Motor neurons with limb-innervating character in the cervical spinal cord are sculpted by apoptosis based on the Hox code in chick embryo.

In the developing chick embryo, a certain population of motor neurons (MNs) in the non-limb-innervating cervical spinal cord undergoes apoptosis between embryonic days 4 and 5. However, the characteristics of these apoptotic MNs remain undefined. Here, by examining the spatiotemporal profiles of apoptosis and MN subtype marker expression in normal or apoptosis-inhibited chick embryos, we found that this apoptotic population is distinguishable by Foxp1 expression. When apoptosis was inhibited, the Foxp1+ MNs survived and showed characteristics of lateral motor column (LMC) neurons, which are of a limb-innervating subtype, suggesting that cervical Foxp1+ MNs are the rostral continuation of the LMC. Knockdown and misexpression of Foxp1 did not affect apoptosis progression, but revealed the role of Foxp1 in conferring LMC identity on the cervical MNs. Furthermore, ectopic expression of Hox genes that are normally expressed in the brachial region prevented apoptosis, and directed Foxp1+ MNs to LMC neurons at the cervical level. These results indicate that apoptosis in the cervical spinal cord plays a role in sculpting Foxp1+ MNs committed to LMC neurons, depending on the Hox expression pattern.

The morphological basis of the development of the chick embryo immune system.

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.

Neither altered incubation temperature during fetal development nor preferred rearing temperature improves leg bone characteristics of broilers.

The present study evaluated whether broiler femoral and tibiotarsal characteristics (as assessed at slaughter age) could be improved if birds were reared under their preferred temperature and whether continuous high or low incubation temperature during the fetal period improves bone characteristics of broilers reared under heat stress or thermal preference. Broiler breeder eggs were incubated from day 13 until hatching under cold (36 °C), control (37.5 °C), or hot (39 °C) temperatures. Under these conditions, the eggshell temperatures were 37.4 ± 0.1°C, 37.8 ± 0.15°C, and 38.8 ± 0.3°C, respectively. Then, broiler chicks were reared under control, preferred (determined previously in thermal preference test), or high temperatures. At day 42 of age, the broilers were weighed and euthanized, and femora and tibiotarsi collected to measure weight, length, diaphysis perimeter, breaking strength, maximum flexion, rigidity, ash, phosphorus, and calcium. Rearing under the preferred temperature did not affect broiler body weight or femoral and tibiotarsal characteristics (P > 0.05). In contrast, high rearing temperature, decreased the body weight, mineral contents of both bones, femoral breaking strength, and tibiotarsal rigidity (P < 0.05). Regarding incubation temperature effects, egg exposure to cold and hot temperatures during the fetal period minimized or avoided a few effects of high rearing temperature, such as those on femoral and tibiotarsal morphological characteristics, mineral composition, and mechanical properties at slaughter age (P < 0.05), but not all. In conclusion, rearing under the preferred broiler temperature did not improve the bone characteristics, and the negative effects of high rearing temperature on bone development were minimized but not completely prevented by high or low temperature incubation during the fetal period.

A reviewed chronology of primordial germ cells migration in the chick embryo.

INTRODUCTION: Primordial Germ Cells (PGCs) are present in all sexually reproducing animals. They differentiate into spermatozoa or oocytes and are therefore responsible for the transmission of genetic and epigenetic information across generations. In birds, PGCs are first observed in the center of the blastodisc at stage Eyal-Giladi X. With the formation of the primitive streak, germ cells are translocated anteriorly to the germinal crescent. At stage Hamburger- Hamilton 10-12, they enter the vasculature before migrating through the dorsal mesentery towards the genital ridges. MATERIAL AND METHODS: Embryos from stages Hamburger-Hamilton (HH) 16 to 22 were collected. Blood samples were taken from the dorsal aorta and from the heart in order to perform blood smears and PAS staining. Embryos were dissected and fixed in Serra's medium. Sections were placed on slides for PAS staining. A sample of each embryo was collected for DNA extraction and PCR in order to determine the sex of the embryos. RESULTS: PGCs were observed in blood circulation until stage HH 20 on blood smears and until stage HH 19 on histological sections. The first PGCs arrived in the genital ridges were observed from stage HH 17. A few germ cells were still migrating in the dorsal mesentery at stage HH 22. The aim of this study was to review the chronology of the migration of PGCs in chick embryos.

