Hematopoietic-specific heterozygous loss of Dnmt3a exacerbates colitis-associated colon cancer.

Clonal hematopoiesis (CH) is defined as clonal expansion of mutant hematopoietic stem cells absent diagnosis of a hematologic malignancy. Presence of CH in solid tumor patients, including colon cancer, correlates with shorter survival. We hypothesized that bone marrow-derived cells with heterozygous loss-of-function mutations of DNMT3A, the most common genetic alteration in CH, contribute to the pathogenesis of colon cancer. In a mouse model that combines colitis-associated colon cancer (CAC) with experimental CH driven by Dnmt3a+/Δ, we found higher tumor penetrance and increased tumor burden compared with controls. Histopathological analysis revealed accentuated colonic epithelium injury, dysplasia, and adenocarcinoma formation. Transcriptome profiling of colon tumors identified enrichment of gene signatures associated with carcinogenesis, including angiogenesis. Treatment with the angiogenesis inhibitor axitinib eliminated the colon tumor-promoting effect of experimental CH driven by Dnmt3a haploinsufficiency and rebalanced hematopoiesis. This study provides conceptually novel insights into non-tumor-cell-autonomous effects of hematopoietic alterations on colon carcinogenesis and identifies potential therapeutic strategies.

Hedgehog signaling via its ligand DHH acts as cell fate determinant during skeletal muscle regeneration.

Successful muscle regeneration relies on the interplay of multiple cell populations. However, the signals required for this coordinated intercellular crosstalk remain largely unknown. Here, we describe how the Hedgehog (Hh) signaling pathway controls the fate of fibro/adipogenic progenitors (FAPs), the cellular origin of intramuscular fat (IMAT) and fibrotic scar tissue. Using conditional mutagenesis and pharmacological Hh modulators in vivo and in vitro, we identify DHH as the key ligand that acts as a potent adipogenic brake by preventing the adipogenic differentiation of FAPs. Hh signaling also impacts muscle regeneration, albeit indirectly through induction of myogenic factors in FAPs. Our results also indicate that ectopic and sustained Hh activation forces FAPs to adopt a fibrogenic fate resulting in widespread fibrosis. In this work, we reveal crucial post-developmental functions of Hh signaling in balancing tissue regeneration and fatty fibrosis. Moreover, they provide the exciting possibility that mis-regulation of the Hh pathway with age and disease could be a major driver of pathological IMAT formation.

A Guide to Examining Intramuscular Fat Formation and its Cellular Origin in Skeletal Muscle.

Fibro-adipogenic progenitors (FAPs) are mesenchymal stromal cells that play a crucial role during skeletal muscle homeostasis and regeneration. FAPs build and maintain the extracellular matrix that acts as a molecular myofiber scaffold. In addition, FAPs are indispensable for myofiber regeneration as they secrete a multitude of beneficial factors sensed by the muscle stem cells (MuSCs). In diseased states, however, FAPs are the cellular origin of intramuscular fat and fibrotic scar tissue. This fatty fibrosis is a hallmark of sarcopenia and neuromuscular diseases, such as Duchenne Muscular Dystrophy. One significant barrier in determining why and how FAPs differentiate into intramuscular fat is effective preservation and subsequent visualization of adipocytes, especially in frozen tissue sections. Conventional methods of skeletal muscle tissue processing, such as snap-freezing, do not properly preserve the morphology of individual adipocytes, thereby preventing accurate visualization and quantification. To overcome this hurdle, a rigorous protocol was developed that preserves adipocyte morphology in skeletal muscle sections allowing visualization, imaging, and quantification of intramuscular fat. The protocol also outlines how to process a portion of muscle tissue for RT-qPCR, enabling users to confirm observed changes in fat formation by viewing differences in the expression of adipogenic genes. Additionally, it can be adapted to visualize adipocytes by whole-mount immunofluorescence of muscle samples. Finally, this protocol outlines how to perform genetic lineage tracing of Pdgfrα-expressing FAPs to study the adipogenic conversion of FAPs. This protocol consistently yields high-resolution and morphologically accurate immunofluorescent images of adipocytes, along with confirmation by RT-qPCR, allowing for robust, rigorous, and reproducible visualization and quantification of intramuscular fat. Together, the analysis pipeline described here is the first step to improving our understanding of how FAPs differentiate into intramuscular fat, and provides a framework to validate novel interventions to prevent fat formation.

