Differentiation in Coprinus lagopus

Fruit-body expansion was studied in Coprinus lagopus (sensu Buller) following surgical procedures. Elongation occurred after denuding mushroom caps of essentially all peripheral scales. Young primordia (1–5 mm) failed to develop after vertical bisection. Older primordia (e. g. 10 mm) expanded 3–4-fold after vertical bisection or quadrisection, underwent autolysis and basidiospore production. An amorphous brown gel in the stipe lumen disappeared during development of bisected primordia. Stipes isolated from primordia expanded autonomously and exhibited negative geotropism when incubated upside down or when the stipe apex was removed. Displacement of charcoal particles dusted on intact stipes revealed the most active zone of expansion to be the upper mid-region of the stipe. Segmented stipes likewise showed most active elongation in the mid-region. Vertically bisected stipes also expanded.     

Axis determination in polyspermic Xenopus laevis eggs

Polyspermic Xenopus laevis eggs can be identified easily because of regions of pigment accumulation and white stripes, which arise by a nocodazole-sensitive process. Eggs containing up to four sperm are capable of forming a single embryonic axis. Dispermic eggs display two regions of pigment accumulation, one around each sperm entry point (SEP), and one white stripe between the SEPs. Such eggs with a 180 degree separation between the SEPs were bisected before first cleavage along the white stripe, creating dorsal and ventral halves in many cases. Each half cleaved and formed a tadpole. When eggs were bisected early in the period of cytoplasmic reorganization (0.5-0.6 normalized time), each half could form a complete tadpole. When eggs were bisected after the period of reorganization (0.8-0.9), often one half formed a tadpole with a complete head but reduced or absent tail and the other half formed a tadpole with a complete tail but reduced or absent head. These results demonstrate that sperm cooperate to give a single embryonic axis in polyspermic eggs and the development of dorsal and ventral egg halves differs after egg reorganization before first cleavage.     

Quick-splitting of bovine embryos

Described is a simplified method of bovine embryo bisection amenable to on-farm embryo transfer. Using a microblade operated by a hand-held micromanipulator, Day 7 bovine embryos were bisected while in the zona pellucida. With a vertical motion, the embryo was pinned between the blade and the bottom of a plastic petri dish and bisected. Demi-embryos were transferred nonsurgically (without zonae pellucidae) into synchronized recipients. Pregnancy rates were normal with 5 13 (38%) and 9 20 (45%) of recipients confirmed pregnant 70 to 80 d after receiving either twin or single half embryos, respectively. This compared to 12 28 (43%) of recipients becoming pregnant from transfer of whole embryos. These data confirm that bovine demi-embryos do not need zonae pellucidae on Day 7 and that simplified field methods of bisection give normal pregnancy results.     

Visual behaviors of the migratory grasshopper,Melanoplus sanguinipes F. (Orthoptera: Acrididae)

The visual behavior of adult Melanoplus sanguinipesF. (Orthoptera: Acrididae) was investigated by placing individuals in the center of an arena and recording their orientation responses to visual targets at the perimeter of the arena. Targets that reflected more 540- to 570-nm light were approached more frequently; however, when reflectance in the 540 -to 570-nm region was combined with reflectance in the 400- to 520-nm region, orientation responses were reduced significantly. This suggests that spectral discrimination in M. sanguinipesinvolves at least two classes of photoreceptors, which respond to different regions of the wavelength spectrum. In addition, grasshoppers oriented to vertical, but not horizontal, contrasting stripes. However, when vertical stripes were added to targets reflecting 520- to 650-nm light, responses to verticals on these targets were not enhanced relative to verticals presented against a target background of 400- to 520-nm + 520- to 650-nm light. Thus, spectral discrimination and vertical stripe fixation appear to be two distinct visual behaviors, controlled by outputs from two classes of photoreceptors and a single class of photoreceptors, respectively, and may be used in different physiological or ecological contexts.     

