Variation in development of rat embryos at the presomite period.

Rat embryos at a single gestational time in the presomite period were studied for their variation in development and their fate after culture. They were explanted at 8 A.M. on day 9 of gestation from timed-pregnant Sprague-Dawley rats which were obtained by mating between 8 and 10 A.M. (plug day = day 0). In the first experiment, a total of 203 embryos from 20 litters were examined for their variation in development. Several dimensions of embryo/egg cylinder were measured and development of various embryonic/extraembryonic structures were assessed using a scoring system that we developed for the present study. Embryos were then divided into different stages of development based on their scores using the staging system that we developed previously. A large variation in developmental stage was demonstrated; the youngest embryo was at the early primitive streak stage with no signs of amniotic folds and the oldest one was at the late neural plate stage with a foregut pocket but without visible somites. No strong correlation was demonstrated between developmental stage and size of embryo/egg cylinder, nor between developmental stage and development of the proamniotic tube, ectoplacental cavity, or allantois. In the second experiment, embryos were explanted at the same time and those at different stages were cultured separately in rotating bottles and their outcomes were compared after 49 hours. The difference in mean somites number of embryos cultured from the mid primitive streak and late neural plate stages was 6.1. This difference corresponds to approximately 10 hours based on the known linear increase of somites number on day 11 of approximately 0.6 somites per hour. These results indicate a large variation in development of presomite period embryos supposedly of the same gestational age and suggest the importance of careful staging at the time of explantation if precision is needed for whole embryo culture experiments.     

Embryonic stages from cleavage to gastrula in the loach Misgurnus anguillicaudatus

Early developmental staging from the zygote stage to the gastrula is a basic step for studying embryonic development and biotechnology. We described the early embryonic development of the loach, Misgurnus anguillicaudatus, based on morphological features and gene expression. Synchronous cleavage was repeated for 9 cycles about every 27 min at 20 degrees C after the first cleavage. After the 10th synchronous cleavage, asynchronous cleavage was observed 5.5 h post-fertilization (hpf), indicating the mid-blastula transition. The yolk syncytial layer (YSL) was formed at this time. Expressions of goosecoid and no tail were detected by whole-mount in situ hybridization from 6 hpf. This time corresponded to the late-blastula period. Thereafter, epiboly started and a blastoderm covered over the yolk cell at 8 hpf. At 10 hpf, the germ ring and the embryonic shield were formed, indicating the stage of early gastrula. Afterward, the epiboly advanced at the rate of 10% of the yolk cell each hour. The blastoderm covered the yolk cell completely at 15 hpf. The embryonic development of the loach resembled that of the zebrafish in terms of morphological change and gene expression. Therefore, it is possible that knowledge of the developmental stages of the zebrafish might be applicable to the loach.     

Prolongation of somitogenesis in two anguilliform species,the Japanese eel Anguilla japonica and pike eel Muraenesox cinereus,with refined descriptions of their early development

The embryonic development of the Japanese eel Anguilla japonica and pike eel Muraenesox cinereus was morphologically investigated with laboratory‐reared specimens to clarify the characteristics of somitogenesis. In A. japonica, somites were first observed at 18 h post fertilization (hpf) when epiboly reached 90%. Somitogenesis progressed at a rate of 1·6 h^?1 at mean ± s.d . 22·6 ± 0·7° C and completed at 107 hpf (3 days post hatching; dph) when total number of somites (ST) reached 114, which corresponds to the species' number of vertebrae (112–119). In M. cinereus, somites were first observed at 14 hpf when epiboly completed. Somitogenesis progressed at a rate of 1·9 h^?1 at mean ± s.d . 24·4 ± 0·2° C and completed at 90 hpf (2 dph) with 149 ± 4 ST, which corresponds to the species' number of vertebrae (142–158). Both species hatched before somitogenesis was completed, at 37 hpf with 47 ST and 42 hpf with 82 ± 4 ST, respectively. The formation of other organs such as the heart, mouth and pectoral fin bud occurred during somitogenesis. Comparison with the development of zebrafish Danio rerio indicates a prolongation of somitogenesis in A. japonica and M. cinereus. Their somitogenesis rates, however, correspond well with that of D. rerio estimated at the same temperature and their developmental stages at hatching are almost equivalent to other fishes having similar yolk sizes. Therefore, the prolongation of somitogenesis in A. japonica and M. cinereus may be accounted for solely by the increased numbers of somites to be formed, not by a slow somitogenesis rate or an acceleration in organogenesis.     

