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The chick immune system is a fundamental model in basic immunology. In birds, the bone marrow derived pluripotent stem cells after entering the circulation, migrate to bursa of Fabricius to benefit from a microenvironment which supports the differentiation and maturation of B lymphocytes by the help of its resident cells and tissues. Delivering sufficient functional B cells is required to maintain their peripheral population and normal peripheral humoral responses. Additionally, bursa acts as an active site for the generation of antibody diversity through gene conversion. Being consisted of 98% B lymphocytes, the organ is occupied by other cell types including T cells, macrophages, eosinophils and mast cells. Thymus, which is an epithelial organ is the main site of T cell development where positive and negative selections contribute to the development of functional and not autoreactive T cell repertoire. Bursectomy and thymectomy are surgical exercises through which the involvement of cells of specific immunity including B cells and T cells can be determined., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The transcription factor Nr4a2 was recently revealed as a very early developmental marker of the claustrum (CL) proper in the mouse. The earliest claustral primordium was identified superficially, dorsal to the olfactory cortex, and was subsequently covered by the Nr4a2-negative cells of the insular cortex. Some tangentially migrating claustral derivatives (subplate cells and some endopiriform elements) also expressed this marker. The present study employs the same genetic marker to explore the presence of a comparable pallial division in chicken in which, in principle, the same pallial sectors exist as in mammals. We were indeed able to delineate an early-developing Nr4a2-positive mantle domain at the expected topologic position within the developing chicken lateral pallium. In the chicken as well as in the turtle (from data in the literature), the earliest postmitotic lateropallial cells likewise express Nr4a2 and occupy a corticoid superficial stratum of the mesopallium, which is clearly comparable in spatial and chronological profile to the mouse CL. Other cells produced in this pallial sector include various tangentially migrating Nr4a2-labeled derivatives as well as Nr4a2-negative and Nr4a2-positive local deeper subpopulations that partially interdigitate, forming mesopallial core and shell populations. We hold that the deep avian and reptilian mesopallial formation developing under the superficial corticoid CL homolog represents a field homolog of the insula, although additional studies are required to underpin this hypothesis., (© 2015 Wiley Periodicals, Inc.)

Competing Interests: The author has declared that no competing interests exist.

From early dinosaurs with as many as nine wrist bones, modern birds evolved to develop only four ossifications. Their identity is uncertain, with different labels used in palaeontology and developmental biology. We examined embryos of several species and studied chicken embryos in detail through a new technique allowing whole-mount immunofluorescence of the embryonic cartilaginous skeleton. Beyond previous controversy, we establish that the proximal-anterior ossification develops from a composite radiale+intermedium cartilage, consistent with fusion of radiale and intermedium observed in some theropod dinosaurs. Despite previous claims that the development of the distal-anterior ossification does not support the dinosaur-bird link, we found its embryonic precursor shows two distinct regions of both collagen type II and collagen type IX expression, resembling the composite semilunate bone of bird-like dinosaurs (distal carpal 1+distal carpal 2). The distal-posterior ossification develops from a cartilage referred to as "element x," but its position corresponds to distal carpal 3. The proximal-posterior ossification is perhaps most controversial: It is labelled as the ulnare in palaeontology, but we confirm the embryonic ulnare is lost during development. Re-examination of the fossil evidence reveals the ulnare was actually absent in bird-like dinosaurs. We confirm the proximal-posterior bone is a pisiform in terms of embryonic position and its development as a sesamoid associated to a tendon. However, the pisiform is absent in bird-like dinosaurs, which are known from several articulated specimens. The combined data provide compelling evidence of a remarkable evolutionary reversal: A large, ossified pisiform re-evolved in the lineage leading to birds, after a period in which it was either absent, nonossified, or very small, consistently escaping fossil preservation. The bird wrist provides a modern example of how developmental and paleontological data illuminate each other. Based on all available data, we introduce a new nomenclature for bird wrist ossifications., Competing Interests: The authors have declared that no competing interests exist.

The poultry industry is a sector of agribusiness which represents an important role in the country's agricultural exports. Therefore, the study about embryogenesis of the domestic chicken (Gallus gallus domesticus) has a great economic importance. The aim of this study was to evaluate embryonic development of the endoderm in chicken (Gallus gallus domesticus). Forty fertilized eggs of domestic chickens, starting from the 1st day of gestation and so on until the 19 days of the incubation were collected from the Granja São José (Amparo, SP, Brazil). Embryos and fetus were fixed in 10% formaldehyde solution, identified, weighed, measured, and subjected to light and scanning electron microscopy. The endoderm originates the internal lining epithelium of the digestive, immune, respiratory systems, and the organs can be visualized from the second day (48 h) when the liver is formed. The formation of the digestive system was complete in the 12th day. Respiratory system organs begin at the fourth day as a disorganized tissue and undifferentiated. Their complete differentiation was observed at the 10 days of incubation, however, until the 19 days the syrinx was not observed. The formation of immune system at 10th day was observed with observation of the spleen, thymus, and cloacal bursa. The study of the organogenesis of the chicken based on germ layers is very complex and underexplored, and the study of chicken embryology is very important due the economic importance and growth of the use of this animal model studies such as genetic studies., (Copyright © 2013 Wiley Periodicals, Inc.)

The objective of this study was to examine the effect of embryonic nutritional enrichment on the development and properties of broiler leg bones (tibia and femur) from the prenatal period until maturity. To accomplish the objective, 300 eggs were divided into 2 groups: a noninjected group (control) and a group injected in ovo with a solution containing minerals, vitamins, and carbohydrates (enriched). Tibia and femur from both legs were harvested from chicks on embryonic days 19 (E19) and 21 (E21) and d 3, 7, 14, 28, and 54 posthatch (n = 8). The bones were mechanically tested (stiffness, maximal load, and work to fracture) and scanned in a micro-computed tomography (μCT) scanner to examine the structural properties of the cortical [cortical area, medullary area, cortical thickness, and maximal moment of inertia (Imax)] and trabecular (bone volume percent, trabecular thickness, and trabecular number) areas. To examine bone mineralization, bone mineral density (BMD) of the cortical area was obtained from the μCT scans, and bones were analyzed for the ash and mineral content. The results showed improved mechanical properties of the enriched group between E19 and d 3 and on d 14 (P < 0.05). Differences in cortical morphology were noted between E19 and d 14 as the enriched group had greater medullary area on E19 (femur), reduced medullary area on E21 (both bones), greater femoral cortical area on d 3, and greater Imax of both bones on d 14 (P < 0.05). The major differences in bone trabecular architecture were that the enriched group had greater bone volume percent and trabecular thickness in the tibia on d 7 and the femur on d 28 (P < 0.05). The pattern of mineralization between E19 and d 54 showed improved mineralization in the enriched group on E19 whereas on d 3 and 7, the control group showed a mineralization advantage, and on d 28 and 54, the enriched group showed again greater mineralization (P < 0.05). In summary, this study demonstrated that in ovo enrichment affects multiple bone properties pre- and postnatally and showed that avian embryos are a good model for studying the effect of embryonic nutrition on natal and postnatal development. Most importantly, the enrichment led to improved mechanical properties until d 14 (roughly third of the lifespan of the bird), a big advantage for the young broiler. Additionally, the improved mineralization and trabecular architecture on d 28 and 54 indicate a potential long-term effect of altering embryonic nutrition.

