bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell‐cycle progression during the development of Dictyostelium discoideum and its relation to the subsequent cell‐sorting in the multicellular structures
Authors: Araki Tsuyoshi , Maeda Yasuo
Published: October 2003

Previous studies have shown that the cell‐cycle phase at the onset of starvation is a naturally occurring variable that is closely involved in the subsequent sorting and differentiation of cells during Dictyostelium development. Here the cell‐cycle progression during the development of D. discoideum Ax‐2 cells and its relation to the subsequent cell‐sorting were analyzed in detail using synchronized cells and their pulse‐labeling by 5′‐bromodeoxyuridine (BrdU). Measurements of cell number and nuclearity provided evidence that about 80% of cells progressed their cell‐cycle after formation of multicellular structures (mounds). Many cells (T7 cells) starved at mid–late G2‐phase (just before the PS‐point from which cells initiate development when starved) progressed to the cell‐cycle after mound formation. In contrast, a less amount of cells (T1 cells) starved at late G2‐phase (just after the PS‐point) progressed through the cell‐cycle after mound formation. The significance of cell‐cycle progression presented here is discussed, with reference to cell differentiation and pattern formation.
Development of irradiated tunicate buds: Is cell division cycle required for morphallaxis?
Authors: Kawamura Kazuo , Hashimoto Koichi , Nakauchi Mitsuaki
Published: October 2003

In the tunicate,Polyandrocarpa misakiensis, transdifferentiation occurs in the multipotent atrial epithelium during morphallactic bud development. Irradiation (10–80 Gy) or aphidicolin (10 μg/mL) blocked this process severely, although the atrial epithelium could form organ placodes. The placodes consisted of cuboidal cells with a high nucleus : cytoplasm ratio and were lacking the alkaline phosphatase antigen from the cell surface, suggesting that the atrial epithelium might undergo dedifferentiation without initiating cell cycling. Irradiated buds could resume organogenesis in temporal accordance with the restoration of mitotic activity. Bud pieces irradiated at 40 Gy were juxtaposed with unirradiated counterparts. In the operated buds, irradiated, non‐dividing cells participated in organogenesis at the site of juxtaposition in cooperation with the unirradiated, dividing cells. These results have shown that inP. misakiensisthe cell division cycle, probably DNA replication, is indispensable for transdifferentiation of the atrial epithelium, although every cell in the organ rudiment need not enter cell cycling. We suggest that homoiogenetic induction occurs between dividing cells and non‐dividing cells.
Effects of vitamin D metabolites on axolotl limb regeneration
Authors: Washabaugh Charles H. , Tsonis Panagiotis A.
Published: October 2003

Vitamin D is essential for normal metabolism of phosphorus and calcium, and differentiation of skeletal elements. 1,25 dihydroxyvitamin‐D3, the biologically active metabolite, acts as an induction/proliferation switch in various cell types and promotes chondrogenesis of chick limb bud mesenchymal cells. The function of vitamin D is mediated through its nuclear receptor, the vitamin D receptor (VDR). The proliferative actions of 1,25(OH)2‐D3 on limb bud mesenchymal cells are similar to the ones produced by retinoids, such as all‐trans retinoic acid (RA) or 9‐cis retinoic acid (9‐cis). The retinoids have been shown to be compounds of extreme importance in the field of limb development and regeneration. In order to examine possible roles of vitamin D metabolites on limb regeneration, the effects of 1,25(OH)2‐D3, 24,25(OH)2‐D3 and KH1060 (a more potent metabolite) alone or in conjunction with all‐trans RA or 9‐cis RA on the regenerating axolotl limb. Vitamin D affects limb morphogenesis by generating abnormalities in skeletal elements. Synergism of vitamin D with retinoic acid in affecting pattern formation is suggested by the results.
The brain neurosecretory cells of the moth Samia cynthia ricini: Immunohistochemical localization and developmental changes of the Samia homologues of the Bombyx prothoracicotropic hormone and bombyxin
Authors: Yagi Yoshimasa , Ishibashi Jun , Nagata Koji , Kataoka Hiroshi , Suzuki Akinori , Mizoguchi Akira , Ishizaki Hironori
Published: October 2003

We produced mouse antisera against synthetic peptides corresponding to the sequences of the Samia cynthia ricini homologues of the Bombyx mori PTTH and bombyxin. Immunohistochemical analyses of the Samia cephalic neuroendocrine system using these antisera were performed to identify the neurosecretory cells (NSC) containing the PTTH and bombyxin homologues and to examine the developmental changes in their amounts in the NSC. The results show that the PTTH and bombyxin homologues are produced by two pairs of dorsolateral and 16 pairs of dorsomedial NSC of Samia brain, respectively, and both are transported to, and released from, the corpora allata. No clear‐cut correlation was found between the fluctuation in the amount of immunoreactive substances in the brain NSC and the endocrinologically anticipated timings of PTTH secretion. From Samia brain extract, two forms of PTTH activity (∼30 kDa and ∼5 kDa) were resolved through Sephadex gel filtration. The ∼30 kDa and ∼5 kDa PTTH seem to represent the PTTH and bombyxin homologues, respectively. We discuss that the ∼30 kDa PTTH homologue is the true PTTH of Samia.
A spatially restricted molecule of the extracellular matrix is contributed both maternally and zygotically in the sea urchin embryo
Authors: Wessel Gary M. , Berg Linnea
Published: October 2003

