Spineless mRNA staining first appears at embryonic stage 8 in a crescent just anterior to the cephalic furrow. This staining develops rapidly into an intense patch. As germ-band extension continues, staining develops in the maxillary, labial, and mandibular segments, followed by expression in a ventral patch in all three thoracic segments. These patches have been identified as the leg anlage by double-labeling for transcripts from Spineless and Aristaless. Spineless mRNA staining also appears in cells of the peripheral nervous system in each abdominal segment. The staining pattern is maintained through germ-band retraction and continues until the deposition of larval cuticle makes it difficult to follow further. To position the Spineless head patch, embryos were stained for both Spineless transcript and Engrailed protein. The posterior boundary of the Spineless antennal patch coincides precisely with the posterior edge of the antennal En stripe. There is a one-to-one correspondence between Spineless- and En-expressing cells for some distance along this stripe, although the En antennal stripe extends ventrally several cells beyond the Spineless patch. The anterior limit of Spineless expression lies just posterior to the En head spot, which delimits the posterior border of the ocular segment. Thus, Spineless mRNA is expressed throughout most or all of the embryonic antennal segment, and is expressed in a segmental, not parasegmental, register (Duncan, 1998).

The expression of spineless in embryos is unexpected, as no embryonic defects have been described for spineless mutants. However, examination of null mutant larvae reveals that the antennal sense organ is misshapen and often sclerotized. In addition, the dorso-medial papilla of the maxillary sense organ, which is thought to be derived from the antennal segment, fails to migrate completely. Examination of spineless mutant embryos stained with the monoclonal antibody 22C10 fails to reveal any defects in the peripheral nervous system (Duncan, 1998).


Spineless mRNA is expressed in the distal portion of the antennal imaginal disc, the tarsal region of each leg disc, and in bristle precursor cells. In leg discs, Spineless mRNA staining is first seen in the late second instar in a central ring that corresponds to the presumptive tarsal region. This ring is transient, and fades out by the late third instar. The Spineless tarsal ring likely corresponds to the tarsal structures deleted in spineless mutants, which include the distal part of the first tarsal segment and the second through fourth tarsal segments. After the tarsal ring fades out, Spineless mRNA becomes expressed in a patch in the anterior-proximal portion of the disc in a region that will give rise to structures of the thorax proper. spineless null alleles show no defects in these structures (Duncan, 1998). Spineless mRNA staining in the antennal disc is first detected during the late second instar. At all times after this, intense staining is seen in an oval patch in the central (distal) portion of the antennal disc. After disc eversion, the limits of intense Spineless mRNA expression coincide precisely with the boundary between the second (AII) and third (AIII) antennal segments. In spineless null mutants, the antenna shows both transformation to leg and tarsal deletion. In this case, the entire AIII segment and arista are affected. Strikingly, the AIII segment in spineless null mutants is unlike any normal appendage segment in that it completely lacks bristles or cuticular hairs. This segment is followed distally by most or all of a fifth tarsal segment terminated by claws (Duncan, 1998).

In the late third instar, spineless is expressed in a small patch in the antennal disc that corresponds to the maxillary palp anlage. Consistent with this, the maxillary palps of null mutants are truncated. This suggests a general requirement for spineless in the development of distal structures in ventral appendages (Duncan, 1998).

In the wing disc, spineless is expressed in the presumptive notum and wing hinge region, and in a ventral stripe. Perhaps related to this expression, the wings of null mutants are held perpendicular to the body and curve ventrally. The haltere disc stains in a similar pattern. Expression is also detected in the genital and labial discs, and in the morphogenetic furrow of the eye disc. Genital, labial, and eye development appear normal in spineless mutants (Duncan, 1998).

At pupariation and disc eversion, stereotyped patterns of single large intensely stained cells are seen in most discs, including the leg. The pattern of labeled cells is identical to that shown by the neuralized enhancer trap A101, indicating that these late spineless expressing cells are sensory organ precursors. At later stages, intense staining is seen in developing bristle cells, but not in the associated socket cells (Duncan, 1998).

Effects of mutation or deletion

Mutations in the spineless-aristapedia (ssa) gene of Drosophila are pleiotropic; their classical manifestations include a reduction in size of all bristles (spineless phenotype), transformation of distal parts of antennae into tarsal segments of the mesothoracic leg (aristapedia phenotype), and, in extreme alleles, fusion of tarsal segments on all six legs and the transformed aristaes. A new allele has been isolated, which is a severe loss-of-function mutation and, in addition to the above-mentioned features, is characterized by amplification of sex combs on the first leg. This phenotype can be caused by a change in the expression of the Sex combs reduced (Scr) gene of the Antp-C. Identification of this phenotype, together with observed variations in the extent of the fusion of tarsal segments in the legs of different segments, raises the possibility that ssa interacts with homeotic genes controlling the identity of segments. This possibility was tested in genetic experiments using flies with loss-of-function mutations in several homeotic genes and flies transformed by heat shock-driven homeotic genes. Analysis of adult phenotypes of different ssa alleles in the background of under-, over-, or ectopic expression of some genes of Bx-C and Ant-C suggests that the ssa product is required to prevent the effect of the homeotic gene products in the distal segments of the appendages (Kuzin, 1997).

