Three distinct roles for notch in Drosophila R7 photoreceptor specification

Receptor tyrosine kinases (RTKs) and Notch (N) proteins are different types of transmembrane receptors that transduce extracellular signals and control cell fate. Here we examine cell fate specification in the Drosophila retina and ask how N acts together with the RTKs Sevenless (Sev) and the EGF receptor (DER) to specify the R7 photoreceptor. The retina is composed of many hundred ommatidia, each of which grows by recruiting surrounding, undifferentiated cells and directing them to particular fates. The R7 photoreceptor derives from a cohort of three cells that are incorporated together following specification of the R2-R5 and R8 photoreceptors. Two cells of the cohort are specified as the R1/6 photoreceptor type by DER activation. These cells then activate N in the third cell (the R7 precursor). By manipulation of N and RTK signaling in diverse combinations we establish three roles for N in specifying the R7 fate. The first role is to impose a block to photoreceptor differentiation; a block that DER activation cannot overcome. The second role, paradoxically, is to negate the first; Notch activation up-regulates Sev expression, enabling the presumptive R7 cell to receive an RTK signal from R8 that can override the block. The third role is to specify the cell as an R7 rather than an R1/6 once RTK signaling has specified the cells as a photoreceptor. We speculate why N acts both to block and to facilitate photoreceptor differentiation, and provide a model for how N and RTK signaling act combinatorially to specify the R1/6 and R7 photoreceptors as well as the surrounding non-neuronal cone cells.     

Involvement of numb in vertebrate retinal development: evidence for multiple roles of numb in neural differentiation and maturation

Cell fate specification is regulated in part by lateral inhibition mediated by Notch signaling. Notch signaling is negatively regulated by Numb, an intrinsic factor that regulates cellular competence. In this study we have examined the involvement of Numb in retinal development, which has been shown to be influenced by Notch signaling. In the developing retina, Numb is asymmetrically distributed towards the ventricular and vitreal poles of different cells. Asymmetric localization is evident not only in mitotic cells but in postmitotic ganglion cells as well, suggesting that the subcellular distribution of Numb may play a role after cells have exited the cell cycle. This is supported by the expression of Numb in terminally differentiated neurons in the adult retina. Although Numb is an intrinsic factor, it is observed that its subcellular distribution is influenced by epigenetic cues such that a higher proportion of cells cultured at high density express Numb asymmetrically. A correlation is observed between asymmetric localization and cellular competence; cells in which Numb is asymmetric differentiate more readily in culture than those that express Numb symmetrically. We have identified alternative splice variants in the developing and adult retina that correspond to isoforms that have been shown to regulate proliferation and differentiation. The dynamic temporal expression patterns of alternative splice variants and isoforms suggest that Numb may influence proliferation and differentiation of retinal progenitors during neurogenesis and maturation of postmitotic neurons. Together, these results demonstrate the complex role of the distribution of Numb within progenitors and postmitotic neurons.     

Cell cycle and cell-fate determination in Drosophila neural cell lineages

"Normal" development requires a finely tuned equilibrium between cell differentiation and cell proliferation. Important issues in development include whether the cell cycle controls the cell-fate determination and whether cell identity in turn regulates cell-cycle progression. Although, these issues are of general biological relevance, stereotyped Drosophila neural lineages are particularly suited to address these questions and have provided insights into the links between cell-cycle progression and cell-fate specification.     

Lethal giant larvae acts together with numb in notch inhibition and cell fate specification in the Drosophila adult sensory organ precursor lineage

