Giuseppe Lupo

Mechanisms of ventral patterning in the vertebrate nervous system

Dorsoventral patterning of the neural tube has a crucial role in shaping the functional organization of the CNS. It is well established that hedgehog signalling plays a key role in specifying ventral cell types throughout the neuroectoderm, and major progress has been made in elucidating how hedgehog signalling works in this ventral specification. In addition, other molecular pathways, including nodal, retinoic acid and fibroblast growth factor signalling, have been identified as important molecular cues for ventral patterning of the spinal cord, telencephalon and eye. Here, we discuss recent advances in this field, highlighting the emerging interplay of these signalling pathways in the molecular specification of ventral patterning at different rostrocaudal levels of the CNS.

Dorsoventral patterning of the Xenopus eye : a collaboration of retinoid, hedgehog and FGF receptor signaling

In the developing spinal cord and telencephalon, ventral patterning involves the interplay of Hedgehog (Hh), Retinoic Acid (RA) and Fibroblast Growth Factor (FGF) signaling. In the eye, ventral specification involves Hh signaling, but the roles of RA and FGF signaling are less clear. By overexpression assays in Xenopus embryos, we found that both RA and FGF receptor (FGFR) signaling ventralize the eye, by expanding optic stalk and ventral retina, and repressing dorsal retina character. Co-overexpression experiments show that RA and FGFR can collaborate with Hh signaling and reinforce its ventralizing activity. In loss-of-function experiments, a strong eye dorsalization was observed after triple inhibition of Hh, RA and FGFR signaling, while weaker effects were obtained by inhibiting only one or two of these pathways. These results suggest that the ventral regionalization of the eye is specified by interactions of Hh, RA and FGFR signaling. We argue that similar mechanisms might control ventral neural patterning throughout the central nervous system.

Tsukushi Functions as an Organizer Inducer by Inhibition of BMP Activity in Cooperation with Chordin

During chick gastrulation, inhibition of BMP signaling is required for primitive streak formation and induction of Hensens node. We have identified a unique secreted protein, Tsukushi (TSK), which belongs to the Small Leucine-Rich Proteoglycan (SLRP) family and is expressed in the primitive streak and Hensens node. Grafts of cells expressing TSK in combination with the middle primitive streak induce an ectopic Hensens node, while electroporation of TSK siRNA inhibits induction of the node. In Xenopus embryos, TSK can block BMP function and induce a secondary dorsal axis, while it can dorsalize ventral mesoderm and induce neural tissue in embryonic explants. Biochemical analysis shows that TSK binds directly to both BMP and chordin and forms a ternary complex with them. These observations indicate that TSK is an essential dorsalizing factor involved in the induction of Hensens node.

The Xenopus Emx genes identify presumptive dorsal telencephalon and are induced by head organizer signals.

We have isolated and studied the expression pattern of Xemx1 and Xemx2 genes in Xenopus laevis. Xemx genes are the homologues of mouse Emx genes, related to Drosophila empty spiracles. They are expressed in selected regions of the developing brain, particularly in the telencephalon, and, outside the brain, in the otic vesicles, olfactory placodes, visceral arches and the developing excretory system. We also report on experiments concerning the tissue and molecular signals responsible for their activation in competent ectoderm. Xemx genes are activated in ectoderm conjugated with head organizer tissue, but not with tail organizer tissue. Furthermore, they are not activated in animal cap either by noggin or by Xnr3, thus suggesting that a different inducer or the integration of several signals may be responsible for their activation.

Tsukushi controls ectodermal patterning and neural crest specification in Xenopus by direct regulation of BMP4 and X-delta-1 activity

In Xenopus, ectodermal patterning depends on a mediolateral gradient of BMP signaling, higher in the epidermis and lower in the neuroectoderm. Neural crest cells are specified at the border between the neural plate and the epidermis, at intermediate levels of BMP signaling. We recently described a novel secreted protein, Tsukushi (TSK), which works as a BMP antagonist during chick gastrulation. Here, we report on the Xenopus TSK gene (X-TSK), and show that it is involved in neural crest specification. X-TSK expression accumulates after gastrulation at the anterior-lateral edges of the neural plate, including the presumptive neural crest region. In gain-of-function experiments, X-TSK can strongly enhance neural crest specification by the dorsolateral mesoderm or X-Wnt8 in ectodermal explants, while the electroporation of X-TSK mRNA in the lateral ectoderm of embryos after gastrulation can induce the expression of neural crest markers in vivo. By contrast, depletion of X-TSK in explants or embryos impairs neural crest specification. Similarly to its chick homolog, X-TSK works as a BMP antagonist by direct binding to BMP4. However, X-TSK can also indirectly regulate BMP4 mRNA expression at the neural plate border via modulation of the Delta-Notch signaling pathway. We show that X-TSK directly binds to the extracellular region of X-delta-1, and modulates Delta-dependent Notch activity. We propose that X-TSK plays a key role in neural crest formation by directly regulating BMP and Delta activities at the boundary between the neural and the non-neural ectoderm.

