InteractiveFly: GeneBrief

: Biological Overview | References

BIOLOGICAL OVERVIEW

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. This work describes 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. 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. It was further shown 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 (Avetisyan, 2021).

A central question in developmental biology is how different cells that originate in the same lineage and develop within the same organ, acquire unique identities, properties and specialized morphologies. One of the common mechanisms involved in cell fate diversification within a cell lineage is asymmetric cell division in which cytoplasmic determinants of the mother cell differentially segregate into one of the two daughter cells. This asymmetry is then translated into differential gene expression and the activation of cell-type-specific gene regulatory networks (GRN) that dictate the differentiation programs of cells with unique properties. The transition from a primary cell fate to the characteristic phenotype of a fully differentiated cell involves complex GRNs in which numerous genes regulate each other's expression. Despite this complexity, genetic analyses in well-characterized developmental systems can often reveal elementary interactions in small GRNs which dictate a specific cell fate, or a specific feature of the differentiating cell (Avetisyan, 2021).

Many of the core components and the central processes underlying asymmetric cell divisions and primary cell fate decisions have been uncovered in studies performed on the central and peripheral nervous system (PNS) of Drosophila. The PNS of Drosophila contains two classes of multicellular sensory organs, external sensory organs and chordotonal organs (ChOs), whose lineages share a similar pattern of asymmetric cell divisions. In both types of organs, the neuron and support cells, which collectively comprise the sensory organ, arise from a single sensory organ precursor cell (SOP) through a sequence of precisely choreographed asymmetric cell divisions. Antagonistic interactions involving Notch and Numb are key regulators of the asymmetry generated between each two sibling cells within these lineages. Unlike the primary cell-fate specification, which has been extensively investigated, the process of terminal differentiation of the post-mitotic progeny is poorly understood. This study has used the larval lateral pentascolopidial ChO (LCh5) as a model system to study cell fate diversification within a sensory lineage (Avetisyan, 2021).

The LCh5 organ is composed of five mechano-sensory units (scolopidia) that are attached to the cuticle via specialized epidermal attachment cells. Each of the five scolopidia originates in a single precursor cell that divides asymmetrically to generate five of the six cell types that construct the mature organ: the neuron, scolopale, ligament, cap, and cap-attachment cell. Three of the five cap-attachment cells are rapidly removed by apoptosis, leaving two cap-attachment cells that anchor the five cap cells to the epidermis. Later in development, following the migration of the LCh5 organ from the dorsal to the lateral region of the segment, a single ligament-attachment cell is recruited from the epidermis to anchor the five ligament cells to the cuticle. The mature LCh5 organ responds to mechanical stimuli generated by muscle contractions that lead to relative displacement of the attachment cells and the consequent shortening of the organ (Avetisyan, 2021).

Very little is known about the unique cell-type-specific differentiation programs that characterize each of the ChO cells, whose morphologies and mechanical properties differ dramatically from each other. To address this issue, focus was placed on the transcription factor Taxi wings/Delilah (Dei), an important regulator of cell adhesion (Egoz-Matia, 2011), which is expressed in the four accessory cell types (cap, ligament, cap-attachment and ligament-attachment) but is excluded from the neuron and the scolopale cell. Even though dei is expressed in all four accessory cells, its expression in these cells is differentially regulated. The transcription of dei in the ChO is controlled by two cis-regulatory modules (CRMs): The dei attachment enhancer, located ~2.5 Kb upstream of the dei transcription start site, drives expression in the cap-attachment and ligament-attachment cells (as well as tendon cells), whereas the deiChO-1353 enhancer, an intronic 1353 bp DNA fragment, drives dei expression specifically in the cap and ligament cells. The dei^attachment enhancer was shown to be activated by the transcription factor Stripe, which is considered a key regulator of tendon cell development and a known determinant of attachment cell identity. This study provides a high-resolution dissection of the deiChO-1353 enhancer and shows that it integrates both activating and repressive cues to drive dei expression in cap and ligament cells while suppressing it in scolopale cells. D-Pax2/Shaven (Sv), which is expressed in both branches of the cell lineage, is a positive regulator of dei, and Prospero (Pros) inhibits dei expression specifically in the scolopale cell. This small GRN is required for the realization of differentiation programs characterizing cap versus scolopale cell fates and is therefore essential for ChO functionality and coordinated larval locomotion (Avetisyan, 2021).

This work has identified a small GRN that governs the alternative differentiation programs of two cousins once removed cells within the ChO lineage - the cap cell and the scolopale cell. Pros and D-Pax2/Sv are direct regulators of dei that together dictate its expression in the cap cell and its repression in the scolopale cell. Both D-Pax2/Sv and Pros exert their effects on dei transcription via a 262 bp chordotonal-specific enhancer (deiChO-262) in which two D-Pax2/Sv and three Pros binding sites were identified (Avetisyan, 2021).

