Regenerative specificity of motor axons when reinnervation is partially suppressed

We asked whether regenerating hindlimb motor axons would innervate inappropriate hindlimb regions if competition from appropriate innervation were prevented. The three ventral roots that innervate the hindlimb in the bullfrog (Rana catesbeiana) tadpole were transected, and the two more rostral roots were ligated to prevent regeneration. The most caudal root, which primarily supplies more distal limb musculature in unoperated tadpoles, was left free to regenerate. The specificity of regeneration was assessed by retrogradely labeling spinal motoneurons with HRP placed in the ventral thigh, a region that receives most of its innervation from the ligated roots. Despite the lack of competition from appropriate innervation, the regenerating root did not provide substantial innervation to proximal limb musculature. The same result was obtained in tadpoles operated upon at stages when regeneration of motor axons is specific and in tadpoles at stages when regenerating motor axons do not reinnervate their appropriate targets (Farel and Bemelmans, 1986), although the mechanisms in each case are likely different.     

Restricted patterns of Hoxd10 and Hoxd11 set segmental differences in motoneuron subtype complement in the lumbosacral spinal cord

During normal vertebrate development, Hoxd10 and Hoxd11 are expressed by differentiating motoneurons in restricted patterns along the rostrocaudal axis of the lumbosacral (LS) spinal cord. To assess the roles of these genes in the attainment of motoneuron subtypes characteristic of LS subdomains, we examined subtype complement after overexpression of Hoxd10 or Hoxd11 in the embryonic chick LS cord and in a Hoxd10 loss-of-function mouse embryo. Data presented here provide evidence that Hoxd10 defines the position of the lateral motor column (LMC) as a whole and, in rostral LS segments, specifically promotes the development of motoneurons of the lateral subdivision of the lateral motor column (LMCl). In contrast, Hoxd11 appears to impart a caudal and medial LMC (LMCm) identity to some motoneurons and molecular profiles suggestive of a suppression of LMC development in others. We also provide evidence that Hoxd11 suppresses the expression of Hoxd10 and the retinoic acid synthetic enzyme, retinaldehyde dehydrogenase 2 (RALDH2). In a normal chick embryo, Hoxd10 and RALDH2 are expressed throughout the LS region at early stages of motoneuron differentiation but their levels decline in Hoxd11-expressing caudal LS segments that ultimately contain few LMCl motoneurons. We hypothesize that one of the roles played by Hoxd11 is to modulate Hoxd10 and local retinoic acid levels and thus, perhaps define the caudal boundaries of the LMC and its subtype complement.     

Vitamin A induced homeotic hindlimb formation on dorsal and ventral sides of regenerating tissue of amputated tails of Japanese brown frog tadpoles

When anuran tadpoles are treated with vitamin A after tail amputation, hindlimb‐like structures can be generated instead of the lost tail part at the amputation site. This homeotic transformation was initially expected to be a key to understanding the body plan of vertebrates. Unfortunately, homeotic limb formation has been reproduced in only some Indian frog species and a European species, but not in experimental anurans such as Xenopus laevis or Rana catesbeiana. Consequently, this fascinating phenomenon has not been well analyzed, especially at the molecular level. In addition, the initial processes of ectopic limb development are also unclear because morphological changes in the early phases have not been analyzed in detail. In this study, we report the induction of homeotic transformation using Japanese brown frogs and present a detailed morphological analysis. Unexpectedly, the ectopic limbs developed not only at the ventral sites, but also at the dorsal sites of the tail regenerates of vitamin A‐treated tadpoles. The relationship between position and axial orientation of ectopic limbs suggested the double duplication of positional value order along the rostral‐caudal axis and the dorsal‐ventral axis of the tail regenerates.     