Retinoic acid receptor signaling regulates choroid fissure closure through independent mechanisms in the ventral optic cup and periocular mesenchyme

Retinoic acid receptor (RAR) signaling is required for morphogenesis of the ventral optic cup and closure of the choroid fissure, but the mechanisms by which this pathway regulates ventral eye development remain controversial and poorly understood. Although previous studies have implicated neural crest–derived periocular mesenchyme (POM) as the critical target of RA action in the eye, we show here that RAR signaling regulates choroid fissure closure in zebrafish by acting on both the ventral optic cup and the POM. We describe RAR-dependent regulation of eight genes in the neuroepithelial cells of the ventral retina and optic stalk and of six genes in the POM and show that these ventral retina/optic stalk and POM genes function independently of each other. Consequently, RAR signaling regulates ventral eye development through two independent, nonredundant mechanisms in different ocular tissues. Furthermore, the identification of two cohorts of genes implicated in ventral eye morphogenesis may help to elucidate the genetic basis of ocular coloboma in humans.

Induction and patterning of the telencephalon in Xenopus laevis

We report an analysis of the tissue and molecular interplay involved in the early specification of the forebrain, and in particular telencephalic, regions of the Xenopus embryo. In dissection/recombination experiments, different parts of the organizer region were explanted at gastrula stage and tested for their inducing/patterning activities on either naive ectoderm or on midgastrula stage dorsal ectoderm. We show that the anterior dorsal mesendoderm of the organizer region has a weak neural inducing activity compared with the presumptive anterior notochord, but is able to pattern either neuralized stage 10.5 dorsal ectoderm or animal caps injected with BMP inhibitors to a dorsal telencephalic fate. Furthermore, we found that a subset of this tissue, the anterior dorsal endoderm, still retains this patterning activity. At least part of the dorsal telencephalic inducing activities may be reproduced by the anterior endoderm secreted molecule cerberus, but not by simple BMP inhibition, and requires the N-terminal region of cerberus that includes its Wnt-binding domain. Furthermore, we show that FGF action is both necessary and sufficient for ventral forebrain marker expression in neuralized animal caps, and possibly also required for dorsal telencephalic specification. Therefore, integration of organizer secreted molecules and of FGF, may account for patterning of the more rostral part of Xenopus CNS.

Tsukushi functions as a Wnt signaling inhibitor by competing with Wnt2b for binding to transmembrane protein Frizzled4

The Wnt signaling pathway is essential for the development of diverse tissues during embryogenesis. Signal transduction is activated by the binding of Wnt proteins to the type I receptor low-density lipoprotein receptor–related protein 5/6 and the seven-pass transmembrane protein Frizzled (Fzd), which contains a Wnt-binding site in the form of a cysteine-rich domain. Known extracellular antagonists of the Wnt signaling pathway can be subdivided into two broad classes depending on whether they bind primarily to Wnt or to low-density lipoprotein receptor–related protein 5/6. We show that the secreted protein Tsukushi (TSK) functions as a Wnt signaling inhibitor by binding directly to the cysteine-rich domain of Fzd4 with an affinity of 2.3 × 10−10 M and competing with Wnt2b. In the developing chick eye, TSK is expressed in the ciliary/iris epithelium, whereas Wnt2b is expressed in the adjacent anterior rim of the optic vesicle, where it controls the differentiation of peripheral eye structures, such as the ciliary body and iris. TSK overexpression effectively antagonizes Wnt2b signaling in chicken embryonic retinal cells both in vivo and in vitro and represses Wnt-dependent specification of peripheral eye fates. Conversely, targeted inactivation of the TSK gene in mice causes expansion of the ciliary body and up-regulation of Wnt2b and Fzd4 expression in the developing peripheral eye. Thus, we uncover a crucial role for TSK as a Wnt signaling inhibitor that regulates peripheral eye formation.

Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures

Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification.

Expression of the Xvax2 gene demarcates presumptive ventral telencephalon and specific visual structures in Xenopus laevis

vax2 is a recently isolated homeobox gene, that plays an important role in controlling the dorso-ventral patterning of the retina. In this paper we present a thorough description of the Xvax2 expression pattern all along Xenopus embryogenesis, and compare this pattern in detail to that shown by Xvax1b and Xpax2, two genes also involved in ventral eye development. At early neurula stages, while Xpax2 starts to be expressed within the eye field, both Xvax2 and Xvax1b are exclusively activated in the presumptive ventral telencephalon. Since midneurula stages, Xvax2 and Xvax1b are also transcribed in the medial aspect of the eye field. At tailbud and tadpole stages, Xvax2, Xvax1b and Xpax2 expression overlaps in the optic stalk and nerve and in the optic disk, while Xvax2 and Xvax1b also display specific activation domains in the ventral retina as well as in the ventral telencephalon and diencephalon. Finally, during metamorphosis a high level of both Xvax2 and Xvax1b transcription is maintained in the optic chiasm. In addition, Xvax1b is transcribed in the ventral hypothalamus and in the hypophysis, whereas a strong Xvax2 expression is retained in the ventral portion of the mature retina.