Following primary cell fate decisions within the ChO lineage, Pros expression becomes restricted to the scolopale cell, whereas D-Pax2/Sv expression becomes restricted to the scolopale and cap cells, similar to its behavior in the external sensory lineages. D-Pax2/Sv activates the expression of dei in the cap cell but is unable to do so in the scolopale cell where Pros is co-expressed. If D-Pax2/Sv activity is compromised, the cap cell fails to express dei and loses some of its differentiation markers, such as the expression of αTub85E. In contrast, if Pros activity is lost, dei is ectopically expressed in the scolopale cell that, as a consequence, acquires some cap cell features including the expression of αTub85E. The observed D-Pax2/Sv- and Pros-associated phenotypes do not reflect genuine cell fate transformations, suggesting that D-Pax2/Sv and Pros do not affect primary cell fate decisions within the ChO lineage. Rather, the observed phenotypes reflect a failure of the cap and scolopale cells to follow the cell type-specific differentiation programs responsible for bringing about their characteristic cellular phenotypes. The D-Pax2/Sv-deficient cap cells fail to express unique differentiation markers (such as αTub85E) and are therefore hardly detectable. It is also possible that the Sv/Pax2-deficient cap cells fail to survive. Thus, the possibility that some of the findings reflect more upstream roles of Sv/D-Pax2 in the specification of cap-cell identity cannot be excluded (Avetisyan, 2021).

The switch between the differentiation programs of cap and scolopale identities cannot be simply explained by the nature of asymmetric cell divisions within the ChO lineage. The effects on the ChO lineage of major regulators of asymmetric cell division, such as Notch and Numb, and the expression pattern of cell differentiation determinants such as Pros and D-Pax2/Sv, were mainly postulated based on knowledge gained by analyzing external sensory lineages. According to the similarity between the lineages, the cap cell parallels the Notch-non-responsive hair (trichogen) cell, whereas the scolopale parallels the Notch- responder sheath (thecogen) cell. Thus, D-Pax2/Sv is expressed in one Notch responder and one non-responder cells in the lineage. The presence of Pros in the Notch-responder cell represses the cap-promoting activity of D-Pax2/Sv. Somewhat similar cousin-cousin cell transformation was found in external sensory organs in the adult where mutations in hamlet transform the sheath cell into a hair cell (parallel to scolopale-to-cap transformation). Ectopic expression of hamlet induced pros expression and repressed the hair shaft-promoting activity of D-Pax2/Sv (Avetisyan, 2021).

In the adult external sensory lineage, Pros was shown to be important for the specification of the pIIb precursor, which gives rise to the neuron and sheath cell (the scolopale counterpart). However, the absence of Pros from the pIIa precursor, which gives rise to the hair and socket cells (the cap and cap-attachment cells counterparts) was even more critical for proper development of this branch of the lineage. This phenomenon is somewhat conserved in the larval ChO. While Pros is required for proper differentiation of the scolopale cell, its absence from the cap cell is critical for adopting the correct differentiation programs within the lineage (Avetisyan, 2021).

Opposing effects of D-Pax2/Sv and Pros activities on cell differentiation have been also identified in the regulation of neuronal versus non-neuronal cell fate decisions in the developing eye, where they play a role in modulating the Notch and Ras signaling pathway. Interestingly, in the R7 equivalence group Pros and D-Pax2/Sv can only alter the cell-type-specific differentiation program of cells that already express the other gene. Similarly, in the ChO lineage, ectopic expression of Pros in the cap and ligament cells transforms the D-Pax2/Sv-positive cap cell toward a scolopale cell identity but does not affect the D-Pax2/Sv-negative ligament cell in a similar fashion, even though the ectopic expression of Pros does repress the transcription of dei in both cell types. Additionally, a loss of Pros activity in the scolopale cell can transform the identity of this cell toward a cap cell identity only in the presence of D-Pax2/Sv (Avetisyan, 2021).

This study has shown that the opposing influences of Pros and D-Pax2/Sv on dei expression is integrated by the deiChO-262 enhancer in both larval and adult ChO lineages. This is the first example of an enhancer that responds to these opposing signals to dictate cell-specific differentiation programs in a sensory lineage. While the identified enhancer is ChO-specific, it is plausible that other enhancers of sensory organ lineage-specific genes encode coupled Pros and D-Pax2/Sv binding sites. The expression of the dei gene in other (non-ChO) organs is regulated via different enhancers. Some of these enhancers are responsible for regulating dei expression in tissues where Pros and Pax2 play opposing roles, such as the eye and wing margin ES organs (the dei^wing+eye enhancer; Nachman , 2015). It is beyond the scope of this work, but in the future, it will be interesting to decipher whether these enhancers also serve as molecular platforms for integrating opposing effects of Pax2 and Pros (Avetisyan, 2021).

This study has identified two D-Pax2/Sv and three Pros binding sites in the deiChO-262 enhancer. Apart from D-Pax2/Sv site 2, none of these sites match the published binding motifs for D-Pax2/Sv or Pros. These results agree with recent studies that showed that many transcription factors function in vivo through low-affinity or suboptimal binding sites that differ from their predicted binding motifs. It was suggested that low-affinity binding sites provide specificity for individual transcription factors belonging to large paralogous families, such as the homeodomain family of transcription factors, that share similar DNA-binding preferences. To compensate for their weak binding capabilities, low-affinity binding sites are often organized in homotypic clusters that can increase the cumulative binding affinity of an enhancer. The findings that the homeodomain transcription factor Pros functions through a cluster of low-affinity binding sites in deiChO-262, may represent another example for the suggested tradeoff between transcription factor binding affinity and specificity (Avetisyan, 2021).

It is not known how Pros opposes the effect of D-Pax2/Sv in the context of dei^ChO-262 to inhibit the expression of dei in scolopale cells. The results suggest that the inhibitory effect of Pros is not mediated through binding competition with D-Pax2/Sv at the D-Pax2/Sv high-affinity site (site 2), since this site does not overlap with a Pros-binding site. The D-Pax2/Sv low-affinity site does overlap with a Pros binding site and mutations in the Pros binding site affect D-Pax2/Sv binding in vitro, however, while being important for robust dei expression, this site is dispensable in the presence of the high-affinity site. It is possible that binding of Pros to the dei^ChO-262 enhancer targets this sequence to a repressed heterochromatin domain as was recently shown for other Pros target genes in differentiating neurons (Avetisyan, 2021).