The transformation of antenna to leg in Drosophila was carried out using ectopically expressed transgenes with heat shock promoters: heat shock Antennapedia, heat shock Ultrabithorax, and heat shock mouse Hox A5. The frequency of transformation of several leg markers was determined in response to Antennapedia protein delivered by heat shock at different times and doses. Stage-specific responses to the transgene, heat shock mouse Hox A5, were also studied. Each marker has its own stage and dose-specific pattern of response. The same marker can pass through a period of high-dose inhibition followed by a dose-independent response and then a positive dose-dependent phase. The heat shock-induced transgenes and spineless aristapedia transform the apterous enhancer trap antenna disc expression pattern toward the pattern found in leg discs. These results are considered in relation to developmental competence: the ability of developing tissue to respond to internal or external influences. The results suggest that all genes tested interact with the same competence system and that at least two classes of mechanisms are associated with antenna to leg transformation: one comprises global mechanisms that permit transformation over approximately 24 hr; the second class of mechanisms act very locally and are responsible for changes in dose response on the order of 4-8 hr (Larsen, 1996).

The Drosophila gene stubarista (sta) encodes the highly conserved putative ribosome-associated protein D-p40. sta maps to cytological position 2A3-B2 on the X chromosome and encodes a protein (D-p40) of 270 amino acids. D-p40 shares 63% identity with the human p40 ribosomal protein. P element-mediated transformation of a 4.4-kb genomic fragment encompassing the 1-kb transcript corresponding to D-p40 was used to rescue both a lethal (sta2) and a viable (sta1) mutation at the stubarista (sta) locus. Developmental analysis of the sta2 mutation implicates a requirement for D-p40 during oogenesis and imaginal development, which is consistent with the expression of sta throughout development. The basis of the sta1 visible phenotype which consists of shortened antennae and bristles has been analyzed. sta1 is a translocation of the 1E1-2 to 2B3-4 region of the X chromosome onto the third chromosome at 89B21-C4. Genetic evidence is provided that Dp(1;3)sta1 is mutant at the spineless (ss) locus and that it is associated with partial D-p40 activity. sta1 acts as a recessive enhancer of ss; reduction in the amount of D-p40 provided by the transposed X chromosomal region of sta1 reveals a haplo-insufficient phenotype of the otherwise recessive ss mutations. This phenomenon is reminiscent of the enhancing effect observed with Minute mutations, one of which, rp49, has previously been shown to encode a ribosomal protein (Melnick, 1993).

The homeotic mutation spineless-aristapedia (ssa) transforms the aristae into second tarsi. Flies with an ssa phenotype also show extremely positive geotaxis as measured in a Hirsch-type geotaxis maze. Other antennal mutants and flies with their aristae amputated do not show such extreme positive geotaxis. Deletion analysis has co-mapped the geotaxis effect with ssa in band 89C on the third chromosome. A biometrical analysis has detected additional genes on the X chromosome that also affect geotaxis (McMillan, 1992).

Loss-of-function mutations in the spineless-aristapedia gene of Drosophila (ssa mutants) cause transformations of the distal antenna to distal second leg, deletions or fusions of the tarsi from all three legs, a general reduction in bristle size, and sterility. Because ssa mutants are pleiotropic, it has been suggested that ss+ has some rather general function and that the ssa antennal transformation is an indirect consequence of perturbations in the expression of other genes that more directly control antennal or second leg identity. A test has been made of whether the ssa transformation results from aberrant expression of Antennapedia (Antp), a homeotic gene thought to specify directly the identity of the second thoracic segment. Antp-ssa mitotic recombination clones in the distal antenna behave identically to Antp+ ssa clones, and are transformed to second leg. This demonstrates that the ssa antennal transformation is independent of Antp+, and suggests that ss+ may itself directly define distal antennal identity. The results also reveal that Antp+ is not required for the development of distal second leg structures, as these develop apparently normally in Antp- ssa antennal clones. It is suggested that ss+ and Antp+ may play similar, but complementary, roles in the distal and proximal portions of appendages, respectively, because Antp- mutations cause deletions or transformations that are restricted to proximal structures, whereas ssa alleles cause similar defects that are distally restricted (Burgess, 1990).

A two-step screen for isolating null mutations of the spineless-aristapedia locus has been performed, and several amorphic mutations, as well as a small deficiency, have been obtained. With the exception of the deficiency, which deletes genes required for viability on either side of the spineless-aristapedia locus, these mutations result in a transformation of only the distal antenna into distal leg, thereby indicating that the spineless-aristapedia gene is required for specifying antennal, as opposed to leg development, in only the distal portion of the antenna. Because this distal region does not appear to be a developmental compartment, it is probable that the spineless-aristapedia gene, unlike several other homeotic genes, is required for maintaining the correct determined state in a population of cells defined by their relative position, not by their ancestry (Struhl, 1992).

The development of the sensory neuron pattern in the antennal disc of Drosophila melanogaster was studied with a neuron-specific monoclonal antibody (22C10). In the wild type, the earliest neurons become visible 3 h after pupariation, much later than in other imaginal discs. They lie in the center of the disc and correspond to the neurons of the adult aristal sensillum. Their axons join the larval antennal nerve and seem to establish the first connection towards the brain. Later on, three clusters of neurons appear in the periphery of the disc. Two of them most likely give rise to the Johnston's organ in the second antennal segment. Neurons of the olfactory third antennal segment are formed only after eversion of the antennal disc (clusters t1-t3). The adult pattern of antennal neurons is established at about 27% of metamorphosis. In the mutant lozenge3 (lz3), which lacks basiconic antennal sensilla, cluster t3 fails to develop. This indicates that, in the wild type, a homogeneous group of basiconic sensilla is formed by cluster t3. The possible role of the lozenge gene in sensillar determination is discussed. The homeotic mutant spineless-aristapedia (ssa) transforms the arista into a leg-like tarsus. Unlike leg discs, neurons are missing in the larval antennal disc of ssa. However, the first neurons differentiate earlier than in normal antennal discs. Despite these changes, the pattern of afferents in the ectopic tarsus appears leg specific, whereas in the non-transformed antennal segments a normal antennal pattern is formed. This suggests that neither larval leg neurons nor early aristal neurons are essential for the outgrowth of subsequent afferents (Lienhard, 1991).

