Control of topographic retinal axon branching by inhibitory membrane-bound molecules.

Retinotopic map development in nonmammalian vertebrates appears to be controlled by molecules that guide or restrict retinal axons to correct locations in their targets. However, the retinotopic map in the superior colliculus (SC) of the rat is developed instead by a topographic bias in collateral branching and arborization. Temporal retinal axons extending across alternating membranes from the topographically correct rostral SC or the incorrect caudal SC of embryonic rats preferentially branch on rostral membranes. Branching preference is due to an inhibitory phosphatidylinositol-linked molecule in the caudal SC. Thus, position-encoding membrane-bound molecules may establish retinotopic maps in mammals by regulating axon branching, not by directing axon growth.

Potential of visual cortex to develop an array of functional units unique to somatosensory cortex.

The identification of specialized areas in the mammalian neocortex, such as the primary visual or somatosensory cortex, is based on distinctions in architectural and functional features. The extent to which certain features that distinguish neocortical areas in rats are prespecified or emerge as a result of epigenetic interactions was investigated. Late embryonic visual cortex transplanted to neonatal somatosensory cortex was later assayed for "barrels," anatomically identified functional units unique to somatosensory cortex, and for boundaries of glycoconjugated molecules associated with barrels. Barrels and boundaries form in transplanted visual cortex and are organized in an array that resembles the pattern in the normal barrelfield. These findings show that different regions of the developing neocortex have similar potentials to differentiate features that distinguish neocortical areas and contribute to their unique functional organizations.

Regulation of area identity in the mammalian neocortex by Emx2 and Pax6.

The contribution of extrinsic and genetic mechanisms in determining areas of the mammalian neocortex has been a contested issue. This study analyzes the roles of the regulatory genes Emx2 and Pax6, which are expressed in opposing gradients in the neocortical ventricular zone, in specifying areas. Changes in the patterning of molecular markers and area-specific connections between the cortex and thalamus suggest that arealization of the neocortex is disproportionately altered in Emx2 and Pax6 mutant mice in opposing manners predicted from their countergradients of expression: rostral areas expand and caudal areas contract in Emx2 mutants, whereas the opposite effect is seen in Pax6 mutants. These findings suggest that Emx2 and Pax6 cooperate to regulate arealization of the neocortex and to confer area identity to cortical cells.

Target control of collateral extension and directional axon growth in the mammalian brain.

Individual neurons in the brain send their axons over considerable distances to multiple targets, but the mechanisms governing this process are unresolved. An amenable system for studying axon outgrowth, branching, and target selection is the mammalian corticopontine projection. This major connection develops from parent corticospinal axons that have already grown past the pons, by a delayed interstitial budding of collateral branches that then grow directly into their target, the basilar pons. When cocultured with explants of developing cortex in three-dimensional collagen matrices, the basilar pons elicits the formation and directional growth of cortical axon collaterals across the intervening matrix. This effect appears to be target-specific and selectively influences neurons in the appropriate cortical layer. These in vitro findings provide evidence that the basilar pons becomes innervated by controlling at a distance the budding and directed ingrowth of cortical axon collaterals through the release of a diffusible, chemotropic molecule.

Regressive events in neurogenesis.

The development of most regions of the vertebrate nervous system includes a distinct phase of neuronal degeneration during which a substantial proportion of the neurons initially generated die. This degeneration primarily adjusts the magnitude of each neuronal population to the size or functional needs of its projection field, but in the process it seems also to eliminate many neurons whose axons have grown to either the wrong target or an inappropriate region within the target area. In addition, many connections that are initially formed are later eliminated without the death of the parent cell. In most cases such process elimination results in the removal of terminal axonal branches and hence serves as a mechanism to "fine-tune" neuronal wiring. However, there are now also several examples of the large-scale elimination of early-formed pathways as a result of the selective degeneration of long axon collaterals. Thus, far from being relatively minor aspects of neural development, these regressive phenomena are now recognized as playing a major role in determining the form of the mature nervous system.

Cortical axons branch to multiple subcortical targets by interstitial axon budding: implications for target recognition and "waiting periods".

We are studying how axons branch in vivo. Individual cortical neurons send axons to both the spinal cord and the basilar pons. Here we show that the corticopontine projection develops by an interstitial budding of collaterals from parent axons rather than a reported mechanism of axon branching, growth cone bifurcation. This mechanism is used regardless of whether the parent axon's postpontine segment, which forms the corticospinal projection, is permanent (motor cortex) or transient (visual cortex). Budding occurs days after the parent axons grow spinally past the pons, accounting for the "waiting period" reported in this system in contrast to an alternative explanation that the growth cones pause outside of their target. Timing and location of pontine collateral budding vary with cortical origin of the parent axon and are correlated with the temporal ordering of axon arrival.