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve (1). Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components (2,3). Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

1. The objective of the study was to investigate the effects of breeder age and egg weight on hatching performance and morphological changes in segments of the small intestine of broiler chicks during a 21 h hatch window. 2. Eggs from Ross broiler breeder flocks aged 29 (young) and 48 weeks (old) were classified as light (LE) or heavy (HE) and incubated at the same conditions. At 475 h of incubation, eggs were checked every 3 h to determine time of external pipping and hatching. The first 42 chicks to emerge from each group were weighed and chick length was measured and 14 chicks from each group were sampled to collect residual yolk and intestine segments. The rest of chicks were placed back in the incubator and chick weight and length were measured individually at 9, 15 and 21 h after chicks hatched. At the end of 21 h, 14 chicks from each group were sampled again and the same procedure was followed. 3. The HE chicks pipped and hatched later than LE, regardless of breeder age. From hatch to the end of the hatch window, chick weight, but not yolk-free chick weight, gradually reduced. Relative residual yolk weight of chicks from both egg weights was similar at hatch, however, yolk sac utilisation was higher for LE chicks during the 21 h post-hatch period. At hatch, jejunum and ileum villus development was very similar for HE and LE chicks but greater development was observed for villus area with an increase in the jejunum villus length, width and goblet cell numbers in HE chicks. 4. The longest jejunum villus and the widest duodenum and jejunum villus were obtained for HE chicks from old breeders indicating that HE chicks from old breeders would have a greater surface area for nutrient absorption.

Vertebrates have achieved great evolutionary success due in large part to the anatomical diversification of their jaw complex, which allows them to inhabit almost every ecological niche. While many studies have focused on mechanisms that pattern the jaw skeleton, much remains to be understood about the origins of novelty and diversity in the closely associated musculature. To address this issue, we focused on parrots, which have acquired two anatomically unique jaw muscles: the ethmomandibular and the pseudomasseter. In parrot embryos, we observe distinct and highly derived expression patterns for Scx, Bmp4, Tgfβ2 and Six2 in neural crest-derived mesenchyme destined to form jaw muscle connective tissues. Furthermore, immunohistochemical analysis reveals that cell proliferation is more active in the cells within the jaw muscle than in surrounding connective tissue cells. This biased and differentially regulated mode of cell proliferation in cranial musculoskeletal tissues may allow these unusual jaw muscles to extend towards their new attachment sites. We conclude that the alteration of neural crest-derived connective tissue distribution during development may underlie the spatial changes in jaw musculoskeletal architecture found only in parrots. Thus, parrots provide valuable insights into molecular and cellular mechanisms that may generate evolutionary novelties with functionally adaptive significance.

The embryonic chick occupies a privileged place among animal models used in developmental studies. Its rapid development and accessibility for visualization and experimental manipulation are just some of the characteristics that have made it a vertebrate model of choice for more than two millennia. Until a few years ago, the inability to perform genetic manipulations constituted a major drawback of this system. However, the completion of the chicken genome project and the development of techniques to manipulate gene expression have allowed this classic animal model to enter the molecular age. Such techniques, combined with the embryological manipulations that this system is well known for, provide a unique toolkit to study the genetic basis of neural development. A major advantage of these approaches is that they permit targeted gene misexpression with extremely high spatiotemporal resolution and over a large range of developmental stages, allowing functional analysis at a level, speed and ease that is difficult to achieve in other systems. This article provides a general overview of the chick as a developmental model focusing more specifically on its application to the study of eye development. Special emphasis is given to the state of the art of the techniques that have made gene gain- and loss-of-function studies in this model a reality. In addition, we discuss some methodological considerations derived from our own experience that we believe will be beneficial to researchers working with this system.

The anterior border of the neural plate, presumed to contain the prospective peripheral portion (roof) of the prospective telencephalon, emerges within a vaguely defined proneural ectodermal region. Fate maps carried out at HH4 in the chick reveal that this region still produces indistinctly neural, placodal and non-neural derivatives; it does not express neural markers. We examined how the definitive anterior border domain of the rostral forebrain becomes established and comes to display a neural molecular profile, whereas local non-neural derivatives become separated. The process, interpreted as a border sharpening mechanism via intercalatory cell movements, was studied using fate mapping, time-lapse microscopy and in situ hybridization. Separation of neural and non-neural domains proceeds along stages HH4-HH4+, is well advanced at HH5, and is accompanied by a novel dorsoventral intercalation, oriented orthogonal to the border, that distributes transitional cells into molecularly distinct neural and non-neural fields. Meanwhile, neuroectodermal Sox2 expression spreads peripherally from the neighbourhood of the node, reaching the nascent anterior border domain at HH5. We also show that concurrent signals from the endodermal layer are necessary to position and sharpen the neural border, and suggest that FGF8 might be a component of this signalling.

The breeding of male layer chickens is currently considered to be highly uneconomical. In Germany alone, 40 to 50 million newly hatched male chickens were killed annually immediately after hatching. Therefore, it is necessary to find a method for sexing chickens early in the embryonic development, preferably before incubation. The genotypic sex of an egg can be determined using information found in the germinal disc, so knowledge of the exact position of the germinal disc is essential for further sexing, or for other actions such as the in ovo injection of agents. Previous studies have shown that the germinal disc is located somewhere on top of the yolk. However, no studies have yet been performed that investigate the influence of time spent in horizontal storage on the position of the germinal disc. Magnetic resonance imaging was chosen to determine this influence on the position of the germinal disc. It was found that eggs placed horizontally for long periods of time before scanning had significant changes in the positions of their germinal discs compared with those of eggs scanned minutes after positioning. The position of the germinal disc in eggs, minutes after horizontal positioning, deviated 14.7 ± 0.6 mm from the maximum vertical plane of the egg (zero position) in the z-direction; eggs scanned after 96 h of horizontal positioning showed a deviation of only 4.9 ± 1.6 mm. The x-axis also exhibited changes in the position of the germinal disc over time. Immediately after horizontal positioning, the eggs showed a deviation of 0.4 ± 0.4 mm in the x-direction, whereas the deviation after 96 h was 2.9 ± 0.5 mm. These results show that horizontal positioning of the egg hours before the measurement is necessary.

