Further data on the development of SRIF-like immunoreactive nerve cell populations in the chick embryo brain stem. I. Medulla and pons.

Further immunocytochemical analysis of the neuroblasts with SRIF-like immunoreactivity (ir) was carried out on the chick embryo medulla and pons. 5 or 100 microns rombencephalon sections were obtained from 60 White Leghorn chick embryos at stages (E = Embryonic days) ranging from E4 1/2 to E18 and incubated with rabbit polyclonal antibodies against synthetic cyclic Somatostatin-14, according to PAP-DAB technique. In the medulla and pons the ir appeared as from E12. From E12 to E13 1/2-E14 the ir distribution gradually changed. From E14 to E18 numbers and spatial arrangement of the positive neuroblast groups did not show substantial changes; in these respects the ir distributional pattern proved to be markedly different from the one observed by the Authors in adult animals. Moreover, from E13 to E15 the positive neuroblast density appeared to be higher than that of positive neurons in adults. These results are consistent with a possible SRIF local regulative role.     

Double-labelling data on somatostatin-like and tyrosine-hydroxylase-like immunoreactivities in chick embryo brain stem. I. Pons and mesencephalon.

With the aim of investigating some factors and mechanisms of the chicken brain development, the same thick sections of brain stems from twelve E13-to-E21-aged chick embryos were sequentially tested with a rabbit anti-Somatostatin antiserum, using a PAP-DAB technique, and with anti-tyrosine hydroxylase (-TH) monoclonal antibodies, using an indirect immuno-fluorescence technique. As regards the pons and mesencephalon, the following main results were obtained. At E21 almost the same distribution of the TH-like immunoreactivity (ir) as at E13 was observed. Neuroblasts in a central, relatively wide region of mesencephalic tegmentum and in the central portion of the pons showed TH-like ir. A co-localization of the 2 immunoreactivities was detected only at E18, within some neuroblasts of the mesencephalic and pontine regions with TH-like ir. It is possible that this transitory co-localization plays a role in the development of the pons and mesencephalon of this species.     

Control of chick tectum territory along dorsoventral axis by Sonic hedgehog

Chick midbrain comprises two major components along the dorsoventral axis, the tectum and the tegmentum. The alar plate differentiates into the optic tectum, while the basal plate gives rise to the tegmentum. It is largely unknown how the differences between these two structures are molecularly controlled during the midbrain development. The secreted protein Sonic hedgehog (Shh) produced in the notochord and floor plate induces differentiation of ventral cell types of the central nervous system. To evaluate the role of Shh in the establishment of dorsoventral polarity in the developing midbrain, we have ectopically expressed Shh unilaterally in the brain vesicles including whole midbrain of E1.5 chick embryos in ovo. Ectopic Shh repressed normal growth of the tectum, producing dorsally enlarged tegmentum region. In addition, the expression of several genes crucial for tectum formation was strongly suppressed in the midbrain and isthmus. Markers for midbrain roof plate were inhibited, indicating that the roof plate was not fully generated. After E5, the tectum territory of Shh-transfected side was significantly reduced and was fused with that of untransfected side. Moreover, ectopic Shh induced a considerable number of SC1-positive motor neurons, overlapping markers such as HNF-3(beta) (floor plate), Isl-1 (postmitotic motor neuron) and Lim1/2. Dopaminergic and serotonergic neurons were also generated in the dorsally extended region. These changes indicate that ectopic Shh changed the fate of the mesencephalic alar plate to that of the basal plate, suppressing the massive cell proliferation that normally occurs in the developing tectum. Taken together our results suggest that Shh signaling restricts the tectum territory by controlling the molecular cascade for tectum formation along dorsoventral axis and by regulating neuronal cell diversity in the ventral midbrain.     

Oculomotor neuroblast migration in the chick embryo in the absence of tecto-tegmental fibers.

