Labeling every neuron in a lineage in the fruit fly olfactory system reveals that every cell is born with a pre-determined cell fate that is invariant and dependent upon neuron birth order
Drosophila brains contain numerous neurons that form complex circuits. These neurons are derived in stereotyped patterns from a fixed number of progenitors, called neuroblasts, and identifying individual neurons made by a neuroblast facilitates the reconstruction of neural circuits. An improved MARCM (mosaic analysis with a repressible cell marker) technique, called twin-spot MARCM, allows one to label the sister clones derived from a common progenitor simultaneously in different colors. It enables identification of every single neuron in an extended neuronal lineage based on the order of neuron birth. Here we report the first example, to our knowledge, of complete lineage analysis among neurons derived from a common neuroblast that relay olfactory information from the antennal lobe (AL) to higher brain centers. By identifying the sequentially derived neurons, we found that the neuroblast serially makes 40 types of AL projection neurons (PNs). During embryogenesis, one PN with multi-glomerular innervation and 18 uniglomerular PNs targeting 17 glomeruli of the adult AL are born. Many more PNs of 22 additional types, including four types of polyglomerular PNs, derive after the neuroblast resumes dividing in early larvae. Although different offspring are generated in a rather arbitrary sequence, the birth order strictly dictates the fate of each post-mitotic neuron, including the fate of programmed cell death. Notably, the embryonic progenitor has an altered temporal identity following each self-renewing asymmetric cell division. After larval hatching, the same progenitor produces multiple neurons for each cell type, but the number of neurons for each type is tightly regulated. These observations substantiate the origin-dependent specification of neuron types. Sequencing neuronal lineages will not only unravel how a complex brain develops but also permit systematic identification of neuron types for detailed structure and function analysis of the brain.
A brain consists of numerous, potentially individually unique neurons that derive from a limited number of progenitors. It has been shown in various model organisms that specific neurons arise in a lineage made by a repeatedly renewing progenitor at specific times of development. However, except in the worm C. elegans, the stereotype of neural development has never been examined in sufficient detail to account for every single neuron derived from a common progenitor. Here we applied a sophisticated genetic mosaic system to mark single neurons in the adult Drosophila brain and simultaneously reveal in which lineage a targeted neuron had arisen and when along the lineage it was made. We have identified each neuron in a lineage of olfactory projection neurons. There are a remarkable 40 types of neurons within this lineage born over two epochs. Strikingly, the birth order strictly dictates the fate of each post-mitotic neuron, including the fate of programmed cell death, such that every neuron type has a unique and invariant cell count. Sequencing an entire neuronal lineage provides definitive evidence for origin-dependent neuron type specification. It further permits a systematic characterization of neuron types for comprehensive circuitry mapping.
Crossveinless-2 (Cv2), Twisted Gastrulation (Tsg) and Chordin (Chd) are components of an extracellular biochemical pathway that regulates Bone Morphogenetic Protein (BMP) activity during dorso-ventral patterning of Drosophila and Xenopus embryos, the formation of the fly wing, and mouse skeletogenesis. Because the nature of their genetic interactions remained untested in the mouse, we generated a null allele for Cv2 which was crossed to Tsg and Chd mutants to obtain Cv2;Tsg and Cv2;Chd compound mutants. We found that Cv2 is essential for skeletogenesis as its mutation caused the loss of multiple bone structures and posterior homeotic transformation of the last thoracic vertebra. During early vertebral development, Smad1 phosphorylation in the intervertebral region was decreased in the Cv2 mutant, even though CV2 protein is normally located in the future vertebral bodies. Because Cv2 mutation affects BMP signaling at a distance, this suggested that CV2 is involved in the localization of the BMP morphogenetic signal. Cv2 and Chd mutations did not interact significantly. However, mutation of Tsg was epistatic to all CV2 phenotypes. We propose a model in which CV2 and Tsg participate in the generation of a BMP signaling morphogenetic field during vertebral formation in which CV2 serves to concentrate diffusible Tsg/BMP4 complexes in the vertebral body cartilage.
