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1.  Differentiated Horizontal Interneurons Clonally Expand to Form Metastatic Retinoblastoma in Mice 
Cell  2007;131(2):378-390.
During neurogenesis, the progression from a progenitor cell to a differentiated neuron is believed to be unidirectional and irreversible. The Rb family of proteins (Rb, p107 and p130) regulates cell cycle exit and differentiation during retinogenesis. Rb and p130 are redundantly expressed in the neurons of the inner nuclear layer (INL) of the retina. We have found that in the adult Rb;p130-deficient retinae p107 compensation prevents ectopic proliferation of INL neurons. However, p107 is haploinsufficient in this process. Differentiated Rb−/−;p107+/−;p130−/− horizontal interneurons re-entered the cell cycle, clonally expanded, and formed metastatic retinoblastoma. Horizontal cells were not affected in Rb+/−;p107−/−;p130−/− or Rb−/−;p107−/−;p130+/− retinae suggesting that one copy of Rb or p130 was sufficient to prevent horizontal proliferation. This is the first demonstration that differentiated neurons can proliferate and form cancer while maintaining their differentiated state including neurites and synaptic connections.
doi:10.1016/j.cell.2007.09.036
PMCID: PMC2203617  PMID: 17956737
horizontal cell; Rb; p107; retinoblastoma; metastasis
2.  Cellular mechanisms underlying spatiotemporal features of cholinergic retinal waves 
The Journal of Neuroscience  2012;32(3):850-863.
Prior to vision, a transient network of recurrently connected cholinergic interneurons, called starburst amacrine cells (SACs), generates spontaneous retinal waves. Despite an absence of robust inhibition, cholinergic retinal waves initiate infrequently and propagate within finite boundaries. Here we combine a variety of electrophysiological and imaging techniques and computational modeling to elucidate the mechanisms underlying these spatial and temporal properties of waves in developing mouse retina. Waves initiate via rare spontaneous depolarizations of SACs. Waves propagate through recurrent cholinergic connections between SACs and volume release of ACh as demonstrated using paired recordings and a cell-based ACh optical sensor. Perforated patch recordings and two-photon calcium imaging reveal that individual SACs have slow afterhyperpolarizations that induce SACs to have variable depolarizations during sequential waves. Using a computational model in which the properties of SACs are based on these physiological measurements, we reproduce the slow frequency, speed, and finite size of recorded waves. This study represents a detailed description of the circuit that mediates cholinergic retinal waves and indicates that variability of the interneurons that generate this network activity may be critical for the robustness of waves across different species and stages of development.
doi:10.1523/JNEUROSCI.5309-12.2012
PMCID: PMC3311224  PMID: 22262883
calcium imaging; development; afterhyperpolarization; pacemaker
3.  Role of ACh-GABA co-transmission in detecting image motion and motion direction 
Neuron  2010;68(6):1159-1172.
Summary
Starburst amacrine cells (SACs) process complex visual signals in the retina using both ACh and GABA, but the synaptic organization and function of ACh-GABA corelease remain unclear. Here, we show that SACs make cholinergic synapses onto On-Off direction-selective ganglion cells (DSGCs) from all directions, but make GABAergic synapses onto DSGCs only from the null direction. ACh and GABA were released differentially in a Ca2+ level-specific manner, suggesting the two transmitters were released from different vesicle populations. Despite the symmetric cholinergic connection, the light-evoked cholinergic input to a DSGC, detected at both light onset and offset, was motion- and direction-sensitive. This input was facilitated by two-spot apparent motion in the preferred direction, but supressed in the null direction, presumably by a GABAergic mechnism. The results revealed a new level of synaptic intricacy in the starburst circuit and suggest differential, yet synergistic, roles of ACh-GABA cotransmission in motion sensitivity and direction selectivity.
doi:10.1016/j.neuron.2010.11.031
PMCID: PMC3094727  PMID: 21172616
4.  Rescue of myogenic defects in Rb-deficient cells by inhibition of autophagy or by hypoxia-induced glycolytic shift 
The Journal of Cell Biology  2010;191(2):291-301.
pRb is required to inhibit apoptosis in myoblasts and autophagy in myotubes but not for activation of the myogenic differentiation program.