Morphogenesis of aligned collagen fibers in the annulus fibrosus: Mammals versus avians.

The mammalian intervertebral disc (IVD) consists of a gel-like, disordered nucleus pulposus (NP) surrounded by a highly ordered collagen structure, the annulus fibrosus (AF). While this concentric array of lamellae has been amply studied, its physical origin is poorly understood. The notochord is a rod-like organ located in the mid-line of the growing embryo and plays an essential role in IVD development. The aim of this study was to elucidate the effect of notochord development on the collagen fiber arrangement evolution in the AF. To that end, we studied IVD development in mouse embryos and compared these observations to those from chicken embryos, which do not form the typical laminar structure around the NP. In mouse, cross-aligned collagen arrangement of the AF forms from the sclerotome upon bulging of the notochord to become NP. By contrast, the notochord in the chicken embryo swells substantially without the physical restrictions of the future vertebrae and thus do not bulge. From these observations, we conclude that physical and geometrical constrictions are essential for the formation of the highly structured AF.

Splitting of circulating red blood cells as an in vivo mechanism of erythrocyte maturation in developing zebrafish, chick and mouse embryos.

Nucleated circulating red blood cells (RBCs) of developing zebrafish, chick and mouse embryos can actively proliferate. While marrow- or organ-mediated erythropoiesis has been widely studied, transforming in vivo processes of circulating RBCs are under little scrutiny. We employed confocal, stereo- and electron microscopy to document the maturation of intravascular RBCs. In zebrafish embryos (32-72 h post-fertilization), RBC splitting in the caudal vein plexus follows a four-step program: (i) nuclear division with continued cytoplasmic connection between somata; (ii) dumbbell-shaped RBCs tangle at transluminal vascular pillars; (iii) elongation; and (iv) disruption of soma-to-soma connection. Dividing RBCs of chick embryos, however, retain the nucleus in one of their somata. Here, RBC splitting acts to pinch off portions of cytoplasm, organelles and ribosomes. Dumbbell-shaped primitive RBCs re-appeared as circulation constituents in mouse embryos. The splitting of circulating RBCs thus represents a biologically relevant mechanism of RBC division and maturation during early vertebrate ontogeny.

Identification and characterisation of microRNAs and Piwi-interacting RNAs in cockerels' spermatozoa by Solexa sequencing.

1. There has been substantial research focused on the roles of microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs) derived from mammalian spermatozoa; however, comparatively little is known about the role of spermatozoa-derived miRNAs and piRNAs within breeding cockerels' spermatozoa. 2. A small RNA library of cockerels' spermatozoa was constructed using Illumina high-throughput sequencing technology. Unique sequences with lengths of 18-26 nucleotides were mapped to miRBase 21.0 and unique sequences with lengths of 25-37 nucleotides were mapped to a piRNA database. A total of 1311 miRNAs and 2448 potential piRNAs were identified. Based on stem-loop qRT-PCR, 8 miRNAs were validated. 3. Potential target genes of the abundant miRNAs were predicted, and further Kyoto Encyclopedia of Genes and Genomes database (KEGG) and Gene Ontology (GO) analyses were performed, which revealed that some candidate miRNAs were involved in the spermatogenesis process, spermatozoa epigenetic programming and further embryonic development. 5. GO and KEGG analyses based on mapping genes of expressed piRNAs were performed, which revealed that spermatozoal piRNAs could play important regulatory roles in embryonic development of offspring. 6. The search for endogenous spermatozoa miRNAs and piRNAs will contribute to a preliminary database for functional and molecular mechanistic studies in embryonic development and spermatozoa epigenetic programming.

The role of embryo movement in the development of the furcula.

