Inactivation of Hedgehog signal transduction in adult astrocytes results in region-specific blood-brain barrier defects.

In this study, we use molecular genetic approaches to clarify the role of the Hedgehog (Hh) pathway in regulating the blood-brain/spinal cord barrier (BBB) in the adult mouse central nervous system (CNS). Our work confirms and extends prior studies to demonstrate that astrocytes are the predominant cell type in the adult CNS that transduce Hh signaling, revealed by the expression of Gli1, a target gene of the canonical pathway that is activated in cells receiving Hh, and other key pathway transduction components. Gli1+ (Hh-responsive) astrocytes are distributed in specific regions of the CNS parenchyma, including layers 4/5/6 of the neocortex, hypothalamus, thalamus, and spinal cord, among others. Notably, although BBB properties in endothelial cells are normally regulated by both paracellular and transcellular mechanisms, conditional inactivation of Hh signaling in astrocytes results in transient, region-specific BBB defects that affect transcytosis but not paracellular diffusion. These findings stand in contrast to prior studies that implicated astrocytes as a source of Sonic hedgehog that limited extravasation via both mechanisms [J. I. Alvarez et al., Science 334, 1727-1731 (2011)]. Furthermore, using three distinct Cre driver lines as well as pharmacological approaches to inactivate Hh-pathway transduction globally in CNS astrocytes, we find that these specific BBB defects are only detected in the rostral hypothalamus and spinal cord but not the cortex or other regions where Gli1+ astrocytes are found. Together, our data show that Gli1+ Hh-responsive astrocytes have regionally distinct molecular and functional properties and that the pathway is required to maintain BBB properties in specific regions of the adult mammalian CNS.

A Shh/Gli-driven three-node timer motif controls temporal identity and fate of neural stem cells.

How time is measured by neural stem cells during temporal neurogenesis has remained unresolved. By combining experiments and computational modeling, we define a Shh/Gli-driven three-node timer underlying the sequential generation of motor neurons (MNs) and serotonergic neurons in the brainstem. The timer is founded on temporal decline of Gli-activator and Gli-repressor activities established through down-regulation of Gli transcription. The circuitry conforms an incoherent feed-forward loop, whereby Gli proteins not only promote expression of Phox2b and thereby MN-fate but also account for a delayed activation of a self-promoting transforming growth factor-ß (Tgfß) node triggering a fate switch by repressing Phox2b. Hysteresis and spatial averaging by diffusion of Tgfß counteract noise and increase temporal accuracy at the population level, providing a functional rationale for the intrinsically programmed activation of extrinsic switch signals in temporal patterning. Our study defines how time is reliably encoded during the sequential specification of neurons.

Signals from the brain and olfactory epithelium control shaping of the mammalian nasal capsule cartilage.

Facial shape is the basis for facial recognition and categorization. Facial features reflect the underlying geometry of the skeletal structures. Here, we reveal that cartilaginous nasal capsule (corresponding to upper jaw and face) is shaped by signals generated by neural structures: brain and olfactory epithelium. Brain-derived Sonic Hedgehog (SHH) enables the induction of nasal septum and posterior nasal capsule, whereas the formation of a capsule roof is controlled by signals from the olfactory epithelium. Unexpectedly, the cartilage of the nasal capsule turned out to be important for shaping membranous facial bones during development. This suggests that conserved neurosensory structures could benefit from protection and have evolved signals inducing cranial cartilages encasing them. Experiments with mutant mice revealed that the genomic regulatory regions controlling production of SHH in the nervous system contribute to facial cartilage morphogenesis, which might be a mechanism responsible for the adaptive evolution of animal faces and snouts.

Atrophin protein RERE positively regulates Notch targets in the developing vertebrate spinal cord.

