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Mechanisms of Asymmetric Stem Cell Division

2008; Cell Press; Volume: 132; Issue: 4 Linguagem: Inglês

10.1016/j.cell.2008.02.007

1097-4172

Juergen A. Knoblich,

Cancer Cells and Metastasis

Stem cells self-renew but also give rise to daughter cells that are committed to lineage-specific differentiation. To achieve this remarkable task, they can undergo an intrinsically asymmetric cell division whereby they segregate cell fate determinants into only one of the two daughter cells. Alternatively, they can orient their division plane so that only one of the two daughter cells maintains contact with the niche and stem cell identity. These distinct pathways have been elucidated mostly in Drosophila. Although the molecules involved are highly conserved in vertebrates, the way they act is tissue specific and sometimes very different from invertebrates. Stem cells self-renew but also give rise to daughter cells that are committed to lineage-specific differentiation. To achieve this remarkable task, they can undergo an intrinsically asymmetric cell division whereby they segregate cell fate determinants into only one of the two daughter cells. Alternatively, they can orient their division plane so that only one of the two daughter cells maintains contact with the niche and stem cell identity. These distinct pathways have been elucidated mostly in Drosophila. Although the molecules involved are highly conserved in vertebrates, the way they act is tissue specific and sometimes very different from invertebrates. A hallmark of all stem cells is the ability to simultaneously generate identical copies of themselves but also to give rise to more differentiated progeny. Work mostly done in the fruitfly, Drosophila, has suggested two different mechanisms by which this can be achieved (Horvitz and Herskowitz, 1992Horvitz H.R. Herskowitz I. Mechanisms of asymmetric cell division: two Bs or not two Bs, that is the question.Cell. 1992; 68: 237-255Abstract Full Text PDF PubMed Google Scholar) (Figure 1). When an intrinsic mechanism is used, regulators of self-renewal are localized asymmetrically during mitosis so that they are inherited by only one of the two daughter cells (Betschinger and Knoblich, 2004Betschinger J. Knoblich J.A. Dare to be different: asymmetric cell division in Drosophila, C. elegans and vertebrates.Curr. Biol. 2004; 14: R674-R685Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, Yu et al., 2006Yu F. Kuo C.T. Jan Y.N. Drosophila neuroblast asymmetric cell division: recent advances and implications for stem cell biology.Neuron. 2006; 51: 13-20Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Already in interphase, cells which undergo such intrinsically asymmetric divisions use apical-basal or planar polarity of the surrounding tissue to set up an axis of polarity. As they enter mitosis, this axis is used to polarize the distribution of protein determinants and to orient the mitotic spindle so that these determinants are inherited by only one of the two daughter cells. Alternatively, the stem cell is in close contact with the stem cell niche and depends on this contact for maintaining the potential to self-renew (Li and Xie, 2005Li L. Xie T. Stem cell niche: structure and function.Annu. Rev. Cell Dev. Biol. 2005; 21: 605-631Crossref PubMed Scopus (503) Google Scholar). By orienting its mitotic spindle perpendicularly to the niche surface, it ensures that only one daughter cell can maintain contact with the stem cell niche and retain the ability to self-renew. In contrast to intrinsically asymmetric cell divisions, which usually follow a predefined developmental program, niche-controlled stem cell divisions offer a high degree of flexibility. Occasionally, the stem cell can divide parallel to the niche, thereby generating two stem cells to increase stem cell number or to compensate for occasional stem cell loss. For this reason, niche mechanisms are more common in adult stem cells, whereas intrinsically asymmetric divisions predominate during development. Collectively, both types of cell division are referred to as asymmetric cell division. An asymmetric cell division is defined as any division