lozenge: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - lozenge

Synonyms -

Cytological map position - 8D8-9

Function - transcription factor

Keywords - eye, peripheral nervous system

Symbol - lz

FlyBase ID:FBgn0002576

Genetic map position - 1-27.7

Classification - AML-1 homolog

Cellular location - nuclear

NCBI links: Precomputed Blast | Entrez Gene |
Recent literature
Wagamitsu, S., Takase, D., Aoki, F. and Suzuki, M. G. (2017). Identification of the Doublesex protein binding sites that activate expression of lozenge in the female genital disc in Drosophila melanogaster. Mech Dev [Epub ahead of print]. PubMed ID: 28087460
Normal sexual differentiation in the genital organs is essential for the animal species that use sexual reproduction. Although it is known that doublesex (dsx) is required for the sexual development of the genitalia in various insect species, the direct target genes responsible for the sexual differentiation of the genitalia have not been identified. The lozenge (lz) gene is expressed in the female genital disc and is essential for developments of spermathecae and accessory glands in Drosophila melanogaster. The female-specific isoform of DSX (DSXF) is required for activating lz expression in the female genital disc. However, it still remains unclear whether the DSXF directly activates the transcription of lz in the female genital disc. This study found two sequences (lz-DBS1 and lz-DBS2) within lz locus that showed high homology to the DSX binding motif identified previously. Competition assays using recombinant DSX DNA-binding domain (DSX-DBD) protein verified that the DSX-DBD protein bound to lz-DBS1 and lz-DBS2 in a sequence-specific manner with lower affinity than to the known DSX binding site in the bric-a-brac 1 (bab1) gene. Reporter gene analyses revealed that a 2.5-kbp lz genomic fragment containing lz-DBS1 and lz-DBS2 drove reporter gene (EGFP) expression in a manner similar to endogenous lz expression in the female genital disc. Mutations in lz-DBS1 alone significantly reduced the area of EGFP-expressing region, while EGFP expression in the female genital disc was abolished when both sites were mutated. These results demonstrated that DSX directly activates female-specific lz expression in the genital disc through lz-DBS1 and lz-DBS2.

In Drosophila, the lozenge gene complex is considered a historically classic loci. It was the first genetic locus to reveal a genetic fine structure, more than a decade before the elegant genetic fine structure analysis of the rII locus in bacteriophage T4 (Green, 1990 and references). Work with lozenge proved that genes could be subdivided into constituent parts, much like atoms can be broken down into more fundamental particles. The occurrence of interallelic recombination allowed the mapping of lozenge alleles to one of four discrete sub-loci: spectacle, krivshenko, lozenge and glossy. With the recent cloning of lozenge, the genetic basis of these sub-loci can now be analyzed.

The involvement of lozenge in the development of the antennal olfactory sense organs is of particular interest both because it is a relatively neglected area of research and because the olfactory sense organs do not show a stereotyped arrangement or fixed cellular composition characteristic of cells of the external sense organs (See Peripheral nervous system). The fixed pattern of external sense organs is found for taste (gustatory) sensilla, located on the labellum, pharynx, leg, wing and female genitalia. Olfactory sensilla are found only on the antenna (Stocker, 1994). In the PNS, all the cells comprising a sensory element are derived from a single progenitor cell (the sensory mother cell). In contrast, cells of the olfactory sensilla (found only in antenna) are not related to a common progenitor by lineage (Ray, 1995).

There are three morphologically distinct types of olfactory sensilla arranged in broad zones on the surface of the third (terminal) antennal segment. There appears to be no strict control of the spacing between neighboring sensilla. Moving from medial to lateral, the three types are: basiconical sensilla, coeloconic sensilla and tricoid sensilla. Olfactory and taste sensilla share a common anatomical design: each consists of a cuticular shaft (hair cell or trichogen), a socket (outermost cell or tormogen), and a sheath cell (glial) that wraps around the multiple neurons of each sensillum. All the sensory neurons from the third antennal segment project to the olfactory lobe in the brain. The cephalic disc (eye-antennal disc) forms the whole of the head capsule in the adult. An "enhancer trap" line A101 is used as a marker of sensillum development, as the reporter enzyme ß-galactosidase is expressed specifically in the sensory progenitors and subsequently in all cells of a developing sensillum. In addition, a monoclonal antibody 22C10 (see Futsch) recognizes all developing and adult neurons and also the sensory progenitors.

