Cytogenetic Analysis of Chromosome 3 in DROSOPHILA MELANOGASTER: Isolation and Characterization of Four New Alleles of the Proboscipedia (pb) Locus

Previous studies on proximal 3R have cytologically localized the dominant homeotic loci Antennapedia (Antp), Multiple Sex Comb (Msc), Nasobemia (Ns), and Extra Sex Comb (Scx). In this study we set out to find the site of the proboscipedia (pb) locus. In order to accomplish this, four new alleles of this homeotic gene were induced with gamma rays. Genetic and cytogenetic analyses have shown that the pb locus resides in polytene chromosome bands 84A1–6, immediately adjacent to the Antp gene complex in 84B1–2. An analysis of the morphology of the proboscis and the dose relationships of the four new alleles have shown that this homeosis is unusual in at least two respects. First, the two different developmental fates realized in the proboscis at 18° (labial palps → arista) and 29° (labial palps → leg) under the influence of pb¹ grown at 18°, while the remaining three are like pb¹ at 29°. Dosage studies reveal that this difference reflects a hypomorphic vs. amorphic condition. Second, like the original, these new alleles produce a prothoracic rather than a mesothoracic leg in the proboscis. Both of these results indicate that pb is unique among the homeotics, and as such it may offer some new insights into developmental processes.     

Timing and heritability of theNasobemia transformation inDrosophila

Summary Nasobemia (Ns) is a dominant homeotic mutant ofDrosophila melanogaster which converts parts or all of the antenna to mesothoracic leg.Ns has a temperature sensitive period between 48 and 60 h. The hypothesis thatNs acts during this period and is not required thereafter to maintain the homeotic transformation to leg was tested by removingNs fromNs/+ cells at different stages of development through X-ray induced somatic recombination. The expression of theNs homeotic transformation in recombinant wild type (+/+) cells increased sharply between 48 and 65 h. In clones induced after 65 h the expression of the leg transformation was equal in large and small +/+ clones. We interpret these results as supporting the hypothesis that transient action ofNs between 48 and 65 h switches antennal cells to a clonally stable leg determined state whose maintenance does not require futherNs action.     

Transdetermination in the homeotic eye--antenna imaginal disc of Drosophila melanogaster

The effects of homeotic mutations on transdetermination in eye-antenna imaginal discs of Drosophila melanogaster were studied. After 12 days of culture in vivo, antenna discs transformed to ventral mesothorax by AntpNs or AntpZ, transdetermined to notum and wing structures four to five times more frequently than the corresponding wild-type antenna discs. Likewise, eye discs transformed to dorsal mesothorax by eyopt transdetermined to leg structures, also extremely frequently (90%). It seems that, during culture, homeotic antenna as well as homeotic eye discs tend to complete the structural inventory of the mesothoracic segment. Transdetermination in the homeotic disc parts is interpreted as a regeneration process which reestablishes an entire segment, i.e., the ventral mesothoracic portion (leg) in the antenna disc regenerates dorsal mesothoracic parts, and the dorsal mesothoracic portion in the eye disc (wing) regenerates ventral mesothoracic parts, respectively. This implies that antenna and leg discs (ventral qualities) as well as eye and wing discs (dorsal qualities) are serially homologous. The transdetermination frequency of the untransformed eye disc to notum and wing structures is enhanced by Antp to the same extent as is the transdetermination frequency of the antenna disc. The first allotypic wing disc structure formed by the eye disc is notum, followed by structures of the anterior wing compartment and finally by posterior wing structures. No evidence for such a sequence was found in the transdetermination pattern of the antenna disc.     

