Modulation of p53 and met expression by Krüppel-like factor 8 regulates zebrafish cerebellar development.

Krüppel-like factor 8 (Klf8) is a zinc-finger transcription factor implicated in cell proliferation, and cancer cell survival and invasion; however, little is known about its role in normal embryonic development. Here, we show that Klf8 is required for normal cerebellar development in zebrafish embryos. Morpholino knockdown of klf8 resulted in abnormal cerebellar primordium morphology and the induction of p53 in the brain region at 24 hours post-fertilization (hpf). Both p53-dependent reduction of cell proliferation and augmentation of apoptosis were observed in the cerebellar anlage of 24 hpf-klf8 morphants. In klf8 morphants, expression of ptf1a in the ventricular zone was decreased from 48 to 72 hpf; on the other hand, expression of atohla in the upper rhombic lip was unaffected. Consistent with this finding, Purkinje cell development was perturbed and granule cell number was reduced in 72 hpf-klf8 morphants; co-injection of p53 MO(sp) or klf8 mRNA substantially rescued development of cerebellar Purkinje cells in klf8 morphants. Hepatocyte growth factor/Met signaling is known to regulate cerebellar development in zebrafish and mouse. We observed decreased met expression in the tectum and rhombomere 1 of 24 hpf-klf8 morphants, which was largely rescued by co-injection with klf8 mRNA. Moreover, co-injection of met mRNA substantially rescued formation of Purkinje cells in klf8 morphants at 72 hpf. Together, these results demonstrate that Klf8 modulates expression of p53 and met to maintain ptf1a-expressing neuronal progenitors, which are required for the appropriate development of cerebellar Purkinje and granule cells in zebrafish embryos.

Role of En2 in the tectal laminar formation of chick embryos.

The chick optic tectum consists of 16 laminae. Here, we report contribution of En2 to laminar formation in chick optic tecta. En2 is specifically expressed in laminae g-j of stratum griseum et fibrosum superficiale (SGFS). Misexpression of En2 resulted in disappearance of En2-expressing cells from the superficial layers (laminae a-f of SGFS), where endogenous En2 is not expressed. Misexpression of En2 before postmitotic cells had left the ventricular layer indicated that En2-misexpressing cells stopped at the laminae of endogenous En2 expression and that they did not migrate into the superficial layers. Induction of En2 misexpression using a tetracycline-inducible system after the postmitotic cells had reached superficial layers also resulted in disappearance of En2-expressing cells from the superficial layers. Time-lapse analysis showed that En2-misexpressing cells migrated back from the superficial layers towards the middle layers, where En2 is strongly expressed endogenously. Our results suggest a potential role of En2 in regulating cell migration and positioning in the tectal laminar formation.

EphA3 expressed in the chicken tectum stimulates nasal retinal ganglion cell axon growth and is required for retinotectal topographic map formation.

BACKGROUND: Retinotopic projection onto the tectum/colliculus constitutes the most studied model of topographic mapping and Eph receptors and their ligands, the ephrins, are the best characterized molecular system involved in this process. Ephrin-As, expressed in an increasing rostro-caudal gradient in the tectum/colliculus, repel temporal retinal ganglion cell (RGC) axons from the caudal tectum and inhibit their branching posterior to their termination zones. However, there are conflicting data regarding the nature of the second force that guides nasal axons to invade and branch only in the caudal tectum/colliculus. The predominant model postulates that this second force is produced by a decreasing rostro-caudal gradient of EphA7 which repels nasal optic fibers and prevents their branching in the rostral tectum/colliculus. However, as optic fibers invade the tectum/colliculus growing throughout this gradient, this model cannot explain how the axons grow throughout this repellent molecule. METHODOLOGY/PRINCIPAL FINDINGS: By using chicken retinal cultures we showed that EphA3 ectodomain stimulates nasal RGC axon growth in a concentration dependent way. Moreover, we showed that nasal axons choose growing on EphA3-expressing cells and that EphA3 diminishes the density of interstitial filopodia in nasal RGC axons. Accordingly, in vivo EphA3 ectodomain misexpression directs nasal optic fibers toward the caudal tectum preventing their branching in the rostral tectum. CONCLUSIONS: We demonstrated in vitro and in vivo that EphA3 ectodomain (which is expressed in a decreasing rostro-caudal gradient in the tectum) is necessary for topographic mapping by stimulating the nasal axon growth toward the caudal tectum and inhibiting their branching in the rostral tectum. Furthermore, the ability of EphA3 of stimulating axon growth allows understanding how optic fibers invade the tectum growing throughout this molecular gradient. Therefore, opposing tectal gradients of repellent ephrin-As and of axon growth stimulating EphA3 complement each other to map optic fibers along the rostro-caudal tectal axis.

