Reprogramming tumor-associated macrophages to outcompete endovascular endothelial progenitor cells and suppress tumor neoangiogenesis.

Tumors develop by invoking a supportive environment characterized by aberrant angiogenesis and infiltration of tumor-associated macrophages (TAMs). In a transgenic model of breast cancer, we found that TAMs localized to the tumor parenchyma and were smaller than mammary tissue macrophages. TAMs had low activity of the metabolic regulator mammalian/mechanistic target of rapamycin complex 1 (mTORC1), and depletion of negative regulator of mTORC1 signaling, tuberous sclerosis complex 1 (TSC1), in TAMs inhibited tumor growth in a manner independent of adaptive lymphocytes. Whereas wild-type TAMs exhibited inflammatory and angiogenic gene expression profiles, TSC1-deficient TAMs had a pro-resolving phenotype. TSC1-deficient TAMs relocated to a perivascular niche, depleted protein C receptor (PROCR)-expressing endovascular endothelial progenitor cells, and rectified the hyperpermeable blood vasculature, causing tumor tissue hypoxia and cancer cell death. TSC1-deficient TAMs were metabolically active and effectively eliminated PROCR-expressing endothelial cells in cell competition experiments. Thus, TAMs exhibit a TSC1-dependent mTORC1-low state, and increasing mTORC1 signaling promotes a pro-resolving state that suppresses tumor growth, defining an innate immune tumor suppression pathway that may be exploited for cancer immunotherapy.

Kidney Nephron Determination.

The nephron is a multifunctional filtration device equipped with an array of sophisticated sensors. For appropriate physiological function in the human and mouse, nephrons must be stereotypically arrayed in large numbers, and this essential structural property that defines the kidney is determined during its fetal development. This review explores the process of nephron determination in the fetal kidney, providing an overview of the foundational literature in the field as well as exploring new developments in this dynamic research area. Mechanisms that ensure that a large number of nephrons can be formed from a small initial number of progenitor cells are central to this process, and the question of how the nephron progenitor cell population balances epithelial differentiation with renewal in the progenitor state is a subject of particular interest. Key growth factor signaling pathways and transcription factor networks are discussed.

Salivary gland function, development, and regeneration.

Salivary glands produce and secrete saliva, which is essential for maintaining oral health and overall health. Understanding both the unique structure and physiological function of salivary glands, as well as how they are affected by disease and injury, will direct the development of therapy to repair and regenerate them. Significant recent advances, particularly in the OMICS field, increase our understanding of how salivary glands develop at the cellular, molecular, and genetic levels: the signaling pathways involved, the dynamics of progenitor cell lineages in development, homeostasis, and regeneration, and the role of the extracellular matrix microenvironment. These provide a template for cell and gene therapies as well as bioengineering approaches to repair or regenerate salivary function.

The growth factor EPIREGULIN promotes basal progenitor cell proliferation in the developing neocortex.

Neocortex expansion during evolution is linked to higher numbers of neurons, which are thought to result from increased proliferative capacity and neurogenic potential of basal progenitor cells during development. Here, we show that EREG, encoding the growth factor EPIREGULIN, is expressed in the human developing neocortex and in gorilla cerebral organoids, but not in the mouse neocortex. Addition of EPIREGULIN to the mouse neocortex increases proliferation of basal progenitor cells, whereas EREG ablation in human cortical organoids reduces proliferation in the subventricular zone. Treatment of cortical organoids with EPIREGULIN promotes a further increase in proliferation of gorilla but not of human basal progenitor cells. EPIREGULIN competes with the epidermal growth factor (EGF) to promote proliferation, and inhibition of the EGF receptor abrogates the EPIREGULIN-mediated increase in basal progenitor cells. Finally, we identify putative cis-regulatory elements that may contribute to the observed inter-species differences in EREG expression. Our findings suggest that species-specific regulation of EPIREGULIN expression may contribute to the increased neocortex size of primates by providing a tunable pro-proliferative signal to basal progenitor cells in the subventricular zone.

Calcium Signaling in the Oligodendrocyte Lineage: Regulators and Consequences.