The effects of the in ovo injection of different carbohydrate solutions on the internal egg temperature (IT), hatchability, and time of hatch of embryonated Ross × Ross 708 broiler hatching eggs were determined. In addition, the BW, liver weight, yolk sac weight (YSW), and yolk-free BW (YFBW) of the embryos on d 19.5 of incubation and of the chicks on day of hatch were determined. Eggs containing live embryos were injected in the amnion on d 18.5 of incubation using an automated multiple-egg injector. Solution injections delivered 1.2 mL of physiological saline (0.85%) alone or with a supplemental carbohydrate. The following supplemental carbohydrates were separately dissolved in saline at a concentration of 0.3 g/mL: glucose, fructose, sucrose, maltose, and dextrin. Temperature transponders were implanted in the air cells of embryonated and nonembryonated eggs after in ovo injection for the detection of IT at 6, 14, and 22 h after injection. The IT of embryonated eggs was significantly greater than that of nonembryonated eggs at all 3 times after the treatment period. Eggs that were injected with saline with or without supplemental carbohydrates experienced a reduction in IT when compared with control eggs whose shells were perforated without solution delivery, and the decrease in IT was associated with a delay in hatch time. Liver weight was negatively related to YSW and positively related to YFBW, and YSW was negatively related to YFBW. Although the saline and carbohydrate solution injections increased chick BW compared with noninjected controls, chick YFBW was decreased in the maltose- and sucrose-injected groups. In conclusion, the injection of 1.2 mL of saline with or without supplemental carbohydrates lowered embryonic metabolism, as reflected by a lower IT and a delay in time of hatch. However, effects of the different carbohydrate solutions on yolk absorption and tissue deposition in yolk-free embryos varied. These results suggest that lower volumes for solutions containing maltose, sucrose, or fructose should be considered for in ovo injection.

Neural crest cells give rise to a diverse range of structures during vertebrate development. These cells initially exist in the dorsal neuroepithelium and subsequently acquire the capacity to migrate. Although studies have documented the importance of adherens junctions in regulating neural crest cell migration, little attention has been paid to tight junctions during this process. We now identify the tight junction protein cingulin as a key regulator of neural crest migration. Cingulin knock-down increases the migratory neural crest cell domain, which is correlated with a disruption of the neural tube basal lamina. Overexpression of cingulin also augments neural crest cell migration and is associated with similar basal lamina changes and an expansion of the premigratory neural crest population. Cingulin overexpression causes aberrant ventrolateral neuroepithelial cell delamination, which is linked to laminin loss and a decrease in RhoA. Together, our results highlight a novel function for cingulin in the neural crest., (Copyright © 2011 Wiley-Liss, Inc.)

We present a broadly applicable procedure for whole-mount imaging of antibody probes in embryonic tissues at microscopic resolutions based on combining a metal-based immunodetection scheme with x-ray microtomography (microCT). The method is generally accessible, relying on standard enzyme-conjugated secondary antibodies and other readily available reagents, and is demonstrated here with microCT visualizations of acetylated α-tubulin in the chick nervous system and of type II collagen in developing limbs. The tomographic images offer complete three-dimensional representations of molecular patterns obtained with immunostaining methods at the level of organ development, with added possibilities to quantify both spatial distributions and varying densities of gene products in situ. This imaging modality bridges a crucial gap in three-dimensional molecular imaging by combining the histological resolutions of confocal microscopy with a greater specimen size range than optical projection tomography, and thus enables a powerful new approach to long-standing issues of skeletogenic pattern formation in vertebrate limbs., (Copyright © 2011 Wiley-Liss, Inc.)

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., (Copyright © 2011 Wiley-Liss, Inc.)

The ability to measure in vivo strain and strain rate in embryonic chick heart is one of the key requirements for understanding the mechanisms of cardiac development. Due to its high temporal and spatial resolution as well as its fast imaging capability, optical coherence tomography (OCT) has the potential to reveal the complex myocardial activity in the living chick heart. We describe a method to evaluate the in vivo strain and strain rate of the myocardium through analyzing the periodic variation of the myocardial wall thickness calculated from real time serial OCT images. The results demonstrate that OCT can be a useful tool to describe the biomechanical characteristics of the embryonic heart.

Many clinically relevant congenital malformations arise during mid to late embryonic stages. This period is challenging to image quantitatively in live embryos, necessitating the use of multiple specimens with increased experimental variability. Here we establish X-ray and blood-pool computed tomography (CT) contrast agent toxicity and teratogenesis thresholds for 3D Micro-CT imaging of live avian embryos. Day 4 chick embryos micro-injected with Visipaque™ (VP) developed for an additional 6 days without defect. X-ray radiation up to 798 mGy was nontoxic. Peak average contrast of 1,060 HU occurred within 1 hr of imaging at 50 μm resolution. VP-enhanced contrast persisted past 24 hr with delayed accumulation in the allantois. Regional volumes of VP-injected embryos were statistically identical to those of fixed embryos perfused with osmium tetroxide. We further quantified longitudinal volumetric morphogenesis of the allantois over 30 hr. These results demonstrate the safety and efficacy of contrast enhanced quantitative micro-CT imaging for live embryos., (Copyright © 2011 Wiley-Liss, Inc.)

The morphogenesis of vertebrate body parts remains an open question. It is not clear whether the existence of different structures, such as a head, can be addressed by fundamental laws of tissue movement and deformation, or whether they are only a sequence of stop-and-go genetic instructions. I have filmed by time-lapse microscopy the formation of the presumptive head territory in chicken embryos. I show that the early lateral evagination of the eye cups and of the mesencephalic plate is a consequence of a sudden change in boundary conditions of the initial cell flow occurring in these embryos. Due to tissue flow, and collision of the two halves of the embryo, the tissue sheet movement is first dipolar, and next quadrupolar. In vivo air puff tonometry reveals a simple visco-elastic behaviour of the living material. The jump from a dipolar to a quadrupolar flow changes the topology of the early morphogenetic field which is observed towards a complex vortex winding with a trail (the eye cups and brain folds). The hydrodynamical model accounts for the discontinuity of the vector field at the moment of collision of the left and right halves of the embryo, at a quantitative level. This suggests a possible mechanism for the morphogenesis of the head of amniotes, as compared to cephalochordates and anamniotes.

The aim of this study was to evaluate the sequence of the histological and stereological changes that occur in diverse components of left growing and right regressing ovaries of Gallus domesticus as well as the variations in the number and size of the different cell subpopulations from 8-day-old chicken embryo to 4-week-old chickens. Results indicate a bilateral asymmetry in favor of the left ovary at all ages studied. The left ovary shows cortex and medulla, but the right ovary has no cortex, only a medulla. Stereological results show in the left ovary an increase in total volume of all components of the ovarian medulla with advancing development. The right ovary follows a similar pattern until the age of 1-day-old chicken, to regress afterward, thereby, reducing the total volume of blood vessels, lacunar channels, and interstitium, but increasing the total volume of interstitial cells as development progresses; consequently, the total volume of interstitial cells is the dominant component in the right ovary of 4-week-old chickens. Results also reveal that the increase in total volume of interstitial cells in the left ovary at all ages and in the right ovary from 8-day-old chicken embryo to 1-day-old chicken is due to hyperplasia and cell hypertrophy of interstitial cells; hence, in the right ovary, cell hypertrophy is the main cause for the increase in the total volume of interstitial cells from 1-week-old to 4-week-old chickens., (Copyright © 2011 Wiley-Liss, Inc.)