How is dei regulated in other ChO cell types? dei is expressed in four out of six cell types comprising the ChO: the cap-attachment and ligament-attachment cells, in which dei transcription is activated by Sr via the dei^attachment regulatory module, and the cap and ligament cells in which the expression of dei is regulated via the deiChO-262 enhancer. This study now shows that D-Pax2/Sv activates dei transcription in the cap cell, and that Pros inhibits its expression in the scolopale cell. The identity of the positive regulator/s of dei in the ligament cell, whose cell-fate is determined by the glial identity genes gcm and repo, and the identity of the negative regulator/s of dei in the neuron remains unknown. Interestingly, the expression of dei was found to be altered in response to ectopic expression of gcm in the embryonic nervous system; its expression was upregulated at embryonic stage 11, but was repressed in embryonic stages 15-16. This observation points to GCM as a potential regulator of dei expression in the ligament cells. Another interesting candidate for repressing dei in the sensory neuron is the transcriptional repressor Lola. Lola has been identified as a putative direct regulator of dei in the Y1H screen and was shown to be required in post-mitotic neurons in the brain for preserving a fully differentiated state of the cells. The possible involvement of Gcm and Lola in the regulation of dei awaits further studies. The observed upregulation of the deiChO-262 reporter in the ligament cells of embryos with mutated Pros-binding sites may reflect an early role of Pros in the pIIb precursor before its restriction to the scolopale cell, which prevents dei expression in the ligament cell (Avetisyan, 2021).

Although the loss of dei in the genetic/cellular milieu of the ligament cell (unlike the cap cell), even when accompanied by ectopic expression of Pros, is not sufficient for transforming ligament cell properties towards those of scolopale cells, it is known that the expression of dei in the ligament cell is critical for its proper development. Ligament-specific knockdown of dei leads to failure of the ligament cells to acquire the right mechanical properties and leads to their dramatic over-elongation. By analysing the locomotion phenotypes of larvae homozygous for a dei null allele and the newly generated cap and ligament-specific deiΔChO allele, it was possible to show that the expression of dei in the cap and ligament cells is crucial for normal locomotion. Thus, it is concluded that the correct expression of dei within the ChO is critical for organ functionality. Surprisingly, the gross morphology of LCh5 of deiΔChO larvae appears normal. Yet, in a way that remains to be elucidated, the Dei-deficient cap and ligament cells fail to correctly transmit the cuticle deformations to the sensory neuron, most likely due to changes in their mechanical properties (Avetisyan, 2021).

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. This paper describes 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 alphaTub85E, beta1Tub56D, Tbce, CCT8, mys, Rac1 and shot, represent putative effectors that link between cell-fate determinants and the realization of cell-specific mechanical properties (Hassan, 2018).

Screening and analysis of Janelia FlyLight project enhancer-Gal4 strains identifies multiple gene enhancers active during hematopoiesis in normal and wasp-challenged Drosophila larvae

A GFP expression screen has been conducted on greater than one thousand Janelia FlyLight Project enhancer-Gal4 lines to identify transcriptional enhancers active in the larval hematopoietic system. A total of 190 enhancers associated with 87 distinct genes showed activity in cells of the third instar larval lymph gland and hemolymph. That is, gene enhancers were active in cells of the lymph gland posterior signaling center (PSC), medullary zone (MZ), and/or cortical zone (CZ), while certain of the transcriptional control regions were active in circulating hemocytes. Phenotypic analyses were undertaken on 81 of these hematopoietic-expressed genes with nine genes characterized in detail as to gain- and loss-of-function phenotypes in larval hematopoietic tissues and blood cells. These studies demonstrated the functional requirement of the cut gene for proper PSC niche formation, the hairy, Btk29A, and E2F1 genes for blood cell progenitor production in the MZ domain, and the longitudinals lacking, dFOXO, kayak, cap-n-collar, and Delilah genes for lamellocyte induction and/or differentiation in response to parasitic wasp challenge and infestation of larvae. Together, these findings contribute substantial information to knowledge of genes expressed during the larval stage of Drosophila hematopoiesis and newly identify multiple genes required for this developmental process (Tokusumi, 2016).

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

To decode how adhesion is regulated in cells stemming from different pedigrees this study analyzed the regulatory region that drives the expression of Delilah, which is a transcription factor that serves as a central determinant of cell adhesion, particularly by inducing expression of βPS-integrin. Activation of dei transcription was shown to be 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, 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 in 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 (Nachman, 2015).

Developmental programs are controlled by gene regulatory networks, which are mediated by enhancers with spatially and temporally restricted activities. Understanding the transcriptional regulatory mechanisms employed to provide common properties to different cell types holds a great challenge. One example is that of adhesive molecules, such as integrin receptors, which are expressed in many different cell types in a tightly regulated fashion. One way to gain insight into how proper levels of integrin expression are achieved in different cells types is by studying its upstream transcriptional regulators. The bHLH transcription factor Dei represents a good candidate for such a study due to its key role in activating the expression of βPS integrin in various cell types each harboring a distinct constellation of transcription factors and cell-signaling components (Nachman, 2015).

