Position of growth cones within the retinal nerve fibre layer of fetal ferrets.

Optic axons are added to the retinal nerve fibre layer of fish along its vitreal border in a chronotopic manner. Likewise, the optic tract of all vertebrate species acquires axons preferentially along the superficial surface of the pathway. We have examined the developing retina of fetal ferrets (Mustela putorius furo) aged between embryonic day 27 (E27) and E34 to see whether a similar segregation of growth cones is apparent within the mammalian retinal nerve fibre layer. The distributions of growth cone, "wrist" (thick trailing portion of the growth cone), axonal, and glial profiles were determined from electron micrographs, and expressed as a percentage of neural profiles for several retinal locations. The retinal nerve fibre layer of fetal ferrets contains radially elongated bundles of fibres composed of axonal, wrist, and growth cone profiles. Glial processes of varying density divide the adjacent bundles, occasionally subdividing them in the plane of the retina, and give rise to endfeet lining the basal lamina and separating the optic axons from the latter. Growth cones within the developing fibre layer represented about 2.4% of profiles at E28, while at later developmental stages (E34), this value fell to about 0.6%. During this period of axonal outgrowth, growth cones were not preferentially segregated toward the vitreal basal lamina or the glial endfeet within the nerve fibre layer. Rather, they were found scattered throughout the axon bundles of the fibre layer. While there were differences in the proportion of immature profiles found within the vitreal half compared to the scleral half of the fibre layer, such that more growth cones and wrists were found vitreally, there was no clear accumulation of them in association with features of the vitreal margin. The present results show that young and old optic axons course together throughout the depth of the nerve fibre layer. A chronotopic mode of pathway genesis such as seen in the optic fibre layer of fish or in the optic tract of mammals is not present in the nerve fibre layer of ferrets. Differences in growth cone behaviour in the optic fibre layer and tract indicate that the mechanisms governing pathway formation differ along its course.     

Transient GABA immunoreactivity in cranial nerves of the chick embryo

The distribution and time course of gamma-aminobutyric acid (GABA) immunoreactivity was investigated in the cranium of the chick embryo from 2 to 16 days of incubation (E2-16). A fraction of nerve fibers transiently stains GABA-positive in all cranial motor nerves and in the vestibular nerve. Cranial motor nerves stain GABA-positive from E4 to E11, including neuromuscular junctions at E8-11; labeled fibers are most frequent in the motor trigeminal root (E6-9.5). Substantial GABA staining is present from E4 to E10 in a subpopulation (1-2%) of vestibular ganglion cells. Their peripheral processes are labeled in the vestibular endorgan, predominantly in the posterior crista. Some GABA-positive fibers are present in the olfactory nerve (after E5) and in the optic nerve (after E9.5); their immunoreactivity persists throughout the period investigated. Transient GABA immunoreactivity follows "pioneer" fiber outgrowth and coincides with the formation of early synaptic contacts. GABA-containing neurons may change their neuronal phenotype (loss of GABA expression) or they may be eliminated by embryological cell death. Periods of cell death were determined in cranial ganglia and motor nuclei by aggregations of pycnotic cells in the same embryonic material. The periods of embryonic cell death partly coincide with transient GABA immunoreactivity. The function(s) of transient GABA expression is unknown. Some lines of evidence suggest that GABA has neurotrophic functions in developing cranial nerves or their target tissue. In the developing neuromuscular junction, GABA may be involved in the regulation of acetylcholine receptors.     

