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PubMed1999

Neurogenin1 and neurogenin2 control two distinct waves of neurogenesis in developing dorsal root ganglia.

Different classes of sensory neurons in dorsal root ganglia (DRG) are generated in two waves: large-diameter trkC+ and trkB+ neurons are born first, followed by small-diameter trkA+ neurons. All such neurons require either neurogenin (ngn) 1 or 2, two neuronal determination genes encoding basic helix-loop-helix (bHLH) transcription factors. ngn2 is required primarily if not exclusively for the generation of trkC+ and trkB+ neurons, whereas the generation of most or all trkA+ neurons requires ngn1. Comparison with previous lineage tracing data in the chick suggests that this dichotomy reflects a requirement for the two ngns in distinct sensory precursor populations. The neurogenesis defect in ngn2(-/-) embryos is transient and later compensated by ngn1-dependent precursors, suggesting that feedback or competitive interactions between these precursors may control the proportion of different neuronal subtypes they normally produce. These data reveal remarkable parallels in the roles of bHLH factors during neurogenesis in the DRG, and myogenesis in the neighboring myotome.

Animals↗
PubMed2026

A conserved Notch-Meis1-Pbx cascade specifies secretory progenitors into spatially diverse intestinal best4 + cells.

best4 + cells are a recently described vertebrate intestinal epithelial cell type. best4 + cells are altered in inflammatory bowel disease and colorectal cancer, suggesting that stimulation of their homeostatic replenishment may have therapeutic potential. However, the development and function of best4 + cells remain unclear. Since mice lack best4 + cells, we established zebrafish as a tractable in vivo model to observe, manipulate, and remove best4 + cells in an organismal context. We dissected best4 + cell developmental regulation in vivo from birth to differentiation and specialization, focusing on factors conserved in best4 + cells across vertebrates. Lineage tracing demonstrated that best4 + cells arise from secretory progenitors, where Notch/Dll4 signaling mediates a decision between best4 + and enterochromaffin cells by triggering meis1b expression. Following specification by meis1b, pbx3a spatially diversifies best4 + cells, which develop regional heterogeneity in gene expression, intracellular pH, and function. In vivo live imaging and removal of best4 + cells showed that best4+ cells sense luminal pH changes and extend dynamic luminal and stromal projections, but are not required to restore global luminal pH after challenge. Altogether, this study experimentally delineates best4 + cell developmental regulation and develops a genetic toolkit to examine their function in vivo, both of which will aid investigating how best4 + cells are altered or can be restored during disease.

Animals↗
PubMed2004

Role of TSC-22 during early embryogenesis in Xenopus laevis.

Transforming growth factor-beta1-stimulated clone 22 (TSC-22) encodes a leucine zipper-containing protein that is highly conserved. During mouse embryogenesis, TSC-22 is expressed at the site of epithelial-mesenchymal interaction. Here, we isolated Xenopus laevis TSC-22 (XTSC-22) and analyzed its function in early development. XTSC-22 mRNA was first detected in the ectoderm of late blastulae. Translational knockdown using XTSC-22 antisense morpholino oligonucleotides (XTSC-22-MO) caused a severe delay in blastopore closure in gastrulating embryos. This was not due to mesoderm induction or convergent-extension, as confirmed by whole-mount in situ hybridization and animal cap assay. Cell lineage tracing revealed that migration of ectoderm cells toward blastopore was disrupted in XTSC-22-depleted embryos, and these embryos had a marked increase in the number of dividing cells. In contrast, cell division was suppressed in XTSC-22 mRNA-injected embryos. Co-injection of XTSC-22-MO and mRNA encoding p27Xic1, which inhibits cell cycle promotion by binding cyclin/Cdk complexes, reversed aberrant cell division. This was accompanied by rescue of the delay in blastopore closure and cell migration. These results indicate that XTSC-22 is required for cell movement during gastrulation though cell cycle regulation.