Drosophila melanogaster females expressing the homoeotic mutation, spineless-aristapedia (ssa), were tested for their ability to hear the song of courting males. Since courtship song increases a females' receptivity to copulation, the frequency of mating within a short observation period was used as a measure of the ability of mutant females to distinguish between singing males and males that were unable to sing. These results show that ssa females, although lacking aristae, can distinguish between the two types of males in that they mated more readily with males that sang. Furthermore, the homoeotic legs of ssa females are not required to be present for the detection of courtship song, since females whose homoeotic legs were removed could still distinguish between singing and non-singing males (McRobert, 1991).

Segmentation is a developmental mechanism that subdivides a tissue into repeating functional units, which can then be further elaborated upon during development. In contrast to embryonic segmentation, Drosophila leg segmentation occurs in a tissue that is rapidly growing in size and thus segmentation must be coordinated with tissue growth. Segmentation of the Drosophila leg, as assayed by expression of the key regulators of segmentation, the Notch ligands and fringe, occurs progressively and this study defines the sequence in which the initial segmental subdivisions arise. The proximal-distal patterning genes homothorax and dachshund are positively required, while Distal-less is unexpectedly negatively required, to establish the segmental pattern of Notch ligand and fringe expression. Two Serrate enhancers that respond to regulation by dachshund are also identified. Together, these studies provide evidence that distinct combinations of the proximal-distal patterning genes independently regulate each segmental ring of Notch ligand and fringe expression and that this regulation occurs through distinct enhancers. These studies thus provide a molecular framework for understanding how segmentation during tissue growth is accomplished (Rauskolb, 2001).

A general theme in patterning during development is the subdivision of tissues initially by genes expressed in broad, partially overlapping domains, which through combinatorial control, subsequently regulate the expression of downstream genes to generate a repeating pattern. The studies presented here demonstrate that leg segmentation follows this same theme. The 'leg gap genes' Hth, Dac, and Distal-less are expressed in broad domains in the leg disc that encompass more than a single segment. Initially expression of these genes is largely nonoverlapping, but as the leg disc grows, the expression patterns of the leg gap genes change such that five different domains of gene expression are established. The analysis of the regulation of Notch ligand and fringe expression during leg development reveals two fundamental aspects of leg development. (1) These leg gap genes are key components in regulating the expression of the molecules controlling segmentation. Indeed, the effect of these leg gap genes on leg segmentation and growth can be accounted for by their regulation of Serrate, Delta and fringe expression. (2) The expression of each ring of Serrate, Delta and fringe is controlled by its own unique combination of regulators, apparently acting through independent enhancers (Rauskolb, 2001).

Most of the tarsus of the Drosophila leg derives from cells expressing Distal-less, but not Dac or Hth. Surprisingly, the studies presented here have shown that Distal-less actually represses Notch ligand expression. This negative regulatory role for Distal-less contrasts with the positive promoting role of Dac and Hth, and further indicates that a distinct molecular mechanism must promote segmentation within the tarsus. One key gene is spineless-aristapedia (ss), since simple, unsegmented tarsi develop in ss mutant flies. Moreover, ss regulates the expression of bric-à-brac (bab), which is also required for the subdivision of the tarsus into individual segments. Together, ss and bab must, in some way, ultimately overcome the repression of Notch ligand and fringe expression by Distal-less. If the sole function of ss and bab is to overcome the inhibitory effects of Distal-less, then in the absence of ss and/or bab, Serrate expression is expected to remain repressed (Rauskolb, 2001).

The Drosophila antenna is a highly derived appendage required for a variety of sensory functions including olfaction and audition. To investigate how this complex structure is patterned, the specific functions of genes required for antenna development were examined. The nuclear factors, Homothorax, Distal-less and Spineless, are each required for particular aspects of antennal fate. Coexpression of Homothorax, necessary for nuclear localization of its ubiquitously expressed partner Extradenticle with Distal-less is required to establish antenna fate. This study tests which antenna patterning genes are targets of Homothorax, Distal-less and/or Spineless. Antennal expression of dachshund, atonal, spalt, and cut requires Homothorax and/or Distal-less, but not Spineless. It is concluded that Distal-less and Homothorax specify antenna fates via regulation of multiple genes. Phenotypic consequences of losing either dachshund or spalt and spalt-related from the antenna are reported. dachshund and spalt/spalt-related are essential for proper joint formation between particular antennal segments. Furthermore, the spalt/spalt-related null antennae are defective in hearing. Hearing defects are also associated with the human diseases Split Hand/Split Foot Malformation and Townes-Brocks Syndrome, which are linked to human homologs of Distal-less and spalt, respectively. It is therefore proposed that there are significant genetic similarities between the auditory organs of humans and flies (Dong, 2002).