Responses of retinal axons in vivo and in vitro to position-encoding molecules in the embryonic superior colliculus.

We show that rat retinal ganglion cell axons exhibit no topographic specificity in growth along the rostral-caudal axis of the embryonic superior colliculus (SC). Position-related, morphological differences are not found between temporal and nasal axon growth cones. However, embryonic retinal axons respond in vitro to a position-dependent molecular property of SC membranes. In vivo, regional specificity in side branching is the earliest indication that axons make topographic distinctions along the rostral-caudal SC axis. Our contrasting in vivo and in vitro results indicate that molecules encoding rostral-caudal position in the SC neither guide nor restrict retinal axon growth, but may promote the development of topographic connections by controlling specificity in the extension or stabilization of branches.

Action of a diffusible target-derived chemoattractant on cortical axon branch induction and directed growth.

Cortical axons innervate their brainstem target, the basilar pons, by the initiation and extension of collateral branches interstitially along their length. To address whether a diffusible pons-derived chemoattractant controls these events, we used cocultures in collagen matrices and time-lapse microscopy. Pontine explants enhanced by 5-fold the de novo initiation of transient branches along cortical axons; most branches were directed toward pons. Of the branches extended toward pons, 2%-3% were stabilized; those extended away were not. Pontine explants also enhanced the stable bifurcation of growth cones and prompted directional changes by growth cone turning and collateral extension. These effects were distance dependent and mimicked by pons-conditioned medium. This evidence indicates that the pons activity promotes branch initiation interstitially along cortical axons, a novel property for a chemoattractant, and provides a directional cue for their growth. These findings suggest that the pons chemoattractant serves as a diffusible target-recognition molecule.

Labeling neural cells using adenoviral gene transfer of membrane-targeted GFP.

We describe an experimental system to visualize the soma and processes of mammalian neurons and glia in living and fixed preparations by using a recombinant adenovirus vector to transfer the jellyfish green fluorescent protein (GFP) into postmitotic neural cells both in vitro and in vivo. We have introduced several modifications of GFP that enhance its fluorescence intensity in mammalian axons and dendrites. This method should be useful for studying the dynamic processes of cell migration and the development of neuronal connections, as well as for analyzing the function of exogenous genes introduced into cells using the adenovirus vector.

Ephrin-A5 (AL-1/RAGS) is essential for proper retinal axon guidance and topographic mapping in the mammalian visual system.

Ephrin-A5 (AL-1/RAGS), a ligand for Eph receptor tyrosine kinases, repels retinal axons in vitro and has a graded expression in the superior colliculus (SC), the major midbrain target of retinal ganglion cells. These properties implicate ephrin-A5 in the formation of topographic maps, a fundamental organizational feature of the nervous system. To test this hypothesis, we generated mice lacking ephrin-A5. The majority of ephrin-A5-/- mice develop to adulthood, are morphologically intact, and have normal anterior-posterior patterning of the midbrain. However, within the SC, retinal axons establish and maintain dense arborizations at topographically incorrect sites that correlate with locations of low expression of the related ligand ephrin-A2. In addition, retinal axons transiently overshoot the SC and extend aberrantly into the inferior colliculus (IC). This defect is consistent with the high level of ephrin-A5 expression in the IC and the finding that retinal axon growth on membranes from wild-type IC is inhibited relative to that on membranes from ephrin-A5-/- IC. These findings show that ephrin-A5 is required for the proper guidance and mapping of retinal axons in the mammalian midbrain.

A POU domain transcription factor-dependent program regulates axon pathfinding in the vertebrate visual system.

Axon pathfinding relies on the ability of the growth cone to detect and interpret guidance cues and to modulate cytoskeletal changes in response to these signals. We report that the murine POU domain transcription factor Brn-3.2 regulates pathfinding in retinal ganglion cell (RGC) axons at multiple points along their pathways and the establishment of topographic order in the superior colliculus. Using representational difference analysis, we identified Brn-3.2 gene targets likely to act on axon guidance at the levels of transcription, cell-cell interaction, and signal transduction, including the actin-binding LIM domain protein abLIM. We present evidence that abLIM plays a crucial role in RGC axon pathfinding, sharing functional similarity with its C. elegans homolog, UNC-115. Our findings provide insights into a Brn-3.2-directed hierarchical program linking signaling events to cytoskeletal changes required for axon pathfinding.

Altered hormonal regulation and blood flow distribution with cardiovascular deconditioning after short-duration head down bed rest.

