Paola Bovolenta

Beyond Wnt inhibition: new functions of secreted Frizzled-related proteins in development and disease

The secreted Frizzled-related proteins (SFRPs) are a family of soluble proteins that are structurally related to Frizzled (Fz) proteins, the serpentine receptors that mediate the extensively used cell-cell communication pathway involving Wnt signalling. Because of their homology with the Wnt-binding domain on the Fz receptors, SFRPs were immediately characterised as antagonists that bind to Wnt proteins to prevent signal activation. Since these initial studies, interest in the family of SFRPs has grown progressively, offering new perspectives on their function and mechanism of action in both development and disease. These studies indicate that SFRPs are not merely Wnt-binding proteins, but can also antagonise one anothers activity, bind to Fz receptors and influence axon guidance, interfere with BMP signalling by acting as proteinase inhibitors, and interact with other receptors or matrix molecules. Furthermore, their expression is altered in different types of cancers, bone pathologies, retinal degeneration and hypophosphatemic diseases, indicating that their activity is fundamental for tissue homeostasis. Here we review some of the debated aspects of SFRP-Wnt interactions and discuss the new and emerging roles of SFRPs.

Sonic hedgehog in CNS development: one signal, multiple outputs

Sonic hedgehog (SHH) is a member of the hedgehog family of signalling molecules. SHH was initially described as a protein secreted from two signalling centres, the notochord and the floor plate. Subsequently, it was identified as a morphogen that is directly responsible for dorso-ventral patterning of the CNS. More recently, additional sites of SHH expression have been identified and multiple actions of SHH during CNS development discovered, including the specification of oligodendrocytes, proliferation of neural precursors and control of axon growth. Despite these various activities, it appears that the SHH signalling pathway is well conserved and that the same mechanisms are utilized to achieve a variety of cellular responses. Therefore, a more precise understanding of the molecular mechanisms that underlie the different responses to SHH signalling is the next step in the study of this molecule and its role in the regulation of neural development.

Nervous system proteoglycans as modulators of neurite outgrowth.

The proteoglycans are multifunctional macromolecules composed of a core polypeptide and a variable number of glycosaminoglycan chains. The structural diversity and complexities of proteoglycan expression in the developing and adult Nervous System underlies the variety of biological functions that these molecules fulfill. Thus, in the Nervous System, proteoglycans regulate the structural organisation of the extracellular matrix, modulate growth factor activities and cellular adhesive and motility events, such as cell migration and axon outgrowth. This review summarises the evidences indicating that proteoglycans have an important role as modulators of neurite outgrowth and neuronal polarity. Special emphasis will be placed on those studies that have shown that proteoglycans of certain subtypes inhibit neurite extension either during the development and/or the regeneration of the vertebrate Central Nervous System.

Cux1 and Cux2 regulate dendritic branching, spine morphology, and synapses of the upper layer neurons of the cortex.

Dendrite branching and spine formation determines the function of morphologically distinct and specialized neuronal subclasses. However, little is known about the programs instructing specific branching patterns in vertebrate neurons and whether such programs influence dendritic spines and synapses. Using knockout and knockdown studies combined with morphological, molecular, and electrophysiological analysis, we show that the homeobox Cux1 and Cux2 are intrinsic and complementary regulators of dendrite branching, spine development, and synapse formation in layer II-III neurons of the cerebral cortex. Cux genes control the number and maturation of dendritic spines partly through direct regulation of the expression of Xlr3b and Xlr4b, chromatin remodeling genes previously implicated in cognitive defects. Accordingly, abnormal dendrites and synapses in Cux2(-/-) mice correlate with reduced synaptic function and defects in working memory. These demonstrate critical roles of Cux in dendritogenesis and highlight subclass-specific mechanisms of synapse regulation that contribute to the establishment of cognitive circuits.

Implication of OTX2 in pigment epithelium determination and neural retina differentiation

The expression pattern of Otx2, a homeobox-containing gene, was analyzed from the beginning of eye morphogenesis until neural retina differentiation in chick embryos. Early on, Otx2 expression was diffuse throughout the optic vesicles but became restricted to their dorsal part when the vesicles contacted the surface ectoderm. As the optic cup forms,Otx2 was expressed only in the outer layer, which gives rise to the pigment epithelium. This early Otx2expression pattern was complementary to that of PAX2, which localizes to the ventral half of the developing eye and optic stalk.Otx2 expression was always observed in the pigment epithelium at all stages analyzed but was extended to scattered cells located in the central portion of the neural retina around stage 22. The number of cells expressing Otx2 transcripts increased with time, following a central to peripheral gradient. Bromodeoxyuridine labeling in combination with immunohistochemistry with anti-OTX2 antiserum and different cell-specific markers were used to determine that OTX2-positive cells are postmitotic neuroblasts undergoing differentiation into several, if not all, of the distinct cell types present in the chick retina. These data indicate thatOtx2 might have a double role in eye development. First, it might be necessary for the early specification and subsequent functioning of the pigment epithelium. Later, OTX2 expression might be involved in retina neurogenesis, defining a differentiation feature common to the distinct retinal cell classes.

Expression pattern of cSix3, a member of the Six/sine oculis family of transcription factors.

