Timing Of Developmental Events Research Articles

Genetic Mapping and Phenotypic Analysis of GstE14 E.4.1 on Eye and Antennae Development in Drosophila melanogaster.

Genetic screens are valuable for identifying novel genes involved in the regulation of developmental processes. To identify genes associated with cell growth regulation in Drosophila melanogaster , a mutagenesis screen was performed. Undergraduate students participating in Fly-CURE phenotypically characterized the E.4.1 mutant which is associated with rough eyes and antennae overgrowth. Following complementation analysis and subsequent genomic sequencing, E.4.1 was identified as a novel mutant allele of GstE14 , a gene involved in ecdysone biosynthesis important for the timing of developmental events. The abnormal eye and antenna phenotypes observed resulting from the loss of GstE14 suggest its role in tissue growth.

Variation and trade-offs in life history traits of the protist parasite Monocystis perplexa (Apicomplexa) in its earthworm host Amynthas agrestis.

The life history of a parasite describes its partitioning of assimilated resources into growth, reproduction, and transmission effort, and its precise timing of developmental events. The life cycle, in contrast, charts the sequence of morphological stages from feeding to the transmission forms. Phenotypic plasticity in life history traits can reveal how parasites confront variable environments within hosts. Within the protist phylum Apicomplexa major clades include the malaria parasites, coccidians, and most diverse, the gregarines (with likely millions of species). Studies on life history variation of gregarines are rare. Therefore, life history traits were examined for the gregarine Monocystis perplexa in its host, the invasive earthworm Amynthas agrestis at three sites in northern Vermont, United States of America. An important value of this system is the short life-span of the hosts, with only seven months from hatching to mass mortality; we were thus able to examine life history variation during the entire life cycle of both host and parasite. Earthworms were collected (N=968 over 33 sample periods during one host season), then parasites of all life stages were counted, and sexual and transmission stages measured, for each earthworm. All traits varied substantially among individual earthworm hosts and across the sites. Across sites, timing of first appearance of infected earthworms, date when transmission stage (oocysts packed within gametocysts) appeared, date when number of both feeding (trophic) cells and gametocysts were at maximum, and date when 100% of earthworms were infected differed from 2-8 weeks, surprising variation for a short season available for parasite development. The maximal size of mating cells varied among hosts and across sites and this is reflected in the number of oocysts produced by the gametocyst. A negative trade-off was observed for the number of oocysts and their size. Several patterns were striking: (1) Prevalence reached 100% at all sites by mid season, only one to three weeks after parasites first appeared in the earthworms. (2) The number of parasites per host was large, reaching 300 × 103 cells in some hosts, and such high numbers were present even when parasites first appeared in the host. (3) At one site, few infected earthworms produced any oocysts. (4) The transmission rate to reach such high density of parasites in hosts needed to be very high for a microbe, from >0.33% to >34.3% across the three sites. Monocystis was one of the first protist parasites to have its life cycle described (early 19th century), but these results suggest the long-accepted life cycle of Monocystis could be incomplete, such that the parasites may be transmitted vertically (within the earthworm's eggs) as well as horizontally (leading to 100% prevalence) and merogony (asexual replication) could be present, not recognized for Monocystis, leading to high parasitemia even very early in the host's season.

Time-lapse imaging: Morphokinetic analysis of in vitro fertilization outcomes

Time-lapse imaging (TLI) allows continuous monitoring of embryo development without disturbing culture conditions by removing embryos from the incubator. The study of embryo kinetics using TLI has given rise to some new markers for embryo selection that are able to document and evaluate embryo morphology and the timing of developmental events through continuous live image tracking. Time-lapse imaging has emerged as a powerful tool for creating predictive models of invitro fertilization outcomes. Fourty-seven articles were included in the present review to investigate the current situation of TLI in invitro fertilization laboratories. Morphokinetics of the embryo during its invitro development have been described using parameters indicative of the different events capable of predicting the ability to reach blastocyst stage, implantation and pregnancy rates, live birth outcome, and embryo ploidy.