The retinoblastoma tumor suppressor (pRb) is thought to orchestrate terminal differentiation by inhibiting cell proliferation and apoptosis and stimulating lineage-specific transcription factors. In this study, we show that in the absence of pRb, differentiating primary myoblasts fuse to form short myotubes that never twitch and degenerate via a nonapoptotic mechanism. The shortened myotubes exhibit an impaired mitochondrial network, mitochondrial perinuclear aggregation, autophagic degradation, and reduced adenosine triphosphate production. Bcl-2 and autophagy inhibitors restore mitochondrial function and rescue muscle degeneration, leading to formation of long, twitching myotubes that express normal levels of muscle-specific proteins and stably exit the cell cycle. A hypoxia-induced glycolytic switch also rescues the myogenic defect after either chronic or acute inactivation of Rb in a hypoxia-inducible factor-1 (HIF-1)–dependent manner. These results demonstrate that pRb is required to inhibit apoptosis in myoblasts and autophagy in myotubes but not to activate the differentiation program, and they also reveal a novel link between pRb and cell metabolism.
doi:10.1083/jcb.201005067
PMCID: PMC2958467  PMID: 20937698
5.  Direction-Selective Circuitry in Rat Retina Develops Independently of GABAergic, Cholinergic and Action Potential Activity 
PLoS ONE  2011;6(5):e19477.
The ON-OFF direction selective ganglion cells (DSGCs) in the mammalian retina code image motion by responding much more strongly to movement in one direction. They do so by receiving inhibitory inputs selectively from a particular sector of processes of the overlapping starburst amacrine cells, a type of retinal interneuron. The mechanisms of establishment and regulation of this selective connection are unknown. Here, we report that in the rat retina, the morphology, physiology of the ON-OFF DSGCs and the circuitry for coding motion directions develop normally with pharmacological blockade of GABAergic, cholinergic activity and/or action potentials for over two weeks from birth. With recent results demonstrating light independent formation of the retinal DS circuitry, our results strongly suggest the formation of the circuitry, i.e., the connections between the second and third order neurons in the visual system, can be genetically programmed, although emergence of direction selectivity in the visual cortex appears to require visual experience.
doi:10.1371/journal.pone.0019477
PMCID: PMC3088673  PMID: 21573161
6.  Coexpression of Normally Incompatible Developmental Pathways in Retinoblastoma Genesis 
Cancer cell  2011;20(2):260-275.
SUMMARY
It is widely believed that the molecular and cellular features of a tumor reflect its cell of origin and can thus provide clues about treatment targets. The retinoblastoma cell of origin has been debated for over a century. Here, we report that human and mouse retinoblastomas have molecular, cellular, and neurochemical features of multiple cell classes, principally amacrine/horizontal interneurons, retinal progenitor cells, and photoreceptors. Importantly, single-cell gene expression array analysis showed that these multiple cell type-specific developmental programs are coexpressed in individual retinoblastoma cells, which creates a progenitor/neuronal hybrid cell. Furthermore, neurotransmitter receptors, transporters, and biosynthetic enzymes are expressed in human retinoblastoma, and targeted disruption of these pathways reduces retinoblastoma growth in vivo and in vitro.
doi:10.1016/j.ccr.2011.07.005
PMCID: PMC3551581  PMID: 21840489
7.  Combined Inactivation of pRB and Hippo Pathways Induces Dedifferentiation in the Drosophila Retina 
PLoS Genetics  2010;6(4):e1000918.
Functional inactivation of the Retinoblastoma (pRB) pathway is an early and obligatory event in tumorigenesis. The importance of pRB is usually explained by its ability to promote cell cycle exit. Here, we demonstrate that, independently of cell cycle exit control, in cooperation with the Hippo tumor suppressor pathway, pRB functions to maintain the terminally differentiated state. We show that mutations in the Hippo signaling pathway, wts or hpo, trigger widespread dedifferentiation of rbf mutant cells in the Drosophila eye. Initially, rbf wts or rbf hpo double mutant cells are morphologically indistinguishable from their wild-type counterparts as they properly differentiate into photoreceptors, form axonal projections, and express late neuronal markers. However, the double mutant cells cannot maintain their neuronal identity, dedifferentiate, and thus become uncommitted eye specific cells. Surprisingly, this dedifferentiation is fully independent of cell cycle exit defects and occurs even when inappropriate proliferation is fully blocked by a de2f1 mutation. Thus, our results reveal the novel involvement of the pRB pathway during the maintenance of a differentiated state and suggest that terminally differentiated Rb mutant cells are intrinsically prone to dedifferentiation, can be converted to progenitor cells, and thus contribute to cancer advancement.