The tumor suppressor genes lethal giant larvae (lgl) and discs large (dlg) act together to maintain the apical basal polarity of epithelial cells in the Drosophila embryo. Neuroblasts that delaminate from the embryonic epithelium require lgl to promote formation of a basal Numb and Prospero crescent, which will be asymmetrically segregated to the basal daughter cell upon division to specify cell fate. Sensory organ precursors (SOPs) also segregate Numb asymmetrically at cell division. Numb functions to inhibit Notch signaling and to specify the fates of progenies of the SOP that constitute the cellular components of the adult sensory organ. We report here that, in contrast to the embryonic neuroblast, lgl is not required for asymmetric localization of Numb in the dividing SOP. Nevertheless, mosaic analysis reveals that lgl is required for cell fate specification within the SOP lineage; SOPs lacking Lgl fail to specify internal neurons and glia. Epistasis studies suggest that Lgl acts to inhibit Notch signaling by functioning downstream or in parallel with Numb. These findings uncover a previously unknown function of Lgl in the inhibition of Notch and reveal different modes of action by which Lgl can influence cell fate in the neuroblast and SOP lineages.     

Reproductive tract interactions contribute to isolation in Drosophila

The process of speciation requires the development of isolating mechanisms that act as barriers to gene flow between incipient species. Such mechanisms can occur at three different levels: precopulatory or behavioral isolation, postcopulatory-prezygotic isolation occurring in the female reproductive tract, or postzygotic isolation resulting in hybrid sterility or inviability. Only by extensively studying all three types of barriers in young species pairs can we begin to understand the evolution of early reproductive incompatibilities, which may be important to the speciation process. Although precopulatory and postzygotic isolation have been well described it is only recently that the female reproductive tract has been intensely examined for possible mechanisms of reproductive isolation (reviewed in refs 1 and 2). The types of isolating mechanisms that develop at this level and their role in speciation, therefore, remain poorly understood.     

The pteridine pathway in zebrafish: regulation and specification during the determination of neural crest cell-fate

This review describes pteridine biosynthesis and its relation to the differentiation of neural crest derivatives in zebrafish. During the embryonic development of these fish, neural crest precursor cells segregate into neural elements, ectomesenchymal cells and pigment cells; the latter then diversifying into melanophores, iridophores and xanthophores. The differentiation of neural cells, melanophores, and xanthophores is coupled closely with the onset of pteridine synthesis which starts from GTP and is regulated through the control of GTP cyclohydrolase I activity. De novo pteridine synthesis in embryos of this species increases during the first 72-h postfertilization, producing H4biopterin, which serves as a cofactor for neurotransmitter synthesis in neural cells and for tyrosine production in melanophores. Thereafter, sepiapterin (6-lactoyl-7,8-dihydropterin) accumulates as yellow pigment in xanthophores, together with 7-oxobiopterin, isoxanthopterin and 2,4,7-trioxopteridine. Sepiapterin is the key intermediate in the formation of 7-oxopteridines, which depends on the availability of enzymes belonging to the xanthine oxidoreductase family. Expression of the GTP cyclohydrolase I gene (gch) is found in neural cells, in melanoblasts and in early xanthophores (xanthoblasts) of early zebrafish embryos but steeply declines in xanthophores by 42-h postfertilization. The mechanism(s) whereby sepiapterin branches off from the GTP-H4biopterin pathway is currently unknown and will require further study. The surge of interest in zebrafish as a model for vertebrate development and its amenability to genetic manipulation provide powerful tools for analysing the functional commitment of neural crest-derived cells and the regulation of pteridine synthesis in mammals.     

The RNA helicase DDX6 regulates cell-fate specification in neural stem cells via miRNAs

In neural stem cells (NSCs), the balance between stem cell maintenance and neuronal differentiation depends on cell-fate determinants such as TRIM32. Previously, we have shown that TRIM32 associates with the RNA-induced silencing complex and increases the activity of microRNAs such as Let-7a. However, the exact mechanism of microRNA regulation by TRIM32 during neuronal differentiation has yet to be elucidated. Here, we used a mass spectrometry approach to identify novel protein–protein interaction partners of TRIM32 during neuronal differentiation. We found that TRIM32 associates with proteins involved in neurogenesis and RNA-related processes, such as the RNA helicase DDX6, which has been implicated in microRNA regulation. We demonstrate, that DDX6 colocalizes with TRIM32 in NSCs and neurons and that it increases the activity of Let-7a. Furthermore, we provide evidence that DDX6 is necessary and sufficient for neuronal differentiation and that it functions in cooperation with TRIM32.     

