Correlation of expression patterns of homothorax, dachshund, and Distal-less with the proximodistal segmentation of the cricket leg bud

We describe the expression pattern of Gryllus homothorax (Gbhth) and dachshund (Gbdac), a cricket homologue of Drosophila homothorax and dachshund, together with localization of Distal-less or Extradenticle protein during leg development. We correlated their expression patterns with the morphological segmentation of the leg bud. The boundary of Gbhth/GbDll subdivision is correlated with the segment boundary of the future trochanter/femur at early stages. Gbdac expression subdivides the leg bud into the presumptive femur and more distal region. During the leg proximodistal formation, although the early expression patterns of GbDll, Gbdac, and Gbhth significantly differ from those of Drosophila imaginal disc, their expression patterns in the fully segmented Gryllus leg were similar to those in the Drosophila late third instar disc.     

Involvement of canonical Wnt/Wingless signaling in the determination of the positional values within the leg segment of the cricket Gryllus bimaculatus

The cricket Gryllus bimaculatus is a hemimetabolous insect whose nymphs possess the ability to regenerate amputated legs. Previously, we showed that Gryllus orthologues of Drosophila hedgehog (Gb'hh), wingless (Gb'wg) and decapentaplegic (Gb'dpp) are expressed during leg regeneration and play essential roles in the establishment of the proximal-distal axis. Here, we examined their roles during intercalary regeneration: when a distally amputated tibia with disparate positional values is placed next to a proximally amputated host, intercalary growth occurs in order to regenerate the missing part. In this process, we examined expression patterns of Gb'hh and Gb'wg. We found that expressions of Gb'hh and Gb'wg were induced in a regenerate and the host proximal to the amputated region, but not in the grafted donor distal to the regenerate. This directional induction occurs even in the reversed intercalation. Because these results are consistent with a distal-to-proximal respecification of the regenerate, Gb'wg may be involved in the re-establishment of the positional values in the regenerate. Furthermore, we found that no regeneration occurs when Gb'armadillo (the orthologue of beta-catenin) was knocked down by RNA interference. These results indicate that the canonical Wnt/Wingless signaling pathway is involved in the process of leg regeneration and determination of positional information in the leg segment.     

Intercalary regeneration of symmetrical thighs in the axolotl,Ambystoma mexicanum

Intercalary regeneration of stylopodial and zeugopodial skeletal elements takes place in axolotl limbs composed of normal wrist blastemas autografted or homografted to double half-anterior or half-posterior thighs. Analysis of the morphological pattern of the skeleton and, in homografts, of pigmentation pattern, shows that the intercalated elements are derived from the host double half-thigh. Intercalary regeneration from double half-posterior thighs is expected since they normally can undergo complete proximal-distal regeneration, but is not necessarily expected from double half-anterior thighs, since they normally do not regenerate more distal segments. These results demonstrate that (1) cells of double half-anterior thighs are not inherently incapable of undergoing distal transformation, (2) cells of a distal blastema grafted to a more proximal level do not form patterns proximal to their level of origin, and (3) there is an inhibitory interaction between blastema cells derived from double half-anterior thighs that is expressed after simple amputation, but not when these cells are in contact with a more distal, normal blastema. Using these and other data, a three-dimensional boundary model of limb regeneration is proposed.     

Requirements of Lim1, a Drosophila LIM-homeobox gene, for normal leg and antennal development

