Publications

Carbamazepine (CBZ)is an anticonvulsant drug used for epilepsy and other disorders. Prescription of CBZ during pregnancy increases the risk for congenital malformations. CBZ is ubiquitous in effluents and persistent during wastewater treatment. Thus, it is re-introduced into agricultural ecosystems upon irrigation with reclaimed wastewater. People consuming produce irrigated with reclaimed wastewater were found to be exposed to CBZ. However, environmental concentrations of CBZ (μg L−1)are magnitudes lower than its therapeutic levels (μg ml−1), raising the question of whether and how environmental levels of CBZ affect embryonic development. The chick embryo is a powerful and highly sensitive amniotic model system that enables to assess environmental contaminants in the living organism. Since the chick embryonic development is highly similar to mammalians, yet, it develops in an egg, toxic effects can be directly analyzed in a well-controlled system without maternal influences. This research utilized the chick embryo to test whether CBZ is embryo-toxic by using morphological, cellular, molecular and imaging strategies. Three key embryonic stages were monitored: after blastulation (st.1HH), gastrulation/neurulation (st.8HH)and organogenesis (st.15HH). Here we demonstrate that environmental relevant concentrations of CBZ impair morphogenesis in a dose- and stage- dependent manner. Effects on gastrulation, neural tube closure, differentiation and proliferation were exhibited in early stages by exposing embryos to CBZ dose as low as 0.1 μg L−1. Quantification of developmental progression revealed a significant difference in the total score obtained by CBZ-treated embryos compared to controls (up to 5-fold difference, p < 0.05). Yet, defects were unnoticed as embryos passed gastrulation/neurulation. This study provides the first evidence for teratogenic effect of environmental-relevant concentrations of CBZ in amniotic embryos that impair early but not late stages of development. These findings call for in-depth risk analysis to ensure that the environmental presence of CBZ and other drugs is not causing irreversible ecological and public-health damages.

Neural stem cells (NSCs) are self-renewing multipotent cells that line the neural-tube and generate all the nervous system. Understanding NSC biology is fundamental for neurodevelopmental research and therapy. Many studies emphasized the need to culture NSCs, which are typically purified from mammalian embryonic/adult brains. These sources are somewhat limited in terms of quantity, availability and animal ethical guidelines. Therefore, new sources are needed. The chick is a powerful system for experimental embryology which contributed enormously to neurodevelopmental concepts. Its accessibility, genetic/molecular manipulations, and homology to other vertebrates, makes it valuable for developmental biology research. Recently, we identified a population of NSCs in the chick hindbrain. It resides in rhombomere-boundaries, expresses Sox2 and generates progenitors and neurons. Here, we investigated whether these cells can recapitulate hindbrain development in culture. By developing approaches to propagate and image cells, manipulate their growth-conditions and separate them into subpopulations, we demonstrate the ordered formation of multipotent and self-renewing neurospheres that maintain regional identity and display differential stem/differentiation/proliferation properties. Live imaging revealed new cellular dynamics in the culture. Collectively, these NSC cultures reproduce major aspects of hindbrain development in-vitro, proposing the chick as a model for culturing hindbrain-NSCs that can be directly applied to other neural-tube domains and species.

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 flockfs 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.

While the history of developmental biology in Israel is relatively short, its impact is far-reaching, so we wanted to present a concise perspective on the Israeli developmental biology community, past-present-future. This community has undergone a wonderful, nearly exponential growth over the last three decades. How exactly did this happen? There are approximately fifty research groups that focus on developmental biology questions in Israel today that are members of the Israel Society of Developmental Biology (IsSDB; http://issdb.org/). The community has representative groups in a plethora of model systems, such as Nematostella, C. elegans, Drosophila, sea urchin, ascidians, zebrafish, Xenopus, chick and mouse, as well as plants, representing all the major universities and their branches, which include Bar-Ilan University, Ben-Gurion University of the Negev, The Hebrew University of Jerusalem, The University of Haifa, Technion - Israel Institute of Technology, Tel Aviv University and the Weizmann Institute of Science.