Balancing of ephrin/Eph forward and reverse signaling as the driving force of adaptive topographic mapping.

The retinotectal projection, which topographically maps retinal axons onto the tectum of the midbrain, is an ideal model system with which to investigate the molecular genetics of embryonic brain wiring. Corroborating Sperry's seminal hypothesis, ephrin/Eph counter-gradients on both retina and tectum were found to represent matching chemospecificity markers. Intriguingly, however, it has never been possible to reconstitute topographically appropriate fiber growth in vitro with these cues. Moreover, experimentally derived molecular mechanisms have failed to provide explanations as to why the mapping adapts to grossly diverse targets in some experiments, while displaying strict point-to-point specificity in others. In vitro, ephrin-A/EphA forward, as well as reverse, signaling mediate differential repulsion to retinal fibers, instead of providing topographic guidance. We argue that those responses are indicative of ephrin-A and EphA being members of a guidance system that requires two counteracting cues per axis. Experimentally, we demonstrate by introducing novel double-cue stripe assays that the simultaneous presence of both cues indeed suffices to elicit topographically appropriate guidance. The peculiar mechanism, which uses forward and reverse signaling through a single receptor/ligand combination, entails fiber/fiber interactions. We therefore propose to extend Sperry's model to include ephrin-A/EphA-based fiber/fiber chemospecificity, eventually out-competing fiber/target interactions. By computational simulation, we show that our model is consistent with stripe assay results. More importantly, however, it not only accounts for classical in vivo evidence of point-to-point and adaptive topographic mapping, but also for the map duplication found in retinal EphA knock-in mice. Nonetheless, it is based on a single constraint of topographic growth cone navigation: the balancing of ephrin-A/EphA forward and reverse signaling.

Sonic hedgehog (Shh)-Gli signaling controls neural progenitor cell division in the developing tectum in zebrafish.

Despite considerable progress, the mechanisms that control neural progenitor differentiation and behavior, as well as their functional integration into adult neural circuitry, are far from being understood. Given the complexity of the mammalian brain, non-mammalian models provide an excellent model to study neurogenesis, including both the cellular composition of the neurogenic microenvironment, and the factors required for precursor growth and maintenance. In particular, we chose to address the question of the control of progenitor proliferation by Sonic hedgehog (Shh) using the zebrafish dorsal mesencephalon, known as the optic tectum (OT), as a model system. Here we show that either inhibiting pharmacologically or eliminating hedgehog (Hh) signaling by using mutants that lack essential components of the Hh pathway reduces neural progenitor cell proliferation affecting neurogenesis in the OT. On the contrary, pharmacological gain-of-function experiments result in significant increase in proliferation. Importantly, Shh-dependent function controls neural progenitor cell behavior as sox2-positive cell populations were lost in the OT in the absence of Hh signaling, as evidenced in slow-muscle-omitted (smu) mutants and with timed cyclopamine inhibition. Expressions of essential components of the Hh pathway reveal for the first time a late dorsal expression in the embryonic OT. Our observations argue strongly for a role of Shh in neural progenitor biology in the OT and provide comparative data to our current understanding of progenitor/stem cell mechanisms that place Shh as a key niche factor in the dorsal brain.

Early expression of axon guidance molecules in the embryonic chick mesencephalon and pretectum.