This study investigated a hypothesized relationship between the migration of the oculomotor complex, particularly the ventromedial subnucleus, and the presence of the tecto-tegmental fiber system in the chick embryo. It has been suggested that the presence of these fibers at the time of the initiation and continuation of neuroblast migration in this system might serve as some sort of stimulus influencing or guiding this migration. In order to examine this hypothesis, the dorsal midbrain was ablated in stage 12 embryos, thus removing the source of the tecto-tegmental fibers prior to the development of the oculomotor complex. The results indicated that in the complete absence of the tecto-tegmental fibers, all oculomotor subnuclei migrated normally, attained their normal terminal locations, and appeared to differentiate normally. These results are discussed in relation to other studies of possible extracellular influences during early neurogenesis and the possible importance of within-system versus without-system influences is considered.     

Gonadotropin-releasing hormone (GnRH) pathways and reproductive control in elasmobranchs

Synopsis Immunoreactive (ir) gonadotropin-releasing hormone (GnRH) is localized in many neurons of the terminal nerve (TN) and midbrain tegmentum, while few ir-cells are observed in the preoptic area and ventral hypothalamus. The paucity of preoptic ir-cells may relate to an unusual feature of the elasmobranch pituitary, i.e. a lack of portal control of gonadotropin-producing cells. TN and midbrain GnRH-ir neurons may be major sources of GnRH used to modulate or otherwise control both pituitary and brain cells via delivery through the systemic circulation. These ir-nuclei also appear to directly innervate CNS regions (the preoptic area, habenula and clasper control area of the spinal cord) involved in sexual functions. Important regulatory mechanisms, represented by interactions between GnRH pathways and sex-steroid concentrating neurons, are likely to occur in the preoptic area, habenula and midbrain tegmentum.     

Differential expression of the keratan sulphate proteoglycan,keratocan, during chick corneal embryogenesis

Keratan sulphate (KS) proteoglycans (PGs) are key molecules in the connective tissue matrix of the cornea of the eye, where they are believed to have functional roles in tissue organisation and transparency. Keratocan, is one of the three KS PGs expressed in cornea, and is the only one that is primarily cornea-specific. Work with the developing chick has shown that mRNA for keratocan is present in early corneal embryogenesis, but there is no evidence of protein synthesis and matrix deposition. Here, we investigate the tissue distribution of keratocan in the developing chick cornea as it becomes compacted and transparent in the later stages of development. Indirect immunofluorescence using a new monoclonal antibody (KER-1) which recognises a protein epitope on the keratocan core protein demonstrated that keratocan was present at all stages investigated (E10–E18), with distinct differences in localisation and organisation observed between early and later stages. Until E13, keratocan appeared both cell-associated and in the stromal extracellular matrix, and was particularly concentrated in superficial tissue regions. By E14 when the cornea begins to become transparent, keratocan was located in elongate arrays, presumably associated along collagen fibrils in the stroma. This fibrillar label was still concentrated in the anterior stroma, and persisted through E15–E18. Presumptive Bowman’s layer was evident as an unlabelled subepithelial zone at all stages. Thus, in embryonic chick cornea, keratocan, in common with sulphated KS chains in the E12–E14 developmental period, exhibits a preferential distribution in the anterior stroma. It undergoes a striking reorganisation of structure and distribution consistent with a role in relation to stromal compaction and corneal transparency. E. Claire Gealy and Briedgeen C. Kerr were joint first authors.     

Expression of Lrrn1 marks the prospective site of the zona limitans thalami in the early embryonic chicken diencephalon

An unknown chicken gene selected from a published substractive hybridization screen (GenBank Accession No. ; [Christiansen, J.H., Coles, E.G., Robinson, V., Pasini, A., Wilkinson, D.G., 2001. Screening from a subtracted embryonic chick hindbrain cDNA library: identification of genes expressed during hindbrain, midbrain and cranial neural crest development. Mech. Dev. 102, 119-133.]) was deemed of interest because of its dynamic pattern of expression across the forebrain and midbrain regions. A 528bp fragment cloned from early chick embryo cDNA and used for in situ hybridization corresponded to part of the 3' untranslated region of the chicken gene Leucine-rich repeat neuronal protein 1 (Lrrn1). The expression of this gene, mapped in the embryonic chick brain between stages HH10 and HH26, apparently preconfigures the zona limitans thalami site before overt formation of this boundary structure. Apart of colateral expression in the forebrain, midbrain and hindbrain basal plate, the most significant expression of Lrrn1 was found early on across the entire alar plate of midbrain and forebrain (HH10). This unitary domain soon divides at HH14 into a rostral part, across alar secondary prosencephalon and prospective alar prosomere 3 (prethalamus; caudal limit at the prospective zona limitans), and a caudal part in alar prosomere 1 (pretectum) and midbrain. The rostral forebrain domain later downregulates gradually most extratelencephalic signal of Lrrn1, but the rostral shell of zona limitans retains expression longer. Expression in the caudal alar domain also changes by downregulation within its pretectal subdomain. Caudally, the midbrain domain ends at the isthmo-mesencephalic junction throughout the studied period. Embryonic Lrrn1 signal also appears in the somites and in the otic vesicle.     

