Development of spontaneous motility in chick embryos. Normal development and the activating effect of strychnine and picrotoxin.

The longitudinal development of spontaneous motility in chick embryos was studied by Kovach's method (Kovach 1970) from the 10th day of incubation up to hatching, in completely intact eggs. From the 10th to 12th day of incubation, very low amplitude movements of a burst character predominated in spontaneous motility. From the 13th day, both low and high amplitude movements could be distinguished. From the 18th day, high amplitude movements alternating with intervals of motor inactivity preponderated. This discontinuous motility, which was most pronounced on the 20th day of incubation, changed to periodic strong hatching movements. Reduction of spontaneous motility after the 17th day of incubation was not confirmed. Strychnine already activated spontaneous motility in 11-day embryos, but typical convulsions did not appear until the 15th incubation day. With picrotoxin, motility was likewise stimulated in 11-day embryos and paroxysmal activation did not occur until the 15th incubation day. In older embryos, convulsions were gradually succeeded by a continuous increase in spontaneous motility. The effect of picrotoxin had a much longer latent period than the effect of strychnine.     

Development of the interaction of GABA and oxazepam on spontaneous motility in chick embryos

The author studied the development of the interaction of GABA and oxazepam on embryonic spontaneous motility in chick embryos during the second half of incubation. In 13-day-old embryos the two substances already potentiated each other's action, despite the fact that GABA, by itself, did not yet have an inhibitory effect. In older embryos this potentiation increased until spontaneous motor activity was almost completely depressed.     

Neuroethological approaches to the study of motor development in chicks: achievements and challenges.

Chicks and chick embryos provide a useful model system for the study of issues related to the development of motor behaviors. EMG and kinematic analyses of leg movements have been used to provide new data on the organization of embryonic motility. These data suggest that the circuitry needed to produce a basic, coordinated motor pattern is available early in development. This circuitry then appears to be retained throughout life. Evidence from analysis of EMG patterns and leg deafferentation studies suggest that the output of this basic circuit can be modulated by sensory input to produce the motor patterns of later behaviors, such as hatching and walking. If the same circuitry is present throughout life, then mechanisms for initiation and termination of particular behaviors must be available to ensure that specific behaviors are turned on and off at appropriate times. For example, hatching can be turned on by a specific sensory signal: proprioceptive signals from the bent neck. In addition to reviewing current research on the development of chick motor behaviors, methodological considerations and suggestions for future research are presented.     

Development of coordinated movement in chicks: II. Temporal analysis of hindlimb muscle synergies at embryonic day 10 in embryos with spinal gap transections.

Spinal neural circuits can recruit muscles to produce organized patterns of activity early in embryonic development. In a previous study, using multichannel electromyographic (EMG) recordings, we characterized burst parameters for these patterns in the legs of chick embryos during spontaneous motility in ovo at embryonic days (E) 9 and E10 (Bradley and Bekoff, 1990). Results of the study suggested both neural and biomechanical factors play an important role in the development of coordinated limb movements. In this study, to explore the contribution of descending neural inputs to the control of leg movements during motility, we applied similar methods to characterize motor patterns produced by the spinal cord in the absence of descending inputs. Thoracic spinal gap transections were performed at E2 and EMG patterns were recorded at E10. Several EMG features for chronic spinal embryos were similar to those for normal embryos and demonstrate that lumbar spinal circuits can be correctly assembled to control limb movements in the absence of connectivity with more rostral neural structures during early differentiation processes. However, certain aspects of the EMG patterns in chronic spinal embryos were different from patterns in normal embryos and provide support for conclusions drawn earlier by Oppenheim (1975). Specifically, our data support the view that propriospinal and/or supraspinal inputs function to regulate the timing of cyclic limb movements controlled by spinal neural circuits. Finally, we consider the possible long-term effects of chronic spinal gap transections as compared to acute spinal transections on the development of motility.     

