Melina Rapacioli

uPA-uPAR Molecular Complex is Involved in Cell Signaling During Neuronal Migration and Neuritogenesis

Background: In the development of the central nervous system (CNS), neuronal migration and neuritogenesis are crucial processes for establishing functional neural circuits. This relies on the regulation exerted by several signaling molecules, which play important roles in axonal growth and guidance. The urokinase‐type plasminogen activator (uPA)—in association with its receptor—triggers extracellular matrix proteolysis and other cellular processes through the activation of intracellular signaling pathways. Even though the uPA‐uPAR complex is well characterized in nonneuronal systems, little is known about its signaling role during CNS development. Results: In response to uPA, neuronal migration and neuritogenesis are promoted in a dose‐dependent manner. After stimulation, uPAR interacts with α5‐ and β1‐integrin subunits, which may constitute an αβ‐heterodimer that acts as a uPA‐uPAR coreceptor favoring the activation of multiple kinases. This interaction may be responsible for the uPA‐promoted phosphorylation of focal adhesion kinase (FAK) and its relocation toward growth cones, triggering cytoskeletal reorganization which, in turn, induces morphological changes related to neuronal migration and neuritogenesis. Conclusions: uPA has a key role during CNS development. In association with its receptor, it orchestrates both proteolytic and nonproteolytic events that govern the proper formation of neural networks. Developmental Dynamics 243:676–689, 2014.

Developmental pattern of NADPH‐diaphorase positive neurons in chick optic tectum is sensitive to changes in visual stimulation

The chick retinotectal system is a suitable model to investigate the mechanisms involved in the establishment of synaptic connections in whose refinement nitric oxide was implicated. The purpose of this work was to describe the developmental pattern of the nitric oxide synthase (NOS)‐positive neurons as well as to determine if it is sensitive to changes in visual stimulation. The NADPH‐diaphorase histochemical method was used to describe and quantify NOS neurons in normally stimulated and subnormally stimulated chickens. Nine types of NOS neurons were identified; seven of them express NOS until adulthood, while two of them show only a transient expression. The developmental pattern of NOS neurons follows the process of laminar segregation. It can be divided into three phases. The first includes the onset of NOS expression in periventricular neurons and the formation of a deep network of NOS fibers during early development. These neurons do not show any significant change in subnormally stimulated animals. The second phase includes the appearance of two transient NOS populations of bipolar neurons that occupy the intermediate layers during the optic fibers ingrowth. One of them significantly changes in subnormally stimulated chicks. The third phase occurs when the transitory expression of bipolar neurons decreases. It includes NOS expression in six neuronal populations that innervate the superficial retinorecipient layers. Most of these cells suffer plastic changes in subnormally stimulated chicks. The diversity of neuronal types with regard to their morphology, location, and sensitivity to visual stimulation strongly suggests that they serve different functions. J. Comp. Neurol. 494:1007–1030, 2006.

The chick optic tectum developmental stages. A dynamic table based on temporal‐ and spatial‐dependent histogenetic changes: A structural, morphometric and immunocytochemical analysis

Development is often described as temporal sequences of developmental stages (DSs). When tables of DS are defined exclusively in the time domain they cannot discriminate histogenetic differences between different positions along a spatial reference axis. We introduce a table of DSs for the developing chick optic tectum (OT) based on time‐ and space‐dependent changes in quantitative morphometric parameters, qualitative histogenetic features and immunocytochemical pattern of several developmentally active molecules (Notch1, Hes5, NeuroD1, β‐III‐Tubulin, synaptotagmin‐I and neurofilament‐M). Seven DSs and four transitional stages were defined from ED2 to ED12, when the basic OT cortical organization is established, along a spatial developmental gradient axis extending between a zone of maximal and a zone of minimal development. The table of DSs reveals that DSs do not only progress as a function of time but also display a spatially organized propagation along the developmental gradient axis. The complex and dynamic character of the OT development is documented by the fact that several DSs are simultaneously present at any ED or any embryonic stage. The table of DSs allows interpreting how developmental cell behaviors are temporally and spatially organized and explains how different DSs appear as a function of both time and space. The table of DSs provides a reference system to characterize the OT corticogenesis and to reliably compare observations made in different specimens. J. Morphol. 2011.