The extracellular matrix of the early sea urchin embryo is known to have an important functional role in morphogenesis and in the regulation of cell type specific gene expression. We have undertaken an immuno‐cDNA screen to identify the constituents of the embryonic blastocoelic‐extracellular matrix. Here we describe a newly identified member of the extracellular matrix that we have designated ECM 3. The transcript encoding ECM 3 is approximately 9.5 kb in length and partial DNA sequence contains no significant similarity to other sequences in the Genbank. This transcript is present in eggs and early embryos, and early in gastrulation the transcript accumulation increases approximately 25 fold. In situ RNA hybridization shows that the mRNA is present uniformly throughout eggs and early embryos, but beginning at mesenchyme blastula stage, RNA accumulation is selective to cells of the ectoderm except at the animal pole, where ECM 3 mRNA is greatly reduced. In this species, Lytechinus variegatus, the animal pole ectoderm is the site of fusion with the invaginating endoderm during formation of the mouth. In situ analysis of protein expression using a monospecific polyclonal antisera made against recombinant ECM 3 polypeptides shows that during gastrulation the ECM 3 protein accumulates selectively in the basal lamina and blastocoelar regions adjacent to the ectoderm in all regions except for the ectoderm at the animal pole. The ECM 3 protein is not detected in other regions of the blastocoel e.g. adjacent to the endoderm. ECM 3 is also contributed maternally; the ECM 3 protein is synthesized during oogenesis and stored in oocytes within membrane‐bound vesicles in the vicinity of Golgi complexes. Following fertilization ECM 3 is selectively secreted basally into the nascent blastocoel. No accumulation is detected in apical regions of the blastomeres or in the hyaline layer/apical lamina. This newly described molecule of the extracellular matrix thus demonstrates expression regulated both by secretion and by cell type specific gene expression, and shows a correlation between a microenvironment of the extracellular matrix and a morphogenetic event.
Development of Notophthalmus viridescens embryos
Authors: Khan Paul A. , Liversage Richard A.
Published: October 2003

Although the North American, adult, red‐spotted newt has been an excellent model in the study of appendage regeneration at the tissue and cell levels, experiments involving embryonic forms have been essentially impossible to perform at the molecular level due to the great difficulty in breeding newts in the laboratory. Recently, we reported our methods to successfully spawn many thousands of embryos of Notophthalmus viridescens for developmental studies on a year round basis. As no detailed examination of embryogenesis of this amphibian exists, we provide a scanning electron microscopic overview of N. viridescens development. Furthermore, we chronicle the life cycle of the newt, when produced under laboratory conditions, which also undergoes two metamorphoses as newts in the wild.
Injection of myo‐inositol reverses the effects of lithium on sea urchin blastomeres
Authors: Livingston Brian T. , Wilt Fred H.
Published: October 2003

Lithium is known to cause sea urchin blastomeres destined to give rise to epithelium rather than to differentiate into gut or skeleton. While it has been proposed that lithium alters development by interfering with the inositol‐tris phosphate‐protein kinase C (IP3‐PKC) signaling pathway, the mechanism of action of lithium in sea urchins has remained elusive. Here we describe experiments that examine the hypothesis that lithium exerts its effect on sea urchin embryos via the IP3‐PKC pathway. We make use of methods developed to isolate epithelial precursor cells from the animal hemisphere of cleavage 16‐cell stage embryos. Pairs of cells were isolated and one of each pair was injected with either myo‐inositol or its inactive isomer, epi‐inositol. Rhodamine dextran was co‐injected as a lineage tracer to follow the fate of injected cells. We demonstrate that injected myo‐inositol, but not epi‐inositol, can reverse the effects of lithium on sea urchin blastomeres. This is direct evidence that lithium affects the IP3‐PKC pathway in sea urchins, and that this pathway plays an important role in cell fate determination.
Genetic regulation of mec‐3 gene expression implicated in the specification of the mechanosensory neuron cell types in Caenorhabditis elegans
Authors: Mitani Shohei
Published: October 2003

The mec‐3 gene, a member of the LIM‐homeodomain transcription factors, is required for touch receptor, FLP and PVD neurons to differentiate in the nematode Caenorhabditis elegans. Stably integrated transgenic strains with mec‐3‐lacZ fusion were generated by irradiating UV light to an unstable transgenic strain with the extrachromosomal DNA. Expression patterns of the mec‐3‐lacZ fusion were examined in mutant backgrounds (lin‐4, lin‐14, egl‐44, egl‐46 and sem‐4 genes) which alter touch receptor‐specific gene expression. In the lin‐4 mutant background, ectopic mec‐3‐lacZ positive AVM/PVM‐like cells were observed in 9% of the animals. By contrast, in the lin‐14 mutant background, mec‐3‐lacZ staining in AVM/PVM cells was lost in 86% of the animals. In the egl‐44 and egl‐46 mutant backgrounds, expression pattern was the same as wild‐type animals. In the sem‐4 mutant background, more than half of the animals (54–69%) had ectopic staining cells in the tail in addition to the wild‐type staining pattern. The modes of action of these genetically interacting genes in the differentiation of mechanosensory neurons are proposed.
Overlapping origins of pharyngeal arch crest cells on the postotic hind‐brain
Authors: Shigetani Yasuyo , Aizawa Shinichi , Kuratani Shigeru
Published: October 2003