that gives rise to two sister cells that have different fates—a feature that can be recognized by differences in size, morphology, gene expression pattern, or the number of subsequent cell divisions undergone by the two daughter cells (Horvitz and Herskowitz, 1992Horvitz H.R. Herskowitz I. Mechanisms of asymmetric cell division: two Bs or not two Bs, that is the question.Cell. 1992; 68: 237-255Abstract Full Text PDF PubMed Google Scholar). Although some stem cells—in particular hematopoietic and embryonic stem (ES) cells—do not quite fit this definition when kept in culture, it is safe to assume that most, if not all, stem cells undergo asymmetric cell divisions when they are in their natural environment. In Drosophila, neuroblasts and ovarian stem cells are well-studied examples for the intrinsic and extrinsic mode of asymmetric cell division, respectively. Although these simple categories may not apply as exclusively to mammalian stem cells and both pathways seem to be combined in some cell types, they provide a conceptual framework that will help us to understand the complexity of mammalian stem cell biology. Below, I describe the anatomy and molecular machineries of asymmetric cell division in Drosophila neuroblasts and ovarian germline stem cells and use neural, muscle, and hematopoietic stem cells as examples to illustrate the similarities and differences in higher organisms (see Table 1 for a summary of the model systems described).Table 1Model Systems for Asymmetric Cell DivisionMother CellDaughter Cell TypesPolarity CueMechanism of Unequal Fate SpecificationDrosophila sensory organ precursor cellFour cell types forming external sensory organs: socket, hair, sheath, neuronPlanar polarityAsymmetric segregation of Numb results in differential Notch regulationDrosophila neuroblastNeuroblast, ganglion mother cellEpithelial polarityAsymmetric segregation of Numb, Prospero, and Brat results in self-renewal versus cell-cycle exitDrosophila ovarian germline stem cellStem cell, cystoblastNiche architectureDiffusible signal (Dpp and Gbb) from stem cell nicheMouse brain progenitor cellsProgenitor cell, neuron (occasionally: intermediate/basal progenitor)Apical-basal polarity of neuro-epitheliumUnidentified segregating determinant or apical membrane compartment or basal fiberMouse muscle satellite cellsStem cell (Myf5−), committed progenitor (Myf5+)Unclear, maybe integrin contact with basal laminaSegregating determinant (Numb), signal from basal lamina or bothMouse hematopoietic stem cellHematopoietic stem cell, committed progenitorSignal from stem cell niche (blood vessel or osteoblast)Different levels of Notch signaling (maybe induced by Numb segregation)Mouse T-lymphocytesEffector T cell, memory T cellImmunological synapseUnequal segregation of Numb, CD8 and Interferon γ receptor Open table in a new tab Drosophila sensory organ precursor (SOP) cells and neuroblasts (the progenitors of the peripheral and central nervous system, respectively) are well-studied examples of intrinsically asymmetric cell divisions (Figure 2). SOP cells give rise to the four cells present in external sensory organs (Figure 2A). Although they are not stem cells, SOP cells have revealed many of the fundamental principles for asymmetric cell division. This is mainly due to their simple and highly reproducible lineage: SOP cells delaminate from a polarized epithelium and then divide into an anterior pIIb and a posterior pIIa cell. After SOP division, pIIa and pIIb divide once more to generate the two outer and the two inner cells of the organ, respectively. Asymmetry in all of these divisions is generated by different levels of Notch activity in the two daughter cells (Schweisguth, 2004Schweisguth F. Notch signaling activity.Curr. Biol. 2004; 14: R129-R138Abstract Full Text Full Text PDF PubMed Google Scholar, Le Borgne et al., 2005Le Borgne R. Bardin A. Schweisguth F. The roles of receptor and ligand endocytosis in regulating Notch signaling.Development. 