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

Gene name - spineless

Synonyms - spineless-aristapedia (ssa)

Cytological map position - 89C1--89C4

Function - transcription factor

Keywords - antenna, legs

Symbol - ss

FlyBase ID: FBgn0003513

Genetic map position - 3-58.5

Classification - bHLH PAS domain protein

Cellular location - presumably nuclear, possibly also cytoplasmic

NCBI links: Precomputed BLAST | Entrez Gene

Originally reported by Bridges in 1914, spineless plays a central role in defining the distal regions of both the antenna and leg. spineless encodes the closest known homolog of the mammalian dioxin receptor, a transcription factor of the bHLH-PAS family. Loss-of-function alleles of spineless cause three major phenotypes: transformation of distal antenna to leg, deletion of distal leg (tarsal) structures, and reduction in size of most bristles. Consistent with these phenotypes, spineless is expressed in the distal portion of the antennal imaginal disc, the tarsal region of each leg disc, and in bristle precursor cells (Duncan, 1998).

Is there a connection between the antennal and leg transformation, that is some evolutionary or developmental logic to this phenomenon? The following essay explores this question. The deeper question of the relationship between the function of spineless in insect morphogenesis and its relation to the function of the dioxin receptor in vertebrates, is not be dealt with here.

Whenspineless was ectopically expressed in Drosophila, a few flies survived to the pharate adult stage. In this way, the effects of ectopic spineless expression on adult structures have been studied. The results indicate that spineless is a primary determinant of distal antennal identity. Ectopic expression causes transformation of the maxillary palp and distal leg to distal antenna. Transformation of the maxillary palps varies from essentially no effect to an almost complete transformation to the third antennal segment and arista (bristle like structures at the tip of the antenna). Palps are also often deleted. Surprisingly, ectopic spineless induces ectopic antennal structures in the rostral membrane between the maxillary palp and the normal antenna. These range from small patches of third antennal segment and arista to entire ectopic antennae, and are always arranged in mirror symmetry to the normal antenna. Ectopic spineless expression also causes transformation of the distal leg to arista. In some cases, aristal-claw intermediates are present, indicating that aristae can arise by transformation of claws. More proximal antennal structures are never present in the leg. Ectopic expression of spineless also causes the deletion of medial leg structures. More distal tarsal segments are usually unaffected, with the exception of transformation of claw to arista. Ectopic spineless expression also causes deletion of the central part of the wing, and induces a zone of polarity reversal in the abdominal tergites. A few scattered bristles are also induced in the wing blade (Duncan, 1998).

Ectopic expression of the Antennapedia gene can cause a complete transformation of distal antenna to second leg. It was suspected that the loss of function spineless mutation that caused antennal transformation to leg might have resulted from the ectopic activation of Antp+. To test this, dual mutant Antp;spineless clones were studied in the distal antenna. Surprisingly, these are indistinguishable from Antp+;spineless clones, and are transformed to second leg tarsus. This demonstrates that the mutant spineless antennal transformation is independent of Antp+, and leads to the belief that spineless controls distal antennal identity directly (Duncan, 1998).

In addition to specifying the distal antenna, spineless also specifies the tarsus. The tarsus appears to develop without input from the Antennapedia complex and bithorax complex genes. spineless is first expressed in the tarsal region in the late second instar. Staining increases in this region through the early third instar, and then gradually declines. As far as is known, spineless is the only disc-patterning gene that shows such transient expression. Forcing the tarsal segment to undergo premature metamorphosis results in an unsegmented tarsal region. Thus, it would appear that tarsal development occurs in two phases: first, a uniform tarsal region is established, and then this region is subdivided into specialized segments. It has been suggested that spineless is responsible for the first of these phases, whereas downstream genes are responsible for the second. The timing of maximal spineless expression, a temperature-sensitive period for spineless function, and the finding that spineless expression is not segmentally modulated within the tarsal ring are all consistent with this view (Duncan, 1998).

It is generally considered that the arthropod antenna evolved from a leg-like locomotory appendage, a view that has received support from work on Drosophila homeotic and segmentation genes. It is also generally accepted that unsegmented tarsi are ancestral in the hexapods, because simple tarsi resembling those present in spineless mutant Drosophila occur in crustaceans and primitive hexapods. Thus, both the transformation of antenna to leg and the tarsal deletions caused by spineless mutations appear to be atavistic (representing an ancestoral form), suggesting that spineless played an important role in the evolution of distal limb structures in the arthropods. Because antennal specialization occurred very early in arthropod evolution, it is likely that the first function of spineless was antennal specification, and that spineless was recruited into tarsal development much later, during the evolution of the hexapods. Antennae are often elongated appendages used for probing the environment, and it is plausible that as part of antennal specification, spineless came to have an appendage elongation function. Transient expression of this function could then have served to extend other limbs, including the legs. This evolutionary sequence predicts that spineless homologs will prove to be expressed in the antenna, but not the legs, of crustaceans and primitive hexapods, but in both locations in the insects (Duncan, 1998).

