A study on the regenerative potential of partially excised mouse embryonic fore-limb bud

Summary The ability of day E10 mouse fore-limb bud to regulate following the removal of a portion of limb tissue was investigated. A longitudinal strip of tissue, two to three somites in width and extending from the base of the limb bud to its distal tip, was excised. The embryos were then maintained in a roller culture system for periods of 6 h, 12 h or 24 h post-operatively prior to fixation and subsequent examination. The embryos were examined with scanning electron microscopy (SEM) and light microscopy. SEM revealed that about two thirds of the operated limbs grossly restored their overall morphology. The sequence of morphological changes involved in the restoration process is described. The ability of the restored limb bud to develop an apical ectodermal ridge (AER) is shown in histological sections.     

Development of a mouse model for studying the effect of embryo culture on embryonic stem cell derivation

For most of the derived human embryonic stem cell (ESC) lines thus far, the majority of human embryos used have been frozen in liquid nitrogen at or prior to the compacting stage for up to 10 years before human ESC derivation. As such they were grown in media that were relatively simple in their formulation compared with those used today. Here we report that culture of mouse embryos in media similar to these produces blastocysts in which both the inner cell mass cell number and the number of ESC progenitor cells (epiblast cells) in the inner cell mass are reduced compared with blastocysts cultured in a purpose-designed sequential (G1/G2) system commonly used today. Embryos cultured in a simple medium were less likely to attach and generate outgrowths. Further, these outgrowths had increased metabolic activity, which has been linked to differentiation, and altered gene expression. Culture of embryos in a simple medium to the compacting stage followed by culture in G2 to the blastocyst stage reduced some of these effects. However, none were improved to the level seen for culture in G1/G2. These results highlight the influence of embryo culture on embryo quality and pluripotency, which is a key factor in determining ESC isolation efficiencies.     

Endogenous electric current is associated with normal development of the vertebrate limb.

A steady ionic current is driven out of both developing and regenerating amphibian limbs. In the developing limbs of anurans and urodeles, focal outwardly directed current (0.5-2 microA/cm(2)) predicts the location of mesenchyme accumulations producing the early bud. Here, we report measurements of a similar outwardly directed ionic current associated with the development of the limb bud in the mouse and chick embryo by using a noninvasive, self-referencing electrode for the measurement of extracellular current. In both the mouse and chick embryo, flank currents were usually inwardly directed - the direction of Na(+) uptake by ectoderm. Outward currents associated with the mouse limb bud ranged from 0.04-10.8 microA/cm(2). Mouse limb bud and flank currents were similar to those measured in amphibian larvae, because they were reversibly collapsed and/or reversed by application of 30 microM amiloride, a Na(+) channel blocker. Unlike the amphibian embryos, flank ectoderm adjacent to the mouse limb bud in the anterior/posterior axis was usually associated with outwardly directed ionic current. This raises the possibility of a different, or changing, gradient of extracellular voltage experienced by mesenchyme cells in this plane of development than that observed in other regions of the limb bud. In the chick flank caudal to the somites, a striking reversal of the inwardly directed flank currents to very large ( approximately 100 microA/cm(2)) outwardly directed currents occurred three developmental stages before limb bud formation. We tested the relevance of this outwardly directed ionic current to limb formation in the chick embryo by reversing it by using an artificially applied "countercurrent" pulled through a microelectrode inserted just beneath the caudal ectoderm of the embryo. This application was performed for approximately 6 hr 2.5-3 developmental stages before hindlimb bud formation. This method resulted in abnormal limb formation by the tenth day of gestation in some embryos, whereas all control embryos developed normally. These data suggest an early physiological control of limb development.     

