The chick blastoderm during diapause, a landmark for optimization of preincubation storage conditions.

At the time of oviposition, the chicken embryo is in its blastodermal stage. The blastoderm displays the unique ability to undergo developmental arrest at low temperatures in a process called "embryonic diapause." In the wild, diapause occurs in freshly laid eggs until the last egg of the clutch has been laid, providing an evolutionary advantage to hens that can synchronously hatch their eggs. The poultry industry utilizes the diapause phenomenon to store eggs before incubation, thereby mitigating their logistic problems. The embryos can only be stored at particular embryonic stages-termed "diapause developmental window" (DW)-if they are to continue to develop normally thereafter. Both cellular and molecular mechanisms define the limits of this DW which broadly comply with onset of blastulation to early gastrulation. Storage conditions affect the cellular and molecular characteristics of the embryo during this window and their ability to successfully resume development (SRD). At storage temperatures of ~12°C to 18°C, embryos can undergo diapause for a short period (up to 7 days (d)) without affecting SRD. However, following longer period of diapause (up to 28 d), embryo stored at ~12°C, but not at ~18°C, can resume development normally. Moreover, eggs can be heated before or during the storage period which will lead to their commencing in development; however, unlike the non-heated embryos, the storage temperature for heated embryos, which are more advance in developing, is not clear. Thus, based on SRD, this review brings evidence supporting the notion that a lower storage temperature is beneficial for early-stage blastoderms whereas a higher storage temperature is favorable for later-stage/gastrulating embryos. Our understanding of the molecular mechanisms underlying the relationship between storage temperature and development stage within the DW is rather limited. However, it is expected to become relevant in light of the effect of selective breeding of modern avian birds on the advancement of embryonic development stage. Thus, this review discusses parameters that are regulated during the DW and affect SRD, and presents the need to adopt new storage techniques. The pre-managerial decision of required duration of storage with manipulation of storage temperature in the currently used storage techniques may improve SRD characteristics.

Effects of storage conditions on hatchability, embryonic survival and cytoarchitectural properties in broiler from young and old flocks.

Storing eggs at low temperature prior to incubation is common practice in the broiler hatchery industry; however, prolonged storage (beyond 7 d) is known to increase early embryonic mortality and reduce chick quality and performance. To better understand the basis of this mortality, we previously published milestone criteria to evaluate morphological and cellular properties of the freshly laid embryo. Using these criteria, in the present study we checked the effects of storage at 18°C and 12°C for up to 28 d on hatchability and chick quality. Furthermore, using a 3D high-resolution episcopic microscopy (HREM) imaging system combined with standard and confocal microscopy and cell viability markers, we analyzed the effects of the different storage conditions on embryonic developmental stage, cytoarchitectural properties, mitotic index and cell survival. A total of 1,483 eggs from a young flock were divided in 2 groups, 18°C and 12°C, and stored for 7, 14, 21, and 28 d. Following storage, randomly selected 1,222 eggs were incubated, and the hatched chicks were evaluated for chick quality parameters. Nonhatched eggs were also analyzed to determine the stage of embryonic mortality. The remaining 261 eggs were isolated and analyzed for developmental stage, cytoarchitecture, mitotic index, and cell death following storage. Hatchability rates beyond 7 d of storage at 12°C were significantly improved compared to 18°C, and chick quality remained high. Similar results were obtained for an old flock's eggs (n = 1,350). Analyzing the embryos, at each time point, we found that at 12°C, the developmental progression during storage slows significantly, mitotic index-which at this temperature may indicate mitotic arrest-increases and the rate of early apoptosis is half than at 18°C. Moreover, the HREM system and histological sections showed that embryos stored at 18°C for prolonged times undergo dramatic cytoarchitectural changes that may be maladaptive to resuming normal development after diapause. We thus demonstrate the usefulness of the milestone criteria for predicting and studying the storage conditions that will allow for better performance in hatchery practice.

Cellular and morphological characterization of blastoderms from freshly laid broiler eggs.

