Exploring the potential of in vivo reprogramming for studying embryonic development, tissue regeneration, and organismal aging.

Forced expression of a specific set of transcription factors can reprogram terminally differentiated cells and convert them into induced pluripotent stem cells that correspond to cells in the inner cell mass of the developing embryo. It is now recognized that the scope of the reprogramming factors extends far beyond the stem cell biology. Studies using mouse models demonstrated that the induction of the reprogramming factors promotes cellular reprogramming in vivo. Closer inspection of these mice has revealed that expression of the reprogramming factors results in unique consequences that are not seen when cells are reprogrammed ex vivo, and can provide insights into development, tissue regeneration, cancer, and aging.

Function of bidirectional sensitivity in the otolith organs established by transcription factor Emx2.

Otolith organs of the inner ear are innervated by two parallel afferent projections to the brainstem and cerebellum. These innervations were proposed to segregate across the line of polarity reversal (LPR) within each otolith organ, which divides the organ into two regions of hair cells (HC) with opposite stereociliary orientation. The relationship and functional significance of these anatomical features are not known. Here, we show regional expression of Emx2 in otolith organs, which establishes LPR, mediates the neuronal segregation across LPR and constitutes the bidirectional sensitivity function. Conditional knockout (cKO) of Emx2 in HCs lacks LPR. Tmie cKO, in which mechanotransduction was abolished selectively in HCs within the Emx2 expression domain also lacks bidirectional sensitivity. Analyses of both mutants indicate that LPR is specifically required for mice to swim comfortably and to traverse a balance beam efficiently, but LPR is not required for mice to stay on a rotating rod.

The oncogene-dependent resistance to reprogramming unveils cancer therapeutic targets.

The resistance to transcription factor-mediated reprogramming into pluripotent stem cells is one of the distinctive features of cancer cells. Here we dissect the profiles of reprogramming factor binding and the subsequent transcriptional response in cancer cells to reveal its underlying mechanisms. Using clear cell sarcomas (CCSs), we show that the driver oncogene EWS/ATF1 misdirects the reprogramming factors to cancer-specific enhancers and thereby impairs the transcriptional response toward pluripotency that is otherwise provoked. Sensitization to the reprogramming cue is observed in other cancer types when the corresponding oncogenic signals are pharmacologically inhibited. Exploiting this oncogene dependence of the transcriptional "stiffness," we identify mTOR signaling pathways downstream of EWS/ATF1 and discover that inhibiting mTOR activity substantially attenuates the propagation of CCS cells in vitro and in vivo. Our results demonstrate that the early transcriptional response to cell fate perturbations can be a faithful readout to identify effective therapeutics targets in cancer cells.

MYCL-mediated reprogramming expands pancreatic insulin-producing cells.

ß cells have a limited capacity for regeneration, which predisposes towards diabetes. Here, we show that, of the MYC family members, Mycl plays a key role in proliferation of pancreatic endocrine cells. Genetic ablation of Mycl causes a reduction in the proliferation of pancreatic endocrine cells in neonatal mice. By contrast, the expression of Mycl in adult mice stimulates the proliferation of ß and α cells, and the cells persist after withdrawal of Mycl expression. A subset of the expanded α cells give rise to insulin-producing cells after this withdrawal. Transient Mycl expression in vivo is sufficient to normalize the hyperglycaemia of diabetic mice. In vitro expression of Mycl similarly provokes active replication in islet cells, even in those from aged mice. Finally, we show that MYCL stimulates the division of human adult cadaveric islet cells. Our results demonstrate that the induction of Mycl alone expands the functional ß-cell population, which may provide a regenerative strategy for ß cells.

Generation of mice for evaluating endogenous p16Ink4a protein expression.

The cyclin-dependent kinase inhibitor p16Ink4a plays a central role in cellular senescence in vitro. Although previous studies suggested cellular senescence is integrated in the systemic mechanisms of organismal aging, the localization and the dynamics of p16Ink4a in tissues remain poorly understood, which hinders uncovering the role of p16Ink4a under the in vivo context. One of the reasons is due to the lack of reliable reagents; as we also demonstrate here that commonly used antibodies raised against human p16INK4A barely recognize its murine ortholog. Here we generated a mouse model, in which the endogenous p16Ink4a is HA-tagged at its N-terminus, to explore the protein expression of p16Ink4a at the organismal level. p16Ink4a was induced at the protein level along the course of senescence in primary embryonic fibroblasts derived from the mice, consistently to its transcriptional level. Remarkably, however, p16Ink4a was not detected in the tissues of the mice exposed to pro-senescence conditions including genotoxic stress and activation of oncogenic signaling pathways, indicating that there is only subtle p16Ink4a proteins induced. These results in our mouse model highlight the need for caution in evaluating p16Ink4a protein expression in vivo.

