Differential proliferation regulates multi-tissue morphogenesis during embryonic axial extension: integrating viscous modeling and experimental approaches.

A major challenge in biology is to understand how mechanical interactions and cellular behavior affect the shapes of tissues and embryo morphology. The extension of the neural tube and paraxial mesoderm, which form the spinal cord and musculoskeletal system, respectively, results in the elongated shape of the vertebrate embryonic body. Despite our understanding of how each of these tissues elongates independently of the others, the morphogenetic consequences of their simultaneous growth and mechanical interactions are still unclear. Our study investigates how differential growth, tissue biophysical properties and mechanical interactions affect embryonic morphogenesis during axial extension using a 2D multi-tissue continuum-based mathematical model. Our model captures the dynamics observed in vivo by time-lapse imaging of bird embryos, and reveals the underestimated influence of differential tissue proliferation rates. We confirmed this prediction in quail embryos by showing that decreasing the rate of cell proliferation in the paraxial mesoderm affects long-term tissue dynamics, and shaping of both the paraxial mesoderm and the neighboring neural tube. Overall, our work provides a new theoretical platform upon which to consider the long-term consequences of tissue differential growth and mechanical interactions on morphogenesis.

Signaling pathways and regulatory networks in quail skeletal muscle development: insights from whole transcriptome sequencing.

Quail, as an advantageous avian model organism due to its compact size and short reproductive cycle, holds substantial potential for enhancing our understanding of skeletal muscle development. The quantity of skeletal muscle represents a vital economic trait in poultry production. Unraveling the molecular mechanisms governing quail skeletal muscle development is of paramount importance for optimizing meat and egg yield through selective breeding programs. However, a comprehensive characterization of the regulatory dynamics and molecular control underpinning quail skeletal muscle development remains elusive. In this study, through the application of HE staining on quail leg muscle sections, coupled with preceding fluorescence quantification PCR of markers indicative of skeletal muscle differentiation, we have delineated embryonic day 9 (E9) and embryonic day 14 (E14) as the start and ending points, respectively, of quail skeletal muscle differentiation. Then, we employed whole transcriptome sequencing to investigate the temporal expression profiles of leg muscles in quail embryos at the initiation of differentiation (E9) and upon completion of differentiation (E14). Our analysis revealed the expression patterns of 12,012 genes, 625 lncRNAs, 14,457 circRNAs, and 969 miRNAs in quail skeletal muscle samples. Differential expression analysis between the E14 and E9 groups uncovered 3,479 differentially expressed mRNAs, 124 lncRNAs, 292 circRNAs, and 154 miRNAs. Furthermore, enrichment analysis highlighted the heightened activity of signaling pathways related to skeletal muscle metabolism and intermuscular fat formation, such as the ECM-receptor interaction, focal adhesion, and PPAR signaling pathway during E14 skeletal muscle development. Conversely, the E9 stage exhibited a prevalence of pathways associated with myoblast proliferation, exemplified by cell cycle processes. Additionally, we constructed regulatory networks encompassing lncRNA‒mRNA, miRNA‒mRNA, lncRNA‒miRNA-mRNA, and circRNA-miRNA‒mRNA interactions, thus shedding light on their putative roles within quail skeletal muscle. Collectively, our findings illuminate the gene and non-coding RNA expression characteristics during quail skeletal muscle development, serving as a foundation for future investigations into the regulatory mechanisms governing non-coding RNA and quail skeletal muscle development in poultry production.

Retinoic acid influences the timing and scaling of avian wing development.

A fundamental question in biology is how embryonic development is timed between different species. To address this problem, we compared wing development in the quail and the larger chick. We reveal that pattern formation is faster in the quail as determined by the earlier activation of 5'Hox genes, termination of developmental organizers (Shh and Fgf8), and the laying down of the skeleton (Sox9). Using interspecies tissue grafts, we show that developmental timing can be reset during a critical window of retinoic acid signaling. Accordingly, extending the duration of retinoic acid signaling switches developmental timing between the quail and the chick and the chick and the larger turkey. However, the incremental growth rate is comparable between all three species, suggesting that the pace of development primarily governs differences in the expansion of the skeletal pattern. The widespread distribution of retinoic acid could coordinate developmental timing throughout the embryo.

