Non-Invasive PPG-Based System for Continuous Heart Rate Monitoring of Incubated Avian Embryo.

The chicken embryo is a widely used experimental animal model in many studies, including in the field of developmental biology, of the physiological responses and adaptation to altered environments, and for cancer and neurobiology research. The embryonic heart rate is an important physiological variable used as an index reflecting the embryo's natural activity and is considered one of the most difficult parameters to measure. An acceptable measurement technique of embryonic heart rate should provide a reliable cardiac signal quality while maintaining adequate gas exchange through the eggshell during the incubation and embryonic developmental period. In this paper, we present a detailed design and methodology for a non-invasive photoplethysmography (PPG)-based prototype (Egg-PPG) for real-time and continuous monitoring of embryonic heart rate during incubation. An automatic embryonic cardiac wave detection algorithm, based on normalised spectral entropy, is described. The developed algorithm successfully estimated the embryonic heart rate with 98.7% accuracy. We believe that the system presented in this paper is a promising solution for non-invasive, real-time monitoring of the embryonic cardiac signal. The proposed system can be used in both experimental studies (e.g., developmental embryology and cardiovascular research) and in industrial incubation applications.

Effects of thermal manipulation of eggs on the response of jejunal mucosae to posthatch chronic heat stress in broiler chickens.

In this study, the aim was to investigate effects of chronic heat stress (CHS) on the mRNA levels of proinflammatory cytokines (interleukin [IL]-6, IL-8, IL-1ß, and tumor necrosis factor alpha [TNF-α]), toll-like receptors (TLR2 and TLR4), heat shock proteins (Hsp70, heat shock transcription factor [HSF]-1, and HSF3) and antioxidant enzymes (catalase, glutathione peroxidase, NADPH oxidase, and superoxide-dismutase) in the jejunal mucosae of broiler chickens subjected to thermal manipulation (TM) during embryogenesis. TM was carried out at 39°C and 65% relative humidity (RH) for 18 h daily from embryonic days 10 to 18. Control group was incubated at 37.8°C and 56% RH. CHS was induced by raising the temperature to 35°C for 7 D throughout posthatch days 28 to 35. On post-hatch-day 28 (day zero of CHS) and after 1, 3, 5, and 7 D of CHS, the jejunal mucosae were collected from both groups to evaluate the mRNA levels by real-time reverse transcription-PCR analysis. On day zero of CHS, the mRNA levels of antioxidant enzymes, TLRs, HSF3, IL-1ß, and TNF-α were not significantly different between TM and control groups, while the levels of IL-6, IL-8, and HSF1 were lower and the level of Hsp70 was higher in TM. However, during CHS, the mRNA levels of antioxidant enzymes, IL-1ß, TNF-α, TLR4, and HSF1 were significantly lower in TM than in controls, while the levels of TLR2 and IL-8 were significantly higher in TM than in controls. In addition, TM led to significant increase of mRNA levels of IL-6 and HSF3 after 1 D and Hsp70 after 3 D of CHS and to significant decrease of mRNA levels of IL-6 after 3 and 5 D, HSF3 after 7 D, and Hsp70 after 5 D of CHS. Results of this study suggest that TM led to altered posthatch antioxidant, immunological, and Hsp response to CHS in the jejunal mucosae of broiler chickens, probably indicating that TM may mitigate the adverse effects of CHS.

Effects of pre-hatch thermal manipulation and post-hatch acute heat stress on the mRNA expression of interleukin-6 and genes involved in its induction pathways in 2 broiler chicken breeds.

In response to heat stress, interleukin-6 (IL-6) expression is upregulated in broiler chickens. The main aim of the present study was to evaluate the cumulative effects of thermal manipulation (TM) and subsequent acute heat stress (AHS) on the mRNA expression of IL-6 and genes involved in its induction pathways. The studied genes include IL-6, IL-1ß, TNF-α, TLR2, TLR4, NFκB50, NFκB65, Hsp70, and HSF3 in the spleen and liver tissues. TM was carried out at 39°C for 18 h and 65% relative humidity during days 10 to 18 of embryonic development, while AHS was stimulated by raising the temperature to 40°C for 7 h on post-hatch day 28. During AHS at 0, 1, 3, 5, and 7 h, the spleen and liver were collected from all groups to measure the mRNA expression by relative-quantitation real-time RT-PCR, and the blood was collected to measure plasma IL-6 level. TM significantly reduced Tc during AHS for both breeds from 1 to 7 h time intervals. TM resulted in enhanced basal mRNA expression of IL-6, HSF3, and Hsp70, but decreased the basal mRNA level of TLR4. During heat stress, TM enhanced the expression dynamics of Hsp70, HSF3, IL-6, IL-1ß, TNF-α, TLR2, TLR4, NFκB50, and NFκB65. The results of the current study indicate that TM enhanced the heat tolerance through improving the protective immunological response to heat stress by enhancing the expression of IL-6 and modulating the expression of genes important in its induction pathways.