BMP; Crossveinless-2; Chordin; Twisted Gastrulation; Tolloid; vertebra; morphogenetic field; cartilage; pattern formation
Hemangioblasts are bi-potential precursors for blood and endothelial cells (BCs and ECs). Existence of the hemangioblast in vivo by its strict definition, i.e. a clonal precursor giving rise to these two cell types after division, is still debated. Using a combination of mitotic figure analysis, cell labeling and long-term cell tracing, we show that, in chicken, cell division does not play a major role during the entire ventral mesoderm differentiation process after gastrulation. One eighth of cells do undergo at least one round of division, but mainly give rise to daughter cells contributing to the same lineage. Approximately 7% of the dividing cells that contribute to either the BC or EC lineage meet the criteria of true hemangioblasts, with one daughter cell becoming a BC and the other an EC. Our data suggest that hemangioblast-type generation of BC/EC occurs, but is not used as a major mechanism during early chicken development. It remains unclear, however, whether hemangioblast-like progenitor cells play a more prominent role in later development.
A comprehensive understanding of the brain requires analysis of individual neurons. Here we describe twin-spot MARCM for high-resolution lineage tracing by independent labeling of paired sister clones. We determine patterns of neurogenesis and the influences of lineage on neuron-type specification. Notably, neural progenitors may yield intermediate precursors that make one, two, or more neurons. Furthermore, neurons acquire stereotyped projections according to their temporal position within various brain sublineages. Twin-spot MARCM also permits birth dating of mutant clones, enabling the detection of a single temporal fate that requires chinmo in a sublineage of six Drosophila central complex neurons. In sum, twin-spot MARCM can reveal the developmental origins of neurons and the mechanisms that underlie cell fate.
Asymmetric division of progenitor/stem cells generates both self-renewing and differentiating progeny and is fundamental to development and regeneration. How this process is regulated in the vertebrate brain remains incompletely understood. Here we use time-lapse imaging to track radial glia progenitor behavior in the developing zebrafish brain. We find that asymmetric division invariably generates a basal self-renewing daughter and an apical differentiating sibling. Gene expression and genetic mosaic analysis further show that the apical daughter is the source of Notch ligand that is essential to maintain higher Notch activity in the basal daughter. Notably, establishment of this intra-lineage and directional Notch signaling requires the intrinsic polarity regulator Partitioning defective protein-3 (Par-3), which segregates the fate determinant Mind bomb unequally to the apical daughter, thereby restricting the self-renewal potential to the basal daughter. These findings reveal with single-cell resolution how self-renewal and differentiation become precisely segregated within asymmetrically dividing neural progenitor/stem lineages.
single cell imaging analysis in vivo; proliferation; cancer; dysplasia; neural stem cell; clonal analysis; in vivo lineage tracing; interkinetic nuclear migration (INM)
A high-resolution neuronal lineage analysis in the Drosophila antennal lobe reveals the complexity of lineage development and Notch signaling in cell fate specification.
Binary cell fate decisions allow the production of distinct sister neurons from an intermediate precursor. Neurons are further diversified based on the birth order of intermediate precursors. Here we examined the interplay between binary cell fate and birth-order-dependent temporal fate in the Drosophila lateral antennal lobe (lAL) neuronal lineage. Single-cell mapping of the lAL lineage by twin-spot mosaic analysis with repressible cell markers (ts-MARCM) revealed that projection neurons (PNs) and local interneurons (LNs) are made in pairs through binary fate decisions. Forty-five types of PNs innervating distinct brain regions arise in a stereotyped sequence; however, the PNs with similar morphologies are not necessarily born in a contiguous window. The LNs are morphologically less diverse than the PNs, and the sequential morphogenetic changes in the two pairs occur independently. Sanpodo-dependent Notch activity promotes and patterns the LN fates. By contrast, Notch diversifies PN temporal fates in a Sanpodo-dispensable manner. These pleiotropic Notch actions underlie the differential temporal fate specification of twin neurons produced by common precursors within a lineage, possibly by modulating postmitotic neurons' responses to Notch-independent transcriptional cascades.