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 highresolution 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-byside 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.

Neural crest cells (NCCs) are a transient population of neuroectodermal-originated cells that populate the dorsal neural tube (dNT), before migrating and giving rise to multiple cell lineages in the developing embryo. Prior to their migration, NCCs undergo epithelial-to-mesen-chymal-transition (EMT) through which they lose cell contacts and detach from the dNT to invade their surrounding environment. Multiple signals and transcription factors have been identified to regulate these events. Yet, less is known regarding effectors that act downstream to execute the actual NCC separation and migration. Matrix metalloproteinases (MMPs) are a family of proteases that degrade the extracellular matrix as well as other pericellular proteins during processes of tissue remodeling, angiogenesis and metastasis. Previously, we and others have demonstrated the role of the gelatinases MMP2 and MMP9 during the onset of NCC migration. Several evidences link the cleavage and activation of these secreted gelatinases to the activity of membrane-type MMPs (MT-MMP), such as MMP14 and MMP16, which are tethered to plasma membrane and affect various cellular behaviors. The aim of this study was to investigate whether MMP16 acts in NCCs. Here we demonstrate the expression of MMP16 mRNA and protein in cranial NCCs in avian embryos. Knockdown of MMP16 inhibited NCC migration. This inhibition was rescued by the addition of recombinant MMP16, which was also sufficient to increase proper NCC migration. Furthermore, excess MMP16 caused enhanced NCC EMT, concomitant with degradation of dNT-related proteins, laminin and N-cadherin. Altogether, these results uncover MMP16 as a new effector participating in EMT and in the migration of NCCs.

Neural crest cells (NCCs) are transient cell populations that are initially residing at the dorsal-most part of the neural tube of the developing vertebrate embryo. At well-defined time points, NCCs detach from the neural tube as they undergo epithelial-to-mesenchymal transition (EMT) and migrate in distinct pathways to their final destinations. There, this unique cell population differentiates into a great variety of cell types including bone and cartilage tissues of the head and face, connective tissue of the heart, skin melanocytes, adipocytes, enteric neurons, and most of the peripheral sensory neurons, glia, and Schwann cells. Matrix metalloproteinases (MMPs) are a large family of matrix-degrading enzymes, which are divided into several subfamilies according to their structure and substrate specificity. The gelatinases subfamily, which includes MMP-2 and MMP-9 solely, is the most investigated group. Both MMP-2 and MMP-9 were previously reported to be expressed in embryonic NCCs and to have a role in their EMT and migration processes. In this review we present the most recent data regarding the role of MMP-2 and MMP-9 in embryonic NCCs and in their various derivatives in later embryonic stages and in adults.

Background: Compartment boundaries are an essential developmental mechanism throughout evolution, designated to act as organizing centers and to regulate and localize differently fated cells. The hindbrain serves as a fascinating example for this phenomenon as its early development is devoted to the formation of repetitive rhombomeres and their well-defined boundaries in all vertebrates. Yet, the actual role of hindbrain boundaries remains unresolved, especially in amniotes. Results: Here, we report that hindbrain boundaries in the chick embryo consist of a subset of cells expressing the key neural stem cell (NSC) gene Sox2. These cells co-express other neural progenitor markers such as Transitin (the avian Nestin), GFAP, Pax6 and chondroitin sulfate proteoglycan. The majority of the Sox2+ cells that reside within the boundary core are slow-dividing, whereas nearer to and within rhombomeres Sox2+ cells are largely proliferating. In vivo analyses and cell tracing experiments revealed the contribution of boundary Sox2+ cells to neurons in a ventricular-to-mantle manner within the boundaries, as well as their lateral contribution to proliferating Sox2+ cells in rhombomeres. The generation of boundary-derived neurospheres from hindbrain cultures confirmed the typical NSC behavior of boundary cells as a multipotent and self-renewing Sox2+ cell population. Inhibition of Sox2 in boundaries led to enhanced and aberrant neural differentiation together with inhibition in cell-proliferation, whereas Sox2 mis-expression attenuated neurogenesis, confirming its significant function in hindbrain neuronal organization. Conclusions: Data obtained in this study deciphers a novel role of hindbrain boundaries as repetitive pools of neural stem/progenitor cells, which provide proliferating progenitors and differentiating neurons in a Sox2-dependent regulation.