As with Dll and hth loss-of-function mutants, loss of spineless (ss) also results in antenna to leg transformations. The genetic relationship among these genes was investigated. The expression of both Dll and hth appears relatively normal in the ss null antennal disc. It is therefore concluded that ss is not required for either the activation or the maintenance of Dll or hth expression in the antenna. It has been reported that Dll is required for the antennal expression of ss. To test whether Hth is also required to activate antennal ss expression, the effect of ectopic hth was examined. Ectopic Hth where Dll is expressed, for example in the wing pouch and leg disc, can activate ss expression. Conversely, loss of hth in the antenna results in loss of ss expression. Taken together, these results indicate that ss functions downstream of both Dll and hth in the antenna (Dong, 2002).

ss is expressed in a circular pattern in the antenna covering the presumptive a2 through the arista. In the leg disc, ss is transiently expressed in a ring pattern in the presumptive tarsal region and subsequently becomes restricted to leg bristle precursors. Consistent with the ss expression domain, cuticular defects in ss null mutants can be found from a2 through the arista. These include the elongation of a2, loss of olfactory sensilla from a3, and transformation of a4, a5, and arista to tarsal segments (Dong, 2002).

The large differences in the expression patterns of these genes between the antenna and the leg begs the question of whether these differences are due to differential regulation by antenna-determining genes such as Dll and hth. To test whether Dll or hth are responsible for the antenna-specific expression patterns of these genes, the effects on their patterns were examined in Dll and hth loss-of-function mutants. Whether Dll and hth are regulating their antenna-specific targets via ss was tested by examining their expression in ss null antennal discs (Dong, 2002).

The antennal dac expression domain expands in Dll hypomorphs and in hth null clones. This expansion of dac expression in Dll and hth mutant antennae resembles the leg pattern of dac expression. In contrast, in the ss null antenna, there appears to be neither expansion nor reduction of dac expression. The only detectable difference in the ss null antennal disc is overgrowth in the central (distal) area such that the ring of dac expression has a larger radius. This correlates with the transformation phenotype of the ss null arista into a tarsus, which is a larger structure. Since the expression of dac relative to other genes appears normal in ss null antennae, ss is not thought to regulate dac (Dong, 2002).

The expression of ato is required for the formation of the JO. The JO is a structure unique to the antenna and is required to sense sound vibrations transmitted from the arista. ato function is generally associated with neuronal differentiation, so it is interesting that cuticular defects are associated with ato null antennae. It may be that formation of the JO is required for the normal morphology of the a2/a3 joint. The circular outline of the a2/a3 joint is lost in hth and Dll loss-of-function mutants, but is present in ss null mutants. Consistent with this, the antennal expression of ato is lost in hth null clones and in Dll hypomorphs, but persists in the ss null antenna discs. Thus although ss null mutants exhibit cuticular defects in a2 and a3, the a2/a3 joint to which the JO is attached is present. It is noted that the Dll hypomorphic combination used, DllGAL4/Dll3, does not lead to loss of a2. Thus the absence of ato expression in these antennae is not due to death of the cells that would normally express it (Dong, 2002).

Dll and hth are required for the expression of sal in the antenna. sal expression does not appear to be affected in ss null antenna. The fact that Ss is not required for the expression of either ato or sal in a2 is consistent with the observation that the a2/a3 joint is still present in the ss null antenna (Dong, 2002).

This study serves to initiate an understanding of the different roles that these homeotic genes are playing in antenna specification. During imaginal disc development, the expression of Dll and ss is found from a2, a3, a4, a5 and arista. Expression of hth is dynamic and retracts from the distal-most segments by late third instar, but hth is expressed and cell-autonomously required throughout the antenna from a1 through to the arista (Dong, 2002).

Since ss is not required to activate antenna-specific expression of genes such as sal/salr and ato that are involved in antenna differentiation, the question arises as to what ss does do in the antenna. ss represses tarsus and tarsal claw organ formation in the antenna. Since loss of ss also leads to loss of olfactory sensillae on a3, ss probably potentiates the formation of these sensillae, either cooperating with or mediating Dll and hth activities in a3. Similarly, since ectopic expression of ss elsewhere in the body can lead to the formation of ectopic aristae, ss may also cooperate with or mediate Dll and hth activities in arista differentiation (Dong, 2002).

sal and salr, like ato, are required for normal auditory functions. Since both Dll and hth are required for the antennal expression of ato and sal, Dll and hth mutant antennae are also hearing defective. In contrast, ss null antennae exhibit normal expression of both ato and sal and normal morphology of the a2/a3 joint, leading to the idea that ss mutants are likely to be functional in audition (Dong, 2002).

Stochastic spineless expression creates the retinal mosaic for colour vision

Drosophila colour vision is achieved by R7 and R8 photoreceptor cells present in every ommatidium. The fly retina contains two types of ommatidia, called 'pale' and 'yellow', defined by different rhodopsin pairs expressed in R7 and R8 cells. Similar to the human cone photoreceptors, these ommatidial subtypes are distributed stochastically in the retina. The choice between pale versus yellow ommatidia is made in R7 cells, which then impose their fate onto R8. The Drosophila dioxin receptor Spineless is both necessary and sufficient for the formation of the ommatidial mosaic. A short burst of spineless expression at mid-pupation in a large subset of R7 cells precedes rhodopsin expression. In spineless mutants, all R7 and most R8 cells adopt the pale fate, whereas overexpression of spineless is sufficient to induce the yellow R7 fate. Therefore, this study suggests that the entire retinal mosaic required for colour vision is defined by the stochastic expression of a single transcription factor, Spineless (Wernet, 2006).

The ability to discriminate between colours has evolved independently in vertebrates and invertebrates. However, despite the obvious differences in eye development and design, both flies and humans have developed retinal mosaics where classes of photoreceptor cells (PRs) with different spectral sensitivity are randomly distributed. The compound eye of Drosophila consists of ~800 optical units (ommatidia), each containing eight PRs in addition to accessory cells. In each ommatidium, the six 'outer PRs' (R1-R6) function like the vertebrate rod cells, as they are required for motion detection in dim light. These cells express the broad-spectrum rhodopsin, Rh1. The 'inner PRs' (R7 and R8) may be viewed as the equivalent of the colour-sensitive vertebrate cone cells, which express a range of different rhodopsin molecules (Wernet, 2006).