The Drosophila brain develops from a limited number of neural stem cells that produce a series of ganglion mother cells (GMCs) that divide once to produce a pair of neurons in a defined order, termed a neuronal lineage. Here, we provide a detailed lineage map for the neurons derived from the Drosophila lateral antennal lobe (lAL) neuroblast. The lAL lineage consists of two distinct hemilineages, generated through differential Notch signaling in the two GMC daughters, to produce one projection neuron (PN) paired with a local interneuron (LN). Both hemilineages yield distinct cell types in the same sequence, although the temporal identity (birth-order-dependent fate) changes are regulated independently between projection neurons and local interneurons, such that a series of analogous local interneurons may co-derive with different projection neurons and vice versa. We also find that Notch signaling can transform a class of nonantennal lobe projection neurons into antennal lobe projection neurons. These findings suggest that Notch signaling not only modulates temporal fate but itself plays a role in the distinction of antennal lobe versus nonantennal lobe neurons.
Asymmetric positioning of the mitotic spindle before cytokinesis can produce different-sized daughter cells that have distinct fates. Here, we found an asymmetric division in the Caenorhabditis elegans Q neuroblast lineage that began with a centered spindle but generated different-sized daughters, the smaller (anterior) of which underwent apoptosis. During this division, more myosin II accumulated anteriorly, suggesting that asymmetric contractile forces might produce different-sized daughters. Indeed, partial inactivation of anterior myosin by chromophore-assisted laser inactivation created a more symmetric division and allowed the survival and differentiation of the anterior daughter. Thus, the balance of myosin activity on the two sides of a dividing cell can govern the size and fate of the daughters.
Genetic screens in the fruit fly Drosophila melanogaster have identified a class of neoplastic tumor suppressor genes (endocytic nTSGs) that encode proteins that localize to endosomes and facilitate the trafficking of membrane-bound receptors and adhesion molecules into the degradative lysosome. Loss of endocytic nTSGs transforms imaginal disc epithelia into highly proliferative, invasive tissues that fail to differentiate and display defects in cellular apicobasal polarity, adhesion and tissue architecture. As vertebrate homologs of some Drosophila nTSGs are linked to tumor formation, identifying molecular changes in signaling associated with nTSG loss could inform understanding of neoplastic transformation in vertebrates. Here, we show that mutations in genes that act at multiple steps of the endolysosomal pathway lead to autonomous activation of the Sav/Wts/Hpo (SWH) transcriptional effector Yki (YAP/TAZ in vertebrates) and the Jun N-terminal kinase (JNK), which is known to promote Yki activity in cells with disrupted polarity. Yki and JNK activity are elevated by mutations at multiple steps in the endolysosomal pathway, including mutations in the AP-2σ gene, which encodes a component of the AP-2 adaptor complex that recruits cargoes into clathrin-coated pits for subsequent internalization. Moreover, reduction of JNK activity can decrease elevated Yki signaling caused by altered endocytosis. These studies reveal a broad requirement for components of the endocytic pathway in regulating SWH and JNK outputs and place Drosophila endocytic nTSGs into a network that involves two major signaling pathways implicated in oncogenesis.
Drosophila; endocytic tumor suppressor; Yki; JNK; Tsg101; AP-2; Hippo
Small cell lung cancer (SCLC), a special type of lung cancer, is reputed to carry a poor prognosis. The morbidity of SCLC is increasing in China and other countries. A variety of DNA alterations associated with non-small cell lung cancer (NSCLC) have been described. However, genetic and epigenetic changes of SCLC are not well established. Few studies have demonstrated that epigenetic silencing of key tumor suppressor genes (TSGs) is pivotal to initiation and development of SCLC. Recently, promoter methylation of many TSGs have been identified in SCLC. These novel TSGs are potential tumor biomarkers for early diagnosis and prognostic prediction. Moreover, epigenetic promoter methylation of TSGs could be a target of intervention with a wide prospect of clinical application. This review summarizes recent studies on promoter methylation of TSGs in SCLC and aims to provide better understanding of the promoter methylation in tumorigenesis and progression of SCLC.
