Embryo Movement Research Articles

Rethinking In Vitro Embryo Culture: New Developments in Culture Platforms and Potential to Improve Assisted Reproductive Technologies1

The preponderance of research toward improving embryo development in vitro has focused on manipulation of the chemical soluble environment, including altering basic salt composition, energy substrate concentration, amino acid makeup, and the effect of various growth factors or addition or subtraction of other supplements. In contrast, relatively little work has been done examining the physical requirements of preimplantation embryos and the role culture platforms or devices can play in influencing embryo development within the laboratory. The goal of this review is not to reevaluate the soluble composition of past and current embryo culture media, but rather to consider how other controlled and precise factors such as time, space, mechanical interactions, gradient diffusions, cell movement, and surface interactions might influence embryo development. Novel culture platforms are being developed as a result of interdisciplinary collaborations between biologists and biomedical, material, chemical, and mechanical engineers. These approaches are looking beyond the soluble media composition and examining issues such as media volume and embryo spacing. Furthermore, methods that permit precise and regulated dynamic embryo culture with fluid flow and embryo movement are now available, and novel culture surfaces are being developed and tested. While several factors remain to be investigated to optimize the efficiency of embryo production, manipulation of the embryo culture microenvironment through novel devices and platforms may offer a pathway toward improving embryo development within the laboratory of the future.

Small molecule screening in zebrafish: swimming in potential drug therapies

Phenotype-driven chemical genetic screens in zebrafish have become a proven approach for both dissection of developmental mechanisms and discovery of potential therapeutics. A library of small molecules can be arrayed into multiwell plates containing zebrafish embryos. The embryo becomes a whole organism in vivo bioassay that can produce a phenotype upon treatment. Screens have been performed that are based simply on the morphology of the embryo. Other screens have scored complex phenotypes using whole mount in situ hybridization, fluorescent transgenic reporters, and even tracking of embryo movement. The availability of many well-characterized zebrafish mutants has also enabled the discovery of chemical suppressors of genetic phenotypes. Importantly, the application of chemical libraries that already contain FDA-approved drugs has allowed the rapid translation of hits from zebrafish chemical screens to clinical trials.

Fish and Chips: a microfluidic perfusion platform for monitoring zebrafish development

We have developed a multi-channel microfluidic perfusion platform for culturing zebrafish embryos and capturing live images of various tissues and organs inside the embryo. The Fish and Chips was micro-fabricated in silicon and glass for reproducibility and accuracy of the microfluidic structure. The microfluidic platform consists of three parts: a microfluidic gradient generator, a row of eight fish tanks, in which the fish embryos are individually placed, and eight output channels. The fluidic gradient generator supports dose-dependent drug and chemical studies. A unique perfusion system ensures a uniform and constant flow of media to the fish tank while the wastes are efficiently removed. The fish tanks restrict the embryo movements, except rotationally, for live imaging of internal tissues and organs. The embryos showed developmental abnormalities under the influence of the drug valproic acid (VPA).

Inverted selective plane illumination microscopy ( i SPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans

The Caenorhabditis elegans embryo is a powerful model for studying neural development, but conventional imaging methods are either too slow or phototoxic to take full advantage of this system. To solve these problems, we developed an inverted selective plane illumination microscopy (iSPIM) module for noninvasive high-speed volumetric imaging of living samples. iSPIM is designed as a straightforward add-on to an inverted microscope, permitting conventional mounting of specimens and facilitating SPIM use by development and neurobiology laboratories. iSPIM offers a volumetric imaging rate 30× faster than currently used technologies, such as spinning-disk confocal microscopy, at comparable signal-to-noise ratio. This increased imaging speed allows us to continuously monitor the development of C, elegans embryos, scanning volumes every 2 s for the 14-h period of embryogenesis with no detectable phototoxicity. Collecting ∼25,000 volumes over the entirety of embryogenesis enabled in toto visualization of positions and identities of cell nuclei. By merging two-color iSPIM with automated lineaging techniques we realized two goals: (i) identification of neurons expressing the transcription factor CEH-10/Chx10 and (ii) visualization of their neurodevelopmental dynamics. We found that canal-associated neurons use somal translocation and amoeboid movement as they migrate to their final position in the embryo. We also visualized axon guidance and growth cone dynamics as neurons circumnavigate the nerve ring and reach their targets in the embryo. The high-speed volumetric imaging rate of iSPIM effectively eliminates motion blur from embryo movement inside the egg case, allowing characterization of dynamic neurodevelopmental events that were previously inaccessible.