The developing hind‐brain of vertebrates consists of segmental units called rhombomeres. Although crest cells emigrate from the hind‐brain, they are subsequently subdivided into several cell populations that are attached to restricted regions of the hind‐brain. At the preotic level, only even‐numbered rhombomeres are accompanied by crest cells, while the odd‐numbered ones are not. At the postotic level, such the birhombomeric repetition becomes obscure. In order to map the origins and distributions of postotic crest cells, focal injections of Dil were made into various axial levels of the postotic neural tube. Cephalic crest cells at the postotic level first form a single cell population deposited by cells along the dorsolateral pathway. They are called the circumpharyngeal crest cells (CP cells) and are secondarily subdivided into each pharyngeal arch ectomesenchyme. The neural tube extending from r5 to the somite 3/4 boundary gave rise to CP cells. The neuraxial origins of each pharyngeal ectomesenchyme extended for more than three somite lengths, most of which overlapped with the other. Unlike in the preotic region, there is no segmental registration between neuraxial levels and pharyngeal arches. Caudal portions of the CP cell population show a characteristic distribution pattern that circumscribes the postotic pharyngeal arches caudally. Heterotopic transplantation of the Dil‐labeled neural crest into the somite 3 level resulted in a distribution of labeled cells similar to that of CP cells, suggesting that the pattern of distribution depends upon dynamic modification of the body wall associated with pharyngeal arch formation.
Changes in chorion proteins induced by the exudate released from the egg cortex at the time of fertilization in the teleost, Oryzias latipes
Authors: Iwamatsu Takashi , Shibata Yasushi , Kanie Toshihiro
Published: October 2003

The chorion of unfertilized medaka Oryzias latipes eggs consists of two major proteins (77–73 and 49 kDa) and a minor 150 kDa protein. Upon fertilization, these major chorion proteins are polymerized to insoluble high molecular weight proteins via the temporary formation of several new proteins (132, 114, 62 and 61 kDa). Increasing chorion toughness is closely related to the formation of high molecular weight proteins and the increasing insolubility of the chorion proteins. The changes in chorion proteins and hardening could be induced in vitro in isolated chorions by an egg exudate, which includes cortical alveolar contents. The effects of temperature and pH on the egg exudate‐induced changes in chorion proteins were examined in the present study. The major proteins could be digested by proteolytic enzymes. The 49 kDa protein was PAS‐positive. Analysis with polyclonal antibodies against the major proteins demonstrated that the temporarily formed 62 and 61 kDa proteins were derived from the 77–73 kDa protein and that higher molecular weight proteins, newly formed in the process of chorion hardening, contained the same epitopes as did the 77–73 and 49 kDa proteins. The results suggest that the changes in chorion proteins of the medaka egg at the time of fertilization can be induced by an enzyme(s) released from the egg cortex into the perivitelline space.
Activation of newt eggs in the absence of Ca2+ activity by treatment with cycloheximide or D2O
Authors: Iwao Yasuhiro , Masui Yoshio
Published: October 2003

Unfertilized eggs of urodeles that exhibit physiological polyspermy are difficult to activate by ordinary egg‐activating agents, such as pricking and Ca2+ ionophores, that easily activate monospermic anuran eggs. Therefore, we have tested the effects of other agents that cause egg activation in non‐amphibian species in order to investigate the mechanism of egg activation in urodeles. We have found that cycloheximide (a protein synthesis inhibitor), D2O (that induces microtubule polymerization) and 6‐DMAP (a protein kinase inhibitor) caused activation of unfertilized eggs of the newt, Cynops pyrrhogaster. The cell cycle, arrested at meiotic metaphase II, was resumed to form the second polar body accompanied by a loss of maturation promoting factor and cytostatic factor activity. The treated eggs underwent abnormal cleavage. These results indicate that protein synthesis followed by protein phosphorylation is necessary to maintain M phase in unfertilized Cynops eggs. Unfertilized eggs failed to be activated by pricking, but were activated by the ionophore A23187, but only at a concentration 30 times higher than that required to activate Xenopus eggs. Eggs whose intracellular Ca2+ ions had been chelated by BAPTA could also be activated by either cycloheximide or D2O. Cycloheximide‐ as well as 6‐DMAP‐induced egg activations were not inhibited by nocodazole, a microtubule‐depolymerizing agent. These results suggest that the inhibition of synthesis and phosphorylation of short‐lived proteins acts as an egg activation mechanism, downstream of the site of Ca2+ action and independently of microtubule polymerization.
Spontaneous and sperm‐induced activation of oocytes in LT/Sv strain mice
Authors: Maleszewski Marek , Yanagimachi Ryuzo
Published: October 2003

The oocytes of LT/Sv strain mice are unique in that a high proportion of them (∼40% in this study) are ovulated before reaching metaphase of the second meiotic division (metaphase II). The remaining oocytes of LT/Sv mice are ovulated at metaphase II, as in other strains of mice. When recently ovulated oocytes were cultured in vitro for 11–12 h, those ovulated at metaphase II remained at this stage, whereas those ovulated at metaphase of the first meiotic division (metaphase I) commonly resumed meiosis during in vitro aging. These oocytes extrude the polar body and form a diploid pronucleus. This oocyte activation is not coupled with cortical granule exocytosis. The oocytes ovulated at metaphase II are fully capable of normal fertilization, whereas those ovulated at metaphase I are not. Approximately 50% of metaphase I oocytes penetrated by spermatozoa remain at this stage, and sperm nuclei frequently undergo premature chromosome condensation. Only 13% of spermpenetrated metaphase I oocytes formed a diploid female pronucleus and a haploid male pronucleus by 4 h after insemination. These results demonstrate that the two types of ovulated LT/Sv oocytes have different potentials to undergo either spontaneous or sperm‐induced activation.
Unequal first cleavage in the Tubifex egg: involvement of a monastral mitotic apparatus
Authors: Ishii Ryuichi , Shimizu Takashi
Published: October 2003