The general rule of sensory receptor exclusion also applies to Drosophila ommatidia, where only one rhodopsin gene is expressed by a given PR. The expression of inner PR rhodopsins can be used to distinguish three ommatidial subtypes. Two of the subtypes are distributed randomly throughout the retina: ~30% of ommatidia express ultraviolet-sensitive Rh3 in R7 cells and blue-sensitive Rh5 in R8 cells, and therefore are specialized in the detection of short wavelengths ('pale' ommatidia). The remaining ~70% express another ultraviolet-sensitive opsin (Rh4) in R7 and green-sensitive Rh6 in R8, making them more responsive to longer wavelengths ('yellow' ommatidia). The coupled expression of Rh3/Rh5 or Rh4/Rh6 within the same ommatidium results from communication between R7 and R8. In the dorsal rim area (DRA), a third type of ommatidia exists in which both R7 and R8 express ultraviolet-sensitive Rh3. These ommatidia are used to detect the e-vector of polarized sunlight for orientation. Spatially localized polarized light detectors and stochastically distributed colour-sensitive ommatidia therefore reflect two fundamentally different specification strategies that shape the retinal mosaic of Drosophila (Wernet, 2006).

The current model for specifying colour-sensitive ommatidia combines stochastic and instructive steps. First, a subset of R7 (pale R7, pR7) stochastically chooses Rh3 expression over the 'R7 default', Rh4. Second, these cells then impose the p fate (Rh5) onto R8 (pale R8, pR8) of the same ommatidium (Wernet, 2006).

This study reports the identification of spineless (ss) as a key regulatory gene for establishing the retinal mosaic. ss encodes the Drosophila homologue of the human arylhydrocarbon ('dioxin') receptor, a member of the bHLH-PAS (basic helix-loop-helix- Period-Arnt-Single-minded) family of transcription factors. At mid-pupation, ss is stochastically expressed in a majority of R7 that seem to correspond to the y subtype. ss is both necessary and sufficient to specify the yellow R7 (yR7) fate and subsequently the entire y ommatidia; pR7 cells are thus specified by default, and stochastic expression of ss represents the key regulatory event defining the retinal mosaic required for fly colour vision (Wernet, 2006).

homothorax (hth) has been identified as the key regulatory gene necessary and sufficient for the specification of DRA ommatidia. ss and hth cause similar homeotic phenotypes: that is, complete (hth) or partial (ss, 'aristapedia') transformation of antennae into legs. Therefore, a potential role of ss in ommatidial subtype specification was tested by generating whole-mutant eyes, as well as mitotic clones, lacking ss function using the null allele ssD115.7 and the ey-FLP/FRT technique. Owing to ey-FLP expression in the antennal imaginal disc, ss mutant flies showed a strong aristapedia phenotyp, but lacked any obvious morphological eye phenotype. However, expression of rhodopsin genes was severely affected. In wild-type eyes, Rh3 is found in ~30% of R7 cells, as well as in both R7 and R8 of DRA-ommatidia, whereas the remaining ~70% of R7 contain Rh4. In ss mutant eyes, Rh4 was completely absent, whereas Rh3 was expanded into all R7 cells. The total number of ommatidia was not reduced, indicating that R7 cells were mis-specified into pR7, rather than yR7 being specifically eliminated. ss mutant mitotic clones were morphologically wild type; however, Rh3 was always present in mutant R7 cells (marked by the absence of ß-galactosidase (ß-gal) expression), whereas Rh4 was always lost (Wernet, 2006).

To test whether the R7 ss phenotype was cell autonomous, individual mutant R7 cells were generated using the MARCM technique. All mutant R7 cells [marked by the presence of green fluorescent protein (GFP) expression] contained Rh3 and never Rh4, demonstrating that ss is required cell autonomously in R7 to induce Rh4 expression. DRA ommatidia were correctly specified in ss mutant eyes, since Rh3 was expressed normally in both DRA R7 and R8 cells. Therefore, ss is necessary for the establishment of the yR7 subtype without affecting PR fate specification (Wernet, 2006).

The ommatidial subtypes are first specified in R7, which then instruct R8. Therefore, ss mutant eyes should exhibit a rhodopsin phenotype in R8. In wild types, ~30% of R8 cells contain Rh5, and the remaining ~70% contain Rh6. In ss mutant eyes, the large majority (up to 95%) of R8 contained Rh5, with some R8 still containing Rh6. However, most of these remaining yR8 cells were located in the dorsal third of the eye. In this part of the retina, instruction of pale R8 (pR8) by pR7 is less efficient, resulting in ommatidia with odd-coupled (Rh3/Rh6) rhodopsin expression. In ss mutants, the frequency of such ommatidia was significantly increased in the dorsal region. To test whether the R8 opsin phenotype of ss mutants resulted from the inability of some mutant R7 cells to properly instruct R8, rather than from ss being directly required in R8, sevenless; spineless (sev; ss) double-mutant eyes were generated (Wernet, 2006).

These eyes, which lacked R7 cells, always exhibited the sev single-mutant phenotype, with virtually all R8 cells containing Rh6. This indicates that ss is required in R7 for the formation of the yR7 subtype, and consequently for the formation of yR8, without being directly required in R8 PRs (Wernet, 2006).

