Repopulating Kupffer cells originate directly from hematopoietic stem cells.

BACKGROUND: Kupffer cells (KCs) originate from yolk-sac progenitors before birth. Throughout adulthood, they self-maintain independently from the input of circulating monocytes (MOs) at a steady state and are replenished within 2 weeks after having been depleted, but the origin of repopulating KCs in adults remains unclear. The current paradigm dictates that repopulating KCs originate from preexisting KCs or monocytes, but there remains a lack of fate-mapping evidence. METHODS: We first traced the fate of preexisting KCs and that of monocytic cells with tissue-resident macrophage-specific and monocytic cell-specific fate-mapping mouse models, respectively. Secondly, we performed genetic lineage tracing to determine the type of progenitor cells involved in response to KC-depletion in mice. Finally, we traced the fate of hematopoietic stem cells (HSCs) in an HSC-specific fate-mapping mouse model, in the context of chronic liver inflammation induced by repeated carbon tetrachloride treatment. RESULTS: By using fate-mapping mouse models, we found no evidence that repopulating KCs originate from preexisting KCs or MOs and found that in response to KC-depletion, HSCs proliferated in the bone marrow, mobilized into the blood, adoptively transferred into the liver and differentiated into KCs. Then, in the chronic liver inflammation context, we confirmed that repopulating KCs originated directly from HSCs. CONCLUSION: Taken together, these findings provided in vivo fate-mapping evidence that repopulating KCs originate directly from HSCs, which presents a completely novel understanding of the cellular origin of repopulating KCs and shedding light on the divergent roles of KCs in liver homeostasis and diseases.

Identification and validation of alternative splicing isoforms as novel biomarker candidates in hepatocellular carcinoma.

Alternative splicing (AS) is a transcriptional regulation mechanism that participates in multiple aspects of cancer. The present study aimed to identify differential AS events from tumor and non­tumor samples and investigate the potential of AS as a source of candidate cancer diagnostic biomarkers. Deep RNA sequencing of three paired hepatocellular carcinoma (HCC) tumors and adjacent non­tumors was applied to identify AS events. RT­qPCR was performed on 45 HCC clinical samples to validate the splicing differences. The maximal information coefficient was first used to build an association between clinical features and AS changes. We identified 197 significantly differential skipped exon events, of which only 29% overlapped with the differentially expressed genes. The differentially spliced genes were mainly enriched in HCC­characterized biological processes and pathways, clearly separating tumors from non­tumors. We also validated the statistically significant splicing differences of three AS candidates (CEACAM1 exon 7, VPS29 exon 2 and ISOC2 exon 3). Furthermore, a clinicopathological analysis revealed that carcinoembryonic antigen­related cell adhesion molecule 1 (CEACAM1) exon 7 was significantly correlated with the survival time, and VPS29 exon 2 was associated with cell differentiation stages. In conclusion, the findings of the three AS candidates in the present study could be beneficial in HCC prognosis and new treatment strategies.

Targeting Endothelial Erk1/2-Akt Axis as a Regeneration Strategy to Bypass Fibrosis during Chronic Liver Injury in Mice.

Liver sinusoidal endothelial cells (LSECs) have great capacity for liver regeneration, and this capacity can easily switch to profibrotic phenotype, which is still poorly understood. In this study, we elucidated a potential target in LSECs for regenerative treatment that can bypass fibrosis during chronic liver injury. Proregenerative LSECs can be transformed to profibrotic phenotype after 4 weeks of carbon tetrachloride administration or 10 days of bile duct ligation. This phenotypic alternation of LSECs was mediated by extracellular regulated protein kinases 1 and 2 (Erk1/2)-Akt axis switch in LSECs during chronic liver injury; Erk1/2 was normally associated with maintenance of the LSEC proregenerative phenotype, inhibiting hepatic stellate cell (HSC) activation and promoting tissue repair by enhancing nitric oxide (NO)/reactive oxygen species (ROS) ratio and increasing expression of hepatic growth factor (HGF) and Wingless-type MMTV integration site family member 2 (Wnt2). Alternatively, Akt induced LSEC profibrotic phenotype, which mainly stimulated HSC activation and concomitant senescence by reducing NO/ROS ratio and decreasing HGF/Wnt2 expression. LSEC-targeted adenovirus or drug particle to promote Erk1/2 activity can alleviate liver fibrosis, accelerate fibrosis resolution, and enhance liver regeneration. This study demonstrated that the Erk1/2-Akt axis acted as a switch to regulate the proregenerative and profibrotic phenotypes of LSECs, and targeted therapy promoted liver regeneration while bypassing fibrosis, providing clues for a more effective treatment of liver diseases.