2005; 132: 1751-1762Crossref PubMed Scopus (206) Google Scholar). It is thought that SOP cells inherit epithelial planar polarity and use it to segregate regulators of the Notch signaling pathway into one of the two daughter cells. (A) Sensory organ precursor cells generate the four cells of external sensory organs in two consecutive rounds of asymmetric cell division. (B) Neuroblasts divide into one self-renewing daughter cell and one ganglion mother cell (GMC), which divides only once more into two differentiating neurons. Cellular growth is restricted to the self-renewing daughter cell. (C) The Drosophila larval brain contains ventral nerve chord (VNC, brown), optic lobe (OL, gray), mushroom body (MB, green), and central brain (CB, gray) neuroblasts, (GMCs and neurons, red). A group of dorso-posterior (DP, blue) central brain neuroblasts generates more daughter cells and is particularly susceptible to tumor formation. In contrast to SOP cells, Drosophila neuroblasts undergo multiple rounds of stem cell-like divisions (Figure 2B). During each division, they give rise to a large cell that retains neuroblast properties and a smaller cell that is called the ganglion mother cell (GMC) and divides only once more to generate two differentiating neurons. Neuroblasts come in two flavors; embryonic neuroblasts give rise to the relatively simple nervous system of the larva. They are specified within a monolayered epithelium called the ventral neuroectoderm and delaminate from the epithelium to undergo repeated rounds of asymmetric division along the apical-basal axis. It is thought that certain aspects of epithelial polarity are inherited by the neuroblasts and used to polarize the first mitotic division. Although the reproducible position and the relatively simple lineages of embryonic neuroblasts have made them a valuable system to discover basic principles of asymmetric division, their restricted self-renewal capacity limits their usefulness as a true stem cell model. Mainly for this reason, the field has recently begun to focus on larval neuroblasts. Larval neuroblasts generate the thousands of neurons found in the central nervous system of an adult fly. Unlike embryonic neuroblasts, which become smaller with each division, larval neuroblasts regrow back to their original size after each division and can divide hundreds of times (Ito and Hotta, 1992Ito K. Hotta Y. Proliferation pattern of postembryonic neuroblasts in the brain of Drosophila melanogaster.Dev. Biol. 1992; 149: 134-148Crossref PubMed Google Scholar, White and Kankel, 1978White K. Kankel D.R. Patterns of cell division and cell movement in the formation of the imaginal nervous system in Drosophila melanogaster.Dev. Biol. 1978; 65: 296-321Crossref PubMed Google Scholar) (Figure 2B). Several types of larval neuroblasts can be distinguished based on their position within the larval central nervous system (Figure 2C). In the ventral nerve chord, 30 ventral nerve chord neuroblasts per hemisegment divide repeatedly along the apical-basal axis to form the neurons of the thoracic and abdominal ganglia (Truman and Bate, 1988Truman J.W. Bate M. Spatial and temporal patterns of neurogenesis in the central nervous system of Drosophila melanogaster.Dev. Biol. 1988; 125: 145-157Crossref PubMed Google Scholar). In each of the two brain lobes, approximately 85 central brain neuroblasts give rise to most of the neurons present in the adult brain (Ito and Hotta, 1992Ito K. Hotta Y. Proliferation pattern of postembryonic neuroblasts in the brain of Drosophila melanogaster.Dev. Biol. 1992; 149: 134-148Crossref PubMed Google Scholar). Central brain neuroblasts are heterogeneous in cell cycle length and regulation of self-renewal. In particular, a group of less than 10 dorso-posterior (DP) neuroblasts seems to be particularly susceptible to mutations in tumor suppressor genes (Betschinger et al., 2006Betschinger J. Mechtler K. Knoblich J.A. Asymmetric segregation of the tumor suppressor brat regulates self-renewal in Drosophila neural stem cells.Cell. 2006; 124: 1241-1253Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Compared to other central brain neuroblasts, these precursors generate many more progeny and they might even have a different lineage in which GMCs divide more than once. It is worth noting that much of the earlier experiments on Drosophila larval neuroblasts did not distinguish between these subgroups and might have