The first cleavage in the freshwater oligochaete Tubifex hattai is unequal and meridional, and produces a smaller cell AB and a larger cell CD. This study traces the process of furrow formation, reorganization of cortical F‐actin and the assembly of a mitotic apparatus during this unequal division. Cleavage furrow formation consists of two stages: (i) when eggs are viewed from the animal pole, meridionally running furrows emerge at two points of the egg's equator that are 90° apart from each other and approach the egg axis as they deepen; and (ii) at the midpoint between the equator and the egg center, the bottoms of these furrows link to each other on the animal and vegetal surfaces of the egg and form a continuous ring of constriction in a plane parallel to the egg axis. Egg cortices, isolated during the first step and stained with rhodamine‐phalloidin, show that the bottoms of recently formed furrows are underlaid by a belt of tightly packed actin bundles (i.e. a contractile arc). The transition to the second stage of furrow formation coincides with the conversion of these actin belts into a continuous ring of F‐actin. Whole‐mount immunocytochemistry of microtubules reveals that the first cleavage in Tubifex involves an asymmetric mitotic spindle, which initially possesses an aster at one pole but not the other. This ‘monastral’ spindle is located at the egg's center and orients itself perpendicular to the egg axis. During anaphase, astral rays elongate to reach the cell surface, so that the array of astral microtubules in the plane of the egg's equator covers a sector of 270–300°. In contrast, it is not until the transition to telophase that microtubules emanating from the anastral spindle pole approach the cell margin. If eggs are compressed along the egg axis or forced to elongate, they form monastral spindles and divide unequally. In living compressed eggs, mitotic spindles, which are recognizable as bright streaks at the egg's center, appear not to shift their position along the spindle axis during division, suggesting that without eccentric migration of spindles Tubifex eggs are able to divide unequally. These results suggest that mechanisms that translocate the mitotic spindle eccentrically do not operate in Tubifex eggs during the first cell cycle. The mechanisms that generate asymmetry in spindle organization are discussed in the light of the present results.
Patterning of the cranial nerves in the chick embryo is dependent on cranial mesoderm and rhombomeric metamerism
Authors: Kuratani Shigeru , Aizawa Shinichi
Published: October 2003

The vertebrate peripheral nervous system (PNS) consists of two groups of nerves that have a metamerical series of proximal roots along the body axis: the branchial and spinal nerves. Spinal nerve metamerism is brought about by the presence of somites, while that of the branchial nerves is, in part, intrinsic to rhombomeres, the segmental compartments of the hind‐brain. As the distribution pattern of neural crest cells prefigures the morphology of the PNS, we constructed tissue‐recombinant chick embryos in order to determine factors that might regulate the crest cell distribution pattern. When the segmental plate was transplanted between the hind‐brain and the head mesoderm before crest cell emigration, it developed into ectopic somites that inhibited the dorsolateral migration of crest cells such that formation of the cranial nerve trunks was disturbed. Even so, proximal portions of the nerve roots were intact. An ectopic graft of lateral mesoderm did not inhibit the directional migration of the crest cells, but allowed their ectopic distribution, resulting in the fusion of cranial nerve trunks. When spinal neurectoderm was transplanted into the hind‐brain, the graft behaved like an even‐numbered rhombomere and caused the fusion of cranial nerve roots. The identity of the spinal neurectoderm was preserved in the ectopic site analyzed by the immunolocalization of Hoxb‐5 protein, a spinal cord marker. We conclude that the spatial distribution of cephalic crest cells is regulated by successive processes that act on their proximal and distal distribution. The migratory behavior of crest cells is achieved partly by an embryonic environment that is dependent upon the presence of somitomeres, which do not epithelialize as somites, in the trunk.
Localization of DNA‐synthesizing cells and cell proliferation pattern in developing proventricular (glandular stomach) epithelium of embryonic and hatched chickens
Authors: Jin Da‐Yun , Ishii Yasuo , Yasugi Sadao
Published: October 2003

Localization of DNA‐synthesizing cells in the developing proventricular (glandular stomach) epithelium of embryonic and hatched chickens was investigated. DNA‐synthesizing cells were scattered throughout the proventricular epithelium during all developmental stages studied. The results indicate that there is no clear proliferative zone in the proventricular epithelium of the chicken. The labeling indices (LI) of proventricular epithelial cells were measured. On the 6.5th day of incubation, the LI of glandular epithelium reached 29.5 ± 1.5%. the highest value of all the stages studied. This extremely rapid cell proliferation can be considered to be a driving force for the elongation of the proventricular glands during the following stages. Just after hatching, the LI of both the glandular and luminal (non‐glandular) epithelia significantly increased from those on the 18th day of incubation. It is suggested that the rise in LI possibly reflected proventricular growth to fit in the change in the method of nourishment after hatching. In 2 week old chickens, the LI of both the glandular and luminal epithelia were reduced to approximately 1%. The active production of embryonic chicken pepsinogen in all glandular epithelial cells of the embryonic chicken revealed that proliferation and differentiation are not necessarily exclusive during the embryonic stages of proventricular development.
Thermal tolerance and heat shock protein synthesis during development in the anuran Lepidobatrachus laevis
Authors: Carroll Edward J.
Published: October 2003

