Lineage Restriction Research Articles

Anthropogenic cause of range shifts and gene flow between two grasshopper species revealed by environmental modelling, geometric morphometrics and population genetics

Abstract The range of a species is controlled by biotic and abiotic factors; both could have changed recently due to human activity. We used environmental modelling, morphometric and genetic data to interpret ecological responses at the species boundary of a pair of New Zealand grasshoppers with very different ranges; one widespread (Phaulacridium marginale) and one restricted to semi‐arid central/southern South Island (Phaulacridium otagoense). Climate‐ and habitat‐based distribution models for grasshoppers in the past (last glacial maximum), present and future (2070), in concert with modelling of vegetation patterns imply range and demographic expansion of P. marginale and stability of P. otagoense. mtDNA sequence revealed four main lineages with pronounced differences in genetic diversity and geographical range. The widespread lineage associated with P. marginale revealed a signature of range expansion but regionally restricted lineages were geographically structured at a fine scale. Within the narrow geographical range of P. otagoense, three mtDNA lineages resulted in high diversity, more typical of large stable populations. Geometric analysis of pronotum shape identified individuals from a region of sympatry with mixed characteristics. Mismatch of phenotype, mtDNA lineage and nuclear DNA sequence indicates introgression between grasshopper species now in contact. This appears to be accompanied by P. otagoense range reduction through ecological competition. Deforestation by people starting ∼800 years ago best explains range change and resulting hybridisation of these grasshoppers. Anthropogenic habitat modification can have indirect consequences on insect biodiversity and conservation by enabling introgression between formerly separate populations and species.

Using dated molecular phylogenies to help reconstruct geological, climatic, and biological history: Examples from Colombia

Attempts at historical reconstruction are based on limited data. We are more likely to produce accurate historical reconstructions by utilizing information from diverse sources and pooling data within the relevant research communities which will allow us to build up a moving picture of the geological, climatic, and biological evolution of our planet. We suggest that dated phylogenies of plants can contribute greatly to a better understanding of Earth history. Timing of phylogenetic splits of lowland restricted lineages on either side of the Andes could provide information on the timing of montane uplift and associated climatic changes. The timing of the arrival and diversification of organisms restricted to specific climatic regimes at a particular altitude can provide information on the age at which mountains reached a height adequate for that climate once corrected for global climate changes. As a model for study, we discuss how dated phylogenies in biome rich Colombia may contribute to an understanding of geological and climatic change in north‐western South America. Lowland wet forest restricted lineages separated from the mid‐Miocene, whereas lineages primarily restricted to mid‐altitude cloud forests began to diversify from the mid‐ to late‐Miocene and the majority of high‐altitude Páramo lineages began to diversify during the Pliocene. The age of diversification of altitudinally restricted lineages therefore gives an indication of the age at which particular altitudes may have been reached.

FGF mediated MAPK and PI3K/Akt Signals make distinct contributions to pluripotency and the establishment of Neural Crest.

Early vertebrate embryos possess cells with the potential to generate all embryonic cell types. While this pluripotency is progressively lost as cells become lineage restricted, Neural Crest cells retain broad developmental potential. Here, we provide novel insights into signals essential for both pluripotency and neural crest formation in Xenopus. We show that FGF signaling controls a subset of genes expressed by pluripotent blastula cells, and find a striking switch in the signaling cascades activated by FGF signaling as cells lose pluripotency and commence lineage restriction. Pluripotent cells display and require Map Kinase signaling, whereas PI3 Kinase/Akt signals increase as developmental potential is restricted, and are required for transit to certain lineage restricted states. Importantly, retaining a high Map Kinase/low Akt signaling profile is essential for establishing Neural Crest stem cells. These findings shed important light on the signal-mediated control of pluripotency and the molecular mechanisms governing genesis of Neural Crest.

Fibroblast heterogeneity: implications for human disease.