Projection pattern of sensory neurons in the central nervous system of a homeotic mutation of the moth Manduca sexta

Octopod (Octo) is a mutation of the moth Manduca sexta, which transforms the first abdominal segment (A1) in the anterior direction. Mutant animals are characterized by the appearance of homeotic thoracic-like legs on A1. We exploited this mutation to determine what rules might be used in specifying the fates of sensory neurons located on the body surface of larval Manduca. Mechanical stimulation of homeotic leg sensilla did not cause reflexive movements of the homeotic legs, but elicited responses similar to those observed following stimulation of ventral A1 body wall hairs. Intracellular recordings demonstrated that several of the motoneurons in the A1 ganglion received inputs from the homeotic sensory hairs. The responses of these motoneurons to stimulation of homeotic sensilla resembled their responses to stimulation of ventral body wall sensilla. Cobalt fills revealed that the mutation transformed the segmental projection pattern of only the sensory neurons located on the ventral surface of A1, resulting in a greater number with intersegmental projection patterns typical of sensory neurons found on the thoracic body wall. Many of the sensory neurons on the homeotic legs had intersegmental projection patterns typical of abdominal sensory neurons: an anteriorly directed projection terminating in the third thoracic ganglion (T3). Once this projection reached T3, however, it mimicked the projections of the thoracic leg sensory neurons. These results demonstrate that the same rules are not used in the establishment of the intersegmental and leg-specific projection patterns. Segmental identity influences the intersegmental projection pattern of the sensory neurons of Manduca, whereas the leg-specific projections are consistent with a role for positional information in determining their pattern. © 1995 John Wiley & Sons, Inc.     

Genetic studies of mutations at two loci of Drosophila melanogaster which cause a wide variety of homeotic transformations

Summary The ash-1 locus is in the proximal region of the left arm of the third chromosome of Drosophila melanogaster and the ash-2 locus is in the distal region of the right arm of the third chromosome. Mutations at either locus can cause homeotic transformations of the antenna to leg, proboscis to leg and/or antenna, dorsal prothorax to wing, first and third leg to second leg, haltere to wing, and genitalia to leg and/or antenna. Mutations at the ash-1 locus cause, in addition, transformations of the posterior wing and second leg to anterior wing and second leg, respectively. A similar spectrum of transformations is caused by mutations at yet another third chromosome locus, trithorax. One extraordinary aspect of mutations at all three of these loci is that they cause such a wide variety of transformations. For mutations at both of the loci that we have studied the expression of the homeotic phenotype is both disc-autonomous (as shown by injecting mutant discs into metamorphosing larvae) and cell autonomous (as shown by somatic recombination analysis). The original mutations which identified these two loci, although lethal, manifest variable expressivity and incomplete penetrance of the homeotic phenotype suggesting that they are hypomorphic. The phenotype of double mutants which were synthesized by combining different pairs of those original mutations manifest for two of the four pairs a greater degree of expressivity and slightly more penetrance of the homeotic transformations. This mutual enhancement suggests that the products of both loci interact in the same process. A third double mutant expresses a discless phenotype.Additional alleles have been recovered at both the ash-1 and the ash-2 loci. Some of these alleles as homozygotes or transheterozygotes express the wide range of transformations revealed first by double mutants. One of the alleles at the ash-1 locus when homozygous and several transheterozygous pairs can cause either the homeotic transformation of discs or the absence of those discs. The fact that these two defects, absence of specific discs and homeotic transformations of those same discs can be caused by mutations within a single gene suggests that the activity of the product of this gene is essential for normal imaginal disc cell proliferation. Loss of that activity leads to the absence of discs, whereas, reduction of that activity leads to homeotic transformations.     

Optical releasers of the innate proboscis extension in the hoverfly Eristalis tenax L. (Syrphidae,Diptera)

