Neural Groove Research Articles

Development of handed body asymmetry in mammals.

We have proposed a three step model for the specification of left-right in mammalian embryos. The fundamental assumption is that handedness is imparted by an asymmetrical molecule. Conversion of molecular asymmetry to the cellular level gives a property to one side of the embryo to bias an otherwise random generation of an asymmetrical gradient which can be interpreted by developing organs. Rat embryos, treated at discrete stages, show a window of sensitivity for disruption of handedness, which may reflect the time of conversion/biasing. Heat shock and several chemicals cause left-right inversion in up to 50% of embryos exposed during neural groove formation. Earlier stages are less sensitive; no treatment begun after foregut pocket formation influences asymmetry. Evidence for cellular interactions in left-right specification comes from the apparent rescue of iv/iv mutant embryos in chimeras. We are looking for molecular left-right disparity before morphological asymmetry but detect no differences in two-dimensional protein profiles. Using an indirect measure, we find a right-left gradient of tissue oxygen in embryos at the 20-30 somite stage. This may reflect asymmetrical vasculature, as we have suggested to explain drug-induced asymmetrical limb malformations.

Formation and patterning of the avian neuraxis: one dozen hypotheses.

Formation of the neuraxis is dependent on cell-cell interactions and cell movements beginning during stages of gastrulation. Cell movements bring together new combinations of cells, allowing sequential inductive interactions to occur and leading to the specification of the neural plate and to its ultimate mediolateral (subsequently dorsoventral) and rostrocaudal patterning. Formation of the neural plate involves changes in the shape of its constituent cells and the first appearance of neural-specific cell markers. Shortly after the neural plate forms it undergoes 'shaping', in which the pseudostratified columnar epithelium constituting the neural plate thickens apicobasally, narrows transversely and extends longitudinally. Shaping is driven by three principal intrinsic types of cell behaviour: changes in cell shape, position and number. The next stage of neurulation begins while shaping is underway--bending of the neural plate. Bending involves two main processes, furrowing and folding. Furrowing of the neural plate is associated with the formation of the hinge points; these are localized, longitudinal areas where the neuroepithelium is attached to adjacent tissues and where wedging of neuroepithelial cells occurs. Cell wedging in the median hinge point occurs as a result of inductive interactions with the notochord; such wedging drives furrowing, thereby facilitating subsequent folding. Folding of the neural plate requires extrinsic forces generated largely by the surface ectoderm. Types of cell behaviour that could provide such forces include changes in cell shape, position and number. As a result of shaping and bending of the neural plate, the neural folds are brought into apposition in the dorsal midline. Final closure of the neural groove is mediated by cell surface glycoconjugates coating the apical surfaces of the neural folds. Patterning of the neuraxis begins during shaping of the neural plate and continues throughout stages of neurulation and into early postneurula stages. Patterning probably involves inductive interactions with adjacent tissues and the expression of putative positional identity genes such as homeobox-containing genes.

Apoptosis regulates notochord development in Xenopus

The notochord is the defining characteristic of the chordate embryo and plays critical roles as a signaling center and as the primitive skeleton. In this study we show that early notochord development in Xenopus embryos is regulated by apoptosis. We find apoptotic cells in the notochord beginning at the neural groove stage and increasing in number as the embryo develops. These dying cells are distributed in an anterior to posterior pattern that is correlated with notochord extension through vacuolization. In axial mesoderm explants, inhibition of this apoptosis causes the length of the notochord to approximately double compared to controls. In embryos, however, inhibition of apoptosis decreases the length of the notochord and it is severely kinked. This kinking also spreads from the anterior with developmental stage such that, by the tadpole stage, the notochord lacks any recognizable structure, although notochord markers are expressed in a normal temporal pattern. Extension of the somites and neural plate mirrors that of the notochord in these embryos, and the somites are severely disorganized. These data indicate that apoptosis is required for normal notochord development during the formation of the anterior–posterior axis, and its role in this process is discussed.

Embryogenetics: gene control of the embryogenesis of the eye

In recent years there have been significant advances in our knowledge and understanding of the genetic control of embryonic development. The aim of this paper is to review the current state of knowledge regarding genetic control of embryogenesis, the first stage of ocular development. Products of numerous genes participate in signaling, induction and control during the formation of the neural plate, groove and tube, key events leading to the development of the future eye.