Anticipated age of perimenopausal experiences, stress, satisfaction, and health and well-being: observations from the Women Living Better Survey.

People's expectations about the timing of developmental events, specifically the ages at which they will occur, are influenced by social and cultural contexts. When expected timing and lived experience are divergent, events such as menopause may be associated with greater stress or distress. We hypothesized that being "off-time," that is, experiencing perimenopause-related menstrual cycle changes or symptoms in a timeframe before a person expects them, would lead to worse ratings on measures of stress, satisfaction, and health. Participants responded to the online Women Living Better Survey during March to August 2020; 1,262 met the eligibility criteria for inclusion in hypothesis testing. Being "off-time" referred to experiencing changes related to perimenopause at a younger age than that at which participants anticipated experiencing such changes. Using a one-way analysis of variance (ANOVA), we examined differences in being "off-time" versus "on-time" on seven participant-rated measures: stress (overall and health-related stress), satisfaction with life roles and activities, and well-being and health ratings (interference with daily activities, interference with relationships, "not feeling like myself," and perceived health). Using 2-way ANOVA, we then tested hypothesized differences between being "off-time" and on-time and experiencing perimenopause-related menstrual cycle changes, vasomotor/sleep symptoms, or volatile mood symptoms on the same seven measures. Those who were "off-time" versus "on-time" reported significantly poorer health ratings in a one-way ANOVA. Experiencing more noticeable perimenopause-related menstrual cycle changes was significantly related to greater health stress, overall stress, satisfaction with life roles and activities, interference with daily activities, interference with relationships, and "not feeling like myself" (all P < 0.05), but not to health ratings. Experiencing more bothersome vasomotor symptoms was significantly related to health stress, overall stress, interference with daily activities, interference with relationships, and "not feeling like myself" more of the time and to poorer perceived health (all P < 0.05). There were no significant interaction effects of being "off-time" and experiencing perimenopause-related menstrual cycle changes or vasomotor symptoms. In contrast, having more bothersome volatile mood symptoms significantly affected health stress, overall stress, satisfaction with life roles and activities, interference with daily activities, interference with relationships, "not feeling like myself" more of the time, and perceived health. Finally, there was a significant interaction effect of being "off-time" and volatile mood symptoms on health stress, satisfaction with life roles and activities, and perceived health (all P < 0.05). Being "off-time" alone had little effect on studied measures with the exception of poorer perceived health. Experiencing more noticeable perimenopause-related menstrual cycle changes or having more bothersome vasomotor symptoms influenced several measures, but there were no interactive effects with being "off-time." In contrast, those who were "off-time" and experiencing more bothersome volatile mood symptoms reported greater health stress, lower satisfaction with life roles and activities, and poorer perceived health. These interactive effects of being "off-time" and experiencing volatile mood suggest a need for greater attention to the link between volatile mood and perimenopause. Furthermore, anticipatory guidance for those on the path to menopause should include the possibility of volatile mood symptoms.

Transition to extrauterine life and the modeling of perinatal asphyxia in rats.

Generation of murine models for the study of birth-related pathologies has proven to be a complex and controversial problem. Differences in the relative timing of developmental events of both species have led some researchers to suggest that the rat is born comparatively less developed than the human. The solution proposed to this problem would consist in the delay of the experiments of perinatal asphyxia (PA), usually up to 7-10 days, allowing developmental levels to "equalize" with the human at birth. This solution generates a new set of problems. The developmental milestones in both species follow a divergent temporal pattern. Increasing the age of the rat not only can improve resemblance with humans but also will make the model miss a crucial set of milestones related to birth. During this process, there are specific mechanisms to protect the fetus from neuronal damage, especially those caused by asphyxia. These factors are not present in models where the asphyxia is delayed. In these models, there will be more false positives and more damage that would not be present in humans exposed to PA. This article is categorized under: Cancer > Stem Cells and Development Congenital Diseases > Environmental Factors Neurological Diseases > Environmental Factors.