Early axon tracts in the developing vertebrate brain are established along precise paths. Yet, little is known about axon guidance processes at early stages of rostral brain development. Using whole mount in situ hybridisation in combination with immunohistochemistry, we have analysed the expression patterns of Slits, Netrins, Semaphorins and the respective receptors during the formation of the early axon scaffold, particularly focusing on the pretectal-mesencephalic boundary. Many of these guidance molecules are expressed in close correlation with the growing tracts, and the nuclei of the corresponding neurons often express the respective receptors. The expression patterns of Slits and Netrins implicate them with the positioning of the longitudinal tracts along the dorsoventral axis, while Semaphorins could provide guidance at specific choice points. Our study provides a catalogue of gene expression for future studies on axon guidance mechanisms in the early brain.

The duration of Fgf8 isthmic organizer expression is key to patterning different tectal-isthmo-cerebellum structures.

The isthmic organizer and its key effector molecule, fibroblast growth factor 8 (Fgf8), have been cornerstones in studies of how organizing centers differentially pattern tissues. Studies have implicated different levels of Fgf8 signaling from the mid/hindbrain boundary (isthmus) as being responsible for induction of different structures within the tectal-isthmo-cerebellum region. However, the role of Fgf8 signaling for different durations in patterning tissues has not been studied. To address this, we conditionally ablated Fgf8 in the isthmus and uncovered that prolonged expression of Fgf8 is required for the structures found progressively closer to the isthmus to form. We found that cell death cannot be the main factor accounting for the loss of brain structures near the isthmus, and instead demonstrate that tissue transformation underlies the observed phenotypes. We suggest that the remaining Fgf8 and Fgf17 signaling in our temporal Fgf8 conditional mutants is sufficient to ensure survival of most midbrain/hindbrain cells near the isthmus. One crucial role for sustained Fgf8 function is in repressing Otx2 in the hindbrain, thereby allowing the isthmus and cerebellum to form. A second requirement for sustained Fgf8 signaling is to induce formation of a posterior tectum. Finally, Fgf8 is also required to maintain the borders of expression of a number of key genes involved in tectal-isthmo-cerebellum development. Thus, the duration as well as the strength of Fgf8 signaling is key to patterning of the mid/hindbrain region. By extrapolation, the length of Fgf8 expression could be crucial to Fgf8 function in other embryonic organizers.

Graded levels of FGF protein span the midbrain and can instruct graded induction and repression of neural mapping labels.

Graded guidance labels are widely used in neural map formation, but it is not well understood which potential strategy leads to their graded expression. In midbrain tectal map development, FGFs can induce an entire midbrain, but their protein distribution is unclear, nor is it known whether they may act instructively to produce graded gene expression. Using a receptor-alkaline phosphatase fusion probe, we find a long-range posterior > anterior FGF protein gradient spanning the midbrain. Heparan sulfate proteoglycan (HSPG) is required for this gradient. To test whether graded FGF concentrations can instruct graded gene expression, a quantitative tectal explant assay was developed. Engrailed-2 and ephrin-As, normally in posterior > anterior tectal gradients, showed graded upregulation. Moreover, EphAs, normally in anterior > posterior countergradients, showed coordinately graded downregulation. These results provide a mechanism to establish graded mapping labels and more generally provide a developmental strategy to coordinately induce a structure and pattern its cell properties in gradients.

Apc1-mediated antagonism of Wnt/beta-catenin signaling is required for retino-tectal pathfinding in the zebrafish.

The tumor suppressor Apc1 is an intracellular antagonist of the Wnt/beta-catenin pathway. We examined the effects of an Apc1 loss-of-function mutation on retino-tectal axon pathfinding in zebrafish. In apc mutants, the retina is disorganized and optic nerves portray pathfinding defects at the optic chiasm and do not project properly to the tectum. Wild-type cells, transplanted into mutant retinae, acquire retinal ganglion cell fate and project axons that cross at the mispositioned optic chiasm and extend to the contralateral tectum, suggesting a function of apc1 in axon pathfinding. These defects are caused mainly by stabilization of beta-catenin. These data demonstrate that Apc1 function is required for correct patterning of the retina and proper retinal ganglion axon projections.

Developmental species differences in brain cell cycle rates between northern bobwhite quail (Colinus virginianus) and parakeets (Melopsittacus undulatus): implications for mosaic brain evolution.