Author Summary
The inability to respond to growth inhibitory cues is one acquired trait of a cancer cell. Almost all such signals are eventually routed through the Retinoblastoma (pRB) tumor suppressor pathway. Therefore, inactivation of the pRB pathway is considered to be an early and obligatory event during transformation of a normal cell into a malignant cancer cell. In this study, we found that inactivation of the Hippo pathway makes Rb mutant cells prone to undergo morphological changes and to become less differentiated, progenitor-like cells. Furthermore, we show that this was independent of the failure of Rb mutant cells to properly respond to cell cycle exit cues. These results are significant since, in general, tumors containing progenitor-like cells have a higher potential to progress through later stages of tumorigenesis and to become more aggressive and more deadly. Thus, the inactivation of Rb not only renders cells insensitive to growth inhibitory signals, but also sensitizes cells to revert to a progenitor-like state.
doi:10.1371/journal.pgen.1000918
PMCID: PMC2858677  PMID: 20421993
8.  Muscarinic signaling influences the patterning and phenotype of cholinergic amacrine cells in the developing chick retina 
Background
Many studies in the vertebrate retina have characterized the differentiation of amacrine cells as a homogenous class of neurons, but little is known about the genes and factors that regulate the development of distinct types of amacrine cells. Accordingly, the purpose of this study was to characterize the development of the cholinergic amacrine cells and identify factors that influence their development. Cholinergic amacrine cells in the embryonic chick retina were identified by using antibodies to choline acetyltransferase (ChAT).
Results
We found that as ChAT-immunoreactive cells differentiate they expressed the homeodomain transcription factors Pax6 and Islet1, and the cell-cycle inhibitor p27kip1. As differentiation proceeds, type-II cholinergic cells, displaced to the ganglion cell layer, transiently expressed high levels of cellular retinoic acid binding protein (CRABP) and neurofilament, while type-I cells in the inner nuclear layer did not. Although there is a 1:1 ratio of type-I to type-II cells in vivo, in dissociated cell cultures the type-I cells (ChAT-positive and CRABP-negative) out-numbered the type-II cells (ChAT and CRABP-positive cells) by 2:1. The relative abundance of type-I to type-II cells was not influenced by Sonic Hedgehog (Shh), but was affected by compounds that act at muscarinic acetylcholine receptors. In addition, the abundance and mosaic patterning of type-II cholinergic amacrine cells is disrupted by interfering with muscarinic signaling.
Conclusion
We conclude that: (1) during development type-I and type-II cholinergic amacrine cells are not homotypic, (2) the phenotypic differences between these subtypes of cells is controlled by the local microenvironment, and (3) appropriate levels of muscarinic signaling between the cholinergic amacrine cells are required for proper mosaic patterning.
doi:10.1186/1471-213X-8-13
PMCID: PMC2267169  PMID: 18254959
9.  Regulation of Differentiation by HBP1, a Target of the Retinoblastoma Protein 
Molecular and Cellular Biology  1998;18(8):4732-4743.
Differentiation is a coordinated process of irreversible cell cycle exit and tissue-specific gene expression. To probe the functions of the retinoblastoma protein (RB) family in cell differentiation, we isolated HBP1 as a specific target of RB and p130. Our previous work showed that HBP1 was a transcriptional repressor and a cell cycle inhibitor. The induction of HBP1, RB, and p130 upon differentiation in the muscle C2C12 cells suggested a coordinated role. Here we report that the expression of HBP1 unexpectedly blocked muscle cell differentiation without interfering with cell cycle exit. Moreover, the expression of MyoD and myogenin, but not Myf5, was inhibited in HBP1-expressing cells. HBP1 inhibited transcriptional activation by the MyoD family members. The inhibition of MyoD family function by HBP1 required binding to RB and/or p130. Since Myf5 might function upstream of MyoD, our data suggested that HBP1 probably blocked differentiation by disrupting Myf5 function, thus preventing expression of MyoD and myogenin. Consistent with this, the expression of each MyoD family member could reverse the inhibition of differentiation by HBP1. Further investigation implicated the relative ratio of RB to HBP1 as a determinant of whether cell cycle exit or full differentiation occurred. At a low RB/HBP1 ratio cell cycle exit occurred but there was no tissue-specific gene expression. At elevated RB/HBP1 ratios full differentiation occurred. Similar changes in the RB/HBP1 ratio have been observed in normal C2 differentiation. Thus, we postulate that the relative ratio of RB to HBP1 may be one signal for activation of the MyoD family. We propose a model in which a checkpoint of positive and negative regulation may coordinate cell cycle exit with MyoD family activation to give fidelity and progression in differentiation.
PMCID: PMC109059  PMID: 9671483
10.  Terminal osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarcoma 
The Journal of Cell Biology  2004;167(5):925-934.
The molecular basis for the inverse relationship between differentiation and tumorigenesis is unknown. The function of runx2, a master regulator of osteoblast differentiation belonging to the runt family of tumor suppressor genes, is consistently disrupted in osteosarcoma cell lines. Ectopic expression of runx2 induces p27KIP1, thereby inhibiting the activity of S-phase cyclin complexes and leading to the dephosphorylation of the retinoblastoma tumor suppressor protein (pRb) and a G1 cell cycle arrest. Runx2 physically interacts with the hypophosphorylated form of pRb, a known coactivator of runx2, thereby completing a feed-forward loop in which progressive cell cycle exit promotes increased expression of the osteoblast phenotype. Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts. Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas. Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.
doi:10.1083/jcb.200409187
PMCID: PMC2172443  PMID: 15583032
11.  The role of starburst amacrine cells in visual signal processing 
Visual Neuroscience  2012;29(1):73-81.