Recovery from monocular deprivation in the monkey. III. Reversal of anatomical effects in the visual cortex

Transneuronal autoradiography was used to study the effects of visual deprivation on the ocular dominance stripes in layer IVc of the striated cortex of Erythrocebus patas (Old World) monkeys. The animals were studied after: (a) 21-28 days of monocular deprivation starting at, or within, a few days of birth; (b) the same treatment followed by a further 3, 6, 15 or 126 days of monocular vision through both eyes (reopening). One other monkey was monocularly deprived from birth to 1890 days. In most cases the behaviour of the ocular dominance stripes formed by the initially closed eye was studied. After 24 days of monocular deprivation from birth, the input from the normal eye was distributed uniformly within layer IVc, with no periodicity evident. After 21 days of deprivation, the deprived eye's input formed narrow stripes occupying about 38% of layer IVc in the operculum. Seven months of monocular deprivation reduced this to about 29%. Opening the closed eye after the deprivation produced no change in the area innervated: when periods of 15 or 96 days of binocular vision followed the deprivation, the areas innervated by the initially deprived eye were 26 and 30% respectively. However, in both cases the deprived eye's input formed blobs and spots, rather than uniformly narrow stripes. In contrast to reopening, reverse suturing increased the fraction of layer IVc occupied by input form the initially deprived eye. In the operculum, the effects of reverse suturing appeared to be fully developed after only 6 days of reversal: the initially deprived eye's stripes having expanded to occupy about 50% of layer IVc. A further 9 days' reversal produced little change in this. In the visual cortex in the calcarine fissure, the effect of the initial deprivation ws more severe, and the expansion induced by reverse suturing more pronounced. The initial deprivation caused the stripes to shrink to occupy 24% of layer IVc; after 6 days of reverse sulture the proportion increased to 52%, while after 15 days of reverse suture about 88% of IVc was occupied. These results show that reverse suturing can cause fresh growth of afferent axons in regions of layer IVc from which they had been at least partially removed, either by the normal process of segregation, or as a consequence of monocular deprivation. Taken in conjuction with the findings of the accompanying two papers (Blackemore et al...     

Production of monozygotic twins following the transfer of bisected embryos in the goats

Embryos at the morula, blastocyst and hatched blastocyst stage were obtained from superovulated and naturally ovulated Japanese native goats. They were bisected into halves with a glass needle, and transferred immediately or after culture (for morula) to recipients. None of five does which received bisected morula became pregnant. Three of nine goats became pregnant after transfer of bisected hatched blastocysts, six of eleven recipients became pregnant. Four of them produced monozygotic twins and the remaining two produced singles. The present study demonstrated that the hatched blastocyst is suitable for bisection in the goat.     

The generalization of directional visual stimuli in the honey bee, Apis mellifera

In transfer tests the ability of bees to generalize visual stimuli was tested by using differently inclined stripes and stripe patterns offered on a vertical screen. After having been trained to single stripes or equidistant stripe patterns, which were orientated by α₊ = 45° to the horizontal, the bees had to discriminate between the training direction α₊ and the competition direction α_c = 135° by means of special stripe configurations. These transfer patterns were obtained by varying different stimulus parameters of the original training stripes, for example by (1) reversing contrast between a stripe and the surrounding visual field, (2) changing the ratio of length/width and by this the dimensions of the stripe, and (3) inserting white intervals into the black stripes. In all three test series the bees succeeded in detecting the α₊-direction along a broad range of stimulus variations. As the bees in the transfer tests positively responded to patterns, which on the other side were significantly discriminated from the training pattern (control tests), the information about the direction of the visual cue had been transferred to a new pattern configuration never seen by the bees during the training situation.     

Influence of “Solcoseryl” during culture on the sex-dependent repair of bovine demi-embryos

The purpose of this experiment was to determine the effect of culture conditions on the development of split embryos after bisection and on the sex ratio of resultant bovine demi-embryos. Embryos that had developed into blastocysts on days 6½ to 7 or on days 7½ to 8 from oocytes matured and fertilized in vitro were bisected in BMOC-3 medium supplemented with 33% calf serum. The medium also contained 0%, 0.1% or 1.0% Solcoseryl, a deproteinized hemodialysate product from calf blood. The demi-embryos were first cultured for 4 hours in the same medium in which they had been bisected and then co-cultured with cumulus cells in TCM199 supplemented with 1% calf serum for an additional 20 hr. The rate of production of good to excellent quality demi-embryos obtained from days 6½ to 7 blastocysts was higher than from those on days 7½ to 8. The rate was also significantly improved when blastocysts were bisected in medium containing 0.1% or 1.0% Solcoseryl, compared to the medium without Solcoseryl. Male embryos seemed to recover more rapidly than female embryos, as assessed by morphological quality at 4 hr, although the quality of female embryos had improved by 24 hr. The percentage of males after culture was higher in the medium without Solcoseryl than in its presence. Thus, addition of Solcoseryl at either 0.1% or 1.0% to BMOC-3 medium seemed to improve the production efficiency of good quality demi-embryos, but did not influence the sex ratio. It appears as if female demi-embryos required more time than male embryos to be repaired after bisection. © 1996 Wiley-Liss, Inc.     

Gangliogenesis in leech: morphogenetic processes leading to segmentation in the central nervous system

 Using intracellular lineage tracers to study the main neurogenic lineage (N lineage) of the glossiphoniid leech embryo, we have characterized events leading from continuous columns of segmental founder cells (nf and ns primary blast cells) to discrete, segmentally iterated ganglia. The separation between prospective ganglia was first evident as a fissure between the posterior boundary of nf- and the anterior boundary of ns-derived progeny. We also identified the sublineages of nf-derived cells that contribute parallel stripes of cells to each segment. These stripes of cells project ventrolaterally from the dorsolateral margin of each nascent ganglion to the ventral body wall. The position and orientation of the stripes suggests that they play a role in forming the posterior segmental nerve; they are not coincident with the ganglionic boundary, and they form well after the separation of ganglionic primordia. Previous work has shown that cells in the anterior stripe express the leech engrailed-class gene. Thus, in contrast to the role of cells expressing engrailed in Drosophila, the stripes of N-derived cells expressing an engrailed-class gene in leech do not seem to play a direct role in segmentation or segment polarity. Received: 10 October 1997 / Accepted: 12 December 1997     

Sex-dependent loss of bisected bovine morulae after culture and freezing.

The objective of this study was to determine the post-transfer survival rate of bovine embryos cultured between the time of bisection and freezing. In this experiment 158 morulae were bisected and both portions were cultured for 24-44 h either in vivo after transfer to sheep oviducts (n = 80 morulae) or in vitro (n = 78 morulae) in Ham's F10 medium with 20% fetal calf serum, with bovine oviduct cells or in medium collected from oviduct cultures (conditioned medium). After culture, half of each morula was fixed for cytogenetic sex determination (n = 125) and the other half was frozen. The frozen halves were later thawed and transferred (n = 115) to recipients, who were, if pregnant, slaughtered to determine the sex of the fetus. The culture resulted in better pregnancy rates than those previously reported for embryos frozen immediately after bisection. The sex of 49 (33 males, 16 females) of the fixed demi-morulae was determined, and 38 of the transferred demi-morulae established pregnancies (23 males, 10 females and 5 fetuses that were not recovered). The male:female ratio in in vivo and in vitro culture groups was significantly different from the expected ratio of 1:1 and suggests that manipulation and culture of embryos results in a preferential loss of female embryos.     

In vitro and in vivo survival of bisected sheep embryos derived from frozen-thawed unsorted, and frozen-thawed sex-sorted and refrozen-thawed ram spermatozoa

Ovine IVP embryos were derived from frozen-thawed unsorted and frozen-thawed sex-sorted spermatozoa that had been refrozen and thawed. The embryos were bisected and cultured in vitro, or transferred to recipient ewes to determine their survival in vitro and in vivo. Oocyte progression to the blastocyst stage was similar for unsorted (97/232, 41.8%) and sex-sorted spermatozoa (113/286, 39.5%; P > 0.05). Embryo survival in vitro post-bisection was similar for demi-embryos derived from unsorted and sex-sorted sperm, and embryos bisected at the blastocyst and expanded blastocyst stage (P > 0.05). A higher proportion of recipient ewes were pregnant at Day 63 after transfer of two intact embryos derived from unsorted (17/21, 80.9%) than two demi-embryos derived from unsorted (5/15, 33.3%) or sex-sorted spermatozoa (7/17, 41.2%). The number of fetuses per original embryo at Day 63 did not differ among groups (unsorted intact: 23/42, 54.8%; unsorted demi: 7/15, 46.7%; sex-sorted demi: 10/17, 58.8%) and twin pregnancies were observed in all groups. Embryo survival to term was high, and was not significantly different among intact (unsorted: 22/42, 52.4%) and demi-embryos (unsorted: 4/15, 26.7%; sex-sorted spermatozoa: 7/17, 41.2%; P > 0.05). Dizygotic twins (n = 6 sets) were born after the transfer of two intact embryos derived from unsorted spermatozoa, but only singleton lambs resulted from the transfer of demi-embryos. In conclusion, bisected IVP embryos successfully developed into morphologically normal lambs. However, embryo survival to term was neither increased nor decreased by embryo bisection.     

Orientation by polarized light in the crayfish dorsal light reflex: behavioral and neurophysiological studies

In decapod crustaceans, the dorsal light reflex rotates the eyestalk so that the dorsal retina faces the brightest segment of dorsal visual space. Stepwise displacements of white stripes elicit eyestalk rotations in the same direction as that of the stripe. Conversely, stepwise displacements of black stripes on a white background elicit eyestalk rotations in the opposite direction as that of the stripe. The reversal of the response with contrast inversion distinguishes the dorsal light reflex from an optokinetic reflex. When the visual scene is composed of polarized light, segmented by variations in e-vector orientation, displacement of segments containing near vertical e-vectors elicit responses similar to those elicited by a white stripe. Displacement of polarized stripes containing near horizontal e-vectors elicit eyestalk rotations similar to those elicited by a black stripe. The results are consistent with the use of polarized light in orientation. The stimulus conditions described above were also applied to visual interneurons (sustaining fibers) and oculomotor neurons and the results were generally in accord with the behavior. In the neural studies, it was possible to show that responses to polarized stripe displacements are predictable from the receptive field location and the neuron’s polarization tuning function. John P. Schroeter deceased on September 14, 2006.     

Studies on induced changes in growth patterns and polarity in roots of Vicia faba L.

Summary Roots of Vicia faba were treated with colchicine (0.025%), or IAA (4.7×10^-6 M), or both, for 3 hours and fixed at various intervals over the following 11 days. The axis of spindle orientation and the distribution of mitotic figures, lateral root primordia and xylem vessel elements was examined in the apical 10 mm of median longitudinal sections of these roots.No effect of IAA was found on the orientation of the spindle. However, evidence was obtained indicating that the systems controlling the polarity of cell division and cell expansion differ in some way.The number of lateral root primordia formed was greater in roots treated with IAA or colchicine than in control roots. These primordia were always initiated adjacent to a xylem vessel. Thus, no primordium was closer to the apex than the most apical xylem vessel, suggesting that an endogenous factor involved in primordia initiation is transported in the xylem. The primordia which develop after colchicine treatment grow out as lateral roots; this is in contrast with those which form after IAA treatment and which do not undergo elongation. These results, which it must be emphasized apply only to the apical 1 cm of treated roots, indicate that lateral root primordia become sensitive to IAA at a certain stage in their development. Exogenous IAA acts as an inhibitor.The new meristem, which forms in the primary root apex after colchicine treatment, contains both diploid and polyploid cells, i.e. it was formed from cells that were unaffected and from cells that were affected by colchicine. Following colchicine treatment the size of the meristem shrinks and this can be prevented by treatment with IAA. This and other evidence presented here, suggests that IAA is a factor involved in the control of the size of the apical meristem in normal roots.     

Novel thick filament protein of chicken pectoralis muscle: the 86 kd protein. II. Distribution and localization

Antibodies specific for the novel 86 kd protein purified from chicken pectoralis myofibrils stained by indirect immunofluorescence the middle third of each half A-band of isolated myofibrils and myotubes. Pectoralis muscle 86 kd protein, like pectoralis C-protein, displayed a fibre-type specific distribution by being restricted to fast twitch fibres and absent in slow tonic and heart muscle fibres. This was demonstrated by immunoblotting experiments with tissue extracts and by immunofluorescence labelling of cryosections. In primary cell cultures prepared from embryonic chicken breast muscle, 86 kd protein, C-protein and myomesin were all detected in post-mitotic myoblasts where fluorescence was found in a cross-striated pattern along strands of nascent myofibrils. Fluorescence due to the 86 kd protein was restricted to myofibrils within myotubes and no significant labelling of the sarcoplasm was evident. Glycerinated fast twitch muscle fibres, after incubation with antibodies to 86 kd protein, revealed in each half of the A-band nine distinctly labelled stripes, spaced about 43 nm apart. Simultaneous incubation of fibres with antibodies against 86 kd protein and C-protein showed a co-localization of the seven C-protein stripes (stripes 5 to 11), with seven stripes of 86 kd protein. The two additional stripes (stripes 3 and 4) labelled by anti-86 kd antibody continued towards the M-band at the same periodicity from the last C-protein stripe (stripe 5). Thus, partial co-localization of two different thick filament proteins is demonstrated and the identity of transverse stripes at positions 3 and 4 attributed in part to the presence of the new 86 kd protein.     

Visual control of wing beat frequency in Drosophila

The present study shows that the wing beat frequency of Drosophila is visually controlled and modulated in response to different optomotor stimuli.Whereas rotational large field stimuli do not appear to modulate wing beat frequency, single rotating vertical stripes increase or decrease wing beat frequency when moving back-to-front or front-to-back, respectively. Maximal modulations occur at lateral stripe positions.Expansion stimuli eliciting the landing response cause a marked increase in wing beat frequency. Parameters of this frequency response depend in a graded fashion on certain stimulus properties, and the frequency response co-habituates with the landing response. Several results indicate that the frequency response is an integral component of the landing response, although it can also occur when the characteristic front leg extension is not observed. The complex spatial input integration underlying the frequency response and other motor components of the landing response cannot easily be explained by a system of large field integration units, but might indicate the existence of local expansion detectors.     

Non-periodic cues generate seven ftz stripes in the Drosophila embryo

We have examined the expression pattern of the segmentation gene fushi tarazu (ftz) by in situ hybridization to whole mount embryos using digoxygenin labeled probes. This method has revealed previously undetected stages in the development of the ftz RNA pattern. The ftz stripes arise individually in a distinct, non-linear order along the anterior-posterior axis of the embryo. In addition, the stripes develop differentially along the dorsal-ventral axis; most stripes emerge on the ventral side and then gradually spread dorsally until they surround the entire circumference of the embryo. The order of appearance of ftz stripes is not inversely correlated with the order of appearance of hairy (h) stripes as would be expected if ftz stripes were generated by h repression. Furthermore, the seven ftz stripes are correctly established in embryos carrying mutations in h, eve or runt, with normal expression patterns decaying only after cellularization. Thus, the so called primary pair-rule genes are involved in the refinement rather than establishment of the ftz stripes. The contribution of cis-acting regulatory elements to the ftz pattern was examined. The zebra and upstream elements interact to generate seven correctly positioned stripes at the end of cellularization. However, stripe establishement is not correctly mimicked by any ftz/lac fusion gene: stripes arise in an order drastically different from the endogenous ftz gene suggesting the existence of ftz regulatory elements outside the 10-kb region examined to date. These observations suggest that the ftz pattern is directed by at least two independent regulatory systems: first, stripe establishment is directed by regionally distributed factors that act differentially in individual stripes along both anterior-posterior and dorsal-ventral axes of the egg and, second, stripe refinement and maintenance are mediated by pair-rule gene products that interact with previously identified ftz regulatory elements. This multi-level regulation provides a back-up system that ensures the development of seven stripes in the blastoderm.     

Experimental phenocopy of a minute maternal-effect mutation alters blastoderm determination in embryos of Drosophila melanogaster

Maternal haploinsufficiency for a third chromosome Minute, M(3)i55, lowers rates of protein synthesis by approximately 30% during the syncytial nuclear cycles of early embryogenesis. The maternal effect of Mi55 also produces segmentation defects (denticle belt fusions) in the posterior abdomen of larvae. Furthermore, embryos from Minute mothers show abnormal expression patterns of the segmentation gene fushi tarazu (ftz) at the cellular blastoderm stage of embryogenesis. We developed a computer-aided analysis to describe the deviations in ftz expression which demonstrates that abnormally narrow ftz stripes occur in segment primordia that become fused in the larva. Unexpectedly, an abnormally wide ftz stripe occurs in segment primordia which do not develop abnormally. In addition, Mi55 produces a general narrowing of all ftz- interstripes. We phenocopied the Minute mutation by injecting wild-type embryos with cycloheximide concentrations which decreased protein synthesis rates to levels comparable with those of Minute embryos. Thus, a general decrease in protein synthesis during early embryogenesis leads to abnormal determination of posterior abdominal segment primordia.     

Gradients of Krüppel and knirps gene products direct pair-rule gene stripe patterning in the posterior region of the Drosophila embryo

Abdominal segmentation of the Drosophila embryo requires the activities of the gap genes Krüppel (Kr), knirps (kni), and tailless (tll). They control the expression of the pair-rule gene hairy (h) by activating or repressing independent cis-acting units that generate individual stripes. Kr activates stripe 5 and represses stripe 6, kni activates stripe 6 and represses stripe 7, and tll activates stripe 7. Kr and kni proteins bind strongly to h control units that generate stripes in areas of low concentration of the respective gap gene products and weakly to those that generate stripes in areas of high gap gene expression. These results indicate that Kr and kni proteins form overlapping concentration gradients that generate the periodic pair-rule expression pattern.     

Cortical neurite outgrowth and growth cone behaviors reveal developmentally regulated cues in spinal cord membranes.

Corticospinal axon outgrowth in vivo and the ability to sprout or regenerate after injury decline with age. This developmental decline in growth potential has been correlated with an increase in inhibitory myelin-associated proteins in older spinal cord. However, previous results have shown that sprouting of corticospinal fibers after contralateral lesions begins to diminish prior to myelination, suggesting that a decrease in growth promoting and/or an increase in inhibitory molecules in spinal gray matter may also regulate corticospinal axon outgrowth. To address this possibility, we carried out in vitro experiments to measure neurite outgrowth from explants of 1-day-old hamster forelimb sensorimotor cortex that were plated onto membrane carpets or membrane stripe assays prepared from white or gray matter of 1-to 22-day-old cervical spinal cord. On uniform carpets and in the stripe assays cortical neurites grew robustly on young but not older membranes from both white and gray matter. Mixtures of membranes from 1- and 15-day spinal cord inhibited neurite outgrowth, suggesting that the presence of inhibitory molecules in the 15-day cord overwhelmed permissive or growth promoting molecules in membranes from 1-day cord. Video microscopic observations of growth cone behaviors on membrane stripe assays transferred to glass coverslips supported this view. Cortical growth cones repeatedly collapsed at borders between permissive substrates (laminin or young membrane stripes) and nonpermissive substrates (older membrane stripes). Growth cones either turned away from the older membranes or reduced their growth rates. These results suggest that molecules in both the gray and white matter of the developing spinal cord can inhibit cortical neurite outgrowth.     

牛蛭属及其一新种(颚蛭目,医蛭科)

回顾了医蛭科牛蛭属分类研究过程,目前牛蛭属有8种,其中中国有6种.报告了采自中国广东和海南的牛蛭属1新种:广东牛蛭Poecilobdella guangdongensis sp.nov.,对新种形态特征进行了描述,并与近似种菲牛蛭特征进行比较.