ALCAM (CD166) Is a Surface Marker for Early Murine Cardiomyocytes

ALCAM (activated leukocyte cell adhesion molecule, CD166) belongs to the immunoglobulin superfamily and is involved in axon guidance, hematopoiesis, immune response and tumor metastasis. During embryogenesis, mRNA encoding ALCAM was expressed in the cardiac crescent and the neural groove at embryonic day (E) 7.75 and predominately in the tubular heart at E8.5. A newly generated monoclonal antibody against the ALCAM molecule (ALC-48) exclusively stained cardiomyocytes at E8.25–10.5. However, ALCAM expression was lost by cardiomyocytes by E12.5 and its expression shifts to a variety of organs during later stages. ALCAM was found to be a prominent surface marker for cardiomyocytes in early embryonic hearts. The transient expression of ALCAM during early developmental stages marks specific developmental stages in cardiomyocyte differentiation.

Non-classical presentation of congenital cholesteatoma

Congenital cholesteatoma is a rare entity. It arises from aberrant epithelial remnants left at the time of closure of the neural groove, between the 3rd and 5th week of embryonic life. Otoscopy typically shows a pearly white mass, medial to the intact tympanic membrane, primarily in the anterosuperior quadrant, without a preceding history of otorrhoea, tympanic membrane perforation or surgery. It can present at any age from birth to early adulthood. The most common presenting symptom is hearing loss or facial nerve palsy. We present a non-classical presentation of congenital cholesteatoma with loss of taste sensation and numbness on the anterior two-thirds of the tongue associated with a bulging posterosuperior quadrant of the ipsilateral tympanic membrane in a 52-year-old male. After investigation, the patient underwent surgical management resulting in improvement of his symptoms.

Ultrastructural changes in embryoic neuroepithelial cells caused by passive smoking in golden hamsters at different periods of pregnancy: A randomized controlled trial*

Ultrastructural changes in embryoic neuroepithelial cells caused by passive smoking in golden hamsters at different periods of pregnancy: A randomized controlled trial*

Confinement and clearance of OCT4 in the porcine embryo at stereomicroscopically defined stages around gastrulation

In the areas of developmental biology and embryonic stem cell research, reliable molecular markers of pluripotency and early lineage commitment are sparse in large animal species. In this study, we present morphological and immunohistochemical findings on the porcine embryo in the period around gastrulation, days 8-17 postinsemination, introducing a stereomicroscopical staging system in this species. In embryos at the expanding hatched blastocyst stage, OCT4 is confined to the inner cell mass. Following detachment of the hypoblast, and formation of the embryonic disk, this marker of pluripotency was selectively observed in the epiblast. A prominent crescent-shaped thickening at the posterior region of the embryonic disk marked the first polarization within this structure reflecting incipient cell ingression. Following differentiation of the epiblast, clearance of OCT4 from the three germ layers was observed at defined stages, suggesting correlations to lineage specification. In the endoderm, clearance of OCT4 was apparent from early during its formation at the primitive streak stage. The endoderm harbored progenitors of the "fourth germ layer," the primordial germ cells (PGCs), the only cells maintaining expression of OCT4 at the end of gastrulation. In the ectodermal and mesodermal cell lineages, OCT4 became undetectable at the neural groove and somite stage, respectively. As in the mouse, PGCs showed onset of c-kit expression when located in extraembryonal compartments. They appeared to follow the endoderm during extraembryonal allocation and the mesoderm on return to the genital ridge.