The Notch signaling pathway controls cell fate decision, proliferation, and other biological functions in both vertebrates and invertebrates. Precise regulation of the canonical Notch pathway ensures robustness of the signal throughout development and adult tissue homeostasis. Aberrant Notch signaling results in profound developmental defects and is linked to many human diseases. In this study, we identified the Atrophin family protein RERE (also called Atro2) as a positive regulator of Notch target Hes genes in the developing vertebrate spinal cord. Prior studies have shown that during early embryogenesis in mouse and zebrafish, deficit of RERE causes various patterning defects in multiple organs including the neural tube. Here, we detected the expression of RERE in the developing chick spinal cord, and found that normal RERE activity is needed for proper neural progenitor proliferation and neuronal differentiation possibly by affecting Notch-mediated Hes expression. In mammalian cells, RERE co-immunoprecipitates with CBF1 and Notch intracellular domain (NICD), and is recruited to nuclear foci formed by over-expressed NICD1. RERE is also necessary for NICD to activate the expression of Notch target genes. Our findings suggest that RERE stimulates Notch target gene expression by preventing degradation of NICD protein, thereby facilitating the assembly of a transcriptional activating complex containing NICD, CBF1/RBPjκ in vertebrate, Su(H) in Drosophila melanogaster, Lag1 in C. elegans, and other coactivators.

Tcf7l2/Tcf4 Transcriptional Repressor Function Requires HDAC Activity in the Developing Vertebrate CNS.

The generation of functionally distinct neuronal subtypes within the vertebrate central nervous system (CNS) requires the precise regulation of progenitor gene expression in specific neuronal territories during early embryogenesis. Accumulating evidence has implicated histone deacetylase (HDAC) proteins in cell specification, proliferation, and differentiation in diverse embryonic and adult tissues. However, although HDAC proteins have shown to be expressed in the developing vertebrate neural tube, their specific role in CNS neural progenitor fate specification remains unclear. Prior work from our lab showed that the Tcf7l2/Tcf4 transcription factor plays a key role in ventral progenitor lineage segregation by differential repression of two key specification factors, Nkx2.2 and Olig2. In this study, we found that administration of HDAC inhibitors (Valproic Acid (VPA), Trichostatin-A (TSA), or sodium butyrate) in chick embryos in ovo disrupted normal progenitor gene segregation in the developing neural tube, indicating that HDAC activity is required for this process. Further, using functional and pharmacological approaches in vivo, we found that HDAC activity is required for the differential repression of Nkx2.2 and Olig2 by Tcf7l2/Tcf4. Finally, using dominant-negative functional assays, we provide evidence that Tcf7l2/Tcf4 repression also requires Gro/TLE/Grg co-repressor factors. Together, our data support a model where the transcriptional repressor activity of Tcf7l2/Tcf4 involves functional interactions with both HDAC and Gro/TLE/Grg co-factors at specific target gene regulatory elements in the developing neural tube, and that this activity is required for the proper segregation of the Nkx2.2 (p3) and Olig2 (pMN) expressing cells from a common progenitor pool.

Mammalian Nkx2.2+ perineurial glia are essential for motor nerve development.

BACKGROUND: All vertebrate peripheral nerves connect the central nervous system (CNS) with targets in the periphery and are composed of axons, layers of ensheathing glia and connective tissue. Although the structure of these conduits is well established, very little is known about the origin and developmental roles of some of their elements. One understudied component, the perineurium, ensheaths nerve fascicles and is a component of the blood-nerve-barrier. In zebrafish, the motor nerve perineurium is composed of CNS-derived nkx2.2a(+) perineurial glia, which establish the motor exit point (MEP) during development. To determine if mouse perineurial cells also originate within the CNS and perform a similar function, we created a Nkx2.2:EGFP transgenic reporter line. RESULTS: In conjunction with RNA expression analysis and antibody labeling, we observed Nkx2.2(+) cells along peripheral motor nerves at all stages of development and in adult tissue. Additionally, in mice lacking Nkx2.2, we demonstrate that Nkx2.2(+) perineurial glia are essential for motor nerve development and Schwann cell differentiation. CONCLUSIONS: Our studies reveal that a subset of mouse perineurial cells are CNS-derived, express Nkx2.2, and are essential for motor nerve development. This work highlights an under-appreciated but essential contribution of CNS-derived cells to the development of the mammalian peripheral nervous system (PNS).

The PCP protein Vangl2 regulates migration of hindbrain motor neurons by acting in floor plate cells, and independently of cilia function.

