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Tibial dyschondroplasia (TD) is a prevalent skeletal abnormality associated with rapid growth rate in many avian species. It is characterized by the presence of a nonvascularized, nonmineralized lesion that extends from the epiphyseal growth plate into the metaphysis of the proximal tibiotarsal bones. In this study, we examined the expression of 4 members of the matrix metalloproteinase (MMP) family (MMP-2, -3, -9, and -13) in thiram-induced TD lesions and in the process of recovery from TD, by in situ hybridization analysis and quantitative real-time PCR. A model for the induction and recovery of TD was established, consisting of 3 groups of broilers: (1) thiram group, chicks fed a thiram-enriched diet to induce TD; (2) recovery group, chicks fed a thiram-enriched diet during the first week of the experiment and a normal diet from the second week on; and (3) control group, chicks fed a normal diet throughout the experimental period. In agreement with our previous data, the 4 MMP were diminished in the TD lesion (P < 0.05); however, in the current study we show that the growth plate was able to repair itself and that the MMP reappeared during the process of recovery from TD. Our results strengthen the link between MMP expression and growth-plate impairment, and we suggest that gelatinase activity (MMP-2 and 9) facilitates this process. Key words: avian, growth plate, matrix metalloproteinase, tibial dyschondroplasia doi: 10.2527/jas.2009-2068

Hatchability rates in broilers pose a significant challenge in the poultry industry. Despite advancements in breeding and incubation technology, hatch rates remain suboptimal due to factors like genetics, egg management, environmental stress, nutrition, and breeder age. Understanding the mechanisms behind compromised hatchability is crucial for improving broiler production. Since the embryonic phase accounts for ∼40% of a broiler's lifespan, poor embryonic development significantly contributes to malformations and mortality, adversely affecting both hatching and post-hatching performance. The foundations for proper embryogenesis are established within the first days of incubation during the formation of the three-germ layers and onset of organogenesis. These early days are critical, as malformations acquired during this period may severely affect growth and development of both the embryo and the hatchling chick. However, understanding of the types and prevalence of early embryonic malformations in broiler eggs remains incomplete. Here we present a novel tool for categorizing abnormalities in 3-day-old broiler embryos through a standardized classification system. Systematic mapping of malformation types and severities was conducted using light microscopy combined with High-Resolution Episcopic Microscopy (HREM), resulting in a new 'malformation atlas.' This detailed atlas identified various abnormalities, including lethal defects, axis duplications, neural tube and cardiovascular malformations, growth retardation, and head malposition, many of which are difficult to detect in young stages with traditional methods. To validate this classification tool, we next analyzed the impact of various egg management practices, such as storage and incubation conditions, on malformation types and prevalence in embryos from young and old breeding flocks. The atlas revealed significant variations in the types and occurrences of malformations, influenced by flock age and egg managements. Our findings highlight the value of implementing a novel malformation categorization tool for systematic understanding of poultry embryology. This knowledge could help reduce malformations, enhancing hatchability and improving broiler production efficiency., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background: Osteocalcin is a small protein abundant in the bone extracellular-matrix, that serves as a marker for mature osteoblasts. To become activated, osteocalcin undergoes a specific post-translational carboxylation. Osteocalcin is expressed at advanced stages of embryogenesis and after birth, when bone formation takes place. Neural crest cells (NCCs) are a unique cell population that evolves during early stages of development. While initially NCCs populate the dorsal neural-tube, later they undergo epithelial-to-mesenchymal-transition and migrate throughout the embryo in highly-regulated manner. NCCs give rise to multiple cell types including neurons and glia of the peripheral nervous system, chromaffin cells and skin melanocytes. Remarkably, in the head region, NCCs give rise to cartilage and bone., Finding: Here we report that osteocalcin is detected in cranial NCCs. Analysis of chick embryos at stages of cranial NCC migration revealed that osteocalcin mRNA and protein is expressed in pre-migratory and migratory NCCs in-vivo and ex-vivo. Addition of warfarin, an inhibitor of osteocalcin carboxylation, onto neural-tube explants, reduced the amount of NCC migration. These results provide the first evidence of osteocalcin presence in cranial NCCs, much before they give rise to craniofacial skeleton, and propose its possible involvement in the regulation of NCC migration., (© 2024. The Author(s).)

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., (Copyright © 2023 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

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 neural-differentiation 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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Janssen, Kalev-Altman, Shalit, Sela-Donenfeld and Monsonego-Ornan.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pokhrel, Genin, Sela-Donenfeld and Cinnamon.)

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., Competing Interests: Declaration of Competing Interest The authors have declared that no conflict of interest exists., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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.

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hirsch, Kohl, Wang and Sela-Donenfeld.)

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

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., (© 2020 Federation of American Societies for Experimental Biology.)