An MRI view of a ruptured dermoid cyst

Intracranial dermoid cysts are developmental masses which arise from inclusions of ectodermoidal elements in the neural groove and typically contain fatty components.1 Rupture into the subarachnoid space is rare and may produce chemical meningitis. We report a 44-year-old patient presenting with a single complex …

153 IMMUNOCYTOCHEMICAL STUDIES OF OCT-4, SOX-2, AND β-TUBULIN III EXPRESSION IN EARLY PORCINE EMBRYOS

In the mouse, the transcription factor SOX-2 is known to have at least 2 roles: (1) it acts as a co-factor of the transcription factor OCT-4, the key regulator of pluripotency essential for the development of the inner cell mass/epiblast; and (2) it is involved in the direction of neural development. In this study, we elucidate the localization of SOX-2 in early porcine embryos in relation to that of OCT-4 and the early neuronal marker β-tubulin III. Embryos were flushed from uteri, fixed in 4% paraformaldehyde, and processed for paraffin sectioning. Sections (5 �m) were stained with anti-OCT-3/4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using the ABC-AEC-method and counterstained with hematoxylin, or processed for double immunofluorescent staining using antibodies against SOX-2 (R&D Systems Europa, Ltd., Abington, Oxon, UK) and β-tubulin III (Sigma-Aldrich Denmark A/S, Copenhagen, Denmark) and counterstaining with Hoechst. The embryos were classified as pre-streak (n = 8), primitive streak (n = 4), neural groove (n = 5), and somite (n = 4) stage (Vejlsted et al. 2006 Mol. Reprod. Dev. 73, 709–718). At the early pre-streak stage, SOX-2 and OCT-4 staining was found in the nuclei and a weak β-tubulin III staining in the cytoplasm of all epiblast cells. At the late pre-streak and the primitive streak stage, SOX-2 staining became polarized to the nuclei in the anterior epiblast region, whereas OCT-4 staining was found in all nuclei of the epiblast and of the forming meso- and endoderm. The β-tubulin III staining was restricted to the epiblast and showed no anterior-posterior polarization. At the primitive streak, when cells were involuting to form the meso- and endoderm, SOX-2 staining of nuclei was absent. At the neural groove stage, the SOX-2 and β-tubulin III staining was localized to nuclei and cytoplasm, respectively, of the same cells and observed in the neural plate and groove. A polarization in SOX-2 staining was observed in an anterior-posterior direction. At the somite stage, the SOX-2 and β-tubulin III staining was again localized to the same cells and observed in the neuropores and neural tube. The SOX-2 staining of the neural tube was polarized in a dorso-ventral direction. At the neural groove and somite stage, the OCT-4 staining gradually disappeared from the epiblast, mesoderm, and endoderm except from scattered cells, presumably primordial germ cells, localized in the endoderm. Our results suggest that also in the porcine embryo SOX-2 plays a dual role, being involved in regulation of both pluripotency and neural development.

140 LOCALIZATION OF PRIMORDIAL GERM CELLS IN DAY 21 BOVINE EMBRYOS

In most animal species, germ cell precursors, i.e., primordial germ cells (PGCs), arise from the epiblast and then migrate to the future gonadal ridge during development. At least in the mouse, PGCs may be cultured as embryonic germ cells that remain pluripotent. PGCs are the only cells in which OCT4 expression is maintained after gastrulation. The present study aimed at identifying the localization of PGCs in Day 21 in vivo-derived bovine embryos by immunohistochemical staining against OCT4. Six embryos were obtained after slaughter of superovulated heifers 21 days after insemination. The uterine tracts were flushed and embryos fixed, paraffin-embedded, and processed for immunohistochemistry. Embryos were sagitally sectioned, and selected serial sections were immunohistochemically stained for OCT4 to identify potential PGCs. Two embryos were at the neural groove stage. At this stage of development, the primitive gut had not yet been abstricted from the yolk sac and the allantois was not visible. A weak homogeneous OCT4 staining was localized to nuclei in a well-defined region of the epiblast, which was in the process of a gradual anterior to posterior differentiation into neural and surface ectoderm. Moreover, a strong OCT4 staining was localized to a few scattered cells found in the visceral mesoderm associated with the yolk sac in the region of the endoderm-hypoblast transition at some distance from the embryo proper. Four embryos were at the neural tube/somite stage. At this stage of development, the primitive gut had been defined and only the midgut was connected to the yolk sac. Furthermore, the allantois was visible as an anchor-shaped structure at the posterior end of the embryo. A strong OCT4 staining was found in nuclei of solitary cells in the endoderm and its associated visceral mesoderm of the ventral aspect of the mid and hindgut. The described OCT4 staining corresponds well with previous findings in the pig, in which presumptive PGCs are found in the endoderm epithelium during the neural groove stage. Later, during the early somite stages, they are localized in the endoderm and visceral mesoderm of the yolk sac and allantois, and in later somite stages, they are found in the developing genital ridge. This is, however, the first study to demonstrate the localization of these cells, at least by OCT4 staining, in bovine embryos at the neural groove and neural tube/somite stages.

