Neuromancer1 and Neuromancer2 regulate cell fate specification in the developing embryonic CNS of Drosophila melanogaster.

T-box genes encode a large family of transcription factors that regulate many developmental processes in vertebrates and invertebrates. In addition to their roles in regulating embryonic heart and epidermal development in Drosophila, we provide evidence that the T-box transcription factors neuromancer1 (nmr1) and neuromancer2 (nmr2) play key roles in embryonic CNS development. We verify that nmr1 and nmr2 function in a partially redundant manner to regulate neuronal cell fate by inhibiting even-skipped (eve) expression in specific cells in the CNS. Consistent with their redundant function, nmr1 and nmr2 exhibit overlapping yet distinct protein expression profiles within the CNS. Of note, nmr2 transcript and protein are expressed in identical patterns of segment polarity stripes, defined sets of neuroblasts, many ganglion mother cells and discrete populations of neurons. However, while we observe nmr1 transcripts in segment polarity stripes and specific neural precursors in early stages of CNS development, we first detect Nmr1 protein in later stages of CNS development where it is restricted to discrete subsets of Nmr2-positive neurons. Expression studies identify nearly all Nmr1/2 co-expressing neurons as interneurons, while a single Eve-positive U/CQ motor neuron weakly co-expresses Nmr2. Lineage studies map a subset of Nmr1/2-positive neurons to neuroblast lineages 2-2, 6-1, and 6-2 while genetic studies reveal that nmr2 collaborates with nkx6 to regulate eve expression in the CNS. Thus, nmr1 and nmr2 appear to act together as members of the combinatorial code of transcription factors that govern neuronal subtype identity in the CNS.

Drosophila Lame duck, a novel member of the Gli superfamily, acts as a key regulator of myogenesis by controlling fusion-competent myoblast development.

A hallmark of mature skeletal muscles is the presence of multinucleate muscle fibers. In Drosophila, the formation of muscle syncytia requires the cooperative participation of two types of myoblasts, founder cells and fusion-competent myoblasts. We show that a newly identified gene, lame duck (lmd), has an essential regulatory role in the specification and function of fusion-competent myoblasts. Embryos that lack lmd function show a loss of expression of two key differentiation and fusion genes, Mef2 and sticks-and-stones, in fusion-competent myoblasts and are completely devoid of multinucleate muscle fibers. By contrast, founder cells are specified and retain their capability to differentiate into mononucleate muscle cells. lmd encodes a novel member of the Gli superfamily of transcription factors and is expressed in fusion-competent myoblasts and their precursors in a Wingless- and Notch-dependent manner. The activity of the Lmd protein appears to be additionally controlled by its differential cytoplasmic versus nuclear localization. Results from an independent molecular screen for binding factors to a myoblast-specific Mef2 enhancer further demonstrate that Lmd is a direct transcriptional regulator of Mef2 in fusion-competent myoblasts.

Characterization of a novel subset of cardiac cells and their progenitors in the Drosophila embryo.

The Drosophila heart is a simple organ composed of two major cell types: cardioblasts, which form the simple contractile tube of the heart, and pericardial cells, which flank the cardioblasts. A complete understanding of Drosophila heart development requires the identification of all cell types that comprise the heart and the elucidation of the cellular and genetic mechanisms that regulate the development of these cells. Here, we report the identification of a new population of heart cells: the Odd skipped-positive pericardial cells (Odd-pericardial cells). We have used descriptive, lineage tracing and genetic assays to clarify the cellular and genetic mechanisms that control the development of Odd-pericardial cells. Odd skipped marks a population of four pericardial cells per hemisegment that are distinct from previously identified heart cells. We demonstrate that within a hemisegment, Odd-pericardial cells develop from three heart progenitors and that these heart progenitors arise in multiple anteroposterior locations within the dorsal mesoderm. Two of these progenitors divide asymmetrically such that each produces a two-cell mixed-lineage clone of one Odd-pericardial cell and one cardioblast. The third progenitor divides symmetrically to produce two Odd-pericardial cells. All remaining cardioblasts in a hemisegment arise from two cardioblast progenitors each of which produces two cardioblasts. Furthermore, we demonstrate that numb and sanpodo mediate the asymmetric divisions of the two mixed-lineage heart progenitors noted above.