Effects of oxygen concentration during incubation and broiler breeder age on embryonic heat production,chicken development,and 7-day performance

Older breeder flocks produce eggs with a relatively larger yolk and thereby a higher nutrient availability than young breeder flocks. To optimise nutrient utilisation and embryonic development throughout incubation and posthatch period, embryos originating from older breeder flocks may require a higher oxygen availability. The current study investigated effects of broiler breeder flock age and incubational oxygen concentration on embryonic metabolism and chicken development until 7-day posthatch. Similar sized eggs of a young (28–32 week) or old (55–59 week) Cobb 500 breeder flock were incubated at one of three oxygen concentrations (17%, 21% or 25%) from day 7 of incubation until 6 h after emergence from the eggshell. Posthatch, chickens were reared until 7 days of age. Egg composition at the start of incubation, heat production during incubation, and embryo or chicken development at embryonic day (ED)14 and ED18 of incubation, 6 h after hatch and day 7 posthatch were evaluated. An interaction was found between breeder age and oxygen concentration for yolk-free body mass (YFBM) at ED18. A higher oxygen concentration increased YFBM in the old breeder flock, whereas no difference was found between 21 and 25% oxygen in the young breeder flock. Yolk size was larger in the old compared to the young flock from ED0 until 6 h after hatch. Breeder flock age did not affect YFBM at ED14 and 6 h after hatch nor daily embryonic heat production, but there were some effects on relative organ weights. Chickens of the old compared to the young breeder flock showed a higher weight gain at day 7, but at a similar feed conversion ratio (FCR). A higher oxygen concentration during incubation stimulated embryonic development, especially between 17% and 21% of oxygen, in both flock ages_. Although this growth advantage disappeared at 7 days posthatch, a low oxygen concentration during incubation resulted in a higher FCR at 7 days posthatch. Results indicated that breeder flock age seemed to influence body development, with an advantage for the older breeder flock during the posthatch period. Oxygen concentrations during incubation affected body development during incubation and FCR in the first 7 days posthatch. Although an interaction was found between breeder flock age and oxygen concentration at ED18 of incubation, there was no strong evidence that nutrient availability at the start of incubation (represented by breeder flock ages) affected embryo and chicken development at a higher oxygen concentration.     

Development of a heat shock inducible gfp transgenic zebrafish line by using the zebrafish hsp27 promoter

In the present study, a zebrafish hsp27 promoter was isolated and used to develop heat shock inducible gfp transgenic zebrafish. The endogenous hsp27 mRNAs were constitutively expressed from 4 hpf and increased in several regions of brain, heart and somites in early embryogenesis until 24 hpf. Subsequently, the expression was reduced significantly but maintained in the heart and ears. Heat shock induced hsp27 mRNAs in the blastoderm from 6 hpf and later in somites, branchial arches and several regions of brain. Similarly in hsp27-gfp transgenic zebrafish, constitutive GFP expression was observed from 11 hpf. GFP expression was mainly in the skin cells and increased to the peak level at 7 dpf, followed by a reduction. The constitutive GFP expression in the heart was initiated from 50 hpf and maintained even in the adult fish. After heat shock, GFP expression was mainly induced in the muscle in addition to a mild increase in the skin and heart. The early stages of the embryos were more sensitive than late stages as the time required for induced GFP expression in the muscle is shorter. Thus, the hsp27-gfp transgenic line generally recapitulates the expression pattern and heat shock inducibility of endogenous hsp27 RNAs. We also tested the potential of using the hsp27-gfp transgenic zebrafish embryos for heavy metal induction and demonstrated the inducibility of GFP expression by arsenic; this pattern of induction was also supported by examination of endogenous hsp27 mRNA.     