Small cell lung cancer (SCLC); tumor suppressor gene (TSG); promotor methylation; hypermethylation
Tissue replenishment from stem cells follows a precise cascade of events, during which stem cell daughters first proliferate by mitotic transit amplifying divisions and then enter terminal differentiation. Here we address how stem cell daughters are guided through the early steps of development. In Drosophila testes, somatic cyst cells enclose the proliferating and differentiating germline cells and the units of germline and surrounding cyst cells are commonly referred to as cysts. By characterizing flies with reduced or increased Epidermal Growth Factor (EGF) signaling we show that EGF triggers different responses in the cysts dependent on its dose. In addition to the previously reported requirement for EGF signaling in cyst formation, a low dose of EGF signaling is required for the progression of the germline cells through transit amplifying divisions, and a high dose of EGF signaling promotes terminal differentiation. Terminal differentiation was promoted in testes expressing a constitutively active EGF Receptor (EGFR) and in testes expressing both a secreted EGF and the EGFR in the cyst cells, but not in testes expressing either only EGF or only EGFR. We propose that as the cysts develop, a temporal signature of EGF signaling is created by the coordinated increase of both the production of active ligands by the germline cells and the amount of available receptor molecules on the cyst cells.
In the mammalian brain, neural stem cells divide asymmetrically and often amplify the number of progeny they generate via symmetrically dividing intermediate progenitors. Here we investigate whether specific neural stem cell-like neuroblasts in the brain of Drosophila might also amplify neuronal proliferation by generating symmetrically dividing intermediate progenitors.
Cell lineage-tracing and genetic marker analysis show that remarkably large neuroblast lineages exist in the dorsomedial larval brain of Drosophila. These lineages are generated by brain neuroblasts that divide asymmetrically to self renew but, unlike other brain neuroblasts, do not segregate the differentiating cell fate determinant Prospero to their smaller daughter cells. These daughter cells continue to express neuroblast-specific molecular markers and divide repeatedly to produce neural progeny, demonstrating that they are proliferating intermediate progenitors. The proliferative divisions of these intermediate progenitors have novel cellular and molecular features; they are morphologically symmetrical, but molecularly asymmetrical in that key differentiating cell fate determinants are segregated into only one of the two daughter cells.
Our findings provide cellular and molecular evidence for a new mode of neurogenesis in the larval brain of Drosophila that involves the amplification of neuroblast proliferation through intermediate progenitors. This type of neurogenesis bears remarkable similarities to neurogenesis in the mammalian brain, where neural stem cells as primary progenitors amplify the number of progeny they generate through generation of secondary progenitors. This suggests that key aspects of neural stem cell biology might be conserved in brain development of insects and mammals.
The evolutionarily conserved, secreted protein Twisted gastrulation (Tsg) modulates morphogenetic effects of decapentaplegic (dpp) and its orthologs, the bone morphogenetic proteins 2 and 4 (BMP2/4), in early Drosophila and vertebrate embryos. We have uncovered a role for Tsg at a much later stage of mammalian development, during T cell differentiation in the thymus. BMP4 is expressed by thymic stroma and inhibits the proliferation of CD4−CD8− double-negative (DN) thymocytes and their differentiation to the CD4+CD8+ double-positive (DP) stage in vitro. Tsg is expressed by thymocytes and up-regulated after T cell receptor signaling at two developmental checkpoints, the transition from the DN to the DP and from the DP to the CD4+ or CD8+ single-positive stage. Tsg can synergize with the BMP inhibitor chordin to block the BMP4-mediated inhibition of thymocyte proliferation and differentiation. These data suggest that the developmentally regulated expression of Tsg may allow thymocytes to temporarily withdraw from inhibitory BMP signals.
