Complete Embryogenesis Research Articles

Exposure to heat stress leads to striking clone-specific nymph deformity in pea aphid.

Climatic changes, such as heatwaves, pose unprecedented challenges for insects, as escalated temperatures above the thermal optimum alter insect reproductive strategies and energy metabolism. While thermal stress responses have been reported in different insect species, thermo-induced developmental abnormalities in phloem-feeding pests are largely unknown. In this laboratory study, we raised two groups of first instar nymphs belonging to two clones of the pea aphid Acyrthosiphon pisum, on fava beans Vicia faba. The instars developed and then asexually reproduced under constant exposure to a sub-lethal heatwave (27°C) for 14 days. Most mothers survived but their progenies showed abnormalities, as stillbirths and appendageless or weak nymphs with folded appendages were delivered. Clone N116 produced more deceased and appendageless embryos, contrary to N127, which produced fewer dead and more malformed premature embryos. Interestingly, the expression of the HSP70 and HSP83 genes differed in mothers between the clones. Moreover, noticeable changes in metabolism, e.g., lipids, were also detected and that differed in response to stress. Deformed offspring production after heat exposure may be due to heat injury and differential HSP gene expression, but may also be indicative of a conflict between maternal and offspring fitness. Reproductive altruism might have occurred to ensure some of the genetically identical daughters survive. This is because maintaining homeostasis and complete embryogenesis could not be simultaneously fulfilled due to the high costs of stress. Our findings shine new light on pea aphid responses to heatwaves and merit further examination across different lineages and species.

LSM14B is an Oocyte-Specific RNA-Binding Protein Indispensable for Maternal mRNA Metabolism and Oocyte Development in Mice.

Mammalian oogenesis features reliance on the mRNAs produced and stored during early growth phase. These are essential for producing an oocyte competent to undergo meiotic maturation and embryogenesis later when oocytes are transcriptionally silent. The fate of maternal mRNAs hence ensures the success of oogenesis and the quality of the resulting eggs. Nevertheless, how the fate of maternal mRNAs is determined remains largely elusive. RNA-binding proteins (RBPs) are crucial regulators of oogenesis, yet the identity of the full complement of RBPs expressed in oocytes is unknown. Here, a global view of oocyte-expressed RBPs is presented: mRNA-interactome capture identifies 1396 RBPs in mouse oocytes. An analysis of one of these RBPs, LSM family member 14 (LSM14B), demonstrates that this RBP is specific to oocytes and associated with many networks essential for oogenesis. Deletion of Lsm14b results in female-specific infertility and a phenotype characterized by oocytes incompetent to complete meiosis and early embryogenesis. LSM14B serves as an interaction hub for proteins and mRNAs throughout oocyte development and regulates translation of a subset of its bound mRNAs. Therefore, RNP complexes tethered by LSM14B are found exclusively in oocytes and are essential for the control of maternal mRNA fate and oocyte development.

Substantial rDNA copy number reductions alter timing of development and produce variable tissue-specific phenotypes in C. elegans.

The genes that encode ribosomal RNAs are present in several hundred copies in most eukaryotes. These vast arrays of repetitive ribosomal DNA (rDNA) have been implicated not just in ribosome biogenesis, but also aging, cancer, genome stability, and global gene expression. rDNA copy number is highly variable among and within species; this variability is thought to associate with traits relevant to human health and disease. Here we investigate the phenotypic consequences of multicellular life at the lower bounds of rDNA copy number. We use the model Caenorhabditis elegans, which has previously been found to complete embryogenesis using only maternally provided ribosomes. We find that individuals with rDNA copy number reduced to ∼5% of wild type are capable of further development with variable penetrance. Such individuals are sterile and exhibit severe morphological defects, particularly in post-embryonically dividing tissues such as germline and vulva. Developmental completion and fertility are supported by an rDNA copy number ∼10% of wild type, with substantially delayed development. Worms with rDNA copy number reduced to ∼33% of wild type display a subtle developmental timing defect that was absent in worms with higher copy numbers. Our results support the hypothesis that rDNA requirements vary across tissues and indicate that the minimum rDNA copy number for fertile adulthood is substantially less than the lowest naturally observed total copy number. The phenotype of individuals with severely reduced rDNA copy number is highly variable in penetrance and presentation, highlighting the need for continued investigation into the biological consequences of rDNA copy number variation.