Quantitative distribution of chick neural crest cells during gangliogenesis

Summary We have quantitated the distribution of chick neural crest cells after they have completed early migration and are aggregating into ganglia. Variables tested for an influence on the distribution of cells include stage, level of somites, position in each of the primary body axes, and individual embryo. The 11th–15th cervical somites of embryos at stages 30, 35, and 40 somites (s) incubated for 2.5, 3.0, and 3.5 days were labeled with antibody to HNK-1 to detect neural crest cells, and doubly labeled with antibody to HNK-1 and to the 150 kD neurofilament subunit to detect neural crest-derived neurons. Significantly more neural crest cells appear at older stages, but cells are uniformly distributed among the 11th–15th somites at any given stage. Significant differences in the total number of neural crest cells among three embryos sampled at the same stage indicate that the number of cells is independent of the staging series used. As early as the 35 s stage about one-third of the neural crest cells throughout the somite exhibit NF staining. At the 40 s stage, doubly labeled NF cells, as well as HNK-1 labeled cells, aggregate in a circumscribed portion of the mediolateral axis to form presumptive sensory ganglia in the dorsal region of the somites. Also at 40 s a wave of cell aggregation into sympathetic ganglia proceeds anteroposteriorly along the ventral border of the somitic mesenchyme. The results show a sequence of phenotypic expression beginning with neurofilament antigen, then ganglionic aggregation, and finally, in the case of sympathetic neurons, catecholamine transmitter.     

多疣壁虎胚胎发育分期的形态学特征

脊椎动物发育生物学的研究通常依赖于数量有限的模式生物的形态变化,胚胎发育分期表的建立为物种胚胎发育的一系列过程确立了一个统一的标准,成为研究形态演化的重要工具。本研究对多疣壁虎(Gekko japonicus)28℃孵化条件下的胚胎发育过程进行显微观察,并记录了整个胚胎发育历程。基于多疣壁虎胚胎发育过程中头部、咽、四肢等形态变化及皮肤色素沉积和被鳞的情况,将多疣壁虎胚胎发育分为42个时期。刚排出体外的受精卵,其胚胎发育一般已经发生至28期,该期胚胎头部和躯干分化明显,眼泡、咽弓、心和体节可见;29期前、后肢芽均可见;30期肢芽延长并开始出现分区,31期可见明显肢身,32期四肢均出现肢柱和肢杆的分区;33期咽裂消失,指和趾开始显现;35期指和趾间带退化,指和趾完全形成;36期出现爪;37期爪完全形成;38期皮肤色素沉积明显;39期指、趾底部膨大,形成单行攀瓣;40期身体背部和四肢色素沉积且被鳞明显;41期腹部出现色素沉积且被覆鳞片。42期鼻孔开放,体背整体呈灰棕色。对多疣壁虎卵产出后胚胎28~42期发育期形态学变化进行了详细描述,旨在为蜥蜴类胚胎发育研究提供参考。     

Development of feeding structures in larval fish with different life histories: winter flounder and Atlantic cod

The size at which feeding structures developed and shifts in head proportions occurred, differed between Atlantic cod Gadus morhua and winter flounder Pseudopleuronectes americanus. The sequence and timing of the development of feeding structures may not be dependent on size, but may occur because they are necessary to meet specific requirements offish larvae feeding in the plankton. In early larval stages development of feeding structures was similar in number and type and was necessary for first-feeding in both species. In later stages, significant differences between species occurred in the timing of the development of feeding structures. In cod differentiation of new structures and changes in head proportions occurred at about two-thirds of the way through larval life, which coincided with an increase in growth. In flounder changes in feeding morphology did not occur during the symmetrical larval stage, but occurred only after metamorphosis to the asymmetrical demersal juvenile stage. Differences between cod and flounder in the size at which feeding structures develop may reflect life history adaptations expressed in the duration of the pelagic larval stage, as well as differences in juvenile habitat and feeding ecology.     