Study of the origin of connective tissue sheaths of peripheral nerves in the limb of avian embryos

Summary Quail-chick and chick-quail chimeras were constructed by grafting, isotopically, the limb bud of quail embryos into a chick of the same developmental stage and vice versa, prior to the entry of nerve fibres into the limb.After 5–14 days reincubation of the embryos, the components of the connective tissue sheaths of the peripheral nerves were observed by using Feulgen-Rossenbeck staining and light microscopy, in order to distinguish quail cells and chick cells.In all the chimeras studied, the connective tissue sheaths of peripheral nerves (the epineurium, perineurium, perineural septa and endoneural fibroblasts) were formed from the mesenchyme of the limb bud, while Schwann cells were of host origin. Also the outer and inner capsule of muscle spindles originated from the limb bud mesenchyme.These experiments suggest that the connective tissue sheaths of peripheral nerves (at least in the limb region of avian embryos) are not of neural crest origin, but are formed from limb bud mesenchyme.     

Time from insemination to first cleavage predicts developmental competence of human preimplantation embryos in vitro

BACKGROUND: The absence of reliable markers for the identification of viable embryos for transfer at the early cleavage stage is likely to contribute to the generally low implantation rates and high incidence of multiple gestation in IVF treatment. In this study, we investigate the relationship between timing of first cleavage and the incidence of blastocyst formation in vitro. METHODS: Couples (n = 70) with at least one embryo remaining after transfer were included in the analyses. All embryos (n = 579) were examined for early cleavage at 25 h after insemination. Following embryo transfer, the remaining embryos (n = 426) were cultured until day 7 of development, and assessed for blastocyst formation. RESULTS: Eighty-five embryos (14.7%) cleaved to the 2-cell stage within 25 h of insemination; 26 of these were selected for transfer on day 2. Of the 59 embryos remaining in culture, 19 (32.2%) developed to the blastocyst stage; this was a significantly higher number than was observed in embryos (61/367; 16.6%) that failed to cleave within 25 h of insemination (P < 0.01). Within these two groups of embryos the proportion of hatched blastocysts was 11/59 (18.6%) and 26/367 (7.1%) respectively (P < 0.005). CONCLUSIONS: These findings indicate that early cleavage is indicative of increased developmental potential in human embryos and may be useful as an additional criterion in the selection of embryos for transfer.     

Development of a mouse embryo limb bud cell culture system for the estimation of chemical teratogenic potential

High-density cultures of mouse embryo limb bud cells differentiate and synthesize an extracellular matrix containing sulfated proteoglycans. Since these in vitro events are related to those that occur during fetal development, we have investigated the use of cultured limb bud cells for the analysis of the activities of chemical teratogens. We have established the conditions for the use of the radiochemicals 35SO=4 and 3H-thymidine for the assessment of chemical effects on sulfated proteoglycan and DNA synthesis, respectively, in mouse limb bud cells. By performing double-labeling experiments in the presence of test chemicals, the preferential inhibition of either proteoglycan synthesis or DNA synthesis could be demonstrated for 19 of 22 known mouse teratogens tested. The five nonteratogens tested did not cause significant differences in the inhibition of either macromolecular class. The overall predictive accuracy of the system was approximately 89%, and the false-negative rate was approximately 14.8%. No false positives were observed. These data showed that this cell culture system differentiated between the activities of a limited set of selected mouse teratogens and nonteratogens, suggesting that cultured mouse embryo limb bud cells may constitute the basis for the development of a powerful tool for analyses of chemical teratogenic potential.     

Retinoic acid receptor beta 2 mRNA is elevated by retinoic acid in vivo in susceptible regions of mid-gestation mouse embryos.