Hindbrain dorsal interneurons (HDIs) are implicated in receiving, processing, integrating, and transmitting sensory inputs from the periphery and spinal cord, including the vestibular, auditory, and proprioceptive systems. During development, multiple molecularly defined HDI types are set in columns along the dorsoventral axis, before migrating along well-defined trajectories to generate various brainstem nuclei. Major brainstem functions rely on the precise assembly of different interneuron groups and higher brain domains into common circuitries. Yet, knowledge regarding interneuron axonal patterns, synaptic targets, and the transcriptional control that govern their connectivity is sparse. The dB1 class of HDIs is formed in a district dorsomedial position along the hindbrain and gives rise to the inferior olive nuclei, dorsal cochlear nuclei, and vestibular nuclei. dB1 interneurons express various transcription factors (TFs): the pancreatic transcription factor 1a (Ptf1a), the homeobox TF-Lbx1 and the Lim-homeodomain (Lim-HD), and TF Lhx1 and Lhx5. To decipher the axonal and synaptic connectivity of dB1 cells, we have used advanced enhancer tools combined with conditional expression systems and the PiggyBac-mediated DNA transposition system in avian embryos. Multiple ipsilateral and contralateral axonal projections were identified ascending toward higher brain centers, where they formed synapses in the Purkinje cerebellar layer as well as at discrete midbrain auditory and vestibular centers. Decoding the mechanisms that instruct dB1 circuit formation revealed a fundamental role for Lim-HD proteins in regulating their axonal patterns, synaptic targets, and neurotransmitter choice. Together, this study provides new insights into the assembly and heterogeneity of HDIs connectivity and its establishment through the central action of Lim-HD governed programs.

Background: VICKZ (IGF2BP1,2,3/ZBP1/Vg1RBP/IMP1,2,3) proteins bind RNA and help regulate many RNA-mediated processes. In the midbrain region of early chick embryos, VICKZ is expressed in the neural folds and along the basal surface of the neural epithelium, but, upon neural tube closure, is down-regulated in prospective cranial neural crest (CNC) cells, concomitant with their emigration and epithelial-to-mesenchymal transition (EMT). Electroporation of constructs that modulate cVICKZ expression demonstrates that this down-regulation is both necessary and sufficient for CNC EMT. These results suggest that VICKZ down-regulation in CNC cell-autonomously promotes EMT and migration. Reduction of VICKZ throughout the embryo, however, inhibits CNC migration non-cell-autonomously, as judged by transplantation experiments in Xenopus embryos. Results and Conclusions: Given the positive role reported for VICKZ proteins in promoting cell migration of chick embryo fibroblasts and many types of cancer cells, we have begun to look for specific mRNAs that could mediate context-specific differences. We report here that the laminin receptor, integrin alpha 6, is down-regulated in the dorsal neural tube when CNC cells emigrate, this process is mediated by cVICKZ, and integrin alpha 6 mRNA is found in VICKZ ribonucleoprotein complexes. Significantly, prolonged inhibition of cVICKZ in either the neural tube or the nascent dermomyotome sheet, which also dynamically expresses cVICKZ, induces disruption of these epithelia. These data point to a previously unreported role for VICKZ in maintaining epithelial integrity.