Development of the Paraguayan anuran Lepidobatrachus laevis is unusual in that the larvae are obligate carnivores, facultative cannibals and apparently exist at high environmental temperatures in their natural habitat. In the present study, the effect of environmental temperature on the rate of anuran development was investigated. The larvae have a thermotolerance range of 18°C for normal development between 19 and 37°C. The effect of temperature on the rate of development was dramatic; larvae that were incubated at 36.8°C develop to stage 24 (Gosner) in approximately 9 h compared with 24 h for larvae incubated at 19°C. The ability of larvae to survive heat shock was also examined; larvae did not survive a shock of 45°C for 15 min when it was administered at stages 3, 5, 9, 10 or 20. However, using the same heat shock conditions, 50% survival was observed when larvae were shocked at stage 16. To study protein synthesis during heat shock, larvae were pulsed with [35S]‐methionine during heat shock and labeled proteins were analyzed by electrophoresis under reducing and denaturing conditions. Larvae synthesized two sets of heat‐shock proteins at doublet molecular weights of 83/78 and 62/59 kDa. These proteins were synthesized independently of the stage of development at which the shock was administered or the magnitude of the heat shock.
Organization of the spina bifida neural tube in Splotch (Pax‐3 defective) mouse embryos
Authors: Takahashi Yoshiko
Published: October 2003

The development of the dorsal portion of the spinal cord is adversely affected in a mouse mutant, Splotch (Sp). This mutation is a splicing defect of Pax‐3 mRNA, which encodes a transcription factor. Organization of the neural tube in Sp/Sp mice has been studied with the genes Pax‐3, Wnt 3a, Msx 1 and Msx 2, which are expressed specifically in the dorsal portion of the neural tube. Regionalization of the Sp neural tube is properly preserved along the dorso‐ventral and medio‐lateral axes. This suggests that the severe phenotype of the central nervous system (CNS) in Sp embryos is not caused by gross loss of a particular cell population. The level of expression of the Msx 2 gene (a homeobox gene, previously called Hox 8), whose expression pattern is similar to that of Pax‐3 in the developing neural tube, is reduced in neuroepithelial cells of Sp mutants in the CNS. This suggests that Msx 2 is located downstream to Pax‐3 in a regulatory pathway that is required during the development of the dorsal portion of the neural tube.
Dictyostelium mucoroides cells exhibit cell cycle‐dependent sorting behavior as observed in D. discoideum development
Authors: Amagai Aiko , Maeda Yasuo
Published: October 2003

Evidence has been obtained indicating that the cell's position in the cell cycle at the onset of starvation is a naturally occurring variable closely involved in the subsequent sorting and pattern formation during the development of Dictyostelium discoideum Ax2. It is of interest to know whether a similar phenomenon is also noticed in species other than D. discoideum and also without any treatment of cells for cell synchronization. For this, the sorting behavior of D. mucoroides‐7 (Dm7) cells and its relation to the cell‐cycle phase at the onset of starvation were analyzed, using non‐synchronized Dm7 cells pulse‐labeled with 5′‐bromo‐2‐deoxyuridine (BrdU). The results demonstrate that Dm7 cells starved at the early G2 phase aggregate most rapidly, but are eventually sorted out to the posterior prespore zone of migrating slugs. In contrast, cells starved at the mid late G2 phase exhibited slower aggregation, but were sorted out to the anterior zone (tip), this being basically similar to the sorting behavior of D. discoideum cells. Measurements of cell numbers and nuclearity provided evidence that approximately 80% of cells progressed their cell‐cycle after the formation of multicellular structures (mounds), probably coupling with prespore differentiation as in the case of D. discoideum. Thus, cell cycle‐dependent sorting during Dictyostelium development is most likely to be a common phenomenon in different species.
Profiles of conjugation induction by potassium ions in four different Ca2+ channel mutants of Paramecium caudatum
Authors: Takahashi Mihoko , Shirakura Shin‐ichi
Published: October 2003

Conjugation of Paramecium caudatum among cells of a single mating type can be induced parthenogenetically by exposing them to certain chemical solutions, a process called chemical induction of conjugation. The ionic conditions for chemical induction of conjugation are also the conditions that are required for the induction of the activation of voltage dependent Ca2+ channels in Paramecium. Four mutants controlled by independent gene loci with defects in Ca2+ channels, were subjected to an induction solution containing various concentrations of KCl and 0.01, 0.06 and 0.6 mmol/L CaCl2. Conjugation was able to be induced chemically in all four mutants. However, some locus‐dependent differences were observed in the profiles of induction. Mutants controlled by different alleles but with the same locus were similar in their profile of induction, even when they exhibited opposite phenotypes on the activation of Ca2+ channels. These results suggest that the function of Ca2+ channels is not directly involved in the mechanism of chemical induction of conjugation. The locus dependency observed reflects the different role of each gene controlling Ca2+ channel functions.
Possible involvement of an acylation mechanism in thalidomide‐induced teratogenesis of the newt (Pleurodeles waltl.)
Authors: Audit C. O. , Eger K. , Aimar C.
Published: October 2003

An acylation reaction of biological polyamines by thalidomide has been postulated to explain the teratogenic activity of this drug (Fabro et al. 1965). In a further study, thalidomide has been reported to acylate polyamines at physiological pH; the teratogenic activity of this drug appears to be linked to its high acylating power towards polyamines (Audit 1994).