Amino Acid Sequence↗
PubMed2002

In vivo imaging of quantum dots encapsulated in phospholipid micelles.

Fluorescent semiconductor nanocrystals (quantum dots) have the potential to revolutionize biological imaging, but their use has been limited by difficulties in obtaining nanocrystals that are biocompatible. To address this problem, we encapsulated individual nanocrystals in phospholipid block-copolymer micelles and demonstrated both in vitro and in vivo imaging. When conjugated to DNA, the nanocrystal-micelles acted as in vitro fluorescent probes to hybridize to specific complementary sequences. Moreover, when injected into Xenopus embryos, the nanocrystal-micelles were stable, nontoxic (<5 x 10(9) nanocrystals per cell), cell autonomous, and slow to photobleach. Nanocrystal fluorescence could be followed to the tadpole stage, allowing lineage-tracing experiments in embryogenesis.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1993

Commitment of cell fate in the early zebrafish embryo.

When do single cells in the early zebrafish embryo become irreversibly committed to a specific fate? Work with lineage tracing and fate mapping has shown that the marginal cells of the blastoderm give rise to hypoblast-derived fates (mesoderm and endoderm). However, experiments described here show that these marginal blastoderm cells remain pluripotent and uncommitted throughout the late blastula and early gastrula stages. Embryonic cells become committed to a hypoblast-derived fate at mid-gastrulation. Time-lapse photographic analysis reveals that committed cells, when transplanted heterotopically and heterochronically, can migrate along atypical pathways to reposition themselves within a more correct environment.

Animals↗
PubMed2025

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↗
PubMed2026

Aging and DNA damage are associated with the development of endothelial cell clonal expansion.

Endothelial dysfunction is a hallmark of vascular aging and a key contributor to cardiovascular disease. Although senescence has been widely studied as a terminal endothelial cell fate, recent evidence suggests that clonal expansion, the proliferative expansion of genetically identical cells, may also occur in aged tissues. We sought to determine whether endothelial clonal expansion increases with age, specifically at the atheroprone regions of the aorta, and to evaluate whether DNA damage promotes endothelial cell clonal expansion. Tamoxifen-inducible, endothelial-specific Cdh5-CreERT2 male and female mice were used to quantify clonal expansion in endothelial cells (ECs) across the aortic region in both young (4 mo) and aged (24 mo) mice. We further examined the effect of DNA damage by administering systemic doxorubicin (DOXO) to assess clonal dynamics in different aortic regions. Aging significantly increased EC clone size and the percentage of clonal ECs in atheroprone regions, particularly the minor arch, whereas only clone size increased in nonatheroprone regions. Systemic DOXO administration increased clone size across the aortic region without altering clonal recruitment, indicating selective amplification of preexisting clones. These findings suggest that clonal expansion is promoted by both aging and DNA damage. Clonal expansion may represent an underrecognized mechanism contributing to endothelial homogeneity and vascular remodeling during aging and in response to sublethal genomic stress.NEW & NOTEWORTHY Aging reshapes the vascular endothelium in unexpected ways. Using lineage tracing in mice, we show that endothelial cells undergo age-dependent clonal expansion, particularly in atheroprone regions exposed to disturbed blood flow. This process is amplified by DNA damage and reflects the selective expansion of preexisting clones rather than increased recruitment. Endothelial clonal expansion may represent an underrecognized mechanism driving vascular remodeling during aging and genotoxic stress.

Animals↗
PubMed2006

Cardiac neural crest ablation inhibits compaction and electrical function of conduction system bundles.

