The clonal composition of biramous and uniramous arthropod limbs

We present the first comparative cell lineage analysis of uniramous and biramous limbs of an arthropod, the crustacean Orchestia cavimana. Via single cell labelling of the cells that are involved in limb development, we are able to present the first complete clonal composition of an arthropod limb. We show that the two main branches of crustacean limbs, exopod and endopod, are formed by a secondary subdivision of the growth zone of the main limb axis. Additional limb outgrowths such as exites result from the establishment of new axes. In contrast to general belief, uniramous limbs in Orchestia are not formed by the loss of the exopod but by suppression of the split into exopod and endopod. Our results offer a developmental approach to discriminate between the different kinds of branches of arthropod appendages. This leads to the conclusion that a 'true' biramous limb comprising an endopod and an exopod might have occurred much later in euarthropod evolution than has previously been thought, probably either in the lineage of the Mandibulata or that of the Tetraconata.     

A developmentally based categorization of branching in Trifolium repens L.: influence of nodal roots

This study describes the successive stages of development of branches from axillary buds in fully rooted plants of Trifolium repens grown in near optimal conditions, and the way in which this developmental pathway differs when nodal root formation is prevented as plants grow out from a rooted base. Cuttings of a single genotype were established in a glasshouse with nodal root systems on the two basal phytomers and grown on so that nodal rooting was either permitted (+R) or prevented (-R). In +R plants, axillary tissues could be assigned to one of four developmental categories: unemerged buds, emerged buds, unbranched lateral branches or secondarily branched lateral branches. In -R plants, branch development was retarded, with the retardation becoming increasingly pronounced as the number of -R phytomers on the primary stolon increased. Retarded elongation of the internodes of lateral shoots on -R plants resulted in the formation of a distinct fifth developmental category: short shoots (defined as branches with two or more leaves but with mean internode length equal to, or less than, 10% of that of the immediately proximal internode on the parent stolon) which had reduced phytomer appearance rates but retained the potential to develop into lateral branches. Transfer of +R plants to -R conditions, and vice versa, after 66 d demonstrated that subsequent branch development was wholly under the control of the youngest nodal root present, regardless of the age and number of root systems proximal to it.     

A complex role for distal-less in crustacean appendage development.

The developing leg of Drosophila is initially patterned by subdivision of the leg into proximal and distal domains by the activity of the homeodomain proteins Extradenticle (Exd) and Distal-less (Dll). These early domains of gene expression are postulated to reflect a scenario of limb evolution in which an undifferentiated appendage outgrowth was subdivided into two functional parts, the coxapodite and telopodite. The legs of most arthropods have a more complex morphology than the simple rod-shaped leg of Drosophila. We document the expression of Dll and Exd in two crustacean species with complex branched limbs. We show that in these highly modified limbs there is a Dll domain exclusive of Exd but there is also extensive overlap in Exd and Dll expression. While arthropod limbs all appear to have distinct proximal and distal domains, those domains do not define homologous structures throughout arthropods. In addition, we find a striking correlation throughout the proximal/distal extent of the leg between setal-forming cells and Dll expression. We postulate that this may reflect a pleisiomorphic function of Dll in development of the peripheral nervous system. In addition, our results confirm previous observations that branch formation in multiramous arthropod limbs is not regulated by a simple iteration of the proximal/distal patterning module employed in Drosophila limb development.     

The autopod: Its formation during limb development

The autopod, including the mesopodium and the acropodium, is the most distal part of the tetrapod limb, and developmental mechanisms of autopod formation serve as a model system of pattern formation during development. Cartilage rudiments of the autopod develop after proximal elements have differentiated. The autopod region is marked by a change in the expression of two homeobox genes: future autopod cells are first Hoxa11/Hoxa13 -double-positive and then Hoxa13 -single-positive. The change in expression of these Hox genes is controlled by upstream mechanisms, including the retinoic acid pathway, and the expression of Hoxa13 is connected to downstream mechanisms, including the autopod-specific cell surface property mediated by molecules, including cadherins and ephrins/Ephs, for cell-to-cell communication and recognition. Comparative analyses of the expression of Hox genes in fish fins and tetrapod limb buds support the notion on the origin of the autopod in vertebrates. This review will focus on the cellular and molecular regulation of the formation of the autopod during development and evolutionary developmental aspects of the origin of the autopod.     