This study shows that the ability of the dei gene to respond to a variety of upstream regulators in different cell types, or within a single cell in different stages of its development, is achieved by a modular mechanism of regulation, whereby each regulatory module responds to a different combination of transcription factors and activates Dei expression in a specific spatio-temporal pattern. Using an in vivo reporter assay this study identified six distinct CRMs located within two regions of the dei locus that together recapitulated the full expression pattern of the gene in embryos and pupae. Candidate upstream regulators were assigned to most of these modules based on their known expression patterns and bioinformatics analysis for potential DNA binding sites. Overall it seems that Dei is regulated, directly or indirectly, by the central signaling pathways and transcription factors typical to each cell type in which it is expressed: D-Mef2 in muscle cells, Sr in tendon cells, Bs in the wing intervein cells and so on (Nachman, 2015).

Based on the presented data the expression of dei can be summarized as follows. During embryonic stage 11, dei's expression is induced in the mesoderm by the dei^early-muscle module; this expression continues until stage 15 and is activated by unknown signaling pathways. Concomitantly, during stage 11, expression is induced via the dei^ChO module in the ChO CA, cap and ligament cells. In the cap and ligament cells expression is continuous throughout embryogenesis, whereas in the CA cells, expression ceases during stage 15. It remains to be determined what signaling pathways activate the dei^ChO-induced expression. During stage 14, the dei^attachment module, which is activated directly by Sr, starts to drive gene expression in the ChO CA cells and in tendon cells, and later on, in stage 16, also in ChO LA cells. Continuing into late stage 14, expression induced via the dei^late-muscle module is activated in muscle cells by D-Mef2. In pupal stages, dei's expression is induced in the developing wing and eye through the dei^wing+eye module, possibly under the direct regulation of Bs in the wing and possibly under the regulation of Cut in the cone cells of the eye (Nachman, 2015).

The functionality and modular nature of the identified dei regulatory modules could be inferred from the previously described phenotype of the regulatory allele dei^e01478. This allele harbors a PiggyBac-RB transposon inserted within the dei intron upstream to the minimal dei^ChO fragment. It was previously reported that in homozygous dei^e01478 embryos Dei expression was lost from tendons and ChO attachment cells, whereas it was markedly enhanced in ChO cap and ligament cells, as compared to heterozygous siblings (Egoz-Matia, 2011). It is now known that this insertion leads to inhibition of gene expression from all regulatory modules located upstream to it. The only module located downstream to the dei^e01478 insertion is dei^ChO, which continues to induce expression of dei specifically in the cap and ligament cells in this genetic background (Nachman, 2015).

Inhibition of dei's transcription through the upstream CRMs is probably not caused by the sheer size of the dei^e01478 transposon (5.7 kb). Not only the CRMs were shown to be active irrespective of their orientation or distance from the promoter, the dei^e01478 insertion does not alter the distance between the CRMs and the promoter, with the exception of the dei^ChO module, which remains functional. The PiggyBac-RB transposon harbors a splice acceptor site and thus could potentially cause premature splicing which may render transcription from the dei promoter ineffective. In this scenario, activation of the dei^ChO module is possible through an alternative promoter located between the insertion site and the coding exon. New alternative promoters are continuously being identified and over 40% of fly genes use alternative promoters during fly development (Nachman, 2015).

The cis-regulatory modules of dei, which integrate information derived from the transcription factor landscape of each relevant cell, are scattered along 9.4 kb of DNA located upstream to the transcription start site and within the single intron of the gene. It has been recently estimated that the fly genome contains 50,000-100,000 developmental enhancers and that most of these enhancers drive expression of their neighboring genes in a dynamic tissue-specific pattern. It was also estimated that approximately one third of all enhancers are intragenic and that the majority of fly enhancers reside in the vicinity of their target genes. The dei tissue- and stage-specific enhancers conform to the suggested principles. The locus contains at least five enhancers that regulate expression of the dei transcript in a spatially and temporally restricted pattern. Three of these enhancers are intragenic (Nachman, 2015).

It has been shown that most interactions between enhancers to other enhancers and promoters with similar expression pattern remain largely unchanged during development and that transcription initiation from these enhancer-promoter contacts is mediated by the release of paused polymerase (Ghavi-Helm, 2014). In this respect too, the dei gene conforms to the suggested mode of action. This study has shown for example that there is no switching between embryonic to adult ChO-specific enhancers; a single enhancer induces transcription of dei in ChOs in both embryogenesis and in adult development. In addition, dei was identified as a gene with stalled polymerase, a feature typical of developmental control genes poised for fast activation later in development (Nachman, 2015).

In addition to the new insight gained into the regulation of dei's expression, this work has shed light on a few unknown aspects of ChO development. One of the important findings is the biphasic nature of dei's regulation in ChO CA cells, which probably reflects a change in cell differentiation status. The CA cell, unlike the LA cell, is derived from the ChO lineage and is therefore influenced by the Ato-induced program of ChO development. However, following the primary determination of cell fates, when the ChOs start to stretch and terminal differentiation ensues, the CA cell needs to acquire the typical characteristics of a functional attachment cell, similarly to the LA and tendon cells. As part of this shift in cellular properties the dei^ChO module is switched off and the dei^attachment module is switched on to accommodate the required changes in gene regulation. It is not yet known which transcription factors regulate dei's expression through the dei^ChO module in the primary phase of CA cell development, but it was shown that Sr is the major regulator of the dei^attachment module in later stages of CA cell differentiation. Disruption of the Sr binding sites within the dei^attachment module led to complete loss of reporter expression from the tendon and CA cells, but interestingly, allowed for significant expression in LA cells. This observation points to yet unknown differences in gene regulation networks and developmental pathways between the two types of ChO attachment cells (CA and LA) (Nachman, 2015).