Post-hatching development of the porcine and bovine embryo—defining criteria for expected development in vivo and in vitro

Particular attention has been paid to the pre-hatching period of embryonic development although blastocyst development is a poor indicator of embryo viability. Post-hatching embryonic development in vitro would allow for establishment of more accurate tools for evaluating developmental potential without the need for transfer to recipient animals. Such a system would require (1) definition of milestones of expected post-hatching embryonic development in vivo; and (2) development of adequate culture systems. We propose a stereomicroscopical staging system for post-hatching embryos defining the following stages: (1) Expanded hatched blastocyst stage where the embryo presents an inner cell mass (ICM) covered by trophoblast. (2) Pre-streak stage 1 where the embryonic disc is formed. (3) Pre-streak stage 2 where a crescent-shaped thickening of the caudal portion of the embryonic disk appears. (4) Primitive streak stage where the primitive streak has developed as an axis of cell ingression of cells for meso- and endoderm formation. (5) Neural groove stage where the neural groove is developing from the rostral pole of the embryo along with a proportional shortening of the primitive streak; and (6) Somite stage(s) where paraxial mesoderm gradually condensates to form somites. Post-hatching development of bovine embryos in vitro is compromised and although hatching occurs and elongation can be physically provoked by culture in agarose tunnels, the embryonic disk characterizing the pre-streak stage 1 is never established. Thus, particular focus should be placed on establishing culture conditions that support at least some of the above-mentioned critical phases of development that in vivo occur within the initial two (pig) to three (cattle) weeks.

Intraorbital Epidermoid Tumors: A Case Report and a Review of the Literature

Epidermoid tumors are uncommon lesions making up approximately 1% to 1.5% of all intracranial neoplasms. 1 They originate during the fourth and fifth weeks of embryonic life when the neural plate invaginates in the dorsal midline to form the neural groove and neural folds. The neural folds then form the neural tube, which separates from the overlying ectoderm. When this sequence of development is imperfect, ectopic epithelium may be deposited at sites between the neural canal and skin, resulting in epidermoid and dermoid tumors. 2,3 We report on a case of a large epidermoid tumor involving three cranial bones (sphenoid, zygomatic, and frontal) that extended from the orbit and into the temporal fossa. Report of a Case A 41-year-old white woman with no significant medical history was referred to us by her ophthalmologist with a chief complaint of asymmetry between her eyes since the age of 4 or 5. She stated that her right eye had always “bulged out of the socket” and that this condition had recently worsened. She did not take any daily medications and she reported no allergies. Physical examination was normal except for a noticeable downward and inward proptosis of the right eye. Her vision was 20/20 bilaterally, and extraocular movements were essentially normal. Computed tomography scan revealed a bone-destroying mass in the right lateral orbital wall and orbital roof that extended into the temporal fossa (Fig 1). The mass was isodense with brain tissue and had well-defined borders. Magnetic resonance imaging showed a hypointense signal on T1weighted images and a hyperintense signal on T2-weighted images (Fig 2). Total surgical excision was recommended, and the patient agreed after receiving a full explanation of risks, benefits, and alternatives. Surgical access was obtained through a frontotemporal craniotomy. A pearly white, cheesy mass that had eroded the bone of the pterion and the orbital roof was identified. The thinned bone was carefully excised with rongeurs and the lesion was removed piecemeal with copious irrigation. After the contents of the cyst were evacuated, the diaphanous capsule was carefully peeled from the dura and periorbita. All eroded bone was also removed to decompress the area. The bone flap was replaced, and closure proceeded uneventfully. Histopathologic analysis of the surgical specimen revealed a stratified squamous epithelial lining with abundant keratin debris (Fig 3). No sebaceous glands, sweat glands, or skin appendages were recognized, and thus a diagnosis of epidermoid tumor was confirmed. The patient recovered uneventfully and remains tumor free at 4 months. There has been dramatic reduction in the proptosis.

