Regeneration of segment boundaries in oncopeltus: cell lineage

The segment boundary of Oncopeltus is a straight interface between the cells of two segmental compartments; it coincides with an abrupt change of pigmentation and a groove in the surface of the cuticle. When a segment boundary is burnt it is regenerated by the cells migrating into the wound. We describe the cell lineage of this process: the two masses of migrating cells first contact each other on a convoluted frontier, but this soon straightens. The new boundary forms exactly at the interface between the two cell populations, without a single cell straying across the line. Even when the cautery is asymmetric and, as a consequence, the cells from the different segments meet at an abnormal position, the segment boundary is regenerated at this interface. After extirpation or transplantation ectopic boundaries can be formed within a segment. These new boundaries also act as lineage restrictions and stop mingling of cells across them. Our results support the hypothesis that the integrity and straightness of the compartment boundary depend on cell affinities; the cells in one polyclone adhering more to each other than to the cells of the neighbouring polyclone. There may be a gradient of cell affinity in each segment, with an abrupt change at the segment boundary.     

The LRR proteins capricious and Tartan mediate cell interactions during DV boundary formation in the Drosophila wing

Mechanisms to segregate cell populations play important roles in tissue patterning during animal development. Rhombomeres and compartments in the ectoderm and imaginal discs of Drosophila are examples in which initially homogenous populations of cells come to be separated by boundaries of lineage restriction. Boundary formation depends in part on signaling between the distinctly specified cell populations that comprise compartments and in part on formation of affinity boundaries that prevent intermingling of these cell populations. Here, we present evidence that two transmembrane proteins with leucine-rich repeats, known as Capricious and Tartan, contribute to formation of the affinity boundary between dorsal and ventral compartments during Drosophila wing development.     

Patterns of growth, axonal extension and axonal arborization of neuronal lineages in the developing Drosophila brain

The Drosophila central brain is composed of approximately 100 paired lineages, with most lineages comprising 100-150 neurons. Most lineages have a number of important characteristics in common. Typically, neurons of a lineage stay together as a coherent cluster and project their axons into a coherent bundle visible from late embryo to adult. Neurons born during the embryonic period form the primary axon tracts (PATs) that follow stereotyped pathways in the neuropile. Apoptotic cell death removes an average of 30-40% of primary neurons around the time of hatching. Secondary neurons generated during the larval period form secondary axon tracts (SATs) that typically fasciculate with their corresponding primary axon tract. SATs develop into the long fascicles that interconnect the different compartments of the adult brain. Structurally, we distinguish between three types of lineages: PD lineages, characterized by distinct, spatially separate proximal and distal arborizations; C lineages with arborizations distributed continuously along the entire length of their tract; D lineages that lack proximal arborizations. Arborizations of many lineages, in particular those of the PD type, are restricted to distinct neuropile compartments. We propose that compartments are “scaffolded” by individual lineages, or small groups thereof. Thereby, the relatively small number of primary neurons of each primary lineage set up the compartment map in the late embryo. Compartments grow during the larval period simply by an increase in arbor volume of primary neurons. Arbors of secondary neurons form within or adjacent to the larval compartments, resulting in smaller compartment subdivisions and additional, adult specific compartments.     

Compartment boundaries

The subdivision of proliferating tissues into groups of non-intermingling sets of cells, termed compartments, is a common process of animal development. Signaling between adjacent compartments induces the local expression of morphogens that pattern the surrounding tissue. Sharp and straight boundaries between compartments stabilize the source of such morphogens during tissue growth and, thus, are of crucial importance for pattern formation. Signaling pathways required to maintain compartment boundaries have been identified, yet the physical mechanisms that maintain compartment boundaries remained elusive. Recent data now show that a local increase in actomyosin-based mechanical tension on cell bonds is vital for maintaining compartment boundaries in Drosophila.     