In ovo feeding of L-arginine alters energy metabolism in post-hatch broilers.

This study aimed to investigate the effects of in ovo feeding (IOF) of L-arginine (Arg) on energy metabolism in post-hatch broilers. A total of 720 eggs was randomly assigned to 3 treatments: 1) non-injected control group, 2) 0.75% NaCl diluent-injected control group, and 3) 1.0% Arg solution-injected group. At 17.5 d of incubation, 0.6 mL of each solution was injected into the amniotic fluid of each egg of injected groups. After hatching, 80 male chicks were randomly assigned to each treatment group with 8 replicates per group. The results showed that IOF of Arg increased glycogen and glucose concentrations in the liver and pectoral muscle of broilers at hatch (P < 0.05). The plasma glucose and insulin levels were higher in the Arg group than in the non-injected and diluent-injected control groups (P < 0.05). Meanwhile, IOF of Arg enhanced the hepatic glucose-6-phosphatase (G6P) activity at hatch (P < 0.05). There was no difference in hexokinase (HK) or phosphofructokinase (PFK) enzyme activities in the pectoral muscle in all groups. Further, IOF of Arg increased the phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1,6-bisphosphatase (FBP) mRNA expressions at hatch (P < 0.05). In addition, broilers in the Arg group had a higher mRNA expression of glycogen synthase and a lower expression of glycogen phosphorylase in the liver and pectoral muscles than in the non-injected controls at hatch (P < 0.05). In conclusion, IOF of Arg solution enhanced liver and pectoral muscle energy reserves at hatch, which might be considered as an effective strategy for regulating early energy metabolism in broilers.

In ovo feeding of creatine pyruvate alters energy reserves, satellite cell mitotic activity and myogenic gene expression of breast muscle in embryos and neonatal broilers.

We investigated the effects of in ovo feeding (IOF) of creatine pyruvate (CrPyr) on energy reserves, satellite cell mitotic activity (SCMA) and myogenic gene expression in breast muscle of embryos and neonatal broilers. A total of 960 eggs were randomly allocated into three treatments: 1) non-injected control group, 2) saline group injected with 0.6 mL of physiological saline (0.75%), and 3) CrPyr group injected with 0.6 mL of physiological saline (0.75%) containing 12 mg CrPyr/egg at 17.5 d of incubation. After hatching, a total of 120 male chicks were randomly assigned to each treatment group, with eight replicate sets per group. Selected chicks had body BW close to the average of their pooled group. Our results showed that the total and relative breast muscle weights of broilers subjected to CrPyr treatment were higher than those in the control and saline groups on 19 d of incubation (19 E), the day of hatch, 3 and 7 d post-hatch (P < 0.05). The myofiber diameter and cross-sectional area of individuals in the CrPyr group were higher than those in other treatments on 3 and 7 d post-hatch (P < 0.05). Moreover, IOF of CrPyr increased (P < 0.05) creatine concentrations on 19 E, the day of hatch and 3 d post-hatch, the same treatment increased phosphocreatine concentrations on 19 E. Broilers in the CrPyr group showed higher expression of myogenic differentiation 1 (MyoD) (P < 0.05), myogenin and paired box 7 (Pax7), as well as higher index of SCMA on 3 d post-hatch. However, myostatin mRNA expression in CrPyr-treated broilers was down-regulated on 3 d post-hatch (P < 0.05). These results indicated that IOF of CrPyr increased energy reserves of embryos and SCMA of broilers on 3 d post-hatch, which led to enhanced muscle growth in the late embryos and neonatal broilers. Additionally, IOF of CrPyr increased the activity of satellite cells possibly through up-regulating MyoD, myogenin, and Pax7 mRNA expression and down-regulating myostatin mRNA expression.

