Chick Diencephalon Research Articles

Myeloperoxidase expression in diencephalon is potentially associated with fear-related behavior in chicks of laying hen.

Preventing feather pecking (FP) in adult laying hens is important for the welfare of intensively poultry farming. Fear-related behavior in growing female layer chicks may predict FP in adult hens. In this study, in two representative laying breeds (White Leghorn [WL] and Rhode Island Red [RIR]) that have different FP frequencies, we identified a candidate gene associated with fear-related behavior in chicks and FP in adult hens. In the tonic immobility test and open-field test, the behavioral activity was lower in WL chicks than in RIR chicks (P < 0.01), suggesting that WL chicks were more fearful than RIR chicks. Based on previous studies, 51 genes that have been found to be differentially expressed in the brain between high- and low-FP populations were chosen, and their expression levels were screened in the chick diencephalon. This analysis revealed that myeloperoxidase (MPO) gene expression level was higher in WL chicks than that in RIR chicks (P < 0.05). Furthermore, STRING analysis predicted the gene network including MPO and MPO-related genes and revealed the association of these genes with fear-related behavior. These results suggest that MPO is potentially associated with fear-related behavior in growing female layer chicks and FP in adult hens.

Tissue interactions in the developing chick diencephalon

BackgroundThe developing vertebrate brain is patterned first by global signalling gradients that define crude anteroposterior and dorsoventral coordinates, and subsequently by local signalling centres (organisers) that refine cell fate assignment within pre-patterned regions. The interface between the prethalamus and the thalamus, the zona limitans intrathalamica (ZLI), is one such local signalling centre that is essential for the establishment of these major diencephalic subdivisions by secreting the signalling factor Sonic hedgehog. Various models for ZLI formation have been proposed, but a thorough understanding of how this important local organiser is established is lacking.ResultsHere, we describe tissue explant experiments in chick embryos aimed at characterising the roles of different forebrain areas in ZLI formation. We found that: the ZLI becomes specified unexpectedly early; flanking regions are required for its characteristic morphogenesis; ZLI induction can occur independently from ventral tissues; interaction between any prechordal and epichordal neuroepithelial tissue anterior to the midbrain-hindbrain boundary is able to generate a ZLI; and signals from the dorsal diencephalon antagonise ZLI formation. We further show that a localised source of retinoic acid in the dorsal diencephalon is a likely candidate to mediate this inhibitory signal.ConclusionOur results are consistent with a model where planar, rather than vertical, signals position the ZLI at early stages of neural development and they implicate retinoic acid as a novel molecular cue that determines its dorsoventral extent.

Altered BMP signaling disrupts chick diencephalic development

The diencephalon is the caudal part of the forebrain and is organized into easily identifiable clusters of neurons called nuclei. Neurons in different nuclei project to discrete brain regions. Thus precise organization of the nuclei during forebrain development is necessary to build accurate neural circuits. How diencephalic development is regulated is poorly understood. BMP signaling participates in central nervous system patterning and development at many levels along the neural axis. Based on their expression we hypothesized BMPs play a role in diencephalic development. To test this hypothesis, we electroporated constitutively active and dominant negative forms of type I BMP receptors ( Bmpr1a and Bmpr1b) into the embryonic chick forebrain. Ectopic induction of BMP signaling through constitutively active forms of the type I BMP receptors perturbs the normal gene expression patterns in the diencephalon and increases apoptotic cell death. These defects lead to disorganization of the diencephalic nuclei, suggesting BMP signaling is sufficient to modify diencephalic development. Loss-of-function studies, using dominant negative forms of Bmpr1a and Bmpr1b, indicate type I BMP receptors are necessary for normal eye and craniofacial development. However, they do not appear to be required for normal diencephalic development. In summary, our data indicate that while not necessary, BMP signaling via Bmpr1a and Bmpr1b, is sufficient to modify nuclear organization in the chick diencephalon.

Expression pattern of cLhx2b, cZic1 and cZic3 in the developing chick diencephalon

The diencephalon is the caudal part of the developing forebrain that gives rise to the epithalamus, thalamus, hypothalamus and retina. The mature diencephalon is functionally and anatomically parceled into well-defined nuclei. In an effort to understand how this region of the brain develops we examined the expression of several transcription factors during development. We find that the LIM homeodomain transcription factor, cLhx2b, and the zinc finger transcription factors, cZic1 and cZic3, are predominantly expressed in partially overlapping domains of the chick dorsal diencephalon. Interestingly, a correlation exists between their expression in neuroepithelial progenitor cells at early stages and in the differentiated nuclei at progressively more advanced stages of development.

Differential Expression of Eph Receptors and Ephrins Correlates with the Formation of Topographic Projections in Primary and Secondary Visual Circuits of the Embryonic Chick Forebrain

Repulsion plays a fundamental role in the establishment of a topographic map of the chick retinotectal projections. This has been highlighted by studies demonstrating the role of opposing gradients of the EphA3 receptor tyrosine kinase on retinal axons and two of its ligands, ephrin-A2 and ephrin-A5, in the tectum. We have analyzed the distribution of these two ephrins in other retinorecipient structures in the chick diencephalon and mesencephalon during the period when visual connections are being established. We have found that both ephrin-A2 and ephrin-A5 and their receptors EphA4 and EphA7 are expressed in gradients whose orientation is consistent with the topography of the nasotemporal axis of the respective retinofugal projections. In addition, their distribution suggests that receptor–ligand interactions may be involved in the organization of connections between the different primary visual centers and, thus, in the topographic organization of secondary visual projections. Interestingly, where projections lack a clear topographic representation, a uniform expression of the Eph–ephrin molecules was observed. Finally, we also show that a similar patterning mechanism may be implicated in the transfer of visual information to the telencephalon. These results suggest a conserved function for EphA receptors and their ligands in the elaboration of topographic maps at multiple levels of the visual pathway.

DM-GRASP Is Necessary for Nonradial Cell Migration during Chick Diencephalic Development

Cell migration is fundamental to normal CNS development. Radial migration, along radial glial fibers, has been the principal pathway studied, however, nonradial or tangential cell migration has increasingly been identified at all levels of the CNS. Receptors, cell adhesion molecules, and extracellular matrix molecules have all been shown to participate in radial cell migration. In contrast, the molecular basis of nonradial cell migration has only recently begun to be elucidated. Using replication defective retroviral vectors we have determined the location and time when nonradial cell migration begins in the developing chick diencephalon. We have identified three molecules that are expressed in spatially and temporally restricted domains that are consistent with them playing a role in nonradial cell migration. One of these molecules, DM-GRASP, a transmembrane protein with five extracellular Ig domains, is expressed on the nonradially migrating cells in addition to axons. To test the hypothesis that DM-GRASP participates in guiding nonradial cell migration, we injected a replication-defective retroviral vector used for lineage tracing followed by a DM-GRASP blocking antibody. Embryos injected with the blocking antibody showed a near complete block in nonradial cell migration specifically where DM-GRASP is expressed. Furthermore, morphological analyses revealed disruption of the normal architecture of the diencephalon indicating nonradial cell migration is necessary for normal morphological development of the brain. Our data indicate that DM-GRASP is necessary for nonradial cell migration in the chick diencephalon and have provided a system to further explore the function of nonradial cell migration during CNS development.

The Dispersion of Clonally Related Cells in the Developing Chick Telencephalon

Lineage analysis in the chick telencephalon was carried out using a library of retroviral vectors. Clones were analyzed in posthatch day 14–21 animals for the phenotype and final locations of sibling cells. Clones often contained multiple types of neurons and glia. Clones of more than four cells almost always crossed functional boundaries. They were dispersed primarily along the rostrocaudal axis or in multiple directions, e.g., along the rostrocaudal and mediolateral axes. In order to begin to understand how the final patterns of dispersion were reached, embryonic tissue was examined. Radial migration, apparently supported by radial glial cells, occurred within the proliferative zones in all clones. In contrast to the migration of cells in the mammalian telencephalon, no tangential migration within the proliferative zones was observed at any age examined. However, beginning at embryonic day 4.5, tangential migration in the mantle zone in multiple directions was observed among the majority of clones. This type of migration occurred as soon as a mantle zone became apparent. It appeared that the tangential migration was not along radial glial processes. As in the mammalian telencephalon and chick diencephalon, dispersion among clonally related cells in the chick telencephalon is frequent, is extensive, and results from tangential migration in a variety of directions.

Cell migration in the developing chick diencephalon.

We previously reported that retrovirally marked clones in the mature chick diencephalon were widely dispersed in the mediolateral, dorsoventral and rostrocaudal planes. The current study was undertaken to define the migration routes that led to the dispersion. Embryos were infected between stages 10 and 14 with a retroviral stock encoding alkaline phosphatase and a library of molecular tags. Embryos were harvested 2.5-5.5 days later and the brains were fixed and serially sectioned. Sibling relationships were determined following PCR amplification and sequencing of the molecular tag. On embryonic day 4, all clones were organized in radial columns spanning the neuroepithelium, which was composed primarily of a ventricular zone at this age. No tangential migration was seen in the ventricular zone. On embryonic day 5, most clones remained radial with many cells located in the ventricular zone; however, a few clones had cells migrating perpendicular to the radial column, in either a rostrocaudal or dorsoventral direction. The tangential migration began just beyond the basal limit of the ventricular zone. On embryonic days 6 and 7, many clones had cells migrating perpendicular to the radial column, which spanned from the ventricular to the pial surface. The migrating cells appeared to be aligned along axes that were perpendicular to the radial column. Using a combination of DiI tracing, immunohistochemistry and electron microscopy, we have determined that axonal tracts are present and are aligned with the migrating cells, suggesting that they support the non-radial cell migration. These data indicate that migration along pathways independent of radial glia occur outside of the ventricular zone in more than 50% of the clones in the chick diencephalon.