Potential Effect of Pseudevernia furfuracea (L.) Zopf Extract and Metabolite Physodic Acid on Tumour Microenvironment Modulation in MCF-10A Cells.

Lichens comprise a number of unique secondary metabolites with remarkable biological activities and have become an interesting research topic for cancer therapy. However, only a few of these metabolites have been assessed for their effectiveness against various in vitro models. Therefore, the aim of the present study was to assess the effect of extract Pseudevernia furfuracea (L.) Zopf (PSE) and its metabolite physodic acid (Phy) on tumour microenvironment (TME) modulation, focusing on epithelial-mesenchymal transition (EMT), cancer-associated fibroblasts (CAFs) transformation and angiogenesis. Here, we demonstrate, by using flow cytometry, Western blot and immunofluorescence microscopy, that tested compounds inhibited the EMT process in MCF-10A breast cells through decreasing the level of different mesenchymal markers in a time- and dose-dependent manner. By the same mechanisms, PSE and Phy suppressed the function of Transforming growth factor beta (TGF-ß)-stimulated fibroblasts. Moreover, PSE and Phy resulted in a decreasing level of the TGF-ß canonical pathway Smad2/3, which is essential for tumour growth. Furthermore, PSE and Phy inhibited angiogenesis ex ovo in a quail embryo chorioallantoic model, which indicates their potential anti-angiogenic activity. These results also provided the first evidence of the modulation of TME by these substances.

Developmental events and cellular changes occurred during esophageal development of quail embryos.

The current study focused on the histogenesis of the esophagus in quail embryos. Formation of the gut tube occurred on the 4th day of incubation. Development of the muscular layers occurred in a sequential manner; the inner circular layer on the 7th day, the outer longitudinal layer on the 8th day and the muscularis mucosae on the 9th day. Glandular development began on the 13th day of incubation. The epithelium was pseudostratified columnar that consisted of mucous cells, dendritic cells, and keratinocyte precursors. Epithelial stratification occurred on the 15th day of incubation. We used Mallory trichrome, Weigert-Van Gieson, and Gomori silver stains to visualize fibrous components. Scanned samples showed formation of endoderm and mesoderm on the 5th day of incubation. A layer of myoblasts developed on the 8th day of incubation. Formation of mucosal folds, which contained glandular openings, occurred on the 14th to 17th days of incubation. On the 5th to 8th days of incubation, CD34 and vascular endothelial growth factor (VEGF) positive-mesodermal cells, and telocytes (TCs) were detected. On the 15th day of incubation, CD34 and VEGF positive-telocytes, and fibroblasts, were identified. The current study described the correlations between functional morphology and evolutionary biology.

Telocytes are major constituents of the angiogenic apparatus.

The current study investigated role of telocytes (TCs) in angiogenesis during embryonic development of quail using immunohistochemistry (IHC), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The angiogenic apparatus consisted of TCs, endothelial cells, and macrophages. TCs were identified morphologically by their telopodes and podoms using TEM and SEM and immunohistochemically using CD34, and vascular endothelial growth factor (VEGF). TCs also expressed CD68. TCs formed a three-dimensional network and established direct contact with blood vessels, sprouting endothelial cells, and active macrophages, while exerting their effect through paracrine signaling. VEGF was also expressed by endothelial cells and macrophages. Matrix metalloproteinase-9 (MMP-9) was expressed by TCs, endothelial cells, and macrophages. In conclusion, the expression of VEGF by TCs, endothelial cells, and macrophages is required for the proliferation and migration of endothelial cells and vascular growth. The expression of MMP-9 by TCs, endothelial cells, and macrophages is essential for the degradation of extracellular matrix (ECM) components during neoangiogenesis. Macrophages may facilitate phagocytosis and elimination of the degraded ECM components.