Background: The use of Specific Pathogen Free (SPF) eggs in combination with RCAS retrovirus, a member of the Avian Sarcoma-Leukosis Virus (ASLV) family, is of standard practice to study gene function and development. SPF eggs are certified free of infection by specific pathogen viruses of either exogenous or endogenous origin, including those belonging to the ASLV family. Based on this, SPF embryos are considered to be free of ASLV viral protein expression, and consequently in developmental research studies RCAS infected cells are routinely identified by immunohistochemistry against the ASLV viral proteins p19 and p27. Contrary to this generally accepted notion, observations in our laboratory suggested that certified SPF chicken embryos may endogenously express ASLV viral proteins p19 and p27. Since these observations may have significant implications for the developmental research field we further investigated this possibility., Results: We demonstrate that certified SPF chicken embryos have transcriptionally active endogenous ASLV loci (ev loci) capable of expressing ASLV viral proteins, such as p19 and p27, even when those loci are not capable of producing viral particles. We also show that the extent of viral protein expression in embryonic tissues varies not only among flocks but also between embryos of the same flock. In addition, our genetic screening revealed significant heterogeneity in ev loci composition even among embryos of the same flock., Conclusions: These observations have critical implications for the developmental biology research field, since they strongly suggest that the current standard methodology used in experimental studies using the chick embryo and RCAS vectors may lead to inaccurate interpretation of results. Retrospectively, our observations suggest that studies in which infected cells have been identified simply by pan-ASLV viral protein expression may need to be considered with caution. For future studies, they point to a need for careful selection and screening of the chick SPF lines to be used in combination with RCAS constructs, as well as the methodology utilized for qualitative analysis of experimental results. A series of practical guidelines to ensure research quality animals and accuracy of the interpretation of results is recommended and discussed.

1. The aim of this study was to investigate the effect of maternal rearing on lipid metabolism and lipid metabolism-related gene expression in offspring broilers during embryonic development. 2. One hundred laying Sanhuang breeders were divided into two groups, and either floor-reared or cage-reared on the same diet. Liver and serum samples were extracted on days 14 and 19 of embryonic development and at hatching. The lipid metabolism related gene expressions of acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), malic enzyme (ME), apolipoprotein B100(apoB100), sterol regulating element binding protein (SREBP-1c), carnitine palmitoyltransferase (CPT-1) and peroxisome proliferators-activated receptor (PPARα) genes were determined using real time RT-PCR. 3. The results showed that embryonic weight, liver weight, serum and hepatic total cholesterol (TC) concentration and serum triglyceride (TG) content were not significantly different between the cage-reared group and the floor-reared group during embryonic development. However, embryonic weight, liver weight, serum and hepatic TC concentration and serum TG content in the cage-reared group were significantly higher than in the floor-reared group at hatching. 4. Hepatic ACC, FAS, SREBP-1c, ME and apoB genes expression were not significantly different between the cage-reared and the floor-reared groups during E9 and E14 development. Hepatic ME gene expression in the cage-reared group was higher than in the floor-reared group during E19 development. However, hepatic FAS, SREBP-1c, CPT-1 and PPARα gene expressions in the cage-reared group was higher than in the floor-reared group. 5. A change in the maternal regime could regulate lipid metabolism in offspring broilers during embryonic development, and especially at hatching.

Mechanical forces play an important role during brain development. In the early embryo, the anterior end of the neural tube enlarges and differentiates into the major brain subdivisions, including three expanding vesicles (forebrain, midbrain, and hindbrain) separated by two constrictions. Once the anterior neuropore and the spinal neurocoel occlude, the brain tube undergoes further regional growth and expansion in response to increasing cerebrospinal fluid pressure. Although this is known to be a response to mechanical loads, the mechanical properties of the developing brain remain largely unknown. In this work, we measured regional opening angles (due to residual stress) and stiffness of the embryonic chick brain during Hamburger-Hamilton stages 11-13 (approximately 42-51 h incubation). Opening angles resulting from a radial cut on transverse brain slices were about 40-110 deg (depending on region and stage) and served as an indicator of circumferential residual stress. In addition, using a custom-made microindentation device and finite-element models, we determined regional indentation stiffness and material properties. The results indicate that the modulus is relatively independent of position and stage of development with the average shear modulus being about 220 Pa for stages 11-13 chick brains. Information on the regional material properties of the early embryonic brain will help illuminate the process of early brain morphogenesis.

The capacity to image a growing embryo while simultaneously studying the developmental function of specific molecules provides invaluable information on embryogenesis. However, until recently, this approach was accomplished with difficulty both because of the advanced technology needed and because an easy method of minimizing damage to the embryo was unavailable. Here, we present a novel way of adapting the well-known EC culture of whole chick embryos to time-lapse imaging and to functional molecular studies using blocking agents. The novelty of our method stems from the ability to apply blocking agents ex ovo as well as in ovo. We were able to study the function of a set of molecules by culturing developing embryos ex ovo in tissue culture media containing these molecules or by injecting them underneath the live embryo in ovo. The in ovo preparation is particularly valuable, because it extends the period of time during which the developmental function of the molecule can be studied and it provides an easy, reproducible method for screening a batch of molecules. These new techniques will prove very helpful in visualizing and understanding the role of specific molecules during embryonic morphogenesis, including blood vessel formation., ((c) 2009 Wiley-Liss, Inc.)

In this work we studied the regional expression pattern of the Enc1 gene in the chick embryo telencephalon at intermediate and late stages of development, bearing on architectonic groupings and boundaries of current interest. In general, the Enc1 signal shows a markedly heterogeneous areal pattern of expression throughout the telencephalon; this corroborates data on new pallial and subpallial structures defined recently in the stereotaxic chick brain atlas of Puelles et al. (2007. The chick brain in stereotaxic coodinates. San Diego, CA: Academic Press). For example: a periventricular/central domain is Enc1-negative in the ventral pallium or nidopallium; core and shell nuclei appear in the mesopallium; the redefined caudodorsolateral area shows a characteristic pattern; the limits of the densocellular hyperpallium in the dorsal pallium are illuminated; and the postulated entorhinal cortex area is distinct at the posterior telencephalic pole. Interestingly, Enc1 transcripts are distinctly present in the piriform cortex at the surface of the ventral pallium throughout its longitudinal extent, as well as in the most rostral part of the lateral pallium, implying a layout of this cortex more similar to the situation in mammals than was assumed previously. Separate corticoid superficial strata are labeled by the Enc1 probe in the lateral and dorsal pallial regions. In the subpallium, the expression of Enc1 agrees with the new radial subdivisions defined by Puelles et al. (2007).

Here we show the temporal-spatial orchestration of early heart morphogenesis at cellular level resolution, in vivo, and reconcile conflicting positional fate mapping data regarding the primary heart-forming field(s). We determined the positional fates of precardiac cells using a precision electroporation approach in combination with wide-field time-lapse microscopy in the quail embryo, a warm-blooded vertebrate (HH Stages 4 through 10). Contrary to previous studies, the results demonstrate the existence of a "continuous" circle-shaped heart field that spans the midline, appearing at HH Stage 4, which then expands to form a wide arc of progenitors at HH Stages 5-7. Our time-resolved image data show that a subset of these cardiac progenitor cells do not overlap with the expression of common cardiogenic factors, Nkx-2.5 and Bmp-2, until HH Stage 10, when a tubular heart has formed, calling into question when cardiac fate is specified and by which key factors. Sub-groups and anatomical bands (cohorts) of heart precursor cells dramatically change their relative positions in a process largely driven by endodermal folding and other large-scale tissue deformations. Thus, our novel dynamic positional fate maps resolve the origin of cardiac progenitor cells in amniotes. The data also establish the concept that tissue motion contributes significantly to cellular position fate - i.e., much of the cellular displacement that occurs during assembly of a midline heart tube (HH Stage 9) is NOT due to "migration" (autonomous motility), a commonly held belief. Computational analysis of our time-resolved data lays the foundation for more precise analyses of how cardiac gene regulatory networks correlate with early heart tissue morphogenesis in birds and mammals.