During Drosophila leg development, the distal-most compartment (pretarsus) and its immediate neighbour (tarsal segment 5) are specified by a pretarsus-specific homeobox gene, aristaless, and tarsal-segment-specific Bar homeobox genes, respectively; the pretarsus/tarsal-segment boundary is formed by antagonistic interactions between Bar and pretarsus-specific genes that include aristaless (Kojima, T., Sato, M. and Saigo, K. (2000) Development 127, 769-778). Here, we show that Drosophila Lim1, a homologue of vertebrate Lim1 encoding a LIM-homeodomain protein, is involved in pretarsus specification and boundary formation through its activation of aristaless. Ectopic expression of Lim1 caused aristaless misexpression, while aristaless expression was significantly reduced in Lim1-null mutant clones. Pretarsus Lim1 expression was negatively regulated by Bar and abolished in leg discs lacking aristaless activity, which was associated with strong Bar misexpression in the presumptive pretarsus. No Lim1 misexpression occurred upon aristaless misexpression. The concerted function of Lim1 and aristaless was required to maintain Fasciclin 2 expression in border cells and form a smooth pretarsus/tarsal-segment boundary. Lim1 was also required for femur, coxa and antennal development.     

Composite signalling from Serrate and Delta establishes leg segments in Drosophila through Notch.

The receptor protein NOTCH and its ligands SERRATE and DELTA are involved in many developmental processes in invertebrates and vertebrates alike. Here we show that the expression of the Serrate and Delta genes patterns the segments of the leg in Drosophila by a combination of their signalling activities. Coincident stripes of Serrate and Delta expressing cells activate Enhancer of split expression in adjacent cells through Notch signalling. These cells form a patterning boundary from which a putative secondary signal leads to the development of leg joints. Elsewhere in the tarsal segments, signalling by DELTA and NOTCH is necessary for the development of non-joint parts of the leg. We propose that these two effects result from different thresholds of NOTCH activation, which are translated into different downstream gene expression effects. We propose a general mechanism for creation of boundaries by Notch signalling.     

Correlation of diversity of leg morphology in Gryllus bimaculatus (cricket) with divergence in dpp expression pattern during leg development

Insects can be grouped into mainly two categories, holometabolous and hemimetabolous, according to the extent of their morphological change during metamorphosis. The three thoracic legs, for example, are known to develop through two overtly different pathways: holometabolous insects make legs through their imaginal discs, while hemimetabolous legs develop from their leg buds. Thus, how the molecular mechanisms of leg development differ from each other is an intriguing question. In the holometabolous long-germ insect, these mechanisms have been extensively studied using Drosophila melanogaster. However, little is known about the mechanism in the hemimetabolous insect. Thus, we studied leg development of the hemimetabolous short-germ insect, Gryllus bimaculatus (cricket), focusing on expression patterns of the three key signaling molecules, hedgehog (hh), wingless (wg) and decapentaplegic (dpp), which are essential during leg development in Drosophila. In Gryllus embryos, expression of hh is restricted in the posterior half of each leg bud, while dpp and wg are expressed in the dorsal and ventral sides of its anteroposterior (A/P) boundary, respectively. Their expression patterns are essentially comparable with those of the three genes in Drosophila leg imaginal discs, suggesting the existence of the common mechanism for leg pattern formation. However, we found that expression pattern of dpp was significantly divergent among Gryllus, Schistocerca (grasshopper) and Drosophila embryos, while expression patterns of hh and wg are conserved. Furthermore, the divergence was found between the pro/mesothoracic and metathoracic Gryllus leg buds. These observations imply that the divergence in the dpp expression pattern may correlate with diversity of leg morphology.     

Position-specific and non-colinear expression of the planarian posterior (Abdominal-B-like) gene

Hox genes are pivotal molecules in the control of morphogenesis along the anterior-posterior (AP) axis in various bilaterians. Planarians are key animals for understanding the evolution of the bilaterian body plan. Furthermore, they are also known for their strong regeneration ability and are thought to use the Hox genes in the process of reconstruction of the AP axis. In the present paper, the identification and analysis of expression of two posterior (Abdominal-B-like) genes, DjAbd-Ba and DjAbd-Bb, is reported in the planarian Dugesia japonica. DjAbd-Ba is expressed in the entire tail region and its anterior boundary is the posterior pharyngeal region. In contrast, DjAbd-Bb is expressed in several types of cells throughout the body. During regeneration, the expression of DjAbd-Ba rapidly recovers a pattern similar to that in the normal worm. These findings suggest the possibility that DjAbd-Ba is involved in the specification of the tail region. The anterior boundary of the expression domain of the posterior gene DjAbd-Ba is anterior to the domains of the central genes Plox4-Dj and Plox5-Dj. These expression patterns of planarian Hox genes seem out of the rule of spatial colinearity and may reflect an ancestral feature of bilaterian Hox genes.     

