Guide Hedgehog Signaling Protocols

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  1. About this book
  2. Hedgehog Signaling Protocols - Semantic Scholar
  3. Hedgehog signaling is required at multiple stages of zebrafish tooth development
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Zuzana Koledova. Jason Mercer. Christian Dahmann. David Posada. Home Contact us Help Free delivery worldwide. Free delivery worldwide. Bestselling Series. Harry Potter. Popular Features. New Releases. Description Intended for Molecular Biologists, Biochemists, and Geneticists interested in manipulating and analyzing the Hedgehog Signaling Pathway, this volume offers various detailed chapters on experimental description, including anecdotal pointers and notes.

Other books in this series. Hemostasis and Thrombosis Emmanuel J. Add to basket. Cancer Cytogenetics Thomas S. Immunogenetics Frank T. Natural Products Isolation Satyajit D. Surface Plasmon Resonance Nico J. Plant Pathology Christophe Lacomme. Marine Genomics Sarah J. Metabolic Flux Analysis Jens O.

Vaccinia Virus Jason Mercer. Drosophila Christian Dahmann. Back cover copy In both vertebrates and invertebrates, Hedgehog signaling regulates the development of the tissues and structures of the body. In addition, many cancers and diseases arise as a result of misregulation of the pathway, which makes it an incredibly important system to understand. This volume examines various methods used in the analysis of the Hedgehog signaling pathway, bringing together different lines of analyses into one accessible and comprehensive text.

This volume provides a single source for investigators, presenting several different experimental approaches, which are all varied and broad in nature, as well as procedures for a variety of different model systems. Researchers will find this volume to be an invaluble tool as they work towards deeper understanding of the underlying molecular events that the Hedgehog signaling pathway regulates. Purifying the Hedgehog protein and its variants by Darren P. Baker, Frederick R.

About this book

Taylor and R. For a list of all analyzed genes, see Figure 1—source data 1. Anterior up for H , I. To test for enrichment using this dissection technique, amputated head fragments collected from CIW4 asexual strain S. Head fragments contain cephalic ganglia as well as most major planarian tissue types Hyman, To assess the success of our procedure in enriching CNS-associated transcripts, we examined a panel of 70 genes consisting of both experimentally validated head- and nervous system-enriched genes as well as transcripts predicted to be present in neurons based on sequence similarity to molecules with known roles in neuron biology Figure 1—source data 1.

Overall, members of this collection had an average log 2 -fold enrichment of 2. Broadly expressed neuronal markers syn, chat , and pc2 were only somewhat enriched Figure 1E , consistent with the fact that these genes are also expressed in the peripheral nervous system located throughout the head.

Hedgehog Signaling Protocols - Semantic Scholar

Conversely, genes expressed in cells restricted to the medial CNS, such as hh and the prohormone-encoding gene HH Collins et al. We also examined a number of markers known to be expressed in non-neural cell types and found that whereas most of these genes were depleted in cephalic ganglia libraries, some genes were enriched Figure 1—source data 2. However, the non-neuronal markers frequently used to identify specific cell types in planarians smedwi-1, agat-1, marginal adhesive gland-1 mag-1 , carbonic anhydrase ca , mat, collagen , and myosin heavy chain 6 mhc6 were greatly underrepresented in the CNS-specific sample Figure 1E , Figure 1—source data 2.

Genes expressed in many planarian tissues, such as gapdh and ptc , showed little difference in expression in the cephalic ganglia-versus-head dataset Figure 1E , Figure 1—source data 1. These data indicate that although other tissues cannot be completely eliminated, the dissection protocol greatly enriches for cephalic ganglia transcripts. We found insignificant differences in transcript levels for the broadly expressed housekeeping gene gapdh and the neural genes syn and pc2 Figure 1F.

Hh acts by negatively regulating Patched protein, which in turn is a negative regulator of transcriptional targets of Hh signaling including the ptc gene itself Varjosalo and Taipale, Therefore, reduction of ptc transcript levels in hh RNAi animals was not unexpected. The Hh signaling pathway is required for establishing expression domains of the transcription factors Nk2. Absence of SHH expression in the vertebrate floor plate results in loss of cell types that normally form in these domains Ruiz i Altaba et al.

By contrast, we were unable to find evidence that in intact planarians, which exhibit extensive tissue turnover and new cell type specification, Hh signaling modulates expression domains of orthologous transcription factors. The expression levels of Smed-nkx2 nkx2 , Smed-nkx6 nkx6 , and Smed-pax6b pax6b Scimone et al.

We next conducted expression analysis for cephalic ganglia genes affected by Hh pathway perturbation. Intermediate filaments are cytoskeletal proteins that provide structural support and mechanical stress resistance in a variety of cell types Herrmann et al. FISH analysis revealed that if-1 and cali were co-expressed primarily in cells in the neuropil, the neurite-dense region surrounded by neuron cell bodies Best and Noel, ; Morita and Best, , of both the cephalic ganglia and the ventral nerve cords Figure 2A—B , Figure 2—figure supplement 1A.

Rarely, cells expressing these two genes were also observed outside the neuropil, such as near the periphery of the head Figure 2G , but the localization of these rare peripheral cells varied among animals. These isolated cells also showed high levels of expression of both if-1 and cali as well as mRNA-filled processes, suggesting that they are not an artifact of the in situ hybridization protocol used.

Yellow letters indicate regions detailed in E — G.