Freshly emerged, inexperienced imagos of the hoverfly Eristalis tenax L. extend their proboscis towards small, yellow colour stimuli, such as anther parts and artificial floral guides. The releasing of this behaviour, which is adapted to pollen feeding, was investigated in behavioural tests using white, UV-reflecting artificial flowers with four small screens illuminated with test stimuli serving as artificial floral guides. The releasing of the innate proboscis extension was tested using monochromatic test lights. Within an intensity range from approx. 5·10¹¹ to approx. 10¹⁴ quanta · cm^-2· s^-1, the flies extended their proboscis only towards green and yellow test lights (approx. 520–600 nm). The inhibition of the innate proboscis extension was tested using mixed light stimuli composed of a yellow monochromatic reference light (560 nm, 10¹³ quanta·cm^{-2 -1}) and of a monochromatic test light. When the reference light was mixed with ultraviolet or blue test lights, the releasing of the innate proboscis extension was strongly inhibited, whereas admixing green/yellow light slightly promoted it; admixing red light had no effect. The results indicate that the releasing of the innate proboscis extension is mediated by the photoreceptor type R8y. Other receptor types which could cause the inhibition of the proboscis reaction are discussed.     

A scanning electron microscopy study of the embryonic development of Pycnogonum litorale (Arthropoda,Pycnogonida)

The phylogenetic position of the enigmatic Pycnogonida (sea spiders) is still controversial. This is in part due to a lack of detailed data about the morphology and ontogenesis of this, in many aspects, aberrant group. In particular, studies on the embryonic development of pycnogonids are rare and in part contradictory. Here, we present the first embryological study of a pycnogonid species using scanning electron microscopy (SEM). We describe the late embryogenesis of Pycnogonum litorale from the first visible appendage anlagen to the hatchling in 11 embryonic stages. The three pairs of appendage anlagen gain in length by growth, as well as by extension of furrows into the embryo. The opening of the stomodaeum is located far in front of the anlagen of the chelifores and has a Y‐shaped lumen from the onset. During further embryogenesis, the position of the mouth shifts ventrally, until it is located between the chelifores. The proboscis anlage grows out as a circumoral wall‐like structure, which is initially more pronounced ventrally. Hypotheses about the evolution of the proboscis by fusion of originally separated components are critically discussed, because the proboscis anlage of P. litorale shows no indications of a composite nature. In particular, a participation of post‐cheliforal elements in proboscis formation is rejected by our data. Further, no preoral structure and no stage in proboscis formation was found, which could plausibly be homologized with the labrum of othereuarthropods. Thus, our study supports the assumption of a complete lack of a labrum in Pycnogonida. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Comparative analysis of leg and antenna development in wild-type and homeotic<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis>

The insect leg and antenna are thought to be homologous structures, evolved from a common ancestral appendage. The homeotic transformations of antenna to leg in Drosophila produced by mutation of the Hox gene Antennapedia are position-specific, such that every particular antenna structure is transformed into a specific leg counterpart. This has been taken to suggest that the developmental programmes of these two appendages are still similar. In particular, the mechanisms for the specification of a cell's position within the appendage would be identical, only their interpretation would be different and subject to homeotic gene control. Here we explore the degree of conservation between the developmental programmes of leg and antenna in Drosophila and other dipterans, in wild-type and homeotic conditions. Most of the appendage pattern-forming genes are active in both appendages, and their expression domains are partially conserved. However, the regulatory relationships and interactions between these genes are different, and in fact cells change their expression while undergoing homeotic transformation. Thus, the positional information, and the mechanisms which generate it, are not strictly conserved between leg and antenna; and homeotic genes alter the establishment of positional clues, not only their interpretation. The partial conservation of pattern-forming genes in both appendages ensures a predictable re-specification of positional clues, producing the observed positional specificity of homeotic transformations.     

Projection of sensory neurons from a homeotic mutant appendage,Antennapedia, in Drosophila melanogaster