DMRT1-mediated reprogramming drives development of cancer resembling human germ cell tumors with features of totipotency.

In vivo reprogramming provokes a wide range of cell fate conversion. Here, we discover that in vivo induction of higher levels of OSKM in mouse somatic cells leads to increased expression of primordial germ cell (PGC)-related genes and provokes genome-wide erasure of genomic imprinting, which takes place exclusively in PGCs. Moreover, the in vivo OSKM reprogramming results in development of cancer that resembles human germ cell tumors. Like a subgroup of germ cell tumors, propagated tumor cells can differentiate into trophoblasts. Moreover, these tumor cells give rise to induced pluripotent stem cells (iPSCs) with expanded differentiation potential into trophoblasts. Remarkably, the tumor-derived iPSCs are able to contribute to non-neoplastic somatic cells in adult mice. Mechanistically, DMRT1, which is expressed in PGCs, drives the reprogramming and propagation of the tumor cells in vivo. Furthermore, the DMRT1-related epigenetic landscape is associated with trophoblast competence of the reprogrammed cells and provides a therapeutic target for germ cell tumors. These results reveal an unappreciated route for somatic cell reprogramming and underscore the impact of reprogramming in development of germ cell tumors.

NF-κB dynamics determine the stimulus specificity of epigenomic reprogramming in macrophages.

The epigenome of macrophages can be reprogrammed by extracellular cues, but the extent to which different stimuli achieve this is unclear. Nuclear factor κB (NF-κB) is a transcription factor that is activated by all pathogen-associated stimuli and can reprogram the epigenome by activating latent enhancers. However, we show that NF-κB does so only in response to a subset of stimuli. This stimulus specificity depends on the temporal dynamics of NF-κB activity, in particular whether it is oscillatory or non-oscillatory. Non-oscillatory NF-κB opens chromatin by sustained disruption of nucleosomal histone-DNA interactions, enabling activation of latent enhancers that modulate expression of immune response genes. Thus, temporal dynamics can determine a transcription factor's capacity to reprogram the epigenome in a stimulus-specific manner.

Emx2 regulates hair cell rearrangement but not positional identity within neuromasts.

Each hair cell (HC) precursor of zebrafish neuromasts divides to form two daughter HCs of opposite hair bundle orientations. Previously, we showed that transcription factor Emx2, expressed in only one of the daughter HCs, generates this bidirectional HC pattern (Jiang et al., 2017). Here, we asked whether Emx2 mediates this effect by changing location of hair bundle establishment or positions of HCs since daughter HCs are known to switch positions with each other. We showed this HC rearrangement, redefined as two processes named Rock and Roll, is required for positional acquisition of HCs. Apical protrusion formation of nascent HCs and planar polarity signaling are both important for the Rock and Roll. Emx2 facilitates Rock and Roll by delaying apical protrusion of its nascent HCs but it does not determine HCs' ultimate positions, indicating that Emx2 mediates bidirectional HC pattern by changing the location where hair bundle is established in HCs.

RNA-binding protein Ptbp1 regulates alternative splicing and transcriptome in spermatogonia and maintains spermatogenesis in concert with Nanos3.

PTBP1, a well-conserved RNA-binding protein, regulates cellular development by tuning posttranscriptional mRNA modification such as alternative splicing (AS) or mRNA stabilization. We previously revealed that the loss of Ptbp1 in spermatogonia causes the dysregulation of spermatogenesis, but the molecular mechanisms by which PTBP1 regulates spermatogonium homeostasis are unclear. In this study, changes of AS or transcriptome in Ptbp1-knockout (KO) germline stem cells (GSC), an in vitro model of proliferating spermatogonia, was determined by next generation sequencing. We identified more than 200 differentially expressed genes, as well as 85 genes with altered AS due to the loss of PTBP1. Surprisingly, no differentially expressed genes overlapped with different AS genes in Ptbp1-KO GSC. In addition, we observed that the mRNA expression of Nanos3, an essential gene for normal spermatogenesis, was significantly decreased in Ptbp1-KO spermatogonia. We also revealed that PTBP1 protein binds to Nanos3 mRNA in spermatogonia. Furthermore, Nanos3+/-;Ptbp1+/- mice exhibited abnormal spermatogenesis, which resembled the effects of germ cell-specific Ptbp1 KO, whereas no significant abnormality was observed in mice heterozygous for either gene alone. These data implied that PTBP1 regulates alternative splicing and transcriptome in spermatogonia under different molecular pathways, and contributes spermatogenesis, at least in part, in concert with NANOS3.

