Optic Tectum Research Articles

Microglia phagocytose myelin sheaths to modify developmental myelination.

During development, oligodendrocytes contact and wrap neuronal axons with myelin. Similar to neurons and synapses, excess myelin sheaths are produced and selectively eliminated, but how elimination occurs is unknown. Microglia, the resident immune cells of the CNS, engulf surplus neurons and synapses. To determine if microglia also prune myelin sheaths, we used zebrafish to visualize and manipulate interactions between microglia, oligodendrocytes, and neurons during development. We found that microglia closely associate with oligodendrocytes and specifically phagocytose myelin sheaths. By using a combination of optical, genetic, chemogenetic, and behavioral approaches we revealed that neuronal activity bidirectionally balances microglial association with neuronal cell bodies and myelin phagocytosis in the optic tectum. Furthermore, multiple strategies to deplete microglia caused oligodendrocytes to maintain excessive and ectopic myelin. Our work reveals a neuronal activity-regulated role for microglia in modifying developmental myelin targeting by oligodendrocytes.

Histone deacetylase inhibition promotes regenerative neurogenesis after stab wound injury in the adult zebrafish optic tectum

The central nervous system (CNS) of adult zebrafish is capable of recovering from injury, unlike the CNS of mammals such as humans or rodents. Previously, we established a stab wound injury model of the optic tectum (OT) in the adult zebrafish and showed that the radial glial cells (RG) proliferation and neuronal differentiation contributes to OT regeneration. In the present study, we analyzed the function of histone deacetylases (HDACs) as potential regulators of OT regeneration. The expression of both hdac1 and hdac3 was found to be significantly decreased in the injured OT. In order to analyze the roles of HDACs in RG proliferation and differentiation after injury, we performed pharmacological experiments using the HDAC inhibitor trichostatin A. We found that HDAC inhibition after stab wound injury suppressed RG proliferation but promoted neuronal differentiation. Moreover, HDAC inhibition suppressed the injury-induced decline in expression of Notch signaling target genes, her4.1 and her6 after OT injury. These results suggest that HDACs regulate regenerative neurogenesis through changes in Notch target gene expression by histone deacetylation. HDACs and histone acetylation are promising molecular targets for neuronal regeneration and further studies about the molecular mechanisms behind the regulation of regeneration by histone acetylation are necessary.

Gene disruption of medaka (Oryzias latipes) orthologue for mammalian tissue-type transglutaminase (TG2) causes movement retardation.

Transglutaminases are an enzyme family that catalyses protein cross-linking essential for several biological functions. In the previous studies, we characterized the orthologues of the mammalian transglutaminase family in medaka (Oryzias latipes), an established fish model. Among the human isozymes, tissue-type transglutaminase (TG2) has multiple functions that are involved in several biological phenomena. In this study, we established medaka mutants deficient for the orthologue of human TG2 using the CRISPR/Cas9 and transcription activator-like effector nucleases systems. Although apparent morphological changes in the phenotype were not observed, movement retardation was found in the mutant fish when evaluated by a tank-diving test. Furthermore, comparative immunohistochemistry analysis using in this fish model revealed that orthologue of human TG2 was expressed at the periventricular layer of the optic tectum. Our findings provide novel insight for the relationship between tissue-type transglutaminase and the nervous system and the associated behaviour.

Visuomotor deficiency in panx1a knockout zebrafish is linked to dopaminergic signaling

Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play roles in the nervous system. The analysis of roles in both standard and pathological conditions benefits from a model organism with rapid development and early onset of behaviors. Such a model was developed by ablating the zebrafish panx1a gene using TALEN technology. Here, RNA-seq analysis of 6 days post fertilization larvae were confirmed by Real-Time PCR and paired with testing visual-motor behavior and in vivo electrophysiology. Results demonstrated that loss of panx1a specifically affected the expression of gene classes representing the development of the visual system and visual processing. Abnormal swimming behavior in the dark and the expression regulation of pre-and postsynaptic biomarkers suggested changes in dopaminergic signaling. Indeed, altered visuomotor behavior in the absence of functional Panx1a was evoked through D1/D2-like receptor agonist treatment and rescued with the D2-like receptor antagonist Haloperidol. Local field potentials recorded from superficial areas of the optic tectum receiving input from the retina confirmed abnormal responses to visual stimuli, which resembled treatments with a dopamine receptor agonist or pharmacological blocking of Panx1a. We conclude that Panx1a functions are relevant at a time point when neuronal networks supporting visual-motor functions undergo modifications preparing for complex behaviors of freely swimming fish.

Afferent and efferent connections of the nucleus prethalamicus in the yellowfin goby Acanthogobius flavimanus.

The nucleus prethalamicus (PTh) receives fibers from the optic tectum and then projects to the dorsal telencephalon in the yellowfin goby Acanthogobius flavimanus. However, it remained unclear whether the PTh is a visual relay nucleus, because the optic tectum receives not only visual but also other sensory modalities. Furthermore, precise telencephalic regions receiving prethalamic input remained unknown in the goby. We therefore investigated the full set of afferent and efferent connections of the PTh by direct tracer injections into the nucleus. Injections into the PTh labeled cells in the optic tectum, ventromedial thalamic nucleus, central and medial parts of the dorsal telencephalon, and caudal lobe of the cerebellum. We found that the somata of most tecto-prethalamic neurons are present in the stratum periventriculare. Their dendrites ascend to reach the major retinorecipient layers of the tectum. The PTh is composed of two subnuclei (medial and lateral) and topographic organization was appreciated only for tectal projections to the lateral subnucleus (PTh-l), which also receives sparse retinal projections. In contrast, the medial subnucleus receives fibers only from the medial tectum. We found that the PTh projects to nine subregions in the dorsal telencephalon and four in the ventral telencephalon. Furthermore, cerebellar injections revealed that cerebello-prethalamic fibers cross the midline twice to innervate the PTh-l on both sides. The present study is the first detailed report on the full set of the connections of PTh, which suggests that the PTh relays visual information from the optic tectum to the telencephalon.

Acute toxic effects of chlorpyrifos on pseudobranchial neurosecretory system, brain regions and locomotory behavior of an air-breathing catfish, Heteropneustes fossilis (Bloch 1794)

In the late twentieth century, tremendous use of second-class organophosphate insecticides especially chlorpyrifos (CPF) resulting into heavy accumulation in different non-targeted aquatic species including fishes leads to apparent structural and biochemical changes in different organs and related abnormal behavioral responses. The present study has been undertaken as a pioneer attempt to assess the toxic effects of CPF on histopathological changes in pseudobranchial neurosecretory cells (PNSCs) of a neuroendocrine system of gill region, optic tectum (OT) and cerebellum, biochemical changes (acetylcholinesterase (AChE) activity and antioxidant markers) in the brain and associated locomotory behavioral alterations in air-breathing catfish, Heteropneustes fossilis. The fishes were exposed to CPF concentration of 1.92 mg/l for four days and their locomotor activities were recorded by ANY-MAZE software (Stoelting, Kiel, WI), an automated behavior tracking device. The acute exposure of CPF induced pathological changes in PNSCs, subtle changes in granular cells of the cerebellum and neuroarchitectural pattern of different layers of OT as compared to control. In the CPF exposed brain, AChE activity was significantly decreased while antioxidant enzymatic activity such as SOD activity was increased but CAT activity was substantially decreased. The CPF exposed fishes displayed significantly reduced locomotory activities with symptoms of motionless, loss of equilibrium and erratic movements. This study concludes that acute exposure to CPF for short duration may induce dys-regulation of neurosecretory activity of PNSCs, altered biochemical activity of brain and reduced locomotory/swimming performances in fishes.

An Innate Color Preference Displayed by Xenopus Tadpoles Is Persistent and Requires the Tegmentum.