Impact of acute short-term high thermal stress during early embryogenesis on hatchability, physiological body reaction, and ovarian follicles development of quails.

This experiment aimed to evaluate the impact of continuous and intermittent thermal stress during early embryogenesis on hatchability, physiological body reaction, ovary weight, and follicle development of quails. A total of 540 eggs were divided into 3 equal groups (3 groups × 6 replicates × 30 eggs). In the first group (control), eggs were incubated at normal incubation conditions (37.5°C and 50-55% relative humidity) from day 0 till hatching. In the second group (continuous thermal stress [CTS]), eggs were daily exposed to 39.5°C and 50 to 55% during the early embryogenesis for 3 successive days (E4-E6) for 3 h (12:00-15:00). In the third group (intermittent thermal stress [ITS]), eggs were daily exposed to 39.5°C and 50 to 55% during the early embryogenesis for 90 min (12:00-13:30) then temperature was returned to 37.5°C for 60 min (13:30-14:30) after that the temperature was raised again for 39.5°C for 90 min (14:30-16:00) daily for 3 successive days (E4-E6). The findings showed that the highest relative water loss form egg (RWL/%) at 6 d of incubation was obtained in the CTS group (P ≤ 0.05). The hatchability rate was significantly (P ≤ 0.05) decreased in the thermal-treated groups compared with the control group. The body surface temperature and cloacal temperature in the CTS and ITS groups significantly (P ≤ 0.001) increased compared with the control group. Chick weight (g) at 5 wk old, total weight gain, daily weight gain were significantly lower (P ≤ 0.05) in the CTS group compared with the control group. Triiodothyronine (T3) hormone concentration and globulin level were significantly (P ≤ 0.05) lower in the CTS and ITS groups compared with the control. The ovarian follicle weights (first, second, third, fourth, and fifth) and the diameter of the large follicle (fifth follicle) were significantly (P ≤ 0.01) decreased by increasing incubation temperature. From these findings, it could be concluded that the hatchability and body weight at sexual maturity for quails produced from eggs exposed to CTS and IST were significantly decreased by 8 and 2.1% as well as 2.98 and 2.1%, respectively, compared with the control group.

Quail-chick grafting experiments corroborate that Tbr1-positive eminential prethalamic neurons migrate along three streams into hypothalamus, subpallium and septocommissural areas.

The prethalamic eminence (PThE), a diencephalic caudal neighbor of the telencephalon and alar hypothalamus, is frequently described in mammals and birds as a transient embryonic structure, undetectable in the adult brain. Based on descriptive developmental analysis of Tbr1 gene brain expression in chick embryos, we previously reported that three migratory cellular streams exit the PThE rostralward, targeting multiple sites in the hypothalamus, subpallium and septocommissural area, where eminential cells form distinct nuclei or disperse populations. These conclusions needed experimental corroboration. In this work, we used the homotopic quail-chick chimeric grafting procedure at stages HH10/HH11 to demonstrate by fate-mapping the three predicted tangential migration streams. Some chimeric brains were processed for Tbr1 in situ hybridization, for correlation with our previous approach. Evidence supporting all three postulated migration streams is presented. The results suggested a slight heterochrony among the juxtapeduncular (first), the peripeduncular (next), and the eminentio-septal (last) streams, each of which followed differential routes. A possible effect of such heterochrony on the differential selection of medial to lateral habenular hodologic targets by the migrated neurons is discussed.

Relevance of angiogenesis in autoimmune testis inflammation.

