APOL1-Mediated Cell Injury Involves Disruption of Conserved Trafficking Processes.

APOL1 harbors C-terminal sequence variants (G1 and G2), which account for much of the increased risk for kidney disease in sub-Saharan African ancestry populations. Expression of the risk variants has also been shown to cause injury to podocytes and other cell types, but the underlying mechanisms are not understood. We used Drosophila melanogaster and Saccharomyces cerevisiae to help clarify these mechanisms. Ubiquitous expression of the human APOL1 G1 and G2 disease risk alleles caused near-complete lethality in D. melanogaster, with no effect of the G0 nonrisk APOL1 allele, corresponding to the pattern of human disease risk. We also observed a congruent pattern of cellular damage with tissue-specific expression of APOL1. In particular, expression of APOL1 risk variants in D. melanogaster nephrocytes caused cell-autonomous accumulation of the endocytic tracer atrial natriuretic factor-red fluorescent protein at early stages and nephrocyte loss at later stages. We also observed differential toxicity of the APOL1 risk variants compared with the APOL1 nonrisk variants in S. cerevisiae, including impairment of vacuole acidification. Yeast strains defective in endosomal trafficking or organelle acidification but not those defective in autophagy displayed augmented APOL1 toxicity with all isoforms. This pattern of differential injury by the APOL1 risk alleles compared with the nonrisk alleles across evolutionarily divergent species is consistent with an impairment of conserved core intracellular endosomal trafficking processes. This finding should facilitate the identification of cell injury pathways and corresponding therapeutic targets of interest in these amenable experimental platforms.

A newly identified type of attachment cell is critical for normal patterning of chordotonal neurons.

This work describes unknown aspects of chordotonal organ (ChO) morphogenesis revealed in post-embryonic stages through the use of new fluorescently labeled markers. We show that towards the end of embryogenesis a hitherto unnoticed phase of cell migration commences in which the cap cells of the ventral ChOs elongate and migrate towards their prospective attachment sites. This migration and consequent cell attachment generates a continuous zigzag line of proprioceptors, stretching from the ventral midline to a dorsolateral position in each abdominal segment. Our observation that the cap cell of the ventral-most ChO attaches to a large tendon cell near the midline provides the first evidence for a direct physical connection between the contractile and proprioceptive systems in Drosophila. Our analysis has also provided an answer to a longstanding enigma that is what anchors the neurons of the ligamentless ventral ChOs on their axonal side. We identified a new type of ChO attachment cell, which binds to the scolopale cells of these organs, thus behaving like a ligament cell, but on the other hand exhibits all the typical features of a ChO attachment cell and is critical for the correct anchoring of these organs.

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 ßPS-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.

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

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.

Homothorax plays autonomous and nonautonomous roles in proximodistal axis formation and migration of the Drosophila renal tubules.

The Drosophila Malpighian tubules (MpTs) serve as a functional equivalent of the mammalian renal tubules. The MpTs are composed of two pairs of epithelial tubes that bud from the midgut-hindgut boundary during embryogenesis. The MpT primordia grow, elongate and migrate through the body cavity to assume their final position and shape. The stereotypic pattern of MpT migration is regulated by multiple intrinsic and extrinsic signals, many of which are still obscure. In this work, we implicate the TALE-class homeoprotein Homothorax (Hth) in MpT patterning. We show that in the absence of Hth the tubules fail to rearrange and migrate. Hth plays both autonomous and nonautonomous roles in this developmental process. Within the tubules Hth is required for convergent extension and for defining distal versus proximal cell identities. The difference between distal and proximal cell identities seems to be required for proper formation of the leading loop. Outside the tubules, wide-range mesodermal expression of Hth is required for directing anterior migration. The nonautonomous effects of Hth on MpT migration can be partially attributed to its effects on homeotic determination along the anterior posterior axis of the embryo and to its effects on stellate cell (SC) incorporation into the MpT.

Spatial regulation of cell adhesion in the Drosophila wing is mediated by Delilah, a potent activator of ßPS 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, ßPS integrin. Dei's expression is restricted to intervein territories where it acts as a potent activator of ßPS integrin expression. In the absence of normal Dei activity the level of ßPS 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 ßPS 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.