to be reinvestigated. In addition to the central brain neuroblasts, the fly brain contains the mushroom body and optic lobe neuroblasts. In each brain hemisphere, four mushroom body neuroblasts give rise to 2500 neurons called Kenyon cells that form the learning and memory centers (Ito and Hotta, 1992Ito K. Hotta Y. Proliferation pattern of postembryonic neuroblasts in the brain of Drosophila melanogaster.Dev. Biol. 1992; 149: 134-148Crossref PubMed Google Scholar, Ito et al., 1997Ito K. Awano W. Suzuki K. Hiromi Y. Yamamoto D. The Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually identical set of neurones and glial cells.Development. 1997; 124: 761-771PubMed Google Scholar). To generate this large number of neurons, they start dividing much earlier than central brain neuroblasts and proliferate throughout most of the pupal stages of development. Whereas mushroom body and central brain neuroblasts are already specified during embryogenesis and simply reactivate their proliferation programs during larval stages, optic lobe neuroblasts follow a distinct program of neurogenesis (Egger et al., 2007Egger B. Boone J.Q. Stevens N.R. Brand A.H. Doe C.Q. Regulation of spindle orientation and neural stem cell fate in the Drosophila optic lobe.Neural Development. 2007; 2 (Published online January 5, 2007)https://doi.org/10.1186/1749-8104-2-1Crossref PubMed Scopus (78) Google Scholar). They arise from two multilayered neuroepithelia called the inner- and outer-proliferation centers (White and Kankel, 1978White K. Kankel D.R. Patterns of cell division and cell movement in the formation of the imaginal nervous system in Drosophila melanogaster.Dev. Biol. 1978; 65: 296-321Crossref PubMed Google Scholar). Neuroepithelial cells divide symmetrically in parallel to the epithelial surface. Neuroblasts are generated on the rims of these epithelia. They lose their adherens junctions, turn on neuroblast markers, and start dividing asymmetrically and perpendicularly to the epithelial plane. Following a canonical neuroblast lineage, optic lobe neuroblasts give rise to the neurons in the visual processing centers of the fly brain. The different fate of the two neuroblast daughter cells is thought to be induced by the unequal segregation of several proteins into one of the two daughter cells (Figure 3). Due to their combined activity in specifying daughter cell fate, these proteins are referred to as segregating determinants. Because determinant segregation can even occur in individual cultured neuroblasts, it is thought to be governed by a cell-intrinsic machinery (Broadus and Doe, 1997Broadus J. Doe C.Q. Extrinsic cues, intrinsic cues and microfilaments regulate asymmetric protein localization in Drosophila neuroblasts.Curr. Biol. 1997; 7: 827-835Abstract Full Text Full Text PDF PubMed Google Scholar) (note, however, that partially redundant extrinsic cues exist as well—see below and Siegrist and Doe, 2006Siegrist S.E. Doe C.Q. Extrinsic cues orient the cell division axis in Drosophila embryonic neuroblasts.Development. 2006; 133: 529-536Crossref PubMed Scopus (70) Google Scholar). Before mitosis, the proteins Par-3, Par-6, atypical PKC (aPKC), Inscuteable, Pins, Gαi, and Mud (see below for their individual functions) accumulate on the apical side of the cell cortex (Betschinger and Knoblich, 2004Betschinger J. Knoblich J.A. Dare to be different: asymmetric cell division in Drosophila, C. elegans and vertebrates.Curr. Biol. 2004; 14: R674-R685Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, Suzuki and Ohno, 2006Suzuki A. Ohno S. The PAR-aPKC system: lessons in polarity.J. Cell Sci. 2006; 119: 979-987Crossref PubMed Scopus (336) Google Scholar, Goldstein and Macara, 2007Goldstein B. Macara I.G. The par proteins: fundamental players in animal cell polarization.Dev. Cell. 2007; 13: 609-622Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Although they are preferentially inherited by the apical daughter cell, which remains a neuroblast, they are not thought to influence cell fate directly. Instead, they induce the asymmetric localization of cell fate determinants to the opposite, basal side of the cell and their segregation into the basal GMC. Below, I discuss