Publications

The interplay between neural progenitors and stem cells (NPSCs), and their extracellular matrix (ECM) is a crucial regulatory mechanism that determines their behavior. Nonetheless, how the ECM dictates the state of NPSCs remains elusive. The hindbrain is valuable to examine this relationship, as cells in the ventricular surface of hindbrain boundaries (HBs), which arise between any two neighboring rhombomeres, express the NPSC marker Sox2, while being surrounded with the membrane-bound ECM molecule chondroitin sulphate proteoglycan (CSPG), in chick and mouse embryos. CSPG expression was used to isolate HB Sox2+ cells for RNA-sequencing, revealing their distinguished molecular properties as typical NPSCs, which express known and newly identified genes relating to stem cells, cancer, the matrisome and cell cycle. In contrast, the CSPG- non-HB cells, displayed clear neuraldifferentiation transcriptome. To address whether CSPG is significant for hindbrain development, its expression was manipulated in vivo and in vitro. CSPG manipulations shifted the stem versus differentiation state of HB cells, evident by their behavior and altered gene expression. These results provide further understanding of the uniqueness of hindbrain boundaries as repetitive pools of NPSCs in-between the rapidly growing rhombomeres, which rely on their microenvironment to maintain their undifferentiated state during development.

Fragile X mental retardation protein (FMRP) binds a selected set of mRNAs and proteins to guide neural circuit assembly and regulate synaptic plasticity. Loss of FMRP is responsible for Fragile X syndrome, a neuropsychiatric disorder characterized with auditory processing problems and social difficulty. FMRP actions in synaptic formation, maturation, and plasticity are site-specific among the four compartments of a synapse: presynaptic and postsynaptic neurons, astrocytes, and extracellular matrix. This review summarizes advancements in understanding FMRP localization, signals, and functional roles in axons and presynaptic terminals.

Introduction: Both the calvarial and the cortical bones develop through intramembranous ossification, yet they have very different structures and functions. The calvaria enables the rapid while protected growth of the brain, whereas the cortical bone takes part in locomotion. Both types of bones undergo extensive modeling during embryonic and post-natal growth, while bone remodeling is the most dominant process in adults. Their shared formation mechanism and their highly distinct functions raise the fundamental question of how similar or diverse the molecular pathways that act in each bone type are. Methods: To answer this question, we aimed to compare the transcriptomes of calvaria and cortices from 21-day old mice by bulk RNA-Seq analysis. Results: The results revealed clear differences in expression levels of genes related to bone pathologies, craniosynostosis, mechanical loading and bone-relevant signaling pathways like WNT and IHH, emphasizing the functional differences between these bones. We further discussed the less expected candidate genes and gene sets in the context of bone. Finally, we compared differences between juvenile and mature bone, highlighting commonalities and dissimilarities of gene expression between calvaria and cortices during post-natal bone growth and adult bone remodeling. Discussion: Altogether, this study revealed significant differences between the transcriptome of calvaria and cortical bones in juvenile female mice, highlighting the most important pathway mediators for the development and function of two different bone types that originate both through intramembranous ossification.

Fibronectin fibrillogenesis and mechanosensing both depend on integrin-mediated force transmission to the extracellular matrix. However, force transmission is in itself dependent on fibrillogenesis, and fibronectin fibrils are found in soft embryos where high forces cannot be applied, suggesting that force cannot be the sole initiator of fibrillogenesis. Here, we identify a nucleation step prior to force transmission, driven by fibronectin oxidation mediated by lysyl oxidase enzyme family members. This oxidation induces fibronectin clustering, which promotes early adhesion, alters cellular response to soft matrices, and enhances force transmission to the matrix. In contrast, absence of fibronectin oxidation abrogates fibrillogenesis, perturbs cell-matrix adhesion, and compromises mechanosensation. Moreover, fibronectin oxidation promotes cancer cell colony formation in soft agar as well as collective and single-cell migration. These results reveal a force-independent enzyme-dependent mechanism that initiates fibronectin fibrillogenesis, establishing a critical step in cell adhesion and mechanosensing.

Parturition is the final and essential step for mammalian reproduction. While the uterus is quiescent during pregnancy, fundamental changes arise in the myometrial contractility, inducing fetal expulsion. Extracellular matrix (ECM) remodeling is fundamental for these events. The gelatinases subgroup of matrix metalloproteinases (MMPs), MMP2 and MMP9, participate in uterine ECM remodeling throughout pregnancy and parturition. However, their loss-of-function effect is unknown. Here, we determined the result of eliminating Mmp2 and/or Mmp9 on parturition in vivo, using single- and double-knockout (dKO) mice. The dystocia rates were measured in each genotype, and uterine tissue was collected from nulliparous synchronized females at the ages of 2, 4, 9 and 12 months. Very high percentages of dystocia (40–55%) were found in the Mmp2−/− and dKO females, contrary to the Mmp9−/− and wild-type females. The histological analysis of the uterus and cervix revealed that Mmp2−/− tissues undergo marked structural alterations, including highly enlarged myometrial, endometrial and luminal cavity. Increased collagen deposition was also demonstrated, suggesting a mechanism of extensive fibrosis in the Mmp2−/− myometrium, which may result in dystocia. Overall, this study describes a new role for MMP2 in myometrium remodeling during mammalian parturition process, highlighting a novel cause for dystocia due to a loss in MMP2 activity in the uterine tissue.

Though initially discovered as a proto-oncogene in virally induced mouse mammary tumors, FGF3 is primarily active in prenatal stages, where it is found at various sites at specific times. FGF3 is crucial during development, as its roles include tail formation, inner ear development and hindbrain induction and patterning. FGF3 expression and function are highly conserved in vertebrates, while it also interacts with other FGFs in various developmental processes. Intriguingly, while it is classified as a classical paracrine signaling factor, murine FGF3 was uniquely found to also act in an intracrine manner, depending on alternative translation initiation sites. Corresponding with its conserved role in inner ear morphogenesis, mutations in FGF3 in humans are associated with LAMM syndrome, a disorder that include hearing loss and inner ear malformations. While recent studies indicate of some FGF3 presence in post-natal stages, emerging evidences of its upregulation in various human tumors and cariogenic processes in mouse models, highlights the importance of its close regulation in adult tissues. Altogether, the broad and dynamic expression pattern and regulation of FGF3 in embryonic and adult tissues together with its link to congenital malformations and cancer, calls for further discoveries of its diverse roles in health and disease.

The cortex is a highly organized structure that develops from the caudal regions of the segmented neural tube. Its spatial organization sets the stage for future functional arealization. Here, we suggest using a developmental perspective to describe and understand the etiology of common cortical malformations and their manifestation in the human brain.

The gelatinases, a subgroup of the matrix metalloproteinases (MMPs) superfamily are composed of two members; MMP2 and MMP9. They are known to degrade gelatin among other components of the extracellular matrix. Recently, the two gelatinases were found to be necessary for neural crest cell migration and to compensate for each other loss in these cells. To characterize their involvement in the skeletal system, and to better reveal their individual or common roles, we have generated double knockout (dKO) mice, lacking both MMP2 and MMP9. Comprehensive analysis of the skeleton morphological and mechanical parameters at postnatal day (P) 0, P21, 3 months (M) and 8M of age, revealed an unexpected distinct role for each gelatinase; MMP2 was found to be involved merely in intramembranous ossification which led to a smaller skull and inferior cortical parameters upon its loss, while MMP9 was found to affect only the endochondral ossification process, which led to shorter long-bones in its absence. Importantly, the dKO mice demonstrated a combination of both the skull and long bone phenotypes as found in the single-KOs, and not a severer additive phenotype. Transcriptome analysis on the cortical bone, the growth plate and the skull frontal bone, found many genes that were differentially expressed as a direct or indirect result of MMP-loss, and reinforced the specific and distinct role of each gelatinase in each bone type. Altogether, these results suggest that although both gelatinases share the same substrates and are highly expressed in flat and long bones, they are indispensable and control separately the development of different bones.