Four-dimensional (4-D) imaging of the embryonic heart allows study of cardiac morphology and function in vivo during development. However, 4-D imaging of the embryonic heart using current techniques, including optical coherence tomography (OCT), is limited by the rate of image acquisition. Here, we present a nongated 4-D imaging strategy combined with an efficient postacquisition synchronization procedure that circumvents limitations on acquisition rate. The 4-D imaging strategy acquires a time series of images in B mode at several different locations along the heart, rendering out-of-phase image sequences. Then, our synchronization procedure uses similarity of local structures to find the phase shift between neighboring image sequences, employing M-mode images (extracted from the acquired B-mode images) to achieve computational efficiency. Furthermore, our procedure corrects the phase shifts by considering the phase lags introduced by peristaltic-like contractions of the embryonic heart wall. We applied the 4-D imaging strategy and synchronization procedure to reconstruct the cardiac outflow tract (OFT) of a chick embryo, imaged with OCT at early stages of development (Hamburger-Hamilton stage 18). We showed that the proposed synchronization procedure achieves efficiency without sacrificing accuracy and that the reconstructed 4-D images properly captured the dynamics of the OFT wall motion.

VATER association represents a cluster of Vertebral, Anal, Tracheo-Esophageal, Radial and Renal malformations, and caudal regression syndrome is an entity consisting of a spectrum of congenital anomalies of lower spine and hips associated with genitourinary and lower limb defects. The concurrence of various malformations may be explained by a common defect in blastogenesis, but direct evidence is yet to be accumulated. Here, by the use of autofluorescence and the teratogenic effect of adriamycin, we demonstrated that adriamycin administered to eggs of White Leghorns distributes to the caudal portion of the embryo and foregut epithelium and induces caudal regression and tracheal and pulmonary agenesis. The induction of caudal regression syndrome-like anomaly was developmental stage and dose dependent. Embryos with caudal regression demonstrated tracheoesophageal anomalies, one of the defects included in VATER association. The stages at which anomalies were produced corresponded to that of human embryos between days 22 and 26 (Carnegie stages 10-11). In view of the antitumor activity of adriamycin by intercalating to double-stranded DNA of undifferentiated cells undergoing rapid cell division, it is possible that adriamycin had preferentially attacked cells in the caudal end where pronounced proliferation takes place during this narrow period of greatest susceptibility.

In a first paper, we concluded that the muscular region of the interventricular septum is developed by the trabecular branches and showed evidence that the developing interventricular septum elongates in a direction opposite to that of atria. Nevertheless, to date the literature is lacking precise information on the importance of myocardial proliferation not only in this process but also in the morphogenesis of the ventricular cavities. The aim of this study was to determine the spatial and temporal distribution of high-intensity foci of cycling myocytes in the ventricular region of the heart of chicken embryos during cardiac septation. Histological studies, detection of the proliferating cell nuclear antigen by light and confocal microscopy and flow cytometric analysis were carried out. The results corroborate that the developing interventricular septum grows in a direction opposite to that of atria. A remoulding mechanism that results in fenestrated trabecular sheets and trabecular branching is discussed. Our findings allowed us to summarize the normal morphogenesis of the muscular region of the interventricular septum in a way that is different from that suggested by other researchers.

The knee joint has a highly complex 3-dimensional (3D) morphology that is sculpted at the interface of the forming long bones as they are generated in the embryo. Although it is clear that regulatory genes guide joint formation, the mechanisms that are responsible for morphogenesis of the knee are poorly understood. Certainly the process involves integration across several tissues and physical/mechanical influences from neighbouring tissues are important. We describe the acquisition of shape in the chick knee joint in detail and show that by HH34 the joint already displays shape characteristics of the adult structure. Through imaging developing cartilage, tendons, ligaments and muscle across developmental stages from HH28-34 we have built 3D representations of the forming structure including the various components important in knee formation. We describe the timing of muscle and tendon development in parallel with the refinement of cartilage shape, showing when and where (tendon attachment points) muscle forces are applied to the cartilage elements. Shape begins to emerge as the tendons are forming (HH30-32) but is fully refined (HH34) in the presence of tendons. The resulting integrated 3D representations of the developing knee across time will serve as the foundation for computational analysis of the mechanical environment, and experimental approaches to investigating morphogenetic mechanisms.

After gastrulation, during the process of neurulation, the anterior neural region undergoes important morphological transformations. The almost flat epithelium of the rostral neural plate becomes transformed into a spherical region, the prosencephalic vesicle, in the neural tube. Later in development, two bilateral areas (the optic and telencephalic vesicles) progressively protrude from the prosencephalon, generating the eyes and the cerebral hemispheres, respectively. Although the principal processes of neurulation have been well characterized, the growth patterns and evolution of topological relations between internal prosencephalic regions have not been experimentally analyzed. In order to better characterize morphogenetic transformations of the prosencephalon, we have realized and comparatively analyzed neuroepithelial fate maps before and after neurulation using quail/chick chimerical experiments. Since we have previously reported the fate map of the prosencephalon at the neural plate stage, in the present work we report the corresponding fate map at the neural tube stage. Comparative analysis of the two maps has allowed us to descriptively characterize the morphogenetic transformations of the alar prosencephalic regions during neurulation and to establish the topologic evolution of the principal areas of the vertebrate telencephalon.

Vertebrate cranial neurogenic placodes are relatively simple model systems for investigating the control of sensory neurogenesis. The ophthalmic trigeminal (opV) placode, for which the earliest specific marker is the paired domain homeodomain transcription factor Pax3, forms cutaneous sensory neurons in the ophthalmic lobe of the trigeminal ganglion. We previously showed that Pax3 expression in avian opV placode cells correlates with specification and commitment to a Pax3+, cutaneous sensory neuron fate. Pax3 can act as a transcriptional activator or repressor, depending on the cellular context. We show using mouse Splotch(2H) mutants that Pax3 is necessary for the normal neuronal differentiation of opV placode cells. Using an electroporation construct encoding a Pax3-Engrailed fusion protein, which represses Pax3 target genes, we show that activation of Pax3 target genes is required cell-autonomously within chick opV placode cells for expression of the opV placode markers FGFR4 and Ngn2, maintenance of the preplacodal marker Eya2, expression of Pax3 itself (suggesting that Pax3 autoregulates), neuronal differentiation and delamination. Mis-expression of Pax3 in head ectoderm is sufficient to induce FGFR4 and Ngn2 expression, but neurons do not differentiate, suggesting that additional signals are necessary to enable Pax3+ cells to differentiate as neurons. Mis-expression of Pax3 in the Pax2+ otic and epibranchial placodes also downregulates Pax2 and disrupts otic vesicle closure, suggesting that Pax3 is sufficient to alter the identity of these cells. Overall, our results suggest that activation of Pax3 target genes is necessary but not sufficient for neurogenesis in the opV placode.