H Double FISH for if-1 green and cali magenta in animals following inhibition of a control gene, hh , or ptc. Within the neuropil, cells expressing one or both markers are present at In the head not including the neuropil region, cells expressing one or both markers are present at Anterior up, ventral side shown for A — D , H. Accordingly, the density of cells expressing either or both if-1 and cali in ptc RNAi animals increased slightly inside and considerably outside the neuropil Figure 2H—I. Ectopic expression of these genes outside the neuropil in ptc RNAi animals was observed near the ventral surface of the animal Figure 2—figure supplement 1B , with concentration of expression at the rim of the head Figure 2—figure supplement 1C near where presumptive chemosensory neurons reside Okamoto et al.

Inhibition of ptc results in defective head regeneration; the cephalic ganglia in the anterior blastema appear as masses of cells without any discernable neuropil region. To ensure that ablation of if-1 and cali signal resulted from loss of Hh signaling, we performed RNAi on genes encoding the planarian Gli transcription factors, which are downstream effectors of the Hh pathway Dominguez et al.

Inhibition of gli-1 results in a similar defective tail regeneration phenotype as does inhibition of hh Glazer et al. RNAi of gli-1 resulted in loss of if-1 and cali signal whereas RNAi of gli-2 and gli-3 did not have any discernable effect on expression of if-1 and cali Figure 2—figure supplement 2. We conclude that Hh signaling is required for if-1 and cali expression to be detected in the neuropil.

We identified and examined additional neuronal markers to further assess this possibility. A — N Schematic indicates region of focus. O — W Schematic indicates region of focus. Yellow dotted line delineates borders of the neuropil. Arrowheads denote double-positive cells outside the neuropil. Arrowheads denote double-positive cells.

Lower panels show high magnification images of, from left to right, gs or glut magenta , ptc green , DAPI blue , and merged channels from a representative double-positive cell. Anterior up, ventral side shown for all. Maximum intensity projections shown for I — N.

In addition to neurons, the other predominant cells in the nervous systems of other organisms are glia. Glia act as neuronal support cells by providing trophic support, axon insulation, environmental maintenance, the blood-brain barrier, and synapse pruning Pfeiffer et al. Invertebrate glia have been studied in Drosophila Hartenstein, and C. Electron microscopy performed on transverse sections of the planarian Dugesia tigrina revealed cells distributed throughout the ventral nerve cords with lighter cytoplasmic complexity than neighboring neurons; these have been hypothesized to be planarian glial cells, but such cells had not been previously identified with molecular markers Golubev, ; Morita and Best, Excitatory Amino Acid Transporters, which uptake the neurotransmitter glutamate from the extracellular environment Featherstone, , and Glutamine Synthetase, which metabolizes glutamate into glutamine Anderson and Swanson, , are expressed in vertebrate astrocytes Lehmann et al.

These genes act in concert to allow astrocytes to remove glutamate released during synaptic transmission and prevent excitotoxicity Anderson and Swanson, Smed-gat gat is predicted to encode an ortholog of a family of GABA, creatine, and taurine transporters that are commonly used as invertebrate and vertebrate glia markers Carducci et al. Members of the glucose transporter family are expressed in vertebrate astrocytes Morgello et al.

In vertebrates, members of this family are expressed in oligodendrocytes Hoffmann et al. The expression pattern of trpm was similar to that of gs and gat; trpm was co-expressed with if-1 and cali in the neuropil, and expression was also observed in cells of the ventral parenchyma and pharynx Figure 3N , Figure 3—figure supplement 2F.

Because we observed expression of several of these glia markers outside the neuropil, we performed double FISH analysis to determine whether these genes have overlapping expression in non-neuropil cells. Both if-1 and cali , when ectopically expressed outside the neuropil in ptc RNAi animals, were also co-expressed with these markers see below. To ensure that these cells outside the neuropil were not neurons, we performed double FISH for pc2 with gs or eaat and found no evidence of co-expression Figure 3V,W.

Co-expression of gs, eaat, eaat , and gat indicates that these cells function to reuptake and metabolize neurotransmitters, a role performed in the vertebrate nervous system by astrocytes Anderson and Swanson, Because these cells are embedded in the planarian nervous system and express glial markers rather than neuronal markers, we hypothesize that they are glia. To determine the role of these glia markers in planarian biology, we performed RNAi on gs, eaat, eaat, gat, glut , and trpm. However, we did not observe any morphological or behavioral effects in these animals during normal tissue turnover in uninjured animals and following head and tail amputation.

These peripheral branches varied between animals in extent, number, and location along the AP axis. RNAi of if-1 resulted in complete loss of IF-1 antibody immunolabeling, confirming that labeling was specific Figure 4—figure supplement 1A. A Whole-mount immunofluorescence for IF-1 protein magenta in wild-type untreated animals. B Maximum intensity projection of IF-1 localization magenta in the cephalic ganglia. Depicted region is indicated by top dotted box in panel A. C IF-1 localization magenta in the cephalic ganglion neuropil. Depicted region is indicated by dotted box in panel B.

D IF-1 localization in the lateral ventral parenchyma. Depicted region is indicated by middle dotted box in panel A. E IF-1 localization in the ventral nerve cord. Depicted region is indicated by bottom dotted box in panel A. J — K 3D renderings of confocal stacks of J a synaptic glomerulus in the ventral nerve cord or K an orthogonal branch labeled with anti-IF-1 magenta and anti-Synapsin green.

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Image on left is Synapsin only and image on right is Synapsin and IF L Immunofluorescence of IF-1 magenta and Synapsin green following inhibition of hh, ptc , or a control gene. In control RNAi animals, In hh RNAi animals, 2. In ptc RNