Molecular regulation of mammalian hepatic architecture.

The essential liver exocrine and endocrine functions require a precise spatial arrangement of the hepatic lobule consisting of the central vein, portal vein, hepatic artery, intrahepatic bile duct system, and hepatocyte zonation. This allows blood to be carried through the liver parenchyma sampled by all hepatocytes and bile produced by the hepatocytes to be carried out of the liver through the intrahepatic bile duct system composed of cholangiocytes. The molecular orchestration of multiple signaling pathways and epigenetic factors is required to set up lineage restriction of the bipotential hepatoblast progenitor into the hepatocyte and cholangiocyte cell lineages, and to further refine cell fate heterogeneity within each cell lineage reflected in the functional heterogeneity of hepatocytes and cholangiocytes. In addition to the complex molecular regulation, there is a complicated morphogenetic choreography observed in building the refined hepatic epithelial architecture. Given the multifaceted molecular and cellular regulation, it is not surprising that impairment of any of these processes can result in acute and chronic hepatobiliary diseases. To enlighten the development of potential molecular and cellular targets for therapeutic options, an understanding of how the intricate hepatic molecular and cellular interactions are regulated is imperative. Here, we review the signaling pathways and epigenetic factors regulating hepatic cell lineages, fates, and epithelial architecture.

The mechanistic basis for chromatin regulation by pioneer transcription factors.

Pioneer transcription factors play a primary role in establishing competence for gene expression and initiating cellular programming and reprogramming, and their dysregulation causes severe effects on human health, such as promoting tumorigenesis. Although more than 200 transcription factors are expressed in each cell type, only a small number of transcription factors are necessary to elicit dramatic cell-fate changes in embryonic development and cell-fate conversion. Among these key transcription factors, a subset called "pioneer transcription factors" have a remarkable ability to target nucleosomal DNA, or closed chromatin, early in development, often leading to the local opening of chromatin, thereby establishing competence for gene expression. Although more key transcription factors have been identified as pioneer transcription factors, the molecular mechanisms behind their special properties are only beginning to be revealed. Understanding the pioneering mechanisms will enhance our ability to precisely control cell fate at will for research and therapeutic purposes. This article is categorized under: Biological Mechanisms > Cell Fates Biological Mechanisms > Regulatory Biology Developmental Biology > Lineages.

Enhancer Analyses Using Chicken Embryo Electroporation.

Chicken embryo electroporation is a powerful tool used to identify and analyze enhancers involved in developmental gene regulation. In this chapter, the basic procedures and underlying principles of enhancer analysis using chicken embryo electroporation are described in the following steps: (1) identification of enhancers in a wide genomic region, (2) determination of the full enhancer region, (3) definition of the core enhancer regions, and (4) analysis of a functional transcription factor binding sequences in the core region.

Chromatin Scanning by Dynamic Binding of Pioneer Factors.

Pioneer factors such as FoxA target nucleosomal DNA and initiate cooperative interactions at silent genes during development, cellular reprogramming, and steroid hormone induction. Biophysical studies previously showed that the nuclear mobility of FoxA1 is slower than for many other transcription factors, whereas a new single molecule study (Swinstead et al., 2016, Cell) shows comparable chromatin residence times for FoxA1 and steroid receptors. Despite that steroid receptors engage nucleosome-remodeling complexes, the vast majority of co-bound sites with FoxA are dependent upon FoxA, not vice versa. Taken together, the distinguishing feature of pioneer factors remains nucleosomal access rather than an exceptional residence time in chromatin.

Cell fate control by pioneer transcription factors.

Distinct combinations of transcription factors are necessary to elicit cell fate changes in embryonic development. Yet within each group of fate-changing transcription factors, a subset called 'pioneer factors' are dominant in their ability to engage silent, unmarked chromatin and initiate the recruitment of other factors, thereby imparting new function to regulatory DNA sequences. Recent studies have shown that pioneer factors are also crucial for cellular reprogramming and that they are implicated in the marked changes in gene regulatory networks that occur in various cancers. Here, we provide an overview of the contexts in which pioneer factors function, how they can target silent genes, and their limitations at regions of heterochromatin. Understanding how pioneer factors regulate gene expression greatly enhances our understanding of how specific developmental lineages are established as well as how cell fates can be manipulated.

The Pioneer Transcription Factor FoxA Maintains an Accessible Nucleosome Configuration at Enhancers for Tissue-Specific Gene Activation.

Nuclear DNA wraps around core histones to form nucleosomes, which restricts the binding of transcription factors to gene regulatory sequences. Pioneer transcription factors can bind DNA sites on nucleosomes and initiate gene regulatory events, often leading to the local opening of chromatin. However, the nucleosomal configuration of open chromatin and the basis for its regulation is unclear. We combined low and high levels of micrococcal nuclease (MNase) digestion along with core histone mapping to assess the nucleosomal configuration at enhancers and promoters in mouse liver. We find that MNase-accessible nucleosomes, bound by transcription factors, are retained more at liver-specific enhancers than at promoters and ubiquitous enhancers. The pioneer factor FoxA displaces linker histone H1, thereby keeping enhancer nucleosomes accessible in chromatin and allowing other liver-specific transcription factors to bind and stimulate transcription. Thus, nucleosomes are not exclusively repressive to gene regulation when they are retained with, and exposed by, pioneer factors.

Pioneer transcription factors in cell reprogramming.

A subset of eukaryotic transcription factors possesses the remarkable ability to reprogram one type of cell into another. The transcription factors that reprogram cell fate are invariably those that are crucial for the initial cell programming in embryonic development. To elicit cell programming or reprogramming, transcription factors must be able to engage genes that are developmentally silenced and inappropriate for expression in the original cell. Developmentally silenced genes are typically embedded in "closed" chromatin that is covered by nucleosomes and not hypersensitive to nuclease probes such as DNase I. Biochemical and genomic studies have shown that transcription factors with the highest reprogramming activity often have the special ability to engage their target sites on nucleosomal DNA, thus behaving as "pioneer factors" to initiate events in closed chromatin. Other reprogramming factors appear dependent on pioneer factors for engaging nucleosomes and closed chromatin. However, certain genomic domains in which nucleosomes are occluded by higher-order chromatin structures, such as in heterochromatin, are resistant to pioneer factor binding. Understanding the means by which pioneer factors can engage closed chromatin and how heterochromatin can prevent such binding promises to advance our ability to reprogram cell fates at will and is the topic of this review.

Transcriptional regulatory networks in epiblast cells and during anterior neural plate development as modeled in epiblast stem cells.

Somatic development initiates from the epiblast in post-implantation mammalian embryos. Recent establishment of epiblast stem cell (EpiSC) lines has opened up new avenues of investigation of the mechanisms that regulate the epiblast state and initiate lineage-specific somatic development. Here, we investigated the role of cell-intrinsic core transcriptional regulation in the epiblast and during derivation of the anterior neural plate (ANP) using a mouse EpiSC model. Cells that developed from EpiSCs in one day in the absence of extrinsic signals were found to represent the ANP of ~E7.5 embryos. We focused on transcription factors that are uniformly expressed in the E6.5 epiblast but in a localized fashion within or external to the ANP at E7.5, as these are likely to regulate the epiblast state and ANP development depending on their balance. Analyses of the effects of knockdown and overexpression of these factors in EpiSCs on the levels of downstream transcription factors identified the following regulatory functions: cross-regulation among Zic, Otx2, Sox2 and Pou factors stabilizes the epiblastic state; Zic, Otx2 and Pou factors in combination repress mesodermal development; Zic and Sox2 factors repress endodermal development; and Otx2 represses posterior neural plate development. All of these factors variably activate genes responsible for neural plate development. The direct interaction of these factors with enhancers of Otx2, Hesx1 and Sox2 genes was demonstrated. Thus, a combination of regulatory processes that suppresses non-ANP lineages and promotes neural plate development determines the ANP.

B1 and B2 Sox gene expression during neural plate development in chicken and mouse embryos: universal versus species-dependent features.

Cumulative evidence now indicates pivotal roles for the group B1 Sox genes, Sox1, Sox2 and Sox3 in the genesis and development of neural primordia. Shared functions for the Sox1, Sox2 and Sox3 protein products have also been indicated. This emphasizes the importance and integral role of the group B1 Sox genes in regulating the neural primordia. We here review what is currently known about the expression patterns of both the group B1 Sox genes and the related group B2 Sox21 gene during the embryonic stages when the neural plate develops. These expression profiles are compared between the chicken and mouse embryos, both representatives of amniote species. This comparison indicates a gross conservation of the regulation of individual Sox genes, yet also demonstrates the existence of species-dependent variations, which should be taken into account when data from different species are being compared. To link the expression patterns and transcriptional regulation of these genes, contribution of gene-specific enhancers are discussed. The regulation of B1 Sox genes in the axial stem cells, the common precursors to the posterior neural plate and paraxial mesoderm and located at the posterior end of developing neural plate, is also highlighted in this review. This article thus provides a guide to performing readouts of B1/B2 Sox expression data during neural plate development in amniotes.

The Pou5f1/Pou3f-dependent but SoxB-independent regulation of conserved enhancer N2 initiates Sox2 expression during epiblast to neural plate stages in vertebrates.

The transcription factor Sox2 is a core component of the pluripotency control circuits in the early embryo, and later controls many aspects of neural development. Here, we demonstrate that Sox2 expression in the epiblast (mouse blastoderm) and anterior neural plate (ANP) is determined by the upstream enhancer N2. The mouse enhancer N2 exhibits strong activity in mouse ES cells, epiblast and ANP, and is regulated correctly in chicken and zebrafish embryos. Targeted deletion of this enhancer in mouse embryos caused a large reduction of Sox2 expression to 10% of that of wild-type levels in epiblast and ANP. However, this was tolerated by mouse embryo, probably due to functional compensation by Sox3. The activity of enhancer N2 depends on phylogenetically conserved bipartite POU factor-binding motifs in a 73-bp core sequence that function synergistically, but this activation does not involve Sox2. The major POU factor expressed at the epiblastic stage is Pou5f1 (Oct3/4), while those in the anterior neural plate are Pou3f factors (Oct6, Brn2 etc.). These factors are gradually exchanged during the transition from epiblast to ANP stages in mouse embryos and epiblast stem cells (EpiSC). Consistently, enhancer N2 activity changes from full Pou5f1 dependence to Pou3f dependence during the development of neural plate cells (NPC) from EpiSC, as assessed by specific POU factor knockdown in these cells. Zebrafish mutant embryos completely devoid of Pou5f1 activity failed to activate enhancer N2 and to express Sox2 in the blastoderm and ANP, and these defects were rescued by exogenous supply of pou5f1. Previously, Pou5f1-Sox2 synergism-dependent Sox2 activation through enhancer SRR2 in ES cells has been highlighted, but this mechanism is limited to ES cells and amniotes. In contrast, the enhancer N2-mediated, POU factor-dependent activation of Sox2, without involvement of Sox2, is a phylogenetically conserved core mechanism that functions in gene regulatory networks at early embryonic stages.