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At least 19 recordsLinked to original sources

Paired Single-Cell Transcriptome and DNA Barcode Detection in Zebrafish Using ScarTrace.

ScarTrace is a CRISPR/Cas9-based genetic lineage tracing method that allows for uniquely barcoding the DNA of single cells at a target GFP sequence during developing zebrafish embryos. Single cells from barcoded adult zebrafish can be isolated from various tissues (e.g., marrow, brain, eyes, fins), and their transcriptome and barcode sequences are captured by single-cell cDNA amplification and genomic DNA nested PCR, respectively. Computationally, cell type and barcode identification permit clone tracing and lineage tree reconstruction of tissues to unravel fate decisions during embryogenesis.

Animals↗

Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation.

How tissues generate and maintain the correct number of cells is a fundamental problem in biology. In principle, tissue turnover can occur by the differentiation of stem cells, as is well documented for blood, skin and intestine, or by the duplication of existing differentiated cells. Recent work on adult stem cells has highlighted their potential contribution to organ maintenance and repair. However, the extent to which stem cells actually participate in these processes in vivo is not clear. Here we introduce a method for genetic lineage tracing to determine the contribution of stem cells to a tissue of interest. We focus on pancreatic beta-cells, whose postnatal origins remain controversial. Our analysis shows that pre-existing beta-cells, rather than pluripotent stem cells, are the major source of new beta-cells during adult life and after pancreatectomy in mice. These results suggest that terminally differentiated beta-cells retain a significant proliferative capacity in vivo and cast doubt on the idea that adult stem cells have a significant role in beta-cell replenishment.

Aging↗

Human adipose-derived stem cells display myogenic potential and perturbed function in hypoxic conditions.

Here, we enriched a human cell population from adipose tissue that exhibited both mesenchymal plasticity, self-renewal capacity, and a cell-surface marker profile indistinguishable from that of bone marrow-derived mesenchymal stem cells. In addition to adipogenic and osteogenic differentiation, these adipose-derived stem cells displayed skeletal myogenic potential when co-cultured with mouse skeletal myocytes in reduced serum conditions. Physical incorporation of stem cells into multinucleated skeletal myotubes was determined by genetic lineage tracing, whereas human-specific antibody staining was employed to demonstrate functional contribution of the stem cells to a myogenic lineage. To investigate the effects of hypoxia, cells were maintained and differentiated at 2% O(2). In contrast with reports on bone marrow-derived stem cells, both osteogenic and adipogenic differentiation were significantly attenuated. In summary, the relative accessibility of adipose-derived mesenchymal stem cells from human donors provides opportunity for molecular investigation of mechanistic dysfunction in disease settings and may introduce new prospects for cell-based therapy.

Adipocytes↗

Blimp1 defines a progenitor population that governs cellular input to the sebaceous gland.

Epidermal lineage commitment occurs when multipotent stem cells are specified to three lineages: the epidermis, the hair follicle, and the sebaceous gland (SG). How and when a lineage becomes specified remains unknown. Here, we report the existence of a population of unipotent progenitor cells that reside in the SG and express the transcriptional repressor Blimp1. Using cell-culture studies and genetic lineage tracing, we demonstrate that Blimp1-expressing cells are upstream from other cells of the SG lineage. Blimp1 appears to govern cellular input into the gland since its loss leads to elevated c-myc expression, augmented cell proliferation, and SG hyperplasia. Finally, BrdU labeling experiments demonstrate that the SG defects associated with loss of Blimp1 lead to enhanced bulge stem cell activity, suggesting that when normal SG homeostasis is perturbed, multipotent stem cells in the bulge can be mobilized to correct this imbalance.

Animals↗

A cell-autonomous requirement for Cip/Kip cyclin-kinase inhibitors in regulating neuronal cell cycle exit but not differentiation in the developing spinal cord.

