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.

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.

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.

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.

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.

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.

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.

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 Asx's 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 Asx's 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.

Ontogeny of renal P-glycoprotein expression in mice: correlation with digoxin renal clearance.

Digoxin is eliminated mainly by the kidney through glomerular filtration and P-glycoprotein (P-gp) mediated tubular secretion. Toddlers and young children require higher doses of digoxin per kilogram of bodyweight than adults, although the reasons for this have not been elucidated. We hypothesized there is an age-dependant increase in P-gp expression in young children. The objectives of this study were to elucidate age-dependant expression of renal P-gp and its correlation with changes in the clearance rate of digoxin. FVB mice were killed at different ages to prepare total RNA for P-gp expression studies. Semi-quantitative RT-PCR was conducted to analyze mdr1a and mdr1b ontogeny in the kidney at: birth, 7, 14, 21, 28 and 45-d old adults. The pharmacokinetics of digoxin (7 microg/kg) was studied in mice of the same age groups. Newborn and Day 7 levels of both mdr1a and mdr1b were marginal. Day 21 mdr1b levels were significantly higher than both Day 14 and Day 28 levels. Digoxin clearance rates were the highest at Day 21, with significant correlation between P-gp expression and clearance values. Increases in digoxin clearance rates after weaning may be attributed, at least in part, to similar increases in P-gp expression.

A tubule cell model for ifosfamide nephrotoxicity.

Mechanisms leading to ifosfamide (IF)-induced renal damage have not been fully elucidated. Recent work suggests that localized renal tubular metabolism of IF and the production of the nephrotoxic chloroacetaldehyde may lead to nephrotoxicity. Presently no pharmacological method to reduce IF nephrotoxicity has been identified. The objectives of this study were to establish a tubule cell model for IF nephrotoxicity, to verify whether renal proximal tubular cells have the necessary cytochrome P450 (CYP) enzymes to oxidize IF, and whether they can metabolize IF to chloroacetaldehyde. CYP3A, and 2B mRNA and protein were identified in LLCPK-1 cells. The cells metabolized the R- and S-IF enantiomers to their respective 2- and 3-dechloroethylifosfamide metabolites, by-products of chloroacetaldehyde formation. Metabolite production was both time and concentration-dependent. IF did not affect cell viability. In contrast, glutathione-depleted cells showed time and dose-dependent damage. The presence of the relevant CYP enzymes in renal tubular cells along with their ability to metabolize IF to its 2- and 3-dechloroethylifosfamide metabolites suggests that nephrotoxic damage may result from the localized production of chloroacetaldehyde. Glutathione is a major defence mechanism against IF toxicity, thus pharmacological methods for replenishing intracellular glutathione may be effective in modulating IF-induced nephrotoxicity.

Renal ontogeny of ifosfamide nephrotoxicity.

Ifosfamide-induced nephrotoxicity adversely affects the health and well-being of children with cancer. We have recently shown age-dependent nephrotoxicity induced by ifosfamide, with younger children (<3 years) substantially more vulnerable. The mechanisms leading to this age-related ifosfamide-induced renal damage have not been identified. Underlying this work is the hypothesis that renal ontogeny is involved in the expression and activity of the cytochrome P450 (CYP) enzymes responsible for IF metabolism to the nephrotoxic chloroacetaldehyde. We evaluated renal CYP3A and 2B22 activity in pigs between the ages of 1 day and adulthood, as well as the metabolism of ifosfamide by renal microsomes to 2- and 3-dechloroethylifosfamide (2-DCEIF and 3-DCEIF, respectively). Kidney CYP3A messenger RNA expression peaked 15 to 60 days (0.7-76 +/- 0.19 CYP3A/actin ratio; P < 0.001). Subsequently, this level decreased to adult values (0.54 - 0.03 CYP3A/actin ratio; P = 0.04). Similarly, we detected an increase in the ifosfamide-metabolism rate between young (18 +/- 2 pmol/mg protein/min) and adult (12.2 +/- 0.17 pmol/mg protein/min) animals (P = 0.002). Ours is the first documentation of ontogeny of renal CYP3A and of renal ifosfamide metabolism. These data suggest that age-dependent ifosfamide nephrotoxicity is, at least in part, due to ontogeny in the production chloroacetaldehyde.

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.