Epigenetic mechanisms that regulate cell identity

Individual cell fate decisions can vary according to changes in gene expression in response to environmental, developmental, or metabolic cues. This plasticity is tightly regulated during embryonic development and mediated by the exquisitely coordinated activation and repression of groups of genes. Genes that become repressed are immersed in a condensed chromatin environment that renders them refractory to stimulation. This mechanism is responsible for both the loss of cell plasticity during differentiation and the preservation of cell identity. Understanding the molecular events involved in the establishment and maintenance of these restrictive domains will benefit the design of strategies for cellular reprogramming, differentiation, and cancer treatment.     

Notch signaling is not sufficient to define the affinity boundary between dorsal and ventral compartments

The developing limbs of Drosophila are subdivided into distinct cells populations known as compartments. Short-range interaction between cells in adjacent compartments induces expression of signaling molecules at the compartment boundaries. In addition to serving as the sources of long-range signals, compartment boundaries prevent mixing of the adjacent cell populations. One model for boundary formation proposes that affinity differences between compartments are defined autonomously as one aspect of compartment-specific cell identity. An alternative is that the affinity boundary depends on signaling between compartments. Here, we present evidence that the dorsal selector gene apterous plays a role in establishing the dorsoventral affinity boundary that is independent of Notch-mediated signaling between dorsal and ventral cells.     

Compartments and appendage development in Drosophila

The appendages of Drosophila develop from the imaginal discs. During the extensive growth of these discs cell lineages are for the most part unfixed, suggesting a strong role for cell-cell interactions in controlling the final pattern of differentiation. However, during early and middle stages of development, discs are subdivided by strict lineage restrictions into a small number of spatially distinct compartments. These compartments appear to be maintained by stably inheriting states of gene expression; the compartmentspecific expression of two such ‘selector’ - like genes, engrailed and apterous, are critical for anterior-posterior and dorso-ventral compartmentalization, respectively. Recent work suggests that one purpose of compartmentalization is to establish regions of specialized cells near compartment boundaries via intercompartmental induction, using molecules like the hedgehog protein. Thus, compartments can act as organizing centers for patterning within compartments. Evidence for non-compartmental patterning mechanisms will also be discussed.     

Connecting the dots: Melanoma cell of origin,tumor cell plasticity,trans-differentiation,and drug resistance

Melanoma, a lethal malignancy that arises from melanocytes, exhibits a multiplicity of clinico-pathologically distinct subtypes in sun-exposed and non-sun-exposed areas. Melanocytes are derived from multipotent neural crest cells and are present in diverse anatomical locations, including skin, eyes, and various mucosal membranes. Tissue-resident melanocyte stem cells and melanocyte precursors contribute to melanocyte renewal. Elegant studies using mouse genetic models have shown that melanoma can arise from either melanocyte stem cells or differentiated pigment-producing melanocytes depending on a combination of tissue and anatomical site of origin and activation of oncogenic mutations (or overexpression) and/or the repression in expression or inactivating mutations in tumor suppressors. This variation raises the possibility that different subtypes of human melanomas (even subsets within each subtype) may also be a manifestation of malignancies of distinct cells of origin. Melanoma is known to exhibit phenotypic plasticity and trans-differentiation (defined as a tendency to differentiate into cell lineages other than the original lineage from which the tumor arose) along vascular and neural lineages. Additionally, stem cell-like properties such as pseudo-epithelial-to-mesenchymal (EMT-like) transition and expression of stem cell-related genes have also been associated with the development of melanoma drug resistance. Recent studies that employed reprogramming melanoma cells to induced pluripotent stem cells have uncovered potential relationships between melanoma plasticity, trans-differentiation, and drug resistance and implications for cell or origin of human cutaneous melanoma. This review provides a comprehensive summary of the current state of knowledge on melanoma cell of origin and the relationship between tumor cell plasticity and drug resistance.     