In the present study, the action of the thalidomide molecule and its two chemical moieties (phthalimide and glutarimide rings) on Pleurodeles embryonic development has been investigated. The phthalimide moiety, which displays acylating activity, appears to generate Pleurodeles teratogenesis. The occurrence of a correlation between acylating activity and teratogenicity was confirmed using homothalidomide and partially hydrolyzed thalidomide. The glutarimide moiety has been found to act as an enhancer of phthalimide activity and to cause moderate alterations of newt development.

As the acylation of polyamines by thalidomide would deprive the embryo of these essential compounds, the effects of polyamine biosynthesis inhibitors have been compared to those of thalidomide. Both thalidomide and polyamine antimetabolites altered the early cleavage process of the Pleurodeles egg and arrested early development.
A nuclear GFP/β‐galactosidase fusion protein as a marker for morphogenesis in living Drosophila
Authors: Shiga Yasuhiro , Tanaka‐Matakatsu Miho , Hayashi Shigeo
Published: October 2003

A general, non‐invasive method to trace morphogenesis in living Drosophila was developed. To label specific cells, green fluorescence protein (GFP) of jellyfish Aequorea victoria was expressed by the Ga14‐UAS system. Green‐fluorescence from GFP fused to the nuclear localization signal was detectable in polytene larval tissue, but not in diploid tissue. Further fusion to bacterial β‐galactosidase produced GFPN‐lacZ, which fluoresced brightly in several diploid larval and embryonic tissues. GFPN‐lacZ was used to trace dynamic cell movement during the formation of the embryonic tracheal system. These results indicate that GFPN‐lacZ can be used to mark specific cells to study cell movement and gene expression in living animals.
Polyploidization of nuclei in the yolk syncytial layer of the embryo of the medaka, Oryzias latipes, after the halt of mitosis
Authors: Kageyama Tetsuo
Published: October 2003

It has been reported that nuclei repeat parasynchronous mitosis four or five times in the yolk syncytial layer (YSL) of the embryo of the medaka, Oryzias latipes, during the blastula stage and that no mitosis occurs in the YSL after the gastrula stage. The present investigation demonstrated the size of nuclei and the number of nucleoli and their staining properties with DNA binding dye. The results indicate that the YSL nuclei actively transcribe RNA and that their DNA content is greater than that of somatic nuclei. The onset and subsequent time course of polyploidization were examined in embryos stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) by epifluorescence microspectrophotometry from the cessation of mitosis through hatching. Embryos included YSL nuclei whose DNA content spanned from diploid (2C), tetraploid (4C) to octaploid (8C) at the end of the late blastula stage. The last two populations are produced probably by their early cessation of mitosis and the subsequent duplication of DNA without mitosis or by endoreduplication. The frequency distribution of the DNA content examined during epiboly of the blastoderm suggests that each population is duplicated again until the beginning of the gastrula stage and then once more until the end of epiboly. Eventually these nuclei include polyploid DNA between 8C and 64C or more during later embryonic development.
A cyto‐embryological study of gastrulation in the sand dollar, Scaphechinus mirabilis
Authors: Kominami Tetsuya , Masui Mizuko
Published: October 2003

Processes of gastrulation in the sand dollar Scaphechinus mirabilis were compared with those in the sea urchin Hemicentrotus pulcherrimus, which seemed to show a typical pattern of gastrulation. Measurement of the archenteron length clearly demonstrated that invagination processes in H. pulcherrimus are divided into two phases, the primary and secondary invagination. On the other hand, invagination in S. mirabilis was revealed to continue at a constant rate. To see the movement of cells during gastrulation, embryos were labeled with Nile blue. In H. pulcherrimus embryos, labeled cells were observed along the full length of the archenteron, if the embryos had been labeled before and during the primary invagination. Labeled cells were never observed in the embryos stained after the primary invagination. In contrast, labeled cells were always discerned at the basal part of the archenteron in S. mirabilis, even if the embryos were stained after invagination had undergone considerable progress. The number of cells in the archenteron of S. mirabilis embryos increased with the advancement of gastrulation, while the numbers were almost constant in H. pulcherrimus. These results suggest that the cellular basis of gastrulation in S. mirabilis is quite different from that in well‐known species of sea urchins.
Change of cyclin D2 mRNA expression during murine testis development detected by fragmented cDNA subtraction method
Authors: Nakayama Hiroki , Nishiyama Hiroyuki , Higuchi Toshihiro , Kaneko Yoshiyuki , Fukumoto Manabu , Fujita Jun
Published: October 2003