Many animals, especially those that develop externally, are equipped with innate color preferences that promote survival. For example, Xenopus tadpoles are known to phototax most robustly towards mid-spectrum (“green”) wavelengths of light while avoiding shorter (“blue”) wavelengths. The innate preference to phototax towards green likely promotes survival by guiding the tadpoles to green aquatic plants—their source of both food and safety. Here, we characterize the dynamics and circuitry that give rise to this intriguing hard-wired behavior. Using a novel open-field experimental paradigm we found that free-swimming tadpoles indeed spend most of their time in the green portion of the test dish, whether green is pitted against white (brighter than green) or black (darker than green). This preference was modest yet incredibly persistent over time, which, according to the shell game model of predator-prey interactions, minimizes being found by the predator. Furthermore, we found that this innate preference for the color green was experience-independent, and manifested mainly via profoundly slower swimming speeds while in the green region of the test dish. Ablation experiments showed that, at the circuit level, the color-guided swimming behavior requires the tegmentum, but not the optic tectum (OT). Lastly, we determined that exposing tadpoles to the selective serotonin reuptake inhibitor (SSRI) trazodone switched the tadpoles’ preference from color-based to luminance-based, implicating two distinct visual circuits in the tadpole, one that is associated with color-driven behaviors, another associated with luminance-driven behaviors.

NMDARs Translate Sequential Temporal Information into Spatial Maps.

SummarySpatial representations of the sensory world are important for brain function. Timing is an essential component of sensory information. Many brain circuits transform the temporal sequence of input activity into spatial maps; however, the mechanisms underlying this transformation are unclear. Different N-methyl-D-aspartate receptor (NMDAR) response magnitudes result in synaptic potentiation or depression. We asked whether NMDAR response magnitude also affects the transformation of temporal information into directional spatial maps. We quantified retinotectal axon branch dynamics in Xenopus optic tectum in response to temporal sequences of visual stimulation. Reducing NMDAR responses by 50% inverts the spatial distribution of branch dynamics along the rostrocaudal axis in response to temporal patterns of input, suggesting that the magnitude of NMDAR signaling encodes the temporal sequence of inputs and translates the temporal code into a directional spatial map using structural plasticity-based branch dynamics. We discuss how this NMDAR-dependent decoding mechanism retrieves spatial information from sequential afferent activity.

Behavioral and neuronal study of inhibition of return in barn owls

Inhibition of return (IOR) is the reduction of detection speed and/or detection accuracy of a target in a recently attended location. This phenomenon, which has been discovered and studied thoroughly in humans, is believed to reflect a brain mechanism for controlling the allocation of spatial attention in a manner that enhances efficient search. Findings showing that IOR is robust, apparent at a very early age and seemingly dependent on midbrain activity suggest that IOR is a universal attentional mechanism in vertebrates. However, studies in non-mammalian species are scarce. To explore this hypothesis comparatively, we tested for IOR in barn owls (Tyto alba) using the classical Posner cueing paradigm. Two barn owls were trained to initiate a trial by fixating on the center of a computer screen and then turning their gaze to the location of a target. A short, non-informative cue appeared before the target, either at a location predicting the target (valid) or a location not predicting the target (invalid). In one barn owl, the response times (RT) to the valid targets compared to the invalid targets shifted from facilitation (lower RTs) to inhibition (higher RTs) when increasing the time lag between the cue and the target. The second owl mostly failed to maintain fixation and responded to the cue before the target onset. However, when including in the analysis only the trials in which the owl maintained fixation, an inhibition in the valid trials could be detected. To search for the neural correlates of IOR, we recorded multiunit responses in the optic tectum (OT) of four head-fixed owls passively viewing a cueing paradigm as in the behavioral experiments. At short cue to target lags (<100 ms), neural responses to the target in the receptive field (RF) were usually enhanced if the cue appeared earlier inside the RF (valid) and were suppressed if the cue appeared earlier outside the RF (invalid). This was reversed at longer lags: neural responses were suppressed in the valid conditions and were unaffected in the invalid conditions. The findings support the notion that IOR is a basic mechanism in the evolution of vertebrate behavior and suggest that the effect appears as a result of the interaction between lateral and forward inhibition in the tectal circuitry.

