Michael Reber

A relative signalling model for the formation of a topographic neural map.

The highly ordered wiring of retinal ganglion cell (RGC) neurons in the eye to their synaptic targets in the superior colliculus of the midbrain has long served as the dominant experimental system for the analysis of topographic neural maps. Here we describe a quantitative model for the development of one arm of this map—the wiring of the nasal–temporal axis of the retina to the caudal–rostral axis of the superior colliculus. The model is based on RGC–RGC competition that is governed by comparisons of EphA receptor signalling intensity, which are made using ratios of, rather than absolute differences in, EphA signalling between RGCs. Molecular genetic experiments, exploiting a combinatorial series of EphA receptor knock-in and knockout mice, confirm the salient predictions of the model, and show that it both describes and predicts topographic mapping.

Variant Hepatocyte Nuclear Factor 1 expression in the mouse genital tract

Variant Hepatocyte Nuclear Factor 1 (vHNF1/HNF1beta) is a homeodomain-containing transcription factor first expressed in the primitive endoderm and its derivatives, the visceral and parietal endoderm. It is subsequently expressed in epithelial cells of different organs, including the primitive gut and derivatives (liver, pancreas, lung), the kidney, and transiently, in the neural tube. We report here new data concerning vHnf1 expression in the mouse genital tract, using both RNA analyses and our vHnf1 heterozygous mutant mouse line, in which the first coding exon of the vHnf1 gene is replaced by the NLSLacZ reporter gene. Both beta-galactosidase activity and vHnf1 transcripts are detected in epididymus, vas deferens, seminal vesicle, prostate, uterus and oviduct. RNA analysis and in situ hybridization studies demonstrate that vHnf1 transcripts are restricted to the germinal cells of the testis. Unexpectedly, no beta-galactosidase activity is detected in the testis. We further show that, in addition to the somatic transcript, two more abundant vHnf1 transcript variants, which lack exons 1-4, appear in this organ after meiosis.

Functions of HNF1 family members in differentiation of the visceral endoderm cell lineage

The two members of the hepatocyte nuclear factor 1 (HNF1) transcription factor family, HNF1 and variant HNF1 (vHNF1), show a strong homology in their atypical POU-homeodomain and dimerization domain but differ in their transactivation domains. Moreover, two vHNF1 isoforms generated by alternative splicing are present in all tissues expressing this gene. vHnf1-deficient mouse embryos die soon after implantation due to defective visceral endoderm formation, an extraembryonic tissue essential for development and survival of the embryo proper. In contrast, invalidation of Hnf1, which is expressed at later developmental stages than vHnf1, does not lead to embryonic lethality or developmental defects. To examine the specific or potential equivalent functions of vHNF1 isoforms and HNF1 during the process of visceral endoderm differentiation, we stably reexpressed these factors in vHnf1-deficient embryonic stem cells. Analysis of these embryonic stem cells upon differentiation into embryoid bodies shows that vHNF1 isoforms exhibit specific behaviors depending on particular target genes and cooperate in the establishment of a functional visceral endoderm. Furthermore, forced expression of HNF1 in vHnf1-deficient embryonic stem cells fully restores the formation of a mature visceral endoderm with the correct expression profile of early and late markers of this lineage. Thus, in this context, HNF1 functionally replaces both vHNF1 isoforms, suggesting that the different developmental functions of these transcription factors are mainly due to the acquisition of novel expression patterns.

Eph Receptors and Ephrin Ligands in Axon Guidance

The Eph tyrosine kinase receptors (a receptor family named for the expression of Eph in an erythropoietin-producing human hepatocellular carcinoma cell line) make up the largest family of receptor tyrosine kinases. In vertebrates, 14 Eph receptor members have been identified, divided in two sub-groups, the EphAs (EphA1 to A8) and EphBs (EphB1 to B6). Their nine membrane-bound ligands, the ephrins, are also subdivided into the ephrin-As (ephrin-A1 to A6) and ephrin-Bs (ephrin-B1 to B3). The first Eph receptor (EphA1) was identified in 1987, whereas the ephrin ligands were cloned in the mid-90s 1. Eph receptors and ephrins have been found in all animal species analyzed so far, from C. elegans to humans, and are highly conserved through evolution 2. Ephs and ephrins are involved in numerous developmental processes, such as boundary formation, angiogenesis and cell migration. Within the nervous system, Eph signaling regulates the migration pattern of neural crest cells, the boundary formation between hindbrain segments (rhombomeres), the proper formation of the corticospinal tract, the establishment of neural topographic maps and the formation and functional properties of neuronal synapses 1, 3, 4, 5, 6. It is therefore not surprising that nature built a complicated and detailed network of proteins interacting with each other to fine tune each of these important processes. The identity of the receptor or ligand molecule is as important as the structure of the receptor-ligand complex to activate a specific signaling pathway and ultimately elicit the right cell decision.