Embryonic behaviour in the lizard, Lacerta vivipara

The behaviour of embryos of the lizard Lacerta vivipara has been studied in cultured eggs, removed from the mother. Spontaneous movements begin at a time when the embryo is still unresponsive to touch, and about two days before the appearance of reflex responses. The earliest movements consist of lateral flexion which gradually gives place to dorsiventral flexion. Embryonic movements are not necessarily related to contractions of the amnion. The activity of the embryo, as measured by the number of times movements were initiated and the amount of time spent in activity during a half hour period, rises to a plateau and then decreases sharply as the time of hatching approaches. Activity of the tail is prominent, particularly at times when other movements occur in rapid succession. Serpentine movements were not observed in embryos younger than stage 39; some prematurely born animals showed such activity in response to contact with the substratum. During embryonic life any part of the animal may move individually or in combination with any other part, such combined movements being unco-ordinated. In these respects the spontaneous motility of the lizard embryo closely resembles that of the chick. Statistical analysis of the embryonic movements provides evidence of rhythmic activity within the developing central nervous system.     

Development of coordinated movement in chicks: II. Temporal analysis of hindlimb muscle synergies at embryonic day 10 in embryos with spinal gap transections

Spinal neural circuits can recruit muscles to produce organized patterns of activity early in embryonic development. In a previous study, using multichannel electromyographic (EMG) recordings, we characterized burst parameters for these patterns in the legs of chick embryos during spontaneous motility in ovo at embryonic days (E) 9 and E10 (Bradley and Bekoff, 1990). Results of the study suggested both neural and biomechanical factors play an important role in the development of coordinated limb movements. In this study, to explore the contribution of descending neural inputs to the control of leg movements during motility, we applied similar methods to characterize motor patterns produced by the spinal cord in the absence of descending inputs. Thoracic spinal gap transections were performed at E2 and EMG patterns were recorded at E10. Several EMG features for chronic spinal embryos were similar to those for normal embryos and demonstrate that lumbar spinal circuits can be correctly assembled to control limb movements in the absence of connectivity with more rostral neural structures during early differentiation processes. However, certain aspects of the EMG patterns in chronic spinal embryos were different from patterns in normal embryos and provide support for conclusions drawn earlier by Oppenheim (1975). Specifically, our data support the view that propriospinal and/or supraspinal inputs function to regulate the timing of cyclic limb movements controlled by spinal neural circuits. Finally, we consider the possible long-term effects of chronic spinal gap transections as compared to acute spinal transections on the development of motility. © 1992 John Wiley & Sons, Inc.     

Analysis of the origin and development of hatching gland cells by transplantation of the embryonic shield in the fish, Oryzias latipes

Hatching gland cells of the medaka, Oryzias latipes, have been observed to differentiate from the anterior end of the hypoblast, which seems to first involute at the onset of gastrulation. These results suggest that the hatching gland cells of medaka originate from the embryonic shield, the putative organizer of this fish. The present study investigated whether hatching gland cells really originate from the embryonic shield in the medaka. Transplantation experiments with embryonic shield and in situ hybridization detection of hatching enzyme gene expression as a sign of terminal differentiation of the gland cells were carried out. The analysis was performed according to the following processes. First, identification and functional characterization of the embryonic shield region were made by determining the expression of medaka goosecoid gene and its organizer activity. Second, it was confirmed that the embryonic shield had an organizer activity, inducing a secondary embryo, and that the developmental patterns of hatching gland cells in primary and secondary embryos were identical. Finally, the hatching gland cells as identified by hatching enzyme gene expression were found to coincide with the dye-labeled progeny cells of the transplanted embryonic shield. In conclusion, it was determined that hatching gland cells were derived from the embryonic shield that functioned as the organizer in medaka.     

Neuroethological approaches to the study of motor development in chicks: Achievements and challenges

Chicks and chick embryos provide a useful model system for the study of related to the development of motor behaviors. EMG and kinematic analyses of leg movements have been used to provide new data on the organization of embryonic motility. These data suggest that the circuitry needed to produce a basic, coordinated motor pattern is available early in development. This circuitry then appears to be retained throughout life. Evidence from analysis of EMG patterns and leg deafferentation studies suggest that the output of this basic circuit can be modulated by sensory input to produce the motor patterns of later behaviors, such as hatching and walking. If the same circuitry is present throughout life, then mechanisms for initiation and termination of particular behaviors must be available to ensure that specific behaviors are turned on and off at appropriate times. For example, hatching can be turned on by a specific sensory signal: proprioceptive signals from the bent neck. In addition to reviewing current research on the development of chick motor behaviors, methodological considerations and suggestions for future research are presented. © 1992 John Wiley & Sons, Inc.     