Stereological study of the developing distal femoral growth plate

The distal femoral growth plate has a uniquely convoluted structure comprised of four mammillate processes. Factors contributing to the development of these processes and overall plate geometry were explored using three-dimensional image analysis of the canine distal femoral epiphysis. The growth plate at birth remains relatively flat until ossification of the epiphysis begins at 1 week of age. Epiphyseal ossification proceeds eccentrically, projecting in the medial-lateral and anterior-posterior directions. Growth plate activity indexed by [3H]thymidine labeling and plate thickness revealed regional differences in cell proliferation. This was measured as a decreased labeling index and thinning of the growth plate in areas capped by the ossifying epiphysis. The eccentric ossification pattern and associated variations in growth plate activity result in definition of an "intraphyseal" groove and medial-lateral oriented sulcus. The groove and sulcus bisect the plate into four quadrants, giving rise to a convoluted structure composed of four areas of plate elevations termed mammillary processes (MP). By 5 weeks, the pattern of ossification results in greater development of the MP in the anterior-medial quadrant and in decreasing order, in the posterior-medial, anterior, and posterior-lateral quadrants. By 10 weeks, a uniform rate of cell proliferation was observed coincident with completion of ossification of the epiphysis. The data suggest that localized variations in growth plate proliferation are associated with ossification of the epiphysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selection of stereotyped VH81X-{micro}H chains via pre-B cell receptor early in ontogeny and their conservation in adults by marginal zone B cells

The pre-B cell receptor (preBCR) plays critical roles in early B cell differentiation. It has been shown that not all muH chains are capable of pairing with surrogate light (SL) chains to form preBCR. Here, we established a novel system to differentially identify two types of early pre-B cell populations in bone marrow and fetal liver of mice, one producing SL-pairing muH chains and the other producing SL-non-pairing muH chains. The former population accounted for 80% of all the early pre-B cells in adult bone marrow, while it accounted for only 20% of those in fetal liver. Comparison of the two types of pre-B cell populations in fetal liver revealed the structural difference between SL-pairing and -non-pairing muH chains encoded by the V(H)81X segment that was most frequently utilized in fetal liver pre-B cells but rarely expressed by B cells generated in adults. PreBCR played an important role in the positive selection of V(H)81X-muH chains carrying the characteristic sequences of the complementarity-determining region 3 with little or no nibbling or N nucleotide addition, leading to their predominance in neonatal splenic B cells. These fetal-type V(H)81X-muH chains were also detected in adult spleen, but almost exclusively in marginal zone (MZ) B cells in contrast to the adult-type V(H)81X-muH chains. This strongly suggests that neonatally generated and selected B cells expressing the stereotyped V(H)81X-muH chains are maintained in the adult MZ and could function as innate-like lymphocytes.     

A green to red photoconvertible protein as an analyzing tool for early vertebrate development.

Lineage labeling is one of the most important techniques in developmental biology. Most recently, a set of photoactivatable fluorescent proteins originating from marine cnidarians became available. Here, we introduce the application of the green to red photoconvertible protein EosFP as a novel technique to analyze early vertebrate development. Both injection of EosFP mRNA and purified, recombinant EosFP followed by a light-driven green to red conversion allow lineage labeling in virtually any temporal and spatial dimension during embryonic development for at least 2 weeks. Specific staining of cells from nonsurface layers is greatly facilitated by light-driven conversion of EosFP compared with traditional methods. Therefore, green to red photoactivatable proteins promise to be a powerful tool with the potential to satisfy the increasing demand for methods enabling detailed phenotypical analyses after manipulations of morphogenetic events, gene expression, or signal transduction.     

含生长抑素mRNA神经元在移植视网膜发育中的表达

将６０例鼠龄１４ｄＳＤ大鼠视网膜移植到出生后１ｄ大鼠中脑偏左侧处，同时摘除新生鼠的右眼。术后１０ｄ至２２ｄ分别取移植视网膜及其中脑，在相应的年龄取正常视网膜作对照，应用原位杂交组化技术──地高辛标记的生长抑素的ｃＲＮＡ探针检测移植和正常视网膜中含生长抑素ｍＲＮＡ神经元出现时间、发育规律及定位分布。同时用免疫组化方法显示生长抑素免疫反应神经元的形态，以作对照和补充。原位杂交组化实验结果表明：在出生当天的移植视网膜节细胞层已出现阳性表达，出生后４ｄ，外核层也出现小的含生长抑素ｍＲＮＡ神经元，此种神经元随后减少、消失；与此同时内核层及节细胞层内含生长抑素ｍＲＮＡ神经元逐渐增多，至生后１２ｄ接近成年水平。通过对非移植大鼠视网膜进行正常对照观察，移植视网膜与非移植的正常视网膜生后发育的结果相似。生长抑素免疫反应神经元与原位杂交含生长抑素ｍＲＮＡ神经元的表达部位是一致的，但生长抑素免疫反应神经元可见其突起。     