Control over cell cycle exit is fundamental to the normal generation of the wide array of distinct cell types that comprise the mature vertebrate CNS. Here, we demonstrate a critical role for Cip/Kip class cyclin-kinase inhibitory (CKI) proteins in regulating this process during neurogenesis in the embryonic spinal cord. Using immunohistochemistry, we show that all three identified Cip/Kip CKI proteins are expressed in both distinct and overlapping populations of nascent and post-mitotic neurons during early neurogenesis, with p27(Kip1) having the broadest expression, and both p57(Kip2) and p21(Cip1) showing transient expression in restricted populations. Loss- and gain-of-function approaches were used to establish the unique and redundant functions of these proteins in spinal cord neurogenesis. Using genetic lineage tracing, we provide evidence that, in the absence of p57, nascent neurons re-enter the cell cycle inappropriately but later exit to begin differentiation. Analysis of p57(Kip2);p27(Kip1) double mutants, where p21 expression is confined to only a small population of interneurons, demonstrates that Cip/Kip CKI-independent factors initiate progenitor cell cycle exit for the majority of interneurons generated in the developing spinal cord. Our studies indicate that p57 plays a critical cell-autonomous role in timing cell cycle exit at G1/S by opposing the activity of Cyclin D1, which promotes cell cycle progression. These studies support a multi-step model for neuronal progenitor cell cycle withdrawal that involves p57(Kip2) in a central role opposing latent Cyclin D1 and other residual cell cycle promoting activities in progenitors targeted for differentiation.

Animals↗

Contribution of human bone marrow stem cells to individual skeletal myotubes followed by myogenic gene activation.

Much attention is focused on characterizing the contribution of bone marrow (BM)-derived cells to regenerating skeletal muscle, fuelled by hopes for stem cell-mediated therapy of muscle degenerative diseases. Though physical integration of BM stem cells has been well documented, little evidence of functional commitment to myotube phenotype has been reported. This is due to the innate difficulty in distinguishing gene products derived from donor versus host nuclei. Here, we demonstrate that BM-derived stem cells contribute via gene expression following incorporation to skeletal myotubes. By co-culturing human BM-derived mesenchymal stem cells (MSC) with mouse skeletal myoblasts, physical incorporation was observed by genetic lineage tracing and species-specific immunofluorescence. We used a human-specific antibody against the intermediate filament protein nestin, a marker of regenerating skeletal muscle, to identify functional contribution of MSC to myotube formation. Although nestin expression was never detected in MSC, human-specific expression was detected in myotubes that also contained MSC-derived nuclei. This induction of gene expression following myotube integration suggests that bone marrow-derived stem cells can reprogram and functionally contribute to the muscle cell phenotype. We propose that this model of myogenic commitment may provide the means to further characterize functional reprogramming of MSC to skeletal muscle.

Adipocytes↗

Pancreatic cells and their progenitors.

Both type 1 and type 2 diabetes patients would greatly benefit from transplantation of insulin-producing pancreatic beta cells; however, a severe shortage of transplantable beta cells is a major current limitation in the use of such therapy. Understanding the mechanisms by which beta cells are naturally formed is therefore a central challenge for modern pancreas biology, in the hope that insights will be applicable for regenerative cell therapy strategies for diabetes. In particular, the cellular origins of pancreatic beta cells pose an important problem, with significant basic and therapeutic implications. This chapter discusses the current controversy regarding the identity of the cells that give rise to new beta cells in the adult mammal. Whereas numerous models suggest that beta cells can originate from adult stem cells, proposed to reside in the pancreas or in other locations, more recent work indicates that the major source for new beta cells during adult life is the proliferation of preexisting, differentiated beta cells. We present these different views, with emphasis on the methodologies employed. In particular, we focus on genetic lineage tracing using the Cre-lox system in transgenic mice, a technique considered the "gold standard" for addressing in vivo problems of cellular origins.

Adult Stem Cells↗

Hypoxia-activated scleraxis a mediates epicardial progenitor differentiation into a unique cardiac perivascular cell type.

The epicardium provides progenitor cells and paracrine signals essential for heart development and regeneration, yet the mechanisms regulating epicardial cell fate remain poorly understood. Here, we identify the transcription factor Scleraxis a (scxa) as a key regulator of epicardial progenitor differentiation in zebrafish. Single-cell transcriptomics, genetic lineage tracing, and cardiac injury models reveal transient scxa expression in activated epicardial progenitor cells (aEPCs) during developmental coronary angiogenesis and heart regeneration. scxa+ epicardial cells predominantly differentiate into a previously uncharacterized col18a1a+ perivascular population, termed epicardial-derived perivascular mesenchymal cells (Epi-PMCs), which is distinct from pericytes, vascular smooth muscle cells, and mammalian adventitial fibroblasts. Epi-PMCs closely associate with coronary vessels and may contribute to vascular stabilization and remodeling, potentially through collagen XVIII. Loss of scxa increases coronary vessel density. Hypoxia and Hif signaling induce scxa expression, identifying a hypoxia-responsive mechanism that promotes epicardial differentiation toward a vascular-supportive fate during heart development and regeneration.

Animals↗

Ventralized dorsal telencephalic progenitors in Pax6 mutant mice generate GABA interneurons of a lateral ganglionic eminence fate.