Experimental autoimmune orchitis (EAO) is a useful model to study organ-specific autoimmunity and chronic testicular inflammation. This model reflects testicular pathological changes reported in immunological infertility in men. Progression of EAO in rodents is associated with a significantly increased percentage of testicular endothelial cells and interstitial testicular blood vessels, indicating an ongoing angiogenic process. Vascular endothelial growth factor A (VEGFA), the main regulator of physiological and pathological angiogenesis, can stimulate endothelial cell proliferation, chemotaxis and vascular permeability. The aim of this study was to explore the role of VEGFA in the pathogenesis of testicular inflammation. Our results found VEGFA expression in Leydig cells, endothelial cells and macrophages in testis of rats with autoimmune orchitis. VEGFA level was significantly higher in testicular fluid and serum of rats at the end of the immunization period, preceding testicular damage. VEGF receptor (VEGFR) 1 is expressed mainly in testicular endothelial cells, whereas VEGFR2 was detected in germ cells and vascular smooth muscle cells. Both receptors were expressed in testicular interstitial cells. VEGFR2 increased after the immunization period in the testicular interstitium and VEGFR1 was downregulated in EAO testis. In-vivo-specific VEGFA inhibition by Bevacizumab prevented the increase in blood vessel number and reduced EAO incidence and severity. Our results unveil relevance of VEGFA-VEGFR axis during orchitis development, suggesting that VEGFA might be an early marker of testicular inflammation and Bevacizumab a therapeutic tool for treatment of testicular inflammation associated with subfertility and infertility.

PuraMatrix allows differentiation of a broad repertoire of neural and mesenchymal phenotypes from trunk neural crest.

The neural crest (NC) is a transitory embryonic structure of vertebrates that gives rise to an astonishing variety of derivatives, encompassing both neural and mesenchymal cell types. Neural crest cells (NCCs) are an excellent model to study how environmental factors modulate features such as cell multipotentiality and differentiation. Tests with multifunctional substrates that allow NCCs to express their full potential, while promoting cell subcloning, are needed to advance knowledge about NCC self-renewal and to foster future biotechnological approaches. Here we show that a self-assembled peptide named PuraMatrixTM is an excellent substrate that allows the differentiation of NCCs based on the identification of seven different cell types. Depending on the PuraMatrixTM concentration employed, different frequencies and quantities of a given cell type were obtained. It is noteworthy that an enormous quantity and diversity of mesenchymal phenotypes, such as chondrocytes, could be observed. The quantity of adipocytes and osteocytes also increased with the use of mesenchymal differentiation factors (MDF), but PuraMatrixTM alone can support the appearance of these mesenchymal cell types. PuraMatrixTM will promote advances in studies related to multipotentiality, self-renewal and control of NCC differentiation, since it is an extremely simple and versatile material which can be employed for both in vivo and in vitro experiments.

Mechanical Coupling Coordinates the Co-elongation of Axial and Paraxial Tissues in Avian Embryos.

Tissues undergoing morphogenesis impose mechanical effects on one another. How developmental programs adapt to or take advantage of these effects remains poorly explored. Here, using a combination of live imaging, modeling, and microsurgical perturbations, we show that the axial and paraxial tissues in the forming avian embryonic body coordinate their rates of elongation through mechanical interactions. First, a cell motility gradient drives paraxial presomitic mesoderm (PSM) expansion, resulting in compression of the axial neural tube and notochord; second, elongation of axial tissues driven by PSM compression and polarized cell intercalation pushes the caudal progenitor domain posteriorly; finally, the axial push drives the lateral movement of midline PSM cells to maintain PSM growth and cell motility. These interactions form an engine-like positive feedback loop, which sustains a shared elongation rate for coupled tissues. Our results demonstrate a key role of inter-tissue forces in coordinating distinct body axis tissues during their co-elongation.

Simulating flow induced migration in vascular remodelling.