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 (μgL -1 ) are magnitudes lower than its therapeutic levels (μgml -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 0.1μgL -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., (Copyright © 2019. Published by Elsevier Ltd.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

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.

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.

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-mesenchymal-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.

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.

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., (© 2014 Anatomical Society.)

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.

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.

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., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Extracellular matrix mineralization is an essential physiologic process in bone, teeth, and hypertrophic cartilage. Matrix Gla protein (MGP), an inhibitor of mineralization, is expressed by chondrocytes and vascular smooth muscle cells to inhibit calcification of those soft tissues. Tibial dyschondroplasia (TD), a skeletal abnormality apparent as a plug of non-vascularized, non-mineralized, white opaque cartilage in the tibial growth plate of avian species can serve as a good model for studying process and genes involved in matrix mineralization and calcification. In this work, we studied the involvement of MGP in the development of TD, as well as in the processes of spontaneous and induced recovery from this syndrome. First, we found that during normal bone development, MGP is expressed in specific time and locations, starting from wide-spread expression in the yet un-ossified diaphysis during embryonic development, to specific expression in hypertrophic chondrocytes adjacent to the chondro-osseous junction and the secondary ossification center just prior to calcification. In addition, we show that MGP is not expressed in the impaired TD lesion, however when the lesion begins to heal, it strongly express MGP prior to its calcification. Moreover, we show that when calcification is inhibited, a gap is formed between the expression zones of MGP and BMP2 and that this gap is closed during the healing process. To conclude, we suggest that MGP, directly or through interaction with BMP2, plays a role as ossification regulator that acts prior to ossification, rather then simple inhibitor.

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.

This study determined the role of MMP9/gelatinase B during the migration onset of Neural Crest Cells (NCC) in avian embryos. NCC are neuroepithelial progenitors that convert into mesenchyme and migrate along defined paths throughout the embryo. To engage in migration, NCC loose cell contacts, detach from the neural tube and invade the surrounding environment. Multiple signals and transcription factors that regulate these events have been identified. Nevertheless, little is known regarding effectors that act downstream to execute the actual NCC migration. Matrix metalloproteinases (MMPs) compose a large family of enzymes whose principal substrates are basement membranes, adhesion proteins and the extracellular matrix (ECM) components. A major subgroup of MMPs, the gelatinases (MMP9 and 2) are central to many adult physiological and pathological processes, such as tumor metastasis and angiogenesis, in which cell-cell and cell-matrix contacts are degraded to allow migration. As NCC undergo similar processes during development, we hypothesized that MMP9 may also promote the migration of NCC. MMP9 was found to be expressed in delaminating and migrating NCC of both cranial and trunk axial levels. Blocking MMP9 resulted in a dramatic inhibition of NCC delamination and migration, without perturbing specification or survival. This inhibition occurred at regions containing both premigratory and migrating cells, indicative for the central role of MMP9 in executing the detachment of NCC from the neural tube as well as their migration. Conversely, excess MMP9 enhanced mesenchymalization and delamination of NCC and accelerated progenitors to undergo precocious migration. Examination of the mechanistic activity of MMP9 revealed its capability to degrade the adhesion molecule N-cadherin as well as the basement-membrane protein laminin within or around NCC, respectively. Altogether, our study reveals MMP9 as a novel effector which is required for NCC delamination and migration., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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 express multiple genes that differs them from intra-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.

The lack of affordable techniques for gene transfer in birds has inhibited the advancement of molecular studies in avian species. Here we demonstrate a new approach for introducing genes into chicken somatic tissues by administration of a lentiviral vector, derived from the feline immunodeficiency virus (FIV), into the chorioallantoic membrane (CAM) of chick embryos on embryonic day 11. The FIV-derived vectors carried yellow fluorescent protein (YFP) or recombinant alpha-melanocyte-stimulating hormone (α-MSH) genes, driven by the cytomegalovirus (CMV) promoter. Transgene expression, detected in chicks 2 days after hatch by quantitative real-time PCR, was mostly observed in the liver and spleen. Lower expression levels were also detected in the brain, kidney, heart and breast muscle. Immunofluorescence and flow cytometry analyses confirmed transgene expression in chick tissues at the protein level, demonstrating a transduction efficiency of ∼0.46% of liver cells. Integration of the viral vector into the chicken genome was demonstrated using genomic repetitive (CR1)-PCR amplification. Viability and stability of the transduced cells was confirmed using terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assay, immunostaining with anti-proliferating cell nuclear antigen (anti-PCNA), and detection of transgene expression 51 days post transduction. Our approach led to only 9% drop in hatching efficiency compared to non-injected embryos, and all of the hatched chicks expressed the transgenes. We suggest that the transduction efficiency of FIV vectors combined with the accessibility of the CAM vasculature as a delivery route comprise a new powerful and practical approach for gene delivery into somatic tissues of chickens. Most relevant is the efficient transduction of the liver, which specializes in the production and secretion of proteins, thereby providing an optimal target for prolonged study of secreted hormones and peptides.