Retroviral and transgenic lineage-tracing studies have shown that neural crest cells associate with the developing bundles of the ventricular conduction system. Whereas this migration of cells does not provide progenitors for the myocardial cells of the conduction system, the question of whether neural crest affects the differentiation and/or function of cardiac specialized tissues continues to be of interest. Using optical mapping of voltage-sensitive dye, we determined that ventricles from chick embryos in which the cardiac neural crest had been laser ablated did not progress to apex-to-base activation by the expected stage [i.e., Hamburger and Hamilton (HH) 35] but instead maintained basal breakthroughs of epicardial activation consistent with immature function of the conduction system. In direct studies of activation, waves of depolarization originating from the His bundle were found to be uncommon in control hearts from HH34 and HH35 embryos. However, activations propagating from septal base, at or near the His bundle, occurred frequently in hearts from HH34 and HH35 neural crest-ablated embryos. Consistent with His bundle cells maintaining electrical connections with adjacent working myocytes, histological analyses of hearts from neural crest-ablated embryos revealed His bundles that had not differentiated a lamellar organization or undergone a process of compaction and separation from surrounding myocardium observed in controls. Furthermore, measurements on histological sections from optically mapped hearts indicated that, whereas His bundle diameter in control embryos thinned by almost one-half between HH30 and HH34, the His bundle in ablated embryos underwent no such compaction in diameter, maintaining a thickness at HH30, HH32, and HH34 similar to that observed in HH30 controls. We conclude that the cardiac neural crest is required in a novel function involving lamellar compaction and electrical isolation of the basally located His bundle from surrounding myocardium.

Animals↗
PubMed1998

Neural crest cell contribution to the developing circulatory system: implications for vascular morphology?

In this study, the distribution patterns of neural crest (NC) cells (NCCs) in the developing vascular system of the chick were thoroughly studied and examined for a correlation with smooth muscle cell differentiation and vascular morphogenesis. For this purpose, we performed long-term lineage tracing using quail-chick chimera techniques and premigratory NCC infection with a replication-incompetent retrovirus containing the LacZ reporter gene in combination with immunohistochemistry. Results indicate that NCC deposition around endothelial tubes is influenced by anteroposterior positional information from the pharyngeal arterial system. NCCs were shown to be among the first cells to differentiate into primary smooth muscle cells of the arch arteries. At later stages, NCCs eventually differentiated into adventitial fibroblasts and smooth muscle cells and nonmuscular cells of the media and intima. NCCs were distributed in the aortic arch and pulmonary arch arteries and in the brachiocephalic and carotid arteries. The coronary and pulmonary arteries and the descending aorta, however, remained devoid of NCCs. A new finding was that the media of part of the anterior cardinal veins was also determined to be NC-derived. NC-derived elastic arteries differed from non-NC elastic vessels in their cellular constitution and elastic fiber organization, and the NC appeared not to be involved in designating a muscular or elastic artery. Boundaries between NC-infested areas and mesodermal vessel structures were mostly very sharp and tended to coincide with marked changes in vascular morphology, with the exception of an intriguing area in the aortic and pulmonary trunks.

Animals↗
PubMedDate not supplied

The X-gal caution in neural transplantation studies.

Cell transplantation into host brain requires a reliable cell marker to trace lineage and location of grafted cells in host tissue. The lacZ gene encodes the bacterial (E. coli) enzyme beta-galactosidase (beta-gal) and is commonly visualized as a blue intracellular precipitate following its incubation with a substrate, "X gal," in an oxidation reaction. LacZ is the "reporter gene" most commonly employed to follow gene expression in neural tissue or to track the fate of transplanted exogenous cells. If the reaction is not performed carefully-with adequate optimization and individualization of various parameters (e.g.. pH, concentration of reagents, addition of chelators, composition of fixatives) and the establishment of various controls--then misleading nonspecific background X-gal positivity can result, leading to the misidentification of cells. Some of this background results from endogenous nonbacterial beta-gal activity in discrete populations of neurons in the mammalian brain; some results from an excessive oxidation reaction. Surprisingly, few articles have empha sized how to recognize and to eliminate these potential confounding artifacts in order to maximize the utility and credibility of this histochemical technique as a cell marker. We briefly review the phenomenon in general, discuss a specific case that illustrates how an insufficiently scrutinized X-gal positivity can be a pitfall in cell transplantation studies, and then provide recommendations for optimizing the specificity and reliability of this histochemical reaction for discerning E. coli beta-gal activity.

