Embryonic development of the sensory innervation of the antenna of the grasshopper Schistocerca gregaria

The establishment of the sensory nervous system of the antenna of the grasshopper Schistocerca gregaria was examined using immunocytochemical methods and in the light of the appendicular and articulated nature of this structure. The former is demonstrated first by the expression pattern of the segment polarity gene engrailed in the head neuromere innervating the antenna, the deutocerebrum. Engrailed expression is present in identified deutocerebral neuroblasts and, as elsewhere in the body, is continuous with cells of the posterior epithelium of the associated appendage, in this case the antenna. Second, early expression of the glial homeobox gene reversed polarity (repo) in the antenna is by a stereotypic pair of cells at the antenna base, a pattern we show is repeated metamerically for each thoracic appendage of the embryo. Subsequently, three regions of Repo expression (A1, A2, A3) are seen within the antenna, and may represent a preliminary form of articulation. Bromodeoxyuridine incorporation reveals that these regions are sites of intense cell differentiation. Neuron-specific horseradish peroxidase and Lazarillo expression confirm that the pioneers of the ventral and dorsal tracts of the antennal sensory nervous system are amongst these differentiating cells. Sets of pioneers appear simultaneously in several bands and project confluent axons towards the antennal base. We conclude that the sensory nervous system of the antenna is not pioneered from the tip of the antenna alone, but in a stepwise manner by cells from several zones. The early sensory nervous systems of antenna, maxilla and leg therefore follow a similar developmental program consistent with their serially homologous nature.     

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.     

The structure and function of serially homologous leg motor neurons in the locust. II. Physiology

Simultaneous intracellular recordings were made from pairs of motor neurons in the pro- or mesothoracic ganglion of the locust. Though central connections were sought between pairs of motor neurons, none were found. This is in sharp contrast to the findings that flexor and extensor tibiae neurons in the metathoracic ganglion make certain connections between themselves (Hoyle and Burrows, 1973; Heitler and Burrows, 1977a). As the previously mentioned authors believed that the metathoracic flexor-extensor connections were used as part of the motor program for jumping and kicking, the present results strongly support their hypothesis. Common PSPs have been found in a variety of pairs of motor neurons. Of note are common PSPs of the same sign to antagonists. Different innervation patterns have been found for the flexor and extensor muscles. It is proposed that serially homologous motor neurons serving similar functions are, to a first approximation, similar in the locust. Serially homologous motor neurons serving different functions will, in most cases, have altered structures and/or functions.     

Embryonic development of the innervation of the locust extensor tibiae muscle by identified neurons: formation and elimination of inappropriate axon branches

Intracellular dye fills have been used to reveal the pattern of embryonic growth of each of the four neurons which innervate the extensor tibiae muscle (ETi) of the hind leg of the locust. The growth cone of the slow extensor tibiae motoneuron (SETi), the first of the four neurons to leave the central nervous system, pioneers nerve 3 (N3). The fast extensor motoneuron (FETi), the next neuron to grow out, follows earlier outgrowing motoneurons into the periphery in nerve 5 (N5) and then rejoins SETi in N3. As it transfers from N5 to N3, it is transiently dye-coupled to the Tr1 pioneer neuron which spans the gap between the two nerves. It then follows SETi onto the ETi muscle in the femur. The common inhibitory neuron and the dorsal unpaired median neuron (DUMETi) follow SETi and FETi in nerves 3B2 and 5B1, respectively. SETi's growth cone requires almost twice as long to reach ETi as those of the three later motoneurons, all of which follow preexisting neural pathways. At least three of the four developing motoneurons form one or more axon branches not found in the adult. These branches may occur (1) at segmental boundaries; (2) where the nerve, which the growth cone is following, itself branches or the growth cone encounters another nerve; or (3) when the axon continues to grow beyond its target muscle. These findings contrast with the apparent absence of inappropriate axon branches in another developing locust neuromuscular system and during the innervation of zebrafish myotomes, but resemble in some ways the transient production of inappropriate axonal branches reported for embryonic leech motoneurons.     

Common motor mechanisms support body load in serially homologous legs of cockroaches in posture and walking

We studied the mechanisms underlying support of body load in posture and walking in serially homologous legs of cockroaches. Activities of the trochanteral extensor muscle in the front or middle legs were recorded neurographically while animals were videotaped. Body load was increased via magnets attached to the thorax and varied through a coil below the substrate. In posture, tonic firing of the slow trochanteral extensor motoneuron (Ds) in each leg was strongly modulated by changing body load. Rapid load increases produced decreases in body height and sharp increments in extensor firing. The peak of extensor activity more closely approximated the maximum velocity of body displacement than the body position. In walking, extensor bursts in front and middle legs were initiated during swing and continued into the stance phase. Moderate tonic increases in body load elicited similar, specific, phase dependent changes in both legs: extensor firing was not altered in swing but was higher after foot placement in stance. These motor adjustments to load are not anticipatory but apparently depend upon sensory feedback. These data are consistent with previous findings in the hind legs and support the idea that body load is countered by common motor mechanisms in serially homologous legs.     