Influence of light indicators on the development of Clarias gariepinus during early ontogenesis

High-quality and fast fish farming is very relevant and in demand nowadays. The african clary catfish is one of the representatives of aquatic organisms, which makes it possible to study complete embryogenesis in a short time in order to understand what favorable environmental conditions are necessary to obtain a larger volume of marketable fish. The aim of our study is to study the early stage of ontogenesis of Clarias gariepinus under the influence of changes in the degree of illumination of the environment. The material for the study was eggs obtained from females at the age of 2 years. The incubation was carried out on special mesh trays located on the surface of the pools. A total of 91000 eggs were laid for incubation. The eggs were fertilized by artificial insemination. The fertilized eggs were kept at an optimum temperature of 28,5°C. The first group was incubated in the dark, and the second under fluorescent lamps. Every 5-15 minutes, eggs were placed in a Petri dish and the material was counted and photographed using a digital camera.The results of our research showed that 20 minutes after fertilization, the onset of blastodisc formation was recorded in both groups. After 35 minutes, cell cleavage begins. The duration of this period is considered from the moment of cleavage until the complete overgrowth of the embryonic disc with cells. After 80 minutes, further division of the embryonic cell occurs with the formation of a spherical structure - morula, as well as the overgrowth of the yolk with the embryonic layer. Later 15 hours, after fertilization, we found that the development of the embryo occurs evenly, with positive dynamics, regardless of illumination.An analysis of the results obtained allows us to conclude that changes in light parameters do not have a negative effect on the rate of development of the Clarias gariepinus eggs.

ЭМБРИОКУЛЬТУРА IN VITRO В ЭКСПЕРИМЕНТАЛЬНОЙ ОЦЕНКЕ ЗАСУХОУСТОЙЧИВОСТИ ХЛЕБНЫХ ЗЛАКОВ (ОБЗОР)

Drought is an unfavorable combination of meteorological conditions when plants experience a long-term water deficiency both in the air and soil. This is one of the most common abiotic stressors, which leads not only to significant crop losses but also rises threat to food security. Researchers are actively developing ways to breed drought-tolerant cultivars of economically valuable crops, especially cereals – the main food resource. One of the promising areas of biotechnological evaluation of the resistance of existing and newly created cereal genotypes to drought for breeding purposes is the use of culture in vitro. In this case, embryos at the particular stage of development are used as explants (so-called embryo culture in vitro). The review aims to analyze the literature and own data on the production of cereal regenerants in embryo culture in vitro under selective experimental conditions of imitation of physiological drought. It has been shown that in vitro cultivation of immature embryos at a critical stage of relative autonomy is especially promising. This kind of embryo does not depend on the physiological factors of the maternal organism and can autonomously give rise to the fully developed plant under adequate conditions in vitro and later ex vitro. This allows the biotechnologist to obtain regenerants directly, excluding an additional time-consuming stage of the formation of morphogenic calli in vitro. As follows, the time required for expensive experiments is also reduced. Data on the identification of the critical stage of the relative autonomy of the cereal embryogenesis are presented. Criterion (proposed by the authors) for identifying this stage by the ability of the embryos to complete embryogenesis and form the seedlings on a hormone-free medium in vitro and give rise to the full developed regenerants ex vitro has been analyzed. Furthermore, the analysis of the laboratory germination of the obtained caryopses was carried out. It was discovered that in spring soft wheat, for example, such stage, corresponding to the formation of all organs in the embryo, occurs 15 days after pollination. The issues of using relatively autonomous embryos in the biotechnological assessment of the genotype drought resistance under selective conditions in vitro are considered.