Animals↗
PubMed2026

Decoding glioblastoma evolution and heterogeneity through mechanistic modeling: implications for clinical translation.

Glioblastoma (GBM) is one of the most aggressive and lethal primary brain tumors in adults, characterized by dynamic clonal evolution and extensive genomic, cellular, spatial, and microenvironmental heterogeneity. Multi-omics studies have revealed that GBM follows complex evolutionary trajectories involving genetic, epigenetic, transcriptional, and immune-microenvironmental remodeling as tumors grow, adapt to the brain microenvironment, and acquire therapeutic resistance. Increasing evidence suggests that GBM may originate from aberrant neural stem or progenitor cells, including those residing in the subventricular zone, and that glioblastoma stem cells (GSCs) contribute to tumor propagation, heterogeneity, and recurrence. A key conceptual challenge is to reconcile hierarchical cancer stem cell models, in which GSCs are viewed as relatively stable tumor-propagating subpopulations, with dynamic state plasticity models, in which stem-like properties can be reversibly acquired or lost during transitions among proneural-like, mesenchymal-like, invasive, and therapy-tolerant states. Recent advances in single-cell profiling, spatial transcriptomics, lineage tracing, organoid culture, 3D bioprinting, genetically engineered models, and artificial intelligence (AI)-assisted computational modeling have substantially improved the ability to study these processes. However, no currently available model fully recapitulates human GBM heterogeneity, recurrence, treatment history, and tumor-microenvironment interactions. Therefore, model selection should be guided by clearly defined mechanistic questions rather than by reliance on any single platform. This review summarizes current advances in in vitro, ex vivo, in vivo, and computational models for studying GBM evolution and heterogeneity, and discusses how integrated model pipelines may improve preclinical drug testing, treatment-response prediction, and precision neuro-oncology.

Humans↗
PubMed2003

Clonal expansion of ovarian germline stem cells during niche formation in Drosophila.

Stem cell niches are specific regulatory microenvironments formed by neighboring stromal cells. Owing to difficulties in identifying stem cells and their niches in many systems, mechanisms that control niche formation and stem cell recruitment remain elusive. In the Drosophila ovary, two or three germline stem cells (GSCs) have recently been shown to reside in a niche, in which terminal filaments (TFs) and cap cells are two major components. We report that signals from newly formed niches promote clonal expansion of GSCs during niche formation in the Drosophila ovary. After the formation of TFs and cap cells, anterior primordial germ cells (PGCs) adjacent to TFs/cap cells can develop into GSCs at the early pupal stage while the rest directly differentiate. The anterior PGCs are very mitotically active and exhibit two division patterns with respect to cap cells. One of these patterns generates two daughters that both contact cap cells and potentially become GSCs. Our lineage tracing study confirms that one PGC can generate two or three GSCs to occupy a whole niche ('clonal expansion'). decapentaplegic (dpp), the Drosophila homolog of human bone morphogenetic protein 2/4, is expressed in anterior somatic cells of the gonad, including TFs/cap cells. dpp overexpression promotes PGC proliferation and causes the accumulation of more PGCs in the gonad. A single PGC mutant for thick veins, encoding an essential dpp receptor, loses the ability to clonally populate a niche. Therefore, dpp is probably one of the mitotic signals that promote the clonal expansion of GSCs in a niche. This study also suggests that signals from newly formed niche cells are important for expanding stem cells and populating niches.

Animals↗
PubMed2006

her5 expression reveals a pool of neural stem cells in the adult zebrafish midbrain.