Shear stress induces directed endothelial cell (EC) migration in blood vessels leading to vessel diameter increase and induction of vascular maturation. Other factors, such as EC elongation and interaction between ECs and non-vascular areas are also important. Computational models have previously been used to study collective cell migration. These models can be used to predict EC migration and its effect on vascular remodelling during embryogenesis. We combined live time-lapse imaging of the remodelling vasculature of the quail embryo yolk sac with flow quantification using a combination of micro-Particle Image Velocimetry and computational fluid dynamics. We then used the flow and remodelling data to inform a model of EC migration during remodelling. To obtain the relation between shear stress and velocity in vitro for EC cells, we developed a flow chamber to assess how confluent sheets of ECs migrate in response to shear stress. Using these data as an input, we developed a multiphase, self-propelled particles (SPP) model where individual agents are driven to migrate based on the level of shear stress while maintaining appropriate spatial relationship to nearby agents. These agents elongate, interact with each other, and with avascular agents at each time-step of the model. We compared predicted vascular shape to real vascular shape after 4 hours from our time-lapse movies and performed sensitivity analysis on the various model parameters. Our model shows that shear stress has the largest effect on the remodelling process. Importantly, however, elongation played an especially important part in remodelling. This model provides a powerful tool to study the input of different biological processes on remodelling.

Hypoxia-induced downregulation of Sema3a and CXCL12/CXCR4 regulate the formation of the coronary artery stem at the proper site.

BACKGROUND: During the formation of the coronary artery stem, endothelial strands from the endothelial progenitor pool surrounding the conotruncus penetrate into the aortic wall. Vascular endothelial growth factors (VEGFs) as well as CXCL12/CXCR4 signaling are thought to play a role in the formation of the coronary stem. However, the mechanisms regulating how endothelial strands exclusively invade into the aorta remain unknown. METHODS AND RESULTS: Immunohistochemistry showed that before the formation of endothelial strands, Sema3a was highly expressed in endothelial progenitors surrounding the great arteries. At the onset of/during invasion of endothelial strands into the aorta, Sema3a was downregulated and CXCR4 was upregulated in the endothelial strands. In situ hybridization showed that Cxcl12 was highly expressed in the aortic wall compared with in the pulmonary artery. Using avian embryonic hearts, we established two types of endothelial penetration assay, in which coronary endothelial strands preferentially invaded into the aorta in culture. Sema3a blocking peptide induced an excess number of endothelial strands penetrating into the pulmonary artery, whereas recombinant Sema3a inhibited the formation of endothelial strands. In cultured coronary endothelial progenitors, recombinant VEGF protein induced CXCR4-positive endothelial strands, which were capable of being attracted by CXCL12-impregnated beads. Monoazo rhodamine detected that hypoxia was predominant in aortic/subaortic region in ovo and hypoxic condition downregulated the expression of Sema3a in culture. CONCLUSION: Results suggested that hypoxia in the aortic region downregulates the expression of Sema3a, thereby enhancing VEGF activity to induce the formation of CXCR4-positive endothelial strands, which are subsequently attracted into the Cxcl12-positive aortic wall to connect the aortic lumen.

Vitamin E Supplementation and Early Age Heat Conditioning to Alleviate the Negative Effects of Heat Stress in Quail Chicks.

BACKGROUND AND OBJECTIVE: Inclusion of vitamin E in poultry diets and manipulation of temperature at early age has been known to help birds cope with heat stress at later age of their life. This study was conducted to investigate the effects of early age heat conditioning (EHC) and vitamin E addition on the performance and blood parameters to alleviate deleterious impact of heat stress in quail chicks. MATERIAL AND METHODS: Three hundred one-day-old quail chicks were randomly divided into 4 groups of 5 replicates with 15 birds of each. Treatments were: Control, vitamin E: Chicks were fed the basal diet supplemented with 200 IU kg-1 diet vitamin E, EHC: Chicks were exposed to 40±1°C for 2 h at days 7th and 13th of embryogenesis and EHC+vitamin E: Chicks were exposed to 40±1°C for 2 h at days 7th and 13th of embryogenesis and fed the basal diet supplemented with 200 IU kg-1 vitamin E. The experiment lasted from 1-40 days of age. RESULTS: The results indicated that using early age heat conditioning and/or supplementation of vitamin E significantly (p<0.05) improved body weight, feed intake and feed conversion ratio at 40 days of age. Significantly (p<0.05) improvement was observed in blood pH, H/L ratio and Hb concentration, thyroid hormones (T3 and T4), plasma protein fractions and antioxidant status (Total antioxidant capacity, catalase and superoxide dismutase). CONCLUSION: Exposed quail chicks to early age heat conditioning and addition of vitamin E at 200 IU kg-1 diet can effectively alleviate the adverse effects of heat stress.