The pioneering study of Eyal-Giladi and Kochav (EG&K; Eyal-Giladi and Kochav, 1976) on the early developmental stages-from fertilization, through oviposition, to the gastrulation process-set the standard for characterizing chicken embryos, and has been used in numerous studies over the years. During uterine development, the chicken embryo undergoes dramatic changes, extremely rapid cell cycles, massive cell death, and axial determination processes. However, once the egg is laid, the temperature drops and the embryo enters into a diapause-like state. This phenomenon is utilized to store fertile eggs prior to incubation. The ability to resume development to hatching, following storage, relies on several factors, including the number of living cells and the embryonic developmental stage. These factors are highly influenced by the storage conditions-mainly duration and temperature. Thus, to study the effects of storage conditions on embryonic viability, a comprehensive characterization of the starting point-shortly after oviposition-is needed. In this study, we characterized freshly laid broiler eggs from Ross 308 flocks for embryonic developmental stage, total cell count, and cell viability. Using the novel high-resolution episcopic microscopy (HREM) system, we show, for the first time, high-resolution 3D morphological models of blastoderms which allow for highly accurate embryonic staging. Staging was also done under a dissecting microscope thus allowing for a direct side-by-side comparison of the two methods. Analysis of freshly laid blastomeres showed that the total nucleus count increases with developmental stage from ∼60,000 at stage X EG&K to ∼130,000 at stage XIII EG&K, whereas the proportion of mitotic index and dying cells at oviposition are ∼2% and ∼5%, respectively. Moreover, staging embryos from young and old flocks revealed that the blastoderms of the old flocks are more developed. Specifically, the predominant embryonic stages were XI and XII EG&K in young and old flocks, respectively. Collectively, we characterized parameters that can serve to analyze the maladaptive effects of prolonged storage under various conditions on embryo survival.

Expression of matrix metalloproteinases during impairment and recovery of the avian growth plate.

Tibial dyschondroplasia (TD) is a prevalent skeletal abnormality associated with rapid growth rate in many avian species. It is characterized by the presence of a nonvascularized, nonmineralized lesion that extends from the epiphyseal growth plate into the metaphysis of the proximal tibiotarsal bones. In this study, we examined the expression of 4 members of the matrix metalloproteinase (MMP) family (MMP-2, -3, -9, and -13) in thiram-induced TD lesions and in the process of recovery from TD, by in situ hybridization analysis and quantitative real-time PCR. A model for the induction and recovery of TD was established, consisting of 3 groups of broilers: (1) thiram group, chicks fed a thiram-enriched diet to induce TD; (2) recovery group, chicks fed a thiram-enriched diet during the first week of the experiment and a normal diet from the second week on; and (3) control group, chicks fed a normal diet throughout the experimental period. In agreement with our previous data, the 4 MMP were diminished in the TD lesion (P < 0.05); however, in the current study we show that the growth plate was able to repair itself and that the MMP reappeared during the process of recovery from TD. Our results strengthen the link between MMP expression and growth-plate impairment, and we suggest that gelatinase activity (MMP-2 and 9) facilitates this process.

Inhibition of noggin expression in the dorsal neural tube by somitogenesis: a mechanism for coordinating the timing of neural crest emigration.

We have previously shown that axial-dependent delamination of specified neural crest cells is triggered by BMP4 and negatively regulated by noggin. Increasing activity of BMP4 towards the rostral part of the axis is achieved by graded expression of noggin in the dorsal neural tube, the latter being high opposite unsegmented mesoderm, and progressively downregulated facing epithelial and dissociating somites, coinciding in time and axial level with initial delamination of neural crest cells (Sela-Donenfeld, D. and Kalcheim, C. (1999) Development 126, 4749-4762). Here we report that this gradient-like expression of noggin in the neuroepithelium is controlled by the paraxial mesoderm. Deletion of epithelial somites prevented normal downregulation of noggin in the neural tube. Furthermore, partial ablation of either the dorsal half or only the dorsomedial portion of epithelial somites was sufficient to maintain high noggin expression. In contrast, deletion of the segmental plate had no effect. These data suggest that the dorsomedial region of developing somites produces an inhibitor of noggin transcription in the dorsal neural tube. Consistent with this notion, grafting dissociating somites in the place of the unsegmented mesoderm precociously downregulated the expression of noggin and triggered premature emigration of neural crest progenitors from the caudal neural tube. Thus, opposite the unsegmented mesoderm, where noggin expression is high in the neural tube, BMP4 is inactive and neural crest cells fail to delaminate. Upon somitogenesis and further dissociation, the dorsomedial portion of the somite inhibits noggin transcription. Progressive loss of noggin activity releases BMP4 from inhibition, resulting in crest cell emigration. We propose that this inhibitory crosstalk between paraxial mesoderm and neural primordium controls the timing of neural crest delamination to match the development of a suitable mesodermal substrate for subsequent crest migration.

Avian hemangioma retrovirus induces cell proliferation via the envelope (env) gene.