The transcription factor Pax6 is expressed by progenitors in the ventricular zone (VZ) of dorsal telencephalon (dTel), which generate all cortical glutamatergic neurons, but not by progenitors in the medial ganglionic eminence (MGE), which generate cortical GABAergic interneurons (GABA INs), or the lateral ganglionic eminence (LGE), which generate GABA INs that normally migrate to the olfactory bulb. We show that perinatally, Pax6(sey/sey) mice, which lack functional Pax6 protein, have large subpial ectopias in dTel and ventral telencephalon connected by cell streams arising from an aberrant paraventricular ectopia found throughout dTel. The subpial and paraventricular ectopias and connecting cell streams are comprised of postmitotic neurons expressing markers for GABA INs characteristic of a LGE fate. Marker analyses show that dTel VZ progenitors in Pax6 mutants are progressively ventralized, acquiring expression of regulatory genes normally limited to GE progenitors; by midneurogenesis, the entire dTel VZ exhibits ventralization. This ventralization of the dTel VZ is paralleled by the expression of markers for GABA INs superficial to it, suggesting that it ectopically produces GABA INs, leading to their ectopias and a thinner cortical plate due to diminished production of glutamatergic neurons. Genetic lineage tracing demonstrates that the GABA INs comprising the ectopias are from a cortical Emx1 lineage generated in the dTel VZ, definitively showing that dTel progenitors and progeny acquire a ventral, GE, fate in Pax6 mutants. Thus, Pax6 delimits the appropriate proliferative zone for GABA INs and regulates their numbers and distributions by repressing the ventral fates of dTel progenitors and progeny.

Animals↗

Single-generation estimates of individual fitness as proxies for long-term genetic contribution.

Individual fitness is a central evolutionary concept, but the question of how it should be defined in empirical studies of natural selection remains contentious. Using founding cohorts from long-term population studies of two species of individually marked birds (collared flycatcher Ficedula albicollis and Ural owl Strix uralensis), we compared a rate-sensitive (lambdaind) and a rate-insensitive (lifetime reproductive success [LRS]) estimate of individual fitness with an estimate of long-term genetic fitness. The latter was calculated as the number of gene copies present in the population after more than two generations, as estimated by tracing genetic lineages and accounting for the fact that populations were not completely closed. When counting fledglings, rate-insensitive estimates of individual fitness correlated better than rate-sensitive estimates with estimated long-term genetic contribution. When counting recruits, both classes of estimates performed equally well. The results support the contention that simple, rate-insensitive measures of fitness, such as LRS, provide a valid and good estimate of fitness in evolutionary studies of natural populations.

Animals↗

Transient YAP activation uncovers the neurogenic potential of proliferative mammalian Müller glia.

The Hippo pathway effector YAP promotes spontaneous proliferation of Müller glia (MG), suggesting that bypassing Hippo signaling and activating YAP could enhance retinal regeneration. However, whether proliferative adult MGs retain meaningful neurogenic competence remains unclear. Here, using viral delivery of a Hippo-resistant YAP variant to wild-type adult MGs, we achieved transient YAP activation in adult MGs, inducing proliferation followed by cell-cycle withdrawal and differentiation. Intersectional genetic lineage tracing and EdU labeling, combined with transcriptomic analyses, revealed that YAP-activated MGs predominantly regenerate MGs, whereas only a subset gives rise to bipolar cell-like neurons. These results indicate that proliferative MGs acquire a state resembling that of late-stage retinal progenitors, with limited neurogenic lineage potential. We conclude that YAP-activated cell-cycle reentry inefficiently reprograms adult MGs toward photoreceptor or ganglion cell fates. These findings define the limited competence of proliferative adult MGs to contribute to neurogenic fates and provide a rigorous framework for assessing in vivo glial reprogramming strategies.

AAV↗

Islet-derived fibroblast-like cells are not derived via epithelial-mesenchymal transition from Pdx-1 or insulin-positive cells.

As recent studies suggest that newly formed pancreatic beta-cells are a result of self-duplication rather than stem cell differentiation, in vitro expansion of beta-cells presents a potential mechanism by which to increase available donor tissue for cell-based diabetes therapies. Although most studies have found that beta-cells are resilient to substantial in vitro expansion, recent studies have suggested that it is possible to expand these cells through a process referred to as epithelial-mesenchymal transition (EMT). To further substantiate such an expansion mechanism, we used recombination-based genetic lineage tracing to determine the origin of proliferating fibroblast-like cells from cultured pancreatic islets in vitro. We demonstrate, using two culture methods, that EMT does not underlie the appearance of fibroblast-like cells in mouse islet cultures but that fibroblast-like cells appear to represent mesenchymal stem cell (MSC)-like cells akin to MSCs isolated from bone marrow.