BMP4; Twisted gastrulation; thymocyte development; chordin; morphogen
We recently found that leukocytes from thrombospondin-1 (TSP1)-deficient mice exhibit significant reductions in cell surface CD44 relative to those from wild type mice. Because TSG-6 modulates CD44-mediated cellular interactions with hyaluronan, we examined the possibility that TSP1 interacts with TSG-6. We show that recombinant full length human TSG-6 (TSG-6Q) and Link module of TSG-6 (Link_TSG6) bind 125I-TSP1 with comparable affinities. Trimeric recombinant constructs containing the N-modules of TSP1 or TSP2 inhibit binding of TSP1 to TSG-6Q and Link_TSG6, but other recombinant regions of TSP1 do not. Therefore, the N-modules of both TSP1 and TSP2 specifically recognize the Link module of TSG-6. Heparin, which binds to these domains of both proteins, strongly inhibits binding of TSP1 to Link_TSG6 and TSG-6Q, but hyaluronan does not. Inhibition by heparin is due to its binding to TSP1, because heparin also inhibits TSP1 binding to Link_TSG6 mutants deficient in heparin binding. Removal of bound Ca2+ from TSP1 reduces its binding to full-length TSG-6. Binding of TSP1 to Link_TSG6, however, is enhanced by chelating divalent cations. In contrast, divalent cations do not influence binding of the N-terminal region of TSP1 to TSG-6Q. This implies that divalent cation-dependence is due to conformational effects of Ca-binding to the C-terminal domains of TSP1. TSP1 enhances covalent modification of inter-α-trypsin inhibitor by TSG-6 and transfer of its heavy chains to hyaluronan, suggesting a physiological function of TSP1 binding to TSG-6 in regulation of hyaluronan metabolism at sites of inflammation.
Multiple genes involved in endocytosis and endosomal protein trafficking in Drosophila have been shown to function as neoplastic tumor suppressor genes (nTSGs), including Endosomal Sorting Complex Required for Transport-II (ESCRT-II) components vacuolar protein sorting 22 (vps22), vps25, and vps36. However, most studies of endocytic nTSGs have been done in mosaic tissues containing both mutant and non-mutant populations of cells, and interactions among mutant and non-mutant cells greatly influence the final phenotype. Thus, the true autonomous phenotype of tissues mutant for endocytic nTSGs remains unclear. Here, we show that tissues predominantly mutant for ESCRT-II components display characteristics of neoplastic transformation and then undergo apoptosis. These neoplastic tissues show upregulation of c-Jun N-terminal Kinase (JNK), Notch, and Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signaling. Significantly, while inhibition of JNK signaling in mutant tissues partially inhibits proliferation, inhibition of JAK/STAT signaling rescues other aspects of the neoplastic phenotype. This is the first rigorous study of tissues predominantly mutant for endocytic nTSGs and provides clear evidence for cooperation among de-regulated signaling pathways leading to tumorigenesis.
Human plasma fibronectin binds with high affinity to the inflammation-induced secreted protein TSG-6. Fibronectin binds to the CUB_C domain of TSG-6 but not to its Link module. TSG-6 can thus act as a bridging molecule to facilitate fibronectin association with the TSG-6 Link module ligand thrombospondin-1. Fibronectin binding to TSG-6 is divalent cation-independent and is conserved in cellular fibronectins. Based on competition binding studies using recombinant and proteolytic fragments of fibronectin, TSG-6 binding localizes to type III repeats 9–14 of fibronectin. This region of fibronectin contains the Arg-Gly-Asp sequence recognized by α5β1 integrin, but deletion of that sequence does not prevent TSG-6 binding, and TSG-6 does not inhibit cell adhesion on fibronectin substrates mediated by this integrin. This region of fibronectin is also involved in fibronectin matrix assembly, and addition of TSG-6 enhances exogenous and endogenous fibronectin matrix assembly by human fibroblasts. Therefore, TSG-6 is a high affinity ligand that can mediate fibronectin interactions with other matrix components and modulate some interactions of fibronectin with cells.