Neuronal role of taxi is imperative for flight in Drosophila melanogaster.

Extensive studies in Drosophila have led to the elucidation of the roles of many molecular players involved in the sensorimotor coordination of flight. However, the identification and characterisation of new players can add novel perspectives to the process. In this paper, we show that the extant mutant, jumper, is a hypermorphic allele of the taxi/delilah gene, which encodes a transcription factor. The defective flight of jumper flies results from the insertion of an I-element in the 5'-UTR of taxi gene, leading to an over-expression of the taxi. We also show that the molecular lesion responsible for the taxi1 allele results from a 25 bp deletion leading to a shift in the reading frame at the C-terminus of the taxi coding sequence. Thus, the last 20 residues are replaced by 32 disparate residues in taxi1. Both taxi1, a hypomorphic allele, and the CRISPR-Cas9 knock-out (taxiKO) null allele, show a defective flight phenotype. Electrophysiological studies show taxi hypermorphs, hypomorphs, and knock out flies show abnormal neuronal firing. We further show that neuronal-specific knock-down or over-expression of taxi cause a defect in the brain's inputs to the flight muscles, leading to reduced flight ability. Through transcriptomic analysis of the taxiKO fly head, we have identified several putative targets of Taxi that may play important roles in flight. In conclusion, from molecularly characterising jumper to establishing Taxi's role during Drosophila flight, our work shows that the forward genetics approach still can lead to the identification of novel molecular players required for neuronal transmission.

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. Stripe's 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.

Revisiting the Role of ß-Tubulin in Drosophila Development: ß-tubulin60D is not an Essential Gene, and its Novel Pin 1 Allele has a Tissue-Specific Dominant-Negative Impact.

Diversity in cytoskeleton organization and function may be achieved through alternative tubulin isotypes and by a variety of post-translational modifications. The Drosophila genome contains five different ß-tubulin paralogs, which may play an isotype tissue-specific function in vivo. One of these genes, the ß-tubulin60D gene, which is expressed in a tissue-specific manner, was found to be essential for fly viability and fertility. To further understand the role of the ß-tubulin60D gene, we generated new ß-tubulin60D null alleles (ß-tubulin60D M ) using the CRISPR/Cas9 system and found that the homozygous flies were viable and fertile. Moreover, using a combination of genetic complementation tests, rescue experiments, and cell biology analyses, we identified Pin 1 , an unknown dominant mutant with bristle developmental defects, as a dominant-negative allele of ß-tubulin60D. We also found a missense mutation in the Pin1 mutant that results in an amino acid replacement from the highly conserved glutamate at position 75 to lysine (E75K). Analyzing the ß-tubulin structure suggests that this E75K alteration destabilizes the alpha-helix structure and may also alter the GTP-Mg2+ complex binding capabilities. Our results revisited the credence that ß-tubulin60D is required for fly viability and revealed for the first time in Drosophila, a novel dominant-negative function of missense ß-tubulin60D mutation in bristle morphogenesis.

Delilah, prospero, and D-Pax2 constitute a gene regulatory network essential for the development of functional proprioceptors.

Coordinated animal locomotion depends on the development of functional proprioceptors. While early cell-fate determination processes are well characterized, little is known about the terminal differentiation of cells within the proprioceptive lineage and the genetic networks that control them. In this work we describe a gene regulatory network consisting of three transcription factors-Prospero (Pros), D-Pax2, and Delilah (Dei)-that dictates two alternative differentiation programs within the proprioceptive lineage in Drosophila. We show that D-Pax2 and Pros control the differentiation of cap versus scolopale cells in the chordotonal organ lineage by, respectively, activating and repressing the transcription of dei. Normally, D-Pax2 activates the expression of dei in the cap cell but is unable to do so in the scolopale cell where Pros is co-expressed. We further show that D-Pax2 and Pros exert their effects on dei transcription via a 262 bp chordotonal-specific enhancer in which two D-Pax2- and three Pros-binding sites were identified experimentally. When this enhancer was removed from the fly genome, the cap- and ligament-specific expression of dei was lost, resulting in loss of chordotonal organ functionality and defective larval locomotion. Thus, coordinated larval locomotion depends on the activity of a dei enhancer that integrates both activating and repressive inputs for the generation of a functional proprioceptive organ.