Identification of distinct loci for de novo DNA methylation by DNMT3A and DNMT3B during mammalian development.

De novo establishment of DNA methylation is accomplished by DNMT3A and DNMT3B. Here, we analyze de novo DNA methylation in mouse embryonic fibroblasts (2i-MEFs) derived from DNA-hypomethylated 2i/L ES cells with genetic ablation of Dnmt3a or Dnmt3b. We identify 355 and 333 uniquely unmethylated genes in Dnmt3a and Dnmt3b knockout (KO) 2i-MEFs, respectively. We find that Dnmt3a is exclusively required for de novo methylation at both TSS regions and gene bodies of Polycomb group (PcG) target developmental genes, while Dnmt3b has a dominant role on the X chromosome. Consistent with this, tissue-specific DNA methylation at PcG target genes is substantially reduced in Dnmt3a KO embryos. Finally, we find that human patients with DNMT3 mutations exhibit reduced DNA methylation at regions that are hypomethylated in Dnmt3 KO 2i-MEFs. In conclusion, here we report a set of unique de novo DNA methylation target sites for both DNMT3 enzymes during mammalian development that overlap with hypomethylated sites in human patients.

Smarcb1 maintains the cellular identity and the chromatin landscapes of mouse embryonic stem cells.

ES cell (ESC) identity is stably maintained through the coordinated regulation of transcription factors and chromatin structure. SMARCB1, also known as INI1, SNF5, BAF47, is one of the subunits of SWI/SNF (BAF) complexes that play a crucial role in regulating gene expression by controlling chromatin dynamics. Genetic ablation of Smarcb1 in mice leads to embryonic lethality at the peri-implantation stage, indicating that Smarcb1 is important for the early developmental stages. However, the role of SMARCB1 in the maintenance of the ESC identity remains unknown. Here we established mouse ESCs lacking Smarcb1 and investigated the effect of Smarcb1 ablation on the differentiation propensity of ESCs. We found an increased expression of trophectoderm-related genes including Cdx2 in Smarcb1-deficient ESCs. Consistently, they exhibited an extended differentiation propensity into the trophectoderm lineage cells in teratomas. However, although Smarcb1-deficient cells were infrequently incorporated into the trophectoderm cell layer of blastocysts, they failed to contribute to mature placental tissues in vivo. Furthermore, Smarcb1-deficient cells exhibited a premature differentiation in the neural tissue of E14.5 chimeric embryos. Notably, we found that binding motifs for CTCF, which is involved in the maintenance of genomic DNA architecture was significantly enriched in chromatin regions whose accessibility was augmented in Smarcb1-deficient cells, while those for pluripotency factors were overrepresented in regions which have more closed structure in those cells. Collectively, we propose that SMARCB1-mediated remodeling of chromatin landscapes is important for the maintenance and differentiation of ESCs.

Cell-type dependent enhancer binding of the EWS/ATF1 fusion gene in clear cell sarcomas.

Clear cell sarcoma (CCS) is a rare soft tissue sarcoma caused by the EWS/ATF1 fusion gene. Here, we established induced pluripotent stem cells (iPSCs) from EWS/ATF1-controllable murine CCS cells harboring sarcoma-associated genetic abnormalities. Sarcoma-iPSC mice develop secondary sarcomas immediately after EWS/ATF1 induction, but only in soft tissue. EWS/ATF1 expression induces oncogene-induced senescence in most cell types in sarcoma-iPSC mice but prevents it in sarcoma cells. We identify Tppp3-expressing cells in peripheral nerves as a cell-of-origin for these sarcomas. We show cell type-specific recruitment of EWS/ATF1 to enhancer regions in CCS cells. Finally, epigenetic silencing at these enhancers induces senescence and inhibits CCS cell growth through altered EWS/ATF1 binding. Together, we propose that distinct responses to premature senescence are the basis for the cell type-specificity of cancer development.

De Novo DNA Methylation at Imprinted Loci during Reprogramming into Naive and Primed Pluripotency.