FGF17b and FGF18 have different midbrain regulatory properties from FGF8b or activated FGF receptors

Early patterning of the vertebrate midbrain and cerebellum is regulated by a mid/hindbrain organizer that produces three fibroblast growth factors (FGF8, FGF17 and FGF18). The mechanism by which each FGF contributes to patterning the midbrain, and induces a cerebellum in rhombomere 1 (r1) is not clear. We and others have found that FGF8b can transform the midbrain into a cerebellum fate, whereas FGF8a can promote midbrain development. In this study we used a chick electroporation assay and in vitro mouse brain explant experiments to compare the activity of FGF17b and FGF18 to FGF8a and FGF8b. First, FGF8b is the only protein that can induce the r1 gene Gbx2 and strongly activate the pathway inhibitors Spry1/2, as well as repress the midbrain gene Otx2. Consistent with previous studies that indicated high level FGF signaling is required to induce these gene expression changes, electroporation of activated FGFRs produce similar gene expression changes to FGF8b. Second, FGF8b extends the organizer along the junction between the induced Gbx2 domain and the remaining Otx2 region in the midbrain, correlating with cerebellum development. By contrast, FGF17b and FGF18 mimic FGF8a by causing expansion of the midbrain and upregulating midbrain gene expression. This result is consistent with Fgf17 and Fgf18 being expressed in the midbrain and not just in r1 as Fgf8 is. Third, analysis of gene expression in mouse brain explants with beads soaked in FGF8b or FGF17b showed that the distinct activities of FGF17b and FGF8b are not due to differences in the amount of FGF17b protein produced in vivo. Finally, brain explants were used to define a positive feedback loop involving FGF8b mediated upregulation of Fgf18, and two negative feedback loops that include repression of Fgfr2/3 and direct induction of Spry1/2. As Fgf17 and Fgf18 are co-expressed with Fgf8 in many tissues, our studies have broad implications for how these FGFs differentially control development.     

The characteristics of the cell aggregation of the tectum mesencephali in chick embryos and of the cerebral cortex in newborn rats in mixed cultures]

To determine species-specific cells in mixed culture, obtained after simultaneous cultivation of dissociated cells of chick embryo midbrain tegmentum and newborn albino rat cerebral cortex, various DNA amounts were used for testing chick and rat cells. The study of cell structure of the aggregates revealed that in addition to the aggregates consisting of cells belonging to only one species of animals, chimera aggregates also exist made of cells of both species of animals, basically of chick embryo glial cells and newborn rat neurons.     

Spatiotemporal expression of sonic hedgehog signalling molecules in the embryonic mesencephalic dopaminergic neurons

Midbrain dopaminergic neurons (mDA) play an important role in controlling the voluntary motor movement, reward, and emotion-based behaviour. Differentiation of mDA neurons from progenitors depends on several secreted proteins, such as sonic hedgehog (SHH). The present study attempted to elucidate the possible role(s) of some SHH signaling components (Ptch1, Gli1, Gli2 and Gli3) in the spatiotemporal development of mDA neurons along the rostrocaudal axis of the midbrain and their possible roles in differentiation and survival of mDA neurons and the significance of using in vitro models for studying the development of mDA neurons. At E12 and E14, only Ptch1 and Gli1 were expressed in ventrolateral midbrain domains. All examined SHH signalling molecules were not detected in mDA area. Whereas, in MN9D cells, many SHH signalling molecules were expressed and co-localized with the dopaminergic marker; tyrosine hydroxylase (TH), and their expression were upregulated with SHH treatment of the MN9D cells. These results suggest that mDA neurons differentiation and survival might be independent of SHH in the late developmental stages (E12-18). Besides, MN9D cell line is not the ideal in vitro model for investigating the differentiation of mDA and hence, the ventral midbrain primary culture might be favored over MN9D line.     