Left High and Dry: Froth Nesting Allows Eggs of the Anuran Amphibian to Complete Embryogenesis in the Absence of Free-Standing Water

For amphibians that oviposit in temporary aquatic systems, there is a high risk of desiccation-induced offspring mortality when water evaporates prior to the completion of embryo or tadpole development. Such a strong selective pressure has led to the evolution of a variety of traits in reproducing females and their offspring to improve the odds of reproductive success when free-standing water is temporarily available. Herein, we describe an adaptive function of froth nesting in the Sandpaper Frog, Lechriodus fletcheri, as a possible maternal strategy for protecting embryos from the immediate risk of desiccating in highly ephemeral pools that frequently dry prior to hatching. Field observations revealed that embryos located near the core of nests remained alive and continued to develop for several days after becoming stranded out of water due to declining water levels, with embryo viability maintained long enough for additional rainfall to recharge pools and support hatching of tadpoles into water in some cases. In laboratory trials, the proportion of embryos surviving in nests exposed to desiccating conditions was positively correlated with nest volume, while the rate of water loss relative to nest mass declined, both of which are likely a function of reduced surface area relative to volume in larger nests. We suggest that the encapsulation of embryos within an aerated mucus shields them from desiccation by trapping moisture around their external surfaces. As embryos of L. fletcheri complete development rapidly, the ability for the froth nest to protect against desiccation for several days may allow embryogenesis to be completed largely out of water despite the larval phase not being terrestrial. These results suggest froth nesting has played an important role in facilitating this species' use of ephemeral habitats that most other amphibians with aquatic reproductive modes are incapable of exploiting.

Gametophyte and embryonic ontogeny: understanding the reproductive calendar of Cypripedium japonicum Thunb. (Cypripedoideae, Orchidaceae), a lady\u2019s slipper orchid endemic to East Asia

BackgroundThe genus Cypripedium L. is one of the five genera of the subfamily Cypripedioideae, members of which are commonly known as lady’s slipper orchids. Cypripedium japonicum is a perennial herb native to East Asia, specifically China, Japan, and Korea. Due to its limited distribution, the species is included in the Endangered category of the IUCN Red List.ResultsWe investigated gametophyte development, including complete embryogenesis, in C. japonicum. The complete reproductive cycle is presented based on our observations. Anther development begins under the soil, and meiosis of pollen mother cells begins 3 weeks before anthesis, possibly during early April. The megaspore mother cells develop just after pollination in early May and mature in mid–late June. The pattern of embryo sac formation is bisporic, and there are six nuclei: three forming the egg apparatus, two polar nuclei, and an antipodal cell in the mature embryo sac. Triple fertilization results in the endosperm nucleus, which degenerates when the proembryo reaches the eight-to-sixteen-cell stage.ConclusionOur overall comparisons of the features of gametophyte and embryo development in C. japonicum suggest that previous reports on the embryology of Cypripedium are not sufficient for characterization of the entire genus. Based on the available information, a reproductive calendar showing the key reproductive events leading to embryo formation has been prepared.

O-GlcNAcylation Dampens Dpp/BMP Signaling to Ensure Proper Drosophila Embryonic Development.

BMP (bone morphogenetic protein) signaling activity is precisely controlled by both pathway agonists andantagonists. Here, we identify a previously unrecognized BMP signaling antagonist. We demonstrate that the Drosophila BMP type I receptor Sax(Saxophone) functions as a Dpp (Decapentaplegic) receptor in Drosophila embryos, but that its activity is normally inhibited by the O-linked glycosyltransferase Sxc (Super sex combs). In wild-type embryos, Sax activity is inhibited, and the BMP type I receptor Tkv (Thickveins) is the sole conduit for Dpp. In contrast, in sxc mutants, the Dpp signal istransduced by both Tkv and Sax, and elevated Dpp signaling results in embryonic lethality. We also demonstrate that Sxc O-glycosylates Sax andobserve elevated Dpp signaling in response to maternal restriction of dietary sugar. These findings link fertility to nutritive environment and point to Sax signaling as the nutrient-sensitive branch of BMP signaling.