Animals↗

Generation and validation of a Myh11Dre-Spp1Cre intersectional mouse model for lineage tracing of disease-associated smooth muscle cell states.

BACKGROUND: Phenotypic modulation of vascular smooth muscle cells (VSMCs) is a hallmark of vascular remodeling and cardiovascular disease. Recent lineage-tracing and single-cell transcriptomic studies have identified secreted phosphoprotein 1 (SPP1) as a prominent marker associated with disease-associated VSMC states, particularly those linked to fibrotic remodeling and vascular calcification. However, the cellular origins and fate of SPP1-associated VSMC populations remain incompletely understood. METHODS AND RESULTS: We generated a novel Spp1-rSTOPr-Cre (Spp1Cre) knock-in mouse line in which Cre recombinase is expressed from the endogenous Spp1 locus following Dre-mediated excision of a rox-flanked transcriptional STOP cassette. Correct targeting of the knock-in allele was validated by internal, 5' junction, 3' junction, and long-range PCR analyses, as well as Sanger sequencing. To establish an intersectional lineage-tracing strategy, Spp1Cre mice were crossed with Myh11DreERT2 and Rosa26-RSR-LSL-tdTomato-LSL-eGFP reporter mice, enabling permanent labeling of VSMC-derived populations following activation of the endogenous Spp1 locus. Under physiological conditions, eGFP-positive cells were detected at low frequency within the vascular wall and were predominantly negative for the contractile markers ACTA2 and MYH11. As a proof-of-principle application, eGFP-positive cells markedly expanded within atherosclerotic lesions induced by AAV-PCSK9D377Y and high-fat diet feeding. These lineage-traced cells remained largely ACTA2- and MYH11-negative, consistent with a modulated phenotype. Notably, only a minority of eGFP-positive cells expressed SPP1 or fibronectin at the time of analysis, demonstrating the utility of permanent lineage tracing for tracking cells with a history of endogenous Spp1 activation during vascular remodeling. CONCLUSION: We report the generation and validation of a novel Myh11Dre-Spp1Cre intersectional mouse model for lineage tracing of VSMC-derived populations that have activated the endogenous Spp1 locus. This genetic resource provides a valuable platform for investigating the origin, fate, and phenotypic evolution of Spp1-associated VSMC populations during vascular remodeling and cardiovascular disease.

Animals↗

Characterization of a novel subset of cardiac cells and their progenitors in the Drosophila embryo.

The Drosophila heart is a simple organ composed of two major cell types: cardioblasts, which form the simple contractile tube of the heart, and pericardial cells, which flank the cardioblasts. A complete understanding of Drosophila heart development requires the identification of all cell types that comprise the heart and the elucidation of the cellular and genetic mechanisms that regulate the development of these cells. Here, we report the identification of a new population of heart cells: the Odd skipped-positive pericardial cells (Odd-pericardial cells). We have used descriptive, lineage tracing and genetic assays to clarify the cellular and genetic mechanisms that control the development of Odd-pericardial cells. Odd skipped marks a population of four pericardial cells per hemisegment that are distinct from previously identified heart cells. We demonstrate that within a hemisegment, Odd-pericardial cells develop from three heart progenitors and that these heart progenitors arise in multiple anteroposterior locations within the dorsal mesoderm. Two of these progenitors divide asymmetrically such that each produces a two-cell mixed-lineage clone of one Odd-pericardial cell and one cardioblast. The third progenitor divides symmetrically to produce two Odd-pericardial cells. All remaining cardioblasts in a hemisegment arise from two cardioblast progenitors each of which produces two cardioblasts. Furthermore, we demonstrate that numb and sanpodo mediate the asymmetric divisions of the two mixed-lineage heart progenitors noted above.