Regulation of Tak1 alternative splicing by splice-switching oligonucleotides.

Alternative splicing (AS) generates multiple isoforms from a single precursor mRNA, and these isoforms usually exhibit different tissue distributions and functions. Aberrant protein isoforms can lead to abnormalities in protein function and may even result in genetic disorders or cancer. In recent years, splice-switching oligonucleotides (SSOs) have emerged as a promising therapeutic strategy for several neurological diseases, but the efficacy of this strategy in other organs is less reported. In this study, we designed and synthesized SSOs targeting the splicing regulators of exon 12 of the Tak1 gene, inducing variant switching between Tak1-A and Tak1-B. We also designed SSOs capable of knockdown both Tak1 variants by inducing the aberrant splicing of exon 4. The Vivo-morpholino SSOs showed significant splice-switching of Tak1 in mouse liver, with a persistence of at least 10 days after initial SSOs delivery. Bioinformatics analysis indicated a lipid metabolism-related function for Tak1-B but not Tak1-A. The conversion of Tak1-B to Tak1-A consistently led to significant accumulation of lipids in cultured AML12 cells, as well as the dysregulation of several lipid metabolism-related genes in mouse liver. Different functional properties of the two isoforms may explain the conflicting functions previously reported for Tak1. In conclusion, our research clarified the different functions of Tak1 isoforms, and provided an efficient strategy for the functional research of the AS isoforms.

Cell-type-resolved alternative splicing patterns in mouse liver.

Alternative splicing (AS) is an important post-transcriptional regulatory mechanism to generate transcription diversity. However, the functional roles of AS in multiple cell types from one organ have not been reported. Here, we provide the most comprehensive profile for cell-type-resolved AS patterns in mouse liver. A total of 13,637 AS events are detected, representing 81.5% of all known AS events in the database. About 46.2% of multi-exon genes undergo AS from the four cell types of mouse liver: hepatocyte, liver sinusoidal endothelial cell, Kupffer cell and hepatic stellate cell, which regulates cell-specific functions and maintains cell characteristics. We also present a cell-type-specific splicing factors network in these four cell types of mouse liver, allowing data mining and generating knowledge to elucidate the roles of splicing factors in sustaining the cell-type-specialized AS profiles and functions. The splicing switching of Tak1 gene between different cell types is firstly discovered and the specific Tak1 isoform regulates hepatic cell-type-specific functions is verified. Thus, our work constructs a hepatic cell-specific splicing landscape and reveals the considerable contribution of AS to the cell type constitution and organ features.

Discovery of NKCC1 as a potential therapeutic target to inhibit hepatocellular carcinoma cell growth and metastasis.

Metastasis is the essential cause for the high mortality of hepatocellular carcinoma (HCC). In order to investigate the mechanism of metastasis, and to discover therapeutic targets for HCC, the quantitative proteomic technique was applied to characterize the plasma membrane proteins of two HCC cell lines with low (MHCC97L) or high (MHCC97H) metastatic potentials. One of the plasma membrane proteins, sodium-potassium-chloride cotransporter 1 (NKCC1), was upregulated in MHCC97H cell line. Immunohistochemistry result in HCC patients showed that NKCC1 expression was associated with poor differentiation and microvascular invasion. Knockdown of NKCC1 via RNA interference reduced HCC cell proliferation and invasion abilities in vitro and in vivo, whereas over-expression of NKCC1 significantly increased HCC cell proliferation and invasion abilities in vitro and in vivo. Additionally, blocking NKCC1 activity with bumetanide attenuated the proliferation and invasion abilities of HCC cells in vitro and limited the HCC growth in vivo. Further results suggested that NKCC1 promotes the invasion ability via MMP-2 activity, and that the WNK1/OSR1/NKCC1 signal pathway might play roles in HCC metastasis. For the first time, our study demonstrated that NKCC1 plays a role in HCC metastasis, and could be served as a potential target to inhibit HCC cell growth and metastasis.

Proteomic Analysis Reveals Dab2 Mediated Receptor Endocytosis Promotes Liver Sinusoidal Endothelial Cell Dedifferentiation.

Sinusoidal dedifferentiation is a complicated process induced by several factors, and exists in early stage of diverse liver diseases. The mechanism of sinusoidal dedifferentiation is poorly unknown. In this study, we established a NaAsO2-induced sinusoidal dedifferentiation mice model. Liver sinusoidal endothelial cells were isolated and isobaric tag for relative and absolute quantitation (iTRAQ) based proteomic approach was adopted to globally examine the effects of arsenic on liver sinusoidal endothelial cells (LSECs) during the progression of sinusoidal dedifferentiation. In all, 4205 proteins were identified and quantified by iTRAQ combined with LC-MS/MS analysis, of which 310 proteins were significantly changed in NaAsO2 group, compared with the normal control. Validation by western blot showed increased level of clathrin-associated sorting protein Disabled 2 (Dab2) in NaAsO2 group, indicating that it may regulate receptor endocytosis, which served as a mechanism to augment intracellular VEGF signaling. Moreover, we found that knockdown of Dab2 reduced the uptake of VEGF in LSECs, furthermore blocking VEGF-mediated LSEC dedifferentiation and angiogenesis.

Quantitative Proteomic analysis on Activated Hepatic Stellate Cells reversion Reveal STAT1 as a key regulator between Liver Fibrosis and recovery.

Understanding the changes of activated HSCs reversion is an essential step toward clarifying the potential roles of HSCs in the treatment of liver fibrosis. In this study, we chose adipocyte differentiation mixture to induce LX-2 cells for 2 days in vitro as reversion phase, comparing with normal cultured LX-2 cells as activation phase. Mass spectrometric-based SILAC technology was adopted to study differentially expressed proteome of LX-2 cells between reversion and activation. Compared with activated HSCs, 273 proteins showed significant differences in reverted HSCs. The main pathway of up-regulated proteins associated with reversion of HSCs mainly related to oxidation-reduction and lipid metabolism, while the top pathway of down-regulated proteins was found in regulated cytoskeleton formation. Changes in the expression levels of selected proteins were verified by Western blotting analysis, especially STAT1, FLNA, LASP1, and NAMPT proteins. The distinct roles of STAT1 were further analyzed between activated and reverted of HSCs, it was found that STAT1 could affect cell proliferation of HSCs and could be viewed as a key regulator in the reversion of HSCs. Thus, the proteomic analysis could accelerate our understanding of the mechanisms of HSC reversion on cessation of fibrogenic stimuli and provide new targets for antifibrotic liver therapy.

Quantitative Proteomics Analysis of Tissue Interstitial Fluid for Identification of Novel Serum Candidate Diagnostic Marker for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common malignant cancer in the world. The sensitivity of alpha-fetoprotein (AFP) is still inadequate for HCC diagnosis. Tissue interstitial fluid (TIF), as the liquid microenvironment of cancer cells, was used for biomarker discovery in this study. Paired tumor and nontumor TIF samples from 6 HBV-HCC patients were analyzed by a proteomic technique named iTRAQ (isobaric tag for relative and absolute quantitation). Totally, 241 up-regulated proteins (ratio ≥ 1.3, p < 0.05) and 288 down-regulated proteins (ratio ≤ -1.3, p < 0.05) in tumor TIF were identified. Interestingly, proteins in S100 family were found remarkably up-regulated in tumor TIF. One dramatically up-regulated protein S100A9 (ratio = 19) was further validated by ELISA in sera from liver cirrhosis (LC, HCC high risk population) and HCC patients (n = 47 for each group). The level of this protein was significantly elevated in HCC sera compared with LC (p < 0.0001). The area under the curve of this protein to distinguish HCC from LC was 0.83, with sensitivity of 91% (higher than AFP) and specificity of 66%. This result demonstrated the potential of S100A9 as a candidate HCC diagnostic biomarker. And TIF was a kind of promising material to identify candidate tumor biomarkers that could be detected in serum.

SILAC-based quantitative proteomic analysis of secretome between activated and reverted hepatic stellate cells.

Activated hepatic stellate cell (HSC) is the main myofibroblast cell in the liver fibrosis (LF). An important characteristic of the recovery of LF is not only the apoptosis of activated HSCs but also reversal of myofibroblast-like phenotype to a quiescent-like phenotype. Understanding the changes of secreted proteins in the reversion of activated HSCs may provide the broader view of cellular regulatory networks and discover candidate markers or targets for therapeutic strategies of LF. In this study, stable isotope labeling with amino acids (SILAC) combined with linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FT MS) was performed on in vitro activated HSCs and reverted HSCs to obtain a proteomic view of secretory proteins. In total, 330 proteins showed significant differences in reverted HSCs. Among these, 109 upregulated proteins were mainly involved in amino acid metabolism pathway and glucose metabolism pathway using GeneGO/MetaCore software, while 221 downregulated proteins are closely associated with HSCs activation, such as cytoskeleton remodeling, chemokines, and cell adhesion. Additionally, a set of novel proteins associated with HSCs activation and reversion were validated by Western blotting in the cell secretion and in the sera of LF, including vitronectin, laminin beta 1, and ubiquitin conjugation factor E4B. Our study provided the valuable insight into the mechanisms in the reversion of activated HSCs and identified some potential biomarkers of LF in clinical studies. All MS data have been deposited in the ProteomeXchange with identifier PXD000773 (http://proteomecentral.proteomexchange.org/dataset/PXD000773).

Discovery of novel genes and gene isoforms by integrating transcriptomic and proteomic profiling from mouse liver.

Comprehensively identifying gene expression in both transcriptomic and proteomic levels of one tissue is a prerequisite for a deeper understanding of its biological functions. Alternative splicing and RNA editing, two main forms of transcriptional processing, play important roles in transcriptome and proteome diversity and result in multiple isoforms for one gene, which are hard to identify by mass spectrometry (MS)-based proteomics approach due to the relative lack of isoform information in standard protein databases. In our study, we employed MS and RNA-Seq in parallel into mouse liver tissue and captured a considerable catalogue of both transcripts and proteins that, respectively, covered 60 and 34% of protein-coding genes in Ensembl. We then developed a bioinformatics workflow for building a customized protein database that for the first time included new splicing-derived peptides and RNA-editing-caused peptide variants, allowing us to more completely identify protein isoforms. Using this experimentally determined database, we totally identified 150 peptides not present in standard biological databases at false discovery rate of <1%, corresponding to 72 novel splicing isoforms, 43 new genetic regions, and 15 RNA-editing sites. Of these, 11 randomly selected novel events passed experimental verification by PCR and Sanger sequencing. New discoveries of gene products with high confidence in two omics levels demonstrated the robustness and effectiveness of our approach and its potential application into improve genome annotation. All the MS data have been deposited to the iProx ( http://ww.iprox.org ) with the identifier IPX00003601.

A new insight into the impact of different proteases on SILAC quantitative proteome of the mouse liver.

In this study, we examined the use of multiple proteases (trypsin, LysC, tandem LysC/trypsin) on both protein identification and quantification in the Lys-labeled SILAC mouse liver. Our results show that trypsin and tandem LysC/trypsin digestion are superior to LysC in peptides and protein identification while LysC shows advantages in quantification of Lys-labeled proteins. Combination of experimental results from different proteases (LysC and trypsin) enabled a significant increase in the number of identified protein and protein can be quantified. Thus, taking advantage of the complementation of different protease should be a good strategy to improve both qualitative and quantitative proteomics research.

Identification of KAP-1-associated complexes negatively regulating the Ey and ß-major globin genes in the ß-globin locus.

Deregulations of erythroid differentiation may lead to erythroleukemia and other hemoglobinopathies, yet the molecular mechanisms underlying these events are not fully understood. Here, we found that KAP-1-associated complexes contribute to the regulation of the ß-globin locus, the key events of erythroid differentiation. We show that RNAi-mediated knockdown of KAP-1 in mouse erythroleukemia (MEL) cells increases expression of the Ey and ß-major globin genes during hexamethylenebisacetamide (HMBA) induced differentiation process. This indicates that at least part of KAP-1-associated complexes negatively regulates ß-globin gene expression during definitive erythroid differentiation. ChIP-PCR analysis revealed that one or more KAP-1-associated complexes are targeted to the promoter region of the Ey and beta-major globin genes. Since KAP-1 is only a scaffold molecule, there must be some transcriptional regulators allowing its targeted recruitment to the ß-globin locus. To further discover these novel regulators, proteins interacting with KAP-1 were isolated by endogenous immunoprecipitation and identified by LC-ESI-MS/MS. Among the proteins identified, MafK and Zfp445 were studied further. We found that KAP-1 may contribute to the repression of Ey and ß-major globin gene transcription through recruitment to the promoters of these two genes, mediated by the interaction of KAP-1 with either Zfp445 or MafK, respectively.

General trends in the utilization of structural factors contributing to biological complexity.

During evolution, proteins containing newly emerged domains and the increasing proportion of multidomain proteins in the full genome-encoded proteome (GEP) have substantially contributed to increasing biological complexity. However, it is not known how these two potential structural factors are preferentially utilized at given physiological states. Here, we classified proteins according to domain number and domain age and explored the general trends across species for the utilization of proteins from GEP to various certain-state proteomes (CSPs, i.e., all the proteins expressed at certain physiological states). We found that multidomain proteins or only older domain-containing proteins are significantly overrepresented in CSPs compared with GEP, which is a trend that is stronger in multicellular organisms than in unicellular organisms. Interestingly, the strengths of overrepresentation decreased during evolution of multicellular eukaryotes. When comparing across CSPs, we found that multidomain proteins are more overrepresented in complex tissues than in simpler ones, whereas no difference among proteins with domains of different ages is evident between complex and simple tissues. Thus, biological complexity under certain conditions is more significantly realized by diverse domain organization than by the emergence of new types of domain. In addition, we found that multidomain or only older domain-containing proteins tend to evolve slowly and generally are under stronger purifying selection, which may partly result from their general overrepresentation trends in CSPs.

Sample preparation method for isolation of single-cell types from mouse liver for proteomic studies.

It becomes increasingly clear that separation of pure cell populations provides a uniquely sensitive and accurate approach to protein profiling in biological systems and opens up a new area for proteomic analysis. The method we described could simultaneously isolate population of hepatocytes (HCs), hepatic stellate cells (HSCs), Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) by a combination of collagenase-based density gradient centrifugation and magnetic activated cell sorting with high purity and yield for the first time. More than 98% of the isolated HCs were positive for cytokeratin 18, with a viability of 91%. Approximately 97% of the isolated HSCs expressed glial fibrillary acidic protein with a viability of 95%. Nearly 98% of isolated KCs expressed F4/80 with a viability of 94%. And the purity of LSECs reached up to 91% with a viability of 94%. And yield for HCs, HSCs, LSECs and KCs were 6.3, 1.3, 2.6 and 5.0 million per mouse. This systematic isolation method enables us to study the proteome profiling of different types of liver cells with high purity and yield, which is especially useful for sample preparation of Human Liver Proteome Project.

A proton nuclear magnetic resonance metabonomics approach for biomarker discovery in nonalcoholic fatty liver disease.

This study was undertaken to discover novel biomarkers for the noninvasive early diagnosis of nonalcoholic fatty liver disease (NAFLD). A methionine and choline deficient (MCD) diet was used to represent different stages of NAFLD in male C57BL/6 mice. (1)H NMR spectroscopy and principal components analysis (PCA) were used to investigate the time-related biochemical changes in mice sera induced by the MCD diet. Many serum metabolites' concentrations changed between control and MCD-fed mice. Hierarchical cluster analysis (HCA) and artificial neural networks (ANNs) were used to select the least number of metabolites to be used for the noninvasive diagnosis of various stages of NAFLD; four potential biomarkers, serum glucose, lactate, glutamate/glutamine, and taurine were selected. To verify the diagnostic accuracy of these selected metabolites, their serum concentrations were measured in healthy controls (n = 28), NAFLD patients with steatosis (n = 15), steatosis patients with necro-inflammatory disease (n = 11), and NASH patients (n = 6). On the basis of results from MCD-fed mice model, clinical tests, and previous reports, we propose using the levels of the four metabolites for diagnosing NAFLD at various stages. Furthermore, the probability of developing NAFLD at a particular stage was assessed by multinomial logistic regression (MLR) based on the clinical results of the four serum metabolites.

Plasma biomarker screening for liver fibrosis with the N-terminal isotope tagging strategy.

A non-invasive diagnostic approach is crucial for the evaluation of severity of liver disease, treatment decisions, and assessing drug efficacy. This study evaluated plasma proteomic profiling via an N-terminal isotope tagging strategy coupled with liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry measurement to detect liver fibrosis staging. Pooled plasma from different liver fibrosis stages, which were assessed in advance by the current gold-standard of liver biopsy, was quantitatively analyzed. A total of 72 plasma proteins were found to be dysregulated during the fibrogenesis process, and this finding constituted a valuable candidate plasma biomarker bank for follow-up analysis. Validation results of fibronectin by Western blotting reconfirmed the mass-based data. Ingenuity Pathways Analysis showed four types of metabolic networks for the functional effect of liver fibrosis disease in chronic hepatitis B patients. Consequently, quantitative proteomics via the N-terminal acetyl isotope labeling technique provides an effective and useful tool for screening plasma candidate biomarkers for liver fibrosis. We quantitatively monitored the fibrogenesis process in CHB patients. We discovered many new valuable candidate biomarkers for the diagnosis of liver fibrosis and also partly identified the mechanism involved in liver fibrosis disease. These results provide a clearer understanding of liver fibrosis pathophysiology and will also hopefully lead to improvement of clinical diagnosis and treatment.

Quantitative proteomic survey of endoplasmic reticulum in mouse liver.

To gain a better understanding of the critical function of the endoplasmic reticulum (ER) in liver, we carried out a proteomic survey of mouse liver ER. The ER proteome was profiled with a new three-dimensional, gel-based strategy. From 6152 and 6935 MS spectra, 903 and 1042 proteins were identified with at least two peptides matches at 95% confidence in the rough (r) and smooth (s) ER, respectively. Comparison of the rER and sER proteomes showed that calcium-binding proteins are significantly enriched in the sER suggesting that the ion-binding function of the ER is compartmentalized. Comparison of the rat and mouse ER proteomes showed that 662 proteins were common to both, comprising 53.5% and 49.3% of those proteomes, respectively. We proposed that these proteins were stably expressed proteins that were essential for the maintenance of ER function. GO annotation with a hypergeometric model proved this hypothesis. Unexpectedly, 210 unknown proteins and some proteins previously reported to occur in the cytosol were highly enriched in the ER. This study provides a reference map for the ER proteome of liver. Identification of new ER proteins will enhance our current understanding of the ER and also suggest new functions for this organelle.

Characterization of the liver tissue interstitial fluid (TIF) proteome indicates potential for application in liver disease biomarker discovery.

Tissue interstitial fluid (TIF) forms the interface between circulating body fluids and intracellular fluid. Pathological alterations of liver cells could be reflected in TIF, making it a promising source of liver disease biomarkers. Mouse liver TIF was extracted, separated by SDS-PAGE, analyzed by linear ion trap mass spectrometer, and 1450 proteins were identified. These proteins may be secreted, shed from membrane vesicles, or represent cellular breakdown products. They show different profiling patterns, quantities, and possibly modification/cleavage of intracellular proteins. The high solubility and even distribution of liver TIF supports its suitability for proteome analysis. Comparison of mouse liver TIF data with liver tissue and plasma proteome data identified major proteins that might be released from liver to plasma and serve as blood biomarkers of liver origin. This result was partially supported by comparison of human liver TIF data with human liver and plasma proteome data. Paired TIFs from tumor and nontumor liver tissues of a hepatocellular carcinoma patient were analyzed and the profile of subtracted differential proteins supports the potential for biomarker discovery in TIF. This study is the first analysis of the liver TIF proteome and provides a foundation for further application of TIF in liver disease biomarker discovery.

Relationships between hematopoiesis and hepatogenesis in the midtrimester fetal liver characterized by dynamic transcriptomic and proteomic profiles.

In fetal hematopoietic organs, the switch from hematopoiesis is hypothesized to be a critical time point for organogenesis, but it is not yet evidenced. The transient coexistence of hematopoiesis will be useful to understand the development of fetal liver (FL) around this time and its relationship to hematopoiesis. Here, the temporal and the comparative transcriptomic and proteomic profiles were observed during the critical time points corresponding to the initiation (E11.5), peak (E14.5), recession (E15.5), and disappearance (3 ddp) of mouse FL hematopoiesis. We found that E11.5-E14.5 corresponds to a FL hematopoietic expansion phase with distinct molecular features, including the expression of new transcription factors, many of which are novel KRAB (Kruppel-associated box)-containing zinc finger proteins. This time period is also characterized by extensive depression of some liver functions, especially catabolism/utilization, immune and defense, classical complement cascades, and intrinsic blood coagulation. Instead, the other liver functions increased, such as xenobiotic and sterol metabolism, synthesis of carbohydrate and glycan, the alternate and lectin complement cascades and extrinsic blood coagulation, and etc. Strikingly, all of the liver functions were significantly increased at E14.5-E15.5 and thereafter, and the depression of the key pathways attributes to build the hematopoietic microenvironment. These findings signal hematopoiesis emigration is the key to open the door of liver maturation.