This study tested the hypothesis that cardiovascular and hormonal responses to lower body negative pressure (LBNP) would be altered by 4-h head down bed rest (HDBR) in 11 healthy young men. In post-HDBR testing, three subjects failed to finish the protocol due to presyncopal symptoms, heart rate was increased during LBNP compared with pre-HDBR, mean arterial blood pressure was elevated at 0, -10, and -20 mmHg and reduced at -40 mmHg, central venous pressure (CVP) and cardiac stroke volume were reduced at all levels of LBNP. Plasma concentrations of renin, angiotensin II, and aldosterone were significantly lower after HDBR. Renin and angiotensin II increased in response to LBNP only post-HDBR. There was no effect of HDBR or LBNP on norepinephrine while epinephrine tended to increase at -40 mmHg post-HDBR (P = 0.07). Total blood volume was not significantly reduced. Splanchnic blood flow taken from ultrasound measurement of the portal vein was higher at each level of LBNP post-compared with pre-HDBR. The gain of the cardiopulmonary baroreflex relating changes in total peripheral resistance to CVP was increased after HDBR, but splanchnic vascular resistance was actually reduced. These results are consistent with our hypothesis and suggest that cardiovascular instability following only 4-h HDBR might be related to altered hormonal and/or neural control of regional vascular resistance. Impaired ability to distribute blood away from the splanchnic region was associated with reduced stroke volume, elevated heart rate, and the inability to protect mean arterial pressure.

Heterogeneity of responses to orthostatic stress in homozygous twins.

Early analysis into the role of genetics on cardiovascular regulation has been accomplished by comparing blood pressure and heart rate in homozygous twins during unstressed, resting physiological conditions. However, many variables, including cognitive and environmental factors, contribute to the regulation of cardiovascular hemodynamics. Therefore, the purpose of this study was to determine the hemodynamic response of identical twins to an orthostatic stress, ranging from supine rest to presyncope. Heart rate, arterial blood pressure, middle cerebral artery blood velocity, an index of cerebrovascular resistance, cardiac output, total peripheral resistance, and end-tidal carbon dioxide were measured in 16 healthy monozygotic twin pairs. Five minutes of supine resting baseline data were collected, followed by 5 min of 60 degrees head-up tilt. After 5 min of head-up tilt, lower body negative pressure was applied in increments of 10 mmHg every 3 min until the onset of presyncope, at which time the subject was returned to the supine position for a 5-min recovery period. The data indicate that cardiovascular regulation under orthostatic stress demonstrates a significant degree of variance between identical twins, despite similar orthostatic tolerance. As the level of stress increases, so does the difference in the cardiovascular response within a twin pair. The elevated variance with increasing stress may be due to an increase in the role of environmental factors, as the influential role of genetics nears a functional limit. Therefore, although orthostatic tolerance times were very similar between identical twins, the mechanism involved in sustaining cardiovascular function during increasing stress was different.

Do cortical areas emerge from a protocortex?

The adult mammalian neocortex consists of numerous 'areas' distinguished from one another largely on the basis of distinctions in cytoarchitecture and connections. The developing neocortex, though, lacks many of these area-specific distinctions, and is more uniform across its extent. This less differentiated structure, here termed the 'protocortex' undergoes considerable modification after neurogenesis which results in the emergence of well-defined neocortical areas. To what extent, then, are neocortical areas predetermined? This issue is considered in the context of recent findings on the generation of the neocortex and its subsequent parcellation into distinct areas.

Eph receptor tyrosine kinases and their ligands in neural development.

Receptor tyrosine kinases play important roles in many developmental phenomena, including the regulation of cell proliferation, differentiation, and survival. The largest subfamily of receptor tyrosine kinases are the Eph receptors, which are widely expressed in the nervous system. With the recent identification of several Eph ligands, it has become evident that Eph receptors and their ligands are involved in the guidance of retinal axons and in the process of hindbrain segmentation.

Eph receptors and ephrins in neural development.

Ephrins, ligands for the Eph family of receptor tyrosine kinases, are pivotal players in many developmental phenomena in both the central and peripheral nervous systems. Ephrins appear to act typically, but not exclusively, as repellents throughout development to influence axon pathfinding and topographic mapping, as well as restricting cell migration and intermingling. Recent findings are beginning to characterize the function and signaling of ephrins, as well as major roles for them in other tissues.

Development, critical period plasticity, and adult reorganizations of mammalian somatosensory systems.

This review covers recent progress in three major areas of investigation in somatosensory systems: development, developmental plasticity and functional reorganization. Important findings relate to the development of periphery-related patterning in thalamic afferents to somatosensory cortex, the controversial role of neural activity in the development and plasticity of periphery-related afferent patterning in the brainstem and cortex, experience-dependent reorganizations in adult somatosensory cortex, and the locus of these changes.

Combinatorial expression patterns of LIM-homeodomain and other regulatory genes parcellate developing thalamus.

The anatomical and functional organization of dorsal thalamus (dTh) and ventral thalamus (vTh), two major regions of the diencephalon, is characterized by their parcellation into distinct cell groups, or nuclei, that can be histologically defined in postnatal animals. However, because of the complexity of dTh and vTh and difficulties in histologically defining nuclei at early developmental stages, our understanding of the mechanisms that control the parcellation of dTh and vTh and the differentiation of nuclei is limited. We have defined a set of regulatory genes, which include five LIM-homeodomain transcription factors (Isl1, Lhx1, Lhx2, Lhx5, and Lhx9) and three other genes (Gbx2, Ngn2, and Pax6), that are differentially expressed in dTh and vTh of early postnatal mice in distinct but overlapping patterns that mark nuclei or subsets of nuclei. These genes exhibit differential expression patterns in dTh and vTh as early as embryonic day 10.5, when neurogenesis begins; the expression of most of them is detected as progenitor cells exit the cell cycle. Soon thereafter, their expression patterns are very similar to those that we observe postnatally, indicating that unique combinations of these genes mark specific cell groups from the time they are generated to their later differentiation into nuclei. Our findings suggest that these genes act in a combinatorial manner to control the specification of nuclei-specific properties of thalamic cells and the differentiation of nuclei within dTh and vTh. These genes may also influence the pathfinding and targeting of thalamocortical axons through both cell-autonomous and non-autonomous mechanisms.

Differential expression of COUP-TFI, CHL1, and two novel genes in developing neocortex identified by differential display PCR.

Genes that control the specification and differentiation of the functionally specialized areas of the mammalian neocortex are likely expressed across the developing neocortex in graded or restricted patterns. To search for such genes we have performed a PCR-based differential display screen using RNAs from rostral neocortex, which included the primary motor area, and caudal neocortex, which included the primary visual area, of embryonic day 16 rats. We identified 82 differentially expressed gene fragments. Secondary screening by in situ hybridization confirmed that five fragments, representing four genes, are differentially expressed across developing rat neocortex. Two of the genes, chick ovalbumin upstream transcription factor I (COUP-TFI) and close homolog of L1 (CHL1), have been cloned previously, but their differential expression in cortex has not been reported. Sequences from the other two fragments suggest that they represent novel genes. The expression patterns include graded, restricted, and discontinuous expression with abrupt borders that might correlate with those of areas. The differential expression patterns of all four genes are established before the arrival of thalamocortical afferents, suggesting that they are independent of thalamic influence, and could direct or reflect arealization. In addition, COUP-TFI and CHL1 exhibit dynamic expression patterns that undergo substantial changes after thalamocortical afferents invade the cortical plate, suggesting that thalamic axons may influence their later expression. Postnatally, COUP-TFI is most prominently expressed in layer 4, in both rats and mice, and CHL1 is expressed in layer 5. COUP-TFI expression in cortex, and in ventral telencephalon and dorsal thalamus, suggests several possible causes for the loss of layer 4 neurons and the reduced thalamocortical projection reported in COUP-TFI knock-out mice.

Graded and areal expression patterns of regulatory genes and cadherins in embryonic neocortex independent of thalamocortical input.

The differentiation of areas of the mammalian neocortex has been hypothesized to be controlled by intrinsic genetic programs and extrinsic influences such as those mediated by thalamocortical afferents (TCAs). To address the interplay between these intrinsic and extrinsic mechanisms in the process of arealization, we have analyzed the requirement of TCAs in establishing or maintaining graded or areal patterns of gene expression in the developing mouse neocortex. We describe the differential expression of Lhx2, SCIP, and Emx1, representatives of three different classes of transcription factors, and the type II classical cadherins Cad6, Cad8, and Cad11, which are expressed in graded or areal patterns, as well as layer-specific patterns, in the cortical plate. The differential expression of Lhx2, SCIP, Emx1, and Cad8 in the cortical plate is not evident until after TCAs reach the cortex, whereas Cad6 and Cad11 show subtle graded patterns of expression before the arrival of TCAs, which later become stronger. We find that these genes exhibit normal-appearing graded or areal expression patterns in Mash-1 mutant mice that fail to develop a TCA projection. These findings show that TCAs are not required for the establishment or maintenance of the graded and areal expression patterns of these genes and strongly suggest that their regulation is intrinsic to the developing neocortex.