Juxtaposition of two heterotypic monolayer cell cultures of chick limb buds

In chick limb buds, mesenchymal cells of the progress zone (PZ-cells) at different developmental stages segregate one from the other in mixed cell cultures, suggesting they have different cell affinity. In order to learn the possible roles of such differences in the cells, two heterotypic leg PZ-cell populations (cells from stages 25/26 and 20/21) in vitro were juxtaposed to allow them to form the boundary. A method with double cylindrical columns was used to make adjoining monolayer cell cultures. It was shown that heterotypic juxtaposition produced two chondrogenic patterns along the boundary: aggregates of chondrocytes formed by stage 20/21 PZ-cells and a chondrocyte-free band formed by those at stage 25/26. Juxtaposition of PZ-cells and proximal cells also formed these patterns, while that between cells from anterior and posterior PZ formed indistinct patterns along the boundary. Homotypic PZ-cell juxtaposition did not produce these patterns. The results suggest that different cell affinity has a role in the segmentation of cartilage patterns at a point along the proximodistal axis, as well as a role in retaining cells in one area so as not to be recruited to other condensation areas.     

The role of Kupffer cells in rat liver regeneration revealed by cell-specific microarray analysis

Liver regeneration after partial hepatectomy is a process with various types of cells involved. The role of Kupffer cells (KCs) in liver regeneration is still controversial. In this study we isolated KCs from regenerating liver and conducted cell-specific microarray analysis. The results demonstrated that the controversial role of KCs in liver regeneration could be explained with the expression patterns of TGF-α, IL-6, TNF, and possibly IL-18 during liver regeneration. IL-18 may play an important role in negative regulation of liver regeneration. The functional profiles of gene expression in KCs also indicated that KC signaling might play a negative role in cell proliferation: signaling genes were down regulated before cell division. Immune response genes in KCs were also down regulated during liver regeneration, demonstrating similar expression profiles to that of hepatocytes. The expression patterns of key genes in these functional categories were consistent with the temporal functional profiles.     

Molecular patterning mechanism underlying metamorphosis of the thoracic leg in Manduca sexta

The tobacco hornworm Manduca sexta, like many holometabolous insects, makes two versions of its thoracic legs. The simple legs of the larva are formed during embryogenesis, but then are transformed into the more complex adult legs at metamorphosis. To elucidate the molecular patterning mechanism underlying this biphasic development, we examined the expression patterns of five genes known to be involved in patterning the proximal-distal axis in insect legs. In the developing larval leg of Manduca, the early patterning genes Distal-less and Extradenticle are already expressed in patterns comparable to the adult legs of other insects. In contrast, Bric-a-brac and dachshund are expressed in patterns similar to transient patterns observed during early stages of leg development in Drosophila. During metamorphosis of the leg, the two genes finally develop mature expression patterns. Our results are consistent with the hypothesis that the larval leg morphology is produced by a transient arrest in the conserved adult leg patterning process in insects. In addition, we find that, during the adult leg development, some cells in the leg express the patterning genes de novo suggesting that the remodeling of the leg involves changes in the patterning gene regulation.     