those determinants for which functions in Drosophila neural stem cells have been shown. Epithelial apical-basal polarity is used to establish the asymmetric accumulation of Par proteins (Par-3, Par-6, aPKC, red) to the apical cortex. Upon entry into mitosis and the activation of Aurora-A and Polo kinases, the mitotic spindle is oriented by the microtubule binding protein Mud. Mud is recruited apically by Pins and Gαi (green), which in turn associate with Inscuteable (yellow) and Par-3. The asymmetric localization of cell fate determinants (purple) to the cortex opposite Par-3/6 and aPKC requires the phosphorylation of Lgl (blue) by aPKC. Late in mitosis, astral microtubules of the mitotic spindle can redirect cortical polarity (orange arrows) through the telophase rescue pathway, which involves the kinesin Khc-73 (blue) and the protein Discs-large (Dlg, pink). Ultimately, the cell fate determinants Numb, Pros, and Brat (purple) segregate into the small daughter cell with the help of their adaptor proteins Pon and Miranda. In this cell, Numb represses Notch signaling, and Pros regulates transcription; the function of Brat is unknown. The first segregating determinant was called Numb and was actually identified in SOP cells (Rhyu et al., 1994Rhyu M.S. Jan L.Y. Jan Y.N. Asymmetric distribution of numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells.Cell. 1994; 76: 477-491Abstract Full Text PDF PubMed Scopus (456) Google Scholar). In numb mutants, both daughter cells of the SOP assume the fate of the cell that normally does not inherit the Numb protein. Conversely, numb overexpression results in the transformation into the opposite cell fate. Numb acts as a tissue-specific repressor of the Notch pathway (Le Borgne et al., 2005Le Borgne R. Bardin A. Schweisguth F. The roles of receptor and ligand endocytosis in regulating Notch signaling.Development. 2005; 132: 1751-1762Crossref PubMed Scopus (206) Google Scholar, Schweisguth, 2004Schweisguth F. Notch signaling activity.Curr. Biol. 2004; 14: R129-R138Abstract Full Text Full Text PDF PubMed Google Scholar). It binds to the endocytic protein α-Adaptin (Berdnik et al., 2002Berdnik D. Torok T. Gonzalez-Gaitan M. Knoblich J.A. The endocytic protein alpha-Adaptin is required for numb-mediated asymmetric cell division in Drosophila.Dev. Cell. 2002; 3: 221-231Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) and might control the intracellular trafficking of Notch intermediates. When Numb is mutated in the larval brain, the mutant neuroblasts overproliferate and form a tumor-like phenotype (Lee et al., 2006aLee C.Y. Andersen R.O. Cabernard C. Manning L. Tran K.D. Lanskey M.J. Bashirullah A. Doe C.Q. Drosophila Aurora-A kinase inhibits neuroblast self-renewal by regulating aPKC/Numb cortical polarity and spindle orientation.Genes Dev. 2006; 20: 3464-3474Crossref PubMed Scopus (107) Google Scholar, Wang et al., 2006Wang H. Somers G.W. Bashirullah A. Heberlein U. Yu F. Chia W. Aurora-A acts as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts.Genes Dev. 2006; 20: 3453-3463Crossref PubMed Scopus (103) Google Scholar). Lineage analysis shows that this is due to occasional divisions in which a neuroblast still divides into a larger and a smaller daughter cell but both daughter cells eventually show the gene expression and proliferation pattern of a neuroblast. Similar (but not identical) brain phenotypes are observed upon mutation of other segregating determinants and have made Drosophila neuroblasts an ideal model system to investigate the biology of cancer stem cells (Figure 4, see below) (Gonzalez, 2007Gonzalez C. Spindle orientation, asymmetric division and tumour suppression in Drosophila stem cells.Nat. Rev. Genet. 