Automatic and unbiased segmentation and quantification of myofibers in skeletal muscle.

Skeletal muscle has the remarkable ability to regenerate. However, with age and disease muscle strength and function decline. Myofiber size, which is affected by injury and disease, is a critical measurement to assess muscle health. Here, we test and apply Cellpose, a recently developed deep learning algorithm, to automatically segment myofibers within murine skeletal muscle. We first show that tissue fixation is necessary to preserve cellular structures such as primary cilia, small cellular antennae, and adipocyte lipid droplets. However, fixation generates heterogeneous myofiber labeling, which impedes intensity-based segmentation. We demonstrate that Cellpose efficiently delineates thousands of individual myofibers outlined by a variety of markers, even within fixed tissue with highly uneven myofiber staining. We created a novel ImageJ plugin (LabelsToRois) that allows processing of multiple Cellpose segmentation images in batch. The plugin also contains a semi-automatic erosion function to correct for the area bias introduced by the different stainings, thereby identifying myofibers as accurately as human experts. We successfully applied our segmentation pipeline to uncover myofiber regeneration differences between two different muscle injury models, cardiotoxin and glycerol. Thus, Cellpose combined with LabelsToRois allows for fast, unbiased, and reproducible myofiber quantification for a variety of staining and fixation conditions.

Developmental and regenerative paradigms of cilia regulated hedgehog signaling.

Primary cilia are immotile appendages that have evolved to receive and interpret a variety of different extracellular cues. Cilia play crucial roles in intercellular communication during development and defects in cilia affect multiple tissues accounting for a heterogeneous group of human diseases called ciliopathies. The Hedgehog (Hh) signaling pathway is one of these cues and displays a unique and symbiotic relationship with cilia. Not only does Hh signaling require cilia for its function but the majority of the Hh signaling machinery is physically located within the cilium-centrosome complex. More specifically, cilia are required for both repressing and activating Hh signaling by modifying bifunctional Gli transcription factors into repressors or activators. Defects in balancing, interpreting or establishing these repressor/activator gradients in Hh signaling either require cilia or phenocopy disruption of cilia. Here, we will summarize the current knowledge on how spatiotemporal control of the molecular machinery of the cilium allows for a tight control of basal repression and activation states of the Hh pathway. We will then discuss several paradigms on how cilia influence Hh pathway activity in tissue morphogenesis during development. Last, we will touch on how cilia and Hh signaling are being reactivated and repurposed during adult tissue regeneration. More specifically, we will focus on mesenchymal stem cells within the connective tissue and discuss the similarities and differences of how cilia and ciliary Hh signaling control the formation of fibrotic scar and adipose tissue during fatty fibrosis of several tissues.

Disulfide bond-disrupting agents activate the tumor necrosis family-related apoptosis-inducing ligand/death receptor 5 pathway.

Disulfide bond-disrupting agents (DDAs) are a new chemical class of agents recently shown to have activity against breast tumors in animal models. Blockade of tumor growth is associated with downregulation of EGFR, HER2, and HER3 and reduced Akt phosphorylation, as well as the induction of endoplasmic reticulum stress. However, it is not known how DDAs trigger cancer cell death without affecting nontransformed cells. As demonstrated here, DDAs are the first compounds identified that upregulate the TRAIL receptor DR5 through transcriptional and post-transcriptional mechanisms to activate the extrinsic cell death pathway. At the protein level, DDAs alter DR5 disulfide bonding to increase steady-state DR5 levels and oligomerization, leading to downstream caspase 8 and 3 activation. DDAs and TRAIL synergize to kill cancer cells and are cytotoxic to HER2+ cancer cells with acquired resistance to the EGFR/HER2 tyrosine kinase inhibitor Lapatinib. Investigation of the mechanisms responsible for DDA selectivity for cancer cells reveals that DDA-induced upregulation of DR5 is enhanced in the context of EGFR overexpression. DDA-induced cytotoxicity is strongly amplified by MYC overexpression. This is consistent with the known potentiation of TRAIL-mediated cell death by MYC. Together, the results demonstrate selective DDA lethality against oncogene-transformed cells, DDA-mediated DR5 upregulation, and protein stabilization, and that DDAs have activity against drug-resistant cancer cells. Our results indicate that DDAs are unique in causing DR5 accumulation and oligomerization and inducing downstream caspase activation and cancer cell death through mechanisms involving altered DR5 disulfide bonding. DDAs thus represent a new therapeutic approach to cancer therapy.

Omega-3 Fatty Acids Activate Ciliary FFAR4 to Control Adipogenesis.

Adult mesenchymal stem cells, including preadipocytes, possess a cellular sensory organelle called the primary cilium. Ciliated preadipocytes abundantly populate perivascular compartments in fat and are activated by a high-fat diet. Here, we sought to understand whether preadipocytes use their cilia to sense and respond to external cues to remodel white adipose tissue. Abolishing preadipocyte cilia in mice severely impairs white adipose tissue expansion. We discover that TULP3-dependent ciliary localization of the omega-3 fatty acid receptor FFAR4/GPR120 promotes adipogenesis. FFAR4 agonists and ω-3 fatty acids, but not saturated fatty acids, trigger mitosis and adipogenesis by rapidly activating cAMP production inside cilia. Ciliary cAMP activates EPAC signaling, CTCF-dependent chromatin remodeling, and transcriptional activation of PPARγ and CEBPα to initiate adipogenesis. We propose that dietary ω-3 fatty acids selectively drive expansion of adipocyte numbers to produce new fat cells and store saturated fatty acids, enabling homeostasis of healthy fat tissue.

Ciliary Hedgehog Signaling Restricts Injury-Induced Adipogenesis.

Injured skeletal muscle regenerates, but with age or in muscular dystrophies, muscle is replaced by fat. Upon injury, muscle-resident fibro/adipogenic progenitors (FAPs) proliferated and gave rise to adipocytes. These FAPs dynamically produced primary cilia, structures that transduce intercellular cues such as Hedgehog (Hh) signals. Genetically removing cilia from FAPs inhibited intramuscular adipogenesis, both after injury and in a mouse model of Duchenne muscular dystrophy. Blocking FAP ciliation also enhanced myofiber regeneration after injury and reduced myofiber size decline in the muscular dystrophy model. Hh signaling through FAP cilia regulated the expression of TIMP3, a secreted metalloproteinase inhibitor, that inhibited MMP14 to block adipogenesis. A pharmacological mimetic of TIMP3 blocked the conversion of FAPs into adipocytes, pointing to a strategy to combat fatty degeneration of skeletal muscle. We conclude that ciliary Hh signaling by FAPs orchestrates the regenerative response to skeletal muscle injury.

Keratin 79 identifies a novel population of migratory epithelial cells that initiates hair canal morphogenesis and regeneration.

The formation of epithelial tubes underlies the development of diverse organs. In the skin, hair follicles resemble tube-like structures with lumens that are generated through poorly understood cellular rearrangements. Here, we show that creation of the hair follicle lumen is mediated by early outward movement of keratinocytes from within the cores of developing hair buds. These migratory keratinocytes express keratin 79 (K79) and stream out of the hair germ and into the epidermis prior to lumen formation in the embryo. Remarkably, this process is recapitulated during hair regeneration in the adult mouse, when K79(+) cells migrate out of the reactivated secondary hair germ prior to formation of a new hair canal. During homeostasis, K79(+) cells line the hair follicle infundibulum, a domain we show to be multilayered, biochemically distinct and maintained by Lrig1(+) stem cell-derived progeny. Upward movement of these cells sustains the infundibulum, while perturbation of this domain during acne progression is often accompanied by loss of K79. Our findings uncover previously unappreciated long-distance cell movements throughout the life cycle of the hair follicle, and suggest a novel mechanism by which the follicle generates its hollow core through outward cell migration.