Cells of the oligodendrocyte lineage express a wide range of Ca2+ channels and receptors that regulate oligodendrocyte progenitor cell (OPC) and oligodendrocyte formation and function. Here we define those key channels and receptors that regulate Ca2+ signaling and OPC development and myelination. We then discuss how the regulation of intracellular Ca2+ in turn affects OPC and oligodendrocyte biology in the healthy nervous system and under pathological conditions. Activation of Ca2+ channels and receptors in OPCs and oligodendrocytes by neurotransmitters converges on regulating intracellular Ca2+, making Ca2+ signaling a central candidate mediator of activity-driven myelination. Indeed, recent evidence indicates that localized changes in Ca2+ in oligodendrocytes can regulate the formation and remodeling of myelin sheaths and perhaps additional functions of oligodendrocytes and OPCs. Thus, decoding how OPCs and myelinating oligodendrocytes integrate and process Ca2+ signals will be important to fully understand central nervous system formation, health, and function.

Cerebral organoid and mouse models reveal a RAB39b-PI3K-mTOR pathway-dependent dysregulation of cortical development leading to macrocephaly/autism phenotypes.

Dysregulation of early neurodevelopment is implicated in macrocephaly/autism disorders. However, the mechanism underlying this dysregulation, particularly in human cells, remains poorly understood. Mutations in the small GTPase gene RAB39b are associated with X-linked macrocephaly, autism spectrum disorder (ASD), and intellectual disability. The in vivo roles of RAB39b in the brain remain unknown. We generated Rab39b knockout (KO) mice and found that they exhibited cortical neurogenesis impairment, macrocephaly, and hallmark ASD behaviors, which resembled patient phenotypes. We also produced mutant human cerebral organoids that were substantially enlarged due to the overproliferation and impaired differentiation of neural progenitor cells (NPCs), which resemble neurodevelopmental deficits in KO mice. Mechanistic studies reveal that RAB39b interacts with PI3K components and its deletion promotes PI3K-AKT-mTOR signaling in NPCs of mouse cortex and cerebral organoids. The mTOR activity is robustly enhanced in mutant outer radial glia cells (oRGs), a subtype of NPCs barely detectable in rodents but abundant in human brains. Inhibition of AKT signaling rescued enlarged organoid sizes and NPC overproliferation caused by RAB39b mutations. Therefore, RAB39b mutation promotes PI3K-AKT-mTOR activity and alters cortical neurogenesis, leading to macrocephaly and autistic-like behaviors. Our studies provide new insights into neurodevelopmental dysregulation and common pathways associated with ASD across species.

BRCA1 and ELK-1 regulate neural progenitor cell fate in the optic tectum in response to visual experience in Xenopus laevis tadpoles.

In developing Xenopus tadpoles, the optic tectum begins to receive patterned visual input while visuomotor circuits are still undergoing neurogenesis and circuit assembly. This visual input regulates neural progenitor cell fate decisions such that maintaining tadpoles in the dark increases proliferation, expanding the progenitor pool, while visual stimulation promotes neuronal differentiation. To identify regulators of activity-dependent neural progenitor cell fate, we profiled the transcriptomes of proliferating neural progenitor cells and newly differentiated neurons using RNA-Seq. We used advanced bioinformatic analysis of 1,130 differentially expressed transcripts to identify six differentially regulated transcriptional regulators, including Breast Cancer 1 (BRCA1) and the ETS-family transcription factor, ELK-1, which are predicted to regulate the majority of the other differentially expressed transcripts. BRCA1 is known for its role in cancers, but relatively little is known about its potential role in regulating neural progenitor cell fate. ELK-1 is a multifunctional transcription factor which regulates immediate early gene expression. We investigated the potential functions of BRCA1 and ELK-1 in activity-regulated neurogenesis in the tadpole visual system using in vivo time-lapse imaging to monitor the fate of GFP-expressing SOX2+ neural progenitor cells in the optic tectum. Our longitudinal in vivo imaging analysis showed that knockdown of either BRCA1 or ELK-1 altered the fates of neural progenitor cells and furthermore that the effects of visual experience on neurogenesis depend on BRCA1 and ELK-1 expression. These studies provide insight into the potential mechanisms by which neural activity affects neural progenitor cell fate.

miR-26 suppresses adipocyte progenitor differentiation and fat production by targeting Fbxl19.