Ontogenic corticosteroidogenesis of the domestic fowl: response of isolated adrenocortical cells

Ontogenic adrenocortical function of the domestic was investigated using adrenocortical cells isolated from embryonic chicks (18, 19, 20, and 21 days old) and male and female posthatch birds (1 day, 1 week, and 3 weeks old). Production of the predominant corticosteroids secreted by the chicken adrenal gland, corticosterone, cortisol, and aldosterone, was measured by radioimmunoassay after 2-hr incubation of cells with or without steroidogenic agents. Approaching hatch, basal and maximal ACTH-(1-24) (ACTH)-induced corticosteroid production increased steadily and peaked around 1 day posthatch (5-18 times and 3-9 times, respectively, the production values at 18 days embryonic life). Thereafter, corticosteroid production values decreased steadily to 3 weeks posthatch. Corticosterone predominated over the ages studied: Maximal ACTH-induced corticosterone production averaged 52 and 115 times the production values of aldosterone and cortisol, respectively. In addition, maximal ACTH-induced aldosterone production was roughly 2.2 times greater than cortisol production over the ages studied except for a short-lived, disproportionately greater aldosterone production at 1 day posthatch. In addition to perihatch and age-related differences in cellular corticosteroid production, there were also differences in cellular sensitivity to steroidogenic agents as indicated by the differences in half-maximal steroidogenic concentration values (ED50 values) of the steroidogenic agents. Sensitivity to ACTH increased 2.7 times from Day 18 of embryonic life to 1 day posthatch and then decreased steadily to 3 weeks posthatch. In addition, sensitivity to 8-bromo-cAMP (8-Br-cAMP) increased abruptly at 1 day posthatch (nearly 3 times) but then remained constant thereafter. However, a consistent change in cellular sensitivity to 25-hydroxycholesterol was not observed until 3 weeks posthatch (an increase in sensitivity of 3 times that at Day 18 of embryonic life). These data of cellular sensitivity suggest that there were distinct development and maturational alterations in the cellular loci at which ACTH, 8-Br-cAMP, and 25-hydroxycholesterol acted. Thus, during the transition from embryonic to postembryonic life of the domestic fowl, there are alterations in adrenocortical cell steroidogenic capacity and in the function of some cellular loci comprising the corticosteroidogenic pathway.     

Distinct expression patterns of two zebrafish homologues of the human APP gene during embryonic development

The human amyloid protein precursor (APP) gene correlates with early onset of Alzheimer's disease in humans. We have identified two APP homologues in zebrafish, which we call appa and appb. They show a high degree of identity to human APP particularly in the beta APP42 and the transmembrane domain. Widespread expression of both appa and appb was detected from mid-gastrulation until the bud stage. During segmentation, the two genes diverged in their pattern of expression: at 14 h post-fertilisation (hpf) and 18 hpf both genes were expressed rostrally in the prospective CNS, but only appa was found caudally in the paraxial segmental plate and presomitic mesoderm, excluding the midline. In contrast, appb was found caudally in the neural rod at 14 hpf and the developing spinal cord at 18 hpf. Later, at 24 hpf both genes shared common expression domains, namely the telencephalon, the ventral diencephalon, the trigeminal ganglia, and the posterior lateral line ganglia. Unique expression domains for appa were the lens, the otic vesicles and the somites, while appb was expressed in a serially repeated set of nuclei within the hindbrain, the ventral mesencephalon and the motoneurones of the developing spinal cord.     