Akt and FOXO dysregulation contribute to infection-induced wasting in Drosophila

BACKGROUND: Studies in Drosophila have taught us a great deal about how animals regulate the immediate innate immune response, but we still know little about how infections cause pathology. Here, we examine the pathogenesis associated with Mycobacterium marinum infection in the fly. M. marinum is closely related to M. tuberculosis, which causes tuberculosis in people. RESULTS: A microarray analysis showed that metabolism is profoundly affected in M. marinum-infected flies. A genetic screen identified foxo mutants as slower-dying after infection than wild-type flies. FOXO activity is inhibited by the insulin effector kinase Akt; we show that Akt activation is systemically reduced as a result of M. marinum infection. Finally, we show that flies infected with Mycobacterium marinum undergo a process like wasting: They progressively lose metabolic stores, in the form of fat and glycogen. They also become hyperglycemic. In contrast, foxo mutants exhibit less wasting. CONCLUSIONS: In people, many infections--including tuberculosis--can cause wasting, much as we see in Drosophila. Our study is the first examination of the metabolic consequences of infection in a genetically tractable invertebrate and gives insight into the metabolic consequences of mycobacterial infection, implicating impaired insulin signaling as a key mediator of these events. These results suggest that the fly can be used to study more than the immediate innate immune response to infection; it can also be used to understand the physiological consequences of infection and the immune response.     

Sequential phases of cortical specification involve Neurogenin-dependent and -independent pathways

Neocortical projection neurons, which segregate into six cortical layers according to their birthdate, have diverse morphologies, axonal projections and molecular profiles, yet they share a common cortical regional identity and glutamatergic neurotransmission phenotype. Here we demonstrate that distinct genetic programs operate at different stages of corticogenesis to specify the properties shared by all neocortical neurons. Ngn1 and Ngn2 are required to specify the cortical (regional), glutamatergic (neurotransmitter) and laminar (temporal) characters of early-born (lower-layer) neurons, while simultaneously repressing an alternative subcortical, GABAergic neuronal phenotype. Subsequently, later-born (upper-layer) cortical neurons are specified in an Ngn-independent manner, requiring instead the synergistic activities of Pax6 and Tlx, which also control a binary choice between cortical/glutamatergic and subcortical/GABAergic fates. Our study thus reveals an unanticipated heterogeneity in the genetic mechanisms specifying the identity of neocortical projection neurons.     

Patterns of molecular evolution of the germ line specification gene oskar suggest that a novel domain may contribute to functional divergence in Drosophila

In several metazoans including flies of the genus Drosophila, germ line specification occurs through the inheritance of maternally deposited cytoplasmic determinants, collectively called germ plasm. The novel insect gene oskar is at the top of the Drosophila germ line specification pathway, and also plays an important role in posterior patterning. A novel N-terminal domain of oskar (the Long Oskar domain) evolved in Drosophilids, but the role of this domain in oskar functional evolution is unknown. Trans-species transgenesis experiments have shown that oskar orthologs from different Drosophila species have functionally diverged, but the underlying selective pressures and molecular changes have not been investigated. As a first step toward understanding how Oskar function could have evolved, we applied molecular evolution analysis to oskar sequences from the completely sequenced genomes of 16 Drosophila species from the Sophophora subgenus, Drosophila virilis and Drosophila immigrans. We show that overall, this gene is subject to purifying selection, but that individual predicted structural and functional domains are subject to heterogeneous selection pressures. Specifically, two domains, the Drosophila-specific Long Osk domain and the region that interacts with the germ plasm protein Lasp, are evolving at a faster rate than other regions of oskar. Further, we provide evidence that positive selection may have acted on specific sites within these two domains on the D. virilis branch. Our domain-based analysis suggests that changes in the Long Osk and Lasp-binding domains are strong candidates for the molecular basis of functional divergence between the Oskar proteins of D. melanogaster and D. virilis. This molecular evolutionary analysis thus represents an important step towards understanding the role of an evolutionarily and developmentally critical gene in germ plasm evolution and assembly.     