Vangl2, a core component of the Planar Cell Polarity pathway, is necessary for the caudal migration of Facial Branchiomotor (FBM) neurons in the vertebrate hindbrain. Studies in zebrafish suggest that vangl2 functions largely non-cell autonomously to regulate FBM neuron migration out of rhombomere 4 (r4), but the cell-type within which it acts is not known. Here, we demonstrate that vangl2 functions largely in floor plate cells to regulate caudal neuronal migration. Furthermore, FBM neurons fail to migrate caudally in the mouse Gli2 mutant that lacks the floor plate, suggesting an evolutionarily conserved role for this cell type in neuronal migration. Although hindbrain floor plate cilia are disorganized in vangl2 mutant embryos, cilia appear to be dispensable for neuronal migration. Notably, Vangl2 is enriched in the basolateral, but not apical, membranes of floor plate cells. Taken together, our data suggest strongly that Vangl2 regulates FBM neuron migration by acting in floor plate cells, independently of cilia function.

Molecular genetic control of cell patterning and fate determination in the developing ventral spinal cord.

The generation of neuronal diversity in the ventral spinal cord during development is a multistep process that occurs with precise and reproducible spatiotemporal order. The proper functioning of the central nervous system requires that this be carried out with extraordinary precision from the outset. Extrinsic influences such as the secreted Sonic hedgehog (SHH) protein provide positional cues that are read out genetically as specific patterns of gene expression in subsets of dividing progenitors, which is the first overt indication that they have begun to embark upon cell-type-specific differentiation programs. Cells generated from these segregated domains will ultimately share similar properties and functions. Recent work illustrates that SHH, which regulates target genes via the GLI transcription factors, directly controls a subset of progenitor fate determinant genes and that both derepression and activation play a role in shaping the differential response to this morphogen.

In ovo electroporation in embryonic chick spinal cords.

The developing spinal cord is a well-established model system widely used to study the signaling pathways and genetic programs that control neuronal/glial differentiation and neural circuit assembly. This is largely due to the relatively simple organization (compared to other CNS regions) and experimental accessibility of the neural tube, particularly in the chick embryo. In vivo transfection of cells within the developing chick neural tube using in ovo electroporation has emerged as a rapid and powerful experimental technique in that (1) transfected factors can be functionally tested in a spatially and temporally controlled manner and (2) the chick embryo provides a physiologically relevant in vivo environment to conduct biochemical studies such as dual-channel luciferase assay, co-immunoprecipitation (co-IP), and Chromatin Immunoprecipitation (ChIP). In this chapter, we will take an in-depth look at the in ovo electroporation system in embryonic chicken spinal cord. In the following chapter, we will continue by examining the use of in ovo electroporation in the dual-channel luciferase assay as an example of its biochemical application.

Immunofluorescence staining with frozen mouse or chick embryonic tissue sections.

Immunofluorescence (IF), a form of immunohistochemistry (IHC) with specific applications, is commonly used for both basic research and clinical studies, including diagnostics, and involves visualizing the cellular distribution of target molecules (e.g., proteins, DNA, and small molecules) using a microscope capable of exciting and detecting fluorochrome compounds that emit light at specific, largely nonoverlapping wavelengths. The procedure for carrying out IF varies according to the tissue type and methods for processing and preparing tissue (e.g., fixative used to preserve tissue morphology and antigenicity). The protocol presented here provides a general guideline for multichannel IF staining using frozen embryonic mouse or chicken tissue sectioned on a cryostat. In general, the procedure involves the following: (1) fixing freshly dissected tissues in a 4 % paraformaldehyde solution buffered in the physiological pH range, (2) cryopreservation of tissue in a 30 % sucrose solution, (3) embedding and sectioning tissue in Optimal Cutting Temperature (OCT) matrix compound, (4) direct or indirect detection of the target antigen/s using fluorochrome-conjugated antibodies.

In vivo dual luciferase reporter assay with chick neural tube in ovo electroporation system.