Effects of maternal dietary manganese and incubation temperature on hatchability, antioxidant status, and expression of heat shock proteins in chick embryos.

To investigate whether supplementing manganese (Mn) to the maternal diet could reduce the deleterious effect of heat stress on the developing embryo, the hatchability, antioxidant status, and expression of heat shock proteins (HSP) were evaluated in chick embryos under normal and high incubation temperatures. A completely randomized design ( = 6) with 2 maternal dietary Mn treatments (unsupplemented control basal diet versus the basal diet + 120 mg Mn/kg as inorganic Mn) × 2 incubation temperatures (normal, 37.8°C, versus high, 39.0°C) was used. High incubation temperature did not affect ( > 0.19) hatchability and embryo mortality and development but did increase ( < 0.05) activities of heart manganese superoxide dismutase (MnSOD) and liver copper zinc superoxide dismutase and liver MnSOD mRNA and protein levels in embryos. High incubation temperature also decreased ( < 0.003) HSP70 protein level in the heart but had no effects ( > 0.07) in the liver of embryos. Maternal diet with Mn supplementation not only increased ( < 0.05) the hatchability and Mn content ( < 0.001) in the yolk and embryonic tissues and the activity of MnSOD in the heart ( < 0.004) as well as relative liver weight ( < 0.05) under normal incubation temperature but also decreased ( ≤ 0.05) embryo mortality and HSP90 mRNA level in the liver and heart of embryos. Furthermore, under high incubation temperature, maternal diet Mn supplementation increased ( < 0.002) MnSOD protein expression in the liver of embryos but had no effect ( > 0.43) under normal incubation temperature. These results indicated that high incubation temperature induced self-protective responses of chick embryos with a modification of antioxidant status and a depression of HSP70 protein level. Maternal dietary supplementation of Mn could improve the hatchability as well as antioxidant ability to protect against heat challenge in embryos during incubation.

Changes in estrogen receptor expression in the chick thymus during late embryonic development.

In chickens, although estrogen receptors (ER) are reported to be associated with the immunological processes, detailed information about the differences in ER expression in the tissues related to the development of lymphocytes is not fully known, especially during the developmental stage. To learn more about this immunological relationship, we used semi-quantitative polymerase chain reaction method to detect the ER expression levels in the thymus tissues of chicks during the developmental stage. Furthermore, ER-expressing cells were detected by immunohistochemistry. The results of this study show that the expression level of ER increased on embryonic day 16 and decreased on day 20. Furthermore, ER expression was significantly higher in male than in female chickens at day 16. The increased expression on day 16 and decreased level on day 20 were also reproduced in the incidence of immunoreactive cells, although there was a 1-day delay in the elevated incidence of the cells. This study revealed the changes in ER expression and the incidence of ER-positive cells in the thymus of chickens during the developmental stage.

Sexing domestic chicken before hatch: a new method for in ovo gender identification.

Male chicks are an unwanted by-product when producing laying hens. The common practice to kill them directly after they have hatched gives rise to ethical concerns worldwide. The aim of this study was to develop an endocrine method to determine the sex of domestic chicken before hatch. On Days 7 to 10 of incubation, the allantoic fluid from brown layers' eggs (n = 750) was analyzed via enzyme immunoassay for their content of estradiol, estrone sulfate, and testosterone in order to detect gender differences. We successfully established a reliable method for in ovo sex identification on Day 9 of incubation by estrone sulfate measurement in the allantoic fluid. Female embryos displayed significantly higher hormone levels in the allantoic fluid than males (female: median = 0.312 ng/mL; male: median = 0.110 ng/mL; P ≤ 0.001). Our method allows the sexing of domestic chicken at a very early stage of embryonic development, even before the onset of pain perception. The possibility to eliminate eggs containing male embryos on Day 9 of incubation represents a vast improvement compared with culling day-old chicks.

Intra-amniotic administration and dietary inulin affect the iron status and intestinal functionality of iron-deficient broiler chickens.