Mutations in the clustered homeotic genes (HOM-C genes) can cause specific homeotic transformation, suggesting that the HOM-C genes determine segmental identities by acting on different target genes. However, misexpression of several HOM-C genes in the antenna disc causes similar antenna-to-leg transformations. No HOM-C genes are normally expressed in the eye-antenna disc proper. It has been considered that Antp, when ectopically expressed in the eye-antenna disc, suppresses an antenna-determining gene. This study shows that Scr, Antp, Ubx, and abd-A HOM-C genes all exert their effects through a common mechanism: suppression of the transcription of the homothorax (hth) homeobox gene, thereby preventing the nuclear localization of the Extradenticle homeodomain protein. If hth is a key effector suppressed by these four HOM-C genes, addition of hth should reverse the antennal transformations. Coexpression of the hth and HOM-C genes completely or partially reverts the transformation phenotype. It is noted, however, that suppression of hth is probably not the only effect of HOM-C expression in the antenna disc, since Scr, Antp, and Ubx each induce the antenna to transform into leg, showing different segmental characters (i.e., thoracic 1, thoracic 2 and thoracic 3 legs, respectively). Ectopic hth expression can cause duplication of the proximodistal axis of the antenna, suggesting that it is involved in proximodistal development of the antenna (Yao, 1999).

A possible mechanism for the suppression of hth by different HOM-C proteins assumes that the HOM-C proteins compete with a factor required for hth transcription. One candidate protein that fits all of these criteria is Hth itself. The gene spineless exhibits a similar antenna-determining function. It is possible that hth and spineless represent separate pathways specifying antennal identity. Since hth and ss are expressed in the leg discs as well as in the antenna discs, it is not their simple presence that determines antennal identity. What then distinguishes the antenna vs. the leg? One possibility is that the detailed spatial and temporal expression pattern makes the difference. The broader expression pattern of hth in the antenna disc may distinguish the antenna from the leg. It is also possible that the level of spineless makes a difference: high levels of ss correlate with antennal identity and low levels of ss correlate with leg identity. The duplication in the antenna caused by ectopic hth could be explained by the creation of a new proximodistal interface in the distal portion region of the disc. In both antenna and leg discs, Distal-less is expressed in the distal regions and is required for distal development. The roughly complementary expression of hth/nuclear Exd vs. Dll, defines the proximal and distal domains of appendages, respectively. The combined action of Wg and Dpp signaling defines the two domains by activating Dll and repressing hth in the distal domain. Antennal duplication due to ectopic hth could be explained by the juxtaposition of distal (Dll expressing) and proximal (hth expressing) cells (Yao, 1999 and references)


Genomic length - 30 kb

cDNA clone length - 5.2 kb

Exons - 8


Amino Acids - 884

Structural Domains

The Spineless protein shows extensive similarity to the human and murine aryl hydrocarbon receptor (AHR, also known as the dioxin receptor), as well as lower similarity to the human aryl hydrocarbon nuclear translocator, hypoxia-inducible factor 1, and endothelial PAS-1 and the Drosophila proteins Single-minded, Period, and Trachealess. These proteins comprise a recently recognized family distinguished by the PAS domain, which can mediate protein-protein interactions. Most members of the family also contain a bHLH domain. Spineless is most similar to AHR; there is 71% identity between the two proteins in the bHLH region, 45% identity in the PAS domain, and 41% identity overall. The organization of domains within the two proteins is also closely similar. Spineless and AHR share almost twice as much sequence identity as either shares with their next closest relative, Sim. Sequencing of exons reveals that all five of the spineless splice sites within the bHLH and PAS coding sequences are conserved in Ahr. These splice sites lie within conserved codons in the two genes, and separate precisely the same nucleotide positions within these codons. Ahr has three additional splice sites in the PAS domain coding sequence that are not shared with spineless. Three of the five splice junctions of spineless are also shared with sim. The 3' portion of the ORF is rich in opa (CAX) repeats, which encode primarily glutamine or histidine. These repeats extend beyond the ORF for an additional 600-700 nucleotides. Differences between cDNAs indicate alternative splicing (Duncan, 1998).

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

date revised: 15 May 98

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