At the nexus between pattern formation and cell-type specification: the generation of individual neuroblast fates in the Drosophila embryonic central nervous system.

The specification of specific and often unique fates to individual cells as a function of their position within a developing organism is a fundamental process during the development of multicellular organisms. The development of the Drosophila embryonic central nervous system serves as an excellent model system in which to clarify the developmental mechanisms that link pattern formation to cell-type specification. The Drosophila embryonic central nervous system develops from a set of neural stem cells termed neuroblasts. Neuroblasts arise from the ectoderm in an invariant pattern, and each neuroblast acquires a unique fate based on its position within this pattern. Two groups of genes recently have been demonstrated to govern the individual fate specification of neuroblasts. One group, the segment polarity genes, enables neuroblasts that develop in different anteroposterior positions to acquire different fates. The second group, referred to as the columnar genes, ensures that neuroblasts that develop in different dorsoventral domains assume different fates. When integrated, the activities of the segment polarity and columnar genes create a Cartesian coordinate system that bestows unique fates to individual neuroblasts as a function of their position of formation within the ectoderm. BioEssays 1999;21:922-931.

Neural cell fate in rca1 and cycA mutants: the roles of intrinsic and extrinsic factors in asymmetric division in the Drosophila central nervous system.

In the central nervous system (CNS) of Drosophila embryos lacking regulator of cyclin A (rca1) or cyclin A, we observe that several ganglion mother cells (GMCs) fail to divide. Whereas GMCs normally produce two sibling neurons that acquire different fates ('A/B'), non-dividing GMCs differentiate exclusively in the manner of one of their progeny ('B'). In zygotic numb mutants, sibling neuron fate alterations ('A/B' to 'A/A') occur infrequently or do not occur in some sibling pairs; we have determined that depletion of both maternal and zygotic numb causes sibling neurons to acquire equalized fates ('A/A') with near-complete expressivity. In rca1, numb mutant embryos, we observe binary cell fate changes ('B' to 'A') in several GMCs as well. Finally, we have demonstrated that expression of Delta in the mesoderm is sufficient to attain both sibling fates. Our results indicate that the intrinsic determinant Numb is absolutely required to attain differential sibling neuron fates. While the extrinsic factors Notch and Delta are also required to attain both fates, our results indicate that Delta signal can be received from outside the sibling pair.

zfh-1, the Drosophila homologue of ZEB, is a transcriptional repressor that regulates somatic myogenesis.

zfh-1 is a member of the zfh family of proteins, which all contain zinc finger and homeodomains. The roles and mechanisms of action of most family members are still unclear. However, we have shown previously that another member of the family, the vertebrate ZEB protein, is a transcriptional repressor that binds E box sequences and inhibits myotube formation in cell culture assays. zfh-1 is downregulated in Drosophila embryos prior to myogenesis. Embryos with zfh-1 loss-of-function mutation show alterations in the number and position of embryonic somatic muscles, suggesting that zfh-1 could have a regulatory role in myogenesis. However, nothing is known about the nature or mechanism of action of zfh-1. Here, we demonstrate that zfh-1 is a transcription factor that binds E box sequences and acts as an active transcriptional repressor. When zfh-1 expression was maintained in the embryo beyond its normal temporal pattern of downregulation, the differentiation of somatic but not visceral muscle was blocked. One potential target of zfh-1 in somatic myogenesis could be the myogenic factor mef2. mef2 is known to be regulated by the transcription factor twist, and we show here that zfh-1 binds to sites in the mef2 upstream regulatory region and inhibits twist transcriptional activation. Even though there is little sequence similarity in the repressor domains of ZEB and zfh-1, we present evidence that zfh-1 is the functional homologue of ZEB and that the role of these proteins in myogenesis is conserved from Drosophila to mammals.

