels. CsA effectively alleviated Cd-induced mitochondrial dysfunction, blocked mitochondrial membrane potential collapse, and suppressed PINK1/Parkin-mediated mitophagy. Furthermore, CsA treatment reversed the Cd-induced TG accumulation in liver tissues but further increased it in hepatocytes. Taken together, our findings demonstrate (for the first time) the importance of mitochondrial dysfunction and mitophagy via the PINK1/Parkin pathway in Cd-induced disruption of lipid metabolism.

Kruppel-like factor 15 induces the development of mature hepatocyte-like cells from hepatoblasts.

The liver is an important metabolic organ that controls homeostasis in the body. Moreover, it functions as a hematopoietic organ, while its metabolic function is low during development. Hepatocytes, which are parenchymal cells of the liver, acquire various metabolic functions by the maturation of hepatic progenitor cells during the fetal period; however, this molecular mechanism is still unclear. In this study, Kruppel-like factor 15 (KLF15) was identified as a new regulator of hepatic maturation through a comprehensive analysis of the expression of transcriptional regulators in mouse fetal and adult hepatocytes. KLF15 is a transcription factor whose expression in the liver increases from the embryonic stage throughout the developmental process. KLF15 induced the overexpression of liver function genes in mouse embryonic hepatocytes. Furthermore, we found that the expression of KLF15 could also induce the expression of liver function genes in hepatoblasts derived from human induced pluripotent stem cells (iPSCs). Moreover, KLF15 increased the promoter activity of tyrosine aminotransferase, a liver function gene. KLF15 also suppressed the proliferation of hepatoblasts. These results suggest that KLF15 induces hepatic maturation through the transcriptional activation of target genes and cell cycle control.

Mds1CreERT2, an inducible Cre allele specific to adult-repopulating hematopoietic stem cells.

Hematopoietic ontogeny consists of two broad programs: an initial hematopoietic stem cell (HSC)-independent program followed by HSC-dependent hematopoiesis that sequentially seed the fetal liver and generate blood cells. However, the transition from HSC-independent to HSC-derived hematopoiesis remains poorly characterized. To help resolve this question, we developed Mds1CreERT2 mice, which inducibly express Cre-recombinase in emerging HSCs in the aorta and label long-term adult HSCs, but not HSC-independent yolk-sac-derived primitive or definitive erythromyeloid (EMP) hematopoiesis. Our lineage-tracing studies indicate that HSC-derived erythroid, myeloid, and lymphoid progeny significantly expand in the liver and blood stream between E14.5 and E16.5. Additionally, we find that HSCs contribute the majority of F4/80+ macrophages in adult spleen and marrow, in contrast to their limited contribution to macrophage populations in brain, liver, and lungs. The Mds1CreERT2 mouse model will be useful to deconvolute the complexity of hematopoiesis as it unfolds in the embryo and functions postnatally.

A Positive Regulatory Feedback Loop between EKLF/KLF1 and TAL1/SCL Sustaining the Erythropoiesis.

The erythroid Krüppel-like factor EKLF/KLF1 is a hematopoietic transcription factor binding to the CACCC DNA motif and participating in the regulation of erythroid differentiation. With combined use of microarray-based gene expression profiling and the promoter-based ChIP-chip assay of E14.5 fetal liver cells from wild type (WT) and EKLF-knockout (Eklf-/-) mouse embryos, we identified the pathways and direct target genes activated or repressed by EKLF. This genome-wide study together with the molecular/cellular analysis of the mouse erythroleukemic cells (MEL) indicate that among the downstream direct target genes of EKLF is Tal1/Scl. Tal1/Scl encodes another DNA-binding hematopoietic transcription factor TAL1/SCL, known to be an Eklf activator and essential for definitive erythroid differentiation. Further identification of the authentic Tal gene promoter in combination with the in vivo genomic footprinting approach and DNA reporter assay demonstrate that EKLF activates the Tal gene through binding to a specific CACCC motif located in its promoter. These data establish the existence of a previously unknow positive regulatory feedback loop between two DNA-binding hematopoietic transcription factors, which sustains mammalian erythropoiesis.

Quantitative lineage analysis identifies a hepato-pancreato-biliary progenitor niche.