matrix assembly; TSG-6; fibronectin; CUB domains
Asymmetric cell division is a mechanism for generating cell diversity as well as maintaining stem cell homeostasis in both Drosophila and mammals. In Drosophila, larval neuroblasts are stem cell-like progenitors that divide asymmetrically to generate neurons of the adult brain. Mitotic neuroblasts localize atypical protein kinase C (aPKC) to their apical cortex. Cortical aPKC excludes cortical localization of Miranda and its cargo proteins Prospero and Brain tumor, resulting in their partitioning into the differentiating, smaller ganglion mother cell (GMC) where they are required for neuronal differentiation. In addition to aPKC, the kinases Aurora-A and Polo also regulate neuroblast self-renewal, but the phosphatases involved in neuroblast self-renewal have not been identified. Here we report that aPKC is in a protein complex in vivo with Twins, a Drosophila B-type protein phosphatase 2A (PP2A) subunit, and that Twins and the catalytic subunit of PP2A, called Microtubule star (Mts), are detected in larval neuroblasts. Both Twins and Mts are required to exclude aPKC from the basal neuroblast cortex: twins mutant brains, twins mutant single neuroblast mutant clones, or mts dominant negative single neuroblast clones all show ectopic basal cortical localization of aPKC. Consistent with ectopic basal aPKC is the appearance of supernumerary neuroblasts in twins mutant brains or twins mutant clones. We conclude that Twins/PP2A is required to maintain aPKC at the apical cortex of mitotic neuroblasts, keeping it out of the differentiating GMC, and thereby maintaining neuroblast homeostasis.
Drosophila neuroblasts1 and ovarian stem cells2,3 are well characterized models for stem cell biology. In both cell types, one daughter cell self-renews continuously while the other undergoes a limited number of divisions, stops to proliferate mitotically and differentiates. Whereas neuroblasts segregate the Trim–NHL (tripartite motif and Ncl-1, HT2A and Lin-41 domain)-containing protein Brain tumour (Brat) into one of the two daughter cells4-6, ovarian stem cells are regulated by an extracellular signal from the surrounding stem cell niche. After division, one daughter cell looses niche contact. It undergoes 4 transit-amplifying divisions to form a cyst of 16 interconnected cells that reduce their rate of growth and stop to proliferate mitotically. Here we show that the Trim–NHL protein Mei-P26 (refs 7, 8) restricts growth and proliferation in the ovarian stem cell lineage. Mei-P26 expression is low in stem cells but is strongly induced in 16-cell cysts. In mei-P26 mutants, transit-amplifying cells are larger and proliferate indefinitely leading to the formation of an ovarian tumour. Like brat, mei-P26 regulates nucleolar size and can induce differentiation in Drosophila neuroblasts, suggesting that these genes act through the same pathway. We identify Argonaute-1, a component of the RISC complex, as a common binding partner of Brat and Mei-P26, and show that Mei-P26 acts by inhibiting the microRNA pathway. Mei-P26 and Brat have a similar domain composition that is also found in other tumour suppressors and might be a defining property of a new family of microRNA regulators that act specifically in stem cell lineages.
Green fluorescent protein (GFP) and its derivatives are the most widely used molecular reporters for live cell imagining. The development of organelle-specific fusion fluorescent proteins improves the labeling resolution to a higher level. Here we generate a R26 dual fluorescent protein reporter mouse, activated by Cre-mediated DNA recombination, labeling target cells with a chromatin-specific enhanced green fluorescence protein (EGFP) and a plasma membrane-anchored monomeric cherry fluorescent protein (mCherry). This dual labeling allows the visualization of mitotic events, cell shapes and intracellular vesicle behaviors. We expect this reporter mouse to have a wide application in developmental biology studies, transplantation experiments as well as cancer/stem cell lineage tracing.
TSG-6 is an inflammation-induced protein that is produced at pathological
sites, including arthritic joints. In animal models of arthritis, TSG-6
protects against joint damage; this has been attributed to its inhibitory
effects on neutrophil migration and plasmin activity. Here we investigated
whether TSG-6 can directly influence bone erosion. Our data reveal that TSG-6
inhibits RANKL-induced osteoclast differentiation/activation from human and
murine precursor cells, where elevated dentine erosion by osteoclasts derived
from TSG-6-/- mice is consistent with the very severe
arthritis seen in these animals. However, the long bones from unchallenged
TSG-6-/- mice were found to have higher trabecular mass
than controls, suggesting that in the absence of inflammation TSG-6 has a role
in bone homeostasis; we have detected expression of the TSG-6 protein in the
bone marrow of unchallenged wild type mice. Furthermore, we have observed that
TSG-6 can inhibit bone morphogenetic protein-2 (BMP-2)-mediated osteoblast
differentiation. Interaction analysis revealed that TSG-6 binds directly to
RANKL and to BMP-2 (as well as other osteogenic BMPs but not BMP-3) via
composite surfaces involving its Link and CUB modules. Consistent with this,
the full-length protein is required for maximal inhibition of osteoblast
differentiation and osteoclast activation, although the isolated Link module
retains significant activity in the latter case. We hypothesize that TSG-6 has
dual roles in bone remodeling; one protective, where it inhibits RANKL-induced
bone erosion in inflammatory diseases such as arthritis, and the other
homeostatic, where its interactions with BMP-2 and RANKL help to balance
mineralization by osteoblasts and bone resorption by osteoclasts.