Background: Intrauterine growth restriction is associated with an increased future risk for developing cardiovascular diseases. Hypoxia in utero is a common clinical cause of fetal growth restriction. We have previously shown that chronic hypoxia alters cardiovascular development in chick embryos. The aim of this study was to further characterize cardiac disease in hypoxic chick embryos., Methods: Chick embryos were exposed to hypoxia and cardiac structure was examined by histological methods one day prior to hatching (E20) and at adulthood. Cardiac function was assessed in vivo by echocardiography and ex vivo by contractility measurements in isolated heart muscle bundles and isolated cardiomyocytes. Chick embryos were exposed to vascular endothelial growth factor (VEGF) and its scavenger soluble VEGF receptor-1 (sFlt-1) to investigate the potential role of this hypoxia-regulated cytokine., Principal Findings: Growth restricted hypoxic chick embryos showed cardiomyopathy as evidenced by left ventricular (LV) dilatation, reduced ventricular wall mass and increased apoptosis. Hypoxic hearts displayed pump dysfunction with decreased LV ejection fractions, accompanied by signs of diastolic dysfunction. Cardiomyopathy caused by hypoxia persisted into adulthood. Hypoxic embryonic hearts showed increases in VEGF expression. Systemic administration of rhVEGF(165) to normoxic chick embryos resulted in LV dilatation and a dose-dependent loss of LV wall mass. Lowering VEGF levels in hypoxic embryonic chick hearts by systemic administration of sFlt-1 yielded an almost complete normalization of the phenotype., Conclusions/significance: Our data show that hypoxia causes a decreased cardiac performance and cardiomyopathy in chick embryos, involving a significant VEGF-mediated component. This cardiomyopathy persists into adulthood.

The structures that participate in normal ventricular septation, and to what extent they do so, are questions not yet clarified. Even less is known about how much each of the embryonic structures contributes to the topography of the mature interventricular septum (IVS). The aim of the present paper is to investigate the significance of ventricular trabeculations in the normal development of the muscular region (the middle and apical thirds) of the IVS and to determine the direction in which it grows during cardiac septation. Anatomical studies and in vivo labelling were carried out in chicken embryo hearts at stage 18HH, tracing the labels up to stage 36HH. We analysed the results by measuring the distance between the labelled structures at the beginning and end of the experiments. We demonstrate that the muscular region of the septum originates by the fusion of the ventricular trabeculations with evidence that during cardiac development, the IVS as well as the ventricular cavities grow in opposite direction to the atria.

Ten pairs of protrusions, called accessory lobes (ALs), exist at the lateral sides of avian lumbosacral spinal cords. Histological evidence has shown that neurons are present in AL and behavioral evidence suggests that AL acts as a sensory organ of equilibrium during bipedal walking. However, there is little functional evidence to indicate that cells in AL have neuronal functions. To elucidate this point, we developed a method to dissociate cells from chick AL and made electrophysiological recordings with the whole-cell patch clamp technique. Cells dissociated by enzymatic digestion from chick AL contained two major types of cells. One was round with clear cytosol and the other had a round cell body, rich cytosolic structures and some processes. Rapidly activating inward currents and slowly activating outward currents were recorded in response to depolarizing pulses to -10 mV under the voltage clamp configuration only from the latter type of cells. TTX at 100 nM inhibited the inward current by 85%, indicating the functional expression of TTX-sensitive voltage-gated Na(+) channel (VGSC). Activation and inactivation kinetics of the inward currents in AL cells were similar to those of mammalian VGSC. The VGSC-expressing AL cells generated action potentials in response to depolarization under the current clamp configuration. These results clearly indicate that functional neurons expressing fast inactivating and TTXsensitive VGSC which generate action potentials exist in the AL of the chick. These lines of cellular evidence clearly indicate that functional neurons exist in ALs and further support the proposal that the chick ALs function as the sensory organ of equilibrium.

Here, we examine the expression and regulation of the gene BAMBI, a kinase-deficient decoy receptor capable of interacting with type I bone morphogenetic protein (BMP) receptors in avian embryos. Initially, expression was limited to the endoderm during neurula and pharyngula stages. From embryonic day 3.5 (stage 20) and onward, BAMBI expression almost perfectly overlapped with known expression patterns for BMP4, particularly in the face and limbs. We performed bead implant experiments in the face to see which signals could be repressing or promoting expression of BAMBI. Our data point to retinoids and BMPs as being major positive regulators of BAMBI expression; however, fibroblast growth factor 2 acts to repress BAMBI. Furthermore, retinoic acid is likely to act directly on BAMBI as induction occurs in the presence of cycloheximide. The data suggested that BAMBI could be used to regulate Bmp signaling during tissue interactions that are an integral part of facial morphogenesis.

Chicken homologues of prickle-1 (pk-1) and prickle-2 (pk-2) were isolated to gain insight into the extent of planar cell polarity signaling during avian embryogenesis. Bioinformatics analyses demonstrated homology and showed that pk-1 and pk-2 exhibited conserved synteny with ADAMTS20 and ADAMTS9, GON-related zinc metalloproteases. Expression of pk-1 and pk-2 was established during embryogenesis and early organogenesis, using in situ hybridization and sections of chicken embryos. At early stages, pk-1 was expressed in Hensen's node, primitive streak, ventral neural tube, and foregut. In older embryos, pk-1 transcripts were detected in dorsolateral epithelial somites, dorsomedial lip of dermomyotomes, and differentiating myotomes. Furthermore, pk-1 expression was seen in lateral body folds, limb buds, and ventral metencephalon. pk-2 was expressed in Hensen's node and neural ectoderm at early stages. In older embryos, pk-2 expression was restricted to ventromedial epithelial somites, except in the most recently formed somite pair, and limb bud mesenchyme.

The embryonic heart tube consists of an outer myocardial tube, a middle layer of cardiac jelly, and an inner endocardial tube. It is said that tubular hearts pump the blood by peristaltoid contractions. The traditional concept of cardiac peristalsis sees the cyclic deformations of pulsating heart tubes as concentric narrowing and widening of tubes of circular cross-section. We have visualized the cross-sectional deformations of contracting embryonic hearts in chick embryos (HH-stages 9-17) using real-time high-resolution optical coherence tomography. Cardiac contractions are detected from HH-stage 10 onward. During the cardiac cycle, the myocardial tube undergoes concentric narrowing and widening while the endocardial tube undergoes eccentric narrowing and widening, having an elliptic cross-section at end-diastole and a slit-shaped cross-section at end-systole. The eccentric deformation of the endocardial tube is the consequence of an uneven distribution of the cardiac jelly. Our data show that the cyclic deformations of pulsating embryonic heart tubes run other than originally thought. There is evidence that heart tubes of elliptic cross-section might pump blood with a higher mechanical efficiency than those of circular-cross section. The uneven distribution of cardiac jelly seems to prefigure the future AV and cono-truncal endocardial cushions., ((c) 2008 Wiley-Liss, Inc.)