Optogenetic regulation of leg movement in midstage chick embryos through peripheral nerve stimulation

Numerous disorders that affect proper development, including the structure and function of the nervous system, are associated with altered embryonic movement. Ongoing challenges are to understand in detail how embryonic movement is generated and to understand better the connection between proper movement and normal nervous system function. Controlled manipulation of embryonic limb movement and neuronal activity to assess short- and long-term outcomes can be difficult. Optogenetics is a powerful new approach to modulate neuronal activity in vivo. In this study, we have used an optogenetics approach to activate peripheral motor axons and thus alter leg motility in the embryonic chick. We used electroporation of a transposon-based expression system to produce ChIEF, a channelrhodopsin-2 variant, in the lumbosacral spinal cord of chick embryos. The transposon-based system allows for stable incorporation of transgenes into the genomic DNA of recipient cells. ChIEF protein is detectable within 24 h of electroporation, largely membrane-localized, and found throughout embryonic development in both central and peripheral processes. The optical clarity of thin embryonic tissue allows detailed innervation patterns of ChIEF-containing motor axons to be visualized in the living embryo in ovo, and pulses of blue light delivered to the thigh can elicit stereotyped flexures of the leg when the embryo is at rest. Continuous illumination can disrupt full extension of the leg during spontaneous movements. Therefore, our results establish an optogenetics approach to alter normal peripheral axon function and to probe the role of movement and neuronal activity in sensorimotor development throughout embryogenesis.

A Framework for Connecting Gene Expression to Morphogenetic Movements in Embryos

Although much has been learned about genetic networks, cell mechanics, and whole-embryo mechanics through experimental and computational studies, the challenge of connecting these separate bodies of knowledge into an integrated whole remains. Here, we offer a multiscale biochemical-mechanical framework from which such integration might proceed. We identify components of the framework for which quantitative descriptions are currently available, and use the framework to gain insight into convergent extension and gastrulation--crucial tissue movements that occur in early stage amphibian embryos.

Advances in embryo culture platforms: novel approaches to improve preimplantation embryo development through modifications of the microenvironment

The majority of research aimed at improving embryo development in vitro has focused on manipulation of the chemical environment, examining details such as energy substrate composition and impact of various growth factors or other supplements. In comparison, relatively little work has been done examining the physical requirements of preimplantation embryos and the role culture platforms or devices can play in influencing embryo development. Electronic searches were performed using keywords centered on embryo culture techniques using PUBMED through June 2010 and references were searched for additional research articles. Various approaches to in vitro embryo culture that involve manipulations of the physical culture environment are emerging. Novel culture platforms being developed examine issues such as media volume and embryo spacing. Furthermore, methods to permit dynamic embryo culture with fluid flow and embryo movement are now available, and novel culture surfaces are being tested. Although several factors remain to be studied to optimize efficiency, manipulations of the embryo culture microenvironment through novel culture devices may offer a means to improve embryo development in vitro. Reduced volume systems that reduce embryo spacing, such as the well-of-the-well approach, appear beneficial, although more work is needed to verify the source of their true benefit in human embryos. Emerging microfluidic technology appears to be a promising approach. However, along with the work on specialized culture surfaces, more information is required to determine the impact on human embryo development.

Video force microscopy reveals the mechanics of ventral furrow invagination in Drosophila

The absence of tools for mapping the forces that drive morphogenetic movements in embryos has impeded our understanding of animal development. Here we describe a unique approach, video force microscopy (VFM), that allows detailed, dynamic force maps to be produced from time-lapse images. The forces at work in an embryo are considered to be decomposed into active and passive elements, where active forces originate from contributions (e.g., actomyosin contraction) that do mechanical work to the system and passive ones (e.g., viscous cytoplasm) that dissipate energy. In the present analysis, the effects of all passive components are considered to be subsumed by an effective cytoplasmic viscosity, and the driving forces are resolved into equivalent forces along the edges of the polygonal boundaries into which the region of interest is divided. Advanced mathematical inverse methods are used to determine these driving forces. When applied to multiphoton sections of wild-type and mutant Drosophila melanogaster embryos, VFM is able to calculate the equivalent driving forces acting along individual cell edges and to do so with subminute temporal resolution. In the wild type, forces along the apical surface of the presumptive mesoderm are found to be large and to vary parabolically with time and angular position, whereas forces along the basal surface of the ectoderm, for example, are found to be smaller and nearly uniform with position. VFM shows that in mutants with reduced junction integrity and myosin II activity, the driving forces are reduced, thus accounting for ventral furrow failure.

Imaging cell movements in egg-cylinder stage mouse embryos.

Cell movements in the pregastrulation egg cylinder mouse embryo play an important role in patterning. The stereotypic movement of the anterior visceral endoderm converts a proximal-distal axis to an anteroposterior axis by properly positioning the primitive streak. The epiblast at this stage is also characterized by a great deal of cell mixing, about which very little is known. Visualizing such cell movements can help us understand their role in embryonic development. This protocol describes a method to isolate and culture the egg cylinder-stage mouse embryo, as well as an approach for time-lapse imaging of embryos cultured in vivo.

Culture of Bovine Embryos in Polyester Mesh Sections: The Effect of Pore Size and Oxygen Tension on In Vitro Development

The purpose of this study was to assess the feasibility of polyester mesh culture for the in vitro production of bovine embryos, as polyester mesh is an alternative way for tracking individual embryos throughout culture using time-lapse cinematography (TLC). Bovine embryos were isolated during in vitro culture using sections of three different polyethylene terephthalate (PET) mesh products. In vitro matured and fertilized bovine oocytes were cultured in the 217 × 217, 230 × 230 or 238 × 238-μm openings of PET mesh sections or in simple micro-drops (control) for 7 days under either 20% or 5% O(2) tensions. No difference in embryo developmental rates was found between the culture groups in terms of cleavage, blastocyst formation and blastocyst expansion irrespective of O(2) tension. In contrast, under 20% O(2) tension, blastocysts that developed in PET mesh with 217 × 217-μm opening had significantly higher numbers of total and trophectoderm (TE) cells than control embryos; however, the numbers and proportions of inner cell mass (ICM) cells did not differ. Under 5% O(2) tension, no difference was found among the culture groups in the numbers of total, ICM and TE cells in embryos. All three PET mesh products investigated in this study were proven to be effective to prevent embryo movement. The results demonstrate that bovine embryos can be cultured in PET mesh sections without negative side-effects and suggest that embryo distance determined by the mesh affects embryo quality at atmospheric oxygen tension. Polyethylene terephthalate mesh with 217 × 217-μm openings was found to be the most suitable for further application in TLC.

WLS-dependent secretion of WNT3A requires Ser209 acylation and vacuolar acidification

Wnt proteins are secreted post-translationally modified proteins that signal locally to regulate development and proliferation. The production of bioactive Wnts requires a number of dedicated factors in the secreting cell whose coordinated functions are not fully understood. A screen for small molecules identified inhibitors of vacuolar acidification as potent inhibitors of Wnt secretion. Inhibition of the V-ATPase or disruption of vacuolar pH gradients by diverse drugs potently inhibited Wnt/β-catenin signaling both in cultured human cells and in vivo, and impaired Wnt-regulated convergent extension movements in Xenopus embryos. WNT secretion requires its binding to the carrier protein wntless (WLS); we find that WLS is ER-resident in human cells and WNT3A binding to WLS requires PORCN-dependent lipid modification of WNT3A at serine 209. Inhibition of vacuolar acidification results in accumulation of the WNT3A-WLS complex both in cells and at the plasma membrane. Modeling predictions suggest that WLS has a lipid-binding β-barrel that is similar to the lipocalin-family fold. We propose that WLS binds Wnts in part through a lipid-binding domain, and that vacuolar acidification is required to release palmitoylated WNT3A from WLS in secretory vesicles, possibly to facilitate transfer of WNT3A to a soluble carrier protein.

