Adi Salzberg

A Meis family protein caudalizes neural cell fates in Xenopus

A homologue of the Drosophila homothorax (hth) gene, Xenopus Meis3 (XMeis3), was cloned from Xenopus laevis. XMeis3 is expressed in a single stripe of cells in the early neural plate stage. By late neurula, the gene is expressed predominantly in rhombomeres two, three and four, and in the anterior spinal cord. Ectopic expression of RNA encoding XMeis3 protein causes anterior neural truncations with a concomitant expansion of hindbrain and spinal cord. Ectopic XMeis3 expression inhibits anterior neural induction in neuralized animal cap ectoderm explants without perturbing induction of pan-neural markers. In naive animal cap ectoderm, ectopic XMeis3 expression activates transcription of the posteriorly expressed neural markers, but not pan-neural markers. These results suggest that caudalizing proteins, such as XMeis3, can alter A-P patterning in the nervous system in the absence of neural induction. Regionally expressed proteins like XMeis3 could be required to overcome anterior signals and to specify posterior cell fates along the A-P axis.

Nato3 is an evolutionarily conserved bHLH transcription factor expressed in the CNS of Drosophila and mouse

The evolutionarily conserved basic helix-loop-helix (bHLH) transcription factors play important roles during development. Here we report the identification of Nato3 (nephew of atonal fer3) orthologs in Drosophila, C. elegans, mouse, and man, all of which share a high degree of similarity within the bHLH domain. Expression analysis revealed Nato3 transcripts in the central nervous system of both fly and mouse embryos. In the fly, Dnato3 is highly expressed in 9-15h embryos in a few ventral nerve cord cells and a subset of neurons in the brain. In mouse, the MNato3 transcripts were detected from embryonic day 7 until 5 weeks postnatally, with highest levels in the midbrain, thalamus, hypothalamus, pons, and medulla oblongata. In contrast to the brain, expression in the spinal cord was limited to the embryonic stages.

Antizyme is a target of sex-lethal in the drosophila germline and appears to act downstream of hedgehog to regulate sex-lethal and cyclin B

The sex determination master switch, Sex-lethal, has been shown to regulate the mitosis of early germ cells in Drosophila melanogaster. Sex-lethal is an RNA binding protein that regulates splicing and translation of specific targets in the soma, but the germline targets are unknown. In an experiment aimed at identifying targets of Sex-lethal in early germ cells, the RNA encoded by gutfeeling, the Drosophila homolog of Ornithine Decarboxylase Antizyme, was isolated. gutfeeling interacts genetically with Sex-lethal. It is not only a target of Sex-lethal, but also appears to regulate the nuclear entry and overall levels of Sex-lethal in early germ cells. This regulation of Sex-lethal by gutfeeling appears to occur downstream of the Hedgehog signal. We also show that Hedgehog, Gutfeeling, and Sex-lethal function to regulate Cyclin B, providing a link between Sex-lethal and mitosis.

Spatial regulation of cell adhesion in the Drosophila wing is mediated by Delilah, a potent activator of βPS integrin expression.

In spite of our conceptual view of how differential gene expression is used to define different cell identities, we still do not understand how different cell identities are translated into actual cell properties. The example discussed here is that of the fly wing, which is composed of two main cell types: vein and intervein cells. These two cell types differ in many features, including their adhesive properties. One of the major differences is that intervein cells express integrins, which are required for the attachment of the two wing layers to each other, whereas vein cells are devoid of integrin expression. The major signaling pathways that divide the wing to vein and intervein domains have been characterized. However, the genetic programs that execute these two alternative differentiation programs are still very roughly drawn. Here we identify the bHLH protein Delilah (Dei) as a mediator between signaling pathways that specify intervein cell-fate and one of the most significant realizators of this fate, βPS integrin. Deis expression is restricted to intervein territories where it acts as a potent activator of βPS integrin expression. In the absence of normal Dei activity the level of βPS integrin is reduced, leading to a failure of adhesion between the dorsal and ventral wing layers and a consequent formation of wing blisters. The effect of Dei on βPS expression is not restricted to the wing, suggesting that Dei functions as a general genetic switch, which is turned on wherever a sticky cell-identity is determined and integrin-based adhesion is required.