From Mother or Father: Uniparental Embryos Uncover Parent-of-Origin Effects in Humans.

In mammals, both parents make unique contributions to the offspring and maternal and paternal genomes are required for development. Two recent papers in Cell Stem Cell (Leng etal., 2019; Sagi etal., 2019) study uniparental embryos and uniparental embryonic stem cells to interrogate parent-of-origin effects in human embryogenesis.

The essential chloroplast ribosomal protein uL15c interacts with the chloroplast RNA helicase ISE2 and affects intercellular trafficking through plasmodesmata.

Chloroplasts retain part of their ancestral genomes and the machinery for expression of those genomes. The nucleus-encoded chloroplast RNA helicase INCREASED SIZE EXCLUSION LIMIT2 (ISE2) is required for chloroplast ribosomal RNA processing and chloro-ribosome assembly. To further elucidate ISE2's role in chloroplast translation, two independent approaches were used to identify its potential protein partners. Both a yeast two-hybrid screen and a pull-down assay identified plastid ribosomal protein L15, uL15c (formerly RPL15), as interacting with ISE2. The interaction was confirmed invivo by co-immunoprecipitation. Interestingly, we found that rpl15 null mutants do not complete embryogenesis, indicating that RPL15 is an essential gene for autotrophic growth of Arabidopsis thaliana. Arabidopsis and Nicotiana benthamiana plants with reduced expression of RPL15 developed chlorotic leaves, had reduced photosynthetic capacity and exhibited defective chloroplast development. Processing of chloroplast ribosomal RNAs and assembly of ribosomal subunits were disrupted by reduced expression of RPL15. Chloroplast translation was also decreased, reducing accumulation of chloroplast-encoded proteins, in such plants compared to wild-type plants. Notably, knockdown of RPL15 expression increased intercellular trafficking, a phenotype also observed in plants with reduced ISE2 expression. This finding provides further evidence for chloroplast function in modulating intercellular trafficking via plasmodesmata.

Mlh1 is required for female fertility in Drosophila melanogaster: An outcome of effects on meiotic crossing over, ovarian follicles and egg activation

Mismatch repair (MMR) system, a conserved DNA repair pathway, plays crucial role in DNA recombination and is involved in gametogenesis. The impact of alterations in MMR family of proteins (bacterial MutS and MutL homologues) on mammalian fertility is well documented. However, an insight to the role of MMR in reproduction of non-mammalian organisms is limited. Hence, in the present study, we analysed the impact of mlh1 (a MutL homologue) on meiotic crossing over/recombination and fertility in a genetically tractable model, Drosophila melanogaster. Using mlh1e00130 hypomorphic allele, we report female specific adverse reproductive outcome for reduced mlh1 in Drosophila: mlh1e00130 homozygous females had severely reduced fertility while males were fertile. Further, mlh1e00130 females contained small ovaries with large number of early stages as well as significantly reduced mature oocytes, and laid fewer eggs, indicating discrepancies in egg production and ovulation. These observations contrast the sex independent and/or male specific sterility and normal follicular development as well as ovulation reported so far for MMR family proteins in mammals. However, analogous to the role(s) of mlh1 in meiotic crossing over and DNA repair processes underlying mammalian fertility, ovarian follicles from mlh1e00130 females contained significantly increased DNA double strand breaks (DSBs) and reduced synaptonemal complex foci. In addition, large proportion of fertilized eggs display discrepancies in egg activation and fail to proceed beyond stage 5 of embryogenesis. Hence, reduction of the Mlh1 protein level leads to defective oocytes that fail to complete embryogenesis after fertilization thereby reducing female fertility.

An automated microfluidic platform for C. elegans embryo arraying, phenotyping, and long-term live imaging.