2007; 8: 462-472Crossref PubMed Scopus (98) Google Scholar). (A) Normally, neuroblasts divide into one neuroblast daughter (red), which continues to grow, and one GMC (green), which stops cell growth and divides only once more into two neurons (brown). During pupal stages, all neuroblasts stop proliferating, and no mitotic activity exists in adult fly brains. (B) Neuroblasts that lack any of the tumor suppressor genes Brat, Prospero, or Numb or have defects in their asymmetric segregation give rise to tumors. They still divide asymmetrically, but the mutant GMCs do not produce neurons. Instead, they regrow and continue to proliferate like neuroblasts. During pupal stages, these cells do not stop proliferating. Thus, defects in asymmetric cell division lead to the formation of a cell type that proliferates like a neuroblast but is immortal and no longer responds to the hormonal signals that inhibit proliferation during pupal development. Like Numb, the transcription factor Prospero (Pros) segregates asymmetrically in neuroblasts. Although Pros is already present in neuroblasts, it only enters the nucleus once asymmetrically segregated into the GMC (Betschinger and Knoblich, 2004Betschinger J. Knoblich J.A. Dare to be different: asymmetric cell division in Drosophila, C. elegans and vertebrates.Curr. Biol. 2004; 14: R674-R685Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). When Pros is mutated in embryonic neuroblasts, the GMC continues to express neuroblast markers and undergoes multiple rounds of division (Choksi et al., 2006Choksi S.P. Southall T.D. Bossing T. Edoff K. de Wit E. Fischer B.E. van Steensel B. Micklem G. Brand A.H. Prospero acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells.Dev. Cell. 2006; 11: 775-789Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Several cell-cycle regulators including Cyclins A and E and Cdc25 (string in Drosophila) are upregulated and may be responsible for this phenotype (Li and Vaessin, 2000Li L. Vaessin H. Pan-neural Prospero terminates cell proliferation during Drosophila neurogenesis.Genes Dev. 2000; 14: 147-151PubMed Google Scholar). In larval neuroblasts, mutations in Pros cause stem cell-derived tumors (Betschinger et al., 2006Betschinger J. Mechtler K. Knoblich J.A. Asymmetric segregation of the tumor suppressor brat regulates self-renewal in Drosophila neural stem cells.Cell. 2006; 124: 1241-1253Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, Lee et al., 2006cLee C.Y. Wilkinson B.D. Siegrist S.E. Wharton R.P. Doe C.Q. Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal.Dev. Cell. 2006; 10: 441-449Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, Bello et al., 2006Bello B. Reichert H. Hirth F. The brain tumor gene negatively regulates neural progenitor cell proliferation in the larval central brain of Drosophila.Development. 2006; 133: 2639-2648Crossref PubMed Scopus (111) Google Scholar). Pros contains a homeodomain and binds upstream of over 700 genes many of which are involved in neuroblast self-renewal or cell-cycle control. However, Pros can also induce the expression of neural differentiation genes indicating that it can act both as a transcriptional activator and inhibitor (Choksi et al., 2006Choksi S.P. Southall T.D. Bossing T. Edoff K. de Wit E. Fischer B.E. van Steensel B. Micklem G. Brand A.H. Prospero acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells.Dev. Cell. 2006; 11: 775-789Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). More recently, a third important regulator of neuroblast self-renewal has been identified (Lee et al., 2006cLee C.Y. Wilkinson B.D. Siegrist S.E. Wharton R.P. Doe C.Q. Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal.Dev. Cell. 2006; 10: 441-449Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, Bello et al., 2006Bello B. Reichert H. Hirth F. The brain tumor gene negatively regulates neural progenitor cell proliferation in the larval central brain of Drosophila.Development. 2006; 133: 2639-2648Crossref PubMed Scopus (111) Google Scholar, Betschinger et al., 2006Betschinger J. Mechtler K. Knoblich J.A. Asymmetric segregation of the tumor suppressor brat regulates self-renewal in Drosophila neural stem cells.Cell. 2006; 124: 1241-1253Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). This protein is called Brat and was previously shown to act as an inhibitor of ribosome biogenesis and cell growth (Frank et al., 2002Frank D.J. Edgar B.A. Roth M.B. The Drosophila melanogaster gene brain tumor negatively regulates cell growth and ribosomal RNA synthesis.Development. 