Limb Initiation and Development Is Normal in the Absence of the Mesonephros

With rapid progress in understanding the genes that control limb development and patterning interest is becoming focused on the factors that permit the emergence of the limb bud. The current hypothesis is that FGF-8 from the mesonephros induces limb initiation. To test this, the inductive interaction between the Wolffian duct and intermediate mesoderm was blocked rostral to the limb field, preventing mesonephric differentiation while maintaining the integrity of the limb field. The experimental outcome was monitored by following expression ofcSim1andLmx1,molecular markers for the duct and the mesonephros, respectively. Evidence is presented that the intermediate mesoderm undergoes apoptosis when the inductive interaction with the Wolffian duct is blocked.fgf-8expression was undetectable in the mesonephric area of embryos with confirmed absence of mesonephros; nevertheless, limb buds formed and limb development was normal. The mesonephros in general, and specifically itsfgf-8expression, was shown to be unnecessary for limb initiation and development; the hypothesis linking the mesonephros and limb development is not supported. Further studies of axial influences on limb initiation should now concentrate on medial structures such as Hensen's node and paraxial mesoderm; the alternative that no axial influences are required should also be examined.     

Topographic motor projections in the limb imposed by LIM homeodomain protein regulation of ephrin-A:EphA interactions

The formation of topographic neural maps relies on the coordinate assignment of neuronal cell body position and axonal trajectory. The projection of motor neurons of the lateral motor column (LMC) along the dorsoventral axis of the limb mesenchyme constitutes a simple topographic map that is organized in a binary manner. We show that LIM homeodomain proteins establish motor neuron topography by coordinating the mediolateral settling position of motor neurons within the LMC with the dorsoventral selection of axon pathways in the limb. These topographic projections are established, in part, through LIM homeodomain protein control of EphA receptors and ephrin-A ligands in motor neurons and limb mesenchymal cells.     

Rotation of the tectal primordium reveals plasticity of target recognition in retinotectal projection

Retinotectal projection is precisely organized in a retinotopic manner. In normal projection, temporal retinal axons project to the rostral part of the tectum, and nasal axons to the caudal part of the tectum. The two-dimensional relationship between the retina and the tectum offers a useful experimental system for analysis of neuronal target recognition. We carried out rotation of the tectal primordium in birds at an early stage of development, around the 10-somite stage, to achieve a better understanding of the characteristics of target recognition, especially the rostrocaudal specificity of the tectum. Our results showed that temporal retinal axons projected to the rostral part of the rotated tectum, which was originally caudal, and that nasal axons projected to the caudal part of the rotated tectum, which was originally rostral. Therefore, the tectum that had been rotated at the 10-somite stage received normal topographic projection from the retinal ganglion cells. Rostrocaudal specificity of the tectum for target recognition is not determined by the 10-somite stage and is acquired through interactions between the tectal primordium and its surrounding structures.     

Spatial and temporal patterns of cell death in limb bud mesoderm after apical ectodermal ridge removal

A spatiotemporal pattern of cell death occurred in the chick wing and leg bud mesoderm after removal of apical ectodermal ridge at stages 18–20. Cells died in a region extending from the limb bud distal surface to 150–200 μm into the mesoderm. Limb buds from which ridge was removed at later stages in development did not exhibit a spatiotemporal pattern of cell death. In control experiments in which dorsal ectoderm was removed, a pattern of cell death did not occur. Removal of the ridge and part of the 150- to 200-μm zone of prospective cell death resulted in cell death in an area approximately equal to the amount of the zone remaining. After removal of all of the prospective zone of cell death plus the apical ridge, cell death was observed in the remaining limb bud mesoderm. In these limb buds, cell death occurred in a region in which it had not been seen in limb bud with apical ridge alone removed. We conclude that at stages 18–20 the mesodermal cells 150–200 μm beneath the ridge require the apical ridge to survive. More proximal mesodermal cells do not die after ridge removal alone, but apparently require the presence of the more distal mesoderm to survive. Whether this is a requirement for something intrinsic to the distal mesoderm or something it possesses by way of the ridge is unknown. After stage 23, the limb mesoderm cells do not die when the apical ridge is removed. Nevertheless, at the later stages, ridge continues to be required for limb bud proximal-distal elongation and the differentiation of distal limb elements.     