Central projections of sensory neurons from homeotic mutant appendages (Antennapedia) of Drosophila melanogaster were compared with those of wild-type antennae and wild-type legs by means of degeneration and cobalt backfilling methods. Sensory axons originating from wild-type thoracic legs terminate within the ventral ipsilateral half of the corresponding neuropile segment and do not project to the brain. Sensory fibers from the third antennal segment (AIII) of wild-type animals project into the ipsilateral antennal glomerulus (AG) and to a lesser extent into the contralateral AG, whereas those from the second antennal segment terminate principally within the ipsilateral posterior antennal center. The sensory terminals of femur, tibia, and tarsi of the homeotic leg show a distribution very similar to that of the homologous wild-type antennal segment AIII, differing to a minor degree only in the size and precise localization of terminals within the antennal glomeruli. No degenerating axons were evident in ultrastructural examination of neck connectives after removal of homeotic legs. It is thus very improbable that any sensory fibers of the homeotic leg project to normal leg projection areas in the thoracico-abdominal ganglion. Several alternative explanations are offered for the apparent retention of antennal specificity by axons from the transformed appendage.     

Kinematics and motor activity during tethered walking and turning in the cockroach, <Emphasis Type="Italic">Blaberus discoidalis</Emphasis>

When insects turn from walking straight, their legs have to follow different motor patterns. In order to examine such pattern change precisely, we stimulated single antenna of an insect, thereby initiating its turning behavior, tethered over a lightly oiled glass plate. The resulting behavior included asymmetrical movements of prothoracic and mesothoracic legs. The mesothoracic leg on the inside of the turn (in the apparent direction of turning) extended the coxa-trochanter and femur-tibia joints during swing rather than during stance as in walking, while the outside mesothoracic leg kept a slow walking pattern. Electromyograms in mesothoracic legs revealed consistent changes in the motor neuron activity controlling extension of the coxa-trochanter and femur-tibia joints. In tethered walking, depressor trochanter activity consistently preceded slow extensor tibia activity. This pattern was reversed in the inside mesothoracic leg during turning. Also for turning, extensor and depressor motor neurons of the inside legs were activated in swing phase instead of stance. Turning was also examined in free ranging animals. Although more variable, some trials resembled the pattern generated by tethered animals. The distinct inter-joint and inter-leg coordination between tethered turning and walking, therefore, provides a good model to further study the neural control of changing locomotion patterns.     

Behavioral and electrophysiological responses of the blowfly,Phormia regina meigen,to acids

Summary Flies of a standardized degree of thirst were tested for proboscis extension in response to mono- and dicarboxylic acids applied to the tarsi. Presence or lack of extension depended on pH for each acid, but acids of greater chain length were effective in preventing extension at higher pH values than those with shorter chain length (Fig. 1). Concentrated solutions of monocarboxylic acids at low pH values evoked a strange, delayed, hesitant form of proboscis extension which seemed to signify rejection rather than acceptance. It was not abolished by satiation with either water or sucrose (Fig. 2).Electrophysiological tests of tarsal hairs with NaCl and sucrose showed that the corresponding hairs of each type on opposite sides of the same individual leg had almost identical neural responses to a given stimulus (Table). This meant that hairs on one side of a leg could be used as controls for hairs on the other side.Comparisons were made of the neural responses to different pH values of each acid and of the responses to different acids at the same pH. For each acid, as the pH decreased the response increased both in number of neurons firing and in their firing frequency (Fig. 6). There were differences between the responses to different acids at the same pH correlated with the nature of the behavioral response at the pH value tested. In general, responses were greater to mono- than to dicarboxylic acids (Figs. 7–10).Lack of proboscis extension was correlated with the firing of a small spike of regular frequency, especially prominent in responses of D4 hairs, and with firing of the salt receptor. The strange, hesitant extension was correlated with the indiscriminate firing of a large number of neurons at irregular frequencies. It is concluded that flies have available information enabling them to distinguish acids from salts, sugars, and water. The basis for this distinction is at least in part anatomical. Flies also can distinguish between different specific acids at the same pH, depending on the molecular structure of the acids.This work was supported by National Science Foundation grant GB 1472 to Dr. V. G. Dethier and by a National Science Foundation Graduate Fellowship to the author.     