Joint development in the Drosophila leg: cell movements and cell populations

Flexible joints separate the rigid sections of the insect leg, allowing them to move. In Drosophila, the initial patterning of these joints is apparent in the larval imaginal discs from which the adult legs will develop. Here, we describe the later patterning and morphogenesis of the joints, which occurs after pupariation (AP). In the tibial/tarsal joint, the apodeme insertion site provides a fixed marker for the boundary between proximal and distal joint territories (the P/D boundary). Cells on either side of this boundary behave differently during morphogenesis. Morphogenesis begins with the apical constriction of distal joint cells, about 24 h AP. Distal cells then become columnar, causing distal tissue nearest the P/D boundary to fold into the leg. In the last stage of joint morphogenesis, the proximal joint cells closest to the P/D boundary align and elongate to form a "palisade" (a row of columnar cells) over the distal joint cells. The proximal and distal joint territories are characterised by the differential organisation of cytoskeletal and extracellular matrix proteins, and by the differential expression of enhancer trap lines and other gene markers. These markers also define a number of more localised territories within the pupal joint.     

Two msh/msx-related genes, Djmsh1 and Djmsh2, contribute to the early blastema growth during planarian head regeneration

Regeneration in planarians is an intriguing phenomenon, based on the presence of pluripotent stem cells, known as neoblasts. Following amputation, these cells activate mitotic divisions, migrate distally and undergo differentiation, giving rise to the regeneration blastema. We have identified two msh/msx-related genes, Djmsh1 and Djmsh2, which are expressed in distinct cell populations of the planarian Dugesia japonica and activated, with different patterns, during head regeneration. We demonstrate that RNA interference of Djmsh1 or Djmsh2 generates a delay in the growth of cephalic blastema, interfering with the dynamics of mitoses during its initial formation. Our data also reveal that the activity of the two planarian msh genes is required to regulate Djbmp expression during head regeneration. This study identifies, for the first time, a functional association between muscle segment homeobox (MSH) homeoproteins and BMP signaling during stem cell-based regeneration of the planarian head and provides a functional analysis of how msh genes may regulate in vivo the regenerative response of planarian stem cells.     

Expression of the Sex combs reduced protein in Drosophila larvae

We have generated a monoclonal antibody that binds specifically to the protein product of the homeotic Sex combs reduced (Scr) gene of Drosophila, and have mapped the patterns of Scr expression in late third instar larvae. Virtually the entire prothoracic leg imaginal disc expresses the gene, although the levels of expression vary in different disc regions. This heterogeneity does not reflect the compartmental domains defined by engrailed gene expression. Expression is also observed in the cells of the humeral and labial discs, and there is a small patch of Scr-expressing cells in the antenna disc. The gene is expressed in adepithelial cells of the three thoracic leg discs, but not in the wing or haltere discs. In the central nervous system, Scr expression is confined to a narrow band of cells in the subesophageal region of the ventral ganglion. The results are discussed with respect to the known genetic requirements for Scr+ function.     

Extracellular matrix of the regeneration chamber and plasma membranes of the epidermis during leg regeneration in an insect Carausius morosus

An extracellular matrix (ECM) is found in the regeneration chamber during leg regeneration in the stick insect Carausius morosus. The material which surrounds the regenerate is organised into fibrils and it includes proteins distributed in a hydrated polysaccharide gel. The compounds which can be demonstrated are chitin unlinked to proteins, glycoproteins and unsulfated glycosaminoglycans such as hyaluronic acid and/or chondroitin. Molecules related to vertebrate fibronectin and collagen IV were observed on the apical surface of the epidermal cells of the regenerate. During leg regeneration, the basal lamina which normally secures the cells to each other is absent. However a condensation of material on the regenerate epidermal cells ensures their cohesion. The extracellular matrix in the regeneration chamber must be secreted by the cells of retracted epidermis and then by the epidermal cells of the regenerate, until these cells are able to secrete the cuticle for the next instar. The analysis of the epidermal cell surface does not seem to show any localization or any changes during the development of the regenerate.     