Contributions of muscles to terminal-swing knee motions vary with walking speed

Many children with cerebral palsy walk with diminished knee extension during terminal swing, at speeds much slower than unimpaired children. Treatment of these gait abnormalities is challenging because the factors that extend the knee during normal walking, over a range of speeds, are not well understood. This study analyzed a series of three-dimensional, muscle-driven dynamic simulations to determine whether the relative contributions of individual muscles and other factors to angular motions of the swing-limb knee vary with walking speed. Simulations were developed that reproduced the measured gait dynamics of seven unimpaired children walking at self-selected, fast, slow, and very slow speeds (7 subjects×4 speeds=28 simulations). In mid-swing, muscles on the stance limb made the largest net contribution to extension of the swing-limb knee at all speeds examined. The stance-limb hip abductors, in particular, accelerated the pelvis upward, inducing reaction forces at the swing-limb hip that powerfully extended the knee. Velocity-related forces (i.e., Coriolis and centrifugal forces) also contributed to knee extension in mid-swing, though these contributions were diminished at slower speeds. In terminal swing, the hip flexors and other muscles on the swing-limb decelerated knee extension at the subjects’ self-selected, slow, and very slow speeds, but had only a minimal net effect on knee motions at the fastest speeds. Muscles on the stance limb helped brake knee extension at the subjects’ fastest speeds, but induced a net knee extension acceleration at the slowest speeds. These data—which show that the contributions of muscular and velocity-related forces to terminal-swing knee motions vary systematically with walking speed—emphasize the need for speed-matched control subjects when attempting to determine the causes of a patient's abnormal gait.     

Genomic multipotentiality of differentiated somatic cells

Nuclear transplantations from several differentiated somatic cell types into amphibian oocytes and eggs revealed that their genome contains the genes required for the development of prefeeding tadpoles. In addition, erythrocyte nuclei directed the formation of feeding tadpoles (independent organisms) that advanced to larval stages with hind limb buds. Thus, the genome of several differentiated somatic cell types can undergo widespread activation and specify a multiplicity of cell types. Although evidence for the genetic totipotency of differentiated somatic cells is lacking, we speculate that the genetic totipotency of at least some differentiated somatic cell types still remains a tenable hypothesis.     

The role of hind limb flexor muscles during swimming in the toad, Bufo marinus

Most work examining muscle function during anuran locomotion has focused largely on the roles of major hind limb extensors during jumping and swimming. Nevertheless, the recovery phase of anuran locomotion likely plays a critical role in locomotor performance, especially in the aquatic environment, where flexing limbs can increase drag on the swimming animal. In this study, I use kinematic and electromyographic analyses to explore the roles of four anatomical flexor muscles in the hind limb of Bufo marinus during swimming: m. iliacus externus, a hip flexor; mm. iliofibularis and semitendinosus, knee flexors; and m. tibialis anticus longus, an ankle flexor. Two general questions are addressed: (1) What role, if any, do these flexors play during limb extension? and (2) How do limb flexors control limb flexion? Musculus iliacus externus exhibits a large burst of EMG activity early in limb extension and shows low levels of activity during recovery. Both m. iliofibularis and m. semitendinosus are biphasically active, with relatively short but intense bursts during limb extension followed by longer and typically weaker secondary bursts during recovery. Musculus tibialis anticus longus becomes active mid way through recovery and remains active through the start of extension in the next stroke. In conclusion, flexors at all three joints exhibit some activity during limb extension, indicating that they play a role in mediating limb movements during propulsion. Further, recovery is controlled by a complex pattern of flexor activation timing, but muscle intensities are generally lower, suggesting relatively low force requirements during this phase of swimming.     

Power output and work in different muscle groups during ergometer cycling

The aim of this study was to calculate the magnitude of the instantaneous muscular power output at the hip, knee and ankle joints during ergometer cycling. Six healthy subjects pedalled a weight-braked bicycle ergometer at 120 watts (W) and 60 revolutions per minute (rpm). The subjects were filmed with a cine camera, and pedal reaction forces were recorded from a force transducer mounted in the pedal. The muscular work at the hip, knee and ankle joint was calculated using a model based upon dynamic mechanics described elsewhere. The mean peak concentric power output was, for the hip extensors, 74.4 W, hip flexors, 18.0 W, knee extensors, 110.1 W, knee flexors, 30.0 W and ankle plantar flexors, 59.4 W. At the ankle joint, energy absorption through eccentric plantar flexor action was observed, with a mean peak power of 11.4 W and negative work of 3.4 J for each limb and complete pedal revolution. The energy production relationships between the different major muscle groups were computed and the contributions to the total positive work were: hip extensors, 27%; hip flexors, 4%; knee extensors, 39%; knee flexors, 10%; and ankle plantar flexors 20%.     