Animals↗

Hypoxia-inducible factor 2 regulates alveolar regeneration after repetitive injury in three-dimensional cellular and in vivo models.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease in which repetitive epithelial injury and incomplete alveolar repair result in accumulation of profibrotic intermediate/transitional "aberrant" epithelial cell states. The mechanisms leading to the emergence and persistence of aberrant epithelial populations in the distal lung remain incompletely understood. By interrogating single-cell RNA sequencing (scRNA-seq) data from patients with IPF and a mouse model of repeated lung epithelial injury, we identified persistent activation of hypoxia-inducible factor (HIF) signaling in these aberrant epithelial cells. Using mouse genetic lineage-tracing strategies together with scRNA-seq, we found that these disease-emergent aberrant epithelial cells predominantly arose from airway-derived (Scgb1a1-CreER-traced) progenitors and exhibited transcriptional programs of Hif2a activation. In mice treated with repetitive intratracheal bleomycin, deletion of Epas1 (Hif2a) but not Hif1a, from airway-derived progenitors, or administration of the small-molecule HIF2 inhibitor PT-2385, using both prevention and rescue approaches, attenuated experimental lung fibrosis, reduced the appearance of aberrant epithelial cells, and promoted alveolar repair. In mouse alveolar organoids, genetic or pharmacologic inhibition of Hif2 promoted alveolar differentiation of airway-derived epithelial progenitors. In addition, treatment of human distal lung organoids with PT-2385 increased colony-forming efficiency, enhanced protein and transcriptional markers of alveolar type 2 epithelial cell maturation, and prevented the emergence of aberrant epithelial cells. Together, these studies showed that HIF2 activation drives the emergence of aberrant epithelial populations after repetitive injury and that targeted HIF2 inhibition may represent an effective therapeutic strategy to promote functional alveolar repair in IPF and other interstitial lung diseases.

Animals↗

Neuroendocrinology of protochordates: insights from Ciona genomics.

The genome for two species of Ciona is available making these tunicates excellent models for studies on the evolution of the chordates. In this review most of the data is from Ciona intestinalis, as the annotation of the C. savignyi genome is not yet available. The phylogenetic position of tunicates at the origin of the chordates and the nature of the genome before expansion in vertebrates allows tunicates to be used as a touchstone for understanding genes that either preceded or arose in vertebrates. A comparison of Ciona, a sea squirt, to other model organisms such as a nematode, fruit fly, zebrafish, frog, chicken and mouse shows that Ciona has many useful traits including accessibility for embryological, lineage tracing, forward genetics, and loss- or gain-of-function experiments. For neuroendocrine studies, these traits are important for determining gene function, whereas the availability of the genome is critical for identification of ligands, receptors, transcription factors and signaling pathways. Four major neurohormones and their receptors have been identified by cloning and to some extent by function in Ciona: gonadotropin-releasing hormone, insulin, insulin-like growth factor, and cionin, a member of the CCK/gastrin family. The simplicity of tunicates should be an advantage in searching for novel functions for these hormones. Other neuroendocrine components that have been annotated in the genome are a multitude of receptors, which are available for cloning, expression and functional studies.

Animals↗

Development of the zebrafish lymphatic system requires VEGFC signaling.

Lymphangiogenesis results in the formation of a vascular network distinct from arteries and veins that serves to drain interstitial fluid from surrounding tissues and plays a pivotal role in the immune defense of vertebrates as well as in the progression of cancer and other diseases . In mammals, lymph vessels are lined by endothelial cells possibly sprouting from embryonic veins, and their development appears to be critically dependent on the function of PROX1 and VEGFC signaling . The existence of a lymphatic system in teleosts has been a matter of debate for decades. Here we show on the morphological, molecular, and functional levels that zebrafish embryos develop a lymphatic vasculature that serves to retrieve components of the interstitium to the lymph system. We demonstrate the existence of vessels that are molecularly and functionally distinct from blood vessels and show that the development of these vessels depends on Vegfc and VEGFR-3/Flt4 signaling. These findings imply that the molecular components controlling lymphangiogenesis in zebrafish and mammals are conserved and that the zebrafish lymphatic system develops early enough to allow in vivo observations, lineage tracing, and genetic as well as pharmacological screens.

Angiopoietin-2↗

Blimp1 is a critical determinant of the germ cell lineage in mice.

Germ cell fate in mice is induced in pluripotent epiblast cells in response to signals from extraembryonic tissues. The specification of approximately 40 founder primordial germ cells and their segregation from somatic neighbours are important events in early development. We have proposed that a critical event during this specification includes repression of a somatic programme that is adopted by neighbouring cells. Here we show that Blimp1 (also known as Prdm1), a known transcriptional repressor, has a critical role in the foundation of the mouse germ cell lineage, as its disruption causes a block early in the process of primordial germ cell formation. Blimp1-deficient mutant embryos form a tight cluster of about 20 primordial germ cell-like cells, which fail to show the characteristic migration, proliferation and consistent repression of homeobox genes that normally accompany specification of primordial germ cells. Furthermore, our genetic lineage-tracing experiments indicate that the Blimp1-positive cells originating from the proximal posterior epiblast cells are indeed the lineage-restricted primordial germ cell precursors.

Animals↗