Fat storage in adult mammals is a highly regulated process that involves the mobilization of adipocyte progenitor cells (APCs) that differentiate to produce new adipocytes. Here we report a role for the broadly conserved miR-26 family of microRNAs (miR-26a-1, miR-26a-2, and miR-26b) as major regulators of APC differentiation and adipose tissue mass. Deletion of all miR-26-encoding loci in mice resulted in a dramatic expansion of adipose tissue in adult animals fed normal chow. Conversely, transgenic overexpression of miR-26a protected mice from high-fat diet-induced obesity. These effects were attributable to a cell-autonomous function of miR-26 as a potent inhibitor of APC differentiation. miR-26 blocks adipogenesis, at least in part, by repressing expression of Fbxl19, a conserved miR-26 target without a previously known role in adipocyte biology that encodes a component of SCF-type E3 ubiquitin ligase complexes. These findings have therefore revealed a novel pathway that plays a critical role in regulating adipose tissue formation in vivo and suggest new potential therapeutic targets for obesity and related disorders.

Neuronal Activity-Dependent Control of Postnatal Neurogenesis and Gliogenesis.

The addition of new neurons and oligodendroglia in the postnatal and adult mammalian brain presents distinct forms of gray and white matter plasticity. Substantial effort has been devoted to understanding the cellular and molecular mechanisms controlling postnatal neurogenesis and gliogenesis, revealing important parallels to principles governing the embryonic stages. While during central nervous system development, scripted temporal and spatial patterns of neural and glial progenitor proliferation and differentiation are necessary to create the nervous system architecture, it remains unclear what driving forces maintain and sustain postnatal neural stem cell (NSC) and oligodendrocyte progenitor cell (OPC) production of new neurons and glia. In recent years, neuronal activity has been identified as an important modulator of these processes. Using the distinct properties of neurotransmitter ionotropic and metabotropic channels to signal downstream cellular events, NSCs and OPCs share common features in their readout of neuronal activity patterns. Here we review the current evidence for neuronal activity-dependent control of NSC/OPC proliferation and differentiation in the postnatal brain, highlight some potential mechanisms used by the two progenitor populations, and discuss future studies that might advance these research areas further.

Revisiting the lineage contribution of hematopoietic stem and progenitor cells.

For a long time, self-renewing and multipotent hematopoietic stem cells (HSCs) have been thought to make a major contribution to both embryonic and adult hematopoiesis. The canonical hematopoietic hierarchy illustrating HSC self-renewal and multipotency has been established mainly based on invasive functional assays (e.g. transplantation or colony-forming units in the spleen and in culture), which evaluate the cellular potentials of HSCs. With the extensive applications of non-invasive cell fate-mapping strategies, recent lineage tracing-based studies have suggested that not all native hematopoiesis is established via the hierarchical differentiation of HSCs. By contrast, hematopoietic progenitor cells (HPCs) are a dominant contributor to both embryonic and young adult hematopoiesis. These new findings help redefine the cellular origins of embryonic and adult hematopoiesis under native conditions, and emphasize the differences in revealing HSC potential versus HSC fate using distinct approaches during stress and native hematopoiesis. Here, we review recent advances in HPC and HSC development, and provide an updated perspective to incorporate these new findings with our traditional understanding of developmental and adult hematopoiesis.

Heme-deficient primitive red blood cells induce HSPC ferroptosis by altering iron homeostasis during zebrafish embryogenesis.

The crosstalk between hematopoietic lineages is important for developmental hematopoiesis. However, the role of primitive red blood cells (RBCs) in the formation of definitive hematopoietic stem and progenitor cells (HSPCs) is largely unknown. Primitive RBC deficiencies in mammals always lead to early embryonic lethality, but zebrafish lines with RBC deficiencies can survive to larval stage. By taking advantage of a zebrafish model, we find that the survival of nascent HSPCs is impaired in alas2- or alad-deficient embryos with aberrant heme biosynthesis in RBCs. Heme-deficient primitive RBCs induce ferroptosis of HSPCs through the disruption of iron homeostasis. Mechanistically, heme-deficient primitive RBCs cause blood iron-overload via Slc40a1, and an HSPC iron sensor, Tfr1b, mediates excessive iron absorption. Thus, iron-induced oxidative stress stimulates the lipid peroxidation, which directly leads to HSPC ferroptosis. Anti-ferroptotic treatments efficiently reverse HSPC defects in alas2 or alad mutants. HSPC transplantation assay reveals that the attenuated erythroid reconstitution efficiency may result from the ferroptosis of erythrocyte-biased HSPCs. Together, these results illustrate that heme-deficient primitive RBCs are detrimental to HSPC production and may provide potential implications for iron dysregulation-induced hematological malignancies.