Matrix metalloproteinases (MMPs) are required for wound closure and healing during larval leg regeneration in the flour beetle,Tribolium castaneum

Regenerative abilities are found ubiquitously among many metazoan taxa. To compare mechanisms underlying the initial stages of limb regeneration between insects and vertebrates, the roles of matrix metalloproteinases (MMPs) and fibroblast growth factor (FGF) signaling were investigated in the red flour beetle, Tribolium castaneum. RNA interference-mediated knockdown of MMP2 expression delayed wound healing and subsequent leg regeneration. Additionally, pairwise knockdown of MMP1/2 and MMP2/3, but not MMP1/3, resulted in inhibition of wound closure. Wound healing on the dorsal epidermis after injury was also delayed when MMPs were silenced. Our findings show that functionally redundant MMPs play key roles during limb regeneration and wound healing in Tribolium. This MMP-mediated wound healing is necessary for the subsequent formation of a blastema. In contrast, silencing of FGF receptor did not interfere with the initial stages of leg regeneration despite the alterations in tanning of the cuticle. Thus, insects and vertebrates appear to employ similar developmental processes for the initial stages of wound closure during limb regeneration, while the role of FGF in limb regeneration appears to be unique to vertebrates.     

Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes

Drosophila imaginal disc cells can switch fates by transdetermining from one determined state to another. We analyzed the expression profiles of cells induced by ectopic Wingless expression to transdetermine from leg to wing by dissecting transdetermined cells and hybridizing probes generated by linear RNA amplification to DNA microarrays. Changes in expression levels implicated a number of genes: lamina ancestor, CG12534 (a gene orthologous to mouse augmenter of liver regeneration), Notch pathway members, and the Polycomb and trithorax groups of chromatin regulators. Functional tests revealed that transdetermination was significantly affected in mutants for lama and seven different PcG and trxG genes. These results validate our methods for expression profiling as a way to analyze developmental programs, and show that modifications to chromatin structure are key to changes in cell fate. Our findings are likely to be relevant to the mechanisms that lead to disease when homologs of Wingless are expressed at abnormal levels and to the manifestation of pluripotency of stem cells.     

Laminin gamma1 is critical for Schwann cell differentiation, axon myelination, and regeneration in the peripheral nerve

Laminins are heterotrimeric extracellular matrix proteins that regulate cell viability and function. Laminin-2, composed of alpha2, beta1, and gamma1 chains, is a major matrix component of the peripheral nervous system (PNS). To investigate the role of laminin in the PNS, we used the Cre-loxP system to disrupt the laminin gamma1 gene in Schwann cells. These mice have dramatically reduced expression of laminin gamma1 in Schwann cells, which results in a similar reduction in laminin alpha2 and beta1 chains. These mice exhibit motor defects which lead to hind leg paralysis and tremor. During development, Schwann cells that lack laminin gamma1 were present in peripheral nerves, and proliferated and underwent apoptosis similar to control mice. However, they were unable to differentiate and synthesize myelin proteins, and therefore unable to sort and myelinate axons. In mutant mice, after sciatic nerve crush, the axons showed impaired regeneration. These experiments demonstrate that laminin is an essential component for axon myelination and regeneration in the PNS.     

Identification of the increased expression of monocyte chemoattractant protein-1, cathepsin S, UPIX-1, and other genes in dystrophin-deficient mouse muscles by suppression subtractive hybridization

The lack of dystrophin results in muscular dystrophy characterized by degeneration, inflammation, and partial regeneration of skeletal muscles. The fate of these muscles may be determined by the extent of adaptation to the defect and the efficiency of regeneration that is affected by inflammatory cells. We have used suppression subtractive hybridization and quantitative Northern blot analysis to identify differentially expressed genes. Increased expression of murine monocyte chemoattractant protein-1 (JE/MCP-1), cathepsin S, UPIX-1, nmb, cathepsin B, and lysozyme M mRNAs were identified in 2-month-old mdx mouse leg muscles. UPIX-1 is a novel gene. Although it was not expressed in control muscles, it was expressed in control brain, heart, and spleen. JE/MCP-1 and cathepsin S proteins in mdx muscles, as well as JE/MCP-1 protein in the serum of mdx mice were also detected. JE/MCP-1 may be responsible for attraction of inflammatory cells, and cathepsin S, a potent elastolytic protease, may contribute to the remodeling of the extracellular matrix that is required for the migration of these cells to the injured muscles.