Larval legs of mulberry silkworm Bombyx mori are prototypes for the adult legs

Morphological diversity of leg appendages is one of the hallmarks of developmental evolution. Limbs in insects may develop either from their embryonic prototypes or from imaginal discs harbored inside the larva. Bombyx mori (B. mori), a Lepidopteran insect, develops adult wings from larval wing imaginal discs. However, it has been debated whether the adult legs of B. mori arise from imaginal discs or from the larval legs. Here we addressed how the larval legs relate to their adult counterparts. We present the morphological landmarks during early leg development. We used expression of developmental genes like Distalless and extradenticle to mark leg primordia. Finally, we employed classical excision approach to develop a fate map of the adult leg. Excision and ablation of thoracic legs along proximo-distal axis at various times during larval development resulted in the loss of corresponding adult leg segments. Our data suggest that B. mori legs develop from larval appendages rather than leg imaginal discs.     

Developmental morphology of limb reduction in Hemiergis (Squamata: Scincidae): chondrogenesis,osteogenesis, and heterochrony

Digit loss is a common theme in tetrapod evolution that may involve changes in several developmental processes. The skink genus Hemiergis provides an ideal model to study these processes in closely related taxa: within three Western Australian Hemiergis species, digit quantity ranges between two and five. For three consecutive reproductive seasons, gravid females of Hemiergis were collected in the field and their embryos prepared for histological analysis of limb skeletal development (chondrogenesis and osteogenesis). Comparative studies of skeletal developmental morphology demonstrate that limbs with fewer than five digits do not result from a simple truncation of a putative ancestral (five-digit) developmental program. The developmental and adult morphologies in two-, three-, and four-digit Hemiergis are neither predicted nor explained by a simple model of heterochrony involving either chondrogenesis or osteogenesis. In postnatal Hemiergis, digit number and relative limb length do not correlate in a simple linear fashion. Instead, limb size and digit reduction may correlate with substrate conditions and burrowing behavior.     

Limb chondrogenesis of the seepage salamander, Desmognathus aeneus (amphibia: plethodontidae)

Salamanders are infrequently mentioned in analyses of tetrapod limb formation, as their development varies considerably from that of amniotes. However, urodeles provide an opportunity to study how limb ontogeny varies with major differences in life history. Here we assess limb development in Desmognathus aeneus, a direct-developing salamander, and compare it to patterns seen in salamanders with larval stages (e.g., Ambystoma mexicanum). Both modes of development result in a limb that is morphologically indistinct from an amniote limb. Developmental series of A. mexicanum and D. aeneus were investigated using Type II collagen immunochemistry, Alcian Blue staining, and whole-mount TUNEL staining. In A. mexicanum, as each digit bud extends from the limb palette Type II collagen and proteoglycan secretion occur almost simultaneously with mesenchyme condensation. Conversely, collagen and proteoglycan secretion in digits of D. aeneus occur only after the formation of an amniote-like paddle. Within each species, Type II collagen expression patterns resemble those of proteoglycans. In both, distal structures form before more proximal structures. This observation is contrary to the proximodistal developmental pattern of other tetrapods and may be unique to urodeles. In support of previous findings, no cell death was observed during limb development in A. mexicanum. However, apoptotic cells that may play a role in digit ontogeny occur in the limbs of D. aeneus, thereby suggesting that programmed cell death has evolved as a developmental mechanism at least twice in tetrapod limb evolution.     