Starburst amacrine cells (SBACs) within the adult mammalian retina provide the critical inhibition that underlies the receptive field properties of direction-selective ganglion cells (DSGCs). The SBACs generate direction-selective output of GABA that differentially inhibits the DSGCs. We review the biophysical mechanisms that produce directional GABA release from SBACs and test a network model that predicts the effects of reciprocal inhibition between adjacent SBACs. The results of the model simulations suggest that reciprocal inhibitory connections between closely spaced SBACs should be spatially selective, while connections between more widely spaced cells could be indiscriminate. SBACs were initially identified as cholinergic neurons and were subsequently shown to contain release both acetylcholine and GABA. While the role of the GABAergic transmission is well established, the role of the cholinergic transmission remains unclear.
doi:10.1017/S0952523811000393
PMCID: PMC3292856  PMID: 22310373
Retina; Visual system; Direction selectivity; Computational model; Gabaergic inhibition
12.  Synaptotagmin I Regulates Patterned Spontaneous Activity in the Developing Rat Retina via Calcium Binding to the C2AB Domains 
PLoS ONE  2012;7(10):e47465.
Background
In neonatal binocular animals, the developing retina displays patterned spontaneous activity termed retinal waves, which are initiated by a single class of interneurons (starburst amacrine cells, SACs) that release neurotransmitters. Although SACs are shown to regulate wave dynamics, little is known regarding how altering the proteins involved in neurotransmitter release may affect wave dynamics. Synaptotagmin (Syt) family harbors two Ca2+-binding domains (C2A and C2B) which serve as Ca2+ sensors in neurotransmitter release. However, it remains unclear whether SACs express any specific Syt isoform mediating retinal waves. Moreover, it is unknown how Ca2+ binding to C2A and C2B of Syt affects wave dynamics. Here, we investigated the expression of Syt I in the neonatal rat retina and examined the roles of C2A and C2B in regulating wave dynamics.
Methodology/Principal Findings
Immunostaining and confocal microscopy showed that Syt I was expressed in neonatal rat SACs and cholinergic synapses, consistent with its potential role as a Ca2+ sensor mediating retinal waves. By combining a horizontal electroporation strategy with the SAC-specific promoter, we specifically expressed Syt I mutants with weakened Ca2+-binding ability in C2A or C2B in SACs. Subsequent live Ca2+ imaging was used to monitor the effects of these molecular perturbations on wave-associated spontaneous Ca2+ transients. We found that targeted expression of Syt I C2A or C2B mutants in SACs significantly reduced the frequency, duration, and amplitude of wave-associated Ca2+ transients, suggesting that both C2 domains regulate wave temporal properties. In contrast, these C2 mutants had relatively minor effects on pairwise correlations over distance for wave-associated Ca2+ transients.
Conclusions/Significance
Through Ca2+ binding to C2A or C2B, the Ca2+ sensor Syt I in SACs may regulate patterned spontaneous activity to shape network activity during development. Hence, modulating the releasing machinery in presynaptic neurons (SACs) alters wave dynamics.
doi:10.1371/journal.pone.0047465
PMCID: PMC3472990  PMID: 23091625
13.  Domains of E1A that bind p105Rb, p130, and p300 are required to block nerve growth factor-induced neurite growth in PC12 cells. 
Molecular Biology of the Cell  1993;4(4):353-361.
Nerve growth factor (NGF) causes PC12 cells to cease division and undergo sympathetic neuron-like differentiation, including neurite outgrowth. We have tested whether differentiation and division share overlapping control mechanisms in these cells. To do this, we have perturbed the activity of proteins known to participate in cell-cycle regulation by introducing the E1A oncogene or its mutant forms via microinjection into PC12 cells. The E1A protein binds to several putative cell cycle control proteins, including p105Rb (the product of the retinoblastoma susceptibility gene), as well as others of unknown function such as p130, p107, and p300. Similar to previous results, we find that wild-type E1A abrogates NGF-induced neurite extension. However, NGF does cause neurite outgrowth in the presence of E1A mutants known to have greatly reduced binding to either p105Rb and p130 or p300. Our experiments suggest that p105Rb, p130, and p300 may participate either in E1A-mediated inhibition of differentiation or in the NGF signal transduction pathway. We also report here that NGF affects phosphorylation of p105Rb, suggesting that Rb mediates at least some of NGF's effects. Our results raise the possibility that putative cell-cycle control proteins may participate not only in NGF-induced cessation of division but also in differentiation.