Studies of the real-time dynamics of embryonic development require a gentle embryo handling method, the possibility of long-term live imaging during the complete embryogenesis, as well as of parallelization providing a population’s statistics, while keeping single embryo resolution. We describe an automated approach that fully accomplishes these requirements for embryos of Caenorhabditis elegans, one of the most employed model organisms in biomedical research. We developed a microfluidic platform which makes use of pure passive hydrodynamics to run on-chip worm cultures, from which we obtain synchronized embryo populations, and to immobilize these embryos in incubator microarrays for long-term high-resolution optical imaging. We successfully employ our platform to investigate morphogenesis and mitochondrial biogenesis during the full embryonic development and elucidate the role of the mitochondrial unfolded protein response (UPRmt) within C. elegans embryogenesis. Our method can be generally used for protein expression and developmental studies at the embryonic level, but can also provide clues to understand the aging process and age-related diseases in particular.

Adaptive responses to cool climate promotes persistence of a non-native lizard.

Successful establishment and range expansion of non-native species often require rapid accommodation of novel environments. Here, we use common-garden experiments to demonstrate parallel adaptive evolutionary response to a cool climate in populations of wall lizards (Podarcis muralis) introduced from southern Europe into England. Low soil temperatures in the introduced range delay hatching, which generates directional selection for a shorter incubation period. Non-native lizards from two separate lineages have responded to this selection by retaining their embryos for longer before oviposition--hence reducing the time needed to complete embryogenesis in the nest--and by an increased developmental rate at low temperatures. This divergence mirrors local adaptation across latitudes and altitudes within widely distributed species and suggests that evolutionary responses to climate can be very rapid. When extrapolated to soil temperatures encountered in nests within the introduced range, embryo retention and faster developmental rate result in one to several weeks earlier emergence compared with the ancestral state. We show that this difference translates into substantial survival benefits for offspring. This should promote short- and long-term persistence of non-native populations, and ultimately enable expansion into areas that would be unattainable with incubation duration representative of the native range.

Trehalose-6-phosphate synthase 1 is not the only active TPS in Arabidopsis thaliana.

Trehalose metabolism is essential for normal growth and development in higher plants. It is synthesized in a two-step pathway catalysed by TPS (trehalose-6-phosphate synthase) and trehalose phosphatase. Arabidopsis thaliana has 11 TPS or TPS-like proteins, which belong to two distinct clades: class I (AtTPS1-AtTPS4) and class II (AtTPS5-AtTPS11). Only AtTPS1 has previously been shown to have TPS activity. A. thaliana tps1∆ mutants fail to complete embryogenesis and rescued lines have stunted growth and delayed flowering, indicating that AtTPS1 is important throughout the life cycle. In the present study, we show that expression of AtTPS2 or AtTPS4 enables the yeast tps1∆ tps2∆ mutant to grow on glucose and accumulate Tre6P (trehalose 6-phosphate) and trehalose. Class II TPS genes did not complement the yeast mutant. Thus A. thaliana has at least three catalytically active TPS isoforms, suggesting that loss of Tre6P production might not be the only reason for the growth defects of A. thaliana tps1 mutants.

The pea aphid uses a version of the terminal system during oviparous, but not viviparous, development