TheUbx syndrome ofDrosophila: the prothoracic transformation (ppx) is independent ofbx,bxd andpbx

Summary If, early in development, theUbx ⁺ gene is removed by mitotic recombination from cells of the meso-and metathoracic leg primordia, theseUbx ^– cells develop as in the posterior prothoracic leg. We show that this transformation, termedpostprothorax, is a discrete genetic function that is independent of other homeotic transformations such asbx, pbx orbxd, which also result from the inactivation of theUbx gene.     

Genetically displaced sensory neurons in the head of Drosophila project via different pathways into the same specific brain regions

The sensory projection pattern of homoeotic antennal or leg tissue in the proboscis of the mutant proboscipedia in Drosophila melanogaster was studied using orthograde diffusion of cobalt chloride. Both kinds of ectopic nerve fibers terminate in the normal center of the proboscis and in the antennal glomerulus of the brain. These centers are attained by axons which may pass via four different peripheral nerves. In contrast, wild-type proboscis fibers use only one pathway. Depending on the site of entrance into the brain, homoeotic axons may project into the proboscis center first, and then into the antennal glomerulus, or vice versa. Consequently, the tract between these two brain regions may be followed in opposite directions. Ectopic leg axons extending into normal leg centers in the thoracic ganglion have not been found. Projection patterns in proboscipedia are discussed together with those of the leg-like antenna in spineless-aristapedia (R. F. Stocker and P. A. Lawrene, 1981, Dev. Biol.82, 224–237). The fact that displaced antennal and leg neurons project specifically into normal proboscis and antennal centers may reflect the serial homology of antennal, leg, and proboscis neurons, and similar homology of the corresponding centers.     

Loss of <Emphasis Type="Italic">spineless</Emphasis> function transforms the <Emphasis Type="Italic">Tribolium</Emphasis> antenna into a thoracic leg with pretarsal,tibiotarsal, and femoral identity

The Drosophila spineless (ss) gene is regulated downstream of the appendage gene Distal-less (Dll) and is involved in leg and antenna development. Specifically, loss of ss leads to the homeotic transformation of the arista, the distalmost antennal segment, into tarsal identity, and the loss or fusion of distal leg segments. Here we show that the ss homolog from the red flour beetle Tribolium castaneum also homeotically transforms the beetle antenna into leg, but the extent of the transformation is significantly larger than in Drosophila, as the entire antenna (except for the basal antennifer) is transformed into pretarsal, tibiotarsal, and femoral identity; i.e., the transformation comprises the Dll positive area in both appendages. We interpret the antennal phenotype in Tribolium as evidence for a more exclusive role of ss in antennal determination downstream of Dll in the beetle. By contrast, the fact that, in Drosophila ss mutants, only a small portion of the Dll positive area in the antenna is homeotically transformed indicates that Dll uses additional targets to govern the development of the other antennal segments in the fly.     

Developmental analysis of some mutants of the bithorax system ofDrosophila

Summary Mutants in the bithorax system ofDrosophila produce homeotic transformations that affect the mesothoracic, metathoracic and first abdominal segments. In the present report we describe a clonal analysis of the development of those mutants transforming the metathorax and first abdominal segments into mesothorax.The main results indicate that (1) The normal dorsal metathoracic (haltere) disk has similar developmental parameters to the dorsal mesothoracic disk. The main difference is that the initial and final numbers of cells are different in both disks. (2) In flies mutant forBithorax andpostbithorax (which transform the haltere into wing) the transformed haltere disk has the same initial and final number of cells as the normal wing disk. (3) In morphogenetic mosaics homozygousbithorax (andpostbithorax) clones express their genotype autonomously regardless of the genotype of surrounding haltere cells. This autonomy is expressed in a regulation of the number of adult cells per compartment, typical cell affinities and final cuticular differentiation.     