The Effect of Manipulations of Incubation Temperature on Embryonic and Post-hatching Growth of Native Saudi Chickens

Embryonated egg is an ideal, environment in which to investigate the effects of incubation temperature on the development of the chick embryo. The objective of the current study were to investigate the effect of increasing the incubation temperature of chick embryo by 1.2 degrees C for 7 days (ED4 to ED11) on the body movement and mass of native saudi chick embryo. This objective was extended to examine the influence of pre-hatching temperature on post-hatching growth. Therefore, a total of 180 hatching egg of native Saudi chicken divided into two equal groups incubated at temperature 37.5 degrees C. The incubation temperature was raised to 38.7 degrees C from ED4 to ED11 in treated group before being returned to the control group incubation temperature (37.5 degrees C). The study revealed that elevating the incubation temperature of the eggs of native Saudi chicken by just 1.2 degrees C, from 37.5 to 38.7 degrees C, during embryonic days (ED) 4-11 causes significant increase in embryonic movement as demonstrated in day 8 in the chicks incubated at 38.7 degrees C together with an increase in embryonic development, the embryos incubated at higher temperature were heavier in weights and exhibit significantly longer legs than the controls in ED12 and 15. The increase in pre-hatching incubation temperature (38.7 degrees C) did not reveal any significant effects on post-hatching growth or of feed conversion efficiency.

Large-scale in vitro embryo production and pregnancy rates from Bos taurus, Bos indicus, and indicus-taurus dairy cows using sexed sperm

Herein we describe a large-scale commercial program for in vitro production of embryos from dairy Bos taurus, Bos indicus, and indicus-taurus donors, using sexed sperm. From 5,407 OPU, we compared the number of recovered oocytes (n = 90,086), viable oocytes (n = 64,826), and embryos produced in vitro from Gir ( Bos indicus, n = 617), Holstein ( Bos taurus, n = 180), 1/4 Holstein × 3/4 Gir (n = 44), and 1/2 Holstein-Gir (n = 37) crossbred cows, and the pregnancy rate of recipient cows. Viable oocytes were in vitro matured (24 h at 38.8 °C, 5% CO 2 in air) and fertilized by incubating them for 18 to 20 h with frozen-thawed sexed sperm (X-chromosome bearing) from Gir (n = 8) or Holstein (n = 7) sires (2 × 10 6 sperm/dose). Embryos were cultured in similar conditions of temperature and atmosphere as for IVM, with variable intervals of culture (between Days 2 and 5) completed in a portable incubator. All embryos were transferred fresh, after 24 to 72 h of transportation (up to 2,000 km). On average, 16.7 ± 6.3 oocytes (mean ± SEM) were obtained per OPU procedure and 72.0% were considered viable. Total and viable oocytes per OPU procedure were 17.1 ± 4.5 and 12.1 ± 3.9 for Gir cows, 11.4 ± 3.9 and 8.0 ± 2.7 for Holstein cows, 20.4 ± 5.8 and 16.8 ± 5.0 for 1/4 Holstein × 3/4 Gir, and 31.4 ± 5.6 and 24.3 ± 4.7 for 1/2 Holstein-Gir crossbred females (P < 0.01). The mean number of embryos produced by OPU/IVF and the pregnancy rates were 3.2 (12,243/ 3,778) and 40% for Gir cows, 2.1 (2,426/1,138) and 36% for Holstein cows, 3.9 (1,033/267) and 37% for 1/4 Holstein × 3/4 Gir, and 5.5 (1,222/224), and 37% for 1/2 Holstein-Gir. In conclusion, we compared oocyte yield from two levels of indicus-taurus breeds and demonstrated the efficiency of sexed sperm for in vitro embryo production. Culturing embryos during long distance transportation was successful, with potential for international movement of embryos.