The role of ontogeny in wood diversity and evolution.

Evolutionary developmental biology (evo-devo) explores the link between developmental patterning and phenotypic change through evolutionary time. In this review, we highlight the scientific advancements in understanding xylem evolution afforded by the evo-devo approach, opportunities for further engagement, and future research directions for the field. We review evidence that (1) heterochrony-the change in rate and timing of developmental events, (2) homeosis-the ontogenetic replacement of features, (3) heterometry-the change in quantity of a feature, (4) exaptation-the co-opting and repurposing of an ancestral feature, (5) the interplay between developmental and capacity constraints, and (6) novelty-the emergence of a novel feature, have all contributed to generating the diversity of woods. We present opportunities for future research engagement, which combine wood ontogeny within the context of robust phylogenetic hypotheses, and molecular biology.

Time to synchronize our clocks: Connecting developmental mechanisms and evolutionary consequences of heterochrony

Heterochrony, defined as a change in the timing of developmental events altering the course of evolution, was first recognized by Ernst Haeckel in 1866. Haeckel's original definition was meant to explain the observed parallels between ontogeny and phylogeny, but the interpretation of his work became a source of controversy over time. Heterochrony took its modern meaning following the now classical work in the 1970-80s by Steven J. Gould, Pere Alberch, and co-workers. Predicted and described heterochronic scenarios emphasize the many ways in which developmental changes can influence evolution. However, while important examples of heterochrony detected with comparative morphological methods have multiplied, the more mechanistic understanding of this phenomenon lagged conspicuously behind. Considering the rapid progress in imaging and molecular tools available now for developmental biologists, this review aims to stress the need to take heterochrony research to the next level. It is time to synchronize the different levels of heterochrony research into a single analysis flow: from studies on organismal-level morphology to cells to molecules and genes, using complementary techniques. To illustrate how to achieve a more comprehensive understanding of phyletic morphological diversification associated with heterochrony, we discuss several recent case studies at various phylogenetic scales that combine morphological, cellular, and molecular analyses. Such a synergistic approach offers to more fully integrate phylogenetic and ontogenetic dimensions of the fascinating evolutionary phenomenon of heterochrony.

Microbial effects on plant phenology and fitness.

Plant development and the timing of developmental events (phenology) are tightly coupled with plant fitness. A variety of internal and external factors determine the timing and fitness consequences of these life-history transitions. Microbes interact with plants throughout their life history and impact host phenology. This review summarizes current mechanistic and theoretical knowledge surrounding microbe-driven changes in plant phenology. Overall, there are examples of microbes impacting every phenological transition. While most studies have focused on flowering time, microbial effects remain important for host survival and fitness across all phenological phases. Microbe-mediated changes in nutrient acquisition and phytohormone signaling can release plants from stressful conditions and alter plant stress responses inducing shifts in developmental events. The frequency and direction of phenological effects appear to be partly determined by the lifestyle and the underlying nature of a plant-microbe interaction (i.e., mutualistic or pathogenic), in addition to the taxonomic group of the microbe (fungi vs. bacteria). Finally, we highlight biases, gaps in knowledge, and future directions. This biotic source of plasticity for plant adaptation will serve an important role in sustaining plant biodiversity and managing agriculture under the pressures of climate change.

Scalable control of developmental timetables by epigenetic switching networks.

During development, progenitor cells follow timetables for differentiation that span many cell generations. These developmental timetables are robustly encoded by the embryo, yet scalably adjustable by evolution, facilitating variation in organism size and form. Epigenetic switches, involving rate-limiting activation steps at regulatory gene loci, control gene activation timing in diverse contexts, and could profoundly impact the dynamics of gene regulatory networks controlling developmental lineage specification. Here, we develop a mathematical framework to model regulatory networks with genes controlled by epigenetic switches. Using this framework, we show that such epigenetic switching networks uphold developmental timetables that robustly span many cell generations, and enable the generation of differentiated cells in precisely defined numbers and fractions. Changes to epigenetic switching networks can readily alter the timing of developmental events within a timetable, or alter the overall speed at which timetables unfold, enabling scalable control over differentiated population sizes. With their robust, yet flexibly adjustable nature, epigenetic switching networks could represent central targets on which evolution acts to manufacture diversity in organism size and form.