Wnt signaling regulates postembryonic hypothalamic progenitor differentiation.

Previous studies have raised the possibility that Wnt signaling may regulate both neural progenitor maintenance and neuronal differentiation within a single population. Here we investigate the role of Wnt/ß-catenin activity in the zebrafish hypothalamus and find that the pathway is first required for the proliferation of unspecified hypothalamic progenitors in the embryo. At later stages, including adulthood, sequential activation and inhibition of Wnt activity is required for the differentiation of neural progenitors and negatively regulates radial glia differentiation. The presence of Wnt activity is conserved in hypothalamic progenitors of the adult mouse, where it plays a conserved role in inhibiting the differentiation of radial glia. This study establishes the vertebrate hypothalamus as a model for Wnt-regulated postembryonic neural progenitor differentiation and defines specific roles for Wnt signaling in neurogenesis.

Ongoing Notch signaling maintains phenotypic fidelity in the adult exocrine pancreas.

The Notch signaling pathway regulates embryonic development of the pancreas, inhibiting progenitor differentiation into exocrine acinar and endocrine islet cells. The adult pancreas appears to lack progenitor cells, and its mature cell types are maintained by the proliferation of pre-existing differentiated cells. Nonetheless, Notch remains active in adult duct and terminal duct/centroacinar cells (CACs), in which its function is unknown. We previously developed mice in which cells expressing the Notch target gene Hes1 can be labeled and manipulated, by expression of Cre recombinase, and demonstrated that Hes1(+) CACs do not behave as acinar or islet progenitors in the uninjured pancreas, or as islet progenitors after pancreatic duct ligation. In the current study, we assessed the function of Notch signaling in the adult pancreas by deleting the transcription factor partner of Notch, Rbpj, specifically in Hes1(+) cells. We find that loss of Rbpj depletes the pancreas of Hes1-expressing CACs, abrogating their ongoing contribution to growth and homeostasis of more proximal duct structures. Upon Rbpj deletion, CACs undergo a rapid transformation into acinar cells, suggesting that constitutive Notch activity suppresses the acinar differentiation potential of CACs. Together, our data provide direct evidence of an endogenous genetic program to control interconversion of cell fates in the adult pancreas.

Lineage tracing reveals the dynamic contribution of Hes1+ cells to the developing and adult pancreas.

Notch signaling regulates numerous developmental processes, often acting either to promote one cell fate over another or else to inhibit differentiation altogether. In the embryonic pancreas, Notch and its target gene Hes1 are thought to inhibit endocrine and exocrine specification. Although differentiated cells appear to downregulate Hes1, it is unknown whether Hes1 expression marks multipotent progenitors, or else lineage-restricted precursors. Moreover, although rare cells of the adult pancreas express Hes1, it is unknown whether these represent a specialized progenitor-like population. To address these issues, we developed a mouse Hes1(CreERT2) knock-in allele to inducibly mark Hes1(+) cells and their descendants. We find that Hes1 expression in the early embryonic pancreas identifies multipotent, Notch-responsive progenitors, differentiation of which is blocked by activated Notch. In later embryogenesis, Hes1 marks exocrine-restricted progenitors, in which activated Notch promotes ductal differentiation. In the adult pancreas, Hes1 expression persists in rare differentiated cells, particularly terminal duct or centroacinar cells. Although we find that Hes1(+) cells in the resting or injured pancreas do not behave as adult stem cells for insulin-producing beta (ß)-cells, Hes1 expression does identify stem cells throughout the small and large intestine. Together, these studies clarify the roles of Notch and Hes1 in the developing and adult pancreas, and open new avenues to study Notch signaling in this and other tissues.

Exocrine-to-endocrine differentiation is detectable only prior to birth in the uninjured mouse pancreas.