Changes in the number of chromosomes, but also variations in the copy number of chromosomal regions have been described in various pathological conditions, such as cancer and aneuploidy, but also in normal physiological condition. Our classical view of DNA replication and mitotic preservation of the chromosomal integrity is now challenged as new technologies allow us to observe such mosaic somatic changes in copy number affecting regions of chromosomes with various sizes. In order to go further in the understanding of copy number influence in normal condition we could take advantage of the novel strategy called Targeted Asymmetric Sister Chromatin Event of Recombination (TASCER) to induce recombination during the G2 phase so that we can generate deletions and duplications of regions of interest prior to mitosis. Using this approach in the mouse we could address the effects of copy number variation and segmental aneuploidy in daughter cells and allow us to explore somatic mosaics for large region of interest in the mouse.
Chromosomal engineering; Cre/loxP; mosaic genetics; duplication; deletion.
Neural stem cells in Drosophila are currently one of the best model systems for understanding stem cell biology during normal development and during abnormal development of stem cell-derived brain tumors. In Drosophila brain development, the proliferative activity of neural stem cells called neuroblasts gives rise to both the optic lobe and the central brain ganglia, and asymmetric cell divisions are key features of this proliferation. The molecular mechanisms that underlie the asymmetric cell divisions by which these neuroblasts self-renew and generate lineages of differentiating progeny have been studied extensively and involve two major protein complexes, the apical complex which maintains polarity and controls spindle orientation and the basal complex which is comprised of cell fate determinants and their adaptors that are segregated into the differentiating daughter cells during mitosis. Recent molecular genetic work has established Drosophila neuroblasts as a model for neural stem cell-derived tumors in which perturbation of key molecular mechanisms that control neuroblast proliferation and the asymmetric segregation of cell fate determinants lead to brain tumor formation. Identification of novel candidate genes that control neuroblast self-renewal and differentiation as well as functional analysis of these genes in normal and tumorigenic conditions in a tissue-specific manner is now possible through genome-wide transgenic RNAi screens. These cellular and molecular findings in Drosophila are likely to provide valuable genetic links for analyzing mammalian neural stem cells and tumor biology.
Asymmetric division of neural progenitors is a key mechanism by which neuronal diversity in the Drosophila central nervous system is generated. The distinct fates of the daughter cells derived from these divisions are achieved through preferential segregation of the cell fate determinants Prospero and Numb to one of the two daughters. This is achieved by coordinating apical and basal mitotic spindle orientation with the basal cortical localization of the cell fate determinants during mitosis. A complex of apically localized proteins, including Inscuteable (Insc), Partner of Inscuteable (Pins), Bazooka (Baz), DmPar-6, DaPKC, and Gαi, is required to mediate and coordinate basal protein localization with mitotic spindle orientation. Pins, a molecule which directly interacts with Insc, is a key component required for the integrity of this complex; in the absence of Pins, other components become mislocalized or destabilized, and basal protein localization and mitotic spindle orientation are defective. Here we define the functional domains of Pins. We show that the C-terminal region containing the Gαi binding GoLoco motifs is necessary and sufficient for targeting to the neuroblast cortex, which appears to be a prerequisite for apical localization of Pins. The N-terminal tetratricopeptide repeat-containing region of Pins is required for two processes; TPR repeats 1 to 3 plus the C-terminal region are required for apical localization but are insufficient to recruit Insc to the apical cortex, whereas TPR repeats 1 to 7 plus C-terminal Pins can perform both functions. Hence, the abilities of Pins to cortically localize, to apically localize, and to restore Insc apical localization are all separable, and all three capabilities are necessary to mediate asymmetric division. Moreover, the need for N-terminal Pins can be obviated by fusing a minimal Insc functional domain with the C-terminal region of Pins; this chimeric molecule is apically localized and can fulfill the functions of both Insc and Pins.