Despite the importance of the chicken as a model system, our understanding of the development of chicken primordial germ cells (PGCs) is far from complete. Here we characterized the morphology of PGCs at different developmental stages, their migration pattern in the dorsal mesentery of the chicken embryo, and the distribution of the EMA1 epitope on PGCs. The spatial distribution of PGCs during their migration was characterized by immunofluorescence on whole-mounted chicken embryos and on paraffin sections, using EMA1 and chicken vasa homolog antibodies. While in the germinal crescent PGCs were rounded and only 25% of them were labeled by EMA1, often seen as a concentrated cluster on the cell surface, following extravasation and migration in the dorsal mesentery PGCs acquired an elongated morphology, and 90% exhibited EMA1 epitope, which was concentrated at the tip of the pseudopodia, at the contact sites between neighboring PGCs. Examination of PGC migration in the dorsal mesentery of Hamburger and Hamilton stage 20-22 embryos demonstrated a left-right asymmetry, as migration of cells toward the genital ridges was usually restricted to the right, rather than the left, side of the mesentery. Moreover, an examination of another group of cells that migrate through the dorsal mesentery, the enteric neural crest cells, revealed a similar preference for the right side of the mesentery, suggesting that the migratory pathway of PGCs is dictated by the mesentery itself. Our findings provide new insights into the migration pathway of PGCs in the dorsal mesentery, and suggest a link between EMA1, PGC migration and cell-cell interactions. These findings may contribute to a better understanding of the mechanism underlying migration of PGCs in avians.

As the embryo develops, multiple cellular events of division, differentiation, migration, and invasion occur. Cells are formed at specific locations and migrate along different axes to various destinations, by acquiring diverse types of molecular machineries and processes. One such process is the epithelial-to-mesenchymal transition (EMT), in which epithelial cells with highly ordered shapes and contacts transform into mesenchyme in order to start migration. Consequently, these separated cells react to intracellular and extracellular signals to travel through different microenvironments along stereotypical, long-distance migratory routes to their precise homing targets. Different types of proteases are necessary to execute such complex events. One excellent system to evaluate cell movements during embryonic development is the population of neural crest cells. These unique cells are initially formed as part of the neural epithelium, but then they undergo a dramatic EMT after which they extensively migrate and differentiate into various fates including craniofacial skeleton, skin pigments, and peripheral nerves. In this review, we will discuss the central roles of proteases, mainly the family of matrix metalloproteases, in facilitating neural crest cell migration, and propose an integrative model to suggest the orchestrated action of two such proteases in these developmental events.

Neural crest cells (NCCs) migrate throughout the embryo to differentiate into cell types of all germ layers. Initial directed NCC emigration relies on planar cell polarity (PCP), which through the activity of the small GTPases RhoA and Rac governs the actin-driven formation of polarized cell protrusions. We found that the actin binding protein calponin 2 (Cnn2) was expressed in protrusions at the leading edge of migratory NCCs in chicks and frogs. Cnn2 knockdown resulted in NCC migration defects in frogs and chicks and randomized outgrowth of cell protrusions in NCC explants. Morphant cells showed central stress fibers at the expense of the peripheral actin network. Cnn2 acted downstream of Wnt/PCP, as migration defects induced by dominant-negative Wnt11 or inhibition of RhoA function were rescued by Cnn2 knockdown. These results suggest that Cnn2 modulates actin dynamics during NCC migration as an effector of noncanonical Wnt/PCP signaling.

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

During bone development the extracellular matrix (ECM) undergoes extensive modeling and remodeling by different proteases including members of the matrix metalloproteinase (MMP) family. The most dominant MMPs in bone development are the gelatinases MMP-2 and MMP-9, the collagenase MMP-13 and the membrane-bound MT1-MMP. The enzymes are secreted by different cells in the bone microenvironment, including osteocytes, osteoblasts, osteoclasts, chondrocytes, and endothelial cells. In endochondral bone development, MMPs are involved as early as the initial vascularization of the cartilage anlage while later they regulate chondrocytes proliferation, differentiation, and apoptosis at the growth-plate, as well as vascularization at the chondro-osseous junction. At sites of bone resorption the relative importance of MMPs for matrix degradation depends on the bone type: they participate in resorption of calvarial but not long bones while in the latter they are significant for osteoclasts migration and invasion. The importance of MMPs in bone development is emphasized by several bone-related syndromes in human with single mutations in MMP genes. These, together with targeted mutation in animal models shed light on the role of different MMPs in many aspects of bone development. In this view it is not surprising that MMPs also participate in pathological conditions in bones. They play significant role in migration and establishment of tumor metastasis into bone and tumor-induced osteolysis; they are dominant in the degradation of collagen type I during the course of osteoarthritis; and are even involved in fracture repair. In this chapter we summarize the current knowledge regarding the central role of MMPs in bone health and disease.