Congenital spinal dermal sinuses

Neurosurgeons from Children's Memorial Hospital in Chicago present the case reports of 9 children who represent the spectrum of pathologic and clinical findings collectively referred to as congenital spinal “dermal sinuses.” The article could be reprinted in its entirety as a contemporary tutorial. This group of embryonic malformations is common, and their presentations and importance are core knowledge requirements for every pediatrician. The embryologic defect occurs early in fetal life when the ectoblast is differentiated into cutaneous and neural ectoderm. As the neural groove closes to form the medullary tube, cutaneous ectodermal cells may also be invaginated, subsequently developing into intraspinal dermoid or epidermoid growths. If mesodermal defect also occurs, spinal dysraphism results, and a tract may connect the skin directly with the spinal canal. Clinically, defects are recognized in 1 of 3 ways: (1) the finding on routine physical examination of a sinus tract on or near the midline from the tip of the spine to the base of the nose; (2) because of the occurrence of bacterial meningitis or recurrent bacterial meningitis, especially caused by unusual organisms; or (3) when compression or traction on the spinal cord occurs and causes motor weakness, nerve root irritation, or autonomic changes and sphincter dysfunction. The finding of pigmentation, hypertrichosis, hemangioma, lipoma, or intermittent sebaceous discharge also can be the external clue to an almost imperceptible sinus tract. The authors provide astute clinical observations, revealing cases, and excellent discussion of anatomy and embryology—all worth the re-reading. Neurosurgeons from Children's Memorial Hospital in Chicago present the case reports of 9 children who represent the spectrum of pathologic and clinical findings collectively referred to as congenital spinal “dermal sinuses.” The article could be reprinted in its entirety as a contemporary tutorial. This group of embryonic malformations is common, and their presentations and importance are core knowledge requirements for every pediatrician. The embryologic defect occurs early in fetal life when the ectoblast is differentiated into cutaneous and neural ectoderm. As the neural groove closes to form the medullary tube, cutaneous ectodermal cells may also be invaginated, subsequently developing into intraspinal dermoid or epidermoid growths. If mesodermal defect also occurs, spinal dysraphism results, and a tract may connect the skin directly with the spinal canal. Clinically, defects are recognized in 1 of 3 ways: (1) the finding on routine physical examination of a sinus tract on or near the midline from the tip of the spine to the base of the nose; (2) because of the occurrence of bacterial meningitis or recurrent bacterial meningitis, especially caused by unusual organisms; or (3) when compression or traction on the spinal cord occurs and causes motor weakness, nerve root irritation, or autonomic changes and sphincter dysfunction. The finding of pigmentation, hypertrichosis, hemangioma, lipoma, or intermittent sebaceous discharge also can be the external clue to an almost imperceptible sinus tract. The authors provide astute clinical observations, revealing cases, and excellent discussion of anatomy and embryology—all worth the re-reading.

Ultrastructure, development, and homology of insect embryonic cuticles

Ultrastructure and deposition of the cuticles secreted by embryos representing eight insect orders were examined by transmission and scanning electron microscopy. Embryos of the apterygote silverfish Thermobia domestica deposit two embryonic cuticles. Deposition of the first (EC1) is initiated at the beginning of appendage development when the intercalary segment and the neural groove are clearly visible. This cuticle lacks surface microsculpture and consists of an outer epicuticle and an underlying fibrous layer, thought to represent procuticle. At the time of dorsal closure, deposition of a second embryonic cuticle (EC2) begins; this bears sensilla and functions in the first instar larva. In representative embryos of seven pterygote orders (Ephemeroptera, Odonata, Plecoptera, Neuroptera, Coleoptera, Lepidoptera, and Mecoptera), three cuticles were found to be secreted. The first cuticle in pterygotes is homologous to EC1 of T. domestica, but consists solely of outer epicuticle. EC2, the "prolarval cuticle," bears a characteristic surface microsculpture in embryos of some species and egg-teeth and other hatching devices, and consists of outer and inner epicuticles and a more or less reduced procuticle. EC2 is reduced in the embryos of derived endopterygotes, where a procuticle is lacking and the inner epicuticle is reduced. After hatching, when EC2 is shed, the first instar larva is covered by a third embryonic cuticle (EC3), whose deposition was initiated while the insect was still within the egg. Presence of only two embryonic cuticles in cyclorrhaphous flies is due to the total loss of prolarval cuticle. Investigated exopterygote and endopterygote insects excluding flies thus deposit three embryonic cuticles, and their juveniles (exopterygote "nymphs"; endopterygote "larvae") seem to hatch at equivalent stages of development. Differences between the modes of cuticulogenesis in silverfish and pterygote embryos suggest that the apterygote first larval instar was embryonized and became a fully embryonic prolarva in pterygotes.