A DiI-tracing study of the neural connections of the pineal organ in two elasmobranchs (Scyliorhinus canicula and Raja montagui) suggests a pineal projection to the midbrain GnRH-immunoreactive nucleus

The pineal organ of elasmobranchs is an elongated photoreceptive organ. In order to investigate the afferent and efferent connections of the pineal organ of two elasmobranchs, the skate (Raja montagui) and the dogfish (Scyliorhinus canicula), a fluorescent carbocyanine (DiI) was applied to the pineal organ of paraformaldehyde-fixed brains. This application strongly labeled the pineal tract, which formed extensive bilateral projections. In both species, the pinealofugal fibers coursed to the dorsomedial thalamus, the medial pretectal area, the posterior tubercle, and the medial mesencephalic tegmentum and branched profusely in these areas. Application of DiI to the pineal organ also labeled occasional perikarya in the dorsomedial thalamus, posterior commissural region, posterior tubercle, and mesencephalic tegmentum. A comparison of these results with those of immunocytochemical analyses of the dogfish brain with an anti-salmon gonadotropin-releasing hormone (sGnRH) antiserum revealed a close topographical relation between the pineal projections and the midbrain sGnRH-immunoreactive (ir) nucleus, the only structure in the dogfish brain that contained sGnRHir neurons. This and the widespread distribution of sGnRHir fibers in the brain suggest that the midbrain sGnRHir nucleus is a part of the secondary pineal pathways and may be involved in light-mediated pineal regulation of brain function. Although GnRH distribution has not been studied in the skate, a midbrain GnRHir nucleus has been identified in three other elasmobranchs, including a skate relative. The probable existence of direct pineal projections to the GnRHir midbrain nucleus in elasmobranchs and other anamniotes is discussed.     

Appearance and distribution of fetal brain macrophages in mice

Summary A study on the localization of fetal and neonatal brain macrophages of mice from embryonic day 10 (E10) to postnatal day 21 (P21) was carried out immunohistochemically using a monoclonal antibody against a macrophage differentiation antigen (Mac-1) and the labeled avidin-biotin technique. In the central nervous system, the macrophages recognized first were mainly located in the choroid plexuses of the fourth and lateral ventricles at E14. Their number increased at E17–P3 and gradually decreased thereafter. In the cerebral parenchyma, a few macrophages appeared at E14 in the matrix cell layer. They were also detected in the migrating zone at E15, E17 and in the cortical plate at E19. Mapping of positive cells at the stage of neuroblast formation (E15, E17, E19) disclosed the precise distribution of cerebral macrophages. The macrophages that appeared first in the choroid plexuses at E15 may be derived from the subarachnoid vessels, which extend into the stroma of the choroid plexuses when the matrix cell layer invaginates into the lateral ventricle to form the choroid plexuses. Almost all of the macrophages recognized in the cerebral parenchyma disappeared at P9 when the cytoarchitecture seemed to be completed. In the cerebellum, which develops later than the cerebrum, macrophages appeared after birth and were located mainly in the internal granular layer. The brain macrophages always appeared in the regions where cell proliferation and brain remodeling are most active at each stage. These findings suggest that fetal and neonatal brain macrophages may play an important role in scavenging degenerated cells and cell debris during histogenesis of the central nervous system.     

HLH-14 is a C. elegans achaete-scute protein that promotes neurogenesis through asymmetric cell division

Achaete-Scute basic helix-loop-helix (bHLH) proteins promote neurogenesis during metazoan development. In this study, we characterize a C. elegans Achaete-Scute homolog, HLH-14. We find that a number of neuroblasts express HLH-14 in the C. elegans embryo, including the PVQ/HSN/PHB neuroblast, a cell that generates the PVQ interneuron, the HSN motoneuron and the PHB sensory neuron. hlh-14 mutants lack all three of these neurons. The fact that HLH-14 promotes all three classes of neuron indicates that C. elegans proneural bHLH factors may act less specifically than their fly and mammalian homologs. Furthermore, neural loss in hlh-14 mutants results from a defect in an asymmetric cell division: the PVQ/HSN/PHB neuroblast inappropriately assumes characteristics of its sister cell, the hyp7/T blast cell. We argue that bHLH proteins, which control various aspects of metazoan development, can control cell fate choices in C. elegans by regulating asymmetric cell divisions. Finally, a reduction in the function of hlh-2, which encodes the C. elegans E/Daughterless bHLH homolog, results in similar neuron loss as hlh-14 mutants and enhances the effects of partially reducing hlh-14 function. We propose that HLH-14 and HLH-2 act together to specify neuroblast lineages and promote neuronal fate.     

FGF8 can activate Gbx2 and transform regions of the rostral mouse brain into a hindbrain fate.

The mid/hindbrain junction region, which expresses Fgf8, can act as an organizer to transform caudal forebrain or hindbrain tissue into midbrain or cerebellar structures, respectively. FGF8-soaked beads placed in the chick forebrain can similarly induce ectopic expression of mid/hindbrain genes and development of midbrain structures (Crossley, P. H., Martinez, S. and Martin, G. R. (1996) Nature 380, 66-68). In contrast, ectopic expression of Fgf8a in the mouse midbrain and caudal forebrain using a Wnt1 regulatory element produced no apparent patterning defects in the embryos examined (Lee, S. M., Danielian, P. S., Fritzsch, B. and McMahon, A. P. (1997) Development 124, 959-969). We show here that FGF8b-soaked beads can not only induce expression of the mid/hindbrain genes En1, En2 and Pax5 in mouse embryonic day 9.5 (E9.5) caudal forebrain explants, but also can induce the hindbrain gene Gbx2 and alter the expression of Wnt1 in both midbrain and caudal forebrain explants. We also show that FGF8b-soaked beads can repress Otx2 in midbrain explants. Furthermore, Wnt1-Fgf8b transgenic embryos in which the same Wnt1 regulatory element is used to express Fgf8b, have ectopic expression of En1, En2, Pax5 and Gbx2 in the dorsal hindbrain and spinal cord at E10.5, as well as exencephaly and abnormal spinal cord morphology. More strikingly, Fgf8b expression in more rostral brain regions appears to transform the midbrain and caudal forebrain into an anterior hindbrain fate through expansion of the Gbx2 domain and repression of Otx2 as early as the 7-somite stage. These findings suggest that normal Fgf8 expression in the anterior hindbrain not only functions to maintain development of the entire mid/hindbrain by regulating genes like En1, En2 and Pax5, but also might function to maintain a metencephalic identity by regulating Gbx2 and Otx2 expression.     

Assembly of contractile and cytoskeletal elements in developing smooth muscle cells.

Specific developmental changes in smooth muscle were studied in gizzards obtained from 6-, 8-, 10-, 12-, 14-, 16-, 18-, and 20-day chick embryos and from 1- and 7-day posthatch chicks. Myoblasts were actively replicating in tissue from 6-day embryos. Cytoplasmic dense bodies (CDBs) first appeared at Embryonic Day 8 (E8) and were recognized as patches of increased electron density that consisted of actin filaments (AFs), intermediate filaments (IFs), and cross-connecting filaments (CCFs). Although the assembly of CDBs was not synchronized within a cell, the number, size, and electron density of CDBs increased as age increased. Membrane-associated dense bodies (MADBs) also could be recognized at E8. The number and size of MADBs increased as age increased, especially after E16. Filaments with the diameter of thick filaments first appeared at E12. Smooth muscle cells were able to divide as late as E20. The axial intermediate filament bundle (IFB) could first be identified in 1-day posthatch cells and became larger and more prominent in 7-day posthatch cells. Immunogold labeling of 1- and 7-day posthatch cells with anti-desmin showed that the IFB contained desmin IFs. The developmental events during this 23-day period were classified into seven stages, based primarily on the appearance and the growth of contractile and cytoskeletal elements. These stages are myoblast proliferation, dense body appearance, thick filament appearance, dense body growth, muscle cell replication, IFB appearance, and appearance of adult type cells. Smooth muscle cells in each stage express similar developmental characteristics. The mechanism of assembly of myofilaments and cytoskeletal elements in smooth muscle in vivo indicates that myofilaments (AFs and thick filaments) and filament attachment sites (CDBs and MADBs) are assembled before the axial IFB, a major cytoskeletal element.     