An Activating Mutation in ERK Causes Hyperplastic Tumors in a scribble Mutant Tissue in Drosophila.

Receptor tyrosine kinase signaling plays prominent roles in tumorigenesis, and activating oncogenic point mutations in the core pathway components Ras, Raf, or MEK are prevalent in many types of cancer. Intriguingly, however, analogous oncogenic mutations in the downstream effector kinase ERK have not been described or validated in vivo To determine if a point mutation could render ERK intrinsically active and oncogenic, we have assayed in Drosophila the effects of a mutation that confers constitutive activity upon a yeast ERK ortholog and has also been identified in a few human tumors. Our analyses indicate that a fly ERK ortholog harboring this mutation alone (RolledR80S), and more so in conjunction with the known sevenmaker mutation (RolledR80S+D334N), suppresses multiple phenotypes caused by loss of Ras-Raf-MEK pathway activity, consistent with an intrinsic activity that is independent of upstream signaling. Moreover, expression of RolledR80S and RolledR80S+D334N induces tissue overgrowth in an established Drosophila cancer model. Our findings thus demonstrate that activating mutations can bestow ERK with pro-proliferative, tumorigenic capabilities and suggest that Drosophila represents an effective experimental system for determining the oncogenicity of ERK mutants and their response to therapy.

Recruitment of ectodermal attachment cells via an EGFR-dependent mechanism during the organogenesis of Drosophila proprioceptors.

Drosophila proprioceptors (chordotonal organs) are structured as a linear array of four lineage-related cells: a neuron, a glial cell, and two accessory cells, called cap and ligament, between which the neuron is stretched. To function properly as stretch receptors, chordotonal organs must be stably anchored at both edges. The cap cells are anchored to the cuticle through specialized lineage-related attachment cells. However, the mechanism by which the ligament cells at the other edge of the organ attach is not known. Here, we report the identification of specialized attachment cells that anchor the ligament cells of pentascolopidial chordotonal organs (lch5) to the cuticle. The ligament attachment cells are recruited by the approaching ligament cells upon reaching their attachment site, through an EGFR-dependent mechanism. Molecular characterization of lch5 attachment cells demonstrated that they share significant properties with Drosophila tendon cells and with mammalian proprioceptive organs.

Accurate elimination of superfluous attachment cells is critical for the construction of functional multicellular proprioceptors in Drosophila.

Here, we show for the first time that developmental cell death plays a critical role in the morphogenesis of multicellular proprioceptors in Drosophila. The most prominent multicellular proprioceptive organ in the fly larva, the pentascolopidial (LCh5) organ, consists of a cluster of five stretch-responsive sensory organs that are anchored to the cuticle via specialized attachment cells. Stable attachment of the organ to the cuticle is critical for its ability to perceive mechanical stimuli arising from muscle contractions and the resulting displacement of its attachment sites. We now show that five attachment cells are born within the LCh5 lineage, but three of them are rapidly eliminated, normally, by apoptosis. Strong genetic evidence attests to the existence of an autophagic gene-dependent safeguard mechanism that guarantees elimination of the unwanted cells upon perturbation of the apoptotic pathway prior to caspase liberation. The removal of the three superfluous cells guarantees the right ratio between the number of sensory organs and the number of attachment cells that anchor them to the cuticle. This accurate matching seems imperative for the attachment of cell growth and functionality and is thus vital for normal morphogenesis and functionality of the sensory organ.

Selective elimination of cancer cells by the adenovirus E4orf4 protein in a Drosophila cancer model: a new paradigm for cancer therapy.