Inulin, a linear ß-fructan, is present in a variety of plants, with relatively high levels of up to 20% in chicory root. It exhibits prebiotic properties and was shown to enhance mineral absorption. Our objectives were to assess the effect of intra-amniotic administration of inulin at 17 d of incubation on the iron status of broiler chicks (at hatch, 21 d) and to continue to monitor iron status with and without dietary inulin on these hatchlings for 42 d. The study included 3 prehatch treatment groups (n = 30): 1) inulin, inulin solution (4% inulin/0.85% saline); 2) control 1, untreated eggs; and 3) control 2, saline solution (0.85% saline). Solutions were injected into the naturally consumed amniotic fluid of 17-d-old chicken embryos (groups 1, 3). Upon hatch (93% hatchability), and from each group, 10 chicks were killed and their small intestine, liver, and cecum were removed for mRNA abundance of intestinal iron-related transporters, liver ferritin amounts, and bacterial analysis of cecal content, respectively. From the remaining chicks of each group, chicks were allocated to a standard corn-based diet (± 4% inulin, n = 10). During the trial, hemoglobin concentrations and body hemoglobin-Fe values were higher in the inulin group versus controls (P < 0.05). On d 42, birds were anesthetized and their duodenal loops were exposed. A nonocclusive catheter was inserted into the duodenal vein for blood sampling. A solution containing 58Fe (0.1 mg of Fe/10 mM ascorbic acid) added to the digested diet sample was injected into the loop. Blood samples were collected every 5 min and for 90 min postinjection and analyzed by inductively coupled argon-plasma mass spectrometry for 58Fe concentrations. At the end of the procedure, animals were killed and cecum contents and sections of the duodenum and liver were removed. Results showed that 58Fe absorption rates were at times higher in the inulin group versus the other groups. Also, mRNA abundance of DMT1 (an Fe transporter) and ferroportin in addition to liver ferritin amounts were higher (P < 0.05) in the inulin group versus controls. Results indicate that intra-amniotic administration and dietary inulin improved the iron status of iron-deficient broilers.

Expression of the Tgfß2 gene during chick embryogenesis.

We performed a comprehensive analysis of the expression of transforming growth factor (TGF) ß2 during chick embryogenesis from stage 6 to 30 (Hamburger and Hamilton, J Morphol 1951;88:49-92) using in situ hybridization. During cardiogenesis, Tgfß2 was expressed in the endothelial/mesenchymal cells of the valvulo-septal endocardial cushion tissue and in the epicardium until the end of embryogenesis. During the formation of major arteries, Tgfß2 was localized in smooth muscle progenitors but not in the vascular endothelium. During limb development, Tgfß2 was expressed in the mesenchymal cells in the presumptive limb regions at stage 16, and thereafter it was localized in the skeletal muscle progenitors. In addition, strong Tgfß2 expression was seen in the mesenchymal cells in the pharyngeal arches. Tgfß2 mRNA was also detected in other mesoderm-derived tissues, such as the developing bone and pleura. During ectoderm development, Tgfß2 was expressed in the floor plate of the neural tube, lens, optic nerve, and otic vesicle. In addition, Tgfß2 was expressed in the developing gut epithelium. Our results suggest that TGFß2 plays an important role not only in epithelial-mesenchymal interactions but also in cell differentiation and migration and cell death during chick embryogenesis. We also found that chick and mouse Tgfß2 RNA show very similar patterns of expression during embryogenesis. Chick embryos can serve as a useful model to increase our understanding in the roles of TGFß2 in cell-cell interactions, cell differentiation, and proliferation during organogenesis.

Vasoactivity of the gasotransmitters hydrogen sulfide and carbon monoxide in the chicken ductus arteriosus.

Besides nitric oxide (NO) and carbon monoxide (CO), hydrogen sulfide (H(2)S) is a third gaseous messenger that may play a role in controlling vascular tone and has been proposed to serve as an O(2) sensor. However, whether H(2)S is vasoactive in the ductus arteriosus (DA) has not yet been studied. We investigated, using wire myography, the mechanical responses induced by Na(2)S (1 µM-1 mM), which forms H(2)S and HS(-) in solution, and by authentic CO (0.1 µM-0.1 mM) in DA rings from 19-day chicken embryos. Na(2)S elicited a 100% relaxation (pD(2) 4.02) of 21% O(2)-contracted and a 50.3% relaxation of 62.5 mM KCl-contracted DA rings. Na(2)S-induced relaxation was not affected by presence of the NO synthase inhibitor l-NAME, the soluble guanylate cyclase (sGC) inhibitor ODQ, or the K(+) channel inhibitors tetraethylammonium (TEA; nonselective), 4-aminopyridine (4-AP, K(V)), glibenclamide (K(ATP)), iberiotoxin (BK(Ca)), TRAM-34 (IK(Ca)), and apamin (SK(Ca)). CO also relaxed O(2)-contracted (60.8% relaxation) and KCl-contracted (18.6% relaxation) DA rings. CO-induced relaxation was impaired by ODQ, TEA, and 4-AP (but not by L-NAME, glibenclamide, iberiotoxin, TRAM-34 or apamin), suggesting the involvement of sGC and K(V) channel stimulation. The presence of inhibitors of H(2)S or CO synthesis as well as the H(2)S precursor L-cysteine or the CO precursor hemin did not significantly affect the response of the DA to changes in O(2) tension. Endothelium-dependent and -independent relaxations were also unaffected. In conclusion, our results indicate that the gasotransmitters H(2)S and CO are vasoactive in the chicken DA but they do not suggest an important role for endogenous H(2)S or CO in the control of chicken ductal reactivity.