Sonic hedgehog (Shh)/Gli modulates the spatial organization of neuroepithelial cell proliferation in the developing chick optic tectum

BackgroundSonic hedgehog (Shh)/Gli pathway plays an important regulatory role on the neuroepithelial cells (NEc) proliferation in the dorsal regions of the developing vertebrate Central Nervous System. The aim of this paper was to analyze the effect of the Shh/Gli signaling pathway activation on the proliferation dynamics and/or the spatial organization of the NEc proliferation activity during early stages of the developing chick optic tectum (OT). In ovo pharmacological gain and loss of hedgehog function approaches were complemented with in vivo electroporation experiments in order to create ectopic sources of either Shh or Gli activator (GliA) proteins in the OT. NEc proliferating activity was analyzed at ED 4/4.5 by recording the spatial co-ordinates of the entire population of mitotic NEc (mNEc) located along OT dorsal-ventral sections. Several space signals (numerical sequences) were derived from the mNEc spatial co-ordinate records and analyzed by different standardized non-linear methods of signal analysis.ResultsIn ovo pharmacologic treatment with cyclopamine resulted in dramatic failure in the OT expansion while the agonist purmorphamine produced the opposite result, a huge expansion of the OT vesicle. Besides, GliA and Shh misexpressions interfere with the formation of the intertectal fissure located along the dorsal midline. This morphogenetic alteration is accompanied by an increase in the mNEc density. There is a gradient in the response of NEcs to Shh and GliA: the increase in mNEc density is maximal near the dorsal regions and decrease towards the OT-tegmental boundary. Biomathematical analyses of the signals derived from the mNEc records show that both Shh and GliA electroporations change the proliferation dynamics and the spatial organization of the mNEc as revealed by the changes in the scaling index estimated by these methods.ConclusionsThe present results show that the Shh/Gli signaling pathway plays a critical role in the OT expansion and modelling. This effect is probably mediated by a differential mitogenic effect that increases the NEc proliferation and modulates the spatial organization of the NEc proliferation activity.

Development and localisation of GABAA receptor α1, α2, β2 and γ2 subunit mRNA in the chick optic tectum

An in situ hybridisation technique was used to analyse the spatial and temporal pattern of expression of the mRNA encoding the four γ‐aminobutyric acid A (GABAA) receptor subunits (α1, α2, β2, and γ2) in the developing chick optic tectum. As a rule, layer i, layer h, and transient cell compartment 3 (TCC3) show the highest levels of expression, especially of α1, α2 and β2, which undergo striking changes as a function of time. Apart from these common features, the global pattern is highly complex and dynamic. Such complexity derives from the fact that each subunit exhibits a characteristically distinct pattern of expression and the temporal evolution of each differs in the different layers of the tectum. The influence of several developmental cell behaviours such as proliferation, neuronal migration, programmed cell death, and differentiation must be taken into account to understand pattern complexity and dynamics. Our results suggest that differences in the rate of subunit expression, particularly of α1, α2, and β2, could have significant consequences on GABAA receptor complex subunit composition along development and on the functional properties of the GABA neurotransmitter system.

Developmental changes in the spatial pattern of mesencephalic trigeminal nucleus (Mes5) neuron populations in the developing chick optic tectum

The developing mesencephalic trigeminal nucleus (nucleus of the fifth cranial nerve; Mes5) is composed of four neuron populations: 1) the medial group, located at the tectal commissure; 2) the lateral group distributed along the optic tectum hemispheres; 3) a group outside the neural tube; and 4) a population located at the posterior commissure. The present work aims to elucidate the site of appearance, temporal evolution, and spatial distribution of the four Mes5 populations during development. According to detailed qualitative observations Mes5 neurons appear as a primitive unique population along a thin dorsal medial band of the mesencephalon. According to quantitative analyses (changes in cell density along defined reference axes performed as a function of time and space), the definitive spatial pattern of Mes5 neurons results from a process of differential cell movements along the tangential plane of the tectal hemispheres. Radial migration does not have a relevant developmental role. Segregation of medial and lateral group populations depends on the intensity of the lateral displacements. The mesenchymal population appears as an outsider subset of neurons that migrate from the cephalic third of the neural tube dorsal midregion to the mesenchymal compartment. This process, together with the intensive lateral displacements that the insider subset undergoes, contributes to the disappearance of this transient population. We cannot find evidence indicating that neural crest‐derived precursors enter the neural tube and differentiate into Mes5 neurons. Our results can be better interpreted in terms of the notion that a dorsal neural tube progenitor cell population behaves as precursor of both migrating peripheral descendants (neural crest) and intrinsic neurons (Mes5). J. Comp. Neurol. 448:337–348, 2002.

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.