Whether ss was also sufficient to induce the y ommatidial subtype was tested. Overexpression of ss in all developing PRs using a strong LGMR (long glass multiple reporter)-Gal4 driver and UAS-ss (LGMR.ss flies) resulted in a rough eye phenotype, as well as a dramatic rhodopsin phenotype: Rh4 was activated in all PRs throughout the eye (R1-R6 as well as R7 and R8), as revealed by ectopic expression of an Rh4-GFP reporter in many PRs per ommatidium compared with wild type. To avoid the strong phenotype in the eye, ss was misexpressed using the weaker, variegated GMR driver, sGMR (short GMR)-Gal431 (sGMR.ss flies). This led to strong ectopic induction of Rh4 in many PRs without severely affecting retinal morphology. This ectopic induction of Rh4 was also observed in sev mutants, and was thus independent of R7. Rh3 was still detected in some R7 in sGMR.ss flies, presumably due to the lack of variegated Gal4 expression in these cells, whereas Rh4 was expanded to some outer PRs. However, co-localization of Rh3 and Rh4 was never observed, confirming that gain of Rh4 in R7 cells always leads to the exclusion of Rh3. In contrast, gain of Rh4 in outer PRs did not lead to the exclusion of Rh1; frequent coexpression of Rh1 and Rh4 was observed (Wernet, 2006).

Using an Rh4-lacZ reporter construct in LGMR.ss flies, it was found that ß-gal-positive PR axons projected to both lamina and medulla, confirming the expansion of Rh4 into outer PRs. However, Rh4-expressing outer PRs were not transformed into genuine R7 cells, since they maintained their lamina projections. Notably, DRA inner PRs were the only cells not expressing Rh4, suggesting that the DRA fate, specified by the gene hth, antagonizes ss function. Expression of Rh3 and Rh5 was completely lost (including in the DRA, where no rhodopsin was detected), while Rh6 expression was found in most R8 cells. This resulted in R8 coexpressing Rh4 and Rh6, demonstrating that the 'one sensory receptor per cell' rule can be broken in Drosophila PRs, as has been shown in other insects. Therefore, ectopic induction of the yR7 fate by ss specifically excludes the formation of pR7 cells. As a consequence, R8 cells expressing Rh5 are not induced, with most R8 expressing Rh6. Rh6 was never found in outer PRs, supporting the hypothesis that ss is required only in R7 for the choice between Rh3 and Rh4, and not directly in R8 for the Rh6 choice. In LGMR.ss flies, the specification of outer versus inner PRs (markers spalt and seven up) or of R7 versus R8 (prospero and senseless) was normal. Thus, ss acts by segregating ommatidial subtypes downstream of early PR specification events (Wernet, 2006).

Colour PR cell fate determination seems to be a late event in PR differentiation. To test whether ss can transform the R7 fate at late stages of development, the PanR7-Gal4 driver (which is also expressed in DRA R8 cells) was used. Late mis-expression of ss induced the y fate (Rh4) in all R7 cells, whereas Rh3 was absent. Opsin expression in the DRA was also altered, with Homothorax-positive cells (both R7 and R8) now expressing Rh4. Hence, it is possible to reprogramme the R7 fate at later stages of differentiation, as PanR7-Gal4 becomes activated at the time of rhodopsin expression. Surprisingly, expression of R8 rhodopsins outside the DRA was not affected, since the distribution of Rh5 and Rh6 resembled the wild type. As a result, many ommatidia manifested the very unusual coupling of Rh4 in R7 and Rh5 in R8. Therefore, although ss is able to reprogramme all R7 late in development, R8 cannot revert their fate once they have been instructed to become pR8, and they maintain Rh5. Two antagonistic genes expressed in either of the two R8 subtypes have been identified that act together as a molecular consolidation system responsible for this inertia of R8. To confirm that late expression of ss exclusively in R7 is sufficient to transform R7, ss was mis-expressed in ssD115.7 mutants using PanR7-Gal4. This was sufficient to induce Rh4 and to repress Rh3. R8 were again not reprogrammed and exhibited the ss mutant phenotype, with many R8 cells expressing Rh5 (Wernet, 2006).

All of the results presented above strongly indicated that ss must be expressed in the y subtype of R7 at some point during pupal development. Since several attempts to generate an anti-Spineless antibody had failed, in situ hybridization was used to detect ss messenger RNA in the retina at mid-pupation. At ~50% pupation, ss mRNA was detected in four neuronal cells per ommatidial cluster, one PR and three bristle cells. The PR was also labelled by anti-Prospero, confirming its identity as R7. Although the expression levels of ss in bristle cells seemed uniformly high, levels of ss expression varied considerably among R7 cells, ranging from very faint to very strong in 60%-80% of R7. A 1.6 kilobase 'eye enhancer' fragment (sseye) was also identified within the ss promoter that drives PR-specific expression. After crossing ss eye-Gal4 to UAS-ß-gal::NLS (nuclear localization sequence) reporters, PR-specific ss expression was first detected at mid-pupation-that is, approximately one day before rhodopsins are expressed, and before any visible molecular or morphological distinction between ommatidial subtypes. A single PR per ommatidium, which was identified as R7 through co-staining with Prospero, expressed ss. Thus, the ss eye enhancer recapitulates endogenous ss expression in PRs. ss expression was detected in 60%-80% of R7, correlating well with the distribution of Rh4 in adult retina. Like Rh4-expressing ommatidia, ß-gal-positive ommatidia were more abundant in the dorsal half of the eye, and no ß-gal expression was detected in the DRA (marked by Homothorax), where Rh4 is also never expressed. ss eye-Gal4 expression was detectable for only ~2 h at midpupation (Wernet, 2006).