147 IDENTIFICATION OF PRIMORDIAL GERM CELLS IN PORCINE EMBRYOS FROM THE PRIMITIVE STREAK STAGE

In embryonic stem cell research, Oct-4 is one of the most widely used markers of pluripotency. Moreover, at least in the mouse, this marker is restricted to primordial germ cells (PGCs) after gastrulation. Vimentin is often used as a marker of mesoderm/mesenchyme in embryonic tissues and appears to localize to the same embryonic cells as Oct-4, at least in the bovine epiblast. The expression of neither of these markers has been completely addressed in the pig. Therefore, the purpose of the present study was to examine the expression of Oct-4 and vimentin in the porcine epiblast during differentiation and establishment of the three germ layers, i.e. the process of gastrulation. A total of 410 porcine embryos were collected at 8 to 17 days post-insemination from 29 sows of the Danish Landrace breed. Embryos were categorized based on stereo-microscopic observations into the following stages: pre-streak stages 1 and 2, primitive streak stage, neural groove stage, and somite stage. Specimens were fixed at all stages, dehydrated and embedded in paraffin wax. Selected embryos at each stage (n = 28) were completely cut into serial sections for immunohistochemical evaluation of Oct-4 and vimentin. Pre-streak stage 1 embryos were defined by lack of polarization of the embryonic disk, whereas in pre-streak stage 2 embryos a crescent shaped thickening was seen at the posterior pole of the disk. This thickening, marking the first morphological anterior-posterior polarization of the embryo proper, was shown to be a site of incipient ingression of cells from the epiblast. Immunohistochemical analyses localized Oct-4 to nuclei and vimentin to cytoplasm of both founding and ingressing epiblast cells. During formation of mesoderm and endoderm, at the primitive streak stage, solitary Oct-4 positive cells, i.e. potential PGCs, were seen scattered in the endoderm. Cells of the epiblast displayed positive labeling for Oct-4 until specification for the ectoderm cell lineage at the subsequent neural groove stage. In mesoderm, Oct-4 likewise disappeared by the time of formation of the first somites, defining the following somite stage. Thus, at this stage the only cells labeled for Oct-4, i.e. potential PGCs, were seen solitarily scattered in the endoderm. By the 15-somite stage, such cells were no longer visible in the endoderm but were seen located in the mesoderm, spreading from the stalk of the yolk sac and allantois and extending through the mid- and hindgut areas into the incipient genital ridge. Vimentin localized to the mesenchyme and most other derivatives of neural crest and mesodermal origin. In conclusion, based on Oct-4 labeling and distribution pattern, we strongly believe that we have identified the porcine PGCs from the primitive streak stage.