The RNA-binding protein CSDE1 promotes hematopoietic stem and progenitor cell generation via translational control of Wnt signaling.

In vertebrates, the earliest hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of specialized endothelial cells, hemogenic endothelial cells, in the aorta-gonad-mesonephros region through endothelial-to-hematopoietic transition. HSPC generation is efficiently and accurately regulated by a variety of factors and signals; however, the precise control of these signals remains incompletely understood. Post-transcriptional regulation is crucial for gene expression, as the transcripts are usually bound by RNA-binding proteins (RBPs) to regulate RNA metabolism. Here, we report that the RBP protein Csde1-mediated translational control is essential for HSPC generation during zebrafish early development. Genetic mutants and morphants demonstrated that depletion of csde1 impaired HSPC production in zebrafish embryos. Mechanistically, Csde1 regulates HSPC generation through modulating Wnt/ß-catenin signaling activity. We demonstrate that Csde1 binds to ctnnb1 mRNAs (encoding ß-catenin, an effector of Wnt signaling) and regulates translation but not stability of ctnnb1 mRNA, which further enhances ß-catenin protein level and Wnt signal transduction activities. Together, we identify Csde1 as an important post-transcriptional regulator and provide new insights into how Wnt/ß-catenin signaling is precisely regulated at the post-transcriptional level.

Inhibitory SMAD6 interferes with BMP-dependent generation of muscle progenitor cells and perturbs proximodistal pattern of murine limb muscles.

The mechanism of pattern formation during limb muscle development remains poorly understood. The canonical view holds that naïve limb muscle progenitor cells (MPCs) invade a pre-established pattern of muscle connective tissue, thereby forming individual muscles. Here, we show that early murine embryonic limb MPCs highly accumulate pSMAD1/5/9, demonstrating active signaling of bone morphogenetic proteins (BMP) in these cells. Overexpression of inhibitory human SMAD6 (huSMAD6) in limb MPCs abrogated BMP signaling, impaired their migration and proliferation, and accelerated myogenic lineage progression. Fewer primary myofibers developed, causing an aberrant proximodistal muscle pattern. Patterning was not disturbed when huSMAD6 was overexpressed in differentiated muscle, implying that the proximodistal muscle pattern depends on BMP-mediated expansion of MPCs before their differentiation. We show that limb MPCs differentially express Hox genes, and Hox-expressing MPCs displayed active BMP signaling. huSMAD6 overexpression caused loss of HOXA11 in early limb MPCs. In conclusion, our data show that BMP signaling controls expansion of embryonic limb MPCs as a prerequisite for establishing the proximodistal muscle pattern, a process that involves expression of Hox genes.

Cpeb1b-mediated cytoplasmic polyadenylation of shha mRNA modulates zebrafish definitive hematopoiesis.

During vertebrate embryogenesis, hematopoietic stem and progenitor cell (HSPC) production through endothelial-to-hematopoietic transition requires suitable developmental signals, but how these signals are accurately regulated remains incompletely understood. Cytoplasmic polyadenylation, which is one of the posttranscriptional regulations, plays a crucial role in RNA metabolism. Here, we report that Cpeb1b-mediated cytoplasmic polyadenylation is important for HSPC specification by translational control of Hedgehog (Hh) signaling during zebrafish early development. Cpeb1b is highly expressed in notochord and its deficiency results in defective HSPC production. Mechanistically, Cpeb1b regulates hemogenic endothelium specification by the Hedgehog-Vegf-Notch axis. We demonstrate that the cytoplasmic polyadenylation element motif-dependent interaction between Cpeb1b and shha messenger RNA (mRNA) in the liquid-like condensates, which are induced by Pabpc1b phase separation, is required for cytoplasmic polyadenylation of shha mRNA. Intriguingly, the cytoplasmic polyadenylation regulates translation but not stability of shha mRNA, which further enhances the Shha protein level and Hh signal transduction. Taken together, our findings uncover the role of Cpeb1b-mediated cytoplasmic polyadenylation in HSPC development and provide insights into how posttranscriptional regulation can direct developmental signals with high fidelity to translate them into cell fate transition.