vHnf1 Regulates Specification of Caudal Rhombomere Identity in the Chick Hindbrain

The homeobox‐containing gene variant hepatocyte nuclear factor‐1 (vHnf1) has recently been shown to be involved in zebrafish caudal hindbrain specification, notably in the activation of MafB and Krox20 expression. We have explored this regulatory network in the chick by in ovo electroporation in the neural tube. We show that misexpression of vHnf1 confers caudal identity to more anterior regions of the hindbrain. Ectopic expression of mvHnf1 leads to ectopic activation of MafB and Krox20, and downregulation of Hoxb1 in rhombomere 4. Unexpectedly, mvhnf1 strongly upregulates Fgf3 expression throughout the hindbrain, in both a cell‐autonomous and a non‐cell‐autonomous manner. Blockade of FGF signaling correlates with a selective loss of MafB and Krox20 expression, without affecting the expression of vHnf1, Fgf3, or Hoxb1. Based on these observations, we propose that in chick, as in zebrafish, vHnf1 acts with FGF to promote caudal hindbrain identity by activating MafB and Krox20 expression. However, our data suggest differences in the vHnf1 downstream cascade in different vertebrates. Developmental Dynamics 234:567–576, 2005.

Genetic dissection of EphA receptor signaling dynamics during retinotopic mapping

Retinal ganglion cells (RGCs) project axons from their cell bodies in the eye to targets in the superior colliculus of the midbrain. The wiring of these axons to their synaptic targets creates an ordered representation, or “map,” of retinal space within the brain. Many lines of experiments have demonstrated that the development of this map requires complementary gradients of EphA receptor tyrosine kinases and their ephrin-A ligands, yet basic features of EphA signaling during mapping remain to be resolved. These include the individual roles played by the multiple EphA receptors that make up the retinal EphA gradient. We have developed a set of ratiometric “relative signaling” (RS) rules that quantitatively predict how the composite low-nasal-to-high-temporal EphA gradient is translated into topographic order among RGCs. A key feature of these rules is that the component receptors of the gradient—in the mouse, EphA4, EphA5, and EphA6—must be functionally equivalent and interchangeable. To test this aspect of the model, we generated compound mutant mice in which the periodicity, slope, and receptor composition of the gradient are systematically altered with respect to the levels of EphA4, EphA5, and a closely related receptor, EphA3, that we ectopically express. Analysis of the retinotopic maps of these new mouse mutants establishes the general utility of the RS rules for predicting retinocollicular topography, and demonstrates that individual EphA gene products are approximately equivalent with respect to axon guidance and target selection.

Implication of Neuropilin 2/Semaphorin 3F in Retinocollicular Map Formation

Neural representations of the environment within the brain take the form of topographic maps whose formation relies on graded expression of axon guidance molecules. Retinocollicular map formation, from retinal ganglion cells (RGCs) to the superior colliculus (SC) in the midbrain, is mainly driven by Eph receptors and their ligands ephrins. However, other guidance molecules participate in the formation of this map. Here we demonstrate that the receptor Neuropilin‐2 is expressed in an increasing nasal–temporal gradient in RGCs, whereas one of its ligands, Semaphorin3F, but not other Sema3 molecules, presents a graded low‐rostral to high‐caudal expression in the SC when mapping is underway. Neuropilin‐2 and its coreceptor Plexin A1 are present on RGC growth cones. Collapse assays demonstrate that Semaphorin3F induces significant growth cone collapse of temporal, but not nasal, RGCs expressing high levels of Neuropilin‐2. Our results suggest that Neuropilin‐2/Semaphorin3F are new candidates involved in retinotopy formation within the SC. Developmental Dynamics 237:3394–3403, 2008.