Although it was not possible to directly co-stain for ss and Rh4 (which starts to be expressed one day later during pupation), it seems that at mid-pupation a short pulse of ss is deployed in a large subset of R7, which will become yR7 (Wernet, 2006).

Whether a short pulse of ectopic ss expression was able to modify the entire retinal mosaic was tested using a heat shock-Gal4 driver (hs-Gal4) to temporally control ss expression (hs.ss flies). A 30-min heat shock at ~50% pupation indeed resulted in an increase of Rh4 expression with a concomitant reduction of Rh3 in adults. The phenotype varied extensively, from only R7 cells expressing Rh4 (~25% of the flies analysed had Rh4 in most R7), to almost every PR expressing Rh4. In contrast, a 30-min pulse of ss in one-day-old adult flies had no effect. Heat shocks during larval or early pupal stages were lethal. Thus, PRs are extremely sensitive to a short pulse of ss during mid-pupation, at the time when endogenous ss is normally expressed. To further study the mechanism of the stochastic choice between p and y ommatidia, the retinal mosaic was examined in different mutant backgrounds. Flies heterozygous for ssD115.7 had fewer Rh4-expressing R7 cells. Since the ssD115.7 allele affects only the ss coding sequence, heterozygous flies have two functional promoters, only one of which produces a functional protein, suggesting that the non-productive promoter might sequester limiting factor(s) that regulate(s) the expression levels of ss. If this hypothesis is correct, addition of extra copies of the ss promoter should have a similar effect. Indeed, the addition of two functional copies of the ss eye enhancer (ss eye-Gal4) in an otherwise wild-type background also caused a significant reduction of the yR7 subtype. Therefore, the level of Spineless expression is important for the induction of the yR7 fate, which is less efficient in cells where the amount of Spineless is reduced (Wernet, 2006).

Retinal patterning in Drosophila reveals an original mechanism for how PR mosaics can be generated: stochastic expression of a single transcription factor (Spineless) acts as a binary switch that transforms the seemingly homogeneous compound eye into a mosaic, distinguishing p and y subtypes. However, subtype specification and rhodopsin expression can be separated, since ss expression in yR7 has ceased well before the time of rhodopsin expression. Additional factors are therefore required downstream of ss to ensure expression of adult p- and y-specific markers such as rhodopsins and additional screening pigments. A revised two-step model is proposed for the stochastic specification of p and y ommatidia. First, R7 are stochastically divided into two subtypes by the induction of ss in yR7. ss-positive R7 express Rh4, whereas the remaining R7 choose the pR7 fate and express Rh3 by default. Second, only those R7 cells that did not express ss (pR7) retain the ability to induce the pR8 fate (Rh5), whereas yR8 express Rh6 by default. The 'default states' of R7 (Rh3) and R8 (Rh6) therefore belong to opposite subtypes. Expression of R8 rhodopsin genes is maintained by a bistable regulatory loop containing the genes warts and melted34. Notably, the localized specification of polarization-sensitive DRA ommatidia by hth antagonizes the stochastic choice executed by ss, placing these two genes into a new regulatory relationship during retinal patterning. Therefore, the role of the transcription factor Spineless is to generate the retinal mosaic required for fly colour vision by distinguishing yR7 from pR7 cell fates, and preventing R7 from instructing the underlying R8 cells. Mosaic expression of sensory receptors has been described in detail for the olfactory system of both vertebrates and insects, and random PR mosaics have been described for humans and amphibians, as well as insects. Two transcription factors have been shown to regulate the specification of blue versus red/green cone cell fates in mammals. Upon mutation of either - the human nuclear receptor NR2E3 (also known as PNR) or the rodent thyroid hormone ß2 receptor - the number of blue cones is dramatically increased at the expense of green cones, leading to 'enhanced S-cone syndrome'. It should be noted that this retinal phenotype bears important similarity to the altered ommatidial mosaic in Drosophila ss mutants, where long wavelength-sensitive y ommatidia are lost at the expense of the short wavelength-sensitive p type (Wernet, 2006).

The stochastic cell fate choice occurs at the level of the ss promoter: the very short pulse of ss expression at mid-pupation is not only controlled temporally, but its levels are also critical, and only ~70% of R7 receive enough Spineless to commit to the yR7 fate. Elucidating the mechanism that controls ss expression will shed some light into the fascinating process of stochastic gene expression, and the identification of its downstream targets will provide insights into consolidation and maintenance of cell fates (Wernet, 2006).

The bHLH-PAS protein Spineless is necessary for the diversification of dendrite morphology of Drosophila dendritic arborization neurons

Dendrites exhibit a wide range of morphological diversity, and their arborization patterns are critical determinants of proper neural connectivity. How different neurons acquire their distinct dendritic branching patterns during development is not well understood. This study reports that Spineless (Ss), the Drosophila homolog of the mammalian aryl hydrocarbon (dioxin) receptor (Ahr), regulates dendrite diversity in the dendritic arborization (da) sensory neurons. In loss-of-function ss mutants, class I and II da neurons, which are normally characterized by their simple dendrite morphologies, elaborate more complex arbors, whereas the normally complex class III and IV da neurons develop simpler dendritic arbors. Consequently, different classes of da neurons elaborate dendrites with similar morphologies. In its control of dendritic diversity among da neurons, ss likely acts independently of its known cofactor tango and through a regulatory program distinct from those involving cut and abrupt. These findings suggest that one evolutionarily conserved role for Ahr in neuronal development concerns the diversification of dendrite morphology (Kim, 2006).

The ss protein is present at nearly the same level in all da neurons and acts cell-autonomously to dictate their dendritic complexity, while different da neurons exhibit different sensitivity to the level of Ss, and even the bipolar td neuron can respond to elevated ss activity by increasing dendritic complexity (Kim, 2006).