Using subtractive hybridization and polymerase chain reaction, we developed a differential cloning system, the fragmented cDNA subtraction method, that requires only small amounts of materials. The cloning system was used to isolate several cDNA fragments expressed more abundantly in the premeiotic day 3 post‐natal mouse testis than in the adult mouse testis. The isolated cDNA fragments included cDNA encoding the murine cyclin D2. Northern blot and in situ hybridization analyses revealed that, during testis development, cyclin D2 expression was most abundant in the neonatal proliferating Sertoli cells. Those type A spermatogonia that were thought to divide mitotically also expressed cyclin D2 mRNA. Other spermatogenic cells, such as mitotically arrested gonocytes in neonatal testis and meiotically dividing germ cells in adult testis as well as adult Sertoli cells, were negative for the cyclin D2 signal. Adult W/Wv mutant mice lacking germ cells expressed cyclin D2 mRNA in terminally differentiated Sertoli cells. Elimination of germ cells other than the undifferentiated type A spermatogonia by treating wild‐type mice with an anti‐c‐kit monoclonal antibody did not result in the expression of cyclin D2 in Sertoli cells. These results demonstrate that there are lineage‐ and developmental‐specific expression patterns of cyclin D2 mRNA during mouse testis development. At the same time, it is suggested that primitive type A spermatogonia affect the cyclin D2 expression of Sertoli cells.
Ontogeny of low molecular weight stress protein p26 during early development of the brine shrimp, Artemia franciscana
Authors: Jackson Susan A. , Clegg James S.
Published: October 2003

Embryogenesis in the brine shrimp, Artemia sp., occurs by one of two pathways: (i) the direct, uninterrupted development of nauplius larvae within the female or (ii) the production of embryos that arrest development at the gastrula stage and enter diapause. Diapause embryos are released from females into the aqueous environment where they remain in diapause until activated by appropriate environmental cues and resume development. These encysted embryos possess at least one low molecular weight stress protein, which we refer to as p26 and which has been implicated previously in the stress response of activated embryos. We investigated the appearance of p26 in developing diapause embryos in utero and looked for its presence in embryos developing directly into nauplii. We found p26 to be specific to diapause‐destined embryos; it was not detected in direct‐developing embryos. We conclude that p26 is not required for the basic developmental program that produces the nauplius. In diapause‐destined embryos, p26 was first detectable after 3 days of development, at which time the embryos were late gastrulae. This protein continues to increase in amount until the encysted embryos are released, approximately 5 days after fertilization. At the time of release almost all p26 is located in the low speed supernatant fraction, but as released embryos continue diapause, p26 transfers to the pelleted nuclear fraction in increasing amounts. Our working hypothesis views p26 as a molecular chaperone preventing protein denaturation and aggregation under conditions associated with metabolic arrest and other stressful states, which these encysted embryos encounter.
Ultraviolet‐sensitive ooplasmic factors are responsible for the development of an endoderm‐specific alkaline phosphatase in the ascidian embryo
Authors: Marikawa Yusuke , Satoh Noriyuki
Published: October 2003

The ascidian egg contains muscle and endoderm determinants that play critical roles in the specification of muscle and endoderm cells, respectively. Endoderm cells of the ascidian embryo express alkaline phosphatase (AP) as a tissue‐specific enzyme. We obtained egg fragments from the unfertilized eggs of Ciona savignyi by means of centrifugal force. The largest fragment (red fragments) contained the egg nucleus while other small fragments (black, clear and brown fragments) were anucleate. When inseminated, only red fragments developed into partial embryos, which showed only epidermis cell differentiation and, very rarely, AP activity. When red fragments were fused with other fragments, only black fragments promoted AP expression, suggesting that endoderm determinants were concentrated in the black fragments. A lower dose (1500 J/m2) of ultraviolet (UV) light did not eliminate the AP‐promoting ability of black fragments, while this dose significantly repressed the ability to promote the expression of the muscle‐marker. A higher dose (4500 J/m2) of UV light markedly reduced the AP‐promoting activity of black fragments. These results suggest that factors for endodermal AP development are inactivated by UV irradiation, but are more resistant than muscle determinants.
Sperm motility initiation factor is a minor component of the Pacific herring egg chorion
Authors: Griffin Frederick J. , Vines Carol A. , Pillai Murali C. , Yanagimachi Ryuzo , Cherr Gary N.
Published: October 2003

Polyclonal antibodies were generated to the 105 kDa herring sperm motility initiation factor (SMIF) and used to explore the role of SMIF in sperm‐egg interaction. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting with SMIF antibodies, it was demonstrated that SMIF is present as a minor (4–7% of total chorion protein) component of the chorion. The major polypeptides in the chorion migrated at 117 kDa and in a grouping between 48–54 kDa, with other minor bands above and below. The only detectable glycosylated component was the 105 kDa band, which was resolved at two isoelectric points (8.22 and 8.31) after isoelectric focusing gel electrophoresis. Using antibodies to SMIF, fertilization was blocked, sperm motility was inhibited in vitro in the presence of solubilized SMIF and SMIF binding sites on sperm were localized. Lastly, SMIF was localized to the region of the herring egg that encircles the micropyle.
Role of oviductal pars recta extract on interspecific fertilization between anurans
Authors: Whitacre Cecilia M. , Perdigon Gabriela , Fernandez Silvia N. , Miceli Dora C.
Published: October 2003

During their journey through the oviductal pars recta, the vitelline envelope (VE) of Bufo arenarum oocytes encounter structural alterations that make them sensitive to attack by sperm lysin and thus to penetration by sperm cells. The role of pars recta (PR) on the specificity of fertilization between amphibians was analyzed by conditioning Bufo arenarum oocytes with either homologous PR extract (PRE) or Leptodactylus chaquencis PRE. The oocytes were thereafter exposed to sperm lysin preparations from both species. Lysis of the VE only took place when the oocytes were exposed to the homologous PRE. The pattern of protein composition of PRE of these species was strikingly different as shown by Coomassie blue staining of SDS‐PAGE. Moreover, antibodies against PR fluid (PRF) of Bufo arenarum produced seven bands of immunoprecipitation in electrophoresed homologous PRE and only one faint band in Leptodactylus chaquencis PRE. Here we show that: (i) the biological activity of PR from Bufo arenarum and Leptodactylus chaquencis over the VE of Bufo arenarum oocytes is species‐specific; (ii) this specificity seems to be based in differences in protein structure, which was indicated by the fact that proteins from PRE of Leptodactylus chaquencis and Bufo arenarum were antigenically distinct; (iii) the specificity was solely related to PR activity and not to sperm lysin activity since sperm lysin preparations from both species showed comparable activity.
Changes of tropomyosin isoforms during development of cross‐fertilized sea urchin embryos
Authors: Tobita Takayuki , Ishimoda‐Takagi Tadashi
Published: October 2003