2002; 129: 399-407PubMed Google Scholar). Brat is a member of a new conserved protein family that is characterized by the presence of a C-terminal NHL domain, a coiled-coil region and an N-terminal Zinc binding B-box (Slack and Ruvkun, 1998Slack F.J. Ruvkun G. A novel repeat domain that is often associated with RING finger and B-box motifs.Trends Biochem. Sci. 1998; 23: 474-475Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In Drosophila, Brat, Mei-P26, and Dappled are members of this family. Given that all three proteins act as tumor suppressors, growth control might be a common function of this protein family. During embryogenesis, Brat cooperates with Pros to specify GMC fate. Although only a small subset of GMCs is affected in pros mutants, pros/brat double mutants show an almost complete loss of all GMCs (Betschinger et al., 2006Betschinger J. Mechtler K. Knoblich J.A. Asymmetric segregation of the tumor suppressor brat regulates self-renewal in Drosophila neural stem cells.Cell. 2006; 124: 1241-1253Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). In larval brains, brat causes the formation of stem cell-derived tumors consisting almost entirely of large cells expressing neuroblast markers. This has led to the hypothesis that Brat might inhibit cell growth in one of the two neuroblast daughter cells to prevent self-renewal and induce terminal differentiation. The molecular mechanism by which Brat regulates cell growth and cell fate is currently unknown. Brat has a second function in specifying the anterior-posterior body axis and for this function, it binds to Nanos and Pumilio to repress translation of the posterior identity gene hunchback (Sonoda and Wharton, 2001Sonoda J. Wharton R.P. Drosophila Brain Tumor is a translational repressor.Genes Dev. 2001; 15: 762-773Crossref PubMed Scopus (181) Google Scholar). In neuroblasts, however, neither the phenotypes nor the expression patterns of Nanos, Pumilio or Hunchback suggest that Brat acts in a similar manner. Instead, Brat was suggested to be a transcriptional activator of Pros (Lee et al., 2006cLee C.Y. Wilkinson B.D. Siegrist S.E. Wharton R.P. Doe C.Q. Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal.Dev. Cell. 2006; 10: 441-449Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, Bello et al., 2006Bello B. Reichert H. Hirth F. The brain tumor gene negatively regulates neural progenitor cell proliferation in the larval central brain of Drosophila.Development. 2006; 133: 2639-2648Crossref PubMed Scopus (111) Google Scholar) because brat mutant tumors are Pros negative and overexpression of Pros can rescue tumor formation in brat mutants. However, this hypothesis neither explains why brat enhances the pros null mutant phenotype in embryonic neuroblasts nor why it regulates cell growth even in tissues that do not express Pros. Given that brat tumors arise specifically in DP neuroblasts (see above), lower expression levels of Pros in these cells would also explain why the brat tumors are Pros negative and why these cells are particularly susceptible to loss of other tumor suppressors. Clearly, more experiments including the identification of functional binding partners are necessary to determine how Brat acts. In fact, brat orthologs were found to be essential for RNA interference in Caenorhabditis elegans (Kim et al., 2005Kim J.K. Gabel H.W. Kamath R.S. Tewari M. Pasquinelli A. Rual J.F. Kennedy S. Dybbs M. Bertin N. Kaplan J.M. et al.Functional genomic analysis of RNA interference in C. elegans.Science. 2005; 308: 1164-1167Crossref PubMed Scopus (160) Google Scholar) and regulation of micro RNAs could be another very exciting function for this protein family. The asymmetric segregation of Pros, Brat, and Numb is mediated by two adaptor proteins called Miranda and Pon (Partner of Numb) (Betschinger and Knoblich, 2004Betschinger J. Knoblich J.A. Dare to be different: asymmetric cell division in Drosophila, C. elegans and vertebrates.Curr. Biol. 2004; 14: R674-R685Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Miranda is a coiled-coil protein that binds to Pros and Brat. Miranda also binds to the RNA binding protein Staufen which in turn transports pros RNA but is not required for cell-fate specification in neuroblasts. Like the determinants, Miranda localizes asymmetrically and segregates into one of