Current models of vertebrate adult neural stem cells are largely restricted to the rodent forebrain. To extract the general mechanisms of neural stem cell biology, we sought to identify new adult stem cell populations, in other model systems and/or brain areas. The teleost zebrafish appears to be an ideal system, as cell proliferation in the adult zebrafish brain is found in many more niches than in the mammalian brain. As a starting point towards identifying stem cell populations in this system, we used an embryonic neural stem cell marker, the E(spl) bHLH transcription factor Her5. We demonstrate that her5 expression is not restricted to embryonic neural progenitors, but also defines in the adult zebrafish brain a new proliferation zone at the junction between the mid- and hindbrain. We show that adult her5-expressing cells proliferate slowly, self-renew and express neural stem cell markers. Finally, using in vivo lineage tracing in her5:gfp transgenic animals, we demonstrate that the her5-positive population is multipotent, giving rise in situ to differentiated neurons and glia that populate the basal midbrain. Our findings conclusively identify a new population of adult neural stem cells, as well as their fate and their endogenous environment, in the intact vertebrate brain. This cell population, located outside the forebrain, provides a powerful model to assess the general mechanisms of vertebrate neural stem cell biology. In addition, the first transcription factor characteristic of this cell population, Her5, points to the E(Spl) as a promising family of candidate adult neural stem cell regulators.

Animals↗
PubMed2006

Ptf1a determines horizontal and amacrine cell fates during mouse retinal development.

The vertebrate neural retina comprises six classes of neurons and one class of glial cells, all derived from a population of multipotent progenitors. There is little information on the molecular mechanisms governing the specification of cell type identity from multipotent progenitors in the developing retina. We report that Ptf1a, a basic-helix-loop-helix (bHLH) transcription factor, is transiently expressed by post-mitotic precursors in the developing mouse retina. Recombination-based lineage tracing analysis in vivo revealed that Ptf1a expression marks retinal precursors with competence to exclusively produce horizontal and amacrine neurons. Inactivation of Ptf1a leads to a fate-switch in these precursors that causes them to adopt a ganglion cell fate. This mis-specification of neurons results in a complete loss of horizontal cells, a profound decrease of amacrine cells and an increase in ganglion cells. Furthermore, we identify Ptf1a as a primary downstream target for Foxn4, a forkhead transcription factor involved in the genesis of horizontal and amacrine neurons. These data, together with the previous findings on Foxn4, provide a model in which the Foxn4-Ptf1a pathway plays a central role in directing the differentiation of retinal progenitors towards horizontal and amacrine cell fates.

Amacrine Cells↗
PubMed1987

Cytokeratin filament assembly in the preimplantation mouse embryo.

The timing, spatial distribution and control of cytokeratin assembly during mouse early development has been studied using a monoclonal antibody, TROMA-1, which recognizes a 55,000 Mr trophectodermal cytokeratin (ENDO A). This protein was first detected in immunoblots at the 4-cell stage, and became more abundant at the 16-cell stage and later. Immunofluorescence analysis revealed assembled cytokeratin filaments in some 8-cell blastomeres, but not at earlier stages. At the 16-cell stage, filaments were found in both polarized (presumptive trophectoderm; TE) and apolar (presumptive inner cell mass; ICM) cells in similar proportions, although polarized cells possessed more filaments than apolar cells. By the late 32-cell, early blastocyst, stage, all polarized (TE) cells contained extensive filament networks whereas cells positioned inside the embryo tended to have lost their filaments. The presence of filaments in inside cells at the 16-cell stage and in ICM cells was confirmed by immunoelectron microscopy. Lineage tracing techniques demonstrated that those cells in the ICM of early blastocysts which did possess filaments were almost exclusively the progeny of polar 16-cell blastomeres, suggesting that these filaments were directly inherited from outside cells at the 16- to 32-cell transition. Inhibitor studies revealed that proximate protein synthesis but not mRNA synthesis is required for filament assembly at the 8-cell stage. These results demonstrate that there are quantitative rather than qualitative differences in the expression of cytokeratin filaments in the inner cell mass and trophectoderm cells of the mouse embryo.