Embryonic occurrence of ionocytes in the sea bass Dicentrarchus labrax

Because of the permeability of the chorion, sea bass embryos are exposed to seawater before hatching and hence require precocious osmoregulatory processes. Several studies of other species have demonstrated the existence of ion-transporting cells located on the yolk sac membrane of embryos. In these cells, called ionocytes, ion movements are controlled by a pool of transmembrane proteins. Among them, the Na⁺/K⁺-ATPase, an abundant driving enzyme, has been used to reveal the presence or absence of ionocytes. We have immunostained the Na⁺/K⁺-ATPase in sea-bass embryos and shown the presence of the first ionocytes on the yolk sac membrane at stage 12 somites and the occurrence of ionocytes at other sites before hatching. Ionocytes located on the first gill slits have been identified at stage 14 somites. Primitive enteric ionocytes have also been detected at stage 14 somites in the mid and posterior gut. The presence of these cells might be related to the early opening of the gut to perivitelline fluids, both anteriorly by the gill slits and posteriorly by the anus. The role of embryonic ionocytes in osmoregulation before hatching is discussed.     

Motility pattern and lung respiration of embryonic chicks under the influence of L-thyroxine and thiourea

Pressure changes in the air cell and at the egg shell have been used to monitor respiratory and somatic movements of embryonic chicks. During the prehatching period a phase of reduced activity is observed. Pulmonary respiration is initiated during this phase. Exogenous L-thyroxine exerts an accelerating effect on the hatching process and on the onset of the phase of reduced motility and of lung respiration. In thiourea-treated embryos the opposite effects on the hatching process and on the motility and respiration pattern are registered. When, however, the egg shell above the air cell was sealed with glue, times of hatching and of the beginning of lung respiration were similar to those of controls, although pipping the egg shell occurred earlier than normal. It is suggested that the effects of L-thyroxine and thiourea on the hatching process are caused by a premature or delayed onset, respectively, of pulmonary respiration.     

Oxygen, gills, and embryo behavior: mechanisms of adaptive plasticity in hatching.

Many species alter the timing of hatching in response to egg or larval predators, pathogens, or physical risks. This plasticity depends on separation between the onset of hatching competence and physiological limits to embryonic development. I present a framework based on heterokairy to categorize developmental mechanisms and identify traits contributing to and limiting hatching plasticity, then apply it to a case of predator-induced hatching. Red-eyed treefrogs have arboreal eggs, and tadpoles fall into ponds upon hatching. Egg and tadpole predators select for earlier and later hatching, respectively. Embryos hatch up to 30% early in predator attacks, and later if undisturbed. They maintain large external gills throughout the plastic hatching period, delaying gill regression while development otherwise continues. Rapid gill regression occurs upon hatching. Prolonged embryonic development depends on external gills; inducing gill regression causes hatching. External hypoxia retards development, kills eggs, and induces hatching. Nonetheless, embryos develop synchronously and without hatching prematurely across a broad range of perivitelline PO2, from 0.5-12.5 kPa. Embryos exploit spatial variation of PO2 within eggs by positioning gills against patches of air-exposed surface. Respiratory plasticity and oxygen-sensitive behavior appear critical for the hatching plasticity that balances a predation risk trade-off across life stages.     

Changes in the development of spontaneous motility in chick embryos after the chronic administration of GABA

From the 4th to the 16th day of incubation, GABA was administered continuously to chick embryos in a mean dose of 9.04 +/- 0.98 mg/kg e.w./24 h. On the 17th day of incubation, spontaneous motility was evaluated from the frequency of spontaneous movements as resting motility and motility after the acute administration of GABA (100 mg/kg e.w.), bicuculline (1 mg/kg e.w.) and oxazepam (10 mg/kg e.w.). 1) The chronic administration of GABA reduced the spontaneous motor activity of the experimental embryos to 38.4-47.8% of the control value. To obtain this effect it was sufficient to administer GABA between the 4th and the 8th day of incubation. 2) The inhibitory effect of the acute administration of GABA in the experimental embryos was only half its effect in the controls. Conversely, the relative size of bicuculline activation of motility was distinctly greater in the experimental embryos, which were also significantly more sensitive to oxazepam. The results show that GABA has a dual effect during embryogenesis--a) an early effect between the 4th and 8th day of incubation causing a chronic debt in the development of spontaneous motor activity, and b) an inhibitory effect as a central transmitter, which begins to be manifested in embryonic spontaneous motility of chick embryos on about the 15th day of incubation.     