The structure and function of serially homologous leg motor neurons in the locust. I. Anatomy

Twenty-one prothoracic and 17 mesothoracic motor neurons innervating leg muscles have been identified physiologically and subsequently injected with dye from a microelectrode. A tract containing the primary neurites of motor neurons innervating the retractor unquis, levator and depressor tarsus, flexor tibiae, and reductor femora is described. All motor neurons studied have regions in which their dendritic branches overlap with those of other leg motor neurons. Identified, serially homologous motor neurons in the three thoracic ganglia were found to have: (1) cell bodies at similar locations and morphologically similar primary neurites (e.g., flexor tibiae motor neurons), (2) cell bodies at different locations in each ganglion and morphologically different primary neurites in each ganglion (e.g., fast retractor unguis motor neurons), or (3) cell bodies at similar locations and morphologically similar primary neurites but with a functional switch in one ganglion relative to the function of the neurons in the other two ganglia. As an example of the latter, the morphology of the metathoracic slow extensor tibiae (SETi) motor neurons was similar to that of pro- and mesothoracic fast extensor tibiae (FETi) motor neurons. Similarly the metathoracic FETi bears a striking resemblance to the pro- and the mesothoracic SETi. It is proposed that in the metathoracic ganglion the two extensor tibiae motor neurons have switched functions while retaining similar morphologies relative to the structure and function of their pro- and mesothoracic serial homologues.     

Central organization of common inhibitory motoneurons in the locust: role of afferent signals from leg mechanoreceptors

Intracellular recordings of mesothoracic common inhibitory neurons (CI₁, CI₂ and CI₃) were made while tactile hairs of the middle legs of locusts (Locusta migratoria) were mechanically stimulated. Generally the three common inhibitory neurons were excited by stimulation of tactile hairs on the ventral and dorsal surface of femur and tibia. The response pattern of all three CI neurons was similar suggesting that they work as a functional unit. Touching hairs on the dorsal surface of tibia and tarsus in some cases led to inhibition of CIs. The connection between sensory cells of tactile hairs and common inhibitory neurons is polysynaptic.To identify interneurons which mediate afferent signals, simultaneous intracellular recordings from CIs and interneurons were made. Different spiking interneurons were identified which made excitatory or inhibitory monosynaptic connections with CIs. Interneurons with inhibitory input to CIs belonged to the ventral midline group of spiking local interneurons. Behavioral and electrophysiological results indicate that reflex movements of the leg are accompanied by activity of CI neurons. Further it appears that CI activity is inhibited when reflex movements of the leg are actively suppressed by the animal.Abbreviations CI common inhibitor - IN interneuron - LY Lucifer Yellow     

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP¹, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons^2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding⁶ (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER⁷ with a calcium imaging technique⁸ using GCaMP3.0^{1, 9}, I have established an experimental system, where we can monitor activity of neurons in the feeding center – the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca²⁺ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.     

Negative regulation ofUltrabithorax expression byengrailed is required for proper specification of wing development inDrosophila melanogaster

In both vertebrates and invertebrates, homeotic selector genes confer morphological differences along the antero-posterior axis. However, insect wing development is independent of all homeotic gene functions, reflecting the ground plan of an ancestral pterygote, which bore wings on all segments. Dipteran insects such asDrosophila are characterized by a pair of wings in the mesothoracic segment. In all other segments, wing development is essentially repressed by different homeotic genes, although in the metathorax they are modified into a pair of halteres. This necessitates that during development all homeotic genes are to be maintained in a repressed state in wing imaginal discs. In this report we show that (i) the function of the segment polarity geneengrailed (en) is critical to keep the homeotic selector geneUltrabithorax (Ubx) repressed in wing imaginal discs, (ii) normal levels of En in the posterior compartment of haltere discs, however, are not enough to completely repressUbx, and (iii) the repression ofUbx byen is independent of Hedgehog signalling through which the long-range signalling ofen is mediated during wing development. Finally we provide evidence for a possible mechanism by whichen repressesUbx. On the basis of these results we propose thaten has acquired two independent functions during the evolution of dorsal appendages. In addition to its well-known function of conferring posterior fate and inducing long-range signalling to pattern the developing appendages, it maintains wing fate by keepingUbx repressed.