Images
PMCID: PMC300937  PMID: 8507895
14.  The E2FC-DPB Transcription Factor Controls Cell Division, Endoreplication and Lateral Root Formation in a SCFSKP2A-Dependent Manner 
Plant Signaling & Behavior  2007;2(4):273-274.
Cell division is a highly regulated process that has to be coordinated with cell specification and differentiation for proper development and growth of the plants. Cell cycle regulation is carried out by key proteins that control cell cycle entry, progression and exit. This regulation is controlled at different stages such as gene expression, posttranslational modification of proteins and specific proteolysis. The G1/S and the G2/M transitions are critical checkpoints of the cell cycle that are controlled, among others, by the activity of cyclin-dependent kinases (CDK). Different CDK activities, still to be fully identified, impinge on the retinoblastoma (RBR)/E2F/DP pathway as well as on the programmed proteolysis pathway. The specific degradation of proteins through the ubiquitin pathway in plants, highly controlled in time and space, is emerging as a powerful mechanism to regulate the levels and the activity of several proteins, including many cell cycle regulators.
PMCID: PMC2634164  PMID: 19704635
cell cycle; endoreplication; E2F; DP; Ubiquitin; SCF; SKP2; lateral root; Arabidopsis
15.  The Tumor Suppressor Gene Retinoblastoma-1 Is Required for Retinotectal Development and Visual Function in Zebrafish 
PLoS Genetics  2012;8(11):e1003106.
Mutations in the retinoblastoma tumor suppressor gene (rb1) cause both sporadic and familial forms of childhood retinoblastoma. Despite its clinical relevance, the roles of rb1 during normal retinotectal development and function are not well understood. We have identified mutations in the zebrafish space cadet locus that lead to a premature truncation of the rb1 gene, identical to known mutations in sporadic and familial forms of retinoblastoma. In wild-type embryos, axons of early born retinal ganglion cells (RGC) pioneer the retinotectal tract to guide later born RGC axons. In rb1 deficient embryos, these early born RGCs show a delay in cell cycle exit, causing a transient deficit of differentiated RGCs. As a result, later born mutant RGC axons initially fail to exit the retina, resulting in optic nerve hypoplasia. A significant fraction of mutant RGC axons eventually exit the retina, but then frequently project to the incorrect optic tectum. Although rb1 mutants eventually establish basic retinotectal connectivity, behavioral analysis reveals that mutants exhibit deficits in distinct, visually guided behaviors. Thus, our analysis of zebrafish rb1 mutants reveals a previously unknown yet critical role for rb1 during retinotectal tract development and visual function.
Author Summary
Before an organism can execute necessary behavioral responses to environmental stimuli, the underlying neural circuits that regulate these behaviors must be precisely wired during embryonic development. A properly wired neural circuit is the product of a sophisticated collaboration of multiple genetic pathways that orchestrate cell type specification, the extension and growth of the cell processes that connect each circuit component, and the refinement of these connections. In an unbiased genetic screen designed to identify the genes required for proper circuit formation in developing zebrafish embryos, we identified a human disease causing mutation in the retinoblastoma-1 (rb1) gene that disrupts the formation of the zebrafish visual circuit. rb1 canonically functions to regulate the cell cycle, and when mutated the loss of rb1-mediated cell cycle control elicits childhood ocular tumor formation. Genetic models of rb1 have been developed to study the developmental role of rb1 in the retina; however, ectopic cell proliferation and death within the retina have largely precluded the ability to evaluate the formation and integrity of neural circuits connecting the retina with the brain. In this study, through genetic and cellular analysis of a zebrafish rb1 mutant, we reveal a novel role for rb1 in regulating the establishment and functionality of the visual circuitry.
doi:10.1371/journal.pgen.1003106
PMCID: PMC3510048  PMID: 23209449
16.  Mitosis in Neurons: Roughex and APC/C Maintain Cell Cycle Exit to Prevent Cytokinetic and Axonal Defects in Drosophila Photoreceptor Neurons 
PLoS Genetics  2012;8(11):e1003049.
The mechanisms of cell cycle exit by neurons remain poorly understood. Through genetic and developmental analysis of Drosophila eye development, we found that the cyclin-dependent kinase-inhibitor Roughex maintains G1 cell cycle exit during differentiation of the R8 class of photoreceptor neurons. The roughex mutant neurons re-enter the mitotic cell cycle and progress without executing cytokinesis, unlike non-neuronal cells in the roughex mutant that perform complete cell divisions. After mitosis, the binucleated R8 neurons usually transport one daughter nucleus away from the cell body into the developing axon towards the brain in a kinesin-dependent manner resembling anterograde axonal trafficking. Similar cell cycle and photoreceptor neuron defects occurred in mutants for components of the Anaphase Promoting Complex/Cyclosome. These findings indicate a neuron-specific defect in cytokinesis and demonstrate a critical role for mitotic cyclin downregulation both to maintain cell cycle exit during neuronal differentiation and to prevent axonal defects following failed cytokinesis.