Another important observation is that the dei^ChO module is sufficient to drive gene expression in the ligament and cap cells of all ChOs in both embryos and adults (with the possible exception of antennal ChO, unpublished data). This observation points to a hitherto unrecognized similarity in the developmental programs of different types of larval and adult ChOs and suggests that Dei plays a role in the morphogenesis of all ChO's accessory cells. Very little is known about the nature of ChO accessory cells in the adult. Now, the newly identified dei regulatory modules provide a good entry point to the identification of upstream regulators and downstream effectors pertinent to the specification and function of these cells (Nachman, 2015).

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

In spite of conceptual views of how differential gene expression is used to define different cell identities, it is still not understood how different cell identities are translated into actual cell properties. The fly wing is composed of two main cell types, vein and intervein. These two 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 alternative differentiation programs are still very roughly drawn. This study identifies 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. Dei's 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 (Egoz-Matia, 2011).

This study has identified the bHLH transcription factor Dei as an important positive regulator of the expression of βPS, the major β subunit in Drosophila. During embryonic development Dei's expression is confined mainly to cells that adhere strongly to other cells and are able to withstand mechanical strain, for instance, tendon cells that attach body wall muscle to the cuticle. Moreover, when different types of cells arise from within a uniform cell population, or through asymmetric cell division, Dei's expression is restricted to the ‘stickier’ types of cells. For example, in the chordotonal organ lineage, Dei is expressed in the four types of support cells (cap, ligament, cap-attachment and ligament-attachment), but is excluded from the neuron and glia. Similar phenomenon is seen in the developing wing where Dei is expressed only in intervein territories, where the ventral and dorsal layers adhere to each other, and is not expressed in vein cells that do not adhere to cells of the opposite layer. In all these systems, Dei does not function as a selector of cell identity, but it is required to realize the selected fate by activating a developmental program that specifies adhesive properties of cells (Egoz-Matia, 2011).

Although dei's expression has not been characterized in all developmental stages and tissues, published data of various microarray analyses suggest that dei is expressed in other developmental and physiological contexts where up-regulation of βPS integrin is required. For example, dei was up-regulated when larvae were exposed to immune challenge, or when mutant larvae exhibited an increase in lamellocyte cell population. Lamellocytes represent a subset of hemocytes in Drosophila, which differentiate in response to specific immune challenge. The lamellocytes aggregate around large pathogens to form a rigid laminated capsule that confines the pathogen and enables its elimination. This encapsulation process requires members of the integrin family that presumably mediate the lamellocyte's attachment (Egoz-Matia, 2011).

This work focused mainly on the role of dei in intervein cells and showed that dei provides a missing link between the genetic specification of these epithelial cells and their differentiation. The data place dei downstream to the major signaling pathways that divide the wing to regions of veins and interveins and downstream to Bs, which works as a selector of intervein identity. It remains to be determined whether dei is a direct target of Bs, and whether it is a direct regulator of βPS, however the results of the rescue experiment suggest that the effect of Bs on integrin expression is mediated, at least in part, by the activity of Dei (Egoz-Matia, 2011).

The venation phenotypes caused by weak dei alleles could be also attributed to the effects of Dei on βPS expression. Even though vein and intervein territories are established during early stages of wing development, the decision remains plastic for at least 24-h APF. Maintenance of the right fates depends on both vein-specific and intervein-specific genes. Appropriate levels of integrin expression are required for the maintenance of intervein fate, as suggested by the ectopic vein phenotype of certain mys alleles, which is very similar to the venation phenotype of weak dei alleles (Egoz-Matia, 2011).

It is reasonable to assume that Dei regulates multiple target genes in different cells and tissues. However, as for integrins, Dei regulates specifically βPS integrin. No evidence was found for regulation of αPS1 or αPS2, which are expressed differentially in the two wing layers, by Dei. Since βPS is the dimerization partner of both αPS1 and αPS2, by regulating its expression Dei practically affects all integrin-based adhesion processes at both the dorsal and ventral wing layers. The data also suggest that the effects of Dei on integrin-dependent adhesion are not restricted to the wing. Ectopic expression of Dei led to up-regulation of βPS expression in embryonic tissues, whereas loss of Dei's activity caused a reduction in the level of βPS expression in the cone cells of the eye (Egoz-Matia, 2011).

In summary, Dei is thought of as a general switch that turns on βPS integrin expression wherever a sticky cell has to develop. Since such a switch needs to be turned on in different tissues and different developmental and physiological contexts, it is predicted that the dei gene can respond to various signaling pathways and transcription factors. Indeed, analysis of the regulatory region of the dei locus demonstrated that it harbors multiple regulatory modules that respond to different transcription factors working in different developmental contexts (Egoz-Matia, 2011).