PACAP in developing sensory and peripheral organs of the zebrafish, Danio rerio

The anatomical distribution of PACAP-like immunoreactivity was investigated in sensory and peripheral organs of the zebrafish, Danio rerio, during the pharyngula, hatching and larval periods, by using indirect immunofluorescence methods. First PACAP-like immunoreactive (ir) elements appeared during the pharyngula period, at 24 hours post fertilization (hpf), within the most superficial layer of the retina and the dorsal aorta. At 48 hpf, additional ir cells were found in the olfactory placode and esophagus. At 72 hpf (hatching period), PACAP-like immunoreactivity was first detected in the ganglion cell layer of the retina, the otic sensory epithelium, pharyngeal arches, swim bladder and pancreatic progenitor cells. During day 5 of larval development, new groups of ir cells appeared in the liver, whereas no ir elements were observed in the olfactory placode. Subsequently, at day 13 of larval development, additional ir elements were found for the first time in some gut epithelial cells while those previously observed in the retina and otic sensory epithelium were absent. The transient expression of PACAP-like ir material in sensory organs suggests that the peptide could be implicated in neurotrophic activities and neurosensorial connections in the migration and/or differentiation processes. The appearance of PACAP-like ir elements in peripheral organs at different developmental stages, indicates that this peptide could be involved in the control of more specific functions as soon as these peripheral structures begin to operate.     

Expression of the oligopeptide transporter, PepT1, in larval Atlantic cod (Gadus morhua)

The intestinal absorption of di- and tri-peptides generally occurs via the oligopeptide transporter, PepT1. This study evaluates the expression of PepT1 in larval Atlantic cod (Gadus morhua) during the three weeks following the onset of exogenous feeding. Larval Atlantic cod were fed either wild captured zooplankton or enriched rotifers. cDNA was prepared from whole cod larvae preceding first feeding and at 1000 each Tuesday and Thursday for the following three weeks. Spatial and temporal expression patterns of PepT1 mRNA were compared between fish consuming the two prey types using in situ hybridization and quantitative real-time PCR. Results indicated that PepT1 mRNA was expressed prior to the onset of exogenous feeding. In addition, PepT1 was expressed throughout the digestive system except the esophagus and sphincter regions. Expression slightly increased following first-feeding and continued to increase throughout the study for larvae feeding on both prey types. When comparing PepT1 expression in larvae larger than 0.15-mg dry mass with expression levels in larvae prior to feeding, no differences were detected for larvae fed rotifers, but the larvae fed zooplankton had significantly greater PepT1 expression at the larger size. In addition, PepT1 expression in the zooplankton fed larvae larger than 0.15-mg dry mass had significantly greater expression than rotifer fed larvae of a similar weight. Switching prey types did not affect PepT1 expression. These results indicate that Atlantic cod PepT1 expression was slightly different relative to dietary treatment during the three weeks following first-feeding. In addition, PepT1 may play an important role in the larval nutrition since it is widely expressed in the digestive tract.     

团头鲂MSTN基因cDNA结构、表达及过表达对胚胎发育的影响

研究通过cDNA末端快速扩增法(RACE)克隆得到团头鲂生长抑制素(MSTN)基因的cDNA全长并分析了MSTN基因在团头鲂胚胎、成鱼组织中表达以及MSTN基因在胚胎中过表达情况。结果表明团头鲂MSTN基因的cDNA全长为2187 bp, ORF(开放阅读框)大小为1128 bp, 编码376个氨基酸。组织逆转录PCR (RT-PCR)结果显示, MSTN基因在肌肉、脑和精巢组织中大量表达, 肝脏、脾脏和卵巢组织中的少量表达, 肠、腮、心、眼和肾组织中的微量表达。胚胎逆转录PCR (RT-PCR)结果显示, 在0—44 hpf胚胎发育阶段, MSTN基因表达量较低; 而在48—52 hpf胚胎发育阶段, MSTN基因表达量逐渐升高。整胚原位杂交(WISH)结果显示, 胚胎发育的16 hpf时期MSTN基因主要在脊索中表达, 胚胎发育的28 hpf和55 hpf时期MSTN基因在脑中表达。MSTN基因过表达结果显示, 胚胎在体节发生期出现前-后轴拉长, 背-腹轴变短; 脊索发生扭曲, 强烈抑制体节发育而导致不分化等现象。研究为后续团头鲂MSTN基因的功能研究及团头鲂分子育种提供相关参考依据。