Many of the biological effects of retinoic acid are mediated by its nuclear receptors (RAR-alpha, RAR-beta, and RAR-gamma), and each of these three receptors exist in multiple isoforms. As a first step to identify if any of the receptor isoforms are involved in dysmorphogenesis which is induced in mouse embryos after treatment with retinoic acid (RA), we examined the levels of mRNA of several isoforms of each RAR in the limb buds and other embryonic regions of normal and RA-treated embryos. Within 3 to 6 hr after treatment of mice on day 11 of gestation with RA, RAR-beta 2 mRNA levels in the whole embryo increased 7-fold while both RAR-alpha 2 and RAR-gamma 1 mRNA levels were elevated only 2-fold. Since RA treatment of day 11 embryos especially produces limb defects in virtually every embryo, we next examined individual embryonic regions separately. Limb buds showed the highest elevations in RAR-beta 2 mRNA levels (12-fold) compared to a moderate elevation in the head/craniofacial region (8-fold) and a small elevation in the remainder of the body (4-fold). In contrast, RAR-alpha 2 and RAR-gamma 1 mRNA levels were elevated in all these tissues to a similar extent, which amounted to only about a 2-fold increase. Retinol, the precursor of RA in the embryo, was also capable of elevating RAR-beta 2 mRNA levels in the limb bud, but the increase was delayed, apparently indicating that metabolic conversion of retinol to RA preceded the effect on mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

The culture of postimplantation embryos

Culture methods for postimplantation embryos are now widely used in studies of embryo physiology, growth and development. Available methods support growth and development of rat and mouse embryos at all stages of organogenesis. The best results are obtained from embryos between head fold and early limb bud stages; overall growth and differentiation of these embryos in vitro is almost identical to that in vivo. Some examples of the application of postimplantation embryo culture to specific lines of study are given and some possible future developments of the technique are discussed.     

Posterior half amputation of the chick wing bud: the response of the developing vasculature, and subsequent wound healing

Summary Experimental analyses examining pattern formation in the developing chick limb have concentrated on the skeleton, muscles and nerves, and have rarely considered blood vessels. To investigate the relationship between the vasculature and limb development, posterior amputations were performed on 3.5–4 day chick limb-buds. It has been shown that the removal of the posterior half alters the developmental fate of the anterior tissue: it becomes necrotic and fails to differentiate into the complement of skeletal parts predicted by fate maps. The possibility that this developmental failure results from interference with the future arterial supply was examined by Indian ink injection between 3–48 h after operation. Scanning electron microscopy (SEM) and resin histology were used to examine the wound repair at similar post-operative intervals. Results from the Indian ink injections showed that within 6 h of operation a collateral circulation was established by means of a branch from the truncated primary subclavian artery. The capillary density in the operated limbs appeared normal when compared to the contralateral limb. The results support the view that the poor developmental performance of the anterior half is due to removal of the zone of polarising activity (ZPA) rather than to experimentally-induced alteration to the vascular supply. Histological and SEM examination of the wound healing process showed that epithelialization of the cut surface occurred within 24 h, and that the peridermal cells of the bilayered ectoderm appeared to initiate the regrowth. The wound site was not visible 48 h after operation, showing that wound healing at these developmental ages occurs quickly, with no scar tissue formation. These results show that the vasculature in the developing limb is labile, and that the cell death resulting from posterior-half amputation is not due to vascular insufficiency or ischaemia. In addition, this study of wound healing demonstrates the role of the ectoderm in establishing an avascular margin in the subjacent mesenchyme.     

Actin filament organization in aligned prefusion myoblasts

The organization of the actin cytoskeleton in prefusion aligning myoblasts is likely to be important for their shape and interaction. We investigated actin filament organization and polarity by transmission electron microscopy (TEM) in these cells. About 84% of the filaments counted were either found in a subplasmalemma sheet up to 0.5 microm thick that was aligned with the long axis of the cell, or in protrusions. The remaining filaments were found in the cytoplasm, where they were randomly orientated and not organized into bundles. The polarity of the subplasmalemma filaments changed progressively from one end of the cell to the other. At the ends of the cells and in protrusions, the majority of filaments were organized such that their barbed ends faced the tip of the protrusion. We did not find any actin filament bundles or stress fibres in these cells. Time-lapse phase microscopy demonstrated that aligned cells were still actively migrating at the time of our TEM observations, and their direction of movement was restricted to the long axis of the cell group. The ability of these cells to locomote actively in the absence of actin filament bundles suggests that in these cells the subplasmalemma actin sheet contributes not only to cell shape but also to cell locomotion.     