Deep three-photon imaging of the brain in intact adult zebrafish.

Behaviors emerge from activity throughout the brain, but non-invasive optical access in adult vertebrate brains is limited. We show that three-photon (3P) imaging through the head of intact adult zebrafish allows structural and functional imaging at cellular resolution throughout the telencephalon and deep into the cerebellum and optic tectum. With 3P imaging, considerable portions of the brain become non-invasively accessible from embryo to sexually mature adult in a vertebrate model.

Calsequestrins New Calcium Store Markers of Adult Zebrafish Cerebellum and Optic Tectum.

Calcium stores in neurons are heterogeneous in compartmentalization and molecular composition. Danio rerio (zebrafish) is an animal model with a simply folded cerebellum similar in cellular organization to that of mammals. The aim of the study was to identify new endoplasmic reticulum (ER) calcium store markers in zebrafish adult brain with emphasis on cerebellum and optic tectum. By quantitative polymerase chain reaction, we found three RNA transcripts coding for the intra-ER calcium binding protein calsequestrin: casq1a, casq1b, and casq2. In brain homogenates, two isoforms were detected by mass spectrometry and western blotting. Fractionation experiments of whole brain revealed that Casq1a and Casq2 were enriched in a heavy fraction containing ER microsomes and synaptic membranes. By in situ hybridization, we found the heterogeneous expression of casq1a and casq2 mRNA to be compatible with the cellular localization of calsequestrins investigated by immunofluorescence. Casq1 was expressed in neurogenic differentiation 1 expressing the granule cells of the cerebellum and the periventricular zone of the optic tectum. Casq2 was concentrated in parvalbumin expressing Purkinje cells. At a subcellular level, Casq1 was restricted to granular cell bodies, and Casq2 was localized in cell bodies, dendrites, and axons. Data are discussed in relation to the differential cellular and subcellular distribution of other cerebellum calcium store markers and are evaluated with respect to the putative relevance of calsequestrins in the neuron-specific functional activity.

Categorical Signaling of the Strongest Stimulus by an Inhibitory Midbrain Nucleus

The nucleus isthmi pars magnocellularis (Imc), a group of inhibitory neurons in the midbrain tegmentum, is a critical component of the spatial selection network in the vertebrate midbrain. It delivers long-range inhibition among different portions of the space map in the optic tectum (OT), thereby mediating stimulus competition in the OT. Here, we investigate the properties of relative strength-dependent competitive interactions within the Imc, in barn owls of both sexes. We find that when Imc neurons are presented simultaneously with one stimulus inside the receptive field and a second, competing stimulus outside, they exhibit gradual or switch-like response profiles as a function of relative stimulus strength. They do so both when the two stimuli are of the same sensory modality (both visual) or of different sensory modalities (visual and auditory). Moreover, Imc neurons signal the strongest stimulus in a dynamically flexible manner, indicating that Imc responses reflect an online comparison between the strengths of the competing stimuli. Notably, Imc neurons signal the strongest stimulus more categorically, and earlier than the OT. Paired recordings at spatially aligned sites in the Imc and OT reveal that although some properties of stimulus competition, such as the bias of competitive response profiles, are correlated, others such as the steepness of response profiles, are set independently. Our results demonstrate that the Imc is itself an active site of competition, and may be the first site in the midbrain selection network at which stimulus competition is resolved.SIGNIFICANCE STATEMENT This work sheds light on the functional properties of a small group of inhibitory neurons in the vertebrate midbrain that play a key part in how the brain selects a target among competitors. A better understanding of the functioning of these neurons is an important building block for the broader understanding of how distracters are suppressed, and of spatial attention and its dysfunction.