Heartbroken is a specific downstream mediator of FGF receptor signalling in Drosophila.

Drosophila possesses two FGF receptors which are encoded by the heartless and breathless genes. HEARTLESS is essential for early migration and patterning of the embryonic mesoderm, while BREATHLESS is required for proper branching of the tracheal system. We have identified a new gene, heartbroken, that participates in the signalling pathways of both FGF receptors. Mutations in heartbroken are associated with defects in the migration and later specification of mesodermal and tracheal cells. Genetic interaction and epistasis experiments indicate that heartbroken acts downstream of the two FGF receptors but either upstream of or parallel to RAS1. Furthermore, heartbroken is involved in both the HEARTLESS- and BREATHLESS-dependent activation of MAPK. In contrast, EGF receptor-dependent embryonic functions and MAPK activation are not perturbed in heartbroken mutant embryos. A strong heartbroken allele also suppresses the effects of hyperactivated FGF but not EGF receptors. Thus, heartbroken may contribute to the specificity of developmental responses elicited by FGF receptor signalling.

The Drosophila EGF receptor controls the formation and specification of neuroblasts along the dorsal-ventral axis of the Drosophila embryo.

The segmented portion of the Drosophila embryonic central nervous system develops from a bilaterally symmetrical, segmentally reiterated array of 30 unique neural stem cells, called neuroblasts. The first 15 neuroblasts form about 30-60 minutes after gastrulation in two sequential waves of neuroblast segregation and are arranged in three dorsoventral columns and four anteroposterior rows per hemisegment. Each neuroblast acquires a unique identity, based on gene expression and the unique and nearly invariant cell lineage it produces. Recent experiments indicate that the segmentation genes specify neuroblast identity along the AP axis. However, little is known as to the control of neuroblast identity along the DV axis. Here, I show that the Drosophila EGF receptor (encoded by the DER gene) promotes the formation, patterning and individual fate specification of early forming neuroblasts along the DV axis. Specifically, I use molecular markers that identify particular neuroectodermal domains, all neuroblasts or individual neuroblasts, to show that in DER mutant embryos (1) intermediate column neuroblasts do not form, (2) medial column neuroblasts often acquire identities inappropriate for their position, while (3) lateral neuroblasts develop normally. Furthermore, I show that active DER signaling occurs in the regions from which the medial and intermediate neuroblasts will later delaminate. In addition, I demonstrate that the concomitant loss of rhomboid and vein yield CNS phenotypes indistinguishable from DER mutant embryos, even though loss of either gene alone yields minor CNS phenotypes. These results demonstrate that DER plays a critical role during neuroblast formation, patterning and specification along the DV axis within the developing Drosophila embryonic CNS.

Expression pattern of a butterfly achaete-scute homolog reveals the homology of butterfly wing scales and insect sensory bristles.

BACKGROUND: Lepidopteran wing scales are the individual units of wing color patterns and were a key innovation during Lepidopteran evolution. On the basis of developmental and morphological evidence, it has been proposed that the sensory bristles of the insect peripheral nervous system and the wing scales of Lepidoptera are homologous structures. In order to determine if the developmental pathways leading to Drosophila sensory bristle and butterfly scale formation use similar genetic circuitry, we cloned, from the butterfly Precis coenia, a homolog of the Drosophila achaete-scute (AS-C) genes--which encode transcription factors that promote neural precursor formation--and examined its expression pattern during development. RESULTS: During embryonic and larval development, the expression pattern of the AS-C homolog, ASH1, forecasted neural precursor formation. ASH1 was expressed both in embryonic proneural clusters--within which an individual cell retained ASH1 expression, enlarged, segregated, and became a neural precursor--and in larval wing discs in putative sensory mother cells. ASH1 was also expressed in pupal wings, however, in evenly spaced rows of enlarged cells that had segregated from the underlying epidermis but, rather than give rise to neural structures, each cell contributed to an individual scale. CONCLUSIONS: ASH1 appears to perform multiple functions throughout butterfly development, apparently promoting the initial events of selection and formation of both neural and scale precursor cells. The similarity in the cellular and molecular processes of scale and neural precursor formation suggests that the spatial regulation of an AS-C gene was modified during Lepidopteran evolution to promote scale cell formation.