Author Summary
Neurons generally differentiate and never divide again. One barrier to understanding the mechanisms has been the paucity of genetic mutations that result in neuronal cell cycles. Here we show that mutation in three genes lead to cell cycle re-entry by a particular class of developing photoreceptor neurons in the fly retina. Strikingly, these neurons do not complete cell division but only divide their nuclei. The binucleated neurons then typically retain one nucleus in its normal location in the cell body, while transporting the other into the growing axon like other axonal material. Our findings identify Cyclin A regulation as crucial to maintaining cell cycle exit by at least some neurons and identify a neuron-specific defect in cell division as a further barrier to neuron proliferation. Because defects in transporting axonal material have been implicated in the origin of multiple neurodegenerative diseases, our findings also suggest a possible connection between defective cell cycle regulation and neuronal cell death.
doi:10.1371/journal.pgen.1003049
PMCID: PMC3510051  PMID: 23209426
17.  A nutrient-sensitive restriction point is active during retinal progenitor cell differentiation 
Development (Cambridge, England)  2014;141(3):697-706.
In many growing tissues, slowly dividing stem cells give rise to rapidly proliferating progenitors that eventually exit the cell cycle and differentiate. Growth rates are limited by nutrient availability, but it is unclear which steps of the proliferation-differentiation programme are particularly sensitive to fuel supplies. We examined how nutrient deprivation (ND) affects stem and progenitor cells in the ciliary marginal zone (CMZ) of the amphibian retina, a well-characterised neurogenic niche. We show that ND specifically blocks the proliferation and differentiation of progenitor cells through an mTOR-mediated mechanism. By contrast, the identity and proliferation of retinal stem cells are insensitive to ND and mTOR inhibition. Re-feeding starved retinas in vitro rescues both proliferation and differentiation, and activation of mTOR is sufficient to stimulate differentiation even in ND retinas. These results suggest that an mTOR-mediated restriction point operates in vivo to couple nutrient abundance to the proliferation and differentiation programme in retinal progenitor cells.
doi:10.1242/dev.103978
PMCID: PMC3899821  PMID: 24449845
Differentiation; mTOR; Nutrient deprivation; Proliferation; Restriction point; Retina; Xenopus laevis
18.  β-selection-induced proliferation is required for αβ T cell differentiation 
Immunity  2012;37(5):840-853.
Summary
Proliferation and differentiation are tightly coordinated to produce an appropriate number of differentiated cells, and often exhibit an antagonistic relationship. Developing T cells, that arise in the thymus from a minute number of bone marrow-derived progenitors, undergo a major expansion upon pre-TCR expression. The burst of proliferation coincides with differentiation towards the αβ T cell lineage – but the two processes were previously thought to be independent from one another, although both driven by signaling from pre-TCR and Notch receptors. Here we report that proliferation at this step was not only absolutely required for differentiation, but also that its ectopic activation was sufficient to substantially rescue differentiation in the absence of Notch signaling. Consistently, pharmacological inhibition of cell cycle machinery also blocked differentiation in vivo. Thus proliferation step is strictly required prior to differentiation of immature thymocytes.
doi:10.1016/j.immuni.2012.08.020
PMCID: PMC3709258  PMID: 23159226
19.  Skeletal muscle cells lacking the retinoblastoma protein display defects in muscle gene expression and accumulate in S and G2 phases of the cell cycle 
The Journal of Cell Biology  1996;135(2):441-456.
Viral oncoproteins that inactivate the retinoblastoma tumor suppressor protein (pRb) family both block skeletal muscle differentiation and promote cell cycle progression. To clarify the dependence of terminal differentiation on the presence of the different pRb-related proteins, we have studied myogenesis using isogenic primary fibroblasts derived from mouse embryos individually deficient for pRb, p107, or p130. When ectopically expressed in fibroblasts lacking pRb, MyoD induces an aberrant skeletal muscle differentiation program characterized by normal expression of early differentiation markers such as myogenin and p21, but attenuated expression of late differentiation markers such as myosin heavy chain (MHC). Similar defects in MHC expression were not observed in cells lacking either p107 or p130, indicating that the defect is specific to the loss of pRb. In contrast to wild-type, p107- deficient, or p130-deficient differentiated myocytes that are permanently withdrawn from the cell cycle, differentiated myocytes lacking pRb accumulate in S and G2 phases and express extremely high levels of cyclins A and B, cyclin-dependent kinase (Cdk2), and Cdc2, but fail to readily proceed to mitosis. Administration of caffeine, an agent that removes inhibitory phosphorylations on inactive Cdc2/cyclin B complexes, specifically induced mitotic catastrophe in pRb-deficient myocytes, consistent with the observation that the majority of pRb- deficient myocytes arrest in S and G2. Together, these findings indicate that pRb is required for the expression of late skeletal muscle differentiation markers and for the inhibition of DNA synthesis, but that a pRb-independent mechanism restricts entry of differentiated myocytes into mitosis.
PMCID: PMC2121049  PMID: 8896600
20.  Rb and N-ras Function Together To Control Differentiation in the Mouse 
Molecular and Cellular Biology  2003;23(15):5256-5268.
The product of the retinoblastoma tumor suppressor gene (Rb) can control cell proliferation and promote dif-ferentiation. Murine embryos nullizygous for Rb die midgestation with defects in cell cycle regulation, control of apoptosis, and terminal differentiation of several tissues, including skeletal muscle, nervous system, and lens. Previous cell culture-based experiments have suggested that the retinoblastoma protein (pRb) and Ras operate in a common pathway to control cellular differentiation. Here we have tested the hypothesis that the proto-oncogene N-ras participates in Rb-dependent regulation of differentiation by generating and characterizing murine embryos deficient in both N-ras and Rb. We show that deletion of N-ras rescues a unique subset of the developmental defects associated with nullizygosity of Rb, resulting in a significant extension of life span. Rb−/−; N-ras−/− skeletal muscle has normal fiber density, myotube length and thickness, in contrast to Rb-deficient embryos. Additionally, Rb−/−; N-ras−/− muscle shows a restoration in the expression of the late muscle-specific gene MCK, and this correlates with a significant potentiation of MyoD transcriptional activity in Rb−/−; N-ras−/−, compared to Rb−/− myoblasts in culture. The improved differentiation of skeletal muscle in Rb−/−; N-ras−/− embryos occurs despite evidence of deregulated proliferation and apoptosis, as seen in Rb-deficient animals. Our findings suggest that the control of differentiation and proliferation by Rb are genetically separable.
doi:10.1128/MCB.23.15.5256-5268.2003
PMCID: PMC165732  PMID: 12861012
21.  AMPA Receptors Mediate Acetylcholine Release from Starburst Amacrine Cells in the Rabbit Retina 
The light response of starburst amacrine cells is initiated by glutamate released from bipolar cells. To identify the receptors that mediate this response, we used a combination of anatomical and physiological techniques. An in vivo, rabbit eyecup was preloaded with [3H]-choline, and the [3H]-acetylcholine (ACh) released into the superfusate was monitored. A photopic, 3 Hz flashing light increased ACh release, and the selective AMPA receptor antagonist, GYKI 53655, blocked this light-evoked response. Nonselective AMPA/kainate agonists increased the release of ACh, but the specific kainate receptor agonist, SYM 2081, did not increase ACh release. Selective AMPA receptor antagonists, GYKI 53655 or GYKI 52466, also blocked the responses to agonists. We conclude that the predominant excitatory input to starburst amacrine cells is mediated by AMPA receptors. We also labeled lightly fixed rabbit retinas with antisera to choline acetyltransferase (ChAT), AMPA receptor subunits GluR1, GluR2/3, or GluR4, and kainate receptor subunits GluR6/7 and KA2. Labeled puncta were observed in the inner plexiform layer with each of these antisera to glutamate receptors, but only GluR2/3-IR puncta and GluR4-IR puncta were found on the ChAT-IR processes. The same was true of starburst cells injected intracellularly with Neurobiotin, and these AMPA receptor subunits were localized to two populations of puncta. The AMPA receptors are expected to desensitize rapidly, enhancing the sensitivity of starburst amacrine cells to moving or other rapidly changing stimuli.
doi:10.1002/cne.10880
PMCID: PMC3341736  PMID: 14515241
glutamate; ionotropic receptors; localization; cholinergic; interneuron; confocal microscopy
22.  GABAA Receptors Containing the α2 Subunit Are Critical for Direction-Selective Inhibition in the Retina 
PLoS ONE  2012;7(4):e35109.