E4orf4 induces PP2A- and Src-dependent cell death in Drosophila melanogaster and at the same time inhibits classic apoptosis pathways

Significance Expression of the adenovirus protein E4orf4 alone in cultured mammalian cells prompts noncanonical apoptosis that is more efficient in oncogene-transformed cells than in normal cells. Here, E4orf4 activity in a whole organism (Drosophila melanogaster) is described, leading to three significant conclusions: (i) E4orf4-induced cell death is an evolutionarily conserved process; (ii) E4orf4 induces a distinctive mode of cell death, differing from well-characterized cell death mechanisms; and (iii) E4orf4 activates cell death but concomitantly inhibits it, thus minimizing damage to normal tissues. The last finding suggests a possible explanation for the differential effect of E4orf4 in normal and cancer cells. The adenovirus E4orf4 protein regulates the progression of viral infection, and when expressed alone in mammalian tissue culture cells it induces protein phosphatase 2A (PP2A)-B55– and Src-dependent cell death, which is more efficient in oncogene-transformed cells than in normal cells. This form of cell death is caspase-independent, although it interacts with classic caspase-dependent apoptosis. PP2A-B55–dependent E4orf4-induced toxicity is highly conserved in evolution from yeast to mammalian cells. In this work we investigated E4orf4-induced cell death in a whole multicellular organism, Drosophila melanogaster. We show that E4orf4 induced low levels of cell killing, caused by both caspase-dependent and -independent mechanisms. Drosophila PP2A-B55 (twins/abnormal anaphase resolution) and Src64B contributed additively to this form of cell death. Our results provide insight into E4orf4-induced cell death, demonstrating that in parallel to activating caspase-dependent apoptosis, E4orf4 also inhibited this form of cell death induced by the proapoptotic genes reaper, head involution defective, and grim. The combination of both induction and inhibition of caspase-dependent cell death resulted in low levels of tissue damage that may explain the inefficient cell killing induced by E4orf4 in normal cells in tissue culture. Furthermore, E4orf4 inhibited JNK-dependent cell killing as well. However, JNK inhibition did not impede E4orf4-induced toxicity and even enhanced it, indicating that E4orf4-induced cell killing is a distinctive form of cell death that differs from both JNK- and Rpr/Hid/Grim-induced forms of cell death.

Visualization of proprioceptors in Drosophila larvae and pupae.

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis. The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system. Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnstons organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4. In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnstons organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary. Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

The proprioceptive and contractile systems in Drosophila are both patterned by the EGR family transcription factor Stripe

Coordinated locomotion of Drosophila larvae depends on accurate patterning and stable attachment to the cuticle of both muscles and proprioceptors (chordotonal organs). Unlike muscle spindles in mammals, the fly chordotonal organs are not embedded in the body-wall muscles. Yet, the contractile system (muscles and tendons) and the chordotonal organs constitute two parts of a single functional unit that controls locomotion, and thus must be patterned in full coordination. It is not known how such coordination is achieved. Here we show that the positioning and differentiation of the migrating chordotonal organs are instructed by Stripe, the same transcription factor that promotes tendon cell specification and differentiation and is required for normal patterning of the contractile system. Our data demonstrate that although chordotonal organs are patterned in a Stripe-dependent mechanism similarly to muscles, this mechanism is independent of Stripe activity in tendon cells. Thus, the two parts of the locomotive system use similar but independent patterning mechanisms that converge to form a functional unit. Stripe plays at least a dual role in chordotonal development. It is required within the ligament cells for terminal differentiation and proper migration, without which no induction of ligament attachment cells takes place. Stripes activity is then necessary within the recruited cells for their differentiation as attachment cells. Similarly to the biphasic differentiation program of tendons, terminal differentiation of chordotonal attachment cells is associated with sequential activation of the two Stripe isoforms-Stripe B and Stripe A.