The pectoral girdle is a complex structure which varies in its morphology between species. A major component in birds is the furcula, which can be considered equivalent to a fusion of the paired clavicles found in many mammals, and the single interclavicle found in many reptiles. These elements are a remnant of the dermal skeleton and the only intramembranous bones in the trunk. Postnatally, the furcula plays important mechanical roles by stabilising the shoulder joint and acting as a mechanical spring during flight. In line with its mechanical role, previous studies indicate that, unlike many other intramembranous bones, furcula growth during development can be influenced by mechanical stimuli. This study investigated the response of individual aspects of furcula growth to both embryo immobilisation and hypermotility in the embryonic chicken. The impact of altered incubation temperature, which influences embryo motility, on crocodilian interclavicle development was also explored. We employed whole-mount bone and cartilage staining and 3D imaging by microCT to quantify the impact of rigid paralysis, flaccid paralysis and hypermobility on furcula growth in the chicken, and 3D microCT imaging to quantify the impact of reduced temperature (32-28 °C) and motility on interclavicle growth in the crocodile. This revealed that the growth rates of the clavicular and interclavicular components of the furcula differ during normal development. Total furcula area was reduced by total unloading produced by flaccid paralysis, but not by rigid paralysis which maintains static loading of embryonic bones. This suggests that dynamic loading, which is required for postnatal bone adaptation, is not a requirement for prenatal furcula growth. Embryo hypermotility also had no impact on furcula area or arm length. Furcula 3D shape did, however, differ between groups; this was marked in the interclavicular component of the furcula, the hypocleideum. Hypocleideum length was reduced by both methods of immobilisation, and interclavicle area was reduced in crocodile embryos incubated at 28 °C, which are less motile than embryos incubated at 32 °C. These data suggest that the clavicular and interclavicle components of the avian furcula respond differently to alterations in embryo movement, with the interclavicle requiring both the static and dynamic components of movement-related loading for normal growth, while static loading preserved most aspects of clavicle growth. Our data suggest that embryo movement, and the mechanical loading this produces, is important in shaping these structures during development to suit their postnatal mechanical roles.

Lectins selectively label cartilage condensations and the otic neuroepithelium within the embryonic chicken head.

Cartilage morphogenesis during endochondral ossification follows a progression of conserved developmental events. Cells are specified towards a prechondrogenic fate and subsequently undergo condensation followed by overt differentiation. Currently available molecular markers of prechondrogenic and condensing mesenchyme rely on common regulators of the chondrogenic program that are not specific to the tissue type or location. Therefore tissue-specific condensations cannot be distinguished based on known molecular markers. Here, using the chick embryo model, we utilized lectin labeling on serial sections, demonstrating that differential labeling by peanut agglutinin (PNA) and Sambucus nigra agglutinin (SNA) successfully separates adjacently located condensations in the proximal second pharyngeal arch. PNA selectively labels chick middle ear columella and basal plate condensation, whereas SNA specifically marks extracolumella and the ventro-lateral part of the otic capsule. We further extended our study to examine lectin-binding properties of the different parts of the inner ear epithelium, neural tube and notochord. Our results show that SNA labels the auditory and vestibular hair cells of the inner ear, whereas PNA specifically recognizes the statoacoustic ganglion. PNA is also highly specific for the floor plate of the neural tube. Additionally, wheat germ agglutinin (WGA) labels the basement membrane of the notochord and is a marker of the apical-basal polarity of the cochlear duct. Overall, this study indicates that selective lectin labeling is a promising approach to differentiate between contiguously located mesenchymal condensations and subregions of epithelia globally during development.

Transcriptomic analysis of avian digits reveals conserved and derived digit identities in birds.

Morphological characters are the result of developmental gene expression. The identity of a character is ultimately grounded in the gene regulatory network directing development and thus whole-genome gene expression data can provide evidence about character identity. This approach has been successfully used to assess cell-type identity. Here we use transcriptomic data to address a long-standing uncertainty in evolutionary biology, the identity of avian wing digits. Embryological evidence clearly identifies the three wing digits as developing from digit positions 2, 3 and 4 (ref. 6), whereas palaeontological data suggest that they are digits I, II and III. We compare the transcriptomes of the wing and foot digits and find a strong signal that unites the first wing digit with the first foot digit, even though the first wing digit develops from embryological position 2. Interestingly, our transcriptomic data of the posterior digits show a higher degree of differentiation among forelimb digits compared with hindlimb digits. These data show that in the stem lineage of birds the first digit underwent a translocation from digit position 1 to position 2, and further indicate that the posterior wing digits have unique identities contrary to any model of avian digit identity proposed so far.