Broiler egg storage induces cell death and influences embryo quality.

It is well known that egg storage reduces embryo performance, but the fundamental reasons for reduced embryo quality remain unclear. The objective of this study was to investigate possible cellular and molecular mechanisms that might reduce embryo quality after egg storage. Broiler hatching eggs were obtained from the Ross 308 broiler strain, divided into 2 groups, and stored (4 and 14 d) under the same temperature and humidity conditions. Samples of the eggs were used to assess embryo quality by determining daily embryo weight (wet and dry) from 4 to 21 d of incubation. To understand possible cellular and molecular mechanisms that might affect embryo quality, blastoderms (unincubated embryos) were isolated from a sample of eggs from each storage group, dissociated into single cells, and subjected to flow cytometry analysis to differentiate between viable, apoptotic, and necrotic cell populations. Quantitative real-time PCR analysis was used to compare the expression of selected apoptotic genes (Bcl-2 homologous antagonist/killer gene, Bcl-2-associated X gene, Bcl-2-related ovarian killer gene, B-cell lymphoma 2 gene, and B-cell lymphoma xL gene) in blastoderms and embryos (6 d old after incubation). Data were analyzed by the MIXED model procedure of SAS (SAS Institute, Cary, NC), with significance set at P ≤ 0.05. After covariance analysis of initial egg weights, the results showed decreased daily embryo weights (wet and dry), an indication of decreased embryo quality that could affect hatch quality. In addition, a decrease in blastodermal cell viability was associated with an increased percentage of apoptotic cell deaths (P < 0.0001). Expression of pro-apoptotic genes (Bcl-2 homologous antagonist/killer gene, Bcl-2-associated X gene, and Bcl-2-related ovarian killer gene) were upregulated at the blastodermal level as the storage duration increased, but all genes were downregulated after 6 d of incubation. This suggests that an increase in egg storage duration could activate mechanisms of apoptotic cell death at the blastodermal level, which may be one of the molecular mechanisms that leads to reduced daily embryonic weight during incubation. Experimental controls capable of reducing the cellular and molecular mechanisms of egg storage should be used to increase embryo quality.

BMP-induced L-Maf regulates subsequent BMP-independent differentiation of primary lens fibre cells.

Bone morphogenetic protein (BMP) signals are essential for lens development. However, the temporal requirement of BMP activity during early events of lens development has remained elusive. To investigate this question, we have used gain- and loss-of-function analyses in chick explant and intact embryo assays. Here, we show that BMP activity is both required and sufficient to induce L-Maf expression, whereas the onset of δ-crystallin and initial elongation of primary lens fibre cells are BMP-independent. Moreover, before lens placode formation and L-Maf onset, but not after, prospective lens placodal cells can switch to an olfactory placodal fate in response to decreased BMP activity. In addition, L-Maf is sufficient to up-regulate δ-crystallin independent of BMP signals. Taken together, these results show that before L-Maf induction BMP activity is required for lens specification, whereas after L-Maf up-regulation, the early differentiation of primary lens fibre cells occurs independent of BMP signals.

Telomere biology of the chicken: a model for aging research.

Division-dependent telomere shortening correlating with age triggers senescence on a cellular level and telomere dysfunction can facilitate oncogenesis. Therefore, the study of telomere biology is critical to the understanding of aging and cancer. The domestic chicken, a classic model for the study of developmental biology, possesses a telomere genome with highly conserved aspects and distinctive features which make it uniquely suited for the study of telomere maintenance mechanisms, their function and dysfunction. The purpose of this review is to highlight the chicken as a model for aging research, specifically as a model for telomere and telomerase research, and to increase its utility as such by describing developments in the study of chicken telomeres and telomerase in the context of related research in human and mouse.