Amanitins↗
PubMed1990

Neurogenesis in the insect enteric nervous system: generation of premigratory neurons from an epithelial placode.

The enteric plexus (EP) is a major division of the enteric nervous system (ENS) in the moth Manduca sexta and contains a dispersed population of about 360 bipolar neurons, the EP cells. Previously we showed that embryonic EP cells achieve their mature distributions by extensive migration along the gut surface and then display position-specific phenotypes. We now demonstrate that the entire EP cell population is generated from an ectodermal placode that invaginates from the embryonic foregut. Individual EP cells become postmitotic just as they leave the epithelium, but their terminal differentiation is subsequently delayed until after their migratory dispersal. Clonal analysis by injection of lineage-tracing dyes has shown that the EP cell population is derived from a large number of placodal cells, each of which contributes a limited number of neurons to the ENS. Placodally derived clones produce neurons exclusively, while clones arising from cells adjacent to the placode are incorporated into the gut epithelium. These results indicate that neurogenesis in the insect ENS involves a developmental strategy that is distinct from that seen in the insect CNS and which resembles the generation of certain cell classes in the vertebrate nervous system.

Animals↗
PubMed1995

Dorsalizing and neuralizing properties of Xdsh, a maternally expressed Xenopus homolog of dishevelled.

Signaling factors of the Wnt proto-oncogene family are implicated in dorsal axis formation during vertebrate development, but the molecular mechanism of this process is not known. Studies in Drosophila have indicated that the dishevelled gene product is required for wingless (Wnt1 homolog) signal transduction. We demonstrate that injection of mRNA encoding a Xenopus homolog of dishevelled (Xdsh) into prospective ventral mesodermal cells triggers a complete dorsal axis formation in Xenopus embryos. Lineage tracing experiments show that cells derived from the injected blastomere contribute to anterior and dorsal structures of the induced axis. In contrast to its effect on mesoderm, overexpression of Xdsh mRNA in prospective ectodermal cells triggers anterior neural tissue differentiation. These studies suggest that Wnt signal transduction pathway is conserved between Drosophila and vertebrates and point to a role for maternal Xdsh product in dorsal axis formation and in neural induction.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Properties of the dorsal activity found in the vegetal cortical cytoplasm of Xenopus eggs.

The Xenopus egg contains a maternal dorsal determinant that is specifically localized to the vegetal cortex. We have previously shown that vegetal cortical cytoplasm can generate a full dorsal axis when it is injected into ventral vegetal blastomeres of a cleavage-stage embryo. In this study, we have defined further the properties of the dorsal activity. The cortical dorsal activity arises during oocyte maturation after germinal vesicle breakdown. When injected into the four extreme animal pole blastomeres of ultraviolet-ventralized 32-cell embryos, vegetal cortical cytoplasm partially rescued dorsal axial structures. As revealed by lineage tracing, these axial structures formed ectopically from the progeny of the cells that were injected. Injection of animal cortical cytoplasm had no effect. When mid-blastula (stage 8) animal caps from these injected embryos were isolated and cultured, both vegetal cortex-enriched and animal cortex-enriched animal caps produced only epidermis. Therefore vegetal cortex, on its own, is not a mesoderm inducer. Between stage 8 (blastula) and stage 10 (gastrula), a ventral mesoderm-inducing signal spreads from vegetal cells towards the animal pole. We tested whether this natural mesoderm-inducing factor interacts with the activity found in the vegetal cortex. Injection of vegetal cortex enhanced the formation of neural tissue and cement gland when animal caps were isolated at stage 10. When cultured from stage 8 in the presence of the ventral mesoderm-inducing fibroblast growth factor, animal caps enriched in vegetal cortex developed significantly more neural tissue and cement gland than ones enriched in animal cortex. These results indicate that the dorsal activity localized to the egg vegetal cortex alters the response of cells to mesoderm inducers.

Animals↗