N6 -methyladenosine modification of lncRNA Pvt1 governs epidermal stemness.

Dynamic chemical modifications of RNA represent novel and fundamental mechanisms that regulate stemness and tissue homeostasis. Rejuvenation and wound repair of mammalian skin are sustained by epidermal progenitor cells, which are localized within the basal layer of the skin epidermis. N6 -methyladenosine (m6 A) is one of the most abundant modifications found in eukaryotic mRNA and lncRNA (long noncoding RNA). In this report, we survey changes of m6 A RNA methylomes upon epidermal differentiation and identify Pvt1, a lncRNA whose m6 A modification is critically involved in sustaining stemness of epidermal progenitor cells. With genome-editing and a mouse genetics approach, we show that ablation of m6 A methyltransferase or Pvt1 impairs the self-renewal and wound healing capability of skin. Mechanistically, methylation of Pvt1 transcripts enhances its interaction with MYC and stabilizes the MYC protein in epidermal progenitor cells. Our study presents a global view of epitranscriptomic dynamics that occur during epidermal differentiation and identifies the m6 A modification of Pvt1 as a key signaling event involved in skin tissue homeostasis and wound repair.

Amphiregulin Switches Progenitor Cell Fate for Lineage Commitment During Gastric Mucosal Regeneration.

BACKGROUND & AIMS: Isthmic progenitors, tissue-specific stem cells in the stomach corpus, maintain mucosal homeostasis by balancing between proliferation and differentiation to gastric epithelial lineages. The progenitor cells rapidly adopt an active state in response to mucosal injury. However, it remains unclear how the isthmic progenitor cell niche is controlled during the regeneration of damaged epithelium. METHODS: We recapitulated tissue recovery process after acute mucosal injury in the mouse stomach. Bromodeoxyuridine incorporation was used to trace newly generated cells during the injury and recovery phases. To define the epithelial lineage commitment process during recovery, we performed single-cell RNA-sequencing on epithelial cells from the mouse stomachs. We validated the effects of amphiregulin (AREG) on mucosal recovery, using recombinant AREG treatment or AREG-deficient mice. RESULTS: We determined that an epidermal growth factor receptor ligand, AREG, can control progenitor cell lineage commitment. Based on the identification of lineage-committed subpopulations in the corpus epithelium through single-cell RNA-sequencing and bromodeoxyuridine incorporation, we showed that isthmic progenitors mainly transition into short-lived surface cell lineages but are less frequently committed to long-lived parietal cell lineages in homeostasis. However, mucosal regeneration after damage directs the lineage commitment of isthmic progenitors towards parietal cell lineages. During recovery, AREG treatment promoted repopulation with parietal cells, while suppressing surface cell commitment of progenitors. In contrast, transforming growth factor-α did not alter parietal cell regeneration, but did induce expansion of surface cell populations. AREG deficiency impairs parietal cell regeneration but increases surface cell commitment. CONCLUSIONS: These data demonstrate that different epidermal growth factor receptor ligands can distinctly regulate isthmic progenitor-driven mucosal regeneration and lineage commitment.

Molecular divergence of mammalian astrocyte progenitor cells at early gliogenesis.

During mammalian brain development, how different astrocytes are specified from progenitor cells is not well understood. In particular, whether astrocyte progenitor cells (APCs) start as a relatively homogenous population or whether there is early heterogeneity remains unclear. Here, we have dissected subpopulations of embryonic mouse forebrain progenitors using single-cell transcriptome analyses. Our sequencing data revealed two molecularly distinct APC subgroups at the start of gliogenesis from both dorsal and ventral forebrains. The two APC subgroups were marked, respectively, by specific expression of Sparc and Sparcl1, which are known to function in mature astrocytes with opposing activities for regulating synapse formation. Expression analyses showed that SPARC and SPARCL1 mark APC subgroups that display distinct temporal and spatial patterns, correlating with major waves of astrogliogenesis during development. Our results uncover an early molecular divergence of APCs in the mammalian brain and provide a useful transcriptome resource for the study of glial cell specification.