Negative Feedback Regulation of Wnt4 Signaling by EAF1 and EAF2/U19

Previous studies indicated that EAF (ELL-associated factor) family members, EAF1 and EAF2/U19, play a role in cancer and embryogenesis. For example, EAF2/U19 may serve as a tumor suppressor in prostate cancer. At the same time, EAF2/U19 is a downstream factor in the non-canonical Wnt 4 signaling pathway required for eye development in Xenopus laevis, and along with EAF1, contributes to convergence and extension movements in zebrafish embryos through Wnt maintenance. Here, we used zebrafish embryos and mammalian cells to show that both EAF1 and EAF2/U19 were up-regulated by Wnt4 (Wnt4a). Furthermore, we found that EAF1 and EAF2/U19 suppressed Wnt4 expression by directly binding to the Wnt4 promoter as seen in chromatin immunoprecipitation assays. These findings indicate that an auto-regulatory negative feedback loop occurs between Wnt4 and the EAF family, which is conserved between zebrafish and mammalian. The rescue experiments in zebrafish embryos showed that early embryonic development required the maintenance of the appropriate levels of Wnt4a through the feedback loop. Others have demonstrated that the tumor suppressors p63, p73 and WT1 positively regulate Wnt4 expression while p21 has the opposite effect, suggesting that maintenance of appropriate Wnt4 expression may also be critical for adult tissue homeostasis and prevention against tumor initiation. Thus, the auto-regulatory negative feedback loop that controls expression of Wnt4 and EAF proteins may play an important role in both embryonic development and tumor suppression. Our findings provide the first convincing line of evidence that EAF and Wnt4 form an auto-regulatory negative feedback loop in vivo.

Staining and Imaging of Live Rabbit Embryos: Figure 1.

INTRODUCTIONThis protocol describes methods for supravital labeling of cellular proliferation and movement in rabbit embryos as well as for imaging of live cell movements. The techniques use localized labeling of epiblast cells with DiI by injection or by using comprehensive nuclear staining of all cell layers before and during early gastrulation. Because rabbit embryos implant relatively late during embryonic development, gastrulation-stage embryos can be isolated by flushing them from the uterus. Moreover, the embryonic disc is flat and translucent, providing a direct uncompromised overview of gastrulation while the whole blastocyst is protected by the zona pellucida. These properties allow examination of whole blastocysts either under the impact of changing environmental conditions (e.g., medium additives, oxygen concentrations) or following mechanical manipulation of the embryonic disc using a needle penetrating the zona pellucida. Further manipulation (e.g., transplantation) can be performed after removing the embryo from the zona and explantation of the embryonic disc. These techniques allow embryonic growth centers to be identified and single-cell movements to be recorded.

Movement Directionality in Collective Migration of Germ Layer Progenitors

Collective cell migration, the simultaneous movement of multiple cells that are connected by cell-cell adhesion, is ubiquitous in development, tissue repair, and tumor metastasis [1, 2]. It has been hypothesized that the directionality of cell movement during collective migration emerges as a collective property [3, 4]. Here we determine how movement directionality is established in collective mesendoderm migration during zebrafish gastrulation. By interfering with two key features of collective migration, (1) having neighboring cells and (2) adhering to them, we show that individual mesendoderm cells are capable of normal directed migration when moving as single cells but require cell-cell adhesion to participate in coordinated and directed migration when moving as part of a group. We conclude that movement directionality is not a de novo collective property of mesendoderm cells but rather a property of single mesendoderm cells that requires cell-cell adhesion during collective migration.

Wnt5 is required for notochord cell intercalation in the ascidian Halocynthia roretzi