小鼠胚胎体外培养方法的改良

目的改进胚胎培养系统以进一步提高胚胎质量与发育潜能。方法采用内径0.21mm、外径0.28mm的塑料毛细管培养胚胎,将吸有胚胎的毛细管浸在盛有蒸馏水的烧杯内,再将烧杯放置在培养箱里的磁力搅拌器上,允许胚胎在微小的空间内发育并伴随着水的旋转而摆动,更好地模拟了胚胎在输卵管中的运动环境。结果分别对85枚、82枚2-细胞胚胎进行毛细管及微滴培养,其中毛细管培养胚胎的囊胚率以及胚胎细胞数(85.4%,57.0)显著高于微滴培养(36.5%,20.6),囊胚率差异显著(P0.05)。且接种在Matrigel基底膜上形成的内细胞团集落显著增大。结论小鼠胚胎体外培养方法的改良——毛细管培养,促进小鼠植入前胚胎的体外发育。     

Developmental toxicity of indium in cultured rat embryos.

Developmental toxicity of indium was examined using rat embryo culture with reference to toxicokinetics. Rat embryos at day 9.5 of pregnancy were cultured for 48 h under various exposure conditions to indium trichloride. Indium was embryotoxic to cultured rat embryos at concentrations ranging from 25 to 50 microM for 24 h exposure according to the embryonic age, and the exposure concentration was more critical than the exposure time. The embryotoxic concentrations were comparable to the serum concentration at a developmentally toxic dose by intravenous administration in an in vivo experiment. It was considered from these results that the developmental toxicity of indium is a direct effect on the embryo or yolk sac and that weak developmental toxicity of indium by oral administration was due to low exposure concentrations in the embryo.     

Origin of spinal cord meninges, sheaths of peripheral nerves, and cutaneous receptors including Merkel cells

Summary The origin of cells covering the nervous system and the cutaneous receptors was studied using the quail-chick marking technique and light and electron microscopy. In the first experimental series the brachial neural tube of the quail was grafted in place of a corresponding neural tube segment of the chick embryo at HH-stages 10 to 14. In the second series the leg bud of quail embryos at HH-stages 18–20 was grafted in place of the leg bud of the chick embryos of the same stages and vice versa. It was found that all meningeal layers of the spinal cord, the perineurium and the endoneurium of peripheral nerves, as well as the capsular and inner space cells of Herbst sensory corpuscles, develop from the local mesenchymal cells. Schwann cells and cells of the inner core of sensory corpuscles are of neural crest origin. The precursors of Merkel cells migrate similarly to the Schwann cells into the limb bud where they later differentiate. This means that in addition to the Schwann cells and the melanocytes a further neural crest-derived subpopulation of cells enters the limb.     

An in vitro method for analysis of chondrogenesis in limb mesenchyme from individual transgenic (hdf) embryos.

The present study describes a simple, rapid protocol for culture for limb tissue from individual 10.5-day post coitum mouse embryos that supports cartilage differentiation over a six-day period. This technique differs from other commonly used methods utilizing pooled limb tissue in that: 1) forelimbs from individual embryos were used as donor tissue; 2) limb tissue was dissociated by very gentle enzymatic digest (0.1% trypsin, 5 min); and, 3) cell suspensions were plated at a lower density (1 x 10(7) vs. 2 x 10(7) cells/ml) in a reduced volume of 3-5 microl. Under these modified conditions to increase limb cell yield from each embryo, histochemical and immunohistochemical analyses demonstrated reproducible formation of precartilage aggregates and subsequent overt chondrogenesis over a predictable time course. Using this culture protocol, analysis of limb mesenchyme from heterozygous hdf embryos, which bear an insertional mutation of the Cspg2 gene encoding the core protein of the chondroitin sulfate proteoglycan, versican, revealed an overall similar chondrogenic potential to that observed for wild-type littermates. This technique readily enables in vitro culture of limb bud mesenchyme from individual mouse embryos at this developmental stage and may be utilized by investigators to study the effects of the hdf and other transgenic mutations on mammalian limb development in vitro.     

