Unique Cell Fates Research Articles

Wnt3a activates dormant c-Kit(-) bone marrow-derived cells with short-term multilineage hematopoietic reconstitution capacity.

Quiescent cells lacking expression of mature lineage makers and the c-Kit receptor reside in adult bone marrow. Despite their phenotypic similarity to hematopoietic stem cells, these Lin(-)Sca-1(+)c-Kit(-) cells lack myeloid and erythroid potential and long-term hematopoietic repopulating capacity, whereas, recent studies have functionally demonstrated that the Lin(-)Sca-1(+)c-Kit(-) population contains early lymphoid-committed progenitors. Examining the role of Wnt signaling in regulation of this population, we found that c-Kit(-) cells express diverse Wnt receptors and proliferate upon Wnt pathway activation in vitro and in vivo. Stimulation with Wnt3a, but not Wnt5a or Wnt11, promoted c-Kit(-) cells to give rise to myeloid and erythroid progenitors with robust self-renewal capacity measured by clonal replating. In addition, Wnt3a-stimulated c-Kit(-) cells gave rise to all hematopoietic lineages (lymphoid, myeloid, and erythroid) upon transplant into the liver of newborn recipient mice. Our study reveals that Wnt3a activates unique cell fate decisions of dormant c-Kit(-) that promotes short-term multilineage reconstitution capacity in vivo, thereby revealing a unique role for Wnt activation in hematopoiesis. Overall, our results highlight the potential of utilizing signaling molecules known to have instructive roles in regeneration to discover cell subsets residing in adult organisms with unexploited regenerative capacity.

Neuronal Subtype Specification within a Lineage by Opposing Temporal Feed-Forward Loops

Neural progenitors generate distinct cell types at different stages, but the mechanisms controlling these temporal transitions are poorly understood. In the Drosophila CNS, a cascade of transcription factors, the "temporal gene cascade," has been identified that acts to alter progenitor competence over time. However, many CNS lineages display broad temporal windows, and it is unclear how broad windows progress into subwindows that generate unique cell types. We have addressed this issue in an identifiable Drosophila CNS lineage and find that a broad castor temporal window is subdivided by two different feed-forward loops, both of which are triggered by castor itself. The first loop acts to specify a unique cell fate, whereas the second loop suppresses the first loop, thereby allowing for the generation of alternate cell fates. This mechanism of temporal and "subtemporal" genes acting in opposing feed-forward loops may be used by many stem cell lineages to generate diversity.

GATA-1 Represses PU.1/Sfpi-1 Gene Transcription in Erythro-Megakaryocytic Progenitors.

Commitment of multipotential hematopoietic progenitors to unique cell fates is determined by the induction and interplay of specific nuclear factors that reinforce unilineage transcriptional programs. Previously, we reported that loss of transcription factor GATA-1 promotes the expansion of bipotential megakaryocyte erythroid progenitors (MEPs) from embryonic stem cell or fetal liver derived hematopoietic progenitors. These mutant cells, termed G1ME (for Gata-1−Megakaryocyte-Erythroid), proliferate in culture and differentiate into committed megakaryocytes and erythroblasts when GATA-1 activity is restored. To evaluate gene regulation at the MEP stage of hematopoiesis, we performed microarray analysis of G1ME cells before and after GATA-1-induced differentiation. Expression of GATA-1 in G1ME cells induced numerous erythroid and megakaryocytic target genes. In addition, undifferentiated G1ME cells expressed numerous granulocyte and macrophage genes, suggesting that loss of GATA-1 derepresses a myeloid program in MEPs. Myeloid genes were rapidly inhibited upon expression of GATA-1. In particular, mRNA encoding the myeloid transcription factor PU.1 and more than thirty of its downstream targets were repressed by GATA-1. Chromatin immunoprecipitation (ChIP) showed that GATA-1 bound directly to the PU.1 gene (Sfpi1) at the promoter and at a −18kb upstream region coincident with repression. At the same regions, GATA-1 triggered release of both GATA-2, a related transcription factor expressed in multipotential progenitors, and PU.1, a positive autoregulator of its own gene. While GATA-1 is known to inhibit PU.1 functions through direct protein interactions, this work shows that antagonism also occurs at the level of Sfpi1 gene transcription. Together, our findings indicate that GATA-1 not only positively regulates an erythro-megakaryocytic program of gene expression, but also actively restrains myelopoiesis by inhibiting Sfpi1 expression directly. More generally, our work reveals a new mechanism through which cross-antagonism between transcription factors reinforces lineage commitment decisions in hematopoiesis.

Specification of Neuronal Identities by Feedforward Combinatorial Coding

Neuronal specification is often seen as a multistep process: earlier regulators confer broad neuronal identity and are followed by combinatorial codes specifying neuronal properties unique to specific subtypes. However, it is still unclear whether early regulators are re-deployed in subtype-specific combinatorial codes, and whether early patterning events act to restrict the developmental potential of postmitotic cells. Here, we use the differential peptidergic fate of two lineage-related peptidergic neurons in the Drosophila ventral nerve cord to show how, in a feedforward mechanism, earlier determinants become critical players in later combinatorial codes. Amongst the progeny of neuroblast 5–6 are two peptidergic neurons: one expresses FMRFamide and the other one expresses Nplp1 and the dopamine receptor DopR. We show the HLH gene collier functions at three different levels to progressively restrict neuronal identity in the 5–6 lineage. At the final step, collier is the critical combinatorial factor that differentiates two partially overlapping combinatorial codes that define FMRFamide versus Nplp1/DopR identity. Misexpression experiments reveal that both codes can activate neuropeptide gene expression in vast numbers of neurons. Despite their partially overlapping composition, we find that the codes are remarkably specific, with each code activating only the proper neuropeptide gene. These results indicate that a limited number of regulators may constitute a potent combinatorial code that dictates unique neuronal cell fate, and that such codes show a surprising disregard for many global instructive cues.

Specification ofDrosophilaaCC motoneuron identity by a genetic cascade involvingeven-skipped, grainandzfh1

During nervous system development, combinatorial codes of regulators act to specify different neuronal subclasses. However, within any given subclass, there exists a further refinement, apparent in Drosophila and C. elegans at single-cell resolution. The mechanisms that act to specify final and unique neuronal cell fates are still unclear. In the Drosophila embryo, one well-studied motoneuron subclass, the intersegmental motor nerve (ISN), consists of seven unique motoneurons. Specification of the ISN subclass is dependent upon both even-skipped (eve) and the zfh1 zinc-finger homeobox gene. We find that ISN motoneurons also express the GATA transcription factor Grain, and grn mutants display motor axon pathfinding defects. Although these three regulators are expressed by all ISN motoneurons, these genes act in an eve-->grn-->zfh1 genetic cascade unique to one of the ISN motoneurons, the aCC. Our results demonstrate that the specification of a unique neuron, within a given subclass, can be governed by a unique regulatory cascade of subclass determinants.

The Simple Life (of Cortical Progenitors)

Asymmetric cell division plays a major role in the generation of cell diversity during development. In this issue of Neuron, Sun and colleagues present evidence that the epidermal growth factor receptor is asymmetrically distributed in mitotic cerebral cortical precursors, and the resulting unequal inheritance generates offspring with different responsiveness to growth factor and unique cell fates.

The little R cell that could

Drosophila eye development provides an excellent model system to study the role of inter-cellular signaling in the specification of unique cell fates. Behavioral screens by Benzer and his colleagues led to the identification of a gene, Sevenless, a receptor tyrosine kinase (RTK) receptor, required for the specification of the UV sensitive R7 cell. Genetic analysis further showed that the Ras/Raf/MAPK pathway function downstream of Sevenless in the specification of R7 fate. Signaling mediated by another RTK, EGFR and Notch have also been shown to function in either an antagonistic or a synergistic manner in the specification of cell fate during eye development. In some instances, these pathways are linked in a sequential manner by the regulation of the expression of Notch ligand, Delta by EGFR, while in others, these pathways function in a combinatorial fashion on enhancer elements to control target gene expression. In this review, we highlight the elegant genetic strategies used by several laboratories in early elucidation of the Sevenless pathway which helped link the RTK receptor to the Ras/Raf/MAPK cascade and discuss how EGFR and Notch signaling pathways are used in a reiterative manner and by combining in different modes, generate sufficient diversity required for the specification of unique cell fates.

Chromium (VI) Activates Ataxia Telangiectasia Mutated (ATM) Protein: REQUIREMENT OF ATM FOR BOTH APOPTOSIS AND RECOVERY FROM TERMINAL GROWTH ARREST

The ataxia telangiectasia mutated (ATM) protein plays a central role in early stages of DNA double strand break (DSB) detection and controls cellular responses to this damage. Although hypersensitive to ionizing radiation-induced clonogenic lethality, ataxia telangiectasia cells are paradoxically deficient in their ability to undergo ionizing radiation-induced apoptosis. This contradiction illustrates the complexity of the central role of ATM in DNA damage response and the need for further understanding. Certain hexavalent chromium (Cr(VI)) compounds are implicated as occupational respiratory carcinogens at doses that are both genotoxic and cytotoxic. Cr(VI) induces a broad spectrum of DNA damage, but Cr(VI)-induced DSBs have not been reported. Here, we examined the role of ATM in the cellular response to Cr(VI) and found that Cr(VI) activates ATM. We also show that physiological targets of ATM, p53 Ser-15 and Chk2 Thr-68, were phosphorylated by Cr(VI) exposure in an ATM-dependent fashion. We found that ATM-/- cells were markedly resistant to Cr(VI)-induced apoptosis but considerably more sensitive to Cr(VI)-induced clonogenic lethality than wild type cells, indicating that resistance to Cr(VI)-induced apoptosis did not confer a selective survival advantage. However, analysis of long term growth arrest revealed a striking difference: ATM-/- cells were markedly less able to recover from Cr(VI)-induced growth arrest. This indicates that terminal growth arrest is the fate of these apoptosis-resistant cells. In summary, ATM is involved in cellular response to a complex genotoxin that may not directly induce DSBs. Our data suggest that ATM is a major signal initiator for genotoxin-induced apoptosis but, paradoxically, also contributes to maintenance of cell survival by facilitating recovery/escape from terminal growth arrest. The results also strongly suggest that terminal growth arrest is not merely an extended or even irreversible form of checkpoint arrest, but instead an independent and unique cell fate pathway.

Cell Adhesion Molecules during Inner Ear and Hair Cell Development, Including Notch and Its Ligands

Cellular adhesion plays a key role in a number of unique developmental events, including proliferation, cell fate, morphogenesis, neurite outgrowth, fasciculation, and synaptogensis. The number of families of molecules that can mediate cell adhesion and the number of members of each of those families has continued to increase over time. Moreover, the potential for the formation of different pairs of heterodimers with different binding specificities, and for both homo- and hetero-dimeric interactions suggest that a vast number of specific signaling events can be mediated through the expression of different combinations of adhesion factors at different developmental time points. By comparison with the number of known adhesion molecules and their potential effects, our understanding of the role of adhesion in ear development is extremely limited. The patterns of expression for some adhesion molecules have been determined for some aspects of inner ear development. Similarly, with a few exceptions, functional data to indicate the roles of these adhesion molecules are also lacking. However, a consideration of even the limited existing data must lead to the conclusion that adhesion molecules play key roles in all aspects of the development of the auditory system. Unique expression domains for different groups of adhesion molecules within the developing otocyst and ear strongly suggest a role in the determination of different cellular domains. Similarly, the specific expression of adhesion molecules on developing neurites and their target hair cells, suggests a key role for adhesion in the establishment of neuronal connections and possible the development of tonotopy. Finally, the recent demonstration that Cdh23 and Pcdh15 play specific roles in the formation of the hair cell stereociliary bundle provides compelling evidence for the importance of adhesion molecules in the development of stereocilia. With the imminent completion of the mouse genome, it seems likely that the number of adhesion molecules can soon be fixed and that it will then be possible to generate a more comprehensive map of expression of these molecules within the developing inner ear. At the same time, the generation of new transgenic and molecular technologies promises to provide researchers with new tools to examine the specific effects of different adhesion molecules during inner ear development.

