In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms^1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson''s disease, Schizophrenia, depression, and dementia^1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs^6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos^8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points^10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick β-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes¹⁴. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.     

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental manipulations in ovo/in vivo makes the chicken embryonic retina a versatile and very efficient experimental model. Although the chicken retina is easy to target in ovo by intraocular injections or electroporation, the effective and exact concentration of the reagents within the retina may be difficult to fully control. This may be due to variations of the exact injection site, leakage from the eye or uneven diffusion of the substances. Furthermore, the frequency of malformations and mortality after invasive manipulations such as electroporation is rather high.This protocol describes an ex ovo technique for culturing whole retinal explants from chicken embryos and provides a method for controlled exposure of the retina to reagents. The protocol describes how to dissect, experimentally manipulate, and culture whole retinal explants from chicken embryos. The explants can be cultured for approximately 24 hr and be subjected to different manipulations such as electroporation. The major advantages are that the experiment is not dependent on the survival of the embryo and that the concentration of the introduced reagent can be varied and controlled in order to determine and optimize the effective concentration. Furthermore, the technique is rapid, cheap and together with its high experimental success rate, it ensures reproducibleresults. It should be emphasized that it serves as an excellent complement to experiments performed in ovo.     

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons¹, which has been difficult with the conventional method of injection and electroporation at E1.5².     

Sexing of mid‐incubation avian embryos as a management tool for zoological breeding programs

Skewed sex ratios in zoo breeding programs may require housing single birds of an overrepresented gender, increasing demands on limited resources that could otherwise be diverted to breeding pairs or other important species. The ability to selectively incubate and hatch eggs of a desired sex represents a significant improvement in the long‐term management of avian species. This study describes a successful method for in ovo sexing of embryos from stage 30 through 42 of incubation (Hamburger and Hamilton [1951] J Morphol 88:49–92). A 0.01–1 µl blood sample was collected from either the vitelline vessel (VV) or the blood vessels of the chorio–allantoic membrane (CAM) of embryos at stages 14–18 or 30–42, respectively. DNA was isolated from whole blood using the Chelex method (Walsh et al. [1991] Biotechniques 10:506–513; Jensen et al., [2003] Zoo Biol 22:561–571). Sex was determined by PCR amplification using the previously described P2/P8 (Griffiths et al. [1998] Mol Ecol 7:1071–1075) and 1237L/1272H (Kahn et al. [1998] Auk 115:1074–1078) primers or by commercial vendor. Success rate was calculated as the percent of sampled embryos surviving to hatch. Embryos of the undesired sex were not incubated, thus not included in the calculation. There was a considerable difference in success rate when blood was collected from the stage 14–18 VV (0–25%, average 12%) vs. stage 30–42 CAM (33–100%, average 76%). In conclusion, in ovo sexing of embryos between stages 30 and 42 yields acceptable embryo survival rates while providing enough blood for genetic testing. Zoo Biol 31:694‐704, 2012. © 2011 Wiley Periodicals, Inc.     

Cardiac injections of AntagomiRs as a novel tool for knockdown of miRNAs during heart development

Background

Studying microRNA networks during heart development is essential to obtain a better understanding of developmental defects and diseases associated with the heart and to identify novel opportunities for therapeutics. Here we highlight the advantages of chicken embryos as a vertebrate model for studying intermediate processes of heart development. Avians develop a four-chambered heart closely resembling human anatomy and they develop ex utero, which makes them easily accessible. Furthermore, embryos are available all year with a steady supply.

Electroporation of craniofacial mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse ^1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis ^11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers ¹⁴. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (˜100 million years closer) ¹⁵. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.     

Primordial germ cell‐specific expression of eGFP in transgenic chickens

PR domain zinc finger protein 14 (PRDM14) plays an essential role in the development of primordial germ cells (PGCs) in mice. However, its functions in avian species remain unclear. In the present study, we used CRISPR/Cas9 to edit the PRDM14 locus in chickens in order to demonstrate its importance in development. The eGFP gene was introduced into the PRDM14 locus of cultured chicken PGCs to knockout PRDM14 and label PGCs. Chimeric chickens were established by a direct injection of eGFP knocked‐in (gene‐trapped) PGCs into the blood vessels of Hamburger–Hamilton stages (HH‐stages) 13–16 chicken embryos. Gene‐trapped chickens were established by crossing a chimeric chicken with a wild‐type hen with very high efficiency. Heterozygous gene‐trapped chickens grew normally and SSEA‐1‐positive cells expressed eGFP during HH‐stages 13–30. These results indicated the specific expression of eGFP within circulating PGCs and gonadal PGCs. At the blastodermal stage, the ratio of homozygous gene‐trapped embryos obtained by crossing heterozygous gene‐trapped roosters and hens was almost normal; however, all embryos died soon afterward, suggesting the important roles of PRDM14 in chicken early development.     

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing ¹. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth ^2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes ^4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development⁶.The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos⁷. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells^8-10. We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol¹¹. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.     

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudière et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.Download video file.^{(52M, mov)}     

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos^1,2 that we have successfully adapted^3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick⁵. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described⁶. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.To date, in utero electroporation has been most widely used for the analysis of neocortical development^1,2,3,4. More recent studies have targeted the embryonic retina^7,8,9 and thalamus^10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3^rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.Download video file.^{(82M, mov)}     

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols¹. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.     