BACKGROUND: Histological evidence suggests that insulin-producing beta (beta)-cells arise in utero from duct-like structures of the fetal exocrine pancreas, and genetic lineage tracing studies indicate that they are maintained in the adult by self-renewal. These studies have not addressed the origin of the new beta-cells that arise in large numbers shortly after birth, and contradictory lineage tracing results have been published regarding the differentiation potential of duct cells in this period. We established an independent approach to address this question directly. RESULTS: We generated mice in which duct and acinar cells, comprising the exocrine pancreas, can be genetically marked by virtue of their expressing the mucin gene Muc1. Using these mice, we performed time-specific lineage tracing to determine if these cells undergo endocrine transdifferentiation in vivo. We find that Muc1+ cells do give rise to beta-cells and other islet cells in utero, providing formal proof that mature islets arise from embryonic duct structures. From birth onwards, Muc1 lineage-labeled cells are confined to the exocrine compartment, with no detectable contribution to islet cells. CONCLUSIONS: These results argue against a significant contribution by exocrine transdifferentiation to the normal postnatal expansion and maintenance of beta-cell mass. Exocrine transdifferentiation has been proposed to occur during injury and regeneration, and our experimental model is suited to test this hypothesis in vivo.

Lhx2 links the intrinsic and extrinsic factors that control optic cup formation.

A crucial step in eye organogenesis is the transition of the optic vesicle into the optic cup. Several transcription factors and extracellular signals mediate this transition, but whether a single factor links them into a common genetic network is unclear. Here, we provide evidence that the LIM homeobox gene Lhx2, which is expressed in the optic neuroepithelium, fulfils such a role. In Lhx2(-/-) mouse embryos, eye field specification and optic vesicle morphogenesis occur, but development arrests prior to optic cup formation in both the optic neuroepithelium and lens ectoderm. This is accompanied by failure to maintain or initiate the expression patterns of optic-vesicle-patterning and lens-inducing determinants. Of the signaling pathways examined, only BMP signaling is noticeably altered and Bmp4 and Bmp7 mRNAs are undetectable. Lhx2(-/-) optic vesicles and lens ectoderm upregulate Pax2, Fgf15 and Sox2 in response to BMP treatments, and Lhx2 genetic mosaics reveal that transcription factors, including Vsx2 and Mitf, require Lhx2 cell-autonomously for their expression. Our data indicate that Lhx2 is required for optic vesicle patterning and lens formation in part by regulating BMP signaling in an autocrine manner in the optic neuroepithelium and in a paracrine manner in the lens ectoderm. We propose a model in which Lhx2 is a central link in a genetic network that coordinates the multiple pathways leading to optic cup formation.

Retinoic acid is required for endodermal pouch morphogenesis and not for pharyngeal endoderm specification.

Because tissues from all three germ layers contribute to the pharyngeal arches, it is not surprising that all major signaling pathways are involved in their development. We focus on the role of retinoic acid (RA) signaling because it has been recognized for quite some time that alterations in this pathway lead to craniofacial malformations. Several studies exist that describe phenotypes observed upon RA perturbations in pharyngeal arch development; however, these studies did not address whether RA plays multiple roles at distinct time points during development. Here, we report the resulting phenotypes in the hindbrain, the neural crest-derived tissues, and the pharyngeal endoderm when RA synthesis is disrupted during zebrafish gastrulation and pharyngeal arch morphogenesis. Our results demonstrate that RA is required for the post-gastrulation morphogenesis and segmentation of endodermal pouches, and that loss of RA does not affect the length of the pharyngeal ectoderm or medial endoderm along the anterior-posterior axis. We also provide evidence that RA is not required for the specification of pharyngeal pouch endoderm and that the pharyngeal endoderm consists of at least two different cell populations, of which the pouch endoderm is sensitive to RA and the more medial pharyngeal endoderm is not. These results demonstrate that the developmental processes underlying pharyngeal arch defects differ depending on when RA signaling is disturbed during development.