Development of the definitive kidney in albino rat

The current understanding of the kidney development in the pelvis and then a subsequent migration to the lumbar region is disputed. A total of 96 embryos were examined after they were obtained from 24 pregnant rats. A group of 4 pregnant rats were sacrificed at each day over the period from 12 to 17 successive days of pregnancy. An almost equal number of embryos were histologically investigated at each day using the paraffin technique. This was fulfilled by taking serial sections throughout the entire length of the trunk. The 13 days old embryo presented the appearance of the definitive kidney as indicated by the microscopical display of the ureteric bud invading the nephrogenic cord. This microscopic event took place at the lower lumbar region of all examined embryos. Therefore, the present work gives a microscopic evidence that the definitive kidney develops in the lumbar region.     

菟丝子含药血清对大鼠胚胎肢芽形态发育的影响

目的探讨不同剂量菟丝子含药血清对SD大鼠胚胎肢芽形态发育的影响。方法采用中药血清药理学方法制备菟丝子含药血清。取孕第15天胚胎前肢芽,随机分为3个实验组、阴性对照组和阳性对照组。实验组和阴性对照组分别加入10%培养体积的高、中、低剂量菟丝子含药血清和正常血清,阳性对照组加入全反式维甲酸溶液(10 mg/mL)。采用体外肢芽悬浮培养法培养72h后收获肢芽,石蜡包埋、切片、HE染色观察肢芽细胞和软骨形态。每组取30个肢芽,测量肢芽长度。结果经HE染色后,菟丝子含药血清低、中剂量组和阴性对照组肢芽结构完整,细胞排列整齐,软骨轮廓清晰,指突明显。高剂量组和阳性对照组肢芽软骨出现,轮廓较清晰,但指突不明显。分段测量肢芽长度后,与阴性对照组比较,低和中剂量组前肢芽近端a段长度和远端b段长度无显著性差异(P>0.05)。而阳性对照组和高剂量组的肢芽长度显著小于阴性对照组,与之比较差异显著(P<0.05)。结论高剂量菟丝子含药血清对SD大鼠胚胎前肢芽生长有抑制作用,可显著抑制肢芽指突的生长,存在潜在的胚胎发育毒性。     

Ultrastructure of developing muscle in the upper limbs of the human embryo and fetus

Background: The ultrastructure of the myogenesis, which proceeds along with the appearance of muscle-specific proteins and isozymes, has not been fully described in the upper limb of staged human embryos. Methods: Eight human embryos (Carnegie stage 14–22) and two fetuses (11 and 12 weeks of gestation) were fixed with 5% glutaraldehyde, 4% paraformaldehyde, and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.2. The upper limbs were dissected out and processed for transmission electron microscopy, and sections of the biceps brachii muscle were cut and examined. Results: At stage 14, the myoblasts were loosely scattered in the ventral proximal region of the upper limb bud and had a small amount of cytoplasm with a few intracellular organelles. At stage 16, the myoblasts were spindle shaped and oriented parallel to the axis of the upper limb bud. These cells had irregularly shaped nuclei with prominent nucleoli, rough endoplasmic reticulum (ER), and mitochondria, but no myofilaments were observed. At stages 17–19, rough ER, free ribosomes, and mitochondria increased in number and thick and thin filaments with faint Z-lines appeared in the peripheral cytoplasm of the myotube. The plasma membranes of some neighboring myotubes were continuous, suggesting that these cells were in the initial stages of the fusion process. At stage 22, the striated pattern of the myofilaments became evident and tubular structures appeared around them and near the plasma membrane. In the fetus at the 11th week, the basal lamina began to surround the myotubes, and T-tubules with sarcoplasmic reticulum were observed. Dyads and triads were observed in the myotube of the 12th week fetus. Conclusion: These findings suggest that rapid myogenesis occurs during the late embryonic period in human upper limbs and that the ultrastructural characteristics of mature myotubes are established during the early fetal period. © 1995 Wiley-Liss, Inc.