The development of the vertebrate hindbrain requires multiple coordinated signals which act via several pathways. One such signal is Fibroblast Growth Factor (FGF), which is necessary for the patterning of a major transcription factor in the hindbrain, Krox20. However, in the chick, it is still not known which specific FGF ligand is responsible for the regulation of Krox20 and how the signal is dispatched. The most characterized signaling pathway which FGF acts through in the nervous system is the MAPK/Erk1/2 pathway. Nevertheless, a detailed analysis of the hindbrain distribution of various components of this pathway has not been fully described. In this study we present a comprehensive atlas of the FGF ligands, receptors and members of the MAPK/Erk1/2 signaling components in subsequent stages of avian hindbrain development. Moreover, we show that FGF is a major signaling pathway that contributes to the activation of ERK1/2 and expression of the downstream targets Pea3 and Erm. Central to this study, we provide multiple evidence that FGF3 is required for the upregulation of Pea3 that in turn is necessary for Krox20 distribution in rhombomeres 3 and 5. These results show for the first time that Pea3 mediates the FGF3 signal to regulate the hindbrain expression of Krox20., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background: During formation of the vertebrate central nervous system, the hindbrain is organized into segmental units, called rhombomeres (r). These cell-lineage restricted segments are separated by a subpopulation of cells known as boundary cells. Boundary cells display distinct molecular and cellular properties such as an elongated shape, enriched extracellular matrix components and a reduced proliferation rate compared to intra-rhombomeric cells. However, little is known regarding their functions and the mechanisms that regulate their formation., Results: Hindbrain boundary cells express several signaling molecules, such as FGF3, which at earlier developmental stages is transiently expressed in specific rhombomeres. We show that chick embryos that lack boundary cells due to overexpression of truncated EphA4 receptor in the hindbrain have continued segmental expression of FGF3 at stages when it is normally restricted to hindbrain boundaries. Furthermore, surgical ablation of the boundary between r3 and r4, or blocking of the contact of r4 with boundary cells, results in sustained FGF3 expression in this segment., Conclusion: These findings suggest that boundary cells are required for the downregulation of segmental FGF3, presumably mediated by a soluble factor(s) that emanates from boundaries. We propose that this new function of boundary cells enables a switch in gene expression that may be required for stage-specific functions of FGF3 in the developing hindbrain.

A network of molecular interactions is required in the developing vertebrate hindbrain for the formation and anterior-posterior patterning of the rhombomeres. FGF signaling is required in this network to upregulate the expression of the Krox20 and Kreisler segmentation genes, but little is known of how FGF gene expression is regulated in the hindbrain. We show that the dynamic expression of FGF3 in chick hindbrain segments and boundaries is similar to that of the BMP antagonist, follistatin. Consistent with a regulatory relationship between BMP signaling and FGF3 expression, we find that an increase in BMP activity due to blocking of follistatin translation by morpholino antisense oligonucleotides or overexpression of BMP results in strong inhibition of FGF3 expression. Conversely, addition of follistatin leads to an increase in the level of FGF3 expression. Furthermore, the segmental inhibition of BMP activity by follistatin is required for the expression of Krox20, Hoxb1 and EphA4 in the hindbrain. In addition, we show that the maintenance of FGF3 gene expression requires FGF activity, suggestive of an autoregulatory loop. These results reveal an antagonistic relationship between BMP activity and FGF3 expression that is required for correct segmental gene expression in the chick hindbrain, in which follistatin enables FGF3 expression by inhibiting BMP activity.

Eph receptors and ephrins can sharpen domains within developing tissues by mediating repulsion at interfaces. An Eph receptor has now been shown also to regulate cell adhesion within tissue subdivisions.

Delamination of premigratory neural crest cells depends on a balance between BMP/noggin and on successful G1/S transition. Here, we report that BMP regulates G1/S transition and consequent crest delamination through canonical Wnt signaling. Noggin overexpression inhibits G1/S transition and blocking G1/S abrogates BMP-induced delamination; moreover, transcription of Wnt1 is stimulated by BMP and by the developing somites, which concomitantly inhibit noggin production. Interfering with beta-catenin and LEF/TCF inhibits G1/S transition, neural crest delamination and transcription of various BMP-dependent genes, which include Cad6B, Pax3 and Msx1, but not that of Slug, Sox9 or FoxD3. Hence, we propose that developing somites inhibit noggin transcription in the dorsal tube, resulting in activation of BMP and consequent Wnt1 production. Canonical Wnt signaling in turn stimulates G1/S transition and generation of neural crest cell motility independently of its proposed role in earlier neural crest specification.