Commissural neurons of the electrosensory lateral line lobe of Apteronotus leptorhynchus: morphological and physiological characteristics

Extracellular injections of horseradish peroxidase were used to label commissural cells connecting the electrosensory lateral line lobes of the weakly electric fish Apteronotus leptorhynchus. Multiple commissural pathways exist; a caudal commissure is made up of ovoid cell axons, and polymorphic cells' axons project via a rostral commissure. Intracellular recording and labeling showed that ovoid cells discharge spontaneously at high rates, fire at preferred phases to the electric organ discharge, and respond to increased receptor afferent input with short latency partially adapting excitation. Ovoid cell axons ramify extensively in the rostro-caudal direction but are otherwise restricted to a single ELL subdivision. Polymorphic cells are also spontaneously active, but their firing is unrelated to the electric organ discharge waveform. They respond to increased receptor afferent activity with reduced firing frequency and response latency is long. Electrical stimulation of the commissural axons alters the behavior of pyramidal cells in the contralateral ELL. Basilar pyramidal cells are hyperpolarized and nonbasilar pyramidal cells are depolarized by this type of stimulation. The physiological results indicate that the ovoid cells participate in common mode rejection mechanisms and also suggest that the ELLs may function in a differential mode in which spatially restricted electrosensory stimuli can evoke heightened responses.Abbreviations ccELL caudal commissure of the ELL - CE contralaterally excited - DML dorsal molecular layer - ELL electrosensory lateral line lobe - EOD electric organ discharge - HRP horseradish peroxidase - IE ipsilaterally excited - MTI mouth-tail inverted - MTN mouth-tail normal - rcELL rostral commissure of the ELL - TRI transverse inverted - TRN transverse normal     

A distinct patterning mechanism of O and P cell fates in the development of the rostral segments of the leech Helobdella robusta: implications for the evolutionary dissociation of developmental pathway and morphological outcome

Despite a high degree of homonomy in the segmental organization of the ectoderm, the body plan of the leech is divided into two zones based on the distinct cell lineage patterns that give rise to the O/P portion of the segmental ectoderm. In the midbody and caudal segments, each segmental repeat of ectoderm arises in part from one 'o' blast cell and one 'p' blast cell. These two blast cells are positionally specified to distinct O and P fates, and give rise to differentiated descendant cells called O and P pattern elements, respectively. In the rostral segments, each segmental repeat of O and P pattern elements arises from a single 'op' blast cell. Based on their developmental fates and their responses to the ablation of neighboring cells, the granddaughters of the primary op blast cell are categorized into two O-type cells and two P-type cells. The O-type cells do not require the presence of the rest of the op blast cell clone for their normal development. By contrast, normal development of the P-type cells depends upon interactions with the other OP sublineages. Additional experiments showed that the O-type cells are the source of a repressive signal involved in the normal fate specification of the P-type cells. Our data suggest that the cell interactions involved in fate specification differ substantially in the rostral and midbody segments, even though the set of differentiated descendants produced by the rostral OP pathway and the midbody O and P pathways are very similar.     

Dystrophic Changes in Extraocular Muscles after Gamma Irradiation in mdx:utrophin+/? Mice

Extraocular muscles (EOM) have a strikingly different disease profile than limb skeletal muscles. It has long been known that they are spared in Duchenne (DMD) and other forms of muscular dystrophy. Despite many studies, the cause for this sparing is not understood. We have proposed that differences in myogenic precursor cell properties in EOM maintain normal morphology over the lifetime of individuals with DMD due to either greater proliferative potential or greater resistance to injury. This hypothesis was tested by exposing wild type and mdx:utrophin^+/− (het) mouse EOM and limb skeletal muscles to 18 Gy gamma irradiation, a dose known to inhibit satellite cell proliferation in limb muscles. As expected, over time het limb skeletal muscles displayed reduced central nucleation mirrored by a reduction in Pax7-positive cells, demonstrating a significant loss in regenerative potential. In contrast, in the first month post-irradiation in the het EOM, myofiber cross-sectional areas first decreased, then increased, but ultimately returned to normal compared to non-irradiated het EOM. Central nucleation significantly increased in the first post-irradiation month, resembling the dystrophic limb phenotype. This correlated with decreased EECD34 stem cells and a concomitant increase and subsequent return to normalcy of both Pax7 and Pitx2-positive cell density. By two months, normal het EOM morphology returned. It appears that irradiation disrupts the normal method of EOM remodeling, which react paradoxically to produce increased numbers of myogenic precursor cells. This suggests that the EOM contain myogenic precursor cell types resistant to 18 Gy gamma irradiation, allowing return to normal morphology 2 months post-irradiation. This supports our hypothesis that ongoing proliferation of specialized regenerative populations in the het EOM actively maintains normal EOM morphology in DMD. Ongoing studies are working to define the differences in the myogenic precursor cells in EOM as well as the cellular milieu in which they reside.     

T-cadherin expression alternates with migrating neural crest cells in the trunk of the avian embryo.

Trunk neural crest cells and motor axons move in a segmental fashion through the rostral (anterior) half of each somitic sclerotome, avoiding the caudal (posterior) half. This metameric migration pattern is thought to be caused by molecular differences between the rostral and caudal portions of the somite. Here, we describe the distribution of T-cadherin (truncated-cadherin) during trunk neural crest cell migration. T-cadherin, a novel member of the cadherin family of cell adhesion molecules was selectively expressed in the caudal half of each sclerotome at all times examined. T-cadherin immunostaining appeared graded along the rostrocaudal axis, with increasing levels of reactivity in the caudal halves of progressively more mature (rostral) somites. The earliest T-cadherin expression was detected in a small population of cells in the caudal portion of the somite three segments rostral to last-formed somite. This initial T-cadherin expression was observed concomitant with the invasion of the first neural crest cells into the rostral portion of the same somite in stage 16 embryos. When neural crest cells were ablated surgically prior to their emigration from the neural tube, the pattern of T-cadherin immunoreactivity was unchanged compared to unoperated embryos, suggesting that the metameric T-cadherin distribution occurs independent of neural crest cell signals. This expression pattern is consistent with the possibility that T-cadherin plays a role in influencing the pattern of neural crest cell migration and in maintaining somite polarity.     

Sex-ratio adjustment in response to local mate competition is achieved through an alteration of egg size in a haplodiploid spider mite

Sex-ratio adjustments are commonly observed in haplodiploid species. However, the underlying proximate mechanisms remain elusive. We investigated these mechanisms in Tetranychus urticae, a haplodiploid spider mite known to adjust sex ratio in response to the level of local mate competition (LMC). In this species, egg size determines fertilization probability, with larger eggs being more likely to be fertilized, and thus become female. We explored the hypothesis that sex-ratio adjustment is achieved through adjustment of egg size. By using spider mites from a large population, we found that females produced not only a higher proportion of daughters under high levels of LMC, but also larger eggs. Moreover, in populations experimentally evolving under varying levels of LMC, both the proportion of females and the egg size increased with LMC intensity. These results suggest that sex-ratio adjustment in spider mites is mediated by egg size, although the causal relationship remains to be tested.     

Morphological Characterization of Bushy Cells and Their Inputs in the Laboratory Mouse (Mus musculus) Anteroventral Cochlear Nucleus

Spherical and globular bushy cells of the AVCN receive huge auditory nerve endings specialized for high fidelity neural transmission in response to acoustic events. Recent studies in mice and other rodent species suggest that the distinction between bushy cell subtypes is not always straightforward. We conducted a systematic investigation of mouse bushy cells along the rostral-caudal axis in an effort to understand the morphological variation that gives rise to reported response properties in mice. We combined quantitative light and electron microscopy to investigate variations in cell morphology, immunostaining, and the distribution of primary and non-primary synaptic inputs along the rostral-caudal axis. Overall, large regional differences in bushy cell characteristics were not found; however, rostral bushy cells received a different complement of axosomatic input compared to caudal bushy cells. The percentage of primary auditory nerve terminals was larger in caudal AVCN, whereas non-primary excitatory and inhibitory inputs were more common in rostral AVCN. Other ultrastructural characteristics of primary auditory nerve inputs were similar across the rostral and caudal AVCN. Cross sectional area, postsynaptic density length and curvature, and mitochondrial volume fraction were similar for axosomatic auditory nerve terminals, although rostral auditory nerve terminals contained a greater concentration of synaptic vesicles near the postsynaptic densities. These data demonstrate regional differences in synaptic organization of inputs to mouse bushy cells rather than the morphological characteristic of the cells themselves.     

Sites of respiratory rhythmogenesis during development in the tadpole

During ontogeny, amphibian larvae experience a dramatic alteration in the motor act of breathing as the premetamorphic gill breather develops into the postmetamorphic lung ventilator. We tested the hypothesis that the site of lung rhythmogenesis relocates during metamorphosis by recording fictive lung ventilation before and after transecting the in vitro brain stem of pre- and postmetamorphic Rana catesbeiana into four segments. In premetamorphic tadpoles, the two caudalmost brain stem segments combined proved to be the minimum brain stem configuration necessary and sufficient for lung burst generation. In the postmetamorphic counterpart, this function was supplied by the combination of the two rostralmost brain stem segments. In the postmetamorphic brain stem, a 500-microm segment lying just rostral to cranial nerve IX conveys rhythmogenic capability to neighboring rostral or caudal segments. We conclude that lung rhythmogenic capability translocates rostrally during development as the tadpole shifts from gill to lung ventilation.     

Muscular coordination of knee motion during the terminal-swing phase of normal gait

Children with cerebral palsy often walk with diminished knee extension during the terminal-swing phase, resulting in a troublesome "crouched" posture at initial contact and a shortened stride. Treatment of this gait abnormality is challenging because the factors that extend the knee during normal walking are not well understood, and because the potential of individual muscles to limit terminal-swing knee extension is unknown. This study analyzed a series of three-dimensional, muscle-driven dynamic simulations to quantify the angular accelerations of the knee induced by muscles and other factors during swing. Simulations were generated that reproduced the measured gait dynamics and muscle excitation patterns of six typically developing children walking at self-selected speeds. The knee was accelerated toward extension in the simulations by velocity-related forces (i.e., Coriolis and centrifugal forces) and by a number of muscles, notably the vasti in mid-swing (passive), the hip extensors in terminal swing, and the stance-limb hip abductors, which accelerated the pelvis upward. Knee extension was slowed in terminal swing by the stance-limb hip flexors, which accelerated the pelvis backward. The hamstrings decelerated the forward motion of the swing-limb shank, but did not contribute substantially to angular motions of the knee. Based on these data, we hypothesize that the diminished knee extension in terminal swing exhibited by children with cerebral palsy may, in part, be caused by weak hip extensors or by impaired hip muscles on the stance limb that result in abnormal accelerations of the pelvis.     

Recovery Kinetics of Knee Flexor and Extensor Strength after a Football Match

We examined the temporal changes of isokinetic strength performance of knee flexor (KF) and extensor (KE) strength after a football match. Players were randomly assigned to a control (N = 14, participated only in measurements and practices) or an experimental group (N = 20, participated also in a football match). Participants trained daily during the two days after the match. Match and training overload was monitored with GPS devices. Venous blood was sampled and muscle damage was assessed pre-match, post-match and at 12h, 36h and 60h post-match. Isometric strength as well as eccentric and concentric peak torque of knee flexors and extensors in both limbs (dominant and non-dominant) were measured on an isokinetic dynamometer at baseline and at 12h, 36h and 60h after the match. Functional (KF_ecc/KE_con) and conventional (KF_con/KE_con) ratios were then calculated. Only eccentric peak torque of knee flexors declined at 60h after the match in the control group. In the experimental group: a) isometric strength of knee extensors and knee flexors declined (P<0.05) at 12h (both limbs) and 36h (dominant limb only), b) eccentric and concentric peak torque of knee extensors and flexors declined (P<0.05) in both limbs for 36h at 60°/s and for 60h at 180°/s with eccentric peak torque of knee flexors demonstrating a greater (P<0.05) reduction than concentric peak torque, c) strength deterioration was greater (P<0.05) at 180°/s and in dominant limb, d) the functional ratio was more sensitive to match-induced fatigue demonstrating a more prolonged decline. Discriminant and regression analysis revealed that strength deterioration and recovery may be related to the amount of eccentric actions performed during the match and athletes'' football-specific conditioning. Our data suggest that recovery kinetics of knee flexor and extensor strength after a football match demonstrate strength, limb and velocity specificity and may depend on match physical overload and players'' physical conditioning level.