Kakapo, a novel cytoskeletal-associated protein is essential for the restricted localization of the neuregulin-like factor, vein, at the muscle-tendon junction site

In the Drosophila embryo, the correct association of muscle cells with their specific ectodermally derived tendon cells, also known as epidermal muscle attachment or EMA cells, is achieved through reciprocal interactions between these two distinct cell types. Vein, a neuregulin-like factor secreted by the approaching myotube, activates the EGF-receptor signaling pathway within the tendon cells to initiate tendon cell differentiation. kakapo is expressed in the tendons and is essential for muscle-dependent tendon cell differentiation. Kakapo is a large intracellular protein and contains structural domains also found in cytoskeletal-related vertebrate proteins (including plakin, dystrophin, and Gas2 family members). kakapo mutant embryos exhibit abnormal muscle-dependent tendon cell differentiation. The expression of delilah, stripe, and beta1 tubulin is induced in the epidermal attachment cells as a result of the EGF-receptor pathway activation by the neuregulin-like growth factor, Vein (Yarnitzky, 1997). Vein is secreted by mesodermal cells underlying the EMA cells. Vein protein localization is restricted to the muscle-tendon junctional site in wild-type embryos. However, in kak mutant embryos, Vein protein is not localized and appears rather diffuse. This altered pattern of Vein may explain the multiple number of cells expressing delilah and stripe: since Vein is not strictly localized at a given muscle-tendon junction site, it apparently weakly activates the EGF-receptor pathway in neighboring cells as well. It is presumed that the only cells that can respond to the ectopic Vein protein are the competent population of EMA cells, defined by the early expression of stripe. These cells express stripe during early developmental stages in a muscle-independent manner and normally lose their stripe expression by stage 16 of embryonic development. When these competent EMA cells receive the muscle-derived Vein signal, the expression of stripe and delilah is reactivated. It appears that only this population of cells is capable of responding to Vein, since the pattern of the ectopic Stripe- or Delilah-expressing cells in the kak mutant embryos resembles that of the early population of Stripe-expressing cells. The reduced levels of beta1 tubulin mRNA in the mutant tendon cells may also result from the abnormal pattern of Vein localization, since lower levels of Vein may not be sufficient to induce maximal beta1 tubulin expression. It therefore appears that the primary defect in kak mutant embryos stems from the lack of Vein accumulation at the muscle-tendon junctional site (Strumpf, 1998).

To elucidate the function of kak in epidermal muscle attachment (EMA) cell differentiation, an examination was made of the expression of various markers characteristic of tendon cell terminal differentiation, including Stripe, Delilah, and beta1 tubulin mRNA. The expression of the regulatory protein Stripe, a transcription factor of the early growth response (EGR) family, determines the fate of the EMA competent cells at the first phase of tendon cell development. Stripe expression leads to the expression of an array of EMA-specific genes that contribute to the correct guidance of the myotubes. The second phase of tendon cell differentiation depends on inductive interactions between the myotube and the EMA cell. These interactions lead to terminal differentiation of the EMA competent cells into tendon cells, in which high protein levels of Stripe, Groovin (now known as Kakapo), and Alien are maintained, and the transcription of the genes delilah and beta1 tubulin is induced (Strumpf, 1998 and references).

In kak mutants, an excess of EMA cells, marked by the expression of Stripe and Delilah, is observed at a number of sites in the epidermis. This phenotype is particularly notable in domains in which a group of muscles extend together towards neighboring epidermal attachment cells, such as along the ventral segmental border cells, to which the four ventral longitudinal muscles bind. To further study the state of differentiation of the EMA cells in the kak mutant embryos, the expression of the beta1 tubulin gene was examined. In wild-type embryos, the expression of the beta1 tubulin gene is significantly elevated toward the end of tendon cell differentiation. In contrast to the expression of Stripe and Delilah, the mRNA expression of beta1 tubulin in kak mutant embryos is significantly reduced, suggesting that transcription of the latter gene requires different levels of signaling. It is suspected that Vein signaling from mesodermal cells, which is required for terminal differentiation of tendon cells (Yarnitzky, 1997), may be reduced in the mutant embryos; while there is enough signal to trigger Delilah and Stripe expression, the signal is not capable of inducing beta1 tubulin transcription (Strumpf, 1998).

The expression of delilah, stripe, and beta1 tubulin is induced in the epidermal attachment cells as a result of the EGF-receptor pathway activation by the neuregulin-like growth factor, Vein (Yarnitzky, 1997). Vein is secreted by mesodermal cells underlying the EMA cells. Vein protein localization is restricted to the muscle-tendon junctional site in wild-type embryos. However, in kak mutant embryos, Vein protein is not localized and appears rather diffuse. This altered pattern of Vein may explain the multiple number of cells expressing delilah and stripe: since Vein is not strictly localized at a given muscle-tendon junction site, it apparently weakly activates the EGF-receptor pathway in neighboring cells as well. It is presumed that the only cells that can respond to the ectopic Vein protein are the competent population of EMA cells, defined by the early expression of stripe. These cells express stripe during early developmental stages in a muscle-independent manner and normally lose their stripe expression by stage 16 of embryonic development. When these competent EMA cells receive the muscle-derived Vein signal, the expression of stripe and delilah is reactivated. It appears that only this population of cells is capable of responding to Vein, since the pattern of the ectopic Stripe- or Delilah-expressing cells in the kak mutant embryos resembles that of the early population of Stripe-expressing cells. The reduced levels of beta1 tubulin mRNA in the mutant tendon cells may also result from the abnormal pattern of Vein localization, since lower levels of Vein may not be sufficient to induce maximal beta1 tubulin expression. It therefore appears that the primary defect in kak mutant embryos stems from the lack of Vein accumulation at the muscle-tendon junctional site (Strumpf, 1998).