Differential gene expression in CD45 cells at para-aortic foci stage of chicken haematopoiesis.

Para-aortic foci of chicken embryos at 6-7 days of development are considered to provide a microenvironment for haematopoietic stem cell proliferation and initial differentiation similar to that of fetal liver in mammals. Here, we have investigated the genes involved in this process by constructing and analysing a subtractive cDNA library from CD45(+) cells in para-aortic region. Among 394 analysed clones 99 distinct genes were identified by sequence homology search. Classification of the identified genes according to biological processes revealed that innate immunity-related genes are highly expressed at this stage. This can be explained by the presence of yolk sac-derived macrophages in the original tissue sample but also by the indiscriminate expression of multiple lineage-specific genes in haematopoietic stem cells and primitive progenitors. Differentially expressed genes related to transcription, signalling and lymphocyte functions are potential candidates involved in lineage commitment.

Chick tooth induction revisited.

Teeth have been missing from Aves for almost 100 million years. However, it is believed that the avian oral epithelium retains the molecular signaling required to induce odontogenesis, and this has been widely examined using heterospecific recombinations with mouse dental mesenchyme. It has also been argued that teeth can form from the avian oral epithelium owing to contamination of the mouse mesenchyme with mouse dental epithelial cells. To investigate the possibility of tooth formation from chick oral epithelium and the characteristics of possible chick enamel, we applied LacZ transgenic mice during heterospecific recombination and examined the further tooth formation. Transmission electron microscopy was used to identify the two tissues during development after heterospecific recombination. No mixing was detected between chick oral epithelium and mouse dental mesenchyme after 2 days, and secretory ameloblasts with Tomes' processes were observed after 1 week. Teeth were formed after 3 weeks with a single cusp pattern, possibly determined by epithelial factors, which is similar to that of the avian tooth in the late Jurassic period. These recombinant teeth were smaller than mouse molars, whereas perfect structures of both ameloblasts and enamel showed histological characteristics similar to those of mice. Together these observations consistent with previous report that odontogenesis is initially directed by species-specific mesenchymal signals interplaying with common epithelial signals.

Chicken receptor activator of nuclear factor-kappaB ligand induces formation of chicken osteoclasts from bone marrow cells and also directly activates mature osteoclasts.

Receptor activator of nuclear factor-kappaB ligand (RANKL), which functions as a major determinant of osteoclast differentiation and activation, is a type II transmembrane protein and is expressed in osteoblasts-stromal cells. The aim of this study was to clarify the role of chicken RANKL (chRANKL) in chicken osteoclast differentiation and to determine its effect on mature chicken osteoclasts. In the present study, chRANKL protein was first cloned and expressed in Escherichia coli. We then treated chicken bone marrow cells with chRANKL protein and found that it induced the formation of chicken osteoclast-like multinucleated cells in a dose-dependent manner in the presence of human macrophage colony-stimulating factor. Moreover, the addition of chicken osteoprotegerin could block the effect of chRANKL with regard to osteoclast-like multi-nucleated cell formation and bone resorption. Using primary cultures of chicken osteoclasts on bone slices, we also found that bone resorption pits per cell increased with chRANKL concentration in a dose-dependent manner. The chRANKL-treated hens exhibited increased blood Ca(++) levels within 2 h after injection, showing that chRANKL also activates osteoclasts in vivo. These results clearly indicate that the expressed protein is functional and may also be a critical factor for chicken osteoclastogenesis and bone resorption.

Inhibition of electrical activity by retroviral infection with Kir2.1 transgenes disrupts electrical differentiation of motoneurons.