Adult brains differ among species in the proportional sizes of their major subdivisions. For example, the telencephalon occupies 71% of the entire brain in parakeets (Melopsittacus undulatus) but only 54% in quail (Colinus virginianus). In contrast, the tectum is smaller in parakeets than in quail. To determine whether these differences in brain region size arise because of species differences in cell cycle rates, parakeet and quail embryos were collected at various stages of development (HH24-HH37) and stained with antibodies against proliferating cell nuclear antigen (PCNA), which labels all dividing cells, and phosphorylated histone-3 (pH3), which labels M-phase cells. Analysis of pH3+ cell densities and pH3+/PCNA+ cell ratios were used to compare cell cycle rates across stages and species. Cumulative labeling with bromodeoxyuridine (BrdU) was also used to compare cell cycle rates at stages 24 and 28 in quail. We found that telencephalic cell cycle rates lengthen with age in both species, but that they lengthen significantly later in parakeets than in quail. This species difference in cell cycle rates explains, at least partly, why adult parakeets have a proportionately larger telencephalon. Tectal cell cycle rates also remain elevated for a prolonged period of time in parakeets compared to quail. This seems paradoxical at first, given that the parakeet's adult tectum is relatively small. However, the tectum is initially much smaller but then grows more extensively in parakeets than in quail. Thus, species differences in adult brain proportions can be traced back to species differences in cell cycle kinetics.

Comparative analysis of neurogenesis between the core and shell regions of auditory areas in the chick (Gallus gallus domesticus).

Early embryogenesis can reflect constituting organizations and evolutionary origins of brain areas. To determine whether a clear core-versus-shell distinction of neurogenesis that occurs from the auditory midbrain to the telencephalon in the reptile also appears in the bird, a single dose of [(3)H]-thymidine was injected into chick (Gallus gallus domesticus) eggs at some successive embryonic days (E) (from E3 to E10). Towards the end of hatching, [(3)H]-thymidine labeling was examined, and the results were as follows: 1) Neuronal generation in the nucleus intercollicularis (ICo) (shell region) began at E3, whereas neurogenesis began at E4 in the nucleus mesencephalicus lateralis pars dorsalis (MLd) (core region); 2) Neurogenesis initiated at E3 in the nucleus ovoidalis (Ov) shell, but initiated at E4 in the rostral Ov core. In the medial or caudal Ov core, the percentage of heavily-labeled neurons with [(3)H]-thymidine was significantly lower at E3 age group than that in the Ov shell; 3) In field L1 and L3, two flanking regions of the primary telencephalic auditory area (field L2a), neurogenesis started at E5, but started at E6 in field L2a. These data indicate that the onset of embryogenesis began earlier in the auditory shell areas than in the core areas from the midbrain to the telencephalon. These findings provide insight into the organization of auditory nuclei and their evolution in amniotes.

Gli3 coordinates three-dimensional patterning and growth of the tectum and cerebellum by integrating Shh and Fgf8 signaling.

The coordination of anterior-posterior (AP) and dorsal-ventral (DV) patterning of the mesencephalon (mes) and rhombomere 1 (r1) is instrumental for the development of three distinct brain structures: the tectum and cerebellum dorsally and the tegmentum ventrally. Patterning of the mes/r1 is primarily mediated by signaling molecules secreted from two organizers: sonic hedgehog (Shh) from the floor plate (DV) and Fgf8 from the isthmus (AP). Gli3, a zinc-finger transcription factor in the Shh signaling pathway, has been implicated in regulating Fgf8 expression and is therefore a potential candidate for coordinating the action of the two organizers. By inactivating mouse Gli3 at successive embryonic time points in vivo, we uncovered the extent and the underlying mechanism of Gli3 function in the mes/r1. We demonstrate that before E9.0, Gli3 is required for establishing a distinct posterior tectum, isthmus and cerebellum, but does not play a role in the development of the tegmentum. Between E9.0 and E11.0, Gli3 continues to be required for isthmus and cerebellum development, but primarily for defining the cerebellar foliation pattern. We show that Gli3 regulates patterning of the isthmus and cerebellar anlage by confining Fgf8 expression to the isthmus, and attenuates growth of dorsal r1 (before E11.0) and the dorsal mes and isthmus (beyond E11.0) through regulation of cell proliferation and viability. In conclusion, our results show that Gli3 is essential for the coordinated three-dimensional patterning and growth of the dorsal mes/r1.