Cell Fates and the Dilemma of Common Signaling

How can similar signals sent by a receptor tyrosine kinase (RTK) elicit different outcomes (differentiation fates) for various cells? Flores et al . studied the ability of the Drosophila epidermal growth factor receptor (DER), an RTK, to specify the expression of the Drosophila Pax2 ( D-Pax2 ) gene whose expression is restricted to pigment and cone cells within ommatidia. The authors identified an enhancer element for D-Pax2 that is necessary for D-Pax2 expression in cone cells. However, expression required the activity of the transcription factors Suppressor of Hairless (involved in Notch signaling), Pointed (which supplants the effects of the transcription inhibitor Yan), and Lozenge, as well as their cognate DNA binding sites. Removing the ability of any one of these components to function prevented the expression of D-Pax2 . Similarly, Xu et al . studying prospero expression in Drosophila found that although DER signaling was necessary, there was a further requirement for Pointed and Lozenge activity in a prospero enhancer. Thus, in the absence of Pointed or Lozenge binding, the enhancer was unable to mediate the expression of prospero . Finally, Halfon et al . examined the fate switches determined by DER and another RTK termed Heartless (a fibroblast growth factor receptor homolog) by observing the specific expression of even-skipped within the dorsal mesoderm layer. The authors found that Wingless and Decapentaplegic [homologs to Wnt and transforming growth factor β (TGF-β), respectively] were required before RTK activation in order to direct specific gene expression. However, even-skipped expression depended upon the further involvement of the transcription factors Pointed, Mad, and dTCF binding to a specific region within an even-skipped enhancer. As is becoming a recurring theme, the disruption of the binding of any of the transcription factors to the enhancer prevented the specific expression of the gene studied, in this case even-skipped . Thus, these studies demonstrate that the activation of several different signaling pathways impinging on the same genetic end point is important for generating specific gene expression and differentiation into specific cell fates. Simon writes a particularly lucid overview of these papers and their implications. Flores, G.V., Duan, H., Yan, H., Nagaraj, R., Fu, W., Zou, Y., Noll, M., and Banerjee, U. (2000) Combinatorial signaling in the specification of unique cell fates. Cell 103 : 75-85. [Online Journal] Xu, C., Kauffmann, R.C., Zhang, J., Kladny, S., and Carthew, R.W. (2000) Overlapping activators and repressors delimit transcriptional response to receptor tyrosine kinase signals in the Drosophila eye. Cell 103 : 87-97. [Online Journal] Halfon, M.S., Carmena, A., Gisselbrecht, S., Sackerson, C.M., Jiménez, F., Baylies, M.K., and Michelson, A.M. (2000) Ras pathway specificity is determined by the integration of multiple signal-activated and tissue-restricted transcription factors. Cell 103 : 63-74. [Online Journal] Simon, M.A. (2000) Receptor tyrosine kinases: Specific outcomes from general signals. Cell 103 : 13-15. [Online Journal]

Combinatorial Signaling in the Specification of Unique Cell Fates

How multifunctional signals combine to specify unique cell fates during pattern formation is not well understood. Here, we demonstrate that together with the transcription factor Lozenge, the nuclear effectors of the EGFR and Notch signaling pathways directly regulate D-Pax2 transcription in cone cells of the Drosophila eye disc. Moreover, the specificity of D-Pax2 expression can be altered upon genetic manipulation of these inputs. Thus, a relatively small number of temporally and spatially controlled signals received by a set of pluripotent cells can create the unique combinations of activated transcription factors required to regulate target genes and ultimately specify distinct cell fates within this group. We expect that similar mechanisms may specify pattern formation in vertebrate developmental systems that involve intercellular communication.

Krüppel acts as a developmental switch gene that mediates Notch signalling-dependent tip cell differentiation in the excretory organs of Drosophila.

Cell proliferation in the excretory organs of Drosophila, the Malpighian tubules (MT), is under the control of a neural tip cell. This unique cell is singled out from equivalent MT primordial cells in response to Notch signalling. We show that the gene Krüppel (Kr), best known for its segmentation function in the early embryo, is under the control of the Notch-dependent signalling process. Lack-of-function and gain-of-function experiments demonstrate that Kr activity determines the neural fate of tip cells by acting as a direct downstream target of proneural basic helix-loop-helix (bHLH) proteins that are restricted in response to Notch signalling. We have identified a unique cis-acting element that mediates all spatial and temporal aspects of Kr gene expression during MT development. This element contains functional binding sites for the restricted proneural bHLH factors and Fork head protein which is expressed in all MT cells. Our results suggest a mechanism in which these transcription factors cooperate to set up a unique cell fate within an equivalence group of cells by restricting the activity of the developmental switch gene Kr in response to Notch signalling.