27 MORPHOLOGICAL AND IMMUNOHISTOCHEMICAL CHARACTERIZATION OF DAY 21 IVP AND NT BOVINE EMBRYOS

A major limitation of somatic cell nuclear transfer (NT) for the production of cloned calves is that only 1–5% of cloned embryos produce viable calves. The high rate of mortality is attributed to both pre- and post-natal losses and is primarily due to incomplete reprogramming of donor cells. Almost 50% of the pregnancy losses occur in the first trimester of pregnancy, indicating a major disruption in normal embryo development at NT. The aim of this study was to analyze germ layer formation by stereomicroscopy and immunohistochemical techniques for both NT embryos and their in vivo counterparts on Day 21 and to compare deviations from normal embryonic development as a measure of developmental capacity. Blastocyts derived by IVF (n = 20), conventional NT (n = 20), or hand made cloning (HMC; n = 20) were non surgically transferred to each synchronized recipient cow (n = 3). Each group of twenty embryos was transferred to one recipient. Cows were slaughtered on Day 21 and uterine tracts recovered and flushed with phosphate-buffer solution with 10% serum. Recovered Day 21 embryos were fixed in 4% paraformaldehyde, and embedded in paraffin; serial sections were stained with hematoxylin and eosin and evaluated by light microscopy. Immunohistochemical localization of cytokeratin 8 was used as a marker for potential ectoderm, alphafetoprotein for potential endoderm, and vimentin for potential mesoderm. Four IVF (20%; 4/20) embryos, six NT (30%; 6/20) embryos, and ten HMC (50%; 10/20) embryos were recovered following flushing. No obvious morphological differences were seen in the formation of a neural tube, differentiation of mesoderm, and number of somites among IVF (25%; 1/4), NT (33.3%; 2/6), and HMC (20%; 2/10) embryos. Delayed development with respect to the formation of neural groove and mesoderm differentiation was observed in 25% (1/4) of IVF, 16.7% (1/6) of NT, and 30% (3/10) of HMC embryos. In addition, 25% (1/4) of IVF, 16.7% (1/6) of NT, and 33.3% (2/6) of HMC embryos had not initiated gastrulation (i.e. displayed hypoblast and epiblast), suggesting a more substantial developmental delay. The remaining embryos showed gross abnormalities compared to their in vivo counterparts, including degenetration of epiblast and hypoblast cells. Cytokeratin was detected in the trophoblast, ectoderm, hypoblast, and endoderm. Alpha-fetoprotein was detected in the hypoblast while vimentin was seen in the mesoderm. In conclusion, although localization of staining in IVF and cloned embryos was consistent with that of in vivo embryos, the intensity was weaker, suggesting compromised or delayed development. It is also possible that the differences observed were due to the recipient and not to the treatment group. This work was supported by Genetics Australia Cooperative Limited, Victoria, Australia.

Specific regulation of cyclins D1 and D2 by FGF and Shh signaling coordinates cell cycle progression, patterning, and differentiation during early steps of spinal cord development

In the vertebrate embryo, spinal cord elongation requires FGF signaling that promotes the continuous development of the posterior nervous system by maintaining a stem zone of proliferating neural progenitors. Those escaping the caudal neural stem zone, which is expressed to Shh signal, initiate ventral patterning in the neural groove before starting neuronal differentiation in the neural tube. Here we investigated the integration of D-type cyclins, known to govern cell cycle progression under the control of extracellular signals, in the program of spinal cord maturation. In chicken embryo, we find that cyclin D2 is preferentially expressed in the posterior neural plate, whereas cyclin D1 appears in the neural groove. We demonstrated by loss- and gain-of-function experiments that FGF signaling maintains cyclin D2 in the immature caudal neural epithelium, while Shh activates cyclin D1 in the neural groove. Moreover, forced maintenance of cyclin D1 or D2 in the neural tube favors proliferation at the expense of neuronal differentiation. These results contribute to our understanding of how the cell cycle control can be linked to the patterning programs to influence the balance between proliferation and neuronal differentiation in discrete progenitors domains.