External morphology of limb development in the amphipod <Emphasis Type="Italic">Orchestia cavimana</Emphasis> (Crustacea,Malacostraca, Peracarida)

The external morphology of limb development in Orchestia cavimana is examined by scanning electron microscopy and fluorescence staining from the appearance of the first limb buds until hatching. As other amphipods, O. cavimana undergoes direct development and the degree of segmental differentiation shows a more or less continual decrease in anteroposterior direction. Limbs form ventrally as small buds, which elongate and divide into podomeres early in development. This early subdivision largely corresponds with the limb segmentation of the hatchling. When the post-naupliar limbs start to develop, the germ band begins to split into two halves along the midline, so that the trunk limbs transiently occupy a very dorsolateral position. After the germ band has closed again, the differentiation into the characteristic amphipodan tagmata (cephalothorax, pereon, pleon) takes place and the limb podomeres lose their round-shape. The late embryo is covered by a so-called intermediate cuticle, which is formed after an embryonic moult and shed after hatching. The early development of O. cavimana reveals the Anlage of a vestigial seventh pleonic segment that is assumed to belong to the ground pattern of malacostracans, but is retained as a free, limbless segment only in adult Leptostraca. A transient subdivision of the proximal segment of the pleopods suggests the occurrence of a coxa and a basis in these limbs. The mandible attains its upright, adult position via a characteristic bending process that is strikingly similar to that in Archaeognatha (Insecta).     

Muscle precursor cells in the developing limbs of two isopods (Crustacea, Peracarida): an immunohistochemical study using a novel monoclonal antibody against myosin heavy chain

In the hot debate on arthropod relationships, Crustaceans and the morphology of their appendages play a pivotal role. To gain new insights into how arthropod appendages evolved, developmental biologists recently have begun to examine the expression and function of Drosophila appendage genes in Crustaceans. However, cellular aspects of Crustacean limb development such as myogenesis are poorly understood in Crustaceans so that the interpretative context in which to analyse gene functions is still fragmentary. The goal of the present project was to analyse muscle development in Crustacean appendages, and to that end, monoclonal antibodies against arthropod muscle proteins were generated. One of these antibodies recognises certain isoforms of myosin heavy chain and strongly binds to muscle precursor cells in malacostracan Crustacea. We used this antibody to study myogenesis in two isopods, Porcellio scaber and Idotea balthica (Crustacea, Malacostraca, Peracarida), by immunohistochemistry. In these animals, muscles in the limbs originate from single muscle precursor cells, which subsequently grow to form multinucleated muscle precursors. The pattern of primordial muscles in the thoracic limbs was mapped, and results compared to muscle development in other Crustaceans and in insects. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The structure of the normal puparium and in segmentation disorders in Drosophila

In Drosophila puparium is a cuticular mould of external structures formed by the larval cells and can, therefore, preserve traces of developmental defects which appeared in embryogenesis. In this way ontogenetic relationship between embryonic defects and developmental defects in adult individuals can be established. The normal pattern of segmentation in puparia of Drosophila melanogaster was established by comparing segmentation defects in adult flies and corresponding puparia. Boundaries of segment and compartments have been determined, imaginal rudiments of abdominal segments (nests of histoblasts) and rudiments of trachea have been localized on puparia.     

The control of morph development in the parasitic nematode Strongyloides ratti.

The parasitic nematode Strongyloides ratti has a complex life cycle. The progeny of the parasitic females can develop into three distinct morphs, namely directly developing infective third-stage larvae (iL3s), free-living adult males and free-living adult females. We have analysed of the effect of host immune status (an intra-host factor), environmental temperature (an extra-host factor) and their interaction on the proportion of larvae that develop into these three morphs. The results are consistent with the developmental decision of larvae being controlled by at least two discrete developmental switches. One is a sex-determination event that is affected by host immune status and the other is a switch between alternative female morphs that is affected by both host immune status and environmental temperature. These findings clarify the basis of the life cycle of S. ratti and demonstrate how such complex life cycles can result from a combination of simple developmental switches.     

Structure and development of setae on the thoracic limbs of the anostracan crustacean, Thamnocephalus platyurus

Setae are a prominent feature of arthropod limbs. In taxa where the limbs develop during the larval phase, developing setae are an integral part of the developing limb bud and their differentiation cannot easily be separated from the early patterning and formation of the overall limb. Here I describe the morphogenesis and adult setae in a branchiopod crustacean, the anostracan, Thamnocephalus platyurus. The majority of the setae on the limbs are non-innervated plumose setae that are formed from six underlying cells. Because branchiopods are often sampled in comparative studies of limb development, the details of the cellular morphogenesis of their limbs provide a necessary basis for studies of limb patterning.     