Studies based on single cells have revealed vast cellular heterogeneity in stem cell and progenitor compartments, suggesting continuous differentiation trajectories with intermixing of cells at various states of lineage commitment and notable degrees of plasticity during organogenesis1-5. The hepato-pancreato-biliary organ system relies on a small endoderm progenitor compartment that gives rise to a variety of different adult tissues, including the liver, pancreas, gall bladder and extra-hepatic bile ducts6,7. Experimental manipulation of various developmental signals in the mouse embryo has underscored important cellular plasticity in this embryonic territory6. This is reflected in the existence of human genetic syndromes as well as congenital malformations featuring multi-organ phenotypes in liver, pancreas and gall bladder6. Nevertheless, the precise lineage hierarchy and succession of events leading to the segregation of an endoderm progenitor compartment into hepatic, biliary and pancreatic structures have not yet been established. Here we combine computational modelling approaches with genetic lineage tracing to accurately reconstruct the hepato-pancreato-biliary lineage tree. We show that a multipotent progenitor subpopulation persists in the pancreato-biliary organ rudiment, contributing cells not only to the pancreas and gall bladder but also to the liver. Moreover, using single-cell RNA sequencing and functional experiments we define a specialized niche that supports this subpopulation in a multipotent state for an extended time during development. Together these findings indicate sustained plasticity underlying hepato-pancreato-biliary development that might also explain the rapid expansion of the liver while attenuating pancreato-biliary growth.

Transcription Control of Liver Development.

During liver organogenesis, cellular transcriptional profiles are constantly reshaped by the action of hepatic transcriptional regulators, including FoxA1-3, GATA4/6, HNF1α/ß, HNF4α, HNF6, OC-2, C/EBPα/ß, Hex, and Prox1. These factors are crucial for the activation of hepatic genes that, in the context of compact chromatin, cannot access their targets. The initial opening of highly condensed chromatin is executed by a special class of transcription factors known as pioneer factors. They bind and destabilize highly condensed chromatin and facilitate access to other "non-pioneer" factors. The association of target genes with pioneer and non-pioneer transcription factors takes place long before gene activation. In this way, the underlying gene regulatory regions are marked for future activation. The process is called "bookmarking", which confers transcriptional competence on target genes. Developmental bookmarking is accompanied by a dynamic maturation process, which prepares the genomic loci for stable and efficient transcription. Stable hepatic expression profiles are maintained during development and adulthood by the constant availability of the main regulators. This is achieved by a self-sustaining regulatory network that is established by complex cross-regulatory interactions between the major regulators. This network gradually grows during liver development and provides an epigenetic memory mechanism for safeguarding the optimal expression of the regulators.

Nuclear Organization during Hepatogenesis in Zebrafish Requires Uhrf1.

Acquisition of cellular fate during development is initiated and maintained by well-coordinated patterns of gene expression that are dictated by the epigenetic landscape and genome organization in the nucleus. While the epigenetic marks that mediate developmental gene expression patterns during organogenesis have been well studied, less is known about how epigenetic marks influence nuclear organization during development. This study examines the relationship between nuclear structure, chromatin accessibility, DNA methylation, and gene expression during hepatic outgrowth in zebrafish larvae. We investigate the relationship between these features using mutants that lack DNA methylation. Hepatocyte nuclear morphology was established coincident with hepatocyte differentiation at 80 h post-fertilization (hpf), and nuclear shape and size continued to change until the conclusion of outgrowth and morphogenesis at 120 hpf. Integrating ATAC-Seq analysis with DNA methylation profiling of zebrafish livers at 120 hpf showed that closed and highly methylated chromatin occupies most transposable elements and that open chromatin correlated with gene expression. DNA hypomethylation, due to mutation of genes encoding ubiquitin-like, containing PHD and RING Finger Domains 1 (uhrf1) and DNA methyltransferase (dnmt1), did not block hepatocyte differentiation, but had dramatic effects on nuclear organization. Hepatocytes in uhrf1 mutants have large, deformed nuclei with multiple nucleoli, downregulation of nucleolar genes, and a complete lack of the nuclear lamina. Loss of lamin B2 staining was phenocopied by dnmt1 mutation. Together, these data show that hepatocyte nuclear morphogenesis coincides with organ morphogenesis and outgrowth, and that DNA methylation directs chromatin organization, and, in turn, hepatocyte nuclear shape and size during liver development.

A noncoding RNA modulator potentiates phenylalanine metabolism in mice.

The functional role of long noncoding RNAs (lncRNAs) in inherited metabolic disorders, including phenylketonuria (PKU), is unknown. Here, we demonstrate that the mouse lncRNA Pair and human HULC associate with phenylalanine hydroxylase (PAH). Pair-knockout mice exhibited excessive blood phenylalanine (Phe), musty odor, hypopigmentation, growth retardation, and progressive neurological symptoms including seizures, which faithfully models human PKU. HULC depletion led to reduced PAH enzymatic activities in human induced pluripotent stem cell-differentiated hepatocytes. Mechanistically, HULC modulated the enzymatic activities of PAH by facilitating PAH-substrate and PAH-cofactor interactions. To develop a therapeutic strategy for restoring liver lncRNAs, we designed GalNAc-tagged lncRNA mimics that exhibit liver enrichment. Treatment with GalNAc-HULC mimics reduced excessive Phe in Pair -/- and Pah R408W/R408W mice and improved the Phe tolerance of these mice.