Fibroblasts synthesize the extracellular matrix of connective tissue and play an essential role in maintaining the structural integrity of most tissues. Researchers have long suspected that fibroblasts exhibit functional specialization according to their organ of origin, body site, and spatial location. In recent years, a number of approaches have revealed the existence of fibroblast subtypes in mice. Here, we discuss fibroblast heterogeneity with a focus on the mammalian dermis, which has proven an accessible and tractable system for the dissection of these relationships. We begin by considering differences in fibroblast identity according to anatomical site of origin. Subsequently, we discuss new results relating to the existence of multiple fibroblast subtypes within the mouse dermis. We consider the developmental origin of fibroblasts and how this influences heterogeneity and lineage restriction. We discuss the mechanisms by which fibroblast heterogeneity arises, including intrinsic specification by transcriptional regulatory networks and epigenetic factors in combination with extrinsic effects of the spatial context within tissue. Finally, we discuss how fibroblast heterogeneity may provide insights into pathological states including wound healing, fibrotic diseases, and aging. Our evolving understanding suggests that ex vivo expansion or in vivo inhibition of specific fibroblast subtypes may have important therapeutic applications.

Establishing Primary Human Glioblastoma Adherent Cultures from Operative Specimens.

This chapter describes a method for isolation, maintenance, and propagation of primary glioblastoma (GBM) cells in adherent monolayer cultures from patient tumor specimens. This method enables the establishment of GBM cultures with stem or progenitor-like cell characteristics, including self-renewal capacity, differentiation along restricted neural lineages, and tumor-initiating potential when orthotopically injected into immunocompromised mice. This experimentally tractable model system is therefore suitable for a wide variety of analyses in vitro as well as in vivo. Key examples of biological analyses that can be performed using these cells are also described.

HOXA5 protein expression and genetic fate mapping show lineage restriction in the developing musculoskeletal system.

HOX proteins act during development to regulate musculoskeletal morphology. HOXA5 patterns skeletal structures surrounding the cervical-thoracic transition including the vertebrae, ribs, sternum and forelimb girdle. However, the tissue types in which it acts to pattern the skeleton, and the ultimate fates of embryonic cells that activate Hoxa5 expression are unknown. A detailed characterization of HOXA5 expression by immunofluorescence was combined with Cre/LoxP genetic lineage tracing to map the fate of Hoxa5 expressing cells in axial musculoskeletal tissues and in their precursors, the somites and lateral plate mesoderm. HOXA5 protein expression is dynamic and spatially restricted in derivatives of both the lateral plate mesoderm and somites, including a subset of the lateral sclerotome, suggesting a local role in regulating early skeletal patterning. HOXA5 expression persists from somite stages through late development in differentiating skeletal and connective tissues, pointing to a continuous and direct role in skeletal patterning. In contrast, HOXA5 expression is excluded from the skeletal muscle and muscle satellite cell lineages. Furthermore, the descendants of Hoxa5-expressing cells, even after HOXA5 expression has extinguished, never contribute to these lineages. Together, these findings suggest cell autonomous roles for HOXA5 in skeletal development, as well as non-cell autonomous functions in muscle through expression in surrounding connective tissues. They also support the notion that different Hox genes display diverse tissue specificities and locations to achieve their patterning activity.

Connexin 32 and connexin 43 are involved in lineage restriction of hepatic progenitor cells to hepatocytes

BackgroundBi-potential hepatic progenitor cells can give rise to both hepatocytes and cholangiocytes, which is the last phase and critical juncture in terms of sequentially hepatic lineage restriction from any kind of stem cells. If their differentiation can be controlled, it might access to functional hepatocytes to develop pharmaceutical and biotechnology industries as well as cell therapies for end-stage liver diseases.MethodsIn this study, we investigated the influence of Cx32 and Cx43 on hepatocyte differentiation of WB-F344 cells by in vitro gain and loss of function analyses. An inhibitor of Cx32 was also used to make further clarification. To reveal p38 MAPK pathway is closely related to Cxs, rats with 70% partial hepatectomy were injected intraperitoneally with a p38 inhibitor, SB203580. Besides, the effects of p38 MAPK pathway on differentiation of hepatoblasts isolated from fetal rat livers were evaluated by addition of SB203580 in culture medium.ResultsIn vitro gain and loss of function analyses showed overexpression of Connexin 32 and knockdown of Connexin 43 promoted hepatocytes differentiation from hepatic progenitor cells. In addition, in vitro and ex vivo research revealed inhibition of p38 mitogen-activated protein kinase pathway can improve hepatocytes differentiation correlating with upregulation of Connexin 32 expression and downregulation of Connexin 43 expression.ConclusionsHere we demonstrate that Connexins play crucial roles in facilitating differentiation of hepatic progenitors. Our work further implicates that regulators of Connexins and their related pathways might provide new insights to improve lineage restriction of stem cells to mature hepatocytes.