Partitioning maternal and exogenous diet contributions to otolith 87Sr/86Sr in kokanee salmon (Oncorhynchus nerka)

Strontium isotopes (87Sr/86Sr) recorded in the otoliths of Pacific salmon (Oncorhynchus spp.) are commonly used to identify natal origin. For species that migrate at or soon after emergence, the embryonic region of the otolith provides the only record of provenance. However, maternal contribution of Sr from the yolk can confound the isotopic signature of the natal site. We experimentally quantified maternal and exogenous diet contributions to otolith 87Sr/86Sr over embryonic development in kokanee salmon (Oncorhynchus nerka). Eggs from two populations in isotopically distinct lakes were incubated and reared in a common water source. Timing of developmental events and proportional contribution from yolk to otolith 87Sr/86Sr differed significantly between the two populations. We suggest that the magnitude of difference in 87Sr/86Sr between yolk and water, the relative concentrations of Sr and Ca in these isotopic sources, and population-specific effects on otolith growth and composition contribute to this variation. Understanding how these factors affect otolith 87Sr/86Sr could extend the use of otolith geochemistry for determining provenance to species and populations in which natal site rearing is limited.

Manipulation of gametes and embryosin the pig

Summary. Several manipulation techniques including nuclear injections, nuclear transplantation, embryo splitting, chimaera production and sperm injection are discussed with special reference to their application in the pig. The nuclear injection technique is likely to be of greatest use for gene transfer. Gene transfer is feasible for pig embryos. but it is very inefficient. Efficiencies of various steps in the successful production of transgenic offspring as summarized from several published references are as follows: immediate survival of embryos following injection (60%). offspring produced from injected embryos (15%), offspring with an integrated foreign gene (24%) and offspring expressing the integrated foreign gene (60%). The overall efficiency is about 1%. Potential uses for nuclear transplantation are for the production of gynogenetic and androgenetic embryos and for cloning embryos The feasibility of producing androgenetic and gynogenctic embryos will depend on whether in the pig. as in the mouse, the paternal and maternal genomes function differently. Thc feasibility of cloning by nuclear transplantation will depend upon the timing of developmental events in pig embryos and the development of methods for reprogramming nuclei. Pig embryos have been cloned by embryo splitting. Chimaera production and fertilization by sperm injection are likely to be feasible for pig embryos but have not yet found application.

Influence of chronic hyperoxia on the developmental time course of the hypoxic ventilatory response relative to other traits in rats

Newborn mammals exhibit a biphasic hypoxic ventilatory response (HVR) in which an initial increase in ventilation is followed by a decline back toward baseline levels. The magnitude of the secondary decline diminishes with postnatal age, but this transition occurs earlier in rat pups reared in moderate hyperoxia. This pattern is consistent with heterokairy, a form of developmental plasticity in which environmental factors alter the timing of developmental events. The present study investigated whether this plasticity is specific to the HVR or if hyperoxia instead accelerates overall development. Rat pups reared in 60 % O2 (Hyperoxia) exhibited a less biphasic ventilatory response to 12 % O2 than pups reared in 21 % O2 (Control) at 4 days of age (P4) and transitioned to a sustained HVR by P10-11; Control rats exhibited a biphasic HVR at both ages. However, the average ages at which pups attained other key developmental milestones (i.e., fur development at P5, incisor eruption at P9, and eye opening at P15) were similar between treatment groups. Moreover, growth rates and maturation of the metabolic response to cooling were not accelerated, and may have been delayed slightly, relative to Control rats. For example, the capacity for pups to increase their metabolic rate at low ambient temperatures increased with age, but this thermogenic capacity tended to be reduced in Hyperoxia pups at both P4 and P10-11 (i.e., lower CO2 production rates below the lower critical temperature). Collectively, these data support the conclusion that hyperoxia specifically advances the age at which rat pups exhibit a sustained HVR, altering the relative timing of developmental events rather than compressing the entire period of development.