Avian blastoderm can enter into diapause when kept at low temperatures and successfully resume development (SRD) when re-incubated in body temperature. These abilities, which are largely affected by the temperature and duration of the diapause, are poorly understood at the cellular and molecular level. To determine how temperature affects embryonic morphology during diapause, high-resolution episcopic microscopy (HREM) analysis was utilized. While blastoderms diapausing at 12◦C for 28 days presented typical cytoarchitecture, similar to non-diapaused embryos, at 18◦C, much thicker blastoderms with higher cell number were observed. RNAseq was conducted to discover the genes underlying these phenotypes, revealing differentially expressed cell cycle regulatory genes. Among them, WEE1, a negative regulator of G2/M transition, was highly expressed at 12◦C compared to 18◦C. This finding suggested that cells at 12◦C are arrested at the G2/M phase, as supported by bromodeoxyuridine incorporation (BrdU) assay and phospho-histone H3 (pH 3) immunostaining. Inhibition of WEE1 during diapause at 12◦C resulted in cell cycle progression beyond the G2/M and augmented tissue volume, resembling the morphology of 18◦C-diapaused embryos. These findings suggest that diapause at low temperatures leads to WEE1 upregulation, which arrests the cell cycle at the G2/M phase, promoting the perseverance of embryonic cytoarchitecture and future SRD. In contrast, WEE1 is not upregulated during diapause at higher temperature, leading to continuous proliferation and maladaptive morphology associated with poor survivability. Combining HREM-based analysis with RNAseq and molecular manipulations, we present a novel mechanism that regulates the ability of diapaused avian embryos to maintain their cytoarchitecture via cell cycle arrest, which enables their SRD.

The avian embryo has a remarkable ability that allows it to suspend its development during blastulation for a long time at low temperatures, and to resume normal development when incubated. This ability is used by poultry hatcheries to store eggs prior to incubation. We have previously found that this ability correlates with the temperature during storage; embryos recover much better following prolonged storage at 12°C rather than at 18°C. However, the molecular and cellular mechanisms underlying these differences are poorly understood. To successfully resume development following storage, the embryo has to shift from the blastulation phase to gastrulation. Several genes are known to partake in the blastulation-to-gastrulation transition under normal conditions, such as the pluripotency-related genes Inhibitor of DNA Binding 2 (ID2) and NANOG that are expressed during blastulation, and the gastrulation-regulating genes NODAL and Brachyury (TBXT). However, their expression and activity following storage is unknown. To elucidate the molecular mechanisms that initiate the ability to successfully transit from blastulation to gastrulation following storage, embryos were stored for 28 days at 12°C or 18°C, and were assessed either prior to incubation, 12, or 18 h of incubation at 37.8°C. Immediately following storage at 18°C group showed remarkable impaired morphology compared to the blastoderm of the 12°C group and of non-stored control embryos. Concurrently with these, expression of ID2 and NANOG was maintained following storage at 12°C similar to the control group, but was significantly reduced upon storage at 18°C. Nevertheless, when the 18°C-stored embryos were incubated, the morphology and the reduced genes were reverted to resemble those of the 12°C group. At variance, key gastrulation genes, NODAL and its downstream effector Brachyury (TBXT), which were similarly expressed in the control and the 12°C group, were not restored in the 18°C embryos following incubation. Notably, ectopic administration of Activin rescued NODAL and TBXT expression in the 18°C group, indicating that these embryos maintain the potential to initiate. Collectively, this study suggests a temperature-dependent mechanisms that direct the transition from blastulation to gastrulation. These mechanisms promote a successful developmental resumption following prolonged storage at low temperatures.

Unraveling the inner workings of neural circuits entails understanding the cellular origin and axonal pathfinding of various neuronal groups during development. In the embryonic hindbrain, different subtypes of dorsal interneurons (dINs) evolve along the dorsal-ventral (DV) axis of rhombomeres and are imperative for the assembly of central brainstem circuits. dINs are divided into two classes, class A and class B, each containing four neuronal subgroups (dA1-4 and dB1-4) that are born in well-defined DV positions. While all interneurons belonging to class A express the transcription factor Olig3 and become excitatory, all class B interneurons express the transcription factor Lbx1 but are diverse in their excitatory or inhibitory fate. Moreover, within every class, each interneuron subtype displays its own specification genes and axonal projection patterns which are required to govern the stage-by-stage assembly of their connectivity toward their target sites. Remarkably, despite the similar genetic landmark of each dINs subgroup along the anterior-posterior (AP) axis of the hindbrain, genetic fate maps of some dA/dB neuronal subtypes uncovered their contribution to different nuclei centers in relation to their rhombomeric origin. Thus, DV and AP positional information has to be orchestrated in each dA/dB subpopulation to form distinct neuronal circuits in the hindbrain. Over the span of several decades, different axonal routes have been well-documented to dynamically emerge and grow throughout the hindbrain DV and AP positions. Yet, the genetic link between these distinct axonal bundles and their neuronal origin is not fully clear. In this study, we reviewed the available data regarding the association between the specification of early-born dorsal interneuron subpopulations in the hindbrain and their axonal circuitry development and fate, as well as the present existing knowledge on molecular effectors underlying the process of axonal growth.