Genetic control of retinal specification and determination in Drosophila

The Drosophila compound eye has long served as an outstanding model system to study many processes, including cell fate specification, cell division, cell growth and cell death. In addition, exploring the molecular basis of eye specification in Drosophila has identified a set of nuclear factors that trigger the conversion of a group of multipotent epithelial cells into eye primordia. These nuclear factors act in complex networks to regulate retinal specification and appear to be conserved throughout phylogeny. Finally, evidence suggests that these nuclear networks have been co-opted to specify cell fates in other tissues. We review the latest developments in the field of retinal specification in Drosophila and discuss several future directions that remain open for investigation.     

Axis specification in the Drosophila embryo.

Three genetic hierarchies control cell-fate specification in largely distinct regions of the antero-posterior axis of the Drosophila embryo, whereas a single hierarchy specifies dorso-ventral cell fates. Molecular genetic analysis of these hierarchies is leading to increased understanding of the nature of the regulatory circuitry that controls regional cell-fate specification.     

The specification of sensory neuron identity in Drosophila

Different types of sense organs are present on the larva of Drosophila. Several genes that specify the type of sense organ that will form at a particular position have been recently identified. Here we review the functional and molecular analyses of these genes, and summarize the evidence which supports a role in the choice of which type of organ will be formed. Most or all of these genes are required for the appropriate specification of adult as well as larval sense organs, suggesting that the larval and adult systems share many gene requirements. Interestingly, the specifying genes identified so far in the peripheral nervous system are also expressed in subsets of cells in the central nervous system, where they might have similar roles.     

Proximodistal domain specification and interactions in developing Drosophila appendages

The morphological diversification of appendages represents a crucial aspect of animal body plan evolution. The arthropod antenna and leg are homologous appendages, thought to have arisen via duplication and divergence of an ancestral structure (Snodgrass, R. (1935) Book Principles of Insect Morphology. New York: McGraw-Hill). To gain insight into how variations between the antenna and the leg may have arisen, we have compared the epistatic relationships among three major proximodistal patterning genes, Distal-less, dachshund and homothorax, in the antenna and leg of the insect arthropod Drosophila melanogaster. We find that Drosophila appendages are subdivided into different proximodistal domains specified by specific genes, and that limb-specific interactions between genes and the functions of these genes are crucial for antenna-leg differences. In particular, in the leg, but not in the antenna, mutually antagonistic interactions exist between the proximal and medial domains, as well as between medial and distal domains. The lack of such antagonism in the antenna leads to extensive coexpression of Distal-less and homothorax, which in turn is essential for differentiation of antennal morphology. Furthermore, we report that a fundamental difference between the two appendages is the presence in the leg and absence in the antenna of a functional medial domain specified by dachshund. Our results lead us to propose that the acquisition of particular proximodistal subdomains and the evolution of their interactions has been essential for the diversification of limb morphology.     

Cell-cell communication and axis specification in the Drosophila oocyte

Intercellular communication between the somatic and germline cells is vital to development of the Drosophila egg chamber. One critical outcome of this communication is the polarization of the oocyte along the anterior-posterior axis, a process induced by an unknown signal from the somatic follicle cells to the oocyte. The existence of this signal has been inferred from several reports demonstrating that the differentiation and patterning of the follicle cells by the spatially restricted activation of certain cell-signaling pathways is necessary for axis formation in the oocyte. These reports have also provided a framework for understanding how these signaling pathways are integrated to generate the follicle-cell pattern, but the precise role of the follicle cells in anterior-posterior axis formation remains enigmatic. Research has identified several genes that appear to be involved in the polarizing communication from the follicle cells to the oocyte. Interestingly the proteins encoded by most of these genes are associated with the extracellular matrix, suggesting a pivotal role for this complex biological component in the polarizing communication between the follicle cells and the oocyte. This review summarizes the findings in this area, and uses the experimental analyses of these genes to evaluate various models describing the possible nature of the polarizing signal, and the role of these genes in it.