Clones in the chick diencephalon contain multiple cell types and siblings are widely dispersed.

The thalamus, hypothalamus and epithalamus of the vertebrate central nervous system are derived from the embryonic diencephalon. These regions of the nervous system function as major relays between the telencephalon and more caudal regions of the brain. Early in development, the diencephalon morphologically comprises distinct units known as neuromeres or prosomeres. As development proceeds, multiple nuclei, the functional and anatomical units of the diencephalon, derive from the neuromeres. It was of interest to determine whether progenitors in the diencephalon give rise to daughters that cross nuclear or neuromeric boundaries. To this end, a highly complex retroviral library was used to infect diencephalic progenitors. Retrovirally marked clones were found to contain neurons, glia and occasionally radial glia. The majority of clones dispersed in all directions, resulting in sibling cells populating multiple nuclei within the diencephalon. In addition, several distinctive patterns of dispersion were observed. These included clones with siblings distributed bilaterally across the third ventricle, clones that originated in the lateral ventricle, clones that crossed neuromeric boundaries, and clones that crossed major boundaries of the developing nervous system, such as the diencephalon and mesencephalon. These findings demonstrate that progenitor cells in the diencephalon are multipotent and that their daughters can become widely dispersed.

Negative regulatory elements upstream of a novel exon of the neuronal nicotinic acetylcholine receptor alpha 2 subunit gene.

The expression of the nicotinic acetylcholine receptor alpha 2 subunit gene is highly restricted to the Spiriform lateralis nucleus of the Chick diencephalon. As a first step toward understanding the molecular mechanism underlying this regulation, we have investigated the structural and regulatory properties of the 5' sequence of this gene. A strategy based on the ligation of an oligonucleotide to the first strand of the cDNA (SLIC) followed by PCR amplification was used. A new exon was found approximately 3kb upstream from the first coding exon, and multiple transcription start sites of the gene were mapped. Analysis of the flanking region shows many consensus sequences for the binding of nuclear proteins, suggesting that the 1 kb flanking region contains at least a portion of the promoter of the gene. We have analysed the negative regulatory elements present within this region and found that a silencer region located between nucleotide -144 and +76 is active in fibroblasts as well as in neurons. This silencer is composed of six tandem repeat Oct-like motifs (CCCCATGCAAT), but does not bind any member of the Oct family. Moreover these motifs were found to act as a silencer only when they were tandemly repeated. When two, four or five motifs were deleted, the silencer activity of the motifs unexpectedly became an enhancer activity in all cells we have tested.

Immunohistochemical localization of nicotinic acetylcholine receptor subunits in the mesencephalon and diencephalon of the chick (Gallus gallus).

Monoclonal antibodies against two alpha-bungarotoxin-binding subunits (alpha 7 and alpha 8) of the nicotinic acetylcholine receptors (nAChRs) were used as immunohistochemical probes to map their distribution in the chick diencephalon and mesencephalon. The distribution of the alpha 7 and alpha 8 nAChR subunits was compared to the distribution of immunoreactivity produced by a monoclonal antibody against the beta 2 structural subunit of the nAChRs. Structures that contained high numbers of alpha 7-like immunoreactive (LI) somata included the intergeniculate leaflet, nucleus intercalatus thalami, nucleus ovoidalis, organum paraventricularis, nucleus rotundus, isthmic nuclei, nucleus trochlearis, oculomotor complex, nucleus interstitio-pretecto-subpretectalis, stratum griseum centrale of the optic tectum, and nucleus semilunaris. Neuropil staining for alpha 7-LI was intense in the nucleus dorsomedialis hypothalami, nucleus geniculatus lateralis ventralis, griseum tecti, isthmic nuclei, nucleus lentiformis mesencephali, nucleus of the basal optic root, and stratum griseum et fibrosum superficiale of the tectum. High numbers of alpha 8-LI somata were found in the stratum griseum et fibrosum superficiale of the tectum and the nucleus interstitio-pretecto-subpretectalis, and intense neuropil staining for alpha 8-LI was found in the dorsal thalamus, nucleus geniculatus lateralis ventralis, lateral hypothalamus, griseum et fibrosum superficiale of the tectum. High numbers of beta 2-LI somata were found only in the nucleus spiriformis lateralis, whereas neuropil staining for beta 2-LI was intense in the nucleus geniculatus lateralis ventralis, nucleus suprachiasmaticus, nucleus lateralis anterior, nucleus habenularis lateralis, area pretectalis, griseum tecti, nucleus lentiformis mesencephalis, nucleus externus, and nucleus interpeduncularis, and in the stratum griseum centrale, stratum griseum et fibrosum superficiale, and stratum opticum of the tectum. These results indicate that there are major disparities in the localization of the alpha-bungarotoxin-binding alpha 7 and alpha 8 nAChR subunits and the beta 2 structural nAChR subunit in the chick diencephalon and mesencephalon. These nAChR subunits appear, however, to coexist in several regions of the chick brain.