Luciferase reporter systems are widely employed to provide a quantitative readout of gene expression for studies of transcriptional regulation, translation efficiency, and cell signaling. The most common application of luciferase involves transient transfections into cells in vitro or in vivo. In both cases, the normal variability inherent in transfection approaches can introduce significant errors into the data that makes comparison between separate experiments problematic. The dual luciferase reporter assay system (DLR, Promega, WI, USA) is designed to control for this technical issue by using a co-transfection approach with two separate reporter proteins that emit at distinct wavelengths: one from firefly (Photinus pyralis) and the second from Renilla (Renilla reniformis). By normalizing experimental luciferase readings to an internal control transfected under the same conditions, these problems can be largely negated. Here, we describe a method for applying this technique to an in vivo system, the developing chick embryo neural tube. This system provides a physiologically relevant context for functional studies in a spatially and/or temporally controlled manner.

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 (Shh(FP)) 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 Shh(FP) 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 Shh(FP) in regulating the specification and differentiation of glial and epithelial cells in the mouse spinal cord.

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.

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.

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.

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.

A critical role for sFRP proteins in maintaining caudal neural tube closure in mice via inhibition of BMP signaling.

Both the BMP and Wnt pathways have been implicated in directing aspects of dorsal neural tube closure and cell fate specification. However, the mechanisms that control the diverse responses to these signals are poorly understood. In this study, we provide genetic and functional evidence that the secreted sFRP1 and sFRP2 proteins, which have been primarily implicated as negative regulators of Wnt signaling, can also antagonize BMP signaling in the caudal neural tube and that this function is critical to maintain proper neural tube closure and dorsal cell fate segregation. Our studies thus reveal a novel role for specific sFRP proteins in balancing the response of cells to two critical extracellular signaling pathways.

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.

Order in the classroom: graded responses to instructive Hh signaling in the CNS.

In many animals, the secreted Hedgehog (Hh) signaling proteins play important roles during development and in adults. Studies in both flies and vertebrates indicate that Hh functions as a morphogen to elicit different responses at distinct concentration thresholds. In vertebrates, Gli proteins are the primary transcriptional mediators of Hh target genes. However, the mechanisms that implement specific genetic responses to graded Hh-Gli signaling are only just beginning to be understood. In particular, it is unclear whether target gene responses are determined solely by the ambient levels of pathway activity, or if other pathways or factors function to amplify or attenuate the response to this signal to provide an additional level of context that permits a more fine-tuned outcome. Here, I will review recent evidence suggesting that the response of some Hh-Gli target genes in the CNS is regulated by the activity of another important extracellular signal, the canonical Wnt pathway. The possibility that the Hh and Wnt pathways interact at the transcriptional level has broad significance for understanding normal embryogenesis and diagnosing and treating the numerous developmental disorders and cancers that involve these two pathways. Thus, while Hh-Gli signals provide important information, it is likely that they receive assistance from other "instructors".

A cell-autonomous requirement for Cip/Kip cyclin-kinase inhibitors in regulating neuronal cell cycle exit but not differentiation in the developing spinal cord.

Control over cell cycle exit is fundamental to the normal generation of the wide array of distinct cell types that comprise the mature vertebrate CNS. Here, we demonstrate a critical role for Cip/Kip class cyclin-kinase inhibitory (CKI) proteins in regulating this process during neurogenesis in the embryonic spinal cord. Using immunohistochemistry, we show that all three identified Cip/Kip CKI proteins are expressed in both distinct and overlapping populations of nascent and post-mitotic neurons during early neurogenesis, with p27(Kip1) having the broadest expression, and both p57(Kip2) and p21(Cip1) showing transient expression in restricted populations. Loss- and gain-of-function approaches were used to establish the unique and redundant functions of these proteins in spinal cord neurogenesis. Using genetic lineage tracing, we provide evidence that, in the absence of p57, nascent neurons re-enter the cell cycle inappropriately but later exit to begin differentiation. Analysis of p57(Kip2);p27(Kip1) double mutants, where p21 expression is confined to only a small population of interneurons, demonstrates that Cip/Kip CKI-independent factors initiate progenitor cell cycle exit for the majority of interneurons generated in the developing spinal cord. Our studies indicate that p57 plays a critical cell-autonomous role in timing cell cycle exit at G1/S by opposing the activity of Cyclin D1, which promotes cell cycle progression. These studies support a multi-step model for neuronal progenitor cell cycle withdrawal that involves p57(Kip2) in a central role opposing latent Cyclin D1 and other residual cell cycle promoting activities in progenitors targeted for differentiation.