Spontaneous cyclic embryonic movements in humans and guinea pigs

Motility assessment before birth can be used to evaluate the integrity of the nervous system. Sideways bending (SB) of head and/or rump, the earliest embryonic motility in both humans and guinea pigs, can be visualized sonographically. We know from other species that early embryonic motility is cyclic. This study explores the distribution of SB-to-SB intervals in human and guinea pig embryos before the appearance of more complex movements such as general movements. We hypothesized that the activity in both species is cyclic. We made 15-min sonographic recordings of SBs between 5 weeks and 0 days (5wk0d) and 7wk0d conceptional age (CA) in 18 human embryos of uncomplicated IVF pregnancies (term 38 weeks) and in 20 guinea pig embryos between 3wk4d and 4wk0d CA (term 9 weeks). SB-to-SB interval durations were categorized as long (≥10 s) or short (<10 s) intervals. For human embryos, the median values for long and short intervals were 61 s (range, 10-165 s) and 3 s (range, 1-9 s) respectively; for guinea pigs 38 s (range, 10-288 s) and 5 s (range, 1-9 s), respectively. During development, the duration of long intervals decreased while the number of short intervals increased for both species. The earliest embryonic motility in the human and guinea pig is performed cyclically with distinct developmental milestones. The resemblance of their interval development offers promising possibilities to use the guinea pig as a noninvasive animal model of external influences on motor and neural development.     

Optimization of protocols for microinjection-based delivery of cryoprotective agents into Japanese whiting Sillago japonica embryos

Microinjection has proven useful for introduction of low-permeability cryoprotective agents (CPAs) into fish eggs or embryos for cryopreservation. In this work, we examined the suitable conditions for single or combined microinjection into the perivitelline space (PS) and the yolk mass (YM) of embryos of the Japanese whiting, an alternative marine fish model for embryo cryopreservation studies. The parameters examined were injection volume, CPA type and concentration, vehicle (diluent), and suitable developmental stage. Somites and tail elongation embryos tolerated single or combined injection with 2.1 and 15.6 nl in the PS and YM, respectively, whereas earlier embryonic stages tolerated only up to 8.2 nl in the YM. The injected solutions diffused rapidly throughout the PS and YM and remained contained within each compartment unless in the case of structural damage caused by injection of larger volumes. Yamamoto solution was marginally better as a vehicle for microinjection of CPAs than fish Ringer and phosphate buffer saline whereas ¼ artificial sea water was clearly unsuitable. Ethylene glycol was well tolerated by embryos in all developmental stages whereas 1, 2-propylene glycol was suitable only for early embryonic stages. Overall, microinjection was efficient in delivering high loads of CPAs inside whiting embryos more swiftly than previously obtained for this species by immersion-based impregnation protocols. Embryos microinjected with CPAs showed a decrease in embryo nucleation temperature and an increase in chilling tolerance. CPA-microinjected embryos will provide valuable materials to optimize the remaining parameters that are critical for successful cryopreservation such as cooling and warming strategies.     

Oxygen, gills, and embryo behavior: mechanisms of adaptive plasticity in hatching

Many species alter the timing of hatching in response to egg or larval predators, pathogens, or physical risks. This plasticity depends on separation between the onset of hatching competence and physiological limits to embryonic development. I present a framework based on heterokairy to categorize developmental mechanisms and identify traits contributing to and limiting hatching plasticity, then apply it to a case of predator-induced hatching. Red-eyed treefrogs have arboreal eggs, and tadpoles fall into ponds upon hatching. Egg and tadpole predators select for earlier and later hatching, respectively. Embryos hatch up to 30% early in predator attacks, and later if undisturbed. They maintain large external gills throughout the plastic hatching period, delaying gill regression while development otherwise continues. Rapid gill regression occurs upon hatching. Prolonged embryonic development depends on external gills; inducing gill regression causes hatching. External hypoxia retards development, kills eggs, and induces hatching. Nonetheless, embryos develop synchronously and without hatching prematurely across a broad range of perivitelline PO₂, from 0.5–12.5 kPa. Embryos exploit spatial variation of PO₂ within eggs by positioning gills against patches of air-exposed surface. Respiratory plasticity and oxygen-sensitive behavior appear critical for the hatching plasticity that balances a predation risk trade-off across life stages.     