Independent development of sensory and motor innervation patterns in embryonic chick hindlimbs

Previous studies suggest that sensory axon outgrowth is guided by motoneurons, which are specified to innervate particular target muscles. Here we present evidence that questions this conclusion. We have used a new approach to assess the pathfinding abilities of bona fide sensory neurons, first by eliminating motoneurons after neural crest cells have coalesced into dorsal root ganglia (DRG) and second by challenging sensory neurons to innervate muscles in a novel environment created by shifting a limb bud rostrally. The resulting sensory innervation patterns mapped with the lipophilic dyes DiI and DiA showed that sensory axons projected robustly to muscles in the absence of motoneurons, if motoneurons were eliminated after DRG formation. Moreover, sensory neurons projected appropriately to their usual target muscles under these conditions. In contrast, following limb shifts, muscle sensory innervation was often derived from inappropriate segments. In this novel environment, sensory neurons tended to make more "mistakes" than motoneurons. Whereas motoneurons tended to innervate their embryologically correct muscles, sensory innervation was more widespread and was generally from more rostral segments than normal. Similar results were obtained when motoneurons were eliminated in embryos with limb shifts. These findings show that sensory neurons are capable of navigating through their usual terrain without guidance from motor axons. However, unlike motor axons, sensory axons do not appear to actively seek out appropriate target muscles when confronted with a novel terrain. These findings suggest that sensory neuron identity with regard to pathway and target choice may be unspecified or quite plastic at the time of initial axon outgrowth.     

Postnatal development of autonomic and sensory innervation of thoracic hairy skin in the rat

Summary Histochemical, immunocytochemical, and radioenzymatic techniques were used to examine the neurotransmitter-related properties of the innervation of thoracic hairy skin in rats during adulthood and postnatal development. In the adult, catecholamine-containing fibers were associated with blood vessels and piloerector muscles, and ran in nerve bundles throughout the dermis. The distribution of tyrosine hydroxylase (TH)-immunoreactive (IR) fibers was identical. Neuronal fibers displaying neuropeptide Y (NPY) immunoreactivity were seen in association with blood vessels. Double-labeling studies suggested that most, if not all, NPY-IR fibers were also TH-IR and likewise most, if not all, vessel-associated TH-IR fibers were also NPY-IR. Calcitonin gene-related peptide (CGRP)-IR fibers were observed near and penetrating into the epidermis, in close association with hair follicles and blood vessels, and in nerve bundles. A similar distribution of substance P (SP)-IR fibers was evident. In adult animals treated as neonates with the sympathetic neurotoxin 6-hydroxydopamine, a virtual absence of TH-IR and NPY-IR fibers was observed, whereas the distribution of CGRP-IR and SP-IR fibers appeared unaltered. During postnatal development, a generalized increase in the number, fluorescence intensity, and varicose morphology of neuronal fibers displaying catecholamine fluorescence, NPY-IR, CGRP-IR, and SP-IR was observed. By postnatal day 21, the distribution of the above fibers had reached essentially adult levels, although the density of epidermal-associated CGRP-IR and SP-IR fibers was significantly greater than in the adult. The following were not evident in thoracic hairy skin at any timepoint examined: choline acetyltransferase activity, acetylcholinesterase histochemical staining or immunoreactivity, fibers displaying immunoreactivity to vasoactive intestinal peptide, cholecystokinin, or leucine-enkephalin. The present study demonstrates that the thoracic hairy skin in developing and adult rats receives an abundant sympathetic catecholaminergic and sensory innervation, but not a cholinergic innervation.     

Postnatal development of sympathetic and sensory innervation of the rhesus monkey ovary.

We have used immunofluorescence to study the postnatal development of the sympathetic and sensory innervation to the rhesus monkey (Macaca mulatta) ovary. Sympathetic nerves were identified as adrenergic by their content of tyrosine hydroxylase (TH)-like immunoreactivity and as peptidergic by the presence of neuropeptide Y (NPY). Fibers containing substance P (SP) or calcitonin gene-related peptide (CGRP)-like immunoreactivity were considered as sensory, whereas vasoactive intestinal peptide (VIP)-positive fibers were only defined as peptidergic because VIP may be present in both sympathetic and sensory nerves. Ovaries from neonatal (2-mo-old), juvenile (9-18-mo-old), peripubertal (3-3.5-yr-old), adult (9-14-yr-old), and senescent (20-27-yr-old) monkeys were studied. At all ages, with the exception of senescence, TH-, NPY-, and VIP-containing fibers were associated with follicles in different developmental stages. In peripubertal and adult animals, some primordial follicles were found to be selectively innervated by VIPergic fibers that almost completely encircled each follicle. Both sympathetic and VIP fibers were also detected in the interstitial tissue and associated with the ovarian vasculature at all ages. The number of sympathetic and VIP fibers increased significantly (p < 0.01) between 2 mo and 9-18 mo of age, and again increased (p < 0.01) around the age of puberty (approximately 3 yr of age). After this time, the number of NPY and TH fibers remained constant. Conversely, the number of VIP fibers decreased (p < 0.05) by 9-14 yr of age, but remained constant thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Embryonic development of the insect central complex: insights from lineages in the grasshopper and Drosophila

The neurons of the insect brain derive from neuroblasts which delaminate from the neuroectoderm at stereotypic locations during early embryogenesis. In both grasshopper and Drosophila, each developing neuroblast acquires an intrinsic capacity for neuronal proliferation in a cell autonomous manner and generates a specific lineage of neural progeny which is nearly invariant and unique. Maps revealing numbers and distributions of brain neuroblasts now exist for various species, and in both grasshopper and Drosophila four putatively homologous neuroblasts have been identified whose progeny direct axons to the protocerebral bridge and then to the central body via an equivalent set of tracts. Lineage analysis in the grasshopper nervous system reveals that the progeny of a neuroblast maintain their topological position within the lineage throughout embryogenesis. We have taken advantage of this to study the pioneering of the so-called w, x, y, z tracts, to show how fascicle switching generates central body neuroarchitecture, and to evaluate the roles of so-called intermediate progenitors as well as programmed cell death in shaping lineage structure. The novel form of neurogenesis involving intermediate progenitors has been demonstrated in grasshopper, Drosophila and mammalian cortical development and may represent a general strategy for increasing brain size and complexity. An analysis of gap junctional communication involving serotonergic cells reveals an intrinsic cellular organization which may relate to the presence of such transient progenitors in central complex lineages.     

Embryonic development of the pars intercerebralis/central complex of the grasshopper

 We have studied the embryonic development of the pars intercerebralis/central complex in the brain of the grasshopper using immunocytochemical and histochemical techniques. Expression of the cell-surface antigen lachesin reveals that the neuroblasts of the pars intercerebralis first differentiate from the neuroectoderm at around 26% of embryogenesis. Differentiation of medial and lateral neuroblasts occurs first. By the 28% stage a more or less uniform sheet of 20 neuroblasts has formed. As a result of both cell proliferation and cell translocation, the pars intercerebralis proliferative cluster in each hemisphere expands so that at 30% the most medial neuroblasts lie apposed at the midline. We followed the further development of the pars intercerebralis of each brain hemisphere using bromo-deoxy-uridine incorporation and osmium-ethyl-gallate staining. Within the pars intercerebralis itself, the neuroblasts redistribute into discrete subsets. The neuroblasts of each subset generate clusters of progeny which extend in a stereotypic, subset-specific direction in the brain. We have used this feature to identify one subset of four neuroblasts as being the likely progenitor cells for four clusters of embryonic neurons (W, X, Y, Z) which develop at around 55% of embryogenesis. We show that these progeny project axons via four discrete fascicles (w, x, y, z) into the embryonic central complex. At the single cell level, Golgi impregnation reveals that the axons from these neighbouring cell clusters remain discrete, and those from the same cluster tightly fasciculated, as they project into the central complex, consistent with a modular organization for this brain region. Received: 16 June 1997 / Accepted: 25 June 1997     

Embryonic stem cells alone are able to support fetal development in the mouse

The developmental potential of embryonic stem (ES) cells versus 3.5 day inner cell mass (ICM) was compared after aggregation with normal diploid embryos and with developmentally compromised tetraploid embryos. ES cells were capable of colonizing somatic tissues in diploid aggregation chimeras but less efficiently than ICMs of the same genotype. When ICM in equilibrium with tetraploid and ES in equilibrium with tetraploid chimeras were made, the newborns were almost all completely ICM- or ES-derived, as judged by GPI isozyme analysis, but tetraploid cells were found in the yolk sac endoderm and trophectoderm lineage. Investigation of ES contribution in 13.5 day ES in equilibrium with tetraploid chimeras by DNA in situ hybridization confirmed the complete tetraploid origin of the placenta (except the fetal blood and blood vessels) and the yolk sac endoderm. However, the yolk sac mesoderm, amnion and fetus contained only ES-derived cells. ES-derived newborns failed to survive after birth, although they had normal birthweight and anatomically they appeared normal. This phenomenon remains unexplained at the moment. The present results prove that ES cells are able to support complete fetal development, resulting in ES-derived newborns, and suggest a useful route for studying the development of genetically manipulated ES cells in all fetal lineages.     

Morphology and ultrastructure of the sensory appendages of a kinorhynch introvert

The structure and arrangement of appendages (scalids) on the head of the homalorhagid kinorhynch Kinorhynchus phyllotropis Brown & Higgins, 1983 are named, described and illustrated. In adults of this species, seven rings of external scalids are separated by segment boundary structures from the oral styles and three rings of internal scalids. All of these appendages contain ciliated receptor cells which pass to pores at the scalid tips, and all but the two anterior rings are jointed. All of these appendages are radially arranged in multiples of five, and closely associated with the ten lobed circumoral nerve ring. The most posterior scalid ring consists of fourteen trichoscalids, of which six are longer than the other eight. The six longer trichoscalids are arranged in bilateral symmetry, two to each of the three facets of the body. Similarities between scalid arrangement in Kinorhyncha and Loricifera are discussed.