Is the abnormal differentiation of the epidermal muscle attachment (EMA) cells in kak mutant embryos reflected by the pattern of the somatic musculature? kak mutant embryos at stage 16 of embryonic development were labeled with anti-myosin heavy chain antibody to visualize the somatic muscles, and the muscle pattern was compared with that of wild-type embryos. A significant disruption of the somatic muscle pattern is observed in kak mutant embryos. In many cases, individual myotubes are not oriented correctly, and in some cases the myotube rounds up. Since Kak cannot be detected in myotubes using the available antibodies, it is assumed that the somatic muscle derangement is secondary to the abnormal differentiation of the EMA cells. A similar phenotype is also observed in stripe mutant embryos, in which the EMA cells do not differentiate correctly (Frommer, 1996). The similarity between the stripe and kak muscle phenotype and the reduced beta1 tubulin mRNA expression are consistent with the conclusion that EMA cell differentiation is defective in kak mutants. The correct recognition between the muscle and the tendon cell is essential for arresting the extension of the myotube and establishment of the final pattern of somatic musculature (Yarnitzky, 1997). It appears that the muscle development in kak partial loss of function embryos does not represent a complete loss of function phenotype since a more severe muscle defect is observed in kakV104/DfMK1 embryos (Strumpf, 1998).

How could this intracellular protein affect the localization of Vein at the extracellular matrix surrounding the EMA cell? At least two possibilities, which are not mutually exclusive, are considered. The first is the association of Kak with the unique cytoskeletal network of the EMA cell, which is critical for the cell's polarized organization. Tendon cell polarity may be essential for maintaining the characteristic junctional complexes formed between the basal surfaces of the EMA cell and the muscle cells. The space between these junctional complexes contains many extracellular matrix proteins, some of which may possess a Vein binding function. Impaired tendon cell polarity may lead to the loss of the putative Vein-binding component(s). Alternatively, Kak may be associated with a transmembrane protein(s) responsible for Vein localization either by direct binding or by association with additional extracellular matrix components that may directly bind Vein. Immunoprecipitation experiments with anti-Kak antibody indicated that Kakapo forms protein complexes containing the extracellular protein Tiggrin. These results favor the latter possibility that Kak is directly associated with protein complexes that may be important for Vein binding. The reduced amount of electron-dense material observed at the muscle-tendon junction site in the kak mutant embryos described in Prokop, et al. (1998) is in agreement with both mechanisms mentioned above (Strumpf, 1998).

The excess number of Stripe- and Delilah-expressing cells in the kak mutant embryos may be attributed to the dispersed levels of Vein, which could induce partial activation of the EGF-receptor signaling pathway in neighboring cells. An alternative explanation is that muscle-dependent differentiation of tendon cells may be accompanied by lateral inhibition of neighboring cells. The differentiated tendon cell may activate the Notch-signaling pathway in the surrounding cells. Aberrant contacts between tendon cells and their neighboring EMA competent cells in the kak mutant embryos may prevent efficient lateral inhibition, resulting in an excess of Stripe- and Delilah-expressing cells. An observation that supports this possibility is that an excess in beta1 tubulin-expressing cells is detected in Delta mutant embryos. Delta, a well-characterized Notch ligand, mediates lateral inhibition in a large array of tissues during embryonic and adult development. The lack of Delta may prevent lateral inhibition of the competent EMA cells, leading to their differentiation into beta1 tubulin-expressing cells. The impaired integrity of the epidermis described by Gregory (1998) is consistent with this explanation (Strumpf, 1998).

The Drosophila neuregulin homolog vein mediates inductive interactions between myotubes and their epidermal attachment cells

Inductive interactions between cells of distinct fates underlie the basis for morphogenesis and organogenesis across species. In the Drosophila embryo, somatic myotubes form specific interactions with their epidermal muscle attachment (EMA) cells. The establishment of these interactions is a first step toward further differentiation of the EMA cells into elongated tendon cells containing an organized array of microtubules and microfilaments. The molecular signal for terminal differentiation of tendon cells is the secreted Drosophila neuregulin-like growth factor Vein, produced by the myotubes. Although Vein mRNA is produced by all of the myotubes, Vein protein is secreted and accumulates specifically at the muscle-tendon cell junctional site. In loss-of-function vein mutant embryos, muscle-dependent differentiation of epidermal tendon cells, measured by the level of expression of specific markers (Delilah and beta1 tubulin) is blocked. When Vein is expressed in ectopic ectodermal cells, it induces the ectopic expression of these genes. These results favor the possibility that the Drosophila EGF receptor DER/Egfr expressed by the EMA cells functions as a receptor for Vein. Vein/Egfr binding activates the Ras pathway in the EMA cells leading to the transcription of the tendon-specific genes stripe, delilah, and beta1 tubulin. In Egfr1F26 mutant embryos lacking functional Egfr expression, the levels of Delilah and beta1 Tubulin are very low. The ability of ectopic Vein to induce the expression of Delilah and beta1 Tubulin depends on the presence of functional Egfrs. Activation of the Egfr signaling pathway by either ectopically secreted Spitz, or activated Ras, leads to the ectopic expression of Delilah. These results suggest that inductive interactions between myotubes and their epidermal muscle attachment cells are initiated by the binding of Vein, to the Egfr on the surface of EMA cells (Yarnitzky, 1997).

Reciprocal signaling between Drosophila epidermal muscle attachment cells and their corresponding muscles

The stripe gene is both necessary and sufficient to initiate the developmental program of epidermal muscle attachment (EMA or segmental border) cells. In stripe mutant embryos, these cells do not differentiate correctly. Ectopic expression of Stripe in various epidermal cells transforms these cells into muscle-attachment cells expressing an array of epidermal muscle attachment cell-specific markers. These markers include goovin, delilah, and ß1 tubulin. The EMA-specific genes induced by Stripe can be divided into two groups: genes that follow Stripe ectopic expression in all embryonic stages or genes that can not be detected in early (stage 10-11) or late (older than stage 14) developmental stages, but only in intervening stages. groovin and alien represent the first group (all stages) and delilah and ß1 tubulin represent the second group (intervening stages) and are expressed only in stages 12-14 (Becker, 1997).

