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.     

Effects of Polycomb Group Mutations on the Expression of Ultrabithorax in the Drosophila Visceral Mesoderm

The Polycomb group (PcG) genes encode repressors of many developmental regulatory genes including homeotic genes and are known to act by modifying chromatin structure through complex formation. We describe how Ultrabithorax (Ubx) expression is affected by the PcG mutants in the visceral mesoderm. Mutant embryos of the genes extra sex combs (esc), Polycomb (Pc), additional sex combs (Asx) and pleiohomeotic (pho) were examined. In each mutation, Ubx was ectopically expressed outside of their normal domains along the anterior-posterior axis in the visceral mesoderm, which is consistent with the effect of PcG proteins repressing the homeotic genes in other tissues. All of these four PcG mutations exhibit complete or partial lack of midgut constriction. However, two thirds of esc mutant embryos did not show Ubx expression in parasegment 7 (PS7). Even in the embryos showing ectopic Ubx expression, the level of Ubx expression in the PcG mutations was weaker than that in normal embryos. We suggest that in PcG mutations the ectopic Ubx expression is caused by lack of PcG repressor proteins, while the weaker or lack of Ubx expression is due to the repression of Ubx by Abd-B protein which is ectopically expressed in PcG mutations as well.     

Co-factors and co-repressors of Engrailed: expression in the central nervous system and cerci of the cockroach, <Emphasis Type="Italic">Periplaneta americana</Emphasis>

In the larval cockroach (Periplaneta americana), knockout of Engrailed (En) in the medial sensory neurons of the cercal sensory system changes their axonal arborization and synaptic specificity. Immunocytochemistry has been used to investigate whether the co-repressor Groucho (Gro; vertebrate homolog: TLE) and the co-factor Extradenticle (Exd; vertebrate homolog: Pbx) are expressed in the cercal system. Gro/TLE is expressed ubiquitously in cell nuclei in the embryo, except for the distal pleuropodia. Gro is expressed in all nuclei of the thoracic and abdominal central nervous system (CNS) of first instar larva, although some neurons express less Gro than others. Cercal sensory neurons express Gro protein, which might therefore act as a co-repressor with En. Exd/Pbx is expressed in the proximal portion of all segmental appendages in the embryo, with the exception of the cerci. In the first instar CNS, Exd protein is expressed in subsets of neurons (including dorsal unpaired medial neurons) in the thoracic ganglia, in the first two abdominal ganglia, and in neuromeres A8–A11 of the terminal ganglion. Exd is absent from the cerci. Because Ultrabithorax/Abdominal-A (Ubx/Abd-A) can substitute for Exd as En co-factors in Drosophila, Ubx/Abd-A immunoreactivity has also been investigated. Ubx/Abd-A immunostaining is present in abdominal segments of the embryo and first instar CNS as far caudal as A7 and faintly in the T3 segment. However, Ubx/Abd-A is absent in the cerci and their neurons. Thus, in contrast to its role in Drosophila segmentation, En does not require the co-factors Exd or Ubx/Abd-A in order to control the synaptic specificity of cockroach sensory neurons.I acknowledge the support of NIH R01 NS45547, NIH-SCORE S06 GM0088224, and RCMI G12 RR03051.     

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.     

Over-expression of Ultrabithorax alters embryonic body plan and wing patterns in the butterfly Bicyclus anynana

In insects, forewings and hindwings usually have different shapes, sizes, and color patterns. A variety of RNAi experiments across insect species have shown that the hox gene Ultrabithorax (Ubx) is necessary to promote hindwing identity. However, it remains unclear whether Ubx is sufficient to confer hindwing fate to forewings across insects. Here, we address this question by over-expressing Ubx in the butterfly Bicyclus anynana using a heat-shock promoter. Ubx whole-body over-expression during embryonic and larvae development led to body plan changes in larvae but to mere quantitative changes to adult morphology, respectively. Embryonic heat-shocks led to fused segments, loss of thoracic and abdominal limbs, and transformation of head limbs to larger appendages. Larval heat-shocks led to reduced eyespot size in the expected homeotic direction, but neither additional eyespots nor wing shape changes were observed in forewings as expected of a homeotic transformation. Interestingly, Ubx was found to be expressed in a novel, non-characteristic domain – in the hindwing eyespot centers. Furthermore, ectopic expression of Ubx on the pupal wing activated the eyespot-associated genes spalt and Distal-less, known to be directly repressed by Ubx in the fly?s haltere and leg primordia, respectively, and led to the differentiation of black wing scales. These results suggest that Ubx has been co-opted into a novel eyespot gene regulatory network, and that it is capable of activating black pigmentation in butterflies.     