Anoxic Turtles May Be Blind, but Not Deaf: Responses of the In Vitro Pond Turtle Brain to Visual and Auditory Stimuli During Anoxia

Freshwater pond turtles are among the most anoxia‐tolerant vertebrates, an ability that allows some species to overwinter for months in ice‐locked ponds without oxygen. To determine if anoxic slider turtles can maintain the ability to detect visual and auditory stimuli, we monitored field potentials in an in vitro reduced brainstem while superfusing with either oxygenated or anoxic superfusate. Visual stimuli were recorded using glass recording pipettes placed medial to the optic tectum while flashing light on the eyes. For auditory stimuli, the pipette was placed medial to the eighth cranial nerve (the presumptive cochlear nucleus). In place of sound stimuli, a glass rod with a small polished tip was mounted to a piezo‐electric piston that moved onto the submerged tympanic membrane in brief inward and outward steps. Auditory responses of the brainstem occurred after a very brief latency to each step of the tympanic membrane, while there was a long latency to each light flash in the optic tectum . An optical probe measured the concurrent oxygen tensions under control (oxygenated) and anoxic conditions. After switching from control to anoxic superfusate, there was no substantial change in the amplitude of the averaged auditory response to steps of the tympanic membrane. However, responses recorded in the optic tectum to light flashes while bathed in anoxic superfusate decreased strongly, but recovered upon return to the control superfusate. These results suggest that turtles submerged for long periods in anoxic ponds may retain sensory responses to auditory but not visual stimuli.Support or Funding InformationNational Science Foundation CAREER Award (1253939) to DEW.

Developmental Analysis of GABAA Receptor Gene Expression in Gallus gallus Chick Brain

Fetal Alcohol Spectrum Disorders (FASD) are a class of neurological disorders that arise from both ethanol‐related toxicity and alterations in a variety of molecular pathways during fetal development. One result of embryonic ethanol exposure is overactivation of the GABAA receptors within neuronal cells. Due to the varying subunit composition of these pentameric ionotropic receptors in different brain regions and timepoints during development, this experiment characterized the expression levels of nine GABAA receptor subunits (α1–6, β1–3) in an embryonic chick model. RNA was isolated from three brain tissue regions (cerebellum, forebrain, optic tectum). After, the RNA was converted to cDNA by reverse transcription and the gene transcripts were assessed using RT‐qPCR with specifically designed primers for each GABAA subunit type. With the exception of the α6 subunit, expression of the GABAA receptor genes were consistent throughout all brain tissue types and only showed a slight variation in level of expression when it came to the different developmental time points of E7–E17. α6 was absent in forebrain, and not detected until E15 in cerebellum and optic tectum samples. Based on the data collected, it can be concluded that the nine GABAA subunit genes tested are mostly expressed throughout the chicken embryonic timeline. There is currently limited data as to how their gene expression levels change as the embryo develops. Further research is needed to determine the distinct differences between the different subunit types. These subtle differences between the expression of each GABAA receptor subunit gene will point to a better understanding of the influence ethanol has on the developing embryo in the chick model. This evidence may hold meaning to why alcohol exposure during certain time periods in fetal development for humans prove to have more severe effects on the fetus, like FASD, than during other time periods during embryonic growth.

Response to change in the number of visual stimuli in zebrafish:A behavioural and molecular study

Evidence has shown that a variety of vertebrates, including fish, can discriminate collections of visual items on the basis of their numerousness using an evolutionarily conserved system for approximating numerical magnitude (the so-called Approximate Number System, ANS). Here we combine a habituation/dishabituation behavioural task with molecular biology assays to start investigating the neural bases of the ANS in zebrafish. Separate groups of zebrafish underwent a habituation phase with a set of 3 or 9 small red dots, associated with a food reward. The dots changed in size, position and density from trial to trial but maintained their numerousness, and the overall areas of the stimuli was kept constant. During the subsequent dishabituation test, zebrafish faced a change (i) in number (from 3 to 9 or vice versa with the same overall surface), or (ii) in shape (with the same overall surface and number), or (iii) in size (with the same shape and number). A control group of zebrafish was shown the same stimuli as during the habituation. RT-qPCR revealed that the telencephalon and thalamus were characterized by the most consistent modulation of the expression of the immediate early genes c-fos and egr-1 upon change in numerousness; in contrast, the retina and optic tectum responded mainly to changes in stimulus size.