Far from being a simple sensor, the retina actively participates in processing visual signals. One of the best understood aspects of this processing is the detection of motion direction. Direction-selective (DS) retinal circuits include several subtypes of ganglion cells (GCs) and inhibitory interneurons, such as starburst amacrine cells (SACs). Recent studies demonstrated a surprising complexity in the arrangement of synapses in the DS circuit, i.e. between SACs and DS ganglion cells. Thus, to fully understand retinal DS mechanisms, detailed knowledge of all synaptic elements involved, particularly the nature and localization of neurotransmitter receptors, is needed. Since inhibition from SACs onto DSGCs is crucial for generating retinal direction selectivity, we investigate here the nature of the GABA receptors mediating this interaction. We found that in the inner plexiform layer (IPL) of mouse and rabbit retina, GABAA receptor subunit α2 (GABAAR α2) aggregated in synaptic clusters along two bands overlapping the dendritic plexuses of both ON and OFF SACs. On distal dendrites of individually labeled SACs in rabbit, GABAAR α2 was aligned with the majority of varicosities, the cell's output structures, and found postsynaptically on DSGC dendrites, both in the ON and OFF portion of the IPL. In GABAAR α2 knock-out (KO) mice, light responses of retinal GCs recorded with two-photon calcium imaging revealed a significant impairment of DS responses compared to their wild-type littermates. We observed a dramatic drop in the proportion of cells exhibiting DS phenotype in both the ON and ON-OFF populations, which strongly supports our anatomical findings that α2-containing GABAARs are critical for mediating retinal DS inhibition. Our study reveals for the first time, to the best of our knowledge, the precise functional localization of a specific receptor subunit in the retinal DS circuit.
doi:10.1371/journal.pone.0035109
PMCID: PMC3323634  PMID: 22506070
23.  Drosophila RB Proteins Repress Differentiation-Specific Genes via Two Different Mechanisms▿  
Molecular and Cellular Biology  2010;30(10):2563-2577.
The RB and E2F proteins play important roles in the regulation of cell division, cell death, and development by controlling the expression of genes involved in these processes. The mechanisms of repression by the retinoblastoma protein (pRB) have been extensively studied at cell cycle-regulated promoters. However, little is known about developmentally regulated E2F/RB genes. Here, we have taken advantage of the simplicity of the E2F/RB pathway in flies to inspect the regulation of differentiation-specific target genes. These genes are repressed by dE2F2/RBF and a recently identified RB-containing complex, dREAM/MMB, in a cell type- and cell cycle-independent manner. Our studies indicate that the mechanism of repression differs from that of cell cycle-regulated genes. We find that two different activities are involved in their regulation and that in proliferating cells, both are required to maintain repression. First, dE2F2/RBF and dREAM/MMB employ histone deacetylase (HDAC) activities at promoter regions. Remarkably, we have also uncovered an unconventional mechanism of repression by the Polycomb group (PcG) protein Enhancer of zeste [E(Z)], which is involved in silencing of these genes through the dimethylation of histone H3 Lys27 at nucleosomes located downstream of the transcription start sites (TSS).
doi:10.1128/MCB.01075-09
PMCID: PMC2863701  PMID: 20176807
24.  Mechanisms controlling cell cycle exit upon terminal differentiation 
Current opinion in cell biology  2007;19(6):697-704.
Summary
Coordinating terminal differentiation with permanent exit from the cell cycle is critical for proper organogenesis, yet how the cell cycle is blocked in differentiated tissues remains unclear. Important roles for Retinoblastoma family proteins and Cyclin-dependent kinase inhibitors have been delineated, but in many cases it remains unclear what triggers cell cycle exit. This review focuses on describing recent advances in deciphering how terminal differentiation and exit from the cell cycle are coordinated.
doi:10.1016/j.ceb.2007.10.004
PMCID: PMC2700000  PMID: 18035529
25.  Immunohistochemical evidence of neuronal and glial differentiation in retinoblastoma. 
BACKGROUND--The study sought to investigate the histogenesis of retinoblastoma. METHODS--One hundred specimens of retinoblastomas were examined along with those of 18 astrocytic gliomas and 15 medulloblastomas to compare similarities of glial differentiation in retinoblastoma and the two types of brain tumour. Employing avidin-biotin immunoperoxidase technique, antibodies were applied against neuron specific enolase (NSE), glial fibrillary acidic protein (GFAP), and S-100 protein (S-100). RESULTS--Most rosettes and fleurettes, and some undifferentiated cells in retinoblastomas were NSE positive, but GFAP and S-100 negative. GFAP and S-100 positive cells in retinoblastomas were detected mostly in well differentiated glial cells which were interpreted as reactive or non-neoplastic cells. Some of the GFAP and S-100 positive cells in retinoblastomas were defined as tumour cells that resembled neoplastic astrocytes in astrocytic gliomas and medulloblastomas. CONCLUSION--Retinoblastoma may arise from primitive bipotential or multipotential cells capable of neuronal and glial differentiation.
Images
PMCID: PMC505249  PMID: 7547791

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