Previous studies in C. elegans have demonstrated essential roles for invertebrate homologs of Ahr in neuronal cell fate determination. For example, ahr-1 regulates the differentiation program of a subclass of neurons that contact the pseudocoelomic fluid, and both ahr-1 and aha-1 specify GABAergic neuron cell fate in C. elegans. The dramatic changes in the dendrite morphologies of the da neurons, however, are not due to an all-or-nothing change in cell fate because the da neurons in ss mutants displayed normal class-specific expression patterns of the molecular markers Ab and Cut and normal axon projection patterns characteristic of individual da neurons. However, this also does not assume that a partial cell fate change has not occurred. One reflection of the ss function as a transcription factor is the altered expression levels of GFP in the class I Gal4221 reporter, with increased levels of expression in all class IV neurons and essentially no expression in the dorsal class I neuron ddaD and the ventral class I neuron vpda in ss mutants. It will be of interest to further characterize the genetic basis for this Gal4 reporter, to determine whether this regulation constitutes a partial cell fate alteration or transcriptional regulation of genes downstream from ss in the execution of adjustment of dendritic complexity (Kim, 2006).

There is an emerging theme that ss functions to diversify neuronal differentiation by expanding the photopigment repertoire of R7 photoreceptors in the Drosophila eye and by diversifying da neuron dendritic morphologies. Recent studies have demonstrated that the entire retinal mosaic pattern required for color vision in Drosophila is regulated by ss. In the Drosophila retina, two types of ommatidia form the wild-type retinal mosaic: 'pale' and 'yellow.' In ssD115.7 mutants, the yellow ommatidial subtype is lost and normally yellow R7 cells are misspecified into the pale subtype. As a result, nearly all R7 cells adopt the pale subtype, leading to loss of the retinal mosaic pattern. Thus, the pale R7 subtype represents the R7 'default state' (Kim, 2006 and references therein).

The overall lack of dendritic diversity in the da neurons in ss mutants is suggestive of the hypothesis that ss, an ancient, evolutionarily conserved gene, may act to convert a primordial dendrite pattern (perhaps a default state) to different complexities for different neurons in the peripheral nervous system. The loss-of-function ss phenotype in the da neuron dendrites might reflect such a primordial pattern as the dendrites in the mutant are devoid of specific morphological features that define distinct neuronal subclasses. In support of this notion, dendrites of the different classes of da neurons share similar morphological characteristics and elaborate similar numbers of total branches in ss mutants. The ability of ss to regulate the complexity and diversity of this dendrite pattern, by limiting dendritic branching to shape the simpler arbors of the class I and class II neurons and by promoting class-specific terminal branching to shape the more complex arbors of the class III and class IV neurons, is quite unique. Of the many mutants that affect multiple classes of da neurons, the great majority affect da neurons with simple or complex dendritic arbors the same way; that is, causing them to all become simpler or more complex. The ss phenotype of making simple dendritic arbors more complex and complex arbors simpler is very unusual among the many mutants affecting dendrite complexity. It thus seems likely that the distinct dendritic patterns rely not only on a cohort of gene activities specifying the mechanics of dendrite outgrowth and branching, but also a genetic program that diverts the generic primordial mode of dendritic formation to a diverse range of dendritic patterns (Kim, 2006).

How might spineless exert its functions? Unlike the homeodomain protein Cut, which promotes dendritic complexity in a specific direction, ss functions in an opposing manner in different cell types to regulate dendritic diversity. How might ss function differently in different neuronal cell types? One possibility is that ss is activated by different ligands in different neurons. ss is incapable of binding dioxin and other related compounds, suggesting that other, as yet unidentified ligands are required for its activation. Previous reports have suggested that ss and other invertebrate homologs of Ahr are activated by an endogenous ligand or that no ligand is required at all. Recent studies have shown that Ahr can accumulate in the nucleus upon activation by the second messenger cyclic AMP (cAMP), although it is not yet known whether cAMP signaling can activate ss in Drosophila. Thus, it is conceivable that ss is activated by different upstream factors in different cell types. It will be of interest to test in future studies whether in different neuronal cell types ss is activated by different ligands or upstream second messengers and whether ss acts in concert with regulatory programs for cell fate specification to dictate dendritic complexity (Kim, 2006).

In the canonical Ahr signaling pathway, Ahr requires the appropriate cofactor for its proper function. Members of the bHLH-PAS protein family are able to heterodimerize with other bHLH-PAS proteins. Previous studies have shown that, upon ligand binding, Ahr is translocated to the nucleus, where it heterodimerizes with Arnt to form a transcriptionally active complex. However, tango, the Drosophila homolog of Arnt, is likely not required for the regulation of dendritic morphogenesis, indicating that ss is probably not acting through its canonical signaling pathway to specify dendritic complexity. In Sf9 cells, ss can act independently of tgo to enhance expression of a reporter in the absence of a ligand. Furthermore, Ahr is unable to interact with Arnt upon activation by cAMP. Although Ahr, Arnt, and the Arnt homolog Arnt2 are widely distributed throughout the rat brain, Ahr does not preferentially colocalize with either Arnt or Arnt2. Ahr is also expressed in specific regions of the rat brain where neither Arnt nor Arnt2 is expressed. These studies support the notion that ss can act independently of tgo in certain developmental contexts. Tgo can heterodimerize with other bHLH-PAS proteins in addition to ss. It is conceivable that ss may act with different heterodimerization partners to mediate its different functions in different cell types (Kim, 2006).


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

date revised: 5 August 2016

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