Changes of tropomyosin isoforms during development of the sea urchin, Hemicentrotus pulcherrimus, were investigated using two‐dimensional urea‐shift gel electrophoresis. Tropomyosin isoforms included in the embryos were gradually increased after 2 cell stage and retained at a constant level after gastrula stage. To detect the tropomyosin isoforms derived from zygotic genomes, embryos cross‐fertilized between H. pulcherrimus and Pseudocentrotus depressus gametes were prepared. Since tropomyosin isoforms from H. pulcherrimus eggs and from P. depressus eggs could be distinguished from each other on a two‐dimensional electrophoretic gel, the paternal isoforms of tropomyosin in the cross‐fertilized embryos, which were not included endogenously in the egg, could be regarded as products derived from zygotic genomes. The paternal isoforms of tropomyosin were detected first at around the gastrula stage in embryos cross‐fertilized between H. pulcherrimus sperm and P. depressus eggs and also in the reverse combination of the gamete species. Muscle tropomyosins derived from H. pulcherrimus and P. depressus genomes were similarly detected in cross‐fertilized embryos at the pluteic stage when the muscle tropomyosin appeared in sea urchin embryos.
Two cis‐regulatory elements are essential for the muscle‐specific expression of an actin gene in the ascidian embryo
Authors: Satou Yutaka , Satoh Noriyuki
Published: October 2003

Muscle cells in the anterior and middle part of the ascidian larval tail differentiate in a highly autonomous manner. Because this autonomy is due to so‐called muscle determinants in the myoplasm of fertilized eggs, there is a genetic cascade for muscle cell differentiation in ascidian embryos, which begins with muscle determinants and ends with the expression of muscle‐specific structural genes. In this genetic cascade, an important research subject is the disclosure of the molecular nature of muscle determinants. We approached this issue by going upstream of the cascade by analyzing transcriptional control mechanisms of muscle‐specific structural genes. The HrMA4a actin gene, a muscle‐specific structural gene, has been analyzed for this purpose. It has been demonstrated that a short upstream sequence up to −103 bp from the transcription start site is sufficient for the muscle‐specific expression of HrMA4a. In the present study, we performed a more detailed analysis using the β‐galactosidase gene as a reporter. We demonstrated here that, within the proximal region (−103 to −66) of the HrMA4a gene, there are two short sequences essential to the muscle‐specificity of HrMA4a promoter, one is 9 bp long (5′‐TCGCACTTC‐3′) and the other is 13 bp long (5′‐GTGATAACAACTG‐3′).
Requirement of N‐myc protein down‐regulation for neuronal differentiation in the spinal cord
Authors: Shimono Akihiko , Kitamua Akane , Hanaoka Kazunori , Kondoh Hisato
Published: October 2003

Previous work has indicated that N‐myc expression occurs widely in the developing central nervous system, but its level changes dynamically with region‐ and stage‐specificities. We show in the present report that in the developing spinal cord of the mouse, N‐myc protein expression takes place in the ventricular zone and reaches its maximum at the outermost layer, but is extinct in the intermediate zone, indicating that N‐myc protein is not expressed in mature neurons. We examined the effect of forced, persistent N‐myc expression in development of the spinal cord in order to understand the functional significance of N‐myc down‐regulation. We made embryonic stem (ES) cell lines that constitutively expressed N‐myc at a high level, then produced mouse embryo chimeras with a high contribution of the ES cells. The majority of the chimeras developed to day 12 with normal gross morphology, but in these chimeras neuronal differentiation in the spinal cord was perturbed at the histological level. Intermediate zones and ventral horns were formed, but the expression of N‐CAM and neurofilaments was diminished. Chimeras using β‐galactosidase‐expressing recipient embryos indicated that inhibition of the neuronal differentiation was a cell‐autonomous effect of persistent N‐myc expression. These observations indicate that N‐myc down‐regulation in individual cells is required for full differentiation of neurons.
Amphibian metamorphosis: An exquisite model for hormonal regulation of postembryonic development in vertebrates
Authors: Tata Jamshed R.
Published: October 2003

Metamorphosis in invertebrates and vertebrates is an ideal model for studying mechanisms of postembryonic development regulated by external signals. Amphibian metamorphosis shares many similarities with mammalian development in the perinatal period. The precocious induction in vivo and in culture of amphibian metamorphosis by exogenous thyroid hormones and its retardation or inhibition by prolactin, have allowed the analysis of such characteristic features of postembryonic development as morphogenesis, tissue remodelling, gene reprogramming and programmed cell death. Recent studies on metamorphosis have revealed the important role played by such processes as auto‐ and cross‐regulation of hormone receptor genes and by cell death or apoptosis, as in the maturation of the central nervous system, tissue restructuring and organolysis.