Network-driven spontaneous electrical activity in the chicken spinal cord regulates a variety of developmental processes including neuronal differentiation and formation of neuromuscular structures. In this study we have examined the effect of chronic inhibition of spinal cord activity on motoneuron survival and differentiation. Early spinal cord activity in chick embryos was blocked using an avian replication-competent retroviral vector RCASBP (B) carrying the inward rectifier potassium channel Kir2.1. Chicken embryos were infected with one of the following constructs: RCASBP(B), RCASBP(B)-Kir2.1, or RCASBP(B)-GFP. Infection of chicken embryos at E2 resulted in widespread expression of the viral protein marker p27 gag throughout the spinal cord. Electrophysiological recordings revealed the presence of functional Kir2.1 channels in RCASBP(B)-Kir2.1 but not in RCASBP(B)-infected embryos. Kir2.1 expression significantly reduced the generation of spontaneous motor movements in chicken embryos developing in ovo. Suppression of spontaneous electrical activity was not due to a reduction in the number of surviving motoneurons or the number of synapses in hindlimb muscle tissue. Disruption of the normal pattern of activity in chicken embryos resulted in a significant downregulation in the functional expression of large-conductance Ca(2+)-dependent K(+) channels. Reduction of spinal cord activity also generates a significant acceleration in the inactivation rate of A-type K(+) currents without any significant change in current density. Kir2.1 expression did not affect the expression of voltage-gated Na(+) channels or cell capacitance. These experiments demonstrate that chronic inhibition of chicken spinal cord activity causes a significant change in the electrical properties of developing motoneurons.

Gain- and loss-of-function approaches in the chick embryo.

The chicken embryo has been used as a classical embryological model for studying developmental events because of its ready availability, similarity to the human embryos, and amenability to embryological and surgical manipulations. With the arrival of the molecular era, however, avian embryos presented distinct experimental limitations, largely because of the difficulty of performing targeted mutagenesis or transgenic studies. However, in the last decade and a half, a number of new methods for transient transgenesis have been developed that allow efficient alteration of gene function during early embryonic development. These techniques have made it possible to study the effects of gene inactivation or overexpression on downstream transcriptional regulation as well as on embryonic derivatives. This, together with sequencing of the chicken genome, has allowed the chicken embryo to enter the genomic era. While attempts to establish germ line transgenesis are ongoing, methods for rapid, transient spatiotemporally targeted gene alterations have thus again re-established the chick embryo as an important experimental niche by making it possible to apply genetics in concert with classical embryological techniques. This provides a unique tool to explore the role of developmentally important genes (Ishii and Mikawa, 2005; Itasaki et al., 1999; Krull, 2004; Ogura, 2002; Swartz et al., 2001). Transient transfection methods have allowed for efficient mis- and overexpression of transgenes. For long-term analyses, retrovirally mediated gene transfer has particular advantage. For short-term experiments, electroporation and adenoviral-mediated gene transfer methods provide transient expression, largely because of the short persistence time of the transgene within the cell. More recently, Tol2 transposon-mediated constructs have been employed, allowing for integration into the genome and prolonged expression of the transgene (Sato et al., 2007), see Chapter 14 by Takahashi et al., this volume). These methods today are routinely used for gain-of-function analysis, to overexpress or ectopically express genes of interest (Arber et al., 1999; Barembaum and Bronner-Fraser, 2007; Bel-Vialar et al., 2002). Loss-of-function experiments are also possible using electroporation of dominant-negative constructs that act as competitive inhibitors (Bel-Vialar et al., 2002; Renzi et al., 2000; Suzuki-Hirano et al., 2005), morpholino antisense oligos (Basch et al., 2006; Kos et al., 2001; Sheng et al., 2003) that block translation or splicing, or constructs expressing small interfering or small hairpin RNAs (siRNAs or shRNAs) (Chesnutt and Niswander, 2004; Das et al., 2006; Katahira and Nakamura, 2003). Electroporation as the most popular method of the transient transfection into the chick embryos. Electroporation of chicken embryos involves application of an electric field to the exposed tissue that transiently disrupts the stability of the cell plasma membrane, creating reversible pores through which nucleic acids or their analogues can be readily transported into the cytosol. The use of this method for transfection into the vertebrate embryos has been facilitated by adapting the voltage parameters and the type and the duration of the electric pulse. By applying several successive square pulses at a very low voltage, with long rest periods in between, one can successfully deliver a DNA construct or another small charged particle into the cytoplasm, with minimal cell death, high efficiency of the uptake and good embryonic survival rate. The size limit of the DNA molecule that can be transfected in such a way is not yet known, though it is more likely that the size limitation in this procedure (if any) lies within the practical problems of cloning large fragments into the plasmid. We routinely overexpress constructs containing 3-4 kb inserts and coharboring a GFP or RFP reporter whose translation is initiated from an internal ribosomal entry site (IRES), thus allowing easy detection of the electroporated cells.