Identification of kisspeptin2 cDNA in the catfish Heteropneustes fossilis: Expression profile, in situ localization and steroid modulation

Kisspeptin (Kiss) is considered an upstream regulator of gonadotropin-releasing hormone in mammals but its role in non-mammalian vertebrates is not unequivocally established. In the catfish Heteropneustes fossilis, a 605 bp long cDNA was identified from the brain by cloning as well as by retrieving from the catfish transcriptome database. The open reading frame (ORF, 93–405 bp) codes for a 113 amino acids long precursor protein. Homology and phylogenetic analyses showed that the predicted protein belongs to the vertebrate Kiss2 type with a high degree of conservation in the Kiss2-10 region (FNFNPFGLRF). The kiss2 transcripts were expressed highly in the brain and gonads in a dimorphic manner with a female bias. In the brain, kiss2 transcripts showed regional differences with higher expression in the medulla oblongata and forebrain regions. The kiss2 transcripts showed significant seasonal variations with the highest expression in the brain in spawning phase and in the gonads in prespawning phase. The kiss2 transcripts were localized in the brain (nucleus preopticus, habenular nucleus, nucleus recessus posterioris, nucleus recessus lateralis) and stratum periventriculare (radial glial cells) of optic tectum, pituitary and ovary (follicular layer and germinal vesicle). Ovariectomy (1, 2, 3 and 4 weeks) decreased brain kiss2 mRNA levels and a single injection of estradiol-17β (E2; 0.5 μg/g body weight) in 3- week ovariectomized (OVX) and sham operated fish resulted in an increase in the transcript levels after 24 h. The E2 receptor antagonist Tamoxifen (TMX) produced biphasic effects on the kiss2 expression in the dose- response study. TMX inhibited the expression in the OVX fish, but elicited a stimulatory effect in the OVX + E2-treated fish. Testosterone (T) decreased, and progesterone (P4) inhibited (resting phase) or stimulated (prespawning phase) the transcript level in 3-week OVX fish. In the 3-week sham groups, E2 increased, and TMX, T and P4 inhibited the kiss2 transcript levels. The results suggest that Kiss2 is an important regulator of the brain- pituitary- gonadal- endocrine axis, and in habenular and optic tectum functions.

Advancing zebrafish as a model for studying developmental neurotoxicology

The cover photo shows the developing zebrafish nervous system at 5 days post-fertilization. Axon tracts are labeled with an anti-acetylated alpha tubulin antibody. The image, which was acquired on a Zeiss LSM 880 confocal microscope, is a maximum intensity projection of a z-stack that has been color-coded for depth. Major brain regions such as the olfactory bulb, forebrain, habenula, optic tectum, cerebellum, hindbrain, and eye are identifiable. This image is part of a study (Plavicki Lab, Brown University) focused on understanding the impact of toxicant exposures on brain development and activity with the goal of identifyingenvironmental factors that contribute to the etiology of neurodevelopmental disorders.

Evolution of Epileptiform Activity in Zebrafish by Statistical-Based Integration of Electrophysiology and 2-Photon Ca2+ Imaging.