The neurosteroid dehydroepiandrosterone could improve somatic cell reprogramming

Expression of four major reprogramming transgenes, including Oct4, Sox2, Klf4 and c-myc, in somatic cells enables them to have pluripotency. These cells are iPSC (induced pluripotent stem cell) that currently show the greatest potential for differentiation into cells of the three germ lineages. One of the issues facing the successful reprogramming and clinical translation of iPSC technology is the high rate of apoptosis after the reprogramming process. Reprogramming is a stressful process, and the p53 apoptotic pathway plays a negative role in cell growth and self-renewal. Apoptosis via the p53 pathway serves as a major barrier in nuclear somatic cell reprogramming during iPSC generation. DHEA (dehydroepiandrosterone) is an abundant steroid that is produced at high levels in the adrenal cells, and withdrawal of DHEA increases the levels of p53 in the epithelial and stromal cells, resulting in increased levels of apoptotic cells; meanwhile, DHEA decreases cellular apoptosis. DHEA could improve the efficacy of reprogramming yield due to a decrease in apoptosis via the p53 pathway and an increase in cell viability.     

Compartments in the abdomen of Drosophila and the role of the engrailed locus

A clonal analysis has shown that the dorsal surface of the first abdominal segment of Drosophila melanogaster is subdivided into anterior and posterior compartments. Cells of the posterior compartment grow up to but not beyond the anterior-posterior compartment border within the first abdominal segment and the intersegmental border that defines the boundary between the first and second abdominal segments. Growing within these boundaries, a narrow band of tissue clonally isolated from the adjoining tissue is formed. When these posterior cells are deficient for the engrailed locus, however, neither the compartment nor the segment border is maintained. The implications, that compartmentalization is essential for segmentation, and that all insect segments are subdivided by anterior and posterior compartments, are discussed.     

The Role of DNA Methylation Reprogramming During Sex Determination and Transition in Zebrafish

DNA methylation is a prevalent epigenetic modification in vertebrates, and it has been shown to be involved the regulation of gene expression and embryo development. However, it remains unclear how DNA methylation regulates sexual development, especially in species without sex chromosomes. To determine this, we utilized zebrafish to investigate DNA methylation reprogramming during juvenile germ cell development and adult female-to-male sex transition.We reveal that primordial germ cells(PGCs) undergo significant DNA methylation reprogramming during germ cell development, and the methylome of PGCs is reset to an oocyte/ovary-like pattern at 9 days post fertilization(9 dpf). When DNA methyltransferase(DNMT) activity in juveniles was blocked after 9 dpf, the zebrafish developed into females. We also show that Tet3 is involved in PGC development. Notably, we find that DNA methylome reprogramming during adult zebrafish sex transition is similar to the reprogramming during the sex differentiation from 9 dpf PGCs to sperm. Furthermore, inhibiting DNMT activity can prevent the female-to-male sex transition, suggesting that methylation reprogramming is required for zebrafish sex transition. In summary, DNA methylation plays important roles in zebrafish germ cell development and sexual plasticity.     

Overcoming the hurdles for a reproducible generation of human functionally mature reprogrammed neurons

The advent of cell reprogramming technologies has widely disclosed the possibility to have direct access to human neurons for experimental and biomedical applications. Human pluripotent stem cells can be instructed in vitro to generate specific neuronal cell types as well as different glial cells. Moreover, new approaches of direct neuronal cell reprogramming can strongly accelerate the generation of different neuronal lineages. However, genetic heterogeneity, reprogramming fidelity, and time in culture of the starting cells can still significantly bias their differentiation efficiency and quality of the neuronal progenies. In addition, reprogrammed human neurons exhibit a very slow pace in gaining a full spectrum of functional properties including physiological levels of membrane excitability, sustained and prolonged action potential firing, mature synaptic currents and synaptic plasticity. This delay poses serious limitations for their significance as biological experimental model and screening platform. We will discuss new approaches of neuronal cell differentiation and reprogramming as well as methods to accelerate the maturation and functional activity of the converted human neurons.     

hedgehog and engrailed: pattern formation and polarity in the Drosophila abdomen

Like the Drosophila embryo, the abdomen of the adult consists of alternating anterior (A) and posterior (P) compartments. However the wing is made by only part of one A and part of one P compartment. The abdomen therefore offers an opportunity to compare two compartment borders (A/P is within the segment and P/A intervenes between two segments), and ask if they act differently in pattern formation. In the embryo, abdomen and wing P compartment cells express the selector gene engrailed and secrete Hedgehog protein whilst A compartment cells need the patched and smoothened genes in order to respond to Hedgehog. We made clones of cells with altered activities of the engrailed, patched and smoothened genes. Our results confirm (1) that the state of engrailed, whether 'off' or 'on', determines whether a cell is of A or P type and (2) that Hedgehog signalling, coming from the adjacent P compartments across both A/P and P/A boundaries, organises the pattern of all the A cells. We have uncovered four new aspects of compartments and engrailed in the abdomen. First, we show that engrailed acts in the A compartment: Hedgehog leaves the P cells and crosses the A/P boundary where it induces engrailed in a narrow band of A cells. engrailed causes these cells to form a special type of cuticle. No similar effect occurs when Hedgehog crosses the P/A border. Second, we look at the polarity changes induced by the clones, and build a working hypothesis that polarity is organised, in both compartments, by molecule(s) emanating from the A/P but not the P/A boundaries. Third, we show that both the A and P compartments are each divided into anterior and posterior subdomains. This additional stratification makes the A/P and the P/A boundaries fundamentally distinct from each other. Finally, we find that when engrailed is removed from P cells (of, say, segment A5) they transform not into A cells of the same segment, but into A cells of the same parasegment (segment A6).     

Is a mosaic embryo also a mosaic of communication compartments?

We have studied the pathways of cell communication in embryos of the mollusc Lymnaea stagnalis in which the developmental fate of a cell or a group of cells is known from cell lineage studies. We iontophoretically injected Lucifer Yellow CH and followed the spread of fluorescence between cells interconnected via gap junctions. In early stages all blastomeres appear to be dye-coupled, but later on communication is restricted within compartments. The pattern of cell communication corresponds with the development of compartments with specific cell fates. Dye-spread is limited by communication boundaries which completely or mostly prevent the passage of dye to adjacent compartments with different developmental fates. These boundaries appear progressively during development. Our results suggest that, during the development of Lymnaea, the progressive changes in the pattern of dye spread correspond with the progressive restrictions of the developmental fates of individual cells or groups of cells. We conclude that changes in the pattern of cell communication and in the appearance of communication compartments are not exclusive features of regulative embryos.     

msh specifies dorsal cell fate in the Drosophila wing

Drosophila limbs develop from imaginal discs that are subdivided into compartments. Dorsal-ventral subdivision of the wing imaginal disc depends on apterous activity in dorsal cells. Apterous protein is expressed in dorsal cells and is responsible for (1) induction of a signaling center along the dorsal-ventral compartment boundary (2) establishment of a lineage restriction boundary between compartments and (3) specification of dorsal cell fate. Here, we report that the homeobox gene msh (muscle segment homeobox) acts downstream of apterous to confer dorsal identity in wing development.     

Signalling from hindbrain boundaries regulates neuronal clustering that patterns neurogenesis

During central nervous system development, neural progenitors are patterned to form discrete neurogenic and non-neurogenic zones. In the zebrafish hindbrain, neurogenesis is organised by Fgf20a emanating from neurons located at each segment centre that inhibits neuronal differentiation in adjacent progenitors. Here, we have identified a molecular mechanism that clusters fgf20a-expressing neurons in segment centres and uncovered a requirement for this positioning in the regulation of neurogenesis. Disruption of hindbrain boundary cell formation alters the organisation of fgf20a-expressing neurons, consistent with a role of chemorepulsion from boundaries. The semaphorins Sema3fb and Sema3gb, which are expressed by boundary cells, and their receptor Nrp2a are required for clustering of fgf20a-expressing neurons at segment centres. The dispersal of fgf20a-expressing neurons that occurs following the disruption of boundaries or of Sema3fb/Sema3gb signalling leads to reduced FGF target gene expression in progenitors and an increased number of differentiating neurons. Sema3 signalling from boundaries thus links hindbrain segmentation to the positioning of fgf20a-expressing neurons that regulates neurogenesis.