Influence of different prebiotics and mode of their administration on broiler chicken performance

In the post-antibiotics era, prebiotics are proposed as alternatives to antibiotic growth promoters in poultry production. The goal of this study was to compare in ovo method of prebiotic delivery with in-water supplementation and with both methods combined (in ovo+in-water) in broiler chickens. Two trials were conducted. Trial 1 was carried out to optimize the doses of two prebiotics, DN (DiNovo®, extract of beta-glucans) and BI (Bi²tos, trans-galactooligosaccharides), for in ovo delivery. The estimated parameters were hatchability and bacteriological status of the newly hatched chicks. Prebiotics were dissolved in 0.2 ml of physiological saline, at the doses: 0.18, 0.88, 3.5 and 7.0 mg/embryo; control group (C) was injected in ovo with 0.2 ml of physiological saline. Trial 2 was conducted to evaluate effects of different prebiotics (DN, BI and raffinose family oligosaccharides (RFO)) delivered in ovo, in-water and in a combined way (in ovo+in-water) on broiler chickens performance. The results of the Trial 1 indicated that the optimal dose of DN and BI prebiotics delivered in ovo, that did not reduce chicks’ hatchability, was 0.88 mg/embryo (DN) and 3.5 mg/embryo (BI). Both prebiotics numerically increased number of lactobacilli and bifidobacteria in chicken feces (P>0.05). In Trial 2, all prebiotics (DN, BI and RFO) significantly increased BW gain compared with the C group (P<0.05), especially during the first 21 days of life. However, feed intake and feed conversion ratio were increased upon prebiotics delivery irrespective of method used. Injection of prebiotics in ovo combined with in-water supplementation did not express synergistic effects on broilers performance compared with in ovo injection only. Taken together, those results confirm that single in ovo prebiotics injection into the chicken embryo can successfully replace prolonged in-water supplementation post hatching.     

Selenium promotes adipogenic determination and differentiation of chicken embryonic fibroblasts with regulation of genes involved in fatty acid uptake,triacylglycerol synthesis and lipolysis

Selenium (Se) has been utilized in the differentiation of primary pig and rat preadipocytes, indicating that it may have proadipogenic potential; however, some studies have also demonstrated that Se has antiadipogenic activity. In this study, chicken embryonic fibroblasts (CEFs) were used to investigate the role of Se in adipogenesis in vitro and in ovo. Se supplementation increased lipid droplet accumulation and inhibited proliferation of cultured CEFs isolated from 6-day-old embryos dose-dependently. This suggests that Se may play a role in cell cycle inhibition, thereby promoting the differentiation of fibroblasts to adipocytes. Se did not stimulate adipogenic differentiation of CEFs isolated from 9- to 12-day-old embryos, implying a permissive stage of adipogenic determination by Se at earlier embryonic ages. Microarray analysis comparing control and Se treatments on CEFs from 6-day-old embryos and confirmatory analysis by quantitative real-time polymerase chain reaction revealed that genes involved in adipocyte determination and differentiation, fatty acid uptake and triacylglycerol synthesis were up-regulated. In addition, up-regulation of an anti-lipolytic G0/G1 switch gene 2 and down-regulation of a prolipolytic monoglyceride lipase may lead to inhibition of lipolysis by Se. Both osteogenic and myogenic genes were down-regulated, and several genes related to oxidative stress response during adipogenesis were up-regulated. In ovo injection of Se at embryonic day 8 increased adipose tissue mass by 30% and caused adipocyte hypertrophy in 17-day-old chicken embryos, further supporting the proadipogenic role of Se during the embryonic development of chickens. These results suggest that Se plays a significant role in several mechanisms related to adipogenesis.     

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used^1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay^5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye^7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM^10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice¹³ as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM¹⁴.In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications.Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.Download video file.^{(166M, mp4)}     

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s¹, then followed by vitrification methods developed in the late 1980s². Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4°C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained³, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature⁴.Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos⁵. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.     

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic ^1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis ⁴. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects ⁵. For transmission between insect hosts, the bacteria colonize the intestine of the nematode''s infective juvenile stage ^6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects ^9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems ⁹.We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila¹⁴. Similar methods have been used to investigate other nematode-bacterium associations ^9,15-18and the approach therefore is generally applicable.The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization ^14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues ^14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication ²²or grinding ²³, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes ^21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization ^17,18, and is less laborious than other methods, including sonication ^22,25-27and individual nematode dissection ^28,29.     

Contributions to the study of oviparity–viviparity transition: Placentary structures of Liolaemus elongatus (Squamata: Liolaemidae)

Liolaemus elongatus (Liolaemidae) is a viviparous, mainly lecithotrophic species with placental structures specialized for uptake of oxygen and inorganic nutrient transport. An allantoplacenta and an omphaloplacenta are present during early embryonic stages (25–28) and there is a moderate distension of the uterine wall and major glandular activity in the uterine mucosa and submucosa compared with nonpregnant females. The uterine epithelium increases in height, first as a growth in the height of some dispersed cells localized in all regions of the placenta, and later as groups of cells localized in the periembryonic and central‐abembryonic regions. At embryonic stage 39, the allantoplacenta reaches its maximum extension around the yolk sac. Omphaloplacenta is restricted to the abembryonic zone, the yolk cleft limiting the newly formed isolated vitelline mass. At more advanced embryonic stages (39–42), the blood supply to the allantoplacenta's periembryonic zone increases, matching the profuse allantoic vascularization. At embryonic stage 42, a secondary cleft opens in the main vitelline mass, above the first yolk cleft, and allantoic blood vessels enter into this secondary cleft. This secondary cleft subdivides the vitelline mass into a large embryonic region connected to a much smaller abembryonic region. In L. elongatus most nutritional resources seem to be provided by the yolk that remains attached to the newborn for 2 or 3 days as an external supply. The embryo's wet weight doubles the weight of the decrease observed in vitelline mass. But the dry wet diminishes, evidencing the importance of the exchange of water and inorganic nutrients. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.