Background information. In the embryos of various animals, the body elongates after gastrulation by morphogenetic movements involving convergent extension. The Wnt/PCP (planar cell polarity) pathway plays roles in this process, particularly mediolateral polarization and intercalation of the embryonic cells. In ascidians, several factors in this pathway, including Wnt5, have been identified and found to be involved in the intercalation process of notochord cells.Results. In the present study, the role of the Wnt5 genes, Hr-Wnt5α (Halocynthia roretzi Wnt5α) and Hr-Wnt5β, in convergent extension was investigated in the ascidian H. roretzi by injecting antisense oligonucleotides and mRNAs into single precursor blastomeres of various tissues, including notochord, at the 64-cell stage. Hr-Wnt5α is expressed in developing notochord and was essential for notochord morphogenesis. Precise quantitative control of its expression level was crucial for proper cell intercalation. Overexpression of Wnt5 proteins in notochord and other tissues that surround the notochord indicated that Wnt5α plays a role within the notochord, and is unlikely to be the source of polarizing cues arising outside the notochord. Detailed mosaic analysis of the behaviour of individual notochord cells overexpressing Wnt5α indicated that a Wnt5α-manipulated cell does not affect the behaviour of neighbouring notochord cells, suggesting that Wnt5α works in a cell-autonomous manner. This is further supported by comparison of the results of Wnt5α and Dsh (Dishevelled) knockdown experiments. In addition, our results suggest that the Wnt/PCP pathway is also involved in mediolateral intercalation of cells of the ventral row of the nerve cord (floor plate) and the endodermal strand.Conclusion. The present study highlights the role of the Wnt5α signal in notochord convergent extension movements in ascidian embryos. Our results raise the novel possibility that Wnt5α functions in a cell-autonomous manner in activation of the Wnt/PCP pathway to polarize the protrusive activity that drives convergent extension.

A microfluidic in vitro cultivation system for mechanical stimulation of bovine embryos

This work demonstrates a novel microfluidic in vitro cultivation system for embryos that improves their development using a partially constricted channel that mimics peristaltic muscle contraction. Conventional photolithography and a PDMS replica molding process were used to make straight or constricted microchannels. To investigate the effects of constriction geometry on embryonic development, different constriction widths of the channel were designed. Bovine embryos were loaded and incubated by simply placing them on a tilting machine to provide embryo movement via gravity. The fertilized embryos were cultivated on the microfluidic in vitro cultivation system until the blastocyst, hatching, or hatched blastocyst stages. To confirm the quality of blastocysts in the microfluidic channel, double staining was performed and compared with bovine embryos cultivated by the conventional droplet method. The proportion of eight-cell development among total embryos in the constricted channel (56.7+/-13.7%; mean+/-SD) was superior to that in the straight channel (23.9+/-11.0%). This suggests that the effect of constriction is vital for the early development of bovine embryos in assisted-reproduction research.

Precocious Locomotor Behavior Begins in the Egg: Development of Leg Muscle Patterns for Stepping in the Chick

BackgroundThe chicken is capable of adaptive locomotor behavior within hours after hatching, yet little is known of the processes leading to this precocious skill. During the final week of incubation, chick embryos produce distinct repetitive limb movements that until recently had not been investigated. In this study we examined the leg muscle patterns at 3 time points as development of these spontaneous movements unfolds to determine if they exhibit attributes of locomotion reported in hatchlings. We also sought to determine whether the deeply flexed posture and movement constraint imposed by the shell wall modulate the muscle patterns.Methodology/Principal FindingsSynchronized electromyograms for leg muscles, force and video were recorded continuously from embryos while in their naturally flexed posture at embryonic day (E) 15, E18 and E20. We tested for effects of leg posture and constraint by removing shell wall anterior to the foot. Results indicated that by E18, burst onset time distinguished leg muscle synergists from antagonists across a 10-fold range in burst frequencies (1–10 Hz), and knee extensors from ankle extensors in patterns comparable to locomotion at hatching. However, burst durations did not scale with step cycle duration in any of the muscles recorded. Despite substantially larger leg movements after shell removal, the knee extensor was the only muscle to vary its activity, and extensor muscles often failed to participate. To further clarify if the repetitive movements are likely locomotor-related, we examined bilateral coordination of ankle muscles during repetitive movements at E20. In all cases ankle muscles exhibited a bias for left/right alternation.Conclusions/SignificanceCollectively, the findings lead us to conclude that the repetitive leg movements in late stage embryos are locomotor-related and a fundamental link in the establishment of precocious locomotor skill. The potential importance of differences between embryonic and posthatching locomotion is discussed.

Joint Formation Requires Muscle Formation and Contraction

Embryo movement and muscle contraction affect joint formation, but their precise roles have remained elusive. In this issue of Developmental Cell, Joy Kahn and coworkers now demonstrate that these processes are essential for joint formation. They find that joint progenitor cells are unable to remain committed to their specified cell fate in mutant mice with defects in either muscle formation or contraction.