Dynamics of BMP and Hes1/Hairy1 signaling in the dorsal neural tube underlies the transition from neural crest to definitive roof plate.

BACKGROUND: The dorsal midline region of the neural tube that results from closure of the neural folds is generally termed the roof plate (RP). However, this domain is highly dynamic and complex, and is first transiently inhabited by prospective neural crest (NC) cells that sequentially emigrate from the neuroepithelium. It only later becomes the definitive RP, the dorsal midline cells of the spinal cord. We previously showed that at the trunk level of the axis, prospective RP progenitors originate ventral to the premigratory NC and progressively reach the dorsal midline following NC emigration. However, the molecular mechanisms underlying the end of NC production and formation of the definitive RP remain virtually unknown. RESULTS: Based on distinctive cellular and molecular traits, we have defined an initial NC and a subsequent RP stage, allowing us to investigate the mechanisms responsible for the transition between the two phases. We demonstrate that in spite of the constant production of BMP4 in the dorsal tube at both stages, RP progenitors only transiently respond to the ligand and lose competence shortly before they arrive at their final location. In addition, exposure of dorsal tube cells at the NC stage to high levels of BMP signaling induces premature RP traits, such as Hes1/Hairy1, while concomitantly inhibiting NC production. Reciprocally, early inhibition of BMP signaling prevents Hairy1 mRNA expression at the RP stage altogether, suggesting that BMP is both necessary and sufficient for the development of this RP-specific trait. Furthermore, when Hes1/Hairy1 is misexpressed at the NC stage, it inhibits BMP signaling and downregulates BMPR1A/Alk3 mRNA expression, transcription of BMP targets such as Foxd3, cell-cycle progression, and NC emigration. Reciprocally, Foxd3 inhibits Hairy1, suggesting that repressive cross-interactions at the level of, and downstream from, BMP ensure the temporal separation between both lineages. CONCLUSIONS: Together, our data suggest that BMP signaling is important both for NC and RP formation. Given that these two structures develop sequentially, we speculate that the longer exposure of RP progenitors to BMP compared with that of premigratory NC cells may be translated into a higher signaling level in the former. This induces changes in responsiveness to BMP, most likely by downregulating the expression of Alk3 receptors and, consequently, of BMP-dependent downstream transcription factors, which exhibit spatial complementary expression patterns and mutually repress each other to generate alternative fates. This molecular dynamic is likely to account for the transition between the NC and definitive RP stages and thus be responsible for the segregation between central and peripheral lineages during neural development.

Apical constriction initiates new bud formation during monopodial branching of the embryonic chicken lung.

Branching morphogenesis sculpts the airway epithelium of the lung into a tree-like structure to conduct air and promote gas exchange after birth. In the avian lung, a series of buds emerges from the dorsal surface of the primary bronchus via monopodial branching to form the conducting airways; anatomically, these buds are similar to those formed by domain branching in the mammalian lung. Here, we show that monopodial branching is initiated by apical constriction of the airway epithelium, and not by differential cell proliferation, using computational modeling and quantitative imaging of embryonic chicken lung explants. Both filamentous actin and phosphorylated myosin light chain were enriched at the apical surface of the airway epithelium during monopodial branching. Consistently, inhibiting actomyosin contractility prevented apical constriction and blocked branch initiation. Although cell proliferation was enhanced along the dorsal and ventral aspects of the primary bronchus, especially before branch formation, inhibiting proliferation had no effect on the initiation of branches. To test whether the physical forces from apical constriction alone are sufficient to drive the formation of new buds, we constructed a nonlinear, three-dimensional finite element model of the airway epithelium and used it to simulate apical constriction and proliferation in the primary bronchus. Our results suggest that, consistent with the experimental results, apical constriction is sufficient to drive the early stages of monopodial branching whereas cell proliferation is dispensable. We propose that initial folding of the airway epithelium is driven primarily by apical constriction during monopodial branching of the avian lung.