Acute hypoxia differentially affects the γ-aminobutyric acid type A receptor α1, α2, β2, and γ2 subunit mRNA levels in the developing chick optic tectum: Stage-dependent sensitivity

This investigation analyzes the effect of an acute hypoxic treatment on the level of four (α1, α2, β2, and γ2) subunit mRNAs of the GABAA receptor in layer “i” of the developing chick optic tectum. Our results show that 1 hr of normobaric acute hypoxia significantly changes the subunit mRNA levels. Different subunit mRNAs display different sensitivity to hypoxia: α1, β2, and γ2 mRNAs are highly sensitive, whereas α2 mRNA is almost not affected. The sensitivity of the mRNA levels to hypoxia is stage dependent. The mean percentages of variation produced by the hypoxia in the level of expression of the four subunits were 20% at ED12, 5% at ED16, and only 2% at ED18. These changes in the mean percentages of expression modify the probability of coexpression. In the case of double mRNA combinations, the hypoxia produced a mean variation in the probability of coexpression of 37% at ED12, 8% at ED16, and only 4% at ED18. With regard to the triple subunit mRNAs combinations, the variations were 206% at ED12, 11% at ED16, and only 7% at ED18. The quadruple combination values were 1,500% at ED12, 21% at ED16, and only 11% at ED18. This study demonstrates that the subunit mRNA levels are highly sensitive during the early stages, suggesting that GABAA receptor composition might undergo environment‐dependent plastic changes providing a high degree of plasticity to the GABA neurotransmitter system development.

Morphogenetic and Histogenetic Roles of the Temporal-Spatial Organization of Cell Proliferation in the Vertebrate Corticogenesis as Revealed by Inter-specific Analyses of the Optic Tectum Cortex Development

The central nervous system areas displaying the highest structural and functional complexity correspond to the so called cortices, i.e., concentric alternating neuronal and fibrous layers. Corticogenesis, i.e., the development of the cortical organization, depends on the temporal-spatial organization of several developmental events: (a) the duration of the proliferative phase of the neuroepithelium, (b) the relative duration of symmetric (expansive) versus asymmetric (neuronogenic) sub phases, (c) the spatial organization of each kind of cell division, (e) the time of determination and cell cycle exit and (f) the time of onset of the post-mitotic neuronal migration and (g) the time of onset of the neuronal structural and functional differentiation. The first five events depend on molecular mechanisms that perform a fine tuning of the proliferative activity. Changes in any of them significantly influence the cortical size or volume (tangential expansion and radial thickness), morphology, architecture and also impact on neuritogenesis and synaptogenesis affecting the cortical wiring. This paper integrates information, obtained in several species, on the developmental roles of cell proliferation in the development of the optic tectum (OT) cortex, a multilayered associative area of the dorsal (alar) midbrain. The present review (1) compiles relevant information on the temporal and spatial organization of cell proliferation in different species (fish, amphibians, birds, and mammals), (2) revises the main molecular events involved in the isthmic organizer (IsO) determination and localization, (3) describes how the patterning installed by IsO is translated into spatially organized neural stem cell proliferation (i.e., by means of growth factors, receptors, transcription factors, signaling pathways, etc.) and (4) describes the morpho- and histogenetic effect of a spatially organized cell proliferation in the above mentioned species. A brief section on the OT evolution is also included. This section considers how the differential operation of cell proliferation could explain differences among species.

Optic tectum morphogenesis: A step-by-step model based on the temporal–spatial organization of the cell proliferation. Significance of deterministic and stochastic components subsumed in the spatial organization

Background: Cell proliferation plays an important morphogenetic role. This work analyzes the temporal–spatial organization of cell proliferation as an attempt to understand its contribution to the chick optic tectum (OT) morphogenesis. Results: A morphogenetic model based on space‐dependent differences in cell proliferation is presented. Step1: a medial zone of high mitotic density (mZHMD) appears at the caudal zone. Step2: the mZHMD expands cephalically forming the dorsal curvature and then duplicates into two bilateral ZHMDs (bZHMD). Step3: the bZHMDs move toward the central region of each hemitectum. Step4: the planar expansion of both bZHMD and a relative decrement in the dorsal midline growth produces a dorsal medial groove separating the tectal hemispheres. Step5: a relative caudal displacement of the bZHMDs produces the OT caudal curvature. Numerical sequences derived from records of mitotic cells spatial coordinates, analyzed as stochastic point processes, show that they correspond to 1/f(β) processes. The spatial organization subsumes deterministic and stochastic components. Conclusions: The deterministic component describes the presence of a long‐range influence that installs an asymmetric distribution of cell proliferation, i.e., an asymmetrically located ZHMD that print space‐dependent differences onto the tectal corticogenesis. The stochastic component reveals short‐range anti‐correlations reflecting spatial clusterization and synchronization between neighboring cells. Developmental Dynamics 241:1043–1061, 2012.