The ectopic epidermal muscle attachment cells are capable of attracting somatic myotubes from a limited distance, providing that the myotube has not yet been attached to or been influenced by a closer wild-type attachment cell. Analysis of the relationships between muscle binding and differentiation of the epidermal muscle attachment cell has been performed in mutant embryos in which either loss-of-muscles or ectopic muscles were induced. This analysis indicates that although the initial expression of epidermal muscle-attachment cell-specific genes including stripe and groovin is muscle independent, continuous gene expression is maintained only in epidermal muscle attachment cells that are connected to muscles. Normally, the expression of ß1 tubulin is restricted to the final stage of gene expression in tendon-like cells, supporting the idea of a distinct mechanism regulating gene expression within the tendon cells as a result of muscle interactions. These results suggest that the binding of a somatic muscle to an epidermal muscle attachment cell triggers a signal affecting gene expression in the attachment cell. Thus there exists a reciprocal signaling mechanism between the approaching muscles and the epidermal muscle attachment cells. First the epidermal muscle attachment cells signal the myotubes and induce myotube attraction and adhesion to their target cells. Following this binding, the muscle cells send a reciprocal signal to the epidermal muscle attachment cells inducing their terminal differentiation into tendon-like cells (Becker, 1997).

A novel basic helix-loop-helix protein is expressed in muscle attachment sites of the Drosophila epidermis

This study found that a novel basic helix-loop-helix (bHLH) protein is expressed almost exclusively in the epidermal attachments sites for the somatic muscles of Drosophila melanogaster. A Drosophila cDNA library was screened with radioactively labeled E12 protein, which can dimerize with many HLH proteins. One clone that emerged from this screen encoded a previously unknown protein of 360 amino acids, named delilah, that contains both basic and HLH domains, similar to a group of cellular transcription factors implicated in cell type determination. Delilah protein formed heterodimers with E12 that bind to the muscle creatine kinase promoter. In situ hybridization with the delilah cDNA localized the expression of the gene to a subset of cells in the epidermis which form a distinct pattern involving both the segmental boundaries and intrasegmental clusters. This pattern was coincident with the known sites of attachment of the somatic muscles to tendon cells in the epidermis. delilah expression persists in snail mutant embryos which lack mesoderm, indicating that expression of the gene was not induced by attachment of the underlying muscles. The similarity of this gene to other bHLH genes suggests that it plays an important role in the differentiation of epidermal cells into muscle attachment sites of blood (Armand, 1994).

Search PubMed for articles about Drosophila Delilah

Armand, P., Knapp, A. C., Hirsch, A. J., Wieschaus, E. F. and Cole, M. D. (1994). A novel basic helix-loop-helix protein is expressed in muscle attachment sites of the Drosophila epidermis. Mol Cell Biol 14(6): 4145-4154. PubMed ID: 8196652

Avetisyan, A., Glatt, Y., Cohen, M., Timerman, Y., Aspis, N., Nachman, A., Halachmi, N., Preger-Ben Noon, E. and Salzberg, A. (2021). Delilah, prospero, and D-Pax2 constitute a gene regulatory network essential for the development of functional proprioceptors. Elife 10. PubMed ID: 34964712

Becker, S., et al. (1997). Reciprocal signaling between Drosophila epidermal muscle attachment cells and their corresponding muscles. Development 124(13): 2615-2622. PubMed ID: 9217003

Egoz-Matia, N., Nachman, A., Halachmi, N., Toder, M., Klein, Y. and Salzberg, A. (2011). Spatial regulation of cell adhesion in the Drosophila wing is mediated by Delilah, a potent activator of betaPS integrin expression. Dev Biol 351(1): 99-109. PubMed ID: 21215259

Ghavi-Helm, Y., Klein, F. A., Pakozdi, T., Ciglar, L., Noordermeer, D., Huber, W. and Furlong, E. E. (2014). Enhancer loops appear stable during development and are associated with paused polymerase. Nature 512(7512): 96-100. PubMed ID: 25043061

Hassan, A., Timerman, Y., Hamdan, R., Sela, N., Avetisyan, A., Halachmi, N. and Salzberg, A. (2018). An RNAi Screen Identifies New Genes Required for Normal Morphogenesis of Larval Chordotonal Organs. G3 (Bethesda) 8(6): 1871-1884. PubMed ID: 29678948

Nachman, A., Halachmi, N., Matia, N., Manzur, D. and Salzberg, A. (2015). Deconstructing the complexity of regulating common properties in different cell types: lessons from the delilah gene. Dev Biol 403: 180-191. PubMed ID: 25989022

Tokusumi, T., Tokusumi, Y., Brahier, M. S., Lam, V., Stoller-Conrad, J. R., Kroeger, P. T. and Schulz, R. A. (2017). Screening and Analysis of Janelia FlyLight Project Enhancer-Gal4 Strains Identifies Multiple Gene Enhancers Active During Hematopoiesis in Normal and Wasp-Challenged Drosophila Larvae. G3 (Bethesda) 7(2): 437-448. PubMed ID: 27913635

Yarnitzky, T., Min, L. and Volk, T. (1997). The Drosophila neuregulin homolog vein mediates inductive interactions between myotubes and their epidermal attachment cells. Genes Dev. 11(20):2691-2700. PubMed ID: 9334331

date revised: 15 March, 2022

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