BackgroundIn most species of aphid, female nymphs develop into either sexual or asexual adults depending on the length of the photoperiod to which their mothers were exposed. The progeny of these sexual and asexual females, in turn, develop in dramatically different ways. The fertilized oocytes of sexual females begin embryogenesis after being deposited on leaves (oviparous development) while the oocytes of asexual females complete embryogenesis within the mother (viviparous development). Compared with oviparous development, viviparous development involves a smaller transient oocyte surrounded by fewer somatic epithelial cells and a smaller early embryo that comprises fewer cells. To investigate whether patterning mechanisms differ between the earliest stages of the oviparous and viviparous modes of pea aphid development, we examined the expression of pea aphid orthologs of genes known to specify embryonic termini in other insects.ResultsHere we show that pea aphid oviparous ovaries express torso-like in somatic posterior follicle cells and activate ERK MAP kinase at the posterior of the oocyte. In addition to suggesting that some posterior features of the terminal system are evolutionarily conserved, our detection of activated ERK in the oocyte, rather than in the embryo, suggests that pea aphids may transduce the terminal signal using a mechanism distinct from the one used in Drosophila. In contrast with oviparous development, the pea aphid version of the terminal system does not appear to be used during viviparous development, since we did not detect expression of torso-like in the somatic epithelial cells that surround either the oocyte or the blastoderm embryo and we did not observe restricted activated ERK in the oocyte.ConclusionsWe suggest that while oviparous oocytes and embryos may specify posterior fate through an aphid terminal system, viviparous oocytes and embryos employ a different mechanism, perhaps one that does not rely on an interaction between the oocyte and surrounding somatic cells. Together, these observations provide a striking example of a difference in the fundamental events of early development that is both environmentally induced and encoded by the same genome.

Propagação in vitro de genótipos de alface via embriogênese somática

A propagação in vitro via embriogênese somática é uma alternativa eficiente para a propagação em larga escala do material vegetal. No entanto, não há relatos do desenvolvimento de protocolos completos de embriogênese somática, com produção de plantas de alface. O presente trabalho teve como objetivo estabelecer a propagação in vitro de genótipos de alface, Paris White e Red Salad Bowl, avaliando a indução dos calos embriogênicos, regeneração dos embriões somáticos e posterior conversão em plantas. Para a indução de embriogênese somática, duas fontes de explante (folhas cotiledonares inteiras e seccionadas) foram cultivadas em meio MS+10,75µM de ANA+0,89µM de BA. A proliferação dos calos embriogênicos foi realizada em meio MS+24µM de AIA+0,15µM de BA. Para a maturação dos embriões somáticos e conversão em plantas, utilizou-se meio MS sem regulador de crescimento (semi-sólido sem carvão ativado e com carvão ativado e meio líquido). A fonte do explante seccionada foi estatisticamente superior apenas para o genótipo Paris White. Para a regeneração dos embriões somáticos, observou-se que, no genótipo Paris White, o meio líquido foi superior estatisticamente, quando comparado aos meios semi-sólidos e, para o genótipo Red Salad Bowl, foi o meio semi-sólido com acréscimo de carvão ativado. Tendo em vista o potencial da aplicabilidade comercial da embriogênese somática para a produção em larga escala de plântulas de alface do genótipo Paris White, os protocolos de indução, proliferação, maturação dos embriões somáticos e conversão em plantas, foram adequados, em especial, em sistema líquido.

Targeted mutation of the talpid3 gene in zebrafish reveals its conserved requirement for ciliogenesis and Hedgehog signalling across the vertebrates.

Using zinc-finger nuclease-mediated mutagenesis, we have generated mutant alleles of the zebrafish orthologue of the chicken talpid3 (ta3) gene, which encodes a centrosomal protein that is essential for ciliogenesis. Animals homozygous for these mutant alleles complete embryogenesis normally, but manifest a cystic kidney phenotype during the early larval stages and die within a month of hatching. Elimination of maternally derived Ta3 activity by germline replacement resulted in embryonic lethality of ta3 homozygotes. The phenotype of such maternal and zygotic (MZta3) mutant zebrafish showed strong similarities to that of chick ta3 mutants: absence of primary and motile cilia as well as aberrant Hedgehog (Hh) signalling, the latter manifest by the expanded domains of engrailed and ptc1 expression in the somites, reduction of nkx2.2 expression in the neural tube, symmetric pectoral fins, cyclopic eyes and an ectopic lens. GFP-tagged Gli2a localised to the basal bodies in the absence of the primary cilia and western blot analysis showed that Gli2a protein is aberrantly processed in MZta3 embryos. Zygotic expression of ta3 largely rescued the effects of maternal depletion, but the motile cilia of Kupffer's vesicle remained aberrant, resulting in laterality defects. Our findings underline the importance of the primary cilium for Hh signaling in zebrafish and reveal the conservation of Ta3 function during vertebrate evolution.