Toxicogenomic Assessment of 6-OH-BDE47-Induced Developmental Toxicity in Chicken Embryos.

Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) are analogs of PBDEs with hundreds of possible structures and are frequently detected in the environment. However, the in vivo evidence on the toxicity of OH-PBDEs is still very limited. Here, the developmental toxicity of 6-OH-BDE47, a predominant congener of OH-PBDEs detected in the environment, in chicken embryos was assessed using a toxicogenomic approach. Fertilized chicken eggs were dosed via in ovo administration of 0.006 to 0.474 nmol 6-OH-BDE47/g egg followed by 18 days of incubation. Significant embryo lethality (LD50 = 1.940 nmol/g egg) and increased hepatic somatic index (HSI) were caused by 6-OH-BDE47 exposure. The functional enrichment of differentially expressed genes (DEGs) was associated with oxidative phosphorylation, generation of precursor metabolites and energy, and electron transport chains, which suggest that 6-OH-BDE47 exposure may disrupt the embryo development by altering the function of energy production in mitochondria. Moreover, aryl hydrocarbon receptor (AhR)-mediated responses including up-regulation of CYP1A4 were observed in the livers of embryos exposed to 6-OH-BDE47. Overall, this study confirmed the embryo lethality by 6-OH-BDE47 and further improved the mechanistic understanding of OH-PBDEs-caused toxicity. Ecological risk assessment via application of both no-observed-effect level (NOEL) and the sensitive NOTEL (transcriptional NOEL) suggested that OH-PBDEs might cause ecological risk to wild birds.

A random cell motility gradient downstream of FGF controls elongation of an amniote embryo.

Vertebrate embryos are characterized by an elongated antero-posterior (AP) body axis, which forms by progressive cell deposition from a posterior growth zone in the embryo. Here, we used tissue ablation in the chicken embryo to demonstrate that the caudal presomitic mesoderm (PSM) has a key role in axis elongation. Using time-lapse microscopy, we analysed the movements of fluorescently labelled cells in the PSM during embryo elongation, which revealed a clear posterior-to-anterior gradient of cell motility and directionality in the PSM. We tracked the movement of the PSM extracellular matrix in parallel with the labelled cells and subtracted the extracellular matrix movement from the global motion of cells. After subtraction, cell motility remained graded but lacked directionality, indicating that the posterior cell movements associated with axis elongation in the PSM are not intrinsic but reflect tissue deformation. The gradient of cell motion along the PSM parallels the fibroblast growth factor (FGF)/mitogen-activated protein kinase (MAPK) gradient, which has been implicated in the control of cell motility in this tissue. Both FGF signalling gain- and loss-of-function experiments lead to disruption of the motility gradient and a slowing down of axis elongation. Furthermore, embryos treated with cell movement inhibitors (blebbistatin or RhoK inhibitor), but not cell cycle inhibitors, show a slower axis elongation rate. We propose that the gradient of random cell motility downstream of FGF signalling in the PSM controls posterior elongation in the amniote embryo. Our data indicate that tissue elongation is an emergent property that arises from the collective regulation of graded, random cell motion rather than by the regulation of directionality of individual cellular movements.

Temperature during the last week of incubation. I. Effects on hatching pattern and broiler chicken embryonic organ development.

We investigated the effects of an eggshell temperature (EST) of 35.6, 36.7, 37.8, and 38.9°C applied from d of incubation (E) 15, E17, and E19 on hatching pattern and embryonic organ development. A total of 2,850 first-grade eggs of a 43-week-old Ross 308 broiler breeder flock were incubated at an EST of 37.8°C until E15. From E15, E17, or E19 onward, eggs were incubated at an EST of 35.6, 36.7, 37.8, or 38.9°C. Moment of internal pipping (IP), external pipping (EP), and hatch was determined, and organ development was measured at E15, E17, E19, IP, EP, and hatch. A lower EST extended incubation duration compared to a higher EST. The lower incubation duration was mainly caused by the extended time until IP, whereas time between IP and hatch hardly varied between treatments. Relative heart weight was affected by EST already from 2 d after the start of EST treatment on E15, and effects became more pronounced at longer exposure time to various EST treatments. At hatch, the largest difference in relative heart weight was found between an EST of 35.6 and 38.9°C started at E15 (Δ=64.4%). From E17 onward, EST affected yolk-free body mass (YFBM) and relative stomach weight, where a lower EST resulted in a lower YFBM and relative stomach weight before IP and a higher YFBM and relative stomach weight after IP. From E19 onward, a lower EST resulted in a higher relative liver and spleen weight regardless of start time of treatment. Yolk weight and relative intestine weight were not affected by EST before and at E19, but a higher EST resulted in a higher yolk weight and lower relative intestine weight from IP onward. Based on the higher YFBM and higher relative organ growth found at hatch, we concluded that an EST lower than 37.8°C from E15 onward appears to be beneficial for optimal embryo development.