Spontaneous and evoked postsynaptic potentials in an embryonic neuromuscular system of the lobster,Homarus americanus

The embryonic motor innervation to the deep extensor abdominal muscles was studied in lobster eggs in which reflex twitches and tail flips could be evoked by mechanical stimulation in early embryos. Recordings from impaled fibers during early and later stages of embryonic development revealed spontaneous depolarizing and hyperpolarizing potentials, suggesting the presence of excitatory and inhibitory axons. Stimulation of the extensor motor innervation produced a variety of EPSPs and IPSPs. The depolarizing responses included small and large EPSPs and nonovershooting spikes. Although moderate facilitation of the EPSP was sometimes observed, defacilatation was observed in the majority of fibers of all stages. Spiking could not be evoked by motor axon stimulation in embryos of early stages. These findings indicate that from the outset the deep abdominal extensor neuromuscular system of the lobster is phasic in its response to nerve stimulation and is functional as part of the tail flip reflex at least six months before hatching.     

Bicuculline activation of embryonic spontaneous motility

The activating effect of bicuculline on spontaneous central motor output activity was studied in chick embryos from the 11th to the 19th day of incubation by means of spontaneous motility. When applied onto the embryonic membranes, bicuculline [1 mg X kg-1 egg weight] significantly activated embryonic motility from the 15th day of incubation. In 15-day embryos it increased spontaneous motility 2.5-fold and in 17- and 19-day embryos 3.5-fold. The role of supraspinal factors in the activating effect of bicuculline increased with development. In 15-day embryos it accounted for 56.7% and in 17-day embryos for already 84.6% of the total effect of bicuculline. Antagonism was manifested between bicuculline and the inhibitory amino acids glycine and GABA; in the case of GABA it was quantitatively more pronounced. The results of this study of development of the activating effect of bicuculline and its antagonism with gamma-aminobutyric acid are evaluated from the aspect of the connecting-up and development of central GABA-ergic components in the regulation of embryonic motility.     

Multiple extracellular activities in Drosophila egg perivitelline fluid are required for establishment of embryonic dorsal-ventral polarity.

Twelve maternal effect genes (the dorsal group and cactus) are required for the establishment of the embryonic dorsal-ventral axis in the Drosophila embryo. Embryonic dorsal-ventral polarity is defined within the perivitelline compartment surrounding the embryo by the ventral formation of a ligand for the Toll receptor. Here, by transplantation of perivitelline fluid we demonstrate the presence of three separate activities present in the perivitelline fluid that can restore dorsal-ventral polarity to mutant easter, snake, and sp?tzle embryos, respectively. These activities are not capable of defining the polarity of the dorsal-ventral axis; instead they restore structures according to the intrinsic dorsal-ventral polarity of the mutant embryos. They appear to be involved in the ventral formation of a ligand for the Toll protein. This process requires serine proteolytic activity; the injection of serine protease inhibitors into the perivitelline space of wild-type embryos results in the formation of dorsalized embryos.     

Oxygen consumption during embryonic development of the annual fish Nothobranchius korthausae with special reference to diapause

The oxygen consumption of Nothobranchius korthausae eggs in different developmental stages, including diapause II and III, was measured. Oxygen consumption increases exponentially during embryonic development. In diapause II and III there is a drop in oxygen consumption, which attains a minimal level in diapause II after 3 weeks and in diapause III after 2 weeks. During early development the embryos can escape from hypoxic stress by entering diapause I and II. During late embryogenesis embryos in diapause III can escape from hypoxic stress by hatching. We conclude that survival of annual fish embryos is enhanced during conditions of low oxygen concentration by reduced oxygen consumption rates during diapause.     

Temporal expression of ornithine decarboxylase in developing embryos of Musca domestica

The objective of this study was to partially characterize and follow the temporal expression of the enzyme ornithine decarboxylase (ODC) throughout embryonic and early larval development of Musca domestica. Enzymatically active ODC was shown to be present at detectable levels in the embryos only during the latter stages of embryogenesis. This temporally expressed enzyme displayed maximum activity at the time of hatching, and the activity rapidly declined in the newly hatched larvae. The half-life of ODC activity in extracts at the time of hatching and 30 min after hatching was 57 min and 12 min, respectively. The subunit molecular weight of the embryonic ODC was determined to be 46,000, and the apparent native molecular weight was determined to be 276,000. The concentrations of the polyamines putrescine, spermidine, and spermine also were determined throughout embryogenesis. Spermidine was found to be present in the embryos at about 10-fold higher concentrations than spermine and at about 100-fold higher concentrations than putrescine. These polyamines did not undergo major changes in concentration throughout development of the embryos.