Experimental study of the development of the truncus arteriosus of the chick embryo heart. I. Time of appearance

The time of appearance of the truncus arteriosus was studied in the chick embryo using an in ovo labeling technique. Three hundred embryos at stages 13–18 of Hamburger and Hamilton were selectively labeled at the distal end of the heart tube, using gelatine-india ink label; 122 of these embryos were reincubated and 111 of them reached stages 25–28. In these stages the final location of the label was determined. Only 95 of these embryos showed both a normal heart and a label located in it. The remaining embryos were discarded due to abnormal cardiac morphology or because the label was not found. Embryos labeled at stages 13–14 had label in the conus in 42.8% of the cases and in the boundary between the conus and the truncus arteriosus in 57.1% of the cases. Label placed at stages 15–16 was located in the conus in 6.1% of the cases, in the boundary between the conus and the truncus arteriosus in 44.8% of the cases, and in the truncus arteriosus in 48.9% of the cases. Finally, label placed at stages 17–18 was located in the boundary between the conus and the truncus arteriosus in 18.7% of the cases and in the truncus arteriosus in 81.2% of the cases. Our results permit us to conclude that the truncus arteriosus appears in the chick embryo as early as stages 15–16 of Hamburger and Hamilton (50–56 hours of incubation). © 1993 Wiley-Liss, Inc.     

Determination of total body water by multifrequency bio-electric impedance: development of several models

Multifrequency bio-electronic impedance analysis (MF BIA) measurements are taken from a heterogeneous group of patients, varying in size between obese and slim. The measuring system uses four electrodes: two current and two potential electrodes. Three new models are developed to calculate total body water (TBW) from the BIA data, and the resulting TBW values are compared with TBW determined by D₂O dilution. The results demonstrate that the most simple model provides the best TBW values. For individual patients, TBW can be determined by means of bioimpedance measurement with an accuracy of 3 litres. In the most simple model (model 1), the body is electrically represented by a cylinder, and corrections are made for the amount of fat. This is an extension of the model used by Xitron. In the more advanced models (2 and 3), the body is represented by a cylinder for the trunk, and truncated cones represent the arms and legs. In model 2, ΔTBW amounts to 3 litres. It is shown that the resistance of the trunk is proportional to the square root of the length. In model 3, it is assumed that subcutaneous fat is a poor conductor if electric current. An equation is developed that describes the partition of subcutaneous fat, and the fat layer is then removed from the cones representing arms and legs and from the cylinder that models the trunk.     

Preferences of pre-weanling Long-Evans rats for anal excreta of adult males and females

Long-Evans hooded rat pups were reared with their dam alone, their dam and sire or their dam 12 hrs per day and their sire 12 hrs per day and tested for preferences for anal excreta from adult male and female rats when they were 18 to 20 days of age. In all rearing conditions the anal excreta of the rat's own dam was preferred to that of strange dams and the excreta of both own and strange dams was preferred to that of virgin females. Anal excreta from virgin females was preferred to no odor as was anal excreta of unmated males, strange sires and the rat's own sire. Only rats reared with their dam and sire preferred the odor of their own sire to that of other males. The results suggest that pre-weanling rats may attend to different dimensions of the odors of conspecifics, such as the species odor, maternal odor, and individual odor and that different rearing conditions may influence the odor dimension to which the rat attends.