Sanpodo and Notch act in opposition to Numb to distinguish sibling neuron fates in the Drosophila CNS.

In Drosophila, most neuronal siblings have different fates ('A/B'). Here we demonstrate that mutations in sanpodo, a tropomodulin actin-binding protein homologue, equalize a diverse array of sibling neuron fates ('B/B'). Loss of Notch signaling gives the same phenotype, whereas loss of numb gives the opposite phenotype ('A/A'). The identical effect of removing either sanpodo or Notch function on the fates of sibling CNS neurons indicates that sanpodo may act in the Notch signaling pathway. In addition, sanpodo and numb show dosage-sensitive interactions and epistasis experiments indicate that sanpodo acts downstream of numb. Taken together, these results show that interactions between sanpodo, the Notch signaling pathway and numb enable CNS sibling neurons to acquire different fates.

Biochemical analysis of ++Prospero protein during asymmetric cell division: cortical Prospero is highly phosphorylated relative to nuclear Prospero.

Drosophila neuroblasts are a model system for studying asymmetric cell division. Neuroblasts bud off a series of smaller progeny, called ganglion mother cells (GMCs). An essential regulator of GMC development is the Prospero homeodomain transcription factor: Prospero is asymmetrically localized to the basal cortex of the mitotic neuroblast and partitioned into the newborn GMC. Prospero is translocated into the GMC nucleus, where it is necessary to establish GMC-specific gene expression. Cortical localization of Prospero protein is observed only during mitosis; cortical localization requires entry into mitosis and cortical delocalization requires exit from mitosis. The tight correlation and functional requirement between mitosis and cortical Prospero localization suggests that mitosis-specific posttranslational modifications may be involved in regulating Prospero subcellular localization. Here we use monoclonals recognizing the N-terminal or C-terminal region of Prospero to explore its posttranslational regulation. One- and two-dimensional Western analysis reveal a complex pattern of Prospero isoforms; phosphatase assays show that there are several phosphoisoforms of Prospero. Developmental 2D Western blots, cell fractionation assays, and analysis of a missense prospero mutation show that cortical Prospero protein is highly phosphorylated compared to nuclear Prospero protein. Our results are consistent with two functions of Prospero phosphorylation: (i) phosphorylation may be required for Prospero cortical localization; or (ii) phosphorylation may be a consequence of Prospero cortical localization, in which case it may facilitate subsequent events, such as Prospero cortical release or nuclear localization.

Miranda directs Prospero to a daughter cell during Drosophila asymmetric divisions.

Asymmetric cell division is a general process used in many developmental contexts to create two differently fated cells from a single progenitor cell. Intrinsic mechanisms like the asymmetric transmission of cell-fate determinants during cell division, and extrinsic cell-interaction mechanisms, can mediate asymmetric divisions. During embryonic development of the Drosophila central nervous system, neural stem cells called neuroblasts divide asymmetrically to produce another multipotent neuroblast and a ganglion mother cell (GMC) of more restricted developmental potential. Intrinsic mechanisms promote asymmetric division of neuroblasts: for example, the transcription factor Prospero localizes to the basal cell cortex of mitotic neuroblasts and then segregates exclusively into the GMC, which buds off from the basal side of the neuroblast. In the GMC, Prospero translocates to the nucleus, where it establishes differential gene expression between sibling cells. Here we report the identification of a gene, miranda, which encodes a new protein that co-localizes with Prospero in mitotic neuroblasts, tethers Prospero to the basal cortex of mitotic neuroblasts, directing Prospero into the GMC, and releases Prospero from the cell cortex within GMCs. miranda thus creates intrinsic differences between sibling cells by mediating the asymmetric segregation of a transcription factor into only one daughter cell during neural stem-cell division.

heartless encodes a fibroblast growth factor receptor (DFR1/DFGF-R2) involved in the directional migration of early mesodermal cells in the Drosophila embryo.

After invagination of the mesodermal primordium in the gastrulating Drosophila embryo, the internalized cells migrate in a dorsolateral direction along the overlying ectoderm. This movement generates a stereotyped arrangement of mesodermal cells that is essential for their correct patterning by later position-specific inductive signals. We now report that proper mesodermal cell migration is dependent on the function of a fibroblast growth factor (FGF) receptor encoded by heartless (htl). In htl mutant embryos, the mesoderm forms normally but fails to undergo its usual dorsolateral migration. As a result, cardiac, visceral, and dorsal somatic muscle fates are not induced by Decapentaplegic (Dpp), a transforming growth factor beta family member that is derived from the dorsal ectoderm. Visceral mesoderm can nevertheless be induced by Dpp in the absence of htl function. Ras1 is an important downstream effector of Htl signaling because an activated form of Ras1 partially rescues the htl mutant phenotype. The evolutionary conservation of htl function is suggested by the strikingly similar mesodermal migration and patterning phenotypes associated with FGF receptor mutations in species as diverse as nematode and mouse. These studies establish that Htl signaling provides a vital connection between initial formation of the embryonic mesoderm in Drosophila and subsequent cell-fate specification within this germ layer.

The achaete-scute complex proneural genes contribute to neural precursor specification in the Drosophila CNS.

BACKGROUND: The Drosophila central nervous system (CNS) develops from a segmentally reiterated array of 30 neural precursors. Each precursor acquires a unique identity and goes through a stereotyped cell lineage to produce an invariant family of neurons and/or glia. The proneural genes achaete, scute and lethal of scute are required for neural precursor formation in the Drosophila CNS, and are expressed in overlapping subsets of 'proneural cell clusters' from which a single neural precursor later develops. Vertebrate achaete-scute homologues are expressed early during neurogenesis, and promote neurogenesis, neuronal development and/or differentiation. The Drosophila proneural achaete-scute genes govern neural precursor formation, but their role in specifying neural precursor identity has not been tested. RESULTS: Here, we test the role of the Drosophila achaete-scute genes in specifying neural precursor identity, focusing on the well characterized CNS MP2 precursor. MP2 delaminates from a cluster of achaete-scute-expressing ectodermal cells. In an achaete-scute double mutant, MP2 formation was reduced (to 11-14 %) as expected because of the function of proneural genes in promoting neural precursor formation. Surprisingly, we also observed that the developing MP2 precursors were incorrectly specified and acquired traits characteristic of adjacent neural precursors. In rescue experiments, achaete or scute, but not lethal of scute, completely restored the normal MP2 identity. CONCLUSIONS: These results demonstrate that the achaete-scute complex genes specify aspects of neural precursor identity in the Drosophila CNS. Given the phylogenetically conserved function of these genes, our results raise the possibility that achaete-scute homologues may help specify neural precursor identity in other organisms.

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.

New neuroblast markers and the origin of the aCC/pCC neurons in the Drosophila central nervous system.

Drosophila is an ideal system for identifying genes that control central nervous system (CNS) development. Particularly useful tools include molecular markers for subsets of neural precursors (neuroblasts) and the simple expression pattern of the even-skipped (eve) gene in a subset of neurons. Here we provide additional molecular markers for identified neuroblasts, including several with near single cell specificity. In addition, we use these new markers to trace the development of several eve+ neurons. Our results shows that the eve+ aCC/pCC neurons develop from a different neuroblast than previously thought, and have led us to assign new names for several neuroblasts. These results are supported by DiI cell lineage analysis of neuroblasts identified in vivo.

Tag team specification of a neural precursor in the Drosophila embryonic central nervous system.