125MORPHOLOGICAL CHARACTERIZATION OF DAY 14 AND DAY 21 IVP AND CLONED BOVINE EMBRYO DEVELOPMENT

The establishment of viable embryonic development following in vitro production (IVP) or nuclear transfer by SUZI (subzonal injection) and handmade cloning (HMC) methods is pivotal for practical implementation of embryo technology. The development of cloned embryos is currently impeded by the high rate of pre- and postnatal losses. This project is aimed at developing screening methods using both ultra-structural and molecular markers of development of post-hatching/pre-implantation stage embryos to enable selection of embryos with greater developmental competence. Initially twenty embryos derived by IVP or nuclear transfer (SUZI or HMC) were transferred to six recipient cows on Day 7 (n=120). Embryos were collected on Days 14 and 21 by flushing the uterine tracts of slaughtered cows. Embryos were fixed in 4% paraformaldehyde and processed for paraffin embedding. Serial sections were stained with haematoxylin and eosin and evaluated by light microscopy. On each day of collection, certain characteristics were expected to be observed according to normal in vivo development (Maddox-Hyttel et al., 2003, Reproduction 125, 607–623). On Day 14, IVP embryos (n=6) were ovoid to filamentous in shape. Three embryos had extensive foldings of trophoblast in the process of forming an amniotic cavity. NT-SUZI embryos (n=4) had a mainly round uneven shape. No NT-HMC embryos were recovered. On Day 21, IVP embryos (n=4) more closely resembled in vivo embryos than any other group and formed either a neural groove or a neural groove with somites. NT-SUZI embryos (n=6) were mainly filamentous, with one embryo showing complete development of the neural tube and 5 somite pairs. NT-HMC (n=17) development ranged from ovoid to multi-protrusions of the yolk sac. One embryo displayed the formation of a neural tube. Embryos, irrespective of production method, which had an abnormal morphology, presented only an incomplete hypoblast and severe degeneration of the epiblast. Those embryos that were morphologically comparable to in vivo embryos also displayed developmental stages which ranged from presentation of a primitive streak and formation of a neural tube to having a neural tube, differentiation of mesoderm and somites. At present, the low number of embryos recovered as well as their abnormal development clearly reflect the low success rate to term of live calves. It is possible that no Day 14 NT-HMC embryos were recovered due to collection efficiency not being optimal. It is apparent that IVP and cloned embryos have different developmental time lines as compared to that of in vivo embryos.

Immunohistochemical and ultrastructural characterization of the initial post-hatching development of bovine embryos

The problems of sustaining placenta formation in embryos produced by nuclear transfer have emphasized the need for basic knowledge about epiblast formation and gastrulation in bovine embryos. The aims of this study were to define stages of bovine post-hatching embryonic development and to analyse functional mechanisms of germ-layer formation. Embryos developed in vivo were collected after slaughter from superovulated cows on days 9, 11, 14 and 21 after insemination and processed for transmission electron microscopy (n = 26) or immunohistochemistry (n = 27) for potential germ-layer characterization (cytokeratin 8 for potential ectoderm; alpha-1-fetoprotein for potential endoderm; and vimentin for potential mesoderm). On day 9, the embryos were devoid of zona pellucida and presented a well-defined inner cell mass (ICM), which was covered by a thin layer of trophoblast cells (the Rauber's layer). Formation of the hypoblast from the inside of the ICM was ongoing. On day 11, the Rauber's layer was focally interrupted and adjacent underlying ICM cells formed tight junctions. The hypoblast, which formed a thin confluent cell layer, was separated from the ICM and the tropho-blast by intercellular matrix. The embryos were ovoid to tubular and displayed a confluent hypoblast on day 14. The epiblast was inserted into the trophoblast epithelium and tight junctions and desmosomes were present between adjacent epiblast cells as well as between peripheral epiblast and trophoblast cells. In some embryos, the epiblast was more or less covered by foldings of trophoblast in the process of forming the amniotic cavity. Cytokeratin 8 was localized to the trophoblast and the hypoblast underlying the epiblast; alpha-1-fetoprotein was localized to most hypoblast cells underlying the trophoblast; and vimentin was localized to most epiblast cells. On day 21, the smallest embryos displayed a primitive streak and formation of the neural groove, whereas the largest embryos presented a neural tube, up to 14 somites and allantois development. These embryos depicted the gradual formation of the endoderm, mesoderm and ectoderm as well as differentiation of paraxial, intermediate and lateral plate mesoderm. Cytokeratin 8 was localized to the trophoblast, the hypoblast and the surface and neural ectoderm; and alpha-1-fetoprotein was localized to the hypoblast, but not the definitive endoderm, the intensity increasing with development. Vimentin was initially localized to some, but not all, cells positioned particularly in the ventral region of the primitive streak, to presumptive definitive endoderm cells inserted into the hypoblast, and to mesoderm. In conclusion, within 2 weeks of hatching, bovine embryos complete formation of the hypoblast and the epiblast, establishment of the amniotic cavity, ingression of epiblast cells for primitive streak formation, involution of cells through the node and the streak for endoderm and mesoderm fomation, neurulation and differentiation of the mesoderm. The recruitment of cells from the epiblast to form the primitive streak as well as the endoderm and mesoderm is associated with expression of the intermediate filament vimentin.