Formation of the epicardium requires interactions between alpha(4)beta(1) integrin, and the extracellular matrix. We investigated the role of other integrins expressed by epicardial cells. We detected transcripts for alpha(5), alpha(8), alpha(v), beta(1), beta(3), and beta(5) integrins in the chick proepicardial organ (PE). We demonstrate that alpha(5)beta(1), alpha(8)beta(1), and alpha(v)beta(3) integrins are expressed by chick epicardial mesothelial cells (EMCs). Migration of EMCs in vitro was reduced by RGD-containing peptides. Using adenoviruses expressing an antisense to chick alpha(4) (AdGFPalpha4AS), full-length (Adhalpha4V5), and C-terminal deleted alpha(4) (Adhalpha4DeltaCV5), we found that EMCs were less able to adhere to vitronectin and fibronectin(120) indicating that alpha(4)beta(1) plays a role in regulating EMC adhesion to ligands of alpha(5)beta(1), alpha(8)beta(1), and alpha(v)beta(3). In Adhalpha4DeltaCV5-infected EMCs, alpha(5)beta(1) was diminished in fibrillar adhesions and new FN matrix assembly was abnormal. We propose that cooperation between alpha(4)beta(1) and RGD integrins is important for EMC adhesion and subepicardial matrix formation., ((c) 2008 Wiley-Liss, Inc.)

Rho GTPases play central roles in the control of cell adhesion and migration, cell cycle progression, growth, and differentiation. However, although most of our knowledge of Rho GTPase function comes from the study of the three classic Rho GTPases RhoA, Rac1, and Cdc42, recent studies have begun to explore the expression, regulation, and function of some of the lesser-known members of the Rho GTPase family. In the present study, we cloned the avian orthologues of RhoV (or Chp for Cdc42 homologous protein) and RhoU (or Wrch-1 for Wnt-regulated Cdc42 homolog-1) and examined their expression patterns by in situ hybridization analysis both during early chick embryogenesis and later on, during gastrointestinal tract development. Our data show that both GTPases are detected in the primitive streak, the somites, the neural crest cells, and the gastrointestinal tract with distinct territories and/or temporal expression windows. Although both proteins are 90% identical, our results indicate that cRhoV and cRhoU are distinctly expressed during chicken embryonic development., ((c) 2008 Wiley-Liss, Inc.)

One major component of the extracellular matrix is hyaluronan (HA) which is thought to play a crucial role in the development of different organs including the central nervous system (CNS). HA is bound by specific receptors, CD44 and RHAMM, depending on cell types of CNS. However, data are lacking on the relation of HA to different cell populations in developing CNS. To provide new data about the co-localization of HA with the various cellular structures of the developing spinal cord, we studied the distribution pattern of hyaluronan in chicken embryos at Hamburger-Hamilton (HH) stages 8-39. A biotinylated HA-binding complex was used in combination with immunohistochemistry for proliferating and differentiating neurons. The intensity of the HA signal was determined by digital densitometry from histological sections. We found three mediolaterally oriented layers in the HA distribution pattern in stage HH23: (1) a moderate HA signal was detected in the ventricular zone; (2) strong HA accumulation was measured around Lim1,2-expressing cells (differentiating neurons) and early MNR2-expressing neurons (early motoneurons), corresponding to the intermediate zone; (3) a strong pericellular HA reaction was found around the neurons of the marginal zone. Interestingly, the peripheral nerves did not show HA signals. These findings suggest a crucial role of HA during neuronal development. We propose that HA may be involved in cell migration and axonal growth in the developing spinal cord.

The purpose of this study is to delineate with high frequency ultrasound imaging the normal growth and development of the chick embryo throughout its incubation period. White Leghorn chick embryos were imaged through an opening in the egg air cell from incubation day 0-19 (Hamburger & Hamilton stage 1-45) using a 13 MHz clinical high frequency linear small parts transducer. Multiple anatomic growth parameters were measured. Normal growth was confirmed with Hamburger and Hamilton staging. A timeline was constructed showing when each anatomic growth parameter could be visualized. Means and standard deviations of each parameter were plotted against incubation days studied to create nomograms and numerical tables of normal growth and development of the chick embryo. With this set of data, abnormal growth and development of the chick embryo can now be assessed.

1. Eggs from a layer-type breeder flock (Baladi, King Saud University) between 50 and 63 weeks of age were used in three trials to study the effects of electrical field (EF) during incubation on albumen and yolk heights, incubation temperature, egg weight loss and hatchability traits. The effects of egg size and eggshell characteristics on hatchability traits of eggs incubated under EF were investigated. 2. Eggs were weighed and graded into three weight classes (small, medium, and large). The physical dimensions, eggshell characteristics, and conductance of eggs were examined. The incubator was divided into two compartments for the control and EF treatments. Two aluminium plates were fitted on the inside walls of the EF compartment, face to face, and connected to a step up electric transformer. Eggs were exposed constantly to the EF during the first 18 d of incubation at the level of 30 kV/m, 60 Hz. 3. Egg size influenced the physical dimensions and eggshell characteristics of eggs. Large eggs had higher egg weight, egg surface area, egg volume, eggshell conductance, and eggshell weight and lower yolk weight percentage than medium or small size eggs. Small eggs had lower egg length and higher egg density than large or medium size eggs. Large eggs had higher eggshell thickness than small size eggs. 4. EF incubation of eggs raised incubation temperature by 0.06 degrees C, and increased the percentage of egg weight loss, hatchability, and weight of hatching chicks and reduced the early embryo deaths, and length of incubation by approximately 9.8, 19.6, 1.7, 62.1 and 2.1%, respectively. 5. There was no significant difference between the two incubation treatments in the heights of albumen and yolk of incubated eggs, percentages of late embryo deaths, and pips with live and dead embryos. Hatchability traits were not significantly influenced by egg size. 6. It was concluded that EF incubation of eggs increased hatchability, chick-hatching weight, and reduced the length of incubation of Baladi eggs. Differences in the physical dimensions and eggshell characteristics of eggs did not influence hatchability traits of eggs under EF incubation.

Endocrine cells in chick pancreas were observed to map their distribution during development and to perform morphometric studies starting on embryonic day 5. The ratio of exocrine to endocrine tissues first prevailed in favour of the endocrine ones, and changed abruptly after day 9 when rapid growth of exocrine tissue began. Endocrine tissue was formed of two types of islets. The 'light' (or B) islets were composed of insulin-immunoreactive cells, completed perhaps by a few somatostatin-immunoreactive cells occurring on the periphery. The majority of the somatostatin- and glucagon-immunoreactive cells were present in the 'dark' (or A) islets. Endocrine elements were also scattered as single cells over the pancreas. Sporadically, the endocrine cells established contacts with exocrine ducts. In morphometric analysis, volume density of insulin-, glucagon-, and somatostatin-immunoreactive cells was measured, and ratios were calculated between particular components. The volume density of endocrine cells and their ratio appeared stable in individual lobes but varied significantly between each other. Increase of the glucagon volume density is exponential, whereas insulin increases almost linearly especially in splenic lobe. The process results in the increase of the hormone-immunoreactive cell volume density in favour of glucagon-immunoreactive cells typical for birds.