Hedgehog mediated degradation of Ihog adhesion proteins modulates cell segregation in Drosophila wing imaginal discs

The Drosophila Hedgehog receptor functions to regulate the essential downstream pathway component, Smoothened, and to limit the range of signaling by sequestering Hedgehog protein signal within imaginal disc epithelium. Hedgehog receptor function requires both Patched and Ihog activity, the latter interchangeably encoded by interference hedgehog (ihog) or brother of ihog (boi). Here we show that Patched and Ihog activity are mutually required for receptor endocytosis and degradation, triggered by Hedgehog protein binding, and causing reduced levels of Ihog/Boi proteins in a stripe of cells at the anterior/posterior compartment boundary of the wing imaginal disc. This Ihog spatial discontinuity may contribute to classically defined cell segregation and lineage restriction at the anterior/posterior wing disc compartment boundary, as suggested by our observations that Ihog activity mediates aggregation of otherwise non-adherent cultured cells and that loss of Ihog activity disrupts wing disc cell segregation, even with downstream genetic rescue of Hedgehog signal response.

Genome-Nuclear Lamina Interactions Regulate Cardiac Stem Cell Lineage Restriction

Progenitor cells differentiate into specialized cell types through coordinated expression of lineage-specific genes and modification of complex chromatin configurations. We demonstrate that a histone deacetylase (Hdac3) organizes heterochromatin at the nuclear lamina during cardiac progenitor lineage restriction. Specification of cardiomyocytes is associated with reorganization of peripheral heterochromatin, and independent of deacetylase activity, Hdac3 tethers peripheral heterochromatin containing lineage-relevant genes to the nuclear lamina. Deletion of Hdac3 in cardiac progenitor cells releases genomic regions from the nuclear periphery, leading to precocious cardiac gene expression and differentiation into cardiomyocytes; in contrast, restricting Hdac3 to the nuclear periphery rescues myogenesis in progenitors otherwise lacking Hdac3. Our results suggest that availability of genomic regions for activation by lineage-specific factors is regulated in part through dynamic chromatin-nuclear lamina interactions and that competence of a progenitor cell to respond to differentiation signals may depend upon coordinated movement of responding gene loci away from the nuclear periphery.

Molecular and Functional Characterization of Lymphoid Progenitor Subsets Reveals a Bipartite Architecture of Human Lymphopoiesis

The classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127- and CD127+ early lymphoid progenitors (ELPs). Combining molecular analyses with invitro and invivo functional assays, we demonstrated that CD127- and CD127+ ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127- ELPs comprised precursors of Tcells, marginal zone Bcells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127+ ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis.

Effects of ß-TCP scaffolds on neurogenic and osteogenic differentiation of human embryonic stem cells

Extracellular matrix (ECM) and adhesion molecules play crucial roles in regulating growth and differentiation of stem cells. The current study aimed to investigate the effects of beta-tricalcium phosphate (ß-TCP) scaffolds on differentiation and expression of ECM and adhesion molecules of human embryonic stem cells (hESCs). Undifferentiated hESCs were seeded on ß-TCP scaffolds and cell culture plates and cultured in growth and osteogenic medium for 21 days. Scanning electron microscopy (SEM) displayed adhesion and growth of hESCs on the porous ß-TCP scaffolds. Histological analysis, immunohistochemical staining and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) demonstrated that the scaffolds supported growth and differentiation of hESCs. Expression levels of neural crest related genes (AP2a, FoxD3, HNK1, P75, Sox1, Sox10) and osteoblast-related genes (Runx2, SPP1 and BGLA) on the scaffolds in osteogenic medium were significantly higher than on the scaffolds in growth and cell culture plates in osteogenic medium, respectively (p<0.05). Polymerase chain reaction array experiments demonstrated increased expression of ECM and adhesion molecule-related genes on the scaffolds. In conclusion, osteoconductive scaffolds such as ß-TCP scaffolds promoted differentiation of hESCs, particularly expression of genes related to neural crest stem cell and osteoblastic differentiations. Beta-TCP scaffolds could be an alternative cell culture substrate for neural crest and osteogenic differentiation of hESCs. Optimization of culture medium may be necessary to enhance lineage restriction of hESCs on the ß-TCP scaffolds.

Epigenetic restriction of extraembryonic lineages mirrors the somatic transition to cancer.

In mammals, the canonical somatic DNA methylation landscape is established upon specification of the embryo proper and subsequently disrupted within many cancer types1-4. However, the underlying mechanisms that direct this genome-scale transformation remain elusive, with no clear model for its systematic acquisition or potential developmental utility5,6. Here we analyzed global remethylation from the mouse preimplantation embryo into the early epiblast and extraembryonic ectoderm. We show that these two states acquire highly divergent genomic distributions with substantial disruption of bimodal, CpG density-dependent methylation in the placental progenitor7,8. The extraembryonic epigenome includes specific de novo methylation at hundreds of embryonically-protected CpG island promoters particularly those that are associated with key developmental regulators and orthologously methylated across most human cancer types9. Our data suggest that the evolutionary innovation of extraembryonic tissues may have required cooption of DNA methylation-based suppression as an alternative to the embryonically utilized Polycomb group proteins, which coordinate germlayer formation in response to extraembryonic cues10. Moreover, we establish that this decision is made deterministically downstream of promiscuously utilized, and frequently oncogenic, signaling pathways via a novel combination of epigenetic cofactors. Methylation of developmental gene promoters during tumorigenesis may therefore reflect the misappropriation of an innate trajectory and the spontaneous reacquisition of a latent, developmentally-encoded epigenetic landscape.

Temporal Expression of Wnt1 Defines the Competency State and Terminal Identity of Progenitors in the Developing Cochlear Nucleus and Inferior Colliculus.

The auditory system contains a diverse array of interconnected anatomical structures that mediate the perception of sound. The cochlear nucleus of the hindbrain serves as the initial site of convergence for auditory stimuli, while the inferior colliculus of the midbrain serves as an integration and relay station for all ascending auditory information. We used Genetic Inducible Fate Mapping (GIFM) to determine how the timing of Wnt1 expression is related to the competency states of auditory neuron progenitors. We demonstrate that the Wnt1 lineage defines progenitor pools of auditory neurons in the developing midbrain and hindbrain. The timing of Wnt1 expression specifies unique cell types during embryogenesis and follows a mixed model encompassing a brief epoch of de novo expression followed by rapid and progressive lineage restriction to shape the inferior colliculus. In contrast, Wnt1 fate mapping of the embryonic hindbrain revealed de novo induction of Wnt1 in auditory hindbrain progenitors, which is related to the development of biochemically distinct neurons in the cochlear nucleus. Thus, we uncovered two modes of lineage allocation that explain the relationship between the timing of Wnt1 expression and the development of the cochlear nucleus and the inferior colliculus. Finally, our analysis of Wnt1sw/sw mutant mice demonstrated a functional requirement of Wnt1 for the development of auditory midbrain and hindbrain neurons. Collectively, our study provides a deeper understanding of Wnt1 lineage allocation and function in mammalian brain development.

Asynchronous lineage priming determines commitment to T cell and B cell lineages in fetal liver.

The molecular events that initiate lymphoid-lineage specification remain unidentified because the stages of differentiation during which lineage commitment occurs are difficult to characterize. We isolated fetal liver progenitor cells undergoing restriction of their differentiation potential toward the T cell-innate lymphoid cell lineage or the B cell lineage. Transcripts that defined the molecular signatures of these two subsets were sequentially upregulated in lympho-myeloid precursor cells and in common lymphoid progenitor cells, respectively, and this preceded lineage restriction; this indicates that T cell-versus-B cell commitment is not a binary fate 'decision'. The T cell-bias and B cell-bias transcriptional programs were frequently co-expressed in common lymphoid progenitor cells and were segregated in subsets biased toward T cell differentiation or B cell differentiation, after interleukin 7 (IL-7) signaling that controlled the number of progenitor cells engaging in T cell differentiation versus B cell differentiation.

Midbrain-Hindbrain Boundary Morphogenesis: At the Intersection of Wnt and Fgf Signaling.

A constriction in the neural tube at the junction of the midbrain and hindbrain is a conserved feature of vertebrate embryos. The constriction is a defining feature of the midbrain-hindbrain boundary (MHB), a signaling center that patterns the adjacent midbrain and rostral hindbrain and forms at the junction of two gene expression domains in the early neural plate: an anterior otx2/wnt1 positive domain and a posterior gbx/fgf8 positive domain. otx2 and gbx genes encode mutually repressive transcription factors that create a lineage restriction boundary at their expression interface. Wnt and Fgf genes form a mutually dependent feedback system that maintains their expression domains on the otx2 or gbx side of the boundary, respectively. Constriction morphogenesis occurs after these conserved gene expression domains are established and while their mutual interactions maintain their expression pattern; consequently, mutant studies in zebrafish have led to the suggestion that constriction morphogenesis should be considered a unique phase of MHB development. We analyzed MHB morphogenesis in fgf8 loss of function zebrafish embryos using a reporter driven by the conserved wnt1 enhancer to visualize anterior boundary cells. We found that fgf8 loss of function results in a re-activation of wnt1 reporter expression posterior to the boundary simultaneous with an inactivation of the wnt1 reporter in the anterior boundary cells, and that these events correlate with relaxation of the boundary constriction. In consideration of other results that correlate the boundary constriction with Wnt and Fgf expression, we propose that the maintenance of an active Wnt-Fgf feedback loop is a key factor in driving the morphogenesis of the MHB constriction.

Epigenetic Reprogramming of Lineage-Committed Human Mammary Epithelial Cells Requires DNMT3A and Loss of DOT1L.

SummaryOrganogenesis and tissue development occur through sequential stepwise processes leading to increased lineage restriction and loss of pluripotency. An exception to this appears in the adult human breast, where rare variant epithelial cells exhibit pluripotency and multilineage differentiation potential when removed from the signals of their native microenvironment. This phenomenon provides a unique opportunity to study mechanisms that lead to cellular reprogramming and lineage plasticity in real time. Here, we show that primary human mammary epithelial cells (HMECs) lose expression of differentiated mammary epithelial markers in a manner dependent on paracrine factors and epigenetic regulation. Furthermore, we demonstrate that HMEC reprogramming is dependent on gene silencing by the DNA methyltransferase DNMT3A and loss of histone transcriptional marks following downregulation of the methyltransferase DOT1L. These results demonstrate that lineage commitment in adult tissues is context dependent and highlight the plasticity of somatic cells when removed from their native tissue microenvironment.

Escapees from Rhombomeric Lineage Restriction: Extensive Migration Rostral to the r4/r5 Border of Hox-a3 Expression.

The rhombomeric compartments of the hindbrain are characterized by lineage restriction; cells born in one compartment generally remain there and do not migrate to neighboring rhombomeres. Two well-known exceptions are the substantial migrations of the pontine nuclei and the mammalian facial nucleus. In this study we used Hoxa3-Cre lineage to permanently mark cells that originate in rhombomeres caudal to r4. We found that cells born caudal to the r4/r5 border migrate forwards to a number of different locations in rhombomeres 1-4; the final locations include the interfascicular trigeminal nucleus, the principal trigeminal nucleus, the pontine nuclei, the reticulotegmental nucleus, the ventral nucleus of the lateral lemniscus, and the lateral and medial vestibular nuclei. We suggest that there are numerous exceptions to the principle of rhombomeric lineage restriction that have previously gone unnoticed. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1838-1846, 2017. © 2017 Wiley Periodicals, Inc.

Asymmetrically deployed actomyosin-based contractility generates a boundary between developing leg segments in Drosophila

The formation of complex tissues from simple epithelial sheets requires the regional subdivision of the developing tissue. This is initially accomplished by a sequence of gene regulatory hierarchies that set up distinct fates within adjacent territories, and rely on cross-regulatory interactions to do so. However, once adjacent territories are established, cells that confront one another across territorial boundaries must actively participate in maintaining separation from each other. Classically, it was assumed that adhesive differences would be a primary means of sorting cells to their respective territories. Yet it is becoming clear that no single, simple mechanism is at play. In the few instances studied, an emergent theme along developmental boundaries is the generation of asymmetry in cell mechanical properties. The repertoire of ways in which cells might establish and then put mechanical asymmetry to work is not fully appreciated since only a few boundaries have been molecularly studied. Here, we characterize once such boundary in the develop leg epithelium of Drosophila. The region of the pretarsus / tarsus is a known gene expression boundary that also exhibits a lineage restriction (Sakurai et al., 2007). We now show that the interface comprising this boundary is strikingly aligned compared to other cell interfaces across the disk. The boundary also exhibits an asymmetry for both Myosin II accumulation as well as one of its activators, Rho Kinase. Furthermore, the enrichment correlates with increased mechanical tension across that interface, and that tension is Rho Kinase-dependent. Lastly, interfering with actomyosin contractility, either by depletion of myosin heavy chain or expression of a phosphomimetic variant of regulatory light chain causes defects in alignment of the interfaces. These data suggest strongly that mechanical asymmetries are key in establishing and maintaining this developmental boundary.

Distinct Requirements for FGFR1 and FGFR2 in Primitive Endoderm Development and Exit from Pluripotency

Activation of the FGF signaling pathway during preimplantation development of the mouse embryo is known to be essential for differentiation of the inner cell mass and the formation of the primitive endoderm (PrE). We now show using fluorescent reporter knockin lines that Fgfr1 is expressed in all cell populations of the blastocyst, while Fgfr2 expression becomes restricted to extraembryonic lineages, including the PrE. We further show that loss of both receptors prevents the development of the PrE and demonstrate that FGFR1 plays a more prominent role in this process than FGFR2. Finally, we document an essential role for FGFRs in embryonic stem cell (ESC) differentiation, with FGFR1 again having a greater influence than FGFR2 in ESC exit from the pluripotent state. Collectively, these results identify mechanisms through which FGF signaling regulates inner cell mass lineage restriction and cell commitment during preimplantation development.

Single-Cell Analysis Identifies Distinct Stages of Human Endothelial-to-Hematopoietic Transition

During development, hematopoietic cells originate from endothelium in a process known as endothelial-to-hematopoietic transition (EHT). To study human EHT, we coupled flow cytometry and single-cell transcriptional analyses of human pluripotent stem cell-derived CD34+ cells. The resulting transcriptional hierarchy showed a continuum of endothelial and hematopoietic signatures. At the interface of these two signatures, a unique group of cells displayed both an endothelial signature and high levels of key hematopoietic stem cell-associated genes. This interphase group was validated via sort and subculture as an immediate precursor to hematopoietic cells. Differential expression analyses further divided this population into subgroups, which, upon subculture, showed distinct hematopoietic lineage differentiation potentials. We therefore propose that immediate precursors to hematopoietic cells already have their hematopoietic lineage restrictions defined prior to complete downregulation of the endothelial signature. These findings increase our understanding of the processes of de novo hematopoietic cell generation in the human developmental context.