The adenovirus (Ad) E4orf4 protein contributes to efficient progression of virus infection. When expressed alone E4orf4 induces p53- and caspase-independent cell-death, which is more effective in cancer cells than in normal cells in tissue culture. Cancer selectivity of E4orf4-induced cell-death may result from interference with various regulatory pathways that cancer cells are more dependent on, including DNA damage signaling and proliferation control. E4orf4 signaling is conserved in several organisms, including yeast, Drosophila, and mammalian cells, indicating that E4orf4-induced cell-death can be investigated in these model organisms. The Drosophila genetic model system has contributed significantly to the study of cancer and to identification of novel cancer therapeutics. Here, we used the fly model to investigate the ability of E4orf4 to eliminate cancer tissues in a whole organism with minimal damage to normal tissues. We show that E4orf4 dramatically inhibited tumorigenesis and rescued survival of flies carrying a variety of tumors, including highly aggressive and metastatic tumors in the fly brain and eye discs. Moreover, E4orf4 rescued the morphology of adult eyes containing scrib- cancer clones even when expressed at a much later stage than scrib elimination. The E4orf4 partner protein phosphatase 2A (PP2A) was required for inhibition of tumorigenesis by E4orf4 in the system described here, whereas another E4orf4 partner, Src kinase, provided only minimal contribution to this process. Our results suggest that E4orf4 is an effective anticancer agent and reveal a promising potential for E4orf4-based cancer treatments.

A Change in ECM Composition Affects Sensory Organ Mechanics and Function.

Proprioception requires the transduction of muscle-generated deformations into sensory neuronal impulses. In proprioceptive organs, the mechanical coupling between the sensory neuron and the muscle is mediated by a connective structure composed of accessory cells and an extracellular matrix (ECM). Here, we use the fly chordotonal organ (ChO) to investigate how the mechanical properties of the connective element affect mechanosensing. We show that the loss of Pericardin, a major constituent of the ChO ECM, alters the mechanical properties of the ChO resulting in short-wavelength buckling of the accessory cells upon muscle contraction and low compressive strain within the organ. We explain these results using a simplified theoretical model of an elastic beam interacting with an elastic network under a compressive force. We further demonstrate that the transition from compression to bending interferes with the ability of the accessory cells to propagate muscle-generated deformations correctly to the neuron and hence with proper sensing.

An RNAi Screen Identifies New Genes Required for Normal Morphogenesis of Larval Chordotonal Organs.

The proprioceptive chordotonal organs (ChO) of a fly larva respond to mechanical stimuli generated by muscle contractions and consequent deformations of the cuticle. The ability of the ChO to sense the relative displacement of its epidermal attachment sites likely depends on the correct mechanical properties of the accessory (cap and ligament) and attachment cells that connect the sensory unit (neuron and scolopale cell) to the cuticle. The genetic programs dictating the development of ChO cells with unique morphologies and mechanical properties are largely unknown. Here we describe an RNAi screen that focused on the ChO's accessory and attachment cells and was performed in 2nd instar larvae to allow for phenotypic analysis of ChOs that had already experienced mechanical stresses during larval growth. Nearly one thousand strains carrying RNAi constructs targeting more than 500 candidate genes were screened for their effects on ChO morphogenesis. The screen identified 31 candidate genes whose knockdown within the ChO lineage disrupted various aspects of cell fate determination, cell differentiation, cellular morphogenesis and cell-cell attachment. Most interestingly, one phenotypic group consisted of genes that affected the response of specific ChO cell types to developmental organ stretching, leading to abnormal pattern of cell elongation. The 'cell elongation' group included the transcription factors Delilah and Stripe, implicating them for the first time in regulating the response of ChO cells to developmental stretching forces. Other genes found to affect the pattern of ChO cell elongation, such as αTub85E, ß1Tub56D, Tbce, CCT8, mys, Rac1 and shot, represent putative effectors that link between cell-fate determinants and the realization of cell-specific mechanical properties.

Building functional units of movement-generation and movement-sensation in the embryo.