Complex patterns and networks of genes coordinate rhombomeric identities, hindbrain segmentation and neuronal differentiation and are responsible for later brainstem functions. Pax6 is a highly conserved transcription factor crucial for neuronal development, yet little is known regarding its early roles during hindbrain segmentation. We show that Pax6 expression is highly dynamic in rhombomeres, suggesting an early function in the hindbrain. Utilization of multiple gain-and loss-of-function approaches in chick and mice revealed that loss of Pax6 disrupts the sharp expression borders of Krox20, Kreisler, Hoxa2, Hoxb1 and EphA and leads to their expansion into adjacent territories, whereas excess Pax6 reduces these expression domains. A mutual negative cross-talk between Pax6 and Krox20 allows these genes to be co-expressed in the hindbrain through regulation of the Krox20-repressor gene Nab1 by Pax6. Rhombomere boundaries are also distorted upon Pax6 manipulations, suggesting a mechanism by which Pax6 acts to set hindbrain segmentation. Finally, FGF signaling acts upstream of the Pax6-Krox20 network to regulate Pax6 segmental expression. This study unravels a novel role for Pax6 in the segmental organization of the early hindbrain and provides new evidence for its significance in regional organization along the central nervous system.

Hindbrain dorsal interneurons that comprise the rhombic lip relay sensory information and coordinate motor outputs. The progenitor dA1 subgroup of interneurons, which is formed along the dorsal-most region of the caudal rhombic lip, gives rise to the cochlear and precerebellar nuclei. These centers project sensory inputs toward upper-brain regions. The fundamental role of dA1 interneurons in the assembly and function of these brainstem nuclei is well characterized. However, the precise en route axonal patterns and synaptic targets of dA1 interneurons are not clear as of yet. Novel genetic tools were used to label dA1 neurons and trace their axonal trajectories and synaptic connections at various stages of chick embryos. Using dA1-specific enhancers, two contralateral ascending axonal projection patterns were identified; one derived from rhombomeres 6-7 that elongated in the dorsal funiculus, while the other originated from rhombomeres 2-5 and extended in the lateral funiculus. Targets of dA1 axons were followed at later stages using PiggyBac-mediated DNA transposition. dA1 axons were found to project and form synapses in the auditory nuclei and cerebellum. Investigation of mechanisms that regulate the patterns of dA1 axons revealed a fundamental role of Lim-homeodomain (HD) proteins. Switch in the expression of the specific dA1 Lim-HD proteins Lhx2/9 into Lhx1, which is typically expressed in dB1 interneurons, modified dA1 axonal patterns to project along the routes of dB1 subgroup. Together, the results of this research provided new tools and knowledge to the assembly of trajectories and connectivity of hindbrain dA1 interneurons and of molecular mechanisms that control these patterns.

Compartment boundaries act as organizing centers that segregate adjacent areas into domains of gene expression and regulation, and control their distinct fates via the secretion of signalling factors. During hindbrain development, a specialized cell-population forms boundaries between rhombomeres. These boundary cells demonstrate unique morphological properties and expressmultiple genes that differs them fromintra-rhombomeric cells. Yet, little is known regarding the mechanisms that controls the expression or function of these boundary markers. Multiple components of the FGF signaling system, including ligands, receptors, downstream effectors as well as proteoglycans are shown to localize to boundary cells in the chick hindbrain. These patterns raise the possibility that FGF signaling plays a role in regulating boundary properties. We provide evidence to the role of FGF signaling, particularly the boundary-derived FGF3, in regulating the expression of multiple markers at hindbrain boundaries. These findings enable further characterization of the unique boundary-cell population, and expose a new function for FGFs as regulators of boundary-gene expression in the chick hindbrain.