There are two waves of sensillogenesis in antenna, as indicated by the appearance of ß-gal-positive cells: one takes place at 0-10 hr after puparium formation, and a second at 16-18 hr after puparium formation. Precursors specified during the first wave are uniformly spread over the cephalic disc, and precursors arising in the later time interval are interspersed between the earlier, more mature, sensilla. Each wave does not specify a single type of sense organ, but each of the three types is specified in both waves. Between 130 and 140 precursors express ß-gal in the early wave, and these precursors are termed sensillum "founder cells" (FCs). FCs give rise to the cellular constituents (neural, glial and support) of each sensillium.

During 10- to 16-hr after puparium formation, the number of FCs decreases, to be replaced by groups of two to four cells. These groups are referred to as presensillum clusters (PFCs) since the cells in each such cluster have been found to form a single sensillum. The nuclei of the cells in each group are all equivalent in size and smaller than those of the FCs observed in earlier discs. It is thought that FCs, in spite of their larger nuclei, do not undergo division during the formation of PFCs since no mitotic figures are present. Because of the absence of DNA synthesis and mitotic figures, it is believed that additional cells of the cluster (other than FCs) are recruited from surrounding cells, and not derived from FCs by descent. By 14 hours, one or two cells of each PSC can be stained with MAB22C10. These cells have basally placed nuclei. Cells with basal nuclei are the progenitors of the sensory neurons in each PSC. Since there is no DNA synthesis in the cells on the surface of the antenna during this period, it is concluded that olfactory sense organ progenitors do not replicate DNA before the PSC formation.

Subsequently, between 16 and 17 hours, cells of each PSC undergo mitosis before terminal differentiation, but cells of the PSC divide only once before they undergo terminal differentiation. This means that pairs of cells within each sensillum are "sisters." Three cells among these sisters locate apically to form the support cells; the cell bodies of neuronal cells locate basally. By 18 hours, axons can be seen leaving the PSCs. From one to four neurons innnervate each sensillium. Therefore the total number of cells in a single sensory cluster varies from four to seven, at least one neuron and three support cells (Ray, 1995).

In lozenge mutants, all basiconic sensilla and some of the trichoid sensilla are lacking. MAb22C10 positive precursors of the basiconic sensilla fail to develop. In lz mutants examined at 10 hours, there is a reduction of PSCs to approximately 60, versus 132 in wild type. While a lack of sensilla is apparent all over the antennal surface by 26 hours, it is more pronounced in the region normally occupied by the big basiconic sensilla. This implicates lozenge in the process of determination of founder cells of basiconic sensilla (Ray, 1995).

In eye morphogenesis lozenge is required to specify the R7 equivalence group. lozenge is active in R7 and cone cells, where it functions to silence seven-up. lozenge is silent in R3/R4, allowing for the expression of seven-up, and the specification of R3/R4 cell fates. Lozenge plays a dual rule in R1/R6 cells. As seven-up is required to specify R1/R6 fate, Lozenge cannot act to completely silence seven-up function, but apparently acts to reduce seven-up activity. At the same time, Lozenge activates Bar gene expression in the R1/R6 pair, an essential function required for specification of the fate of that pair. It will be of great interest to discover how lozenge is regulated, as it appears to have a critical role in specification of alternative neural fates (Daga, 1996).

Switching cell fates in the developing Drosophila eye

The developing Drosophila ommatidium is characterized by two distinct waves of pattern formation. In the first wave, a precluster of five cells is formed by a complex cellular interaction mechanism. In the second wave, cells are systematically recruited to the cluster and directed to their fates by developmental cues presented by differentiating precluster cells. These developmental cues are mediated through the receptor tyrosine kinase (RTK) and Notch (N) signaling pathways and their combined activities are crucial in specifying cell type. The transcription factor Lozenge (Lz) is expressed exclusively in second wave cells. In this study Lz was ectopically supplied to precluster cells, and the various RTK/N codes that specify each of three second wave cell fates were concomitantly supplied. This protocol reproduced molecular markers of each of the second wave cell types in first wave precluster cells. Three inferences were drawn; (1) it was confirmed that Lz provides key intrinsic information to second wave cells, and this can now be combined with the RTK/N signaling to provide a cell fate specification code that entails both extrinsic and intrinsic information. (2) the reproduction of each second wave cell type in the precluster confirms the accuracy of the RTK/N signaling code, and (3) RTK/N signaling and Lz need only be presented to the cells for a short period of time in order to specify their fate (Mavromatakis, 2013).