The musculoskeletal and proprioceptive sensory systems exhibit intricate crosstalk between force generation, force sensation and force transmission, all of which are critical for coordinated animal locomotion. Recent developmental studies of the musculoskeletal and proprioceptive units of the invertebrate Drosophila embryo, have revealed several common molecular and structural principles mediating the formation of each of these systems. These common principles, as well as the differences between the developmental programs of the two systems, are discussed. Interestingly, a molecular pathway triggered by the Neuregulin/Vein ligand-dependent activation of the epidermal growth factor receptor (EGFR) pathway, which upregulates the early growth response (EGR)-like transcription factor Stripe, is utilized not only by the Drosophila muscle-tendon and proprioceptive organ-ectoderm attachment, but also by their vertebrate counterparts. An additional theme that has been observed during the development of the musculoskeletal system in both invertebrates and vertebrates is the functional importance of the extracellular matrix and its adhesion receptors. The contribution of mechanical forces to proper junction formation between muscles and tendons and between the sensory cap/ligament cells and their epidermal attachment cells is discussed. The structural and genetic similarities between the musculoskeletal and the proprioceptive systems offer new perspectives as to their common developmental nature.

Loss of thrombospondin reveals a possible role for the extracellular matrix in chordotonal cap cell elongation.

In the Drosophila larva, major proprioceptive input is provided to the brain by sub-epidermal stretch receptors called chordotonal organs (ChO). Similarly to the body wall muscle that needs to be attached on both of its sides to the larval exoskeleton in order to generate movement, the sensory unit of a ChO must be stably anchored to the cuticle on both of its sides in order to sense the relative displacement of body parts. Through an RNAi screen we have identified thrombospondin (Tsp), a secreted calcium binding glycoprotein, as a critical component in the anchoring of ChOs to the cuticle. We show that the Tsp protein starts to accumulate in the extracellular matrix (ECM) surrounding the ChO attachment cells towards the end of embryogenesis and that it becomes highly concentrated at the attachment junction during larval stages. In the absence of Tsp, the ChO's accessory cells fail to form a stable junction with their epidermal attachment cells and organ integrity is not maintained. Tsp is a known player in the establishment of the myotendinous junctions in both invertebrates and vertebrates. Thus, our findings extend the known similarities between muscle-attachment and ChO-attachment cells. In addition to its role in establishing the ChO attachment junctions, Tsp was found to affect ligament cell migration and cap cell elongation. Most interestingly, the Tsp protein was found to decorate the ChO cap cells along their entire length, suggesting that the elongated cap cells are supported by the ECM to which they attach via integrin-based, Tsp-dependent, adhesion plaques. The ECM enwrapping the cap cells is probably important for keeping the cap cells fasciculate and may also provide mechanical support that allows the extremely elongated cells to maintain tension.

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). 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. 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. 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. 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. 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 Johnston's organ in the adult antenna that transduce acoustic to mechanical energy. 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 and Johnston's 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.

Chromosomal binding sites of the homeotic cofactor Homothorax.

The Meis family oncoproteins play a crucial role in leukemogenesis and are highly expressed in other types of cancer as well. The transforming potential of Meis proteins depends on their ability to activate gene expression and therefore, revealing the identity of their target genes is very important. The genome of the fruit fly Drosophila melanogaster contains a single Meis gene, homothorax (hth), which plays multiple roles in embryonic and adult development. Mutations in hth affect the development of numerous embryonic and adult tissues, suggesting that Hth regulates the transcription of a large number of genes. However, it is not known how many genes are regulated directly by Hth and what is the nature of these genes. To address this question, we examined the distribution of the in vivo binding sites of Hth on polytene chromosomes. We found that in the salivary glands (SG) of third instar larvae, Hth binds to approximately 150 chromosomal sites in a very reproducible pattern. More than hundred of these sites were mapped cytologically. Interestingly, Hth accumulates at high levels in some of the most prominent hormone-induced chromosomal puffs, pointing to a possible role of Hth in activation of ecdysone-induced targets. Interfering with the normal transcriptional activity of Hth in larval SGs leads to dramatic reduction in cell size and DNA content implicating Hth in the regulation of cell growth and endoreplication in larval SGs.