Effect of cyclical cold stress during embryonic development on aspects of physiological responses and HSP70 gene expression of chicks.

The objective of the present study was to evaluate the effects of cyclical lower incubation temperature at different embryonic ages on the hatchability, body and organs weights, thyroid hormones, and liver HSP70 gene expression of newly hatched chicks. In a completely randomized design, fertile eggs of a broiler breeder (34 weeks of age) were assigned to three treatment groups with six replicates and 145 eggs per each. The treatment groups were as: control group (C) that eggs were incubated at 37.6°C during the whole incubation period; incubation temperature was decreased to 36°C for 3h per day at embryonic age from 12 to 14 (T1); and incubation temperature was decreased to 36°C for 3h per day at embryonic age from 15 to 17 (T2). No significant difference was found among treatments for hatchability (P>0.05). There were no differences (P>0.05) among treatments for body weight and liver weight, while heart weight of chicks in T1 and T2 groups were significantly higher than the control group (P<0.05). There were no differences (P>0.05) among treatments for the levels of thyroid hormones, however, the levels of both hormones tended to increase in chicks exposed to cold stress (T1 and T2). Chicks in T2 group had higher liver HSP70 gene expression compared with those in T1 and the control group (P<0.05). Cold stress in both incubation periods had no significant effect on the plasma levels of aspartate aminotransferase and alanine aminotransferase. Treatments had no effect on the plasma levels of glucose, cholesterol and triglyceride. The results of this study suggest that cyclical lower incubation temperatures (36°C) at the embryonic age from day 15-17 could induce the liver HSP70 gene expression, without negative effects on the hatchability and body weight of hatched chicks.

The proepicardium keeps a potential for glomerular marker expression which supports its evolutionary origin from the pronephros.

The proepicardium is the embryonic primordium of the epicardium. This transient structure is essential for cardiac development giving rise to the epicardium and supplying the heart with vascular and cardiac connective tissue progenitors. However, their nature and evolutionary origin are poorly-known. We have suggested elsewhere (Pombal et al. Evol. Dev. 10: 210-216, 2008; Cano et al., J. Dev. Biol. 1: 3-19, 2013) that the proepicardium is an evolutionary derivative of the primordium of an ancient external pronephric glomerulus, devoid of its original excretory function. In this study, we describe for the first time expression of two podocyte markers in the chick proepicardium (glepp1 and synaptopodin) and we have shown how these podocyte markers as well as the intermediate mesoderm marker Pax2 are strongly upregulated when the proepicardium is cultured with nephrogenic inducers. Retinoic acid treatment also induced in the proepicardium expression of Hoxb4, a gene which confers to intermediate mesoderm competence to respond to nephrogenic signals. Thus, a latent nephrogenic potential persists in the proepicardium and also that its original glomerular fate can be partially rescued. The transcription factor Wt1, essential for kidney and epicardial development, plays opposite roles in both tissues, inducing epithelial-mesenchymal transition in the proepicardium and promoting epithelialization in the kidneys (Essafi et al., Dev. Cell 21: 559-574, 2011). Consistently with this antithetical function of Wt1, we have observed an upregulation of podocalyxin in the epicardium of mouse embryos with conditional deletion of the Wt1 gene, while this protein is transcriptionally activated by Wt1 in podocytes.

Polyspermy in birds: sperm numbers and embryo survival.