This paper explored three inter-related themes bearing on the nature of the signals that specify the cell types in the Drosophila ommatidium. The ability of a transcription factor to predispose the cellular responses to developmental signals was examined, the accuracy of the signaling code that represents these developmental signals was validated, and it was inferred that both the intrinsic and extrinsic aspects are only required for a brief period of time (Mavromatakis, 2013).

Lz had long been assumed to be a key factor that distinguishes how second wave cells differ from the precluster cells in their response to developmental signals. This paper rigorously tested this concept and reproduced features typical of the three second wave cell types in the R3/4 precluster cells by supplying ectopic Lz along with the appropriate RTK/N cell fate code. It is thus inferred that the presence of Lz in R3/4 precluster cells is sufficient to endow them with the second wave cell fate response repertoire. A number of issues related to these observations and their interpretations are discussed (Mavromatakis, 2013).

Normal R3/4 precursors undergo an N-Dl interaction that results in the R4 precursor experiencing much higher levels of N activity than the R3 precursor. When Lz was supplied to R3/4 precursors (sev.lz), the cell in the R4 position frequently transformed into an R7, consistent with the requirement of high N for R7 specification. Less frequently, both R3/4 precursors adopted the R7 fate, and sometimes it was the cell in the R3 position alone that generated an ectopic R7. These results suggest that in the sev.lz flies the R3/R4 N-Dl interaction does not occur correctly. When the mδ0.5.lacZ reporter line was used as a reporter of N activity (which in wild-type larvae is robustly upregulated in R4 precursors) an erratic pattern was observed, sometimes showing the wild-type pattern, sometimes showing both R3/4 cells with high levels of lacZ expression, and sometimes showing R3 alone with high levels. Hence, by expressing Lz in the R3/4 precursors the cells were not only endowed with second wave response abilities but also they were prevented from executing their N-Dl interactions properly. Indeed, it was only when N was artificially activated to a high level with activated Notch (sev.lz; sev.N**) that the R7 fate was potently induced in both R3/4 precursors (Mavromatakis, 2013).

Native R7s critically require sev gene function; in its absence, they differentiate as cone cells. However, some ectopic R7s were able to differentiate when Lz was provided to the R3/4 precursors, even in the absence of sev (sev0; sev.lz), suggesting that normal R7 specification was not fully reiterated here. Examination of these eye discs suggested that some R3/4 precursors differentiated as R7s whereas others became cone cells. Thus, these cells appear to be on the cusp of the R7/cone cell fate choice, and some cells expressing markers for both cell types were observed. In the cells that became R7s, the presence was inferred of sufficient RTK activity, which was likely to have been supplied by endogenous DER signaling active in the precluster cells. Only when N activity was raised in these cells (sev0; sev.lz; sev.N*) did their full sev dependence for the R7 fate emerge, when all R3/4 precursors differentiated as cone cells (Mavromatakis, 2013).

The sev.N* construct is a very useful activator of the N pathway in developing eye cells. Since N activity drives sev expression, the sev.N* transgene feeds back on itself and promotes its own expression, and by subsequent iterations of this effect the cells are left with potent N activity. This level is still within the physiological range, unlike that produced using Gal4/UAS techniques, and is therefore the choice method for activating the N pathway. The transgene that was routinely use to knock down N activity [sev.Su(H)EnR] has the opposite effect; it reduces its own expression, and mildly compromises N activity. This level of reduction in N activity is usually sufficient to trigger major effects without the disadvantage of the severe downregulation that can accompany the use of Gal4/UAS technology. Since the sev.lz construct would also be downregulated by sev.Su(H)EnR, it was necessary to ectopically express Lz in the precluster using another enhancer element, and to this end the ro.Gal4 line was generated. When UAS.lz was expressed under ro control, the R3/4 precursors frequently differentiated as R7s, and crucially, when N activity was concomitantly reduced [ro.Gal4; UAS.lz; sev.Su(H)EnR] cells displaying R1/6 molecular features were now detected in the R3/4 precursors (Mavromatakis, 2013).

The cells in the R2/5 positions in ro.G4; UAS.lz developing ommatidia appear to develop normally; they express Elav and Ro, but none of the other fate markers. This suggests that R2/5 cells are insensitive to the presence of Lz, and argues that there is a major molecular difference between these cells and the R3/4 precursors. Also noteworthy is the transformation of all lz mutant second wave cells into R3/4 types characterized by the expression of Svp (a marker that is not expressed in R2/5 precursors) and Elav. Thus, it appears that ectopic Lz selectively transforms R3/4 precursors of the precluster to the second wave fate, and second wave cells lacking Lz adopt the R3/4 fate. Hence, it is suspected that Lz might not provide the intrinsic information that distinguishes the second wave cells from precluster cells per se, but rather distinguishes second wave cells from R3/4 types. Experiments to evaluate this view are currently being undertaken (Mavromatakis, 2013).

A counter-argument emerges from the fate of the majority of cells in the R3 positions in sev.lz eyes, which do not switch their fate. Only when N activity is activated or reduced in these cells is a change seen in their fates, and to be sure that the R2/5 cells are insensitive to Lz expression, it would also need to correspondingly vary N activity in them. Experiments to do this using ro.Gal4 produced severely disrupted preclusters, presumably as a result of interference with N function at earlier stages of precluster formation. Since these clusters were largely uninterpretable, the issue of whether R2/5 cells are insensitive to Lz expression remains unresolved (Mavromatakis, 2013).

For many years, the role of N in photoreceptor specification was confusing. In some contexts N appeared to oppose photoreceptor specification and in others N seemed to promote it, and this confusion prevented substantial progress in defining the fate codes that specified the different cell types. In recent work three distinct roles for N in this process were identified, and with that information the cell fate codes for R1/6, R7 and the cone cells were inferred. A major goal of this current work has to been to test this code by its reiteration in the R3/4 cells of the precluster using Lz expression to endow them with second wave cell qualities. In these experiments, each of the cell codes induced the expected cell fates, providing cogent support for the validity of the code (Mavromatakis, 2013).

DER is assumed to be ubiquitously expressed in the eye disc tissue, and its ligand, Spitz, diffusing from precluster cells, is thought to reach more distant cells with time. But the N and Sev signals are regulated in a different manner. Both their ligands are membrane bound, and their receptor activations only occur in immediate neighbors. Dl, the ligand for N, is expressed transiently by differentiating cells, and, accordingly, activates N in neighboring cells for only short periods of time (a few hours). sev is an N response gene and, in consequence, Sev is only expressed in cells for a short period of time. By contrast, Boss, the Sev ligand, is expressed for a prolonged period by the R8 precursor. Thus, both the N and Sev signaling systems are only available to the cells for restricted periods, with this restriction controlled by ligand expression in the N system and by receptor expression in the Sev system (Mavromatakis, 2013).

Although Lz is expressed in the second wave cells in a persistent manner in the eye disc, the experiments suggest that it, like the extrinsic signals, is required only for a brief developmental window. Consider the transformation of sev.lz R3/4 cells to the R7 fate. The sev enhancer is only active in these cells for a few hours and yet a complete transformation of the cells is achieved. The expression of specific cell type markers in the eye disc might erroneously indicate the transformation of a cell when only a transient effect occurs, but the presence of ectopic R7s in the adult retina argues otherwise and suggests that the transformations are potent and permanent. This view is further validated by the rescue of lz mutant second wave cells by the sev.lz transgene. This rescue is complete and is evident by the molecular markers expressed in the disc and by the morphology of the adult cells. Thus, it is inferred that Lz is only required during the same time window when the RTK/N signals are transduced, and it is further inferred that the combined activities of the RTK and N pathways, in concert with Lz, function in a short-lived manner to lock in the fate of the cells. How the presence of ephemeral extrinsic and intrinsic information is molecularly 'remembered' by the cells to allow their appropriate differentiation over a prolonged developmental period remains an intriguing question (Mavromatakis, 2013).


cDNA length - 3.5 kb

Exons - 6


Amino Acids - 828

The central domain of Lozenge contains a region homologous to AML1 (acute myeloid leukemia 1). This region includes the dimerization and DNA binding domain, as well as a putative ATP-binding site that is completely conserved among proteins of this group. There is a region of conserved amino acids of unknown function (VWRPY) at the C-terminus. In the homologous domain, LZ sequence has 71% identity to AML1 and 69% homology to Runt. The LZ sequence also possesses an alanine-rich stretch and a glutamine-rich region in the C-terminal portion of the molecule (Daga, 1996).

lozenge: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 5 MAR 97 

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.