Patterns of intraspecific variation through ontogeny: a case study of the Cretaceous nautilid Eutrephoceras dekayi and modern Nautilus pompilius

AbstractThe magnitude and ontogenetic patterns of intraspecific variation can provide important insights into the evolution and development of organisms. Understanding the intraspecific variation of organisms is also a key to correctly pursuing studies in major fields of palaeontology. However, intraspecific variation has been largely overlooked in ectocochleate cephalopods, particularly nautilids. Furthermore, little is known regarding the evolutionary pattern. Here, we present morphological data for the Cretaceous nautilid Eutrephoceras dekayi (Morton) and the modern nautilid Nautilus pompilius Linnaeus through ontogeny. The data are used to describe conch morphology and to elucidate the evolutionary patterns of intraspecific variation. We discovered a similar overall pattern of growth trajectories and the presence of morphological changes at hatching and maturity in both taxa. We also found that intraspecific variation is higher in earlier ontogeny than in later ontogeny in both taxa. The high variation in earlier ontogeny may imply increased flexibility in changing the timing of developmental events, which probably played an important role in nautilid evolution. We assume that the decrease in variation in later ontogeny reflects developmental constraints. Lastly, we compared the similarity/dissimilarity of ontogenetic patterns of variation between taxa. Results reveal that the similarity/dissimilarity of the ontogenetic pattern differs between E. dekayi and N. pompilius. We conclude that this shift in the ontogenetic pattern of variation may be rooted in changes in the developmental programme of nautilids through time. We propose that studying ontogenetic patterns of intraspecific variation can provide new insights into the evolution and development of organisms.

Craniofacial growth and development in a mouse model of Down syndrome

Down syndrome (DS) is a developmental disorder caused by trisomy of human chromosome 21. Ts65Dn, a widely used mouse model of DS, has facilitated the study of mechanisms underlying the genotypic and phenotypic anomalies associated with DS. Previous studies have shown the adult cranium and brain to be markedly smaller in size in Ts65Dn mice relative to their euploid littermates. To determine how postnatal developmental patterns contribute to cranial variation in trisomic mice, we quantitatively compared growth trajectories from birth (postnatal day 0, P0) to postnatal day 6 (P6) in Ts65Dn and euploid mice. We hypothesized that trisomy affects cranial growth rate in Ts65Dn mice, causing size and shape differences between Ts65Dn and euploid mice. Multivariate regression and principal components analyses of craniofacial shape and size show some subtle differences between the trisomic and euploid mice at P0, but by P6 the trisomic mice are considerably smaller than the euploids. Interestingly, Ts65Dn at P6 are closer in both size and shape to P0 euploid mice than to euploid mice at P6 or newborn Ts65Dn. This could occur through changes in growth rate of Ts65Dn mice, resulting in the trisomic P6 mice retaining features common to younger, typically developing mice. Further research focusing on prenatal ages will provide a better understanding of how differences in timing of developmental events, such as delays in migration and mitosis of cranial neural crest cells in mouse model of DS, contribute to trisomic craniofacial growth and development.Support or Funding InformationR01‐HD038384 (RHR)

Early Maturation of the Hypoxic Ventilatory Response in Hyperoxic Rat Pups: Evidence for Trait‐Specific Heterokairy