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.

Neural crest cells (NCCs) are a unique embryonic cell population that initially reside at the dorsal neural tube but later migrate in the embryo and differentiate into multiple types of derivatives. To acquire motility, NCCs undergo epithelial-to-mesenchymal transition and invade the surrounding extracellular matrix (ECM). Matrix metalloproteases (MMPs) are a large family of proteases which regulate migration of various embryonic and adult cells via ECM remodeling. The gelatinase's subgroup of MMPs is the most studied one due to its key role in metastasis. As it is composed of only two proteases, MMP2 and MMP9, it is important to understand whether each is indispensable or redundant in its biological function. Here we explored the role of the gelatinases in executing NCC migration, by determining whether MMP2 and/or MMP9 regulate migration across species in singular, combined, or redundant manners. Chick and mouse embryos were utilized to compare expression and activity of both MMPs using genetic and pharmacological approaches in multiple in vivo and ex vivo assays. Both MMPs were found to be expressed and active in mouse and chick NCCs. Inhibition of each MMP was sufficient to prevent NCC migration in both species. Yet, NCC migration was maintained in MMP2−/− or MMP9−/− mouse mutants due to compensation between the gelatinases, but reciprocal pharmacological inhibition in each mutant prevented NCC migration. This study reveals for the first time that both gelatinases are expressed in avian and mammalian NCCs, and demonstrates their fundamental and conserved role in promoting embryonic cell migration.

Fragile X mental retardation protein (FMRP) is an RNA-binding protein abundant in the nervous system. Functional loss of FMRP leads to sensory dysfunction and severe intellectual disabilities. In the auditory system, FMRP deficiency alters neuronal function and synaptic connectivity and results in perturbed processing of sound information. Nevertheless, roles of FMRP in embryonic development of the auditory hindbrain have not been identified. Here, we developed high-specificity approaches to genetically track and manipulate throughout development of the Atoh1+ neuronal cell type, which is highly conserved in vertebrates, in the cochlear nucleus of chicken embryos. We identified distinct FMRP-containing granules in the growing axons of Atoh1+ neurons and post-migrating NM cells. FMRP downregulation induced by CRISPR/Cas9 and shRNA techniques resulted in perturbed axonal pathfinding, delay in midline crossing, excess branching of neurites, and axonal targeting errors during the period of circuit development. Together, these results provide the first in vivo identification of FMRP localization and actions in developing axons of auditory neurons, and demonstrate the importance of investigating early embryonic alterations toward understanding the pathogenesis of neurodevelopmental disorders.

The hindbrain is a key relay hub of the central nervous system (CNS), linking the bilaterally symmetric half-sides of lower and upper CNS centers via an extensive network of neural pathways. Dedicated neural assemblies within the hindbrain control many physiological processes, including respiration, blood pressure, motor coordination and different sensations. During early development, the hindbrain forms metameric segmented units known as rhombomeres along the antero-posterior (AP) axis of the nervous system. These compartmentalized units are highly conserved during vertebrate evolution and act as the template for adult brainstem structure and function. TALE and HOX homeodomain family transcription factors play a key role in the initial induction of the hindbrain and its specification into rhombomeric cell fate identities along the AP axis. Signaling pathways, such as canonical-Wnt, FGF and retinoic acid, play multiple roles to initially induce the hindbrain and regulate Hox gene-family expression to control rhombomeric identity. Additional transcription factors including Krox20, Kreisler and others act both upstream and downstream to Hox genes, modulating their expression and protein activity. In this review, we will examine the earliest embryonic signaling pathways that induce the hindbrain and subsequent rhombomeric segmentation via Hox and other gene expression. We will examine how these signaling pathways and transcription factors interact to activate downstream targets that organize the segmented AP pattern of the embryonic vertebrate hindbrain.

Specification of primordial germ cells (PGCs) in all vertebrates takes place in extragonadal sites. This requires migration of PGCs through embryonic tissues towards the genital ridges by both passive and active types of migration. Commonly, colonization in the genital ridges follows migration of the PGCs along the thin tissue of the dorsal mesentery. Here we review the anatomy of the dorsal mesentery, the role it plays in migration of PGCs, and the interactions of PGCs with different cell types, extracellular matrix and signaling pathways that are all essential for attraction and orientation of PGCs along the dorsal mesentery towards the gonad anlage.

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.