Neural tube development depends on notochord-derived sonic hedgehog released into the sclerotome.

Sonic hedgehog (Shh), produced in the notochord and floor plate, is necessary for both neural and mesodermal development. To reach the myotome, Shh has to traverse the sclerotome and a reduction of sclerotomal Shh affects myotome differentiation. By investigating loss and gain of Shh function, and floor-plate deletions, we report that sclerotomal Shh is also necessary for neural tube development. Reducing the amount of Shh in the sclerotome using a membrane-tethered hedgehog-interacting protein or Patched1, but not dominant active Patched, decreased the number of Olig2+ motoneuron progenitors and Hb9+ motoneurons without a significant effect on cell survival or proliferation. These effects were a specific and direct consequence of Shh reduction in the mesoderm. In addition, grafting notochords in a basal but not apical location, vis-à-vis the tube, profoundly affected motoneuron development, suggesting that initial ligand presentation occurs at the basal side of epithelia corresponding to the sclerotome-neural tube interface. Collectively, our results reveal that the sclerotome is a potential site of a Shh gradient that coordinates the development of mesodermal and neural progenitors.

A tensile ring drives tissue flows to shape the gastrulating amniote embryo.

Tissue morphogenesis is driven by local cellular deformations that are powered by contractile actomyosin networks. How localized forces are transmitted across tissues to shape them at a mesoscopic scale is still unclear. Analyzing gastrulation in entire avian embryos, we show that it is driven by the graded contraction of a large-scale supracellular actomyosin ring at the margin between the embryonic and extraembryonic territories. The propagation of these forces is enabled by a fluid-like response of the epithelial embryonic disk, which depends on cell division. A simple model of fluid motion entrained by a tensile ring quantitatively captures the vortex-like "polonaise" movements that accompany the formation of the primitive streak. The geometry of the early embryo thus arises from the transmission of active forces generated along its boundary.

Sterol-O acyltransferase 1 is inhibited by gga-miR-181a-5p and gga-miR-429-3p through the TGFß pathway in endodermal epithelial cells of Japanese quail.

Nutrients are utilized and re-constructed by endodermal epithelial cells (EECs) of yolk sac membrane (YSM) in avian species during embryonic development. Sterol O-acyltransferase 1 (SOAT1) is the key enzyme to convert cholesterol to cholesteryl ester for delivery to growing embryos. During embryonic development, yolk absorption is concomitant with significant changes of SOAT1 mRNA concentration and enzyme activity in YSM. Presence of microRNAs (miRNAs) are observed in the embryonic liver and muscle during avian embryogenesis. However, the expression of miRNAs in YSM during embryogenesis and the involvement of miRNAs in lipid utilization are not known. Using a miRNA sequencing technique, we found several miRNA candidates and confirmed their expression patterns individually by real time PCR. MiRNA candidates were selected based on the expression pattern and their possible roles in inhibiting transforming growth factor beta receptor type 1 (TGFBR1) that would regulate the function of SOAT1. Similar to SOAT1 mRNA, the gga-miR-181a-5p expression was gradually elevated during embryonic development. However, the expression of gga-miR-429-3p in YSM was gradually decreased during embryonic development. The inhibitory effects of gga-miR-181a-5p or gga-miR-429-3p on the potential targets (SOAT1 and TGFBR1) were demonstrated by transient miRNA transfections in EECs. We also found that mutated TGFBR1 3'UTR prevented the direct pairings of gga-miR-181a-5p and gga-miR-429-3p. Treatment of TGFBR1 inhibitor, LY364947, further decreased SOAT1 transcription. Similar results were also observed by the miRNA transfection studies. The results showed the vital participations of gga-miR-181a-5p and gga-miR-429-3p in regulating TGFß pathway, and affecting downstream SOAT1 expression and function in the YSM. This is indicative of possible regulation of avian yolk lipid utilization by changing YSM miRNA expressions.