Evolution of a Novel Appendage Ground Plan in Water Striders Is Driven by Changes in the Hox Gene Ultrabithorax

Water striders, a group of semi-aquatic bugs adapted to life on the water surface, have evolved mid-legs (L2) that are long relative to their hind-legs (L3). This novel appendage ground plan is a derived feature among insects, where L2 function as oars and L3 as rudders. The Hox gene Ultrabithorax (Ubx) is known to increase appendage size in a variety of insects. Using gene expression and RNAi analysis, we discovered that Ubx is expressed in both L2 and L3, but Ubx functions to elongate L2 and to shorten L3 in the water strider Gerris buenoi. Therefore, within hemimetabolous insects, Ubx has evolved a new expression domain but maintained its ancestral elongating function in L2, whereas Ubx has maintained its ancestral expression domain but evolved a new shortening function in L3. These changes in Ubx expression and function may have been a key event in the evolution of the distinct appendage ground plan in water striders.     

How do single homeotic genes control multiple segment identities?

Each of the homeotic genes of the bithorax complex of Drosophila defines the identities of more than one body segment. The mechanisms by which this occurs have been elusive. In a recent report, Castelli-Gair and Akam⁽¹⁾ analyze in detail the control of parasegment 5 and parasegment 6 identities by the bithorax complex gene Ubx. Their results indicate that differences in the spatial and temporal expression patterns of Ubx are critical in determining differences between these parasegments. However, dose effects observed by others indicate that parasegment-specific differences in the level of Ubx expression are also important. For the other BX-C genes, parasegment-specific expression of protein isoforms, or combinatorial control dependent on the expression patterns of other spatially restricted regulators, may also play a role.     

COMPARATIVE FUNCTIONAL ANALYSES OF ULTRABITHORAX REVEAL MULTIPLE STEPS AND PATHS TO DIVERSIFICATION OF LEGS IN THE ADAPTIVE RADIATION OF SEMI‐AQUATIC INSECTS

Invasion of new ecological habitats is often associated with lineage diversification, yet the genetic changes underlying invasions and radiations are poorly understood. Over 200 million years ago, the semi‐aquatic insects invaded water surface from a common terrestrial ancestor and diversified to exploit a wide array of niches. Here, we uncover the changes in regulation and function of the gene Ultrabithorax associated with both the invasion of water surface and the subsequent diversification of the group. In the common ancestor of the semi‐aquatic insects, a novel deployment of Ubx protein in the mid‐legs increased their length, thereby enhancing their role in water surface walking. In derived lineages that specialize in rowing on the open water, additional changes in the timing of Ubx expression further elongated the mid‐legs thereby facilitating their function as oars. In addition, Ubx protein function was selectively reversed to shorten specific rear‐leg segments, thereby enabling their function as rudders. These changes in Ubx have generated distinct niche‐specialized morphologies that account for the remarkable diversification of the semi‐aquatic insects. Therefore, changes in the regulation and function of a key developmental gene may facilitate both the morphological change necessary to transition to novel habitats and fuel subsequent morphological diversification.     

Homeotic gene expression in the locust Schistocerca: An antibody that detects conserved epitopes in ultrabithorax and abdominal-A proteins