Determinants of incubation period: do reptilian embryos hatch after a fixed total number of heart beats?

The eggs of birds typically hatch after a fixed (but lineage-specific) cumulative number of heart beats since the initiation of incubation. Is the same true for non-avian reptiles, despite wide intraspecific variation in incubation period generated by variable nest temperatures? Non-invasive monitoring of embryo heart beat rates in one turtle species (Pelodiscus sinensis) and two lizards (Bassiana duperreyi and Takydromus septentrionalis) show that the total number of heart beats during embryogenesis is relatively constant over a wide range of warm incubation conditions. However, incubation at low temperatures increases the total number of heart beats required to complete embryogenesis, because the embryo spends much of its time at temperatures that require maintenance functions but that do not allow embryonic growth or differentiation. Thus, cool-incubated embryos allocate additional metabolic effort to maintenance costs. Under warm conditions, total number of heart beats thus predicts incubation period in non-avian reptiles as well as in birds (the total number of heart beats are also similar); however, under the colder nest conditions often experienced by non-avian reptiles, maintenance costs add significantly to total embryonic metabolic expenditure.

A UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferase Is Required for Epithelial Tube Formation

Epithelial tubes are essential for the proper function of a diverse array of eukaryotic organs. Here we present a novel class of genes required for maintaining epithelial cell shape, polarity, and paracellular barrier function in the Drosophila embryonic tracheal system. Mutations in one member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family (pgant35A) are recessive lethal and result in tracheal tubes that are irregular in diameter and morphology. Further analysis of the pgant35A mutants reveals diminished levels of the apical determinant Crbs and the luminal marker 2A12, concomitant with increased staining in cytoplasmic vesicles within tracheal cells. GalNAc-containing glycoproteins are severely diminished along the apical region of the tracheal system as well. Tracheal cells become irregular in size and shape, and septate junction proteins are mislocalized to a more apical position. Most notably, paracellular barrier function is lost in the tracheal system of the mutants. Overexpression of wild type pgant35A under control of the trachea-specific breathless (btl) promoter results in partial rescue of the lethality. We propose a model where pgant35A is required to establish proper apical composition of tracheal cells by influencing apical delivery of proteins/glycoproteins. Disruption of the normal apical content results in altered cell morphology and loss of paracellular barrier function. These studies demonstrate a previously unrecognized requirement for mucin-type O-glycosylation in epithelial tube integrity and have obvious implications for epithelial morphogenesis in higher eukaryotes, since a unique ortholog to pgant35A exists in mammals.

Low temperature requirement for embryonic development of Itasenpara bitterlingAcheilognathus longipinnis

The Itasenpara bitterling has an embryonic period up to 7 months, when the embryo experiences large seasonal temperature changes. We examined the temperature requisites for normal development during the embryonic stage. Fertilized eggs reared under any of the constant temperatures ranging from 5 degrees C to 30 degrees C did not achieve complete embryogenesis, and none reached the swim-up stage. The optimum temperature for normal embryonic development was found to be stage-dependent: 10-30 degrees C for fertilization, 15-25 degrees C for hatching, 5 degrees C for the requisite low temperature, 10-15 degrees C for eye pigmentation, and 20-30 degrees C for swim-up. These temperatures correlated well with the embryo's natural environmental conditions. Embryos raised at these temperatures sequentially grew normally, with 70% of the fertilized eggs achieving complete embryogenesis and, for the first time, developed to the swim-up stage. These results indicate that the low temperature, as required by the bitterling embryo, is an essential factor and correlates well with the embryo's natural ambient temperatures. Since the populations of Itasenpara bitterlings have been declining in Japan, this study is the first to provide additional information for successful artificial breeding of this endangered species.