The development of vertebrate and invertebrate nervous systems requires the production of thousands to millions of uniquely specified neurons from progenitor neural stem cells. A central question focuses on the elucidation of the developmental mechanisms that function within neural stem cell lineages to impart unique identities to neurons. A recent report details the roles that two genes, pdm-1 and pdm-2, play within an identified neural stem cell lineage in the Drosophila embryonic central nervous system. The results show that pdm-1 and pdm-2 are coexpressed in an identified neural precursor and function redundantly to specify the fate of this cell. As such this report offers an initial view of the genetic programs that create neural diversity.

Specification of neuroblast identity in the Drosophila embryonic central nervous system by gooseberry-distal.

The Drosophila central nervous system develops from a segmentally reiterated array of 30 unique neural precursors, called neuroblasts. Each neuroblast goes through a stereotyped cell lineage to produce an invariant clone of neural progeny. It is critical to identify the genes that specify neuroblast identity as these genes control the time of formation, gene expression profile, and cell lineage characteristics of each neuroblast. Here we show that the Pax-type gooseberry-distal gene specifies row 5 neuroblast identity. Initially, four rows of neuroblasts form per segment (1, 3, 5, 7) and gooseberry-distal is expressed in row 5 neuroblasts. By using 10 molecular markers, and by following the number and orientation of neuroblast divisions, we show that lack of gooseberry-distal transforms row 5 neuroblasts into row 3 neuroblasts, whereas ubiquitous gooseberry-distal generates the reciprocal transformation. Thus, gooseberry-distal is necessary and sufficient to specify row 5 neuroblast identity autonomously. The 10 genes coordinately regulated by gooseberry-distal are prime candidates for controlling specific aspects of neuroblast identity.

The achaete-scute complex: generation of cellular pattern and fate within the Drosophila nervous system.

In developing embryos, cells receive and interpret positional information as they become organized into discrete patterns and structures. One excellent model for understanding the genetic regulatory mechanisms that pattern cellular fields is the regulation and function of the achaete-scute complex (AS-C) in the developing nervous system of the fruit fly, Drosophila melanogaster. Three structurally homologous proneural genes--achaete (ac), scute (sc), and lethal of scute (l'sc)--are required for neural stem cell formation. In Drosophila, the AS-C genes are initially expressed in patterns of cell clusters at reproducible anteroposterior (AP) and dorsoventral (DV) coordinates that foreshadow where neural precursors arise. In the embryonic central nervous system (CNS), the gene products of AP and DV axis-patterning genes act combinatorially via a large array of cis-regulatory regions scattered throughout the AS-C to generate a segmentally repeated pattern of proneural clusters. Within each cluster (an equivalence group), one cell then retains proneural gene expression and is singled out as the neural stem cell (neuroblast). The neuroblast inhibits the surrounding cells from adopting neural fates (lateral inhibition) through a signaling pathway that is mediated via the action of the proneural and neurogenic genes. The proneural genes therefore represent a nodal point in the patterning of the nervous system. They receive global positional information, transduce it to discrete sets of cells, and trigger local cell interactions that mediate cell fate decisions.

The ventral nervous system defective gene controls proneural gene expression at two distinct steps during neuroblast formation in Drosophila.

Within the Drosophila embryo, the formation of many neuroblasts depends on the functions of the proneural genes of the achaete-scute complex (AS-C): achaete (ac), scute (sc) and lethal of scute (l'sc), and the gene ventral nervous system defective (vnd). Here, we show that vnd controls neuroblast formation, in part, through its regulation of the proneural genes of the AS-C. vnd is absolutely required to activate ac, sc and l'sc gene expression in proneural clusters in specific domains along the medial column of the earliest arising neuroblasts. Using ac-lacZ reporter constructs, we determined that vnd controls proneural gene expression at two distinct steps during neuroblast formation through separable regulatory regions. First, vnd is required to activate proneural cluster formation within the medial column of every other neuroblast row through regulatory elements located 3' to ac; second, through a 5' regulatory region, vnd functions to increase or maintain proneural gene expression in the cell within the proneural cluster that normally becomes the neuroblast. By following neuroblast segregation in vnd mutant embryos, we show that the neuroectoderm forms normally and that the defects in neuroblast formation are specific to particular proneural clusters.