CpG islands (CGIs) including those at imprinting control regions (ICRs) are protected from de novo methylation in somatic cells. However, many cancers often exhibit CGI hypermethylation, implying that the machinery is impaired in cancer cells. Here, we conducted a comprehensive analysis of CGI methylation during somatic cell reprogramming. Although most CGIs remain hypomethylated, a small subset of CGIs, particularly at several ICRs, was often de novo methylated in reprogrammed pluripotent stem cells (PSCs). Such de novo ICR methylation was linked with the silencing of reprogramming factors, which occurs at a late stage of reprogramming. The ICR-preferred CGI hypermethylation was similarly observed in human PSCs. Mechanistically, ablation of Dnmt3a prevented PSCs from de novo ICR methylation. Notably, the ICR-preferred CGI hypermethylation was observed in pediatric cancers, while adult cancers exhibit genome-wide CGI hypermethylation. These results may have important implications in the pathogenesis of pediatric cancers and the application of PSCs.

Human Pluripotent Stem Cell-Derived Tumor Model Uncovers the Embryonic Stem Cell Signature as a Key Driver in Atypical Teratoid/Rhabdoid Tumor.

Atypical teratoid/rhabdoid tumor (AT/RT), which harbors SMARCB1 mutation and exhibits a characteristic histology of rhabdoid cells, has a poor prognosis because of the lack of effective treatments. Here, we establish human SMARCB1-deficient pluripotent stem cells (hPSCs). SMARCB1-deficient hPSC-derived neural progenitor-like cells (NPLCs) efficiently give rise to brain tumors when transplanted into the mouse brain. Notably, activation of an embryonic stem cell (ESC)-like signature confers a rhabdoid histology in SMARCB1-deficient NPLC-derived tumors and causes a poor prognosis. Consistently, we find the activation of the ESC-like gene expression signature and an ESC-like DNA methylation landscape in clinical specimens of AT/RT. Finally, we identify candidate genes that maintain the activation of the ESC-like signature and the growth of AT/RT cells. Collectively, SMARCB1-deficient hPSCs offer the human models for AT/RT, which uncover the role of the activated ESC-like signature in the poor prognosis and unique histology of AT/RT.

Sequential conditioning-stimulation reveals distinct gene- and stimulus-specific effects of Type I and II IFN on human macrophage functions.

Macrophages orchestrate immune responses by sensing and responding to pathogen-associated molecules. These responses are modulated by prior conditioning with cytokines such as interferons (IFNs). Type I and II IFN have opposing functions in many biological scenarios, yet macrophages directly stimulated with Type I or II IFN activate highly overlapping gene expression programs. We hypothesized that a sequential conditioning-stimulation approach would reveal with greater specificity the differential effects of Type I and II IFN on human macrophages. By first conditioning with IFN then stimulating with toll-like receptor ligands and cytokines, followed by genome-wide RNA-seq analysis, we identified 713 genes whose expression was unaffected by IFN alone but showed potentiated or diminished responses to a stimulus after conditioning. For example, responses to the cytokine TNF were restricted by Type II IFN conditioning but potentiated by Type I IFN conditioning. We observed that the effects of IFN were not uniformly pro- or anti-inflammatory, but highly gene-specific and stimulus-specific. By assessing expression levels of key signal transducers and characterizing chromatin accessibility by ATAC-seq, we identify the likely molecular mechanisms underlying Type I and Type II-specific effects, distinguishing between modulation of cytoplasmic signaling networks and the nuclear epigenome that synergistically regulate macrophage immune responses.

A Regulatory Circuit Controlling the Dynamics of NFκB cRel Transitions B Cells from Proliferation to Plasma Cell Differentiation.

Humoral immunity depends on efficient activation of B cells and their subsequent differentiation into antibody-secreting cells (ASCs). The transcription factor NFκB cRel is critical for B cell proliferation, but incorporating its known regulatory interactions into a mathematical model of the ASC differentiation circuit prevented ASC generation in simulations. Indeed, experimental ectopic cRel expression blocked ASC differentiation by inhibiting the transcription factor Blimp1, and in wild-type (WT) cells cRel was dynamically repressed during ASC differentiation by Blimp1 binding the Rel locus. Including this bi-stable circuit of mutual cRel-Blimp1 antagonism into a multi-scale model revealed that dynamic repression of cRel controls the switch from B cell proliferation to ASC generation phases and hence the respective cell population dynamics. Our studies provide a mechanistic explanation of how dysregulation of this bi-stable circuit might result in pathologic B cell population phenotypes and thus offer new avenues for diagnostic stratification and treatment.

Genetic interactions support an inhibitory relationship between bone morphogenetic protein 2 and netrin 1 during semicircular canal formation.