The Chicken as a Model Organism to Study Heart Development.

Heart development is a complex process and begins with the long-range migration of cardiac progenitor cells during gastrulation. This culminates in the formation of a simple contractile tube with multiple layers, which undergoes remodeling into a four-chambered heart. During this morphogenesis, additional cell populations become incorporated. It is important to unravel the underlying genetic and cellular mechanisms to be able to identify the embryonic origin of diseases, including congenital malformations, which impair cardiac function and may affect life expectancy or quality. Owing to the evolutionary conservation of development, observations made in nonamniote and amniote vertebrate species allow us to extrapolate to human. This review will focus on the contributions made to a better understanding of heart development through studying avian embryos-mainly the chicken but also quail embryos. We will illustrate the classic and recent approaches used in the avian system, give an overview of the important discoveries made, and summarize the early stages of cardiac development up to the establishment of the four-chambered heart.

FGF2 Stimulates the Growth and Improves the Melanocytic Commitment of Trunk Neural Crest Cells.

Neural crest cells (NCCs) comprise a population of multipotent progenitors and stem cells at the origin of the peripheral nervous system (PNS) and melanocytes of skin, which are profoundly influenced by microenvironmental factors, among which is basic fibroblast growth factor 2 (FGF2). In this work, we further investigated the role of this growth factor in quail trunk NC morphogenesis and demonstrated its huge effect in NCC growth mainly by stimulating cell proliferation but also reducing cell death, despite that NCC migration from the neural tube explant was not affected. Moreover, following FGF2 treatment, reduced expression of the early NC markers Sox10 and FoxD3 and improved proliferation of HNK1-positive NCC were observed. Since these markers are involved in the regulation of glial and melanocytic fate of NC, the effect of FGF2 on NCC differentiation was investigated. Therefore, in the presence of FGF2, increased proportions of NCCs positives to the melanoblast marker Mitf as well as NCCs double stained to Mitf and BrdU were recorded. In addition, treatment with FGF2, followed by differentiation medium, resulted in increased expression of melanin and improved proportion of melanin-pigmented melanocytes without alteration in the glial marker Schwann myelin protein (SMP). Taken together, these data further reveal the important role of FGF2 in NCC proliferation, survival, and differentiation, particularly in melanocyte development. This is the first demonstration of FGF2 effects in melanocyte commitment of NC and in the proliferation of Mitf-positive melanoblasts. Elucidating the differentiation process of embryonic NCCs brings us a step closer to understanding the development of the PNS and then undertaking the search for advanced technologies to prevent, or treat, injuries caused by NC-related disorders, also known as neurocristopathies.

Transplantation of Neural Tissue: Quail-Chick Chimeras.

Tissue transplantation is an important approach in developmental neurobiology to determine cell fate, to uncover inductive interactions required for tissue specification and patterning as well as to establish tissue competence and commitment. Combined with state-of-the-art molecular approaches, transplantation assays have been instrumental for the discovery of gene regulatory networks controlling cell fate choices and how such networks change over time. Avian species are among the favorite model systems for these approaches because of their accessibility and relatively large size. Here we describe two culture techniques used to generate quail-chick chimeras at different embryonic stages and methods to distinguish graft and donor tissue.