To investigate what role homeotic genes may play in morphological evolution, we are comparing homeotic gene expression in two very different insects, Drosophila (Diptera) and Schistocerca (Orthoptera). In this paper we describe a monoclonal antibody, FP6.87, that recognizes the products of both the Ultrabithorax (Ubx) and abdominal-A (abd-A) genes in Drosophila, via an epitope common to the carboxy terminal region of these two proteins. This antibody recognizes nuclear antigens present in the posterior thorax and abdomen of Schistocerca. We infer that it recognizes the Schistocerca homolog of UBX protein, and probably also of ABD-A. As the distribution of Schistocerca ABD-A protein is already known, we can use this reagent to map the expression of Schistocerca UBX in the thorax and anterior abdomen, where ABD-A is not expressed. Both the general domain, and many of the details, of UBX exp ression are remarkably conserved compared with Drosophila. Thus UBX expression extends back from T2 in the ectoderm (including the CNS), but only from A1 in the mesoderm. As noted for other bithorax complex genes in Schistocerca, expression begins in the abdomen, at or shortly before the time of segmentation. It only later spreads anteriorly to the thorax. For much of embryogene-sis, the expression of UBX in the thoracic epidermis is largely restricted to the T3 limb. Inthis limb, UBX is strikingly regulated, in a complex pattern that reflects limb segmentation. Reviewing these and earlier observations, we conclude that evolutionary changes affect both the precise regulation of homeotic genes within segments, and probably also the spectrum of downstream genes that respond to homeotic gene expression in a given tissue. Overall domains of homeotic gene expression appear to be well conserved between different insect groups, though a change in the extent and timing of homeotic gene expression may underlie the modification of the posterior abdomen in different insect groups. © 1994 Wiley-Liss, Inc.     

Conservation and variation in Ubx expression among chelicerates

SUMMARY Chelicerates are an ancient arthropod group with a distinct body plan composed of an anterior (prosoma) and a posterior portion (opisthosoma). The expression of the Hox gene Ultrabithorax (Ubx) has been examined in a single representative of the chelicerates, the spider Cupiennius salei. In spiders, Ubx expression starts in the second opisthosomal segment (O2). Because the first opisthosomal segment (O1) in spiders is greatly reduced relative to other chelicerates, we hypothesized that the observed Ubx expression pattern might be secondarily modified. Shifts in the anterior boundary of the expression of Ubx have been correlated with functional shifts in morphology within malacostracan crustaceans. Thus, the boundary of Ubx expression between chelicerates with different morphologies in their anterior opisthosoma could also be variable. To test this prediction, we examined the expression patterns of Ubx and abdominal‐A (collectively referred to as UbdA) in two basal chelicerate lineages, scorpions and xiphosurans (horseshoe crabs), which exhibit variation in the morphology of their anterior opisthosoma. In the scorpion Paruroctonus mesaensis, the anterior border of early expression of UbdA is in a few cells in the medial, posterior region of the O2 segment, with a predominant expression in O3 and posterior. Expression later spreads to encompass the whole O2 segment and a ventral, posterior portion of the O1 segment. In the xiphosuran Limulus polyphemus, early expression of UbdA has an anterior boundary in the segment. Later in development, the anterior boundary moves forward one segment to the chilarial (O1) segment. Thus, the earliest expression boundary of UbdA lies within the second opisthosomal segment in all the chelicerates examined. These results suggest that rather than being derived, the spider UbdA expression in O2 likely reflects the ancestral expression boundary. Changes in the morphology of the first opisthosomal segment are either not associated with changes in UbdA expression or correlate with late developmental changes in UbdA expression.     

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.     

Long range repression conferring boundaries of Ultrabithorax expression in the Drosophila embryo.

In an attempt to reconstruct the embryonic expression pattern of the homeotic gene Ultrabithorax (Ubx) by stable integration of fusion constructs, we identified three key control regions called PBX, ABX and BXD. Each of these confers an expression pattern mimicking certain aspects of Ubx expression. The PBX and ABX patterns are limited to the Ubx domain with anterior boundaries at parasegments 6 and 5. In contrast, the BXD pattern extends from head to tail. PBX or ABX expression boundaries are imposed on the BXD pattern, if PBX or ABX is linked to BXD. These boundaries, although not the PBX and ABX expression limits themselves, are dependent on Polycomb function. We conclude that PBX and ABX are recognized by repressors which act across large distances to suppress BXD activity. Stable and heritable Ubx expression boundaries are thus mediated by this process of long range repression.     