One of the first signs of cell differentiation in the Drosophila melanogaster embryo occurs 3 h after fertilization, when discrete groups of cells enter their fourteenth mitosis in a spatially and temporally patterned manner creating mitotic domains (Foe, V. E. and G. M. Odell, 1989, Am. Zool. 29:617-652). To determine whether cell residency in a mitotic domain is determined solely by cell position in this early embryo, or whether cell lineage also has a role, we have developed a technique for directly analyzing the behavior of nuclei in living embryos. By microinjecting fluorescently labeled histones into the syncytial embryo, the movements and divisions of each nucleus were recorded without perturbing development by using a microscope equipped with a high resolution, charge-coupled device. Two types of developmental maps were generated from three-dimensional time-lapse recordings: one traced the lineage history of each nucleus from nuclear cycle 11 through nuclear cycle 14 in a small region of the embryo; the other recorded nuclear fate according to the timing and pattern of the 14th nuclear division. By comparing these lineage and fate maps for two embryos, we conclude that, at least for the examined area, the pattern of mitotic domain formation in Drosophila is determined by the position of each cell, with no effect of cell lineage.
Mitotic recombination is an effective tool for generating mutant clones in somatic tissues. Due to difficulties associated with detecting and quantifying mutant clones in mice, this technique is limited to analysis of growth related phenotypes induced by loss function of tumor suppressor genes. Here, we used the polymorphic CD45.1/CD45.2 alleles on chromosome 1 as pan-hematopoietic markers to track mosaic clones generated through mitotic recombination in developing T cells. We show that lineage specific mitotic recombination can be induced and reliably detected as CD45.1 or CD45.2 homozygous clones from the CD45.1/CD45.2 heterozygous background. We have applied this system in the analysis of a lethal mutation in the Dhx9 gene. Mosaic analysis revealed a stage specific role for Dhx9 during T cell maturation. Thus, the experimental system described in this study offers a practical means for mosaic analysis of germline mutations in the hematopoietic system.
Mitotic recombination; CD45.1/CD45.2; T lymphocyte; Dhx9; Cre/lox
The dopaminergic (DA) neurons present in the central brain of the Drosophila larva are spatially arranged in stereotyped groups that define clusters of bilaterally symmetrical neurons. These clusters have been classified according to anatomical criteria (position of the cell bodies within the cortex and/or projection pattern of the axonal tracts). However, information pertaining to the developmental biology, such as lineage relationship of clustered DA neurons and differential cell subtype-specific molecular markers and mechanisms of differentiation and/or survival, is currently not available.
Using MARCM and twin-spot MARCM techniques together with anti-tyrosine hydroxylase immunoreactivity, we have analyzed the larval central brain DA neurons from a developmental point of view and determined their time of birth, their maturation into a DA neurotransmitter phenotype as well as their lineage relationships. In addition, we have found that the homeodomain containing transcription factor Orthodenticle (Otd) is present in a cluster of clonally related DA neurons in both the larval and adult brain. Taking advantage of the otd hypomorphic mutation ocelliless (oc) and the oc2-Gal4 reporter line, we have studied the involvement of orthodenticle (otd) in the survival and/or cell fate specification of these post-mitotic neurons.
Our findings provide evidence of the presence of seven neuroblast lineages responsible for the generation of the larval central brain DA neurons during embryogenesis. otd is expressed in a defined group of clonally related DA neurons from first instar larvae to adulthood, making it possible to establish an identity relationship between the larval DL2a and the adult PPL2 DA clusters. This poses otd as a lineage-specific and differential marker of a subset of clonally related DA neurons. Finally, we show that otd is required in those DA neurons for their survival.