Elisa Martí

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.

Wnt canonical pathway restricts graded Shh/Gli patterning activity through the regulation of Gli3 expression

Dorsoventral patterning of the vertebrate nervous system is achieved by the combined activity of morphogenetic signals secreted from dorsal and ventral signalling centres. The Shh/Gli pathway plays a major role in patterning the ventral neural tube; however, the molecular mechanisms that limit target gene responses to specific progenitor domains remain unclear. Here, we show that Wnt1/Wnt3a, by signalling through the canonical β-catenin/Tcf pathway, control expression of dorsal genes and suppression of the ventral programme, and that this role in DV patterning depends on Gli activity. Additionally, we show that Gli3 expression is controlled by Wnt activity. Identification and characterization of highly conserved non-coding DNA regions around the human Gli3 gene revealed the presence of transcriptionally active Tcf-binding sequences. These indicated that dorsal Gli3 expression might be directly regulated by canonical Wnt activity. In turn, Gli3, by acting as a transcriptional repressor, restricted graded Shh/Gli ventral activity to properly pattern the spinal cord.

The Sonic hedgehog pathway independently controls the patterning, proliferation and survival of neuroepithelial cells by regulating Gli activity

During CNS development, the proliferation of progenitors must be coordinated with the pattern of neuronal subtype generation. In the ventral neural tube, Sonic hedgehog acts as a long range morphogen to organise the pattern of cell differentiation by controlling the activity of Gli transcription factors. Here, we provide evidence that the same pathway also acts directly at long range to promote the proliferation and survival of progenitor cells. Blockade of Shh signaling or inhibition of Gli activity results in cell autonomous decreases in progenitor proliferation and survival. Conversely, positive Gli activity promotes proliferation and rescues the effects of inhibiting Shh signaling. Analysis of neural cells indicates that Shh/Gli signaling regulates the G1 phase of cell cycle and the expression of the anti-apoptotic factor Bcl2. Furthermore, Shh signaling independently regulates patterning, proliferation and survival of neural cells, thus Shh/Gli activity couples these separate cellular responses of progenitors to coordinate neural development.

Bmp2 antagonizes sonic hedgehog-mediated proliferation of cerebellar granule neurones through Smad5 signalling.

During development of the cerebellum, sonic hedgehog (Shh) is directly responsible for the proliferation of granule cell precursors in the external germinal layer. We have looked for signals able to regulate a switch from the Shh-mediated proliferative response to one that directs differentiation of granule neurones. Bone morphogenetic proteins (BMPs) are expressed in distinct neuronal populations within the developing cerebellar cortex. Bmp2 and Bmp4 are expressed in the proliferating precursors and subsequently in differentiated granule neurones of the internal granular layer, whereas Bmp7 is expressed by Purkinje neurones. In primary cultures, Bmp2 and Bmp4, but not Bmp7, are able to prevent Shh-induced proliferation, thereby allowing granule neuron differentiation. Furthermore, Bmp2 treatment downregulates components of the Shh pathway in proliferating granule cell precursors. Smad proteins, the only known BMP receptor substrates capable of transducing the signal, are also differentially expressed in the developing cerebellum: Smad1 in the external germinal layer and Smad5 in newly differentiated granule neurones. Among them, only Smad5 is phosphorylated in vivo and in primary cultures treated with Bmp2, and overexpression of Smad5 is sufficient to induce granule cell differentiation in the presence of Shh. We propose a model in which Bmp2-mediated Smad5 signalling suppresses the proliferative response to Shh by downregulation of the pathway, and allows granule cell precursor to enter their differentiation programme.

Distinct Sonic Hedgehog signaling dynamics specify floor plate and ventral neuronal progenitors in the vertebrate neural tube

The secreted ligand Sonic Hedgehog (Shh) organizes the pattern of cellular differentiation in the ventral neural tube. For the five neuronal subtypes, increasing levels and durations of Shh signaling direct progenitors to progressively more ventral identities. Here we demonstrate that this mode of action is not applicable to the generation of the most ventral cell type, the nonneuronal floor plate (FP). In chick and mouse embryos, FP specification involves a biphasic response to Shh signaling that controls the dynamic expression of key transcription factors. During gastrulation and early somitogenesis, FP induction depends on high levels of Shh signaling. Subsequently, however, prospective FP cells become refractory to Shh signaling, and this is a prerequisite for the elaboration of their identity. This prompts a revision to the model of graded Shh signaling in the neural tube, and provides insight into how the dynamics of morphogen signaling are deployed to extend the patterning capacity of a single ligand. In addition, we provide evidence supporting a common scheme for FP specification by Shh signaling that reconciles mechanisms of FP development in teleosts and amniotes.

Wnt won the war: Antagonistic role of Wnt over Shh controls dorso-ventral patterning of the vertebrate neural tube

The spinal cord has been used as a model to dissect the mechanisms that govern the patterning of tissues during animal development, since the principles that rule the dorso‐ventral patterning of the neural tube are applicable to other systems. Signals that determine the dorso‐ventral axis of the spinal cord include Sonic hedgehog (Shh), acting as a bona fide morphogenetic signal to determine ventral progenitor identities, and members of the Bmp and the Wnt families, acting in the dorsal neural tube. Although Wnts have been initially recognized as important in proliferation of neural progenitor cells, their role in the dorso‐ventral patterning has been controversial. In this review, we discuss recent reports that show an important contribution of the Wnt canonical pathway in dorso‐ventral pattern formation. These data allow building a model by which the ventralizing activity of Shh is antagonized by Wnt activity through the expression of Gli3, a potent inhibitor of the Shh pathway. Therefore, antagonistic interactions between canonical Wnt, promoting dorsal identities, and Shh pathways, inducing ventral ones, would define the dorso‐ventral patterning of the developing central nervous system. Developmental Dynamics 239:69–76, 2010.

Foxj1 regulates floor plate cilia architecture and modifies the response of cells to sonic hedgehog signalling

Sonic hedgehog signalling is essential for the embryonic development of many tissues including the central nervous system, where it controls the pattern of cellular differentiation. A genome-wide screen of neural progenitor cells to evaluate the Shh signalling-regulated transcriptome identified the forkhead transcription factor Foxj1. In both chick and mouse Foxj1 is expressed in the ventral midline of the neural tube in cells that make up the floor plate. Consistent with the role of Foxj1 in the formation of long motile cilia, floor plate cells produce cilia that are longer than the primary cilia found elsewhere in the neural tube, and forced expression of Foxj1 in neuroepithelial cells is sufficient to increase cilia length. In addition, the expression of Foxj1 in the neural tube and in an Shh-responsive cell line attenuates intracellular signalling by decreasing the activity of Gli proteins, the transcriptional mediators of Shh signalling. We show that this function of Foxj1 depends on cilia. Nevertheless, floor plate identity and ciliogenesis are unaffected in mouse embryos lacking Foxj1 and we provide evidence that additional transcription factors expressed in the floor plate share overlapping functions with Foxj1. Together, these findings identify a novel mechanism that modifies the cellular response to Shh signalling and reveal morphological and functional features of the amniote floor plate that distinguish these cells from the rest of the neuroepithelium.

Dorsal-ventral patterning of the neural tube: a tale of three signals.

Development of the vertebrate nervous system begins with the acquisition of neural identity from the midline dorsal‐ectodermal cells of the gastrulating embryos. The subsequent progressive specification of the neural plate along its anterior–posterior and dorsal–ventral (DV) axes allows the generation of the tremendous variety of neuronal and glial cells that compose the vertebrate central nervous system (CNS). Studies on the development of the spinal cord, the anatomically simplest part of the CNS, have generated most of our current knowledge on the signaling events and the genetic networks that orchestrate the DV patterning of the neural plate. In this review, we discuss the recent advances in our understanding of these events and highlight unresolved questions. We focused our attention on the activity and the integration of the three main instructive cues: Sonic hedgehog, the Wnts and the Bone Morphogenetic Proteins, giving particular attention to the less well understood dorsal signaling events.

Hedgehog activation is required upstream of Wnt signalling to control neural progenitor proliferation

The canonical Wnt and sonic hedgehog (Shh) pathways have been independently linked to cell proliferation in a variety of tissues and systems. However, interaction of these signals in the control of cell cycle progression has not been studied. Here, we demonstrate that in the developing vertebrate nervous system these pathways genetically interact to control progression of the G1 phase of the cell cycle. By in vivo loss-of-function experiments, we demonstrate the absolute requirement of an upstream Shh activity for the regulation of Tcf3/4 expression. In the absence of Tcf3/4, the canonical Wnt pathway cannot activate target gene expression, including that of cyclin D1, and the cell cycle is necessarily arrested at G1. In addition to the control of G1 progression, Shh activity controls the G2 phase through the regulation of cyclin E, cyclin A and cyclin B expression, and this is achieved independently of Wnt. Thus, in neural progenitors, cell cycle progression is co-ordinately regulated by Wnt and Shh activities.

The TGFβ intracellular effector Smad3 regulates neuronal differentiation and cell fate specification in the developing spinal cord

Here we show that Smad3, a transforming growth factor β (TGFβ)/activin signaling effector, is expressed in discrete progenitor domains along the dorsoventral axis of the developing chick spinal cord. Restriction of Smad3 expression to the dP6-p2 and p3 domains together with exclusion from the motoneuron progenitor domain, are the result of the activity of key transcription factors responsible for patterning the neural tube. Smad3-mediated TGFβ activity promotes cell-cycle exit and neurogenesis by inhibiting the expression of Id proteins, and activating the expression of neurogenic factors and the cyclin-dependent-kinase-inhibitor p27kip1. Furthermore, Smad3 activity induces differentiation of selected neuronal subtypes at the expense of other subtypes. Within the intermediate and ventral domains, Smad3 promotes differentiation of ventral interneurons at the expense of motoneuron generation. Consequently, the absence of Smad3 expression from the motoneuron progenitor domain during pattern formation of the neural tube is a prerequisite for the correct generation of spinal motoneurons.