Ontogenic development of three GnRH systems in the brain of a pleuronectiform fish, barfin flounder

A pleuronectiform fish, the barfin flounder Verasper moseri, has three molecular forms of gonadotropin-releasing hormone (GnRH) in the brain, salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and seabream GnRH (sbGnRH). To elucidate the ontogenic origin of the neurons that produce these GnRH molecules, the development of three GnRH systems was examined by in situ hybridization and immunocytochemistry. Neuronal somata that express sGnRH mRNA were detected first in the vicinity of the olfactory epithelium 21 days after hatching (Day 21), and then in the transitional area between the olfactory nerve and olfactory bulb and the terminal nerve ganglion on Day 28. cGnRH-II mRNA-expressing neuronal somata were first identified in the midbrain tegmentum near the ventricle on Day 7. cGnRH-II-immunoreactive (ir) fibers were first found in the brain on Day 7. sbGnRH mRNA-expressing neuronal somata were first detected in the preoptic area on Day 42. sbGnRH-ir fibers were localized in the preoptic area-hypothalamus, and formed a distinctive bundle of axons projecting to the pituitary on Day 70. These results indicate that three forms of GnRH neurons have separate embryonic origins in the barfin flounder as in other perciform fish such as tilapia Oreochromis niloticus and red seabream Pagrus major: sGnRH, cGnRH-II and sbGnRH neurons derive from the olfactory placode, the midbrain tegmentum near the ventricle and the preoptic area, respectively.     

Evidence for a cyclic renewal of lymphocyte precursor cells in the embryonic chick thymus

Experiments involving sequential transplantations of the chick embryonic thymus at E9 to E12 into a first 3-day host quail embryo and then into a second chick host allowed demonstration of the cyclic periodicity of hemopoietic cell seeding of the embryonic thymus. After a first wave of colonization occurring between E6.5 and E8, the thymus becomes refractory to hemopoietic cell entry for about 4 days. It resumes its capacity to be seeded by a second wave of blood-borne stem cells at E12. After a second period of non receptivity starting at E14, a third wave of incoming cells reaches the thymus around E18. Therefore, with a slightly different periodicity, the same cyclic mechanism regulates the renewal of lymphocytes in chick and quail embryos. Quail hemopoietic cells were immunostained in the chimeric thymuses, with a species specific monoclonal antibody (anti-MB1) which recognizes a common surface antigenic determinant on all endothelial and blood cells of the quail (except erythrocytes). Two steps could thus be distinguished in the seeding process. When the thymus becomes receptive for hemopoietic cells, the latter first accumulate in the intrathymic blood vessels before penetrating massively in the thymic parenchyma. The quail chick-chimera system combined with the use of a species- and cell-type-specific antibody provides a unique tool for studying thymic colonization by lymphocyte precursors.     

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms^1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson''s disease, Schizophrenia, depression, and dementia^1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs^6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos^8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points^10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick β-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes¹⁴. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.     

Distribution of the small molecular weight GTP-binding proteins Rac1, Cdc42, RhoA and RhoB in the developing chick retina

Immunohistochemistry was used to determine the distribution of Rac1, Cdc42, RhoA and RhoB GTPases during development of the chick retina. All proteins appear as early as embryonic day 5 (E5) in cells of the vitreal margin, E7–8 in cells of the inner third of the inner nuclear layer and E9–10 in photoreceptors. From E10 until hatching, RhoA, Rac1 and Cdc42 were seen in perikarya and/or processes of amacrine, ganglion cells, and photoreceptors. Rho proteins were also observed in retinal Müller cells, with different distributions. RhoB showed a transient expression, being severely down regulated after E18. The distribution pattern of Rho proteins during the development of the chick retina suggests a concerted role in the differentiation of specific cell types, and probably during synaptogenesis.