Newborn mammals exhibit a biphasic hypoxic ventilatory response (HVR) in which an initial increase in ventilation is followed by a decline back toward baseline levels. The magnitude of the secondary decline diminishes with age, but rat pups reared in moderate hyperoxia make this transition earlier (Bavis et al., J. Appl. Physiol. 109(3): 796–803, 2010). This is consistent with heterokairy, a form of developmental plasticity in which environmental factors alter the rate and/or timing of developmental events. The present study investigated whether this effect is specific to the HVR or if hyperoxia instead accelerates overall development. Rat pups reared in 60% O2 (Hyperoxia) exhibited a less biphasic HVR at both P4 and P10‐11 compared to pups reared in 21% O2 (Control). However, growth curves and the average ages at which pups attained key developmental milestones (i.e., fur development at P5, incisor eruption at P9, and eye opening at P15) were similar between treatment groups. We also studied the relationship between metabolic rate and ambient temperature as another indicator of physiological maturity. The lower critical temperature (LCT) decreased with age, but the LCT was the same for Control and Hyperoxia rats at both P4 (~32°C) and P10‐11 (~30°C). The ability to increase metabolic rate at low ambient temperatures also increased with age, but this thermogenic capacity tended to be reduced in Hyperoxia rats at both P4 and P10‐11 (i.e., lower rates of metabolic CO2 production at temperatures below the LCT). We conclude that hyperoxia accelerates the rate at which the HVR matures without affecting the maturation of other morphological and physiological traits.

Both maternal and embryonic exposure to mild hypoxia influence embryonic development of the intertidal gastropod Littorina littorea.

There is growing evidence that maternal exposure to environmental stressors can alter offspring phenotype and increase fitness. Here, we investigate the relative and combined effects of maternal and developmental exposure to mild hypoxia (65 and 74% air saturation, respectively) on the growth and development of embryos of the marine gastropod Littorina littorea Differences in embryo morphological traits were driven by the developmental environment, whereas the maternal environment and interactive effects of maternal and developmental environment were the main driver of differences in the timing of developmental events. While developmental exposure to mild hypoxia significantly increased the area of an important respiratory organ, the velum, it significantly delayed hatching of veliger larvae and reduced their size at hatching and overall survival. Maternal exposure had a significant effect on these traits, and interacted with developmental exposure to influence the time of appearance of morphological characters, suggesting that both are important in affecting developmental trajectories. A comparison between embryos that successfully hatched and those that died in mild hypoxia revealed that survivors exhibited hypertrophy in the velum and associated pre-oral cilia, suggesting that these traits are linked with survival in low-oxygen environments. We conclude that both maternal and developmental environments shape offspring phenotype in a species with a complex developmental life history, and that plasticity in embryo morphology arising from exposure to even small reductions in oxygen tensions affects the hatching success of these embryos.

The kinetics of grape ripening revisited through berry density sorting

Aim: In this study, ripening and heterogeneity in density-sorted berries were investigated, with the aim of more clearly understanding the kinetics of water uptake and sugar, acid and anthocyanin accumulation in the fruit of a new, disease-resistant hybrid.Methods and results: The 3184-1-9N hybrid, grown in a semi-arid climate in the South of France, was used. Its genotype is the result of several backcrosses of a Muscadinia ´ Vitis vinifera F1 hybrid with several V. vinifera varieties. From the end of the green plateau to the over-ripening stage, single berries were sampled weekly and sorted by density, to monitor the advancement of ripening and the heterogeneity of grapevine fruit development at population level. Fruit firmness, density, fresh weight, primary metabolite content (sugars and organic acids), secondary metabolite content (anthocyanins), potassium content and pH were measured in berries from each density class. The data showed that softening and hexose accumulation occurred before fruit pigmentation. Based on malic acid and anthocyanin concentration relative to sugar concentration in density-sorted grapes, malic acid breakdown or dilution was promoted and anthocyanin accumulation reduced in late- or slow-ripening berries. In the different population structures in two experimental plots, berries with similar sugar concentration showed considerable heterogeneity in terms of volume and anthocyanin content, whereas pH, potassium content and acid content showed much more homogeneous kinetics.Conclusions: During ripening, analysis of density-sorted berries provides useful information about sugar concentration heterogeneity and its relation with organic acid and anthocyanin content. The stage at which grapes can be considered ripe may be precisely determined through periodic determination of mean berry volume or weight, to detect the cessation of phloem unloading and the onset of berry shrivelling. However, ripening and shrivelling berries generally coexist in the harvested population. Asynchronous berry development and berry weight heterogeneity greatly complicate determination of the timing of developmental events that are regulated at the single-berry level. Therefore, analysis of individual berries would seem to be an indispensable component of investigations of berry growth and development.Significance and impact of the study: Plotting water uptake and solute accumulation relative to sugar accumulation (as an internal clock) provides an original way to display grapevine fruit development data. Analysis of density-sorted berries has identified the relatively invariant fruit features, such as the minimum concentration of tartaric acid, the stability of potassium accumulation and pH, and conversely, the huge variations in berry size.