The zebrafish zic2a-zic5 gene pair acts downstream of canonical Wnt signaling to control cell proliferation in the developing tectum.

Wnt growth factors acting through the canonical intracellular signaling cascade play fundamental roles during vertebrate brain development. In particular, canonical Wnt signaling is crucial for normal development of the dorsal midbrain, the future optic tectum. Wnts act both as patterning signals and as regulators of cell growth. In the developing tectum, Wnt signaling is mitogenic; however, the mechanism of Wnt function is not known. As a step towards better understanding this mechanism, we have identified two new Wnt targets, the closely linked zic2a and zic5 genes. Using a combination of in vivo assays, we show that zic2a and zic5 transcription is activated by Tcf/Lef transcription factors in the dorsal midbrain. Zic2a and Zic5, in turn, have essential, cooperative roles in promoting cell proliferation in the tectum, but lack obvious patterning functions. Collectively these findings suggest that Wnts control midbrain proliferation, at least in part, through regulation of two novel target genes, the zic2a-zic5 gene pair.

Lmx1b is essential for Fgf8 and Wnt1 expression in the isthmic organizer during tectum and cerebellum development in mice.

Secreted factors FGF8 and WNT1 are essential either for the inductive activity of the isthmus organizer or for the regionalization of the midbrain-hindbrain boundary (MHB). However, transcriptional regulation of these secreted factors during development remains to be elucidated. Here we show that the LIM homeobox gene Lmx1b is expressed in the anterior embryo as early as E7.5 and its expression becomes progressively restricted to the isthmus at E9.0. Analysis of gene expression in the MHB of the mutant embryos showed that many genes were lost by E9.5. In the MHB of Lmx1b-/- embryos, the expression of Fgf8, which normally occurs at the 4-somite stage, was completely absent, whereas Wnt1 was downregulated before the 4-somite stage. Moreover, transcription factors En1 and Pax2 were also downregulated prior to the 4-somite stage, whereas Gbx2 downregulation occurred at the 4-somite stage. By contrast, Otx2 and Pax6 expression was not affected in Lmx1b-/- embryos. The requirement of specific Lmx1b expression in the MHB was further confirmed by Wnt1-Cre-mediated region-specific conditional knockout of Lmx1b. As a result of these molecular defects, the development of the tectum and cerebellum was severely impaired in Lmx1b-/- mice. Taken together, our results indicate that Lmx1b plays an essential role in the development of the tectum and cerebellum by regulating expression of Fgf8, Wnt1 and several isthmus-related transcription factors in the MHB, and is a crucial component of a cross-regulatory network required for the induction activity of the isthmic organizer in the MHB.

Navigation of trochlear motor axons along the midbrain-hindbrain boundary by neuropilin 2.

Trochlear motor axons project dorsally along the midbrain-hindbrain boundary (MHB) to decussate at the dorsal midline. We report on the roles of neuropilin 2 and its ligands in the molecular mechanisms controlling this trajectory. In chick embryos, neuropilin 2 was expressed in the neuroepithelium of the dorsal isthmus in addition to the trochlear neurons, and Sema3F transcripts were localized along the caudal margin of the midbrain. Misexpression of Sema3F demonstrated that Sema3F displays repulsive activity in vivo that guides the trochlear motor axons along the MHB. An unexpected result was that misexpression of neuropilin 2 canceled the midbrain-evoked repulsion, allowing trochlear motor axons to cross the MHB and invade the tectum. A binding assay with neuropilin 2 ectodomain revealed the existence of neuropilin 2 ligands in the midbrain, which were masked by ectopic neuropilin 2. We therefore propose that neuropilin 2 neutralizes the repulsive activity in order to steer trochlear motor axons towards the dorsal decussation point. Taken together, our results suggest that the interaction of neuropilin 2 with its ligands has crucial roles for establishing trochlear trajectory along the MHB.