DNA methylation safeguards the generation of hematopoietic stem and progenitor cells by repression of Notch signaling.

The earliest hematopoietic stem and progenitor cells (HSPCs) are generated from the ventral wall of the dorsal aorta, through endothelial-to-hematopoietic transition during vertebrate embryogenesis. Notch signaling is crucial for HSPC generation across vertebrates; however, the precise control of Notch during this process remains unclear. In the present study, we used multi-omics approaches together with functional assays to assess global DNA methylome dynamics during the endothelial cells to HSPCs transition in zebrafish, and determined that DNA methyltransferase 1 (Dnmt1) is essential for HSPC generation via repression of Notch signaling. Depletion of dnmt1 resulted in decreased DNA methylation levels and impaired HSPC production. Mechanistically, we found that loss of dnmt1 induced hypomethylation of Notch genes and consequently elevated Notch activity in hemogenic endothelial cells, thereby repressing the generation of HSPCs. This finding deepens our understanding of HSPC specification in vivo, which will provide helpful insights for designing new strategies for HSPC generation in vitro.

Bmp7 drives proximal tubule expansion and determines nephron number in the developing kidney.

The mammalian kidney is composed of thousands of nephrons that are formed through reiterative induction of a mesenchymal-to-epithelial transformation by a population of nephron progenitor cells. The number of nephrons in human kidneys ranges from several hundred thousand to nearly a million, and low nephron number has been implicated as a risk factor for kidney disease as an adult. Bmp7 is among a small number of growth factors required to support the proliferation and self-renewal of nephron progenitor cells, in a process that will largely determine the final nephron number. Once induced, each nephron begins as a simple tubule that undergoes extensive proliferation and segmental differentiation. Bmp7 is expressed both by nephron progenitor cells and the ureteric bud derivative branches that induce new nephrons. Here, we show that, in mice, Bmp7 expressed by progenitor cells has a major role in determining nephron number; nephron number is reduced to one tenth its normal value in its absence. Postnatally, Bmp7 also drives proliferation of the proximal tubule cells, and these ultimately constitute the largest segment of the nephron. Bmp7 appears to act through Smad 1,5,9(8), p38 and JNK MAP kinase. In the absence of Bmp7, nephrons undergo a hypertrophic process that involves p38. Following a global inactivation of Bmp7, we also see evidence for Bmp7-driven growth of the nephron postnatally. Thus, we identify a role for Bmp7 in supporting the progenitor population and driving expansion of nephrons to produce a mature kidney.

ID1 and CEBPA coordinate epidermal progenitor cell differentiation.

The regulatory circuits that coordinate epidermal differentiation during development are still not fully understood. Here, we report that the transcriptional regulator ID1 is enriched in mouse basal epidermal progenitor cells and find ID1 expression to be diminished upon differentiation. In utero silencing of Id1 impairs progenitor cell proliferation, leads to precocious delamination of targeted progenitor cells and enables differentiated keratinocytes to retain progenitor markers and characteristics. Transcriptional profiling suggests that ID1 acts by mediating adhesion to the basement membrane while inhibiting spinous layer differentiation. Co-immunoprecipitation reveals ID1 binding to transcriptional regulators of the class I bHLH family. We localize bHLH Tcf3, Tcf4 and Tcf12 to epidermal progenitor cells during epidermal stratification and establish TCF3 as a downstream effector of ID1-mediated epidermal proliferation. Finally, we identify crosstalk between CEBPA, a known mediator of epidermal differentiation, and Id1, and demonstrate that CEBPA antagonizes BMP-induced activation of Id1. Our work establishes ID1 as a key coordinator of epidermal development, acting to balance progenitor proliferation with differentiation and unveils how functional crosstalk between CEBPA and Id1 orchestrates epidermal lineage progression.