Polyspermy is a major puzzle in reproductive biology. In some taxa, multiple sperm enter the ovum as part of the normal fertilization process, whereas in others, penetration of the ovum by more than one sperm is lethal. In birds, several sperm typically enter the germinal disc, yet only one fuses with the female pronucleus. It is unclear whether supernumerary sperm play an essential role in the avian fertilization process and, if they do, how females regulate the progression of sperm through the oviduct to ensure an appropriate number reach the ovum. Here, we show that when very few sperm penetrate the avian ovum, embryos are unlikely to survive beyond the earliest stages of development. We also show that when the number of inseminated sperm is limited, a greater proportion than expected reach and penetrate the ovum, indicating that females compensate for low sperm numbers in the oviduct. Our results suggest a functional role for supernumerary sperm in the processes of fertilization and early embryogenesis, providing an exciting expansion of our understanding of sperm function in birds.

Exposure of chick embryos to cadmium changes the extra-embryonic vascular branching pattern and alters expression of VEGF-A and VEGF-R2.

Cadmium (Cd) has several industrial applications, and is found in tobacco products, a notable source of human exposure. Vascular endothelial cells are key targets of Cd toxicity. Here, we aim to quantify the alteration to vascular branching pattern following Cd exposure in the chick extra-embryonic membrane (EEM) using fractal analysis, and explore molecular cues to angiogenesis such as VEGF-A and VEGF-R2 expression following Cd treatment. Chicken embryos were incubated for 60 h to Hamburger-Hamilton developmental stage 16-17, then explanted and treated with 50 µL of 50 µmol cadmium acetate (CdAc) or an equivalent volume of equimolar sodium acetate (NaAc). Images of embryos and their area vasculosa (AV) were captured and analyzed at 4 different time points (4, 8, 24 and 48 h) following treatment. Vascular branching in the AV was quantified using its fractal dimension (Df), estimated using a box counting method. Gallinaceous VEGF ELISA was used to measure the VEGF-A concentration in the EEM following treatment, with determination of the relative expression of VEGF-A and VEGF-R2 using quantitative real-time RT-PCR. Vascular branching increased monotonically in the control group at all time points. The anti-angiogenic effect of Cd exposure on the AV was reflected by a significant reduction in Df when compared with controls. Df was more markedly reduced in cultures with abnormal embryos. The expression of VEGF-A protein, and VEGF-A and VEGF-R2 mRNA were reduced in Cd-exposed EEMs. Both molecules contribute to growth, vessel sprouting and branching processes, which supports our findings using fractal analysis.

Subpopulation of proepicardial cells is derived from the somatic mesoderm in the chick embryo.

RATIONALE: The proepicardium (PE) is a transient structure forming at the venous pole of the heart and gives rise to the epicardium, fibroblasts, and smooth muscle cells. The embryological origin of the PE is presently unclear. Asymmetrical formation of the PE on the right inflow tract is a conserved feature of many vertebrate embryos, and in the chicken is under the control of fibroblast growth factor 8 and snail homolog 1. OBJECTIVE: To gain further insight into the process of asymmetrical PE formation, we studied the role of TWIST1 during PE formation in the chick embryo. METHODS AND RESULTS: TWIST1 is asymmetrically expressed on the right side in the somatic mesoderm under the control of snail homolog 1. Fate mapping experiments revealed a contribution of the somatic mesoderm to the PE. After colonization of the heart, this cell lineage gives rise to the epicardium, smooth muscle cells, and potentially fibroblast. Suppression of TWIST1 function in the right coelomic cavity caused a severe disruption of the villous protrusions of the PE and Wilms tumor 1 and transcription factor 21 expression. Rescue with the corresponding mouse cDNA normalized gene expression and PE morphology. Forced expression of TWIST1 on the left side induced ectopic expression domains of Wilms tumor 1 and transcription factor 21. CONCLUSIONS: A significant proportion of the PE has its origin outside of the currently proposed domain in the splanchnic layer of the lateral plate mesoderm. The phenotype in embryos subjected to TWIST1 loss- or gain-of-function suggests an important contribution of somatic mesoderm to the mesothelial cell layer of the PE.