Additional sex combs affects antennal development by means of spatially restricted repression of Antp and Wg

Additional sex combs (Asx) is thought to function in protein complexes of both the Trithorax and Polycomb groups, but very little is known about its developmental roles. Here, we present a detailed analysis of Asxs role in antennal development. We show that loss of Asx in the antennal disc causes a complex phenotype, which consists of distal antenna‐to‐leg transformations and outgrowth of ectopic leg‐like appendages from the Dpp‐expressing domain of the disc. Our analyses suggest that these phenotypes are caused mainly by segment‐specific de‐repression of Antp and expansion of wg expression. We thus conclude that Asx functions normally to repress Antp and to restrict wg expression in specific regions of the developing disc. We also show that, in the absence of Asxs function, Antp expression does not lead to efficient repression of the antennal‐determining gene hth, suggesting that Asx is also required for the repression of hth by Antp. Developmental Dynamics 236:2118–2130, 2007.

The adenovirus E4orf4 protein induces a unique mode of cell death while inhibiting classical apoptosis

The adenovirus E4 open reading frame 4 (E4orf4) protein is a multifunctional viral regulator that contributes to temporal regulation of the progression of viral infection. When expressed outside the context of the virus, E4orf4 induces p53-independent cell death in transformed cells. Oncogenic transformation of primary cells in tissue culture sensitizes them to cell killing by E4orf4,1 indicating that E4orf4 research may have implications for cancer therapy. It has been further reported that E4orf4 induces a caspase-independent, non-classical apoptotic pathway that maintains crosstalk with classical caspase-dependent pathways.2,3 An investigation into the mechanisms involved in E4orf4-induced cell death revealed that E4orf4 interacts with the heterotrimeric protein phosphatase 2A (PP2A) through direct association with its regulatory B subunits, and the interaction mediated by the PP2A Bα/B55 subunit is required for inducing cell death.1 Furthermore, E4orf4 recruits PP2A to a new substrate, the ACF chromatin remodeling factor, which contributes to E4orf4 functions.4 E4orf4 has also been reported to associate with members of the Src kinase family, leading to its Tyr phosphorylation and to deregulation of Src signaling, resulting in enhanced cell death.2 We showed previously that the interaction between E4orf4 and PP2A and its toxic consequences were conserved from yeast to mammals,5 indicating a high degree of evolutionary conservation of the underlying mechanisms. This finding suggested the feasibility of using various model organisms for studying E4orf4-induced cell death. Indeed, our work in yeast revealed a novel E4orf4 partner, Ynd1, a Golgi UDPase that contributes to E4orf4 toxicity.6

Deconstructing the complexity of regulating common properties in different cell types: lessons from the delilah gene.

To understand development we need to understand how transcriptional regulatory mechanisms are employed to confer different cell types with their unique properties. Nonetheless it is also critical to understand how such mechanisms are used to confer different cell types with common cellular properties, such as the ability to adhere to the extracellular matrix. To decode how adhesion is regulated in cells stemming from different pedigrees we analyzed the regulatory region that drives the expression of Dei, which is a transcription factor that serves as a central determinant of cell adhesion, particularly by inducing expression of βPS-integrin. We show that activation of dei transcription is mediated through multiple cis regulatory modules, each driving transcription in a spatially and temporally restricted fashion. Thus the dei gene provides a molecular platform through which cell adhesion can be regulated at the transcriptional level in different cellular milieus. Moreover, we show that these regulatory modules respond, often directly, to central regulators of cell identity in each of the dei-expressing cell types, such as D-Mef2 in muscle cells, Stripe in tendon cells and Blistered in wing intervein cells. These findings suggest that the acquirement of common cellular properties shared by different cell types is embedded within the unique differentiation program dictated to each of these cells by the major determinants of its identity.