We describe the expression pattern of cSix3, a chick homologue of the murine Six3. cSix3 transcripts are expressed from presomitic stages in the most anterior portion of the neural plate. As the neural tube folds and the optic vesicles evaginate, cSix3 is expressed in the optic vesicle and the rostroventral forebrain. At later stages, cSix3 is found in most of the structures derived from the anterior neural plate, i.e. olfactory epithelium, septum, adenohypophysis, hypothalamus and preoptic areas. During eye development, cSix3 expression is first found in the entire optic vesicle and the overlying ectoderm but soon becomes restricted to the prospective neural retina and to the lens placode. In the developing neural retina, cSix3 is expressed in the entire undifferentiated neuroepithelium but is rapidly downregulated, first in the postmitotic photoreceptors and later in the majority of retinal ganglion cells.

Developmental changes in the Ca2+-regulated mitochondrial aspartate–glutamate carrier aralar1 in brain and prominent expression in the spinal cord

Aralar1 and citrin are two isoforms of the mitochondrial carrier of aspartate-glutamate (AGC), a calcium regulated carrier, which is important in the malate-aspartate NADH shuttle. The expression and cell distribution of aralar1 and citrin in brain cells has been studied during development in vitro and in vivo. Aralar1 is the only isoform expressed in neurons and its levels undergo a marked increase during in vitro maturation, which is higher than the increase in mitochondrial DNA in the same time window. The enrichment in aralar1 per mitochondria during neuronal maturation is associated with a prominent rise in the function of the malate-aspartate NADH shuttle. Paradoxically, during in vivo development of rat or mouse brain there is very little postnatal increase in total aralar1 levels per mitochondria. This is explained by the fact that astrocytes develop postnatally, have aralar1 levels much lower than neurons, and their increase masks that of aralar1. Aralar1 mRNA and protein are widely expressed throughout neuron-rich areas in adult mouse CNS with clear enrichments in sets of neuronal nuclei in the brainstem and, particularly, in the ventral horn of the spinal cord. These aralar1-rich neurons represent a subset of the cytochrome oxidase-rich neurons in the same areas. The presence of aralar1 could reflect a tonic activity of these neurons, which is met by the combination of high malate-aspartate NADH shuttle and respiratory chain activities.

Chapter 31: CNS glial scar tissue: a source of molecules which inhibit central neurite outgrowth

Publisher Summary The chapter describes the interactions between purified gliotic membranes and embryonic central nervous system (CNS) explants. Gliotic membranes have different effects on neurite outgrowth, depending on whether the lesion is isomorphic or anisomorphic. The partial characterization of a proteoglycan associated with isomorphic gliotic membranes, which appears to inhibit central neurite outgrowth is also described. The major component of the glial scar is reactive glia. Reactive glia formation is the most general and stereotyped reaction of the CNS to any type of insult. From a neuropathological point of view, gliosis can be classified as anisomorphic or isomorphic, based on whether the eliciting stimulus is an open injury. Plasma membranes isolated from fresh tissue maintain the characteristics of the living cell surface; therefore, plasma membranes are isolated from glial scar tissue to study directly the interaction of growing axons with the scar cellular components, predominantly reactive astrocytes and microglial cells.

SFRP1 regulates the growth of retinal ganglion cell axons through the Fz2 receptor

Axon growth is governed by the ability of growth cones to interpret attractive and repulsive guidance cues. Recent studies have shown that secreted signaling molecules known as morphogens can also act as axon guidance cues. Of the large family of Wnt signaling components, only Wnt4 and Wnt5 seem to participate directly in axon guidance. Here we show that secreted Frizzled-related protein 1 (SFRP1), a proposed Wnt signaling inhibitor, can directly modify and reorient the growth of chick and Xenopus laevis retinal ganglion cell axons. This activity does not require Wnt inhibition and is modulated by extracellular matrix molecules. Intracellularly, SFRP1 function requires Gα protein activation, protein synthesis and degradation, and it is modulated by cyclic nucleotide levels. Because SFRP1 interacts with Frizzled-2 (Fz2) and interference with Fz2 expression abolishes growth cone responses to SFRP1, we propose a previously unknown function for this molecule: the ability to guide growth cone movement via the Fz2 receptor.

Six3 and Six6 activity is modulated by members of the groucho family

Six3 and Six6 are two genes required for the specification and proliferation of the eye field in vertebrate embryos, suggesting that they might be the functional counterparts of the Drosophila gene sine oculis (so). Phylogenetic and functional analysis have however challenged this idea, raising the possibility that the molecular network in which Six3 and Six6 act may be different from that described for SO. To address this, we have performed yeast two-hybrid screens, using either Six3 or Six6 as a bait. In this paper, we report the results of the latter screen that led to the identification of TLE1 (a transcriptional repressor of the groucho family) and AES (a potential dominant negative form of TLE proteins) as cofactors for both SIX6 and SIX3. Biochemical and mutational analysis shows that the Six domain of both SIX3 and SIX6 strongly interact with the QD domain of TLE1 and AES, but that SIX3 also interacts with TLE proteins via the WDR domain. Tle1 and Aes are expressed in the developing eye of medaka fish (Oryzias latipes) embryos, overlapping with the distribution of both Six3 and Six6. Gain-of-function studies in medaka show a clear synergistic activity between SIX3/SIX6 and TLE1, which, on its own, can expand the eye field. Conversely, AES alone decreases the eye size and abrogates the phenotypic consequences of SIX3/6 over-expression. These data indicate that both Tle1 and Aes participate in the molecular network that control eye development and are consistent with the view that both Six3 and Six6 act in combination with either Tle1 and/or Aes.