Double anterior chick limb buds and models for cartilage rudiment specification

Most models for the specification of the skeletal elements in the developing limb bud are based on a chemical specification well before overt cartilage differentiation. By contrast, a physico-mechanical model proposes that the process of condensation--an early feature of cartilage differentiation--is itself the basis for patterning the elements. The models thus make quite different predictions as to when the rudiments are specified. Double anterior limb buds have been constructed at stages earlier than condensation, with the expectation that, if specification of the humerus occurs before cartilage condensation, then limbs containing two humeri should develop, since the presumptive humerus lies largely in the anterior region. The development of anterior and posterior parts, on their own, was in general, consistent with the fate map; both developed a humerus that was thinner than normal. Double anterior limbs developed two humeri in 28% of cases and a much thicker humerus in 39%. These results strongly support models based on an early specification of limb rudiments and cannot be accounted for by the physical model. Double anterior limbs in which the two parts were from different stages, developed such that a digit 3 could lie adjacent to the radius, giving further striking evidence for early specification and local autonomy of development.     

Analysis of inflorescence organogenesis in eastern gamagrass, Tripsacum dactyloides (Poaceae): the wild type and the gynomonoecious gsf1 mutant

Inflorescence organogenesis of a wild-type and a gynomonoecious (pistillate) mutant in Tripsacum dactyloides was studied using scanning electron microscopy. SEM (scanning electron microscope) analysis indicated that wild-type T. dactyloides (Eastern gamagrass) expressed a pattern of inflorescence organogenesis that is observed in other members of the subtribe Tripsacinae (Zea: maize and teosinte), family Poaceae. Branch primordia are initiated acropetally along the rachis of wild-type inflorescences in a distichous arrangement. Branch primordia at the base of some inflorescences develop into long branches, which themselves produce an acropetal series of distichous spikelet pair primordia. All other branch primordia function as spikelet pair primordia and bifurcate into pedicellate and sessile spikelet primordia. In all wild-type inflorescences development of the pedicellate spikelets is arrested in the proximal portion of the rachis, and these spikelets abort, leaving two rows of solitary sessile spikelets. Organogenesis of spikelets and florets in wild-type inflorescences is similar to that previously described in maize and the teosintes. Our analysis of gsf1 mutant inflorescences reveals a pattern of development similar to that of the wild type, but differs from the wild type in retaining (1) the pistillate condition in paired spikelets along the distal portion of the rachis and (2) the lower floret in sessile spikelets in the proximal region of the rachis. The gsf1 mutation blocks gynoecial tissue abortion in both the paired-spikelet and the unpaired-spikelet zone. This study supports the hypothesis that both femaleness and maleness in Zea and Tripsacum inflorescences are derived from a common developmental pathway. The pattern of inflorescence development is not inconsistent with the view that the maize ear was derived from a Tripsacum genomic background.     

A new stem craniate from the Ordovician of Morocco and the search for the sister group of the craniata

The investigation of the development of the trigeminal jaw adductor musculature in the turtle Chelydra serpentina documents the early aggregation of muscle rudiments around the innervating nerve branches, probably a consequence of inductive interaction. This may explain the early continuity of the intramandibularis with the intermandibularis muscle. Several aspects of muscle development differ in the turtle as compared to lizards. These differences highlight the fact that conjectures of homology, based on a static topographical correspondence of adult structures, cannot capture the dynamics of the developmental process. The intramandibularis muscle of turtles, comparable to that of crocodiles, represents a plesiomorphous structure which is not homologous to the intramandibularis muscle of lacertoid lizards, a derived feature of the Lacertoidea. A derived feature of the chelonian jaw adductor musculature is the posterodorsal expansion of the external adductor along a supraoccipital crest, developing according to a pattern of Haeckelian recapitulation. Muscle development serves to corroborate the concept of a monophyletic Eureptilia, including diapsids and synapsids, as opposed to the (paraphyletic) Anapsida. The impact of the differentiation of the external adductor into a pulley system on cranial kinesis is analysed in biomechanical terms.