The study of sources and spatiotemporal evolution of ictal bursts is critical for the mechanistic understanding of epilepsy and for the validation of anti-epileptic drugs. Zebrafish is a powerful vertebrate model representing an excellent compromise between system complexity and experimental accessibility. We performed the quantitative evaluation of the spatial recruitment of neuronal populations during physiological and pathological activity by combining local field potential (LFP) recordings with simultaneous 2-photon Ca2+ imaging. We developed a method to extract and quantify electrophysiological transients coupled with Ca2+ events and we applied this tool to analyze two different epilepsy models and to assess the efficacy of the anti-epileptic drug valproate. Finally, by cross correlating the imaging data with the LFP, we demonstrated that the cerebellum is the main source of epileptiform transients. We have also shown that each transient was preceded by the activation of a sparse subset of neurons mostly located in the optic tectum.

Specificity and application of SOX2 antibody

Sox2 is known to play an important role in maintaining the totipotency and self-renewal of embryonic stem cells. The purpose of this study was to prepare an anti-chicken Sox2 polyclonal antibody using prokaryotic expression techniques, to evaluate its specificity and to use it to investigate the expression and distribution of Sox2 in the chicken brain and lungs. The chicken Sox2 gene was amplified and subcloned to a pET-30a vector to construct a prokaryotic expression vector, pET-Sox2. A His-Sox2 fusion protein was expressed, purified, and used to prepare an antichicken Sox2 polyclonal antibody. Western blotting revealed that the antichicken Sox2 antibody could specifically bind not only to the purified His-Sox2 fusion protein but also to the endogenous Sox2 protein in the testes of chicken, showing a distinct dose-dependent relationship between antigen and Sox2 antibody. Indirect immunofluorescent staining of Sox2-overexpressing cells showed strong nuclear and diffuse cytoplasmic immunoreactivity for Sox2 in the antichicken Sox2 antibody-staining cells. A CRISPR/Cas9 effector system-mediated Sox2 knockdown assay indicated that Sox2 expression in HEK 293T cells was downregulated in the presence of doxycycline but upregulated in the absence of doxycycline. In addition, cryosectioning and immunohistochemical staining illustrated that most spermatogonia in the seminiferous tubules, and a small number of Sertoli and Leydig cells, were positive for Sox2. The antichicken Sox2 antibody was also successfully used to investigate the expression and distribution of Sox2 in the chicken cerebellar cortex, optic tectum, cerebral cortex, and lungs. The results of this study confirmed the specificity of the antichicken Sox2 polyclonal antibody, which will be available for the study of biological functions of the chicken Sox2 gene and the self-renewal mechanisms of chicken pluripotent stem cells.

Lhx2/9 and Etv1 Transcription Factors have Complementary roles in Regulating the Expression of Guidance Genes slit1 and sema3a

During neural network development, growing axons read a map of guidance cues expressed in the surrounding tissue that lead the axons toward their targets. In particular, Xenopus retinal ganglion axons use the cues Slit1 and Semaphorin 3a (Sema3a) at a key guidance decision point in the mid-diencephalon in order to continue on to their midbrain target, the optic tectum. The mechanisms that control the expression of these cues, however, are poorly understood. Extrinsic Fibroblast Growth Factor (Fgf) signals are known to help coordinate the development of the brain by regulating gene expression. Here, we propose Lhx2/9 and Etv1 as potential downstream effectors of Fgf signalling to regulate slit1 and sema3a expression in the Xenopus forebrain. We find that lhx2/9 and etv1 mRNAs are expressed complementary to and within slit1/sema3a expression domains, respectively. Our data indicate that Lhx2 functions as an indirect repressor in that lhx2 overexpression within the forebrain downregulates the mRNA expression of both guidance genes, and in vitro lhx2/9 overexpression decreases the activity of slit1 and sema3a promoters. The Lhx2-VP16 constitutive activator fusion reduces sema3a promoter function, and the Lhx2-En constitutive repressor fusion increases slit1 induction. In contrast, etv1 gain of function transactivates both guidance genes in vitro and in the forebrain. Based on these data, together with our previous work, we hypothesize that Fgf signalling promotes both slit1 and sema3a expression in the forebrain through Etv1, while using Lhx2/9 to limit the extent of expression, thereby establishing the proper boundaries of guidance cue expression.