Topographic mapping in dorsoventral axis of the Xenopus retinotectal system depends on signaling through ephrin-B ligands.

Ephrin-B and EphB are distributed in matching dorsoventral gradients in the embryonic Xenopus visual system with retinal axons bearing high levels of ligand (dorsal) projecting to tectal regions with high receptor expression (ventral). In vitro stripe assays show that dorsal retinal axons prefer to grow on EphB receptor stripes supporting an attractive guidance mechanism. In vivo disruption of EphB/ephrin-B function by application of exogenous EphB or expression of dominant-negative ephrin-B ligand in dorsal retinal axons causes these axons to shift dorsally in the tectum, while misexpression of wild-type ephrin-B in ventral axons causes them to shift ventrally. These dorsoventral targeting errors are consistent with the hypothesis that an attractive mechanism that requires ephrin-B cytoplasmic domain is critical for retinotectal mapping in this axis.

Tectal graft modulation of audiogenic seizures in Long-Evans rat.

Audiogenic seizure (AGS) activity can be induced in the seizure-resistant Long-Evans rat by postnatal priming. This study examined the effects of unilateral lesions of the inferior colliculus (IC) and implantation of tectal grafts on AGS components. Animals were primed with a 10-kHz tone burst at 120 dB on postnatal day 14 and tested for AGS susceptibility on day 28, and then two groups were unilaterally lesioned including animals receiving embryonic day 16-17 grafts of caudal tectum. Subsequently, animals were repeatedly tested for wild running and clonic-tonic convulsion components of AGS. The results demonstrate that unilaterally grafted animals with partial IC lesions showed significant reduction in the incidence of clonus expression with greater terminal uniphasic wild running behavior. These effects were stronger than in animals with comparable unilateral lesions alone. Many neurons in graft cases were in direct contact with host tissues to provide a substrate for tissue interactions previously demonstrated to promote neuron survival and remediate IC functions.

[The prenatal development of the tectum mesencephali and substantia nigra in man]. / Prenatal'noe razvitie pokryshki srednego mozga i chernogo veshchestva u cheloveka.

The development of tectum of the midbrain was studied in situ in human 6-11 wks embryos. Using electron microscopy and immunocytochemistry processes that occur in the anlage of tectum of the midbrain at early stages of formation of dopaminergic populations of cells were followed up. Growth of pallium zone due to migration of cells from ventricular zone and their differentiation is proportional to growth of the terms of embryonal development. Proliferating cells were located not only in ventricular zone. Expression of tyrosine hydroxylase was found in 6.5 wks embryos in the fraction of cells that migrated from ventricular zone and was observed only during neuroblast migration. Cells migrate along the radial glia fibres in dorsoventral direction. Microgliocytes with short processes near to vessels located in the vicinity of ventricular zone were found in 7 wks embryos for the first time. Peculiarities of morphogenetic processes participating in anlage of tectum of the midbrain and possible role of microgliocytes in development of embryonal nerve tissue are discussed.

MR imaging of the posterior fossa structures of human embryos and fetuses.

There have been few reports on MR imaging of the developing human fetal brain. The aim of this article is to establish a standard atlas of developing fetal brain, focusing in particular on posterior fossa structures. Eighty-eight formalin-fixed embryos and fetuses were examined using 1.5 Tesla MR units. Specimens ranged from Carnegie stage 17 to 28 gestational weeks. The morphologic changes in developing cerebellum, cerebellar fissures, pontine flexure, fourth ventricle, and cerebral aqueduct were observed in each developmental period. The height of the fourth ventricle and cerebral aqueduct and the thickness of the tectum and the tegmentum of the midbrain were measured. We obtained detailed MR images of the developmental changes in posterior fossa structures and produced an atlas of these images. Our study showed that the period of visualization of cerebellar structures and fissures was later on MR imaging than described in past anatomical and embryological studies. In addition, the sudden decrease in height of the fourth ventricle and the cerebral aqueduct found in our study might reflect the presence of communication between the fourth ventricle and subarachnoid space.