The Effects of Different Diets and Transgenerational Stress on Acyrthosiphon pisum Development.

Despite the fact that sap-feeding hemipterans are major agricultural pests, little is known about the pea aphid’s (Acyrthosiphon pisum) nymphal development, compared to other insect models. Given our limited understanding of A. pisum nymphal development and variability in the naming/timing of its developmental events between different environmental conditions and studies, here, we address developmental knowledge gaps by elucidating how diet impacts A. pisum nymphal development for the LSR1 strain when it develops on its universal host plant (Vicia faba), isolated leaves, and artificial diet. Moreover, we test how plant age and transgenerational stressors, such as overcrowding and low plant vigor, can affect nymphal development. We also validate a morphological method to quickly confirm the life stage of each nymphal instar within a mixed population. Overall, we found extremely high variation in the timing of developmental events and a significant delay in nymphal (~5–25-h/instar) and pre-reproductive adult (~40-h) development when reared on isolated leaves and artificial diets, compared to intact host plants. Also, delays in development were observed when reared on older host plants (~9–17-h/event, post 2nd instar) or when previous generations were exposed to overcrowding on host plants (~20-h delay in nymph laying) compared to controls.

Temporal regulation of Lin28a during mammalian neurulation contributes to neonatal body size control.

The timing of developmental events is tightly regulated along a time axis for normal development. Although the RNA-binding protein Lin28a plays a crucial role in the regulation of developmental timing in Caenorhabditis elegans, how the timing of Lin28a expression affects the rate and/or duration of developmental events during mammalian development remains to be addressed. In this study, we discovered that the timing and the duration of Lin28a expression affect embryonic growth. During the neurulation stage of mouse development, endogenous Lin28a levels start to drop. When Lin28a expression was maintained transiently using the inducible tetracycline-regulated gene expression (Tet-ON) system [doxycycline (Dox)-inducible Lin28a transgenic (iLin28a Tg) mice] with Dox administration at E8.5 and E9.5, it resulted in neonatal lethality, increased body weight (organomegaly), and an increased number of caudal vertebrae at birth. On the other hand, Lin28a induction only at E8.5 caused neonatal lethality and organomegaly, but did not affect the caudal vertebra number. Of note, although Dox treatment before or after neurulation still caused neonatal lethality, it neither caused organomegaly nor the increased caudal vertebra number in iLin28a Tg neonates. Temporal regulation of Lin28a expression during neurulation affects developmental events such as cessation of axial elongation and embryonic growth in mice.

Heterochronic Phenotype Analysis of Hypodermal Seam Cells in Caenorhabditis elegans.

Heterochrony refers to changes in the timing of developmental events, and it is precisely regulated in the organisms by the heterochronic genes such as C. elegans lin-4 and let-7. Mutations in these genes cause precocious or retarded development of certain cell lineages. With well-defined cell lineages, C. elegans is one of the best model systems to study heterochronic genes, since the subtle changes in the development of cell lineages can be easily identified. Among the different cell types in C. elegans, hypodermal seam cells and their lineages are well known to be maintained by lin-14, whose expression level is regulated by two miRNA genes, lin-4 and let-7, at the larval stages. Therefore, analyzing the heterochronic phenotype of hypodermal seam cells in C. elegans could yield detailed insights into the status of the miRNA pathway. Here we describe the assay protocol to analyze the heterochronic phenotypes of C. elegans hypodermal seam cells, which can be used as a reliable method to study the miRNA pathway.