Compartmental modulation of abdominal Hox expression by engrailed and sloppy-paired patterns the fly ectoderm

In Drosophila, segmentation genes partition the early embryo into reiterative segments along the anterior-posterior axis, while Hox genes assign segments their identities. Each segment is also subdivided into distinct anterior (A) and posterior (P) compartments based on the expression of the engrailed (en) segmentation gene. Differences in Hox expression often correlate with compartmental boundaries, but the genetic basis for these differences is not well understood. In this study, we extend previous results to describe a genetic circuit that controls the differential expression of two Hox genes, Ultrabithorax (Ubx) and abdominal-A (abd-A), within the A and P compartments of the abdominal ectoderm. Consistent with earlier findings, we show that en is essential for high Abd-A levels and low Ubx levels in the P compartment, whereas sloppy-paired (slp) is required for high Ubx levels in the A compartment. Overall, these results demonstrate that the compartmental expression of Ubx and abd-A is established through a repressive regulatory network between en, slp, Ubx and abd-A. We also show that abd-A expression in the P compartment is important for the formation of abdominal-specific cell types, suggesting that en and slp modulation of Hox expression within the A and P compartments is essential for embryonic patterning.     

Expression patterns of the homeotic genes Scr, Antp, Ubx, and abd-A during embryogenesis of the cricket Gryllus bimaculatus

We have studied embryogenesis of the two-spotted cricket Gryllus bimaculatus as an example of a hemimetabolous, intermediate germ insect, which is a phylogenetically basal insect and may retain primitive features. We observed expression patterns of the orthologs of the Drosophila homeotic genes, Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx) and abdominal-A (abd-A) during embryogenesis and compared the expression patterns of these genes with the more basal thysanuran insect, Thermobia domestica (the firebrat), and the derived higher dipteran insect, Drosophila melanogaster. Although Scr is expressed commonly in the presumptive posterior maxillary and labial segment in all three insects, the thoracic expression domains vary. Antp is expressed similarly in the three thoracic segments, the limbs, and the anterior abdominal region among these three insects. The early Antp expression in the firebrat and cricket obeys a segmental register in all three thoracic segments, while in Drosophila its initial expression appears in parasegments 4 and 6. Ubx is expressed in the metathoracic (T3) and abdominal segments similarly in the three insects, whereas the expression pattern in the T3 leg differs among them. abd-A is expressed in the posterior compartment of the first abdominal segment and the remaining abdominal segments in all three insects, although its posterior border varies among them.     

Patterns of temperature sensitivity in Contrabithorax/Ultrabithorax heterozygotes of Drosophila

Contrabithorax (Cbx) is a dominant homeotic mutant of Drosophila which transforms wings to halteres, while Ultrabithorax (Ubx) is a dominant mutant which transforms halteres to wings. Therefore $\text{Cbx}\text{Ubx}$ flies carry dominant homeotic mutants engaged in opposing transformations. This article reports that $\text{Cbx}\text{TM2 Ubx}{}^{130}$ is temperature sensitive. At 29°C, flies express strong Cbx transformation of wings, and minor Ubx transformation of halteres. Larvae shifted to 17°C prior to 72 hr express strong Ubx transformation of halteres toward wings, and slight Cbx transformation of wings. Seventeen-degree temperature-pulse experiments show that the $\text{Cbx}\text{TM2 Ubx}{}^{130}$ system is temperature sensitive continuously during embryonic and larval life. Expression of the Cbx transformation in left and right wings is highly correlated in all conditions studied, as is expression of the Ubx transformation in left and right halteres, but the Cbx transformation in wings and Ubx transformation in halteres can be negatively correlated or uncorrelated. The temperature sensitivity in $\text{Cbx}\text{TM2 Ubx}{}^{130}$ is not found in $\text{Cbx}\text{Ubx}{}^{\mathrm{61D}}$, and Ubx is only weakly expressed in $\text{Sb}\text{TM2}$Ubx¹³⁰ flies. These results show that Cbx in trans to Ubx can enhance Ubx expression, although Cbx also causes an opposing transformation to Ubx; that the $\text{Cbx}\text{Ubx}$ system acts in both the mesothorax and metathorax to modulate their phenotypes; and that both transformations are broadly temperature sensitive through embryonic and larval life. This suggests that the $\text{Cbx}\text{TM2 Ubx}{}^{130}$ system functions continuously during embryonic and larval development to maintain mesothoracic and metathoracic commitments. The results are interpreted in terms of a $\text{Cbx}\text{Ubx}$ feedback loop which maintains the mesothorax in a state of low $\text{Cbx}\text{Ubx}$ activity, and metathorax in a state of high $\text{Cbx}\text{Ubx}$ activity.