Differential expression of the neuronal acetylcholine receptor α2 subunit gene during chick brain development

In situ hybridization histochemistry reveals localized expression of the nicotinic acetylcholine receptor (nAChR) α2 subunit mRNA restricted to the lateral spiriform nucleus cleus (SpL) of the chick diencephalon. The α2 nAChR transcripts are not detected in immature SpL neurons at 4.5-5 days of embryonic development. They begin to accumulate in the SpL at embryonic day 11 and increase until the newborn stage. Specific α2 cDNA amplification by the polymerase chain reaction shows that during this period, the absolute content of α2 mRNA increases about 20-fold. The expression of the α2 nAChR gene is thus developmentally regulated and appears concomitant with the entry of cholinergic fibers into the SpL, as demonstrated by choline acetyltransferase immunohistochemistry.

Acetylcholinesterase-histochemical differential staining of subdivisions within the nucleus rotundus in the chick

Histochemical mapping of AChE activity in the chick diencephalon shows differential staining of several subregions within the nucleus rotundus. The topography and extent of these subdivisions were studied in transverse, horizontal and sagittal sections. A correlation with rotundic hodologic subdivisions reported in the literature is feasible, whereas several other chemoarchitectonic or functional markers show a homogeneous distribution throughout the n. rotundus. Moreover, cholinergic markers do not detect cholinergic afferents within the rotundic neuropile. Late embryonic appearance of the AChE heterogeneity suggests a modulation of neuropile AChE levels subsequent to synaptogenetic adjustment of differential hodology.

Retinal axons innervate the embryonic chick diencephalon in an in-vivo-like pattern in explant co-cultures.

Explants of chick embryo diencephalon co-cultured with explants of retina display areas of complex neuropil containing large retinal-like endings which establish synaptic contact with conventional and presynaptic dendrites. Transection of fibre bundles linking retinal to diencephalic explants results in the degeneration of endings of this type, suggesting that axons of extrinsic (retinal) origin innervate the diencephalic explants in an in-vivo-like manner.

Neurogenesis in the chick ventral lateral geniculate and ectomammillary nuclei: relationship of soma size to birthdate.

This study had 3 objectives: to determine if there are gradients of neuron production in the chick diencephalon and mesencephalon, to determine if large neurons are produced before small neurons, and to determine the period of neurogenesis in the ventral lateral geniculate nucleus (GLv) and ectomammillary nucleus (EMN). Tritiated thymidine autoradiography and a cumulative labeling analysis were used in determining neuron birthdates. In our studies the diencephalon, mesencephalon, and EMN did not exhibit clearcut gradients of neuron proliferation. The GLv possessed an inside to outside laminar gradient and a more subtle caudal to rostral gradient of neurogenesis. There was a consistent negative correlation between neuron size and embryonic age among nuclear groups of the diencephalon, in general, and within the GLv and EMN in particular. The neurons of the GLv and EMN were produced from the third to the seventh day of incubation. It was concluded that there is an irregular sequence of production of nuclear groups in the diencephalon and mesencephalon rather than a series of recognizable neurogenetic gradients. Neuron size, however, is an important factor in predicting the order of neuron production.

Escape, hiding, and freezing behaviour elicited by electrical stimulation of the chick diencephalon.

A largely medial escape, hide and freeze (EHF) system has been plotted in the chick diencephalon, from which escape or freezing can be elicited peripherally as alternative responses. Supra-threshold stimulation in the core of the system yields escape mixed with freezing and hiding.The EHF system starts in the rostral anterior hypothalamus and runs back through the medial dorsal hypothalamus. A lateral extension occurs at the entry of the hypothalamic component of the occipito-mesencephalic tract. Posterior to this point the system shows a ventralward extension which does not include the putative nu. ventromedialis, but coincides instead with medial and periventricular fibres. The pre-optic area, lateral hypothalamus and mamillary area are all free of e, h and f sites.The EHF system thus corresponds well in distribution with the mammalian defensive threat-escape system. In both mammal and bird, similar behaviour is elicitable from both the diencephalic escape system and the central mesencephalic grey. The two are probably connected in the bird also by periventricular routes, some part of which can already be identified by e, h and f sites. Other properties of the EHF system, such as its role in the control of vocalisation, and its activation by non-reinforcement have been discussed elsewhere.