Several years ago, a field strain retrovirus, avian hemangioma virus (AHV), was isolated from hemangioma tumors in layer hens. Sequence analysis indicated that the AHV genome contains the three prototypic retroviral genes, gag, pol, and env, and is devoid of an oncogene. In cultured endothelial cells, however, AHV induced a significant cytopathic effect through a typical apoptotic cascade. We now demonstrate that AHV also induces cell proliferation and anchorage-independent growth of BSC-1 epithelial cells and NIH-3T3 fibroblasts. This was shown by measurements of (1) cell viability, (2) DNA synthesis, (3) flow cytometry analysis of the cell DNA content, and (4) clonogenic efficiency of the infected cells. Anchorage-independent cell growth was demonstrated by colony formation in soft agar. Moreover, the AHV env gene was cloned into a MuLV-based retroviral vector, and infection of NIH-3T3 cells with this vector induced cell proliferation as well as clonogenic growth. These results suggest that AHV, which is devoid of an oncogene, is a pleiotropic activator capable of inducing either apoptosis or cellular proliferation, depending on the infected cell type.

Regulation of the onset of neural crest migration by coordinated activity of BMP4 and Noggin in the dorsal neural tube.

For neural crest cells to engage in migration, it is necessary that epithelial premigratory crest cells convert into mesenchyme. The mechanisms that trigger cell delamination from the dorsal neural tube remain poorly understood. We find that, in 15- to 40-somite-stage avian embryos, BMP4 mRNA is homogeneously distributed along the longitudinal extent of the dorsal neural tube, whereas its specific inhibitor noggin exists in a gradient of expression that decreases caudorostrally. This rostralward reduction in signal intensity coincides with the onset of emigration of neural crest cells. Hence, we hypothesized that an interplay between Noggin and BMP4 in the dorsal tube generates graded concentrations of the latter that in turn triggers the delamination of neural crest progenitors. Consistent with this suggestion, disruption of the gradient by grafting Noggin-producing cells dorsal to the neural tube at levels opposite the segmental plate or newly formed somites, inhibited emigration of HNK-1-positive crest cells, which instead accumulated within the dorsal tube. Similar results were obtained with explanted neural tubes from the same somitic levels exposed to Noggin. Exposure to Follistatin, however, had no effect. The Noggin-dependent inhibition was overcome by concomitant treatment with BMP4, which when added alone, also accelerated cell emigration compared to untreated controls. Furthermore, the observed inhibition of neural crest emigration in vivo was preceded by a partial or total reduction in the expression of cadherin-6B and rhoB but not in the expression of slug mRNA or protein. Altogether, these results suggest that a coordinated activity of Noggin and BMP4 in the dorsal neural tube triggers delamination of specified, slug-expressing neural crest cells. Thus, BMPs play multiple and discernible roles at sequential stages of neural crest ontogeny, from specification through delamination and later differentiation of specific neural crest derivatives.

Injury models of the vascular endothelium: apoptosis and loss of thromboresistance induced by a viral protein.

Endothelial injury caused by viruses usually involves viral replication or transformation. We report a novel mechanism of endothelial damage by a toxic viral protein. We have isolated a new retrovirus from hemangiosarcomas which appeared among layer hens. The isolated avian hemangiosarcoma virus (AHV) is capable of inducing hemangiomas in hens in-vivo and causes a cytopathic effect (CPE) and loss of thromboresistance in cultured bovine aortic endothelial cells (BAEC). These effects do not require viral replication and can be induced by purified AHV envelop glycoprotein (gp85). AHV causes CPE in BAEC through a typical programmed cell death (apoptosis). Quiescent G0/G1-BAEC are much more sensitive to AHV induced apoptosis than actively dividing cells. These experiments demonstrate the capacity of viral proteins to affect the integrity and functionality of vascular endothelial cells.

Programmed endothelial cell death induced by an avian hemangioma retrovirus is density dependent.

Hemangiomas are localized tumors of vascular cells which appear frequently in humans and animals, and their mode of induction is unknown. Recently, a new field strain of avian retrovirus, avian hemangioma virus (AHV), was isolated from spontaneous hemangiomas in layer hens. Sequence analysis of the AHV genome revealed the presence of three prototypic retroviral genes, gag, pol, and env, but no oncogenes. AHV was capable of inducing hemangiomas in hens in vivo, but it induced a strong cytopathic effect in cultured endothelial cells. The AHV envelope glycoprotein, gp85, was found to be responsible for the cell-killing effect. Four independent lines of experimental evidence indicated that AHV induces a cytopathic effect through a typical programmed cell death, apoptosis: (i) morphological changes in cells visualized by light microscopy, (ii) nuclear condensation and fragmentation indicated by 4',6-diamidino-2- phenylindole staining, (iii) intranucleosomal degradation of DNA demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining, and (iv) flow cytometry analysis of the DNA content of the infected cells. Quiescent endothelial G0/G1 cells were much more sensitive to AHV-induced apoptosis than actively dividing cells, suggesting that the AHV ability to induce apoptosis is dependent on the proliferative state of the infected cells.