Mutations in Notch2, Jagged1 or homologs of the Hairy-related transcriptional repressor Hey2 cause congenital malformations involving the non-chamber atrioventricular canal (AVC) and inner curvature (IC) regions of the heart, but the underlying mechanisms have not been investigated. By manipulating signaling directly within the developing chick heart, we demonstrated that Notch2, Hey1 and Hey2 initiate a signaling cascade that delimits the non-chamber AVC and IC regions. Specifically, misactivation of Notch2 signaling, or misexpression of either Hey1 or Hey2, repressed Bmp2. Because Jagged (also known as Serrate in non-mammalian species) ligands were found to be present in prospective chamber myocardium, these data support the model that Notch2 and Hey proteins cause the progressive restriction of Bmp2 expression to within the developing AVC and IC, where it is essential for differentiation. Misactivation or inhibition of Notch2 specifically induced or inhibited Hey1, respectively, but these manipulations did not affect Hey2, implicating Hey1 as the direct mediator of Notch2. Bmp2 within the developing AVC and IC has been shown to induce Tbx2, and we found that Tbx2 misexpression inhibited the expression of both Hey1 and Hey2. Tbx2, therefore, is envisaged to constitute a feedback loop that sharpens the border with the developing AVC and IC by delimiting Hey gene expression to within prospective chamber regions. Analysis of the loss-of-function phenotype in mouse embryos homozygous for targeted disruption of Hey2 revealed an expanded AVC domain of Bmp2. Similarly, zebrafish gridlock (Hey2 homolog) mutant embryos showed ectopic expression of Bmp4, which normally marks AVC myocardium in this species. Thus, Hey pathway regulation of cardiac Bmp appears to be an evolutionarily conserved mechanism to delimit AVC and IC fate, and provides a potential mechanistic explanation for cardiac malformations caused by mutations in Serrate/Jagged1 and Notch signaling components.

To better understand the formation and adult organization of the avian pallium, we studied the expression patterns of gamma-aminobutyric acid (GABA), calbindin (CB), calretinin (CR), and neuronal nitric oxide synthase (nNOS) in the hippocampal formation and hyperpallium of developing and adult chicks. Each marker showed a specific spatiotemporal expression pattern and was expressed in a region (area)-specific but dynamic manner during development. The combinatorial expression of these markers was very useful for identifying and following the development of subdivisions of the chicken hippocampal formation and hyperpallium. In the hyperpallium, three separate radially arranged subdivisions were present since early development showing distinct expression patterns: the apical hyperpallium (CB-rich); the intercalated hyperpallium (nNOS-rich, CB-poor); the dorsal hyperpallium (nNOS-poor, CB-moderate). Furthermore, a novel division was identified (CB-rich, CR-rich), interposed between hyper- and mesopallium and related to the lamina separating both, termed laminar pallial nucleus. This gave rise at its surface to part of the lateral hyperpallium. Later in development, the interstitial nucleus of the apical hyperpallium became visible as a partition of the apical hyperpallium. In the hippocampal formation, at least five radial divisions were observed, and these were compared with the divisions proposed recently in adult pigeons. Of note, the corticoid dorsolateral area (sometimes referred as caudolateral part of the parahippocampal area) contained CB immunoreactivity patches coinciding with Nissl-stained cell aggregates, partially resembling the patches described in the mammalian entorhinal cortex. Each neurochemical marker was present in specific neuronal subpopulations and axonal networks, providing insights into the functional maturation of the chicken pallium.

We have investigated the role of Sonic hedgehog (Shh) in the development of facial structures by depriving chicken embryos of the most anterior sources of this morphogen, including the prechordal plate and the anterior ventral endoderm of the foregut, before the onset of neural crest cell (NCC) migration to the first branchial arch (BA1). The entire forehead, including the foregut endoderm, was removed at 5- to 10-somite stage (ss), which led to the absence of the lower jaw when the operation was performed before 7-ss. If the embryos were deprived of their forehead at 8- to 10-ss, they were later on endowed with a lower beak. In embryos that were operated on early, the NCCs migrated normally to BA1 but were subjected to massive apoptosis a few hours later. Cell death did not occur when forehead excision was performed at a later stage. In this case, onward expression of Shh in the ventral foregut endoderm extended caudally over the excision limit, and we hypothesized that absence of Shh production by the endoderm in embryos that were operated on early could be responsible for the NCC apoptosis and the failure of BA1 development. We thus provided exogenous Shh to the embryos that were operated on before 7-ss. In this case, the development of the lower jaw was rescued. Therefore, Shh derived from the ventral foregut endoderm ensures the survival of NCCs at a critical stage of BA1 development.

Cartilage canals have been extensively investigated, in particular as to their mode of formation, morphologic distribution, function and fate. We studied the morphological pattern of the cartilage canals of the right upper chondroepiphysis of the tibia of chick embryos, Hamburger-Hamilton stages 35-42, in serial sagittal wax sections and in reconstructions made with AutoCAD software. The spatial arrangement of the canals is presented in a series of drawings made according to computerized images. The canals were penetrated from three distinct surfaces: the anterior and superior surfaces of the tubercle and the posterior surfaces of the medial and lateral condyles. Immediately after entering, nearly all the canals were extended toward the medial or lateral aspects of the chondroepiphysis, from which no canals took their origin. All of the cartilage canals connected with the perichondrium, and their branching did not follow a specific pattern. The condylar canals did not unite with the tubercular ones.

RNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo.

The chick retinotectal system is a suitable model to investigate the mechanisms involved in the establishment of synaptic connections in whose refinement nitric oxide was implicated. The purpose of this work was to describe the developmental pattern of the nitric oxide synthase (NOS)-positive neurons as well as to determine if it is sensitive to changes in visual stimulation. The NADPH-diaphorase histochemical method was used to describe and quantify NOS neurons in normally stimulated and subnormally stimulated chickens. Nine types of NOS neurons were identified; seven of them express NOS until adulthood, while two of them show only a transient expression. The developmental pattern of NOS neurons follows the process of laminar segregation. It can be divided into three phases. The first includes the onset of NOS expression in periventricular neurons and the formation of a deep network of NOS fibers during early development. These neurons do not show any significant change in subnormally stimulated animals. The second phase includes the appearance of two transient NOS populations of bipolar neurons that occupy the intermediate layers during the optic fibers ingrowth. One of them significantly changes in subnormally stimulated chicks. The third phase occurs when the transitory expression of bipolar neurons decreases. It includes NOS expression in six neuronal populations that innervate the superficial retinorecipient layers. Most of these cells suffer plastic changes in subnormally stimulated chicks. The diversity of neuronal types with regard to their morphology, location, and sensitivity to visual stimulation strongly suggests that they serve different functions., (J. Comp. Neurol. 494:1007-1030, 2006. (c) 2005 Wiley-Liss, Inc.)