The differentiation of the serotonergic neurons in the Drosophila ventral nerve cord depends on the combined function of the zinc finger proteins Eagle and Huckebein.

The Drosophila ventral nerve cord (vNC) derives from a stereotyped population of neural stem cells, neuroblasts (NBs), each of which gives rise to a characteristic cell lineage. The mechanisms leading to the specification and differentiation of these lineages are largely unknown. Here we analyse mechanisms leading to cell differentiation within the NB 7-3 lineage. Analogous to the grasshopper, NB 7-3 is the progenitor of the Drosophila vNC serotonergic neurons. The zinc finger protein Eagle (Eg) is expressed in NB 7-3 just after delamination and is present in all NB 7-3 progeny until late stage 17. DiI cell lineage tracing and immunocytochemistry reveal that eg is required for normal pathfinding of interneuronal projections and for restricting the cell number in the thoracic NB 7-3 lineage. Moreover, eg is required for serotonin expression. Ectopic expression of Eg protein forces specific additional CNS cells to enter the serotonergic differentiation pathway. Like NB 7-3, the progenitor(s) of these ectopic cells express Huckebein (Hkb), another zinc finger protein. However, their progenitors do not express engrailed (en) as opposed to the NB 7-3 lineage, where en acts upstream of eg. We conclude that eg and hkb act in concert to determine serotonergic cell fate, while en is more distantly involved in this process by activating eg expression. Thus, we provide the first functional evidence for a combinatorial code of transcription factors acting early but downstream of segment polarity genes to specify a unique neuronal cell fate.

Nuclear LIM interactor, a rhombotin and LIM homeodomain interacting protein, is expressed early in neuronal development.

LIM domain-containing transcription factors, including the LIM-only rhombotins and LIM-homeodomain proteins, are crucial for cell fate determination of erythroid and neuronal lineages. The zinc-binding LIM domains mediate protein-protein interactions, and interactions between nuclear LIM proteins and transcription factors with restricted expression patterns have been demonstrated. We have isolated a novel protein, nuclear LIM interactor (NLI), that specifically associates with a single LIM domain in all nuclear LIM proteins tested. NLI is expressed in the nuclei of diverse neuronal cell types and is coexpressed with a target interactor islet-1 (Isl1) during the initial stages of motor neuron differentiation, suggesting the mutual involvement of these proteins in the differentiation process. The broad range of interactions between NLI and LIM-containing transcription factors suggests the utilization of a common mechanism to impart unique cell fate instructions.

Neurogenesis in the insect central nervous system

Recent advances provide an increasingly sophisticated understanding of Drosophila CNS development. First, genes have been identified that specify unique neuroblast cell fates. Second, neuroblasts have been found to use a novel mechanism for asymmetric localization of proteins into one daughter cell at mitosis. Third, a gene controlling the choice between glial/neuronal determination has been discovered. And finally, new cell-lineage methods are being used to determine the entire lineage of identified neuroblasts, including axon projections and synaptic contacts.

Asymmetric localization of numb autonomously determines sibling neuron identity in the Drosophila CNS

The central nervous system (CNS) represents an excellent model system for examining how a multitude of unique cell fates are specified. We find that asymmetric localization of the numb protein autonomously controls a binary cell fate decision in the Drosophila CNS. The simplest lineage in the Drosophila CNS is that of the MP2 precursor: it divides unequally to generate the dMP2 and vMP2 neurons. Both are interneurons but project in different directions: dMP2 projects its axon posteriorly while vMP2 projects anteriorly. During MP2 mitosis, numb is localized into dMP2 and excluded from vMP2. Loss of numb transforms dMP2 into vMP2, whereas ectopic numb produces the opposite transformation of vMP2 into dMP2. Thus, numb is asymmetrically localized in the dividing MP2 and is necessary and sufficient to autonomously specify dMP2 neuronal identity.