Michael P. Matise

Gli genes in development and cancer

With the realization that many proto-oncogenes and tumor suppressor genes are expressed and have important functions during mammalian development, it is clear that cancer often involves the inappropriate activation of genetic pathways used during normal development. A signaling cascade that has been of considerable interest to both developmental and cancer biologists involves the Hedgehog (Hh) family of secreted proteins. To date, the only transcription factors shown to be directly downstream of Hh are the zinc-finger containing proteins Cubitus interruptus (Ci) and Gli, in flies and vertebrates, respectively. The identification of many of the genes and proteins involved in Hh signaling has come largely from genetic and biochemical studies in Drosophila. Ci mediates Hh signaling through a Hh-dependent set of protein modifications that alter the activity of Ci on Hh target genes. Recent evidence suggests vertebrate Gli proteins may be similarly regulated. The interest in this pathway has taken on added importance with the identification of mutations in Hh pathway genes, including Gli genes, in several human developmental disorders and cancers. We discuss models for how Gli proteins mediate Hh signaling in both vertebrate development and cancers.

Transduction of graded Hedgehog signaling by a combination of Gli2 and Gli3 activator functions in the developing spinal cord.

The three vertebrate Gli proteins play a central role in mediating Hedgehog (Hh)-dependent cell fate specification in the developing spinal cord; however, their individual contributions to this process have not been fully characterized. In this paper, we have addressed this issue by examining patterning in the spinal cord of Gli2;Gli3 double mutant embryos, and in chick embryos transfected with dominant activator forms of Gli2 and Gli3. In double homozygotes, Gli1 is also not expressed; thus, all Gli protein activities are absent in these mice. We show that Gli3 contributes activator functions to ventral neuronal patterning, and plays a redundant role with Gli2 in the generation of V3 interneurons. We also show that motoneurons and three classes of ventral neurons are generated in the ventral spinal cord in double mutants, but develop as intermingled rather than discrete populations. Finally, we provide evidence that Gli2 and Gli3 activators control ventral neuronal patterning by regulating progenitor segregation. Thus, multiple ventral neuronal types can develop in the absence of Gli function, but require balanced Gli protein activities for their correct patterning and differentiation.

A homeodomain feedback circuit underlies step-function interpretation of a Shh morphogen gradient during ventral neural patterning

The deployment of morphogen gradients is a core strategy to establish cell diversity in developing tissues, but little is known about how small differences in the concentration of extracellular signals are translated into robust patterning output in responding cells. We have examined the activity of homeodomain proteins, which are presumed to operate downstream of graded Shh signaling in neural patterning, and describe a feedback circuit between the Shh pathway and homeodomain transcription factors that establishes non-graded regulation of Shh signaling activity. Nkx2 proteins intrinsically strengthen Shh responses in a feed-forward amplification and are required for ventral floor plate and p3 progenitor fates. Conversely, Pax6 has an opposing function to antagonize Shh signaling, which provides intrinsic resistance to Shh responses and is important to constrain the inductive capacity of the Shh gradient over time. Our data further suggest that patterning of floor plate cells and p3 progenitors is gated by a temporal switch in neuronal potential, rather than by different Shh concentrations. These data establish that dynamic, non-graded changes in responding cells are essential for Shh morphogen interpretation, and provide a rationale to explain mechanistically the phenomenon of cellular memory of morphogen exposure.