Defective response inhibition and collicular noradrenaline enrichment in mice with duplicated retinotopic map in the superior colliculus.

The superior colliculus is a hub for multisensory integration necessary for visuo-spatial orientation, control of gaze movements and attention. The multiple functions of the superior colliculus have prompted hypotheses about its involvement in neuropsychiatric conditions, but to date, this topic has not been addressed experimentally. We describe experiments on genetically modified mice, the Isl2-EphA3 knock-in line, that show a well-characterized duplication of the retino-collicular and cortico-collicular axonal projections leading to hyperstimulation of the superior colliculus. To explore the functional impact of collicular hyperstimulation, we compared the performance of homozygous knock-in, heterozygous knock-in and wild-type mice in several behavioral tasks requiring collicular activity. The light/dark box test and Go/No-Go conditioning task revealed that homozygous mutant mice exhibit defective response inhibition, a form of impulsivity. This defect was specific to attention as other tests showed no differences in visually driven behavior, motivation, visuo-spatial learning and sensorimotor abilities among the different groups of mice. Monoamine quantification and gene expression profiling demonstrated a specific enrichment of noradrenaline only in the superficial layers of the superior colliculus of Isl2-EphA3 knock-in mice, where the retinotopy is duplicated, whereas transcript levels of receptors, transporters and metabolic enzymes of the monoaminergic pathway were not affected. We demonstrate that the defect in response inhibition is a consequence of noradrenaline imbalance in the superficial layers of the superior colliculus caused by retinotopic map duplication. Our results suggest that structural abnormalities in the superior colliculus can cause defective response inhibition, a key feature of attention-deficit disorders.

Estimating the location and size of retinal injections from orthogonal images of an intact retina

Background To study the mapping from the retina to the brain, typically a small region of the retina is injected with a dye, which then propagates to the retina’s target structures. To determine the location of the injection, usually the retina is dissected out of the eye, flattened and then imaged, causing tears and stretching of the retina. The location of the injection is then estimated from the image of the flattened retina. Here we propose a new method that avoids dissection of the retina.ResultsWe have developed IntactEye, a software package that uses two orthogonal images of the intact retina to locate focal injections of a dye. The two images are taken while the retina is still inside the eye. This bypasses the dissection step, avoiding unnecessary damage to the retina, and speeds up data acquisition. By using the native spherical coordinates of the eye, we avoid distortions caused by interpreting a curved structure in a flat coordinate system. Our method compares well to the projection method and to the Retistruct package, which both use the flattened retina as a starting point. We have tested the method also on synthetic data, where the injection location is known. Our method has been designed for analysing mouse retinas, where there are no visible landmarks for discerning retinal orientation, but can also be applied to retinas from other species.Conclusions IntactEye allows the user to precisely specify the location and size of a retinal injection from two orthogonal images taken of the eye. We are solving the abstract problem of locating a point on a spherical object from two orthogonal images, which might have applications outside the field of neuroscience.

A molecular mechanism for the topographic alignment of convergent neural maps

Sensory processing requires proper alignment of neural maps throughout the brain. In the superficial layers of the superior colliculus of the midbrain, converging projections from retinal ganglion cells and neurons in visual cortex must be aligned to form a visuotopic map, but the basic mechanisms mediating this alignment remain elusive. In a new mouse model, ectopic expression of ephrin-A3 (Efna3) in a subset of retinal ganglion cells, quantitatively altering the retinal EFNAs gradient, disrupts cortico-collicular map alignment onto the retino-collicular map, creating a visuotopic mismatch. Genetic inactivation of ectopic EFNA3 restores a wild-type cortico-collicular map. Theoretical analyses using a new mapping algorithm model both map formation and alignment, and recapitulate our experimental observations. The algorithm is based on an initial sensory map, the retino-collicular map, which carries intrinsic topographic information, the retinal EFNAs, to the superior colliculus. These EFNAs subsequently topographically align ingrowing visual cortical axons to the retino-collicular map. DOI: http://dx.doi.org/10.7554/eLife.20470.001