The semicircular canals of the mammalian inner ear are derived from epithelial pouches in which epithelial cells in the central region of each pouch undergo resorption, leaving behind the region at the rim to form a tube-shaped canal. Lack of proliferation at the rim and/or over-clearing of epithelial cells in the center of the pouch can obliterate canal formation. Otic-specific knockout of bone morphogenetic protein 2 (Bmp2) results in absence of all three semicircular canals; however, the common crus and ampullae housing the sensory tissue (crista) are intact. The lack of Bmp2 causes Ntn1 (which encodes netrin 1), which is required for canal resorption, to be ectopically expressed at the canal rim. Ectopic Ntn1 results in reduction of Dlx5 and Lmo4, which are required for rim formation. These phenotypes can be partially rescued by removing one allele of Ntn1 in the Bmp2 mutants, indicating that Bmp2 normally negatively regulates Ntn1 for canal formation. Additionally, non-resorption of the canal pouch in Ntn1-/- mutants is partially rescued by removing one allele of Bmp2 Thus, reciprocal inhibition between Bmp2 and netrin 1 is involved in canal formation of the vestibule.

Dorsoventral differences in cAMP levels and correlated changes in the subcellular distribution of the PKA catalytic domain, provide further evidence that PKA signaling coordinates dorsoventral patterning of the otocyst.

Dorsoventral (DV) patterning of the otocyst gives rise to formation of the morphologically and functionally complex membranous labyrinth composed of unique dorsal and ventral sensory organs. DV patterning results from extracellular signaling by secreted growth factors, which presumably form reciprocal concentration gradients across the DV axis of the otocyst. Previous work suggested a model in which two important growth factors, bone morphogenetic protein (BMP) and SHH, undergo crosstalk through an intersecting pathway to coordinate DV patterning. cAMP-dependent protein kinase A (PKA) lies at the heart of this pathway. Here, we provide further evidence that PKA signaling coordinates DV patterning, showing that both BMPs and SHH regulate cAMP levels, with BMPs increasing levels in the dorsal otocyst and SHH decreasing levels in the ventral otocyst. This, in turn, results in regional changes in the subcellular distribution of the catalytic domain of PKA, as well as DV regulation of PKA activity, increasing it dorsally and decreasing it ventrally. These new results fill an important gap in our previous understanding of how ligand signaling acts intracellularly during otocyst DV patterning and early morphogenesis, thereby initiating the series of events leading to formation of the inner ear sensory organs that function in balance and hearing.

Hearing crosstalk: the molecular conversation orchestrating inner ear dorsoventral patterning.

The inner ear is a structurally and functionally complex organ that functions in balance and hearing. It originates during neurulation as a localized thickened region of rostral ectoderm termed the otic placode, which lies adjacent to the developing caudal hindbrain. Shortly after the otic placode forms, it invaginates to delineate the otic cup, which quickly pinches off of the surface ectoderm to form a hollow spherical vesicle called the otocyst; the latter gives rise dorsally to inner ear vestibular components and ventrally to its auditory component. Morphogenesis of the otocyst is regulated by secreted proteins, such as WNTs, BMPs, and SHH, which determine its dorsoventral polarity to define vestibular and cochlear structures and sensory and nonsensory cell fates. In this review, we focus on the crosstalk that occurs among three families of secreted molecules to progressively polarize and pattern the developing otocyst. WIREs Dev Biol 2018, 7:e302. doi: 10.1002/wdev.302 This article is categorized under: Establishment of Spatial and Temporal Patterns > Gradients Signaling Pathways > Cell Fate Signaling Vertebrate Organogenesis > From a Tubular Primordium: Non-Branched.

Distinct functions for netrin 1 in chicken and murine semicircular canal morphogenesis.

The vestibular system of the inner ear detects head position using three orthogonally oriented semicircular canals; even slight changes in their shape and orientation can cause debilitating behavioral defects. During development, the canals are sculpted from pouches that protrude from the otic vesicle, the embryonic anlage of the inner ear. In the center of each pouch, a fusion plate forms where cells lose their epithelial morphology and the basement membrane breaks down. Cells in the fusing epithelia intercalate and are removed, creating a canal. In mice, fusion depends on the secreted protein netrin 1 (Ntn1), which is necessary for basement membrane breakdown, although the underlying molecular mechanism is unknown. Using gain-of-function approaches, we found that overexpression of Ntn1 in the chick otic vesicle prevented canal fusion by inhibiting apoptosis. In contrast, ectopic expression of the same chicken Ntn1 in the mouse otic vesicle, where apoptosis is less prominent, resulted in canal truncation. These findings highlight the importance of apoptosis for tissue morphogenesis and suggest that Ntn1 may play divergent cellular roles despite its conserved expression during canal morphogenesis in chicken and mouse.