Molecular Control of Spinal Accessory Motor Neuron/Axon Development in the Mouse Spinal Cord

Within the developing vertebrate spinal cord, motor neuron subtypes are distinguished by the settling positions of their cell bodies, patterns of gene expression, and the paths their axons follow to exit the CNS. The inclusive set of cues required to guide a given motor axon subtype from cell body to target has yet to be identified, in any species. This is attributable, in part, to the unavailability of markers that demarcate the complete trajectory followed by a specific class of spinal motor axons. Most spinal motor neurons extend axons out of the CNS through ventral exit points. In contrast, spinal accessory motor neurons (SACMNs) project dorsally directed axons through lateral exit points (LEPs), and these axons assemble into the spinal accessory nerve (SAN). Here we show that an antibody against BEN/ALCAM/SC1/DM-GRASP/MuSC selectively labels mouse SACMNs and can be used to trace the pathfinding of SACMN axons. We use this marker, together with a battery of transcription factor-deficient or guidance cue/receptor-deficient mice to identify molecules required for distinct stages of SACMN development. Specifically, we find that Gli2 is required for the initial extension of axons from SACMN cell bodies, and that netrin-1 and its receptor Dcc are required for the proper dorsal migration of these cells and the dorsally directed extension of SACMN axons toward the LEPs. Furthermore, in the absence of the transcription factor Nkx2.9, SACMN axons fail to exit the CNS. Together, these findings suggest molecular mechanisms that are likely to regulate key steps in SACMN development.

Spatial and temporal requirements for sonic hedgehog in the regulation of thalamic interneuron identity.

In caudal regions of the diencephalon, sonic hedgehog (Shh) is expressed in the ventral midline of prosomeres 1-3 (p1-p3), which underlie the pretectum, thalamus and prethalamus, respectively. Shh is also expressed in the zona limitans intrathalamica (zli), a dorsally projecting spike that forms at the p2-p3 boundary. The presence of two Shh signaling centers in the thalamus has made it difficult to determine the specific roles of either one in regional patterning and neuronal fate specification. To investigate the requirement of Shh from a focal source of expression in the ventral midline of the diencephalon, we used a newly generated mouse line carrying a targeted deletion of the 525 bp intronic sequence mediating Shh brain enhancer-1 (SBE1) activity. In SBE1 mutant mice, Shh transcription was initiated but not maintained in the ventral midline of the rostral midbrain and caudal diencephalon, yet expression in the zli was unaffected. In the absence of ventral midline Shh, rostral thalamic progenitors (pTH-R) adopted the molecular profile of a more caudal thalamic subtype (pTH-C). Surprisingly, despite their early mis-specification, neurons derived from the pTH-R domain continued to migrate to their proper thalamic nucleus, extended axons along their normal trajectory and expressed some, but not all, of their terminal differentiation markers. Our results, and those of others, suggest a model whereby Shh signaling from distinct spatial and temporal domains in the diencephalon exhibits unique and overlapping functions in the development of discrete classes of thalamic interneurons.

Floor plate-derived sonic hedgehog regulates glial and ependymal cell fates in the developing spinal cord

Cell fate specification in the CNS is controlled by the secreted morphogen sonic hedgehog (Shh). At spinal cord levels, Shh produced by both the notochord and floor plate (FP) diffuses dorsally to organize patterned gene expression in dividing neural and glial progenitors. Despite the fact that two discrete sources of Shh are involved in this process, the individual contribution of the FP, the only intrinsic source of Shh throughout both neurogenesis and gliogenesis, has not been clearly defined. Here, we have used conditional mutagenesis approaches in mice to selectively inactivate Shh in the FP (ShhFP) while allowing expression to persist in the notochord, which underlies the neural tube during neurogenesis but not gliogenesis. We also inactivated Smo, the common Hh receptor, in neural tube progenitors. Our findings confirm and extend prior studies suggesting an important requirement for ShhFP in specifying oligodendrocyte cell fates via repression of Gli3 in progenitors. Our studies also uncover a connection between embryonic Shh signaling and astrocyte-mediated reactive gliosis in adults, raising the possibility that this pathway is involved in the development of the most common cell type in the CNS. Finally, we find that intrinsic spinal cord Shh signaling is required for the proper formation of the ependymal zone, the epithelial cell lining of the central canal that is also an adult stem cell niche. Together, our studies identify a crucial late embryonic role for ShhFP in regulating the specification and differentiation of glial and epithelial cells in the mouse spinal cord.

Prox1 regulates a transitory state for interneuron neurogenesis in the spinal cord.

Proper central nervous system (CNS) function depends critically on the generation of functionally distinct neuronal types in specific and reproducible positions. The generation of neuronal diversity during CNS development involves a fine balance between dividing neural progenitors and the differentiated neuronal progeny that they produce. However, the molecular mechanisms that regulate these processes are still poorly understood. Here, we show that the Prox1 transcription factor, which is expressed transiently and specifically in spinal interneurons, plays an important role in neurogenesis. Using both gain‐ and loss‐of‐function approaches, we find that Prox1 is capable of driving neuronal precursors out of the cell cycle and can initiate limited expression of neuronal proteins. Using RNAi approaches, we show that Prox1 function is required to execute a neurogenic differentiation program downstream of Mash1 and Ngn2. Our studies demonstrate an important, spinal interneuron‐specific role for Prox1 in controlling steps required for both cell‐cycle withdrawal and differentiation. Developmental Dynamics 237:393–402, 2008.

Tcf/Lef repressors differentially regulate Shh-Gli target gene activation thresholds to generate progenitor patterning in the developing CNS

During neural tube development, Shh signaling through Gli transcription factors is necessary to establish five distinct ventral progenitor domains that give rise to unique classes of neurons and glia that arise in specific positions along the dorsoventral axis. These cells are generated from progenitors that display distinct transcription factor gene expression profiles in specific domains in the ventricular zone. However, the molecular genetic mechanisms that control the differential spatiotemporal transcriptional responses of progenitor target genes to graded Shh-Gli signaling remain unclear. The current study demonstrates a role for Tcf/Lef repressor activity in this process. We show that Tcf3 and Tcf7L2 (Tcf4) are required for proper ventral patterning and function by independently regulating two Shh-Gli target genes, Nkx2.2 and Olig2, which are initially induced in a common pool of progenitors that ultimately segregate into unique territories giving rise to distinct progeny. Genetic and functional studies in vivo show that Tcf transcriptional repressors selectively elevate the strength and duration of Gli activity necessary to induce Nkx2.2, but have no effect on Olig2, and thereby contribute to the establishment of their distinct expression domains in cooperation with graded Shh signaling. Together, our data reveal a Shh-Gli-independent transcriptional input that is required to shape the precise spatial and temporal response to extracellular morphogen signaling information during lineage segregation in the CNS.

A Dorsal Elaboration in the Spinal Cord

Functionally distinct types of neurons develop in stereotypical positions in the vertebrate spinal cord. The mechanisms that generate this diversity have been well studied in the ventral and intermediate regions of the spinal cord, while dorsal cells have received less attention. In this issue of Neuron, two papers focusing on dorsal interneuron development level the playing field.

Sonic hedgehog signaling in the developing CNS where it has been and where it is going.

Sonic Hedgehog (Shh) is one of three mammalian orthologs of the Hedgehog (Hh) family of secreted proteins first identified for their role in patterning the Drosophila embryo. In this review, we will highlight some of the outstanding questions regarding how Shh signaling controls embryonic development. We will mainly consider its role in the developing mammalian central nervous system (CNS) where the pathway plays a critical role in orchestrating the specification of distinct cell fates within ventral regions, a process of exquisite complexity that is necessary for the proper wiring and hence function of the mature system. Embryonic development is a process that plays out in both the spatial and the temporal dimensions, and it is becoming increasingly clear that our understanding of Shh signaling in the CNS is grounded in an appreciation for the dynamic nature of this process. In addition, any consideration of Hh signaling must by necessity include a consideration of data from many different model organisms and systems. In many cases, the extent to which insights gained from these studies are applicable to the CNS remains to be determined, yet they provide a strong framework in which to explore its role in CNS development. We will also discuss how Shh controls cell fate diversification through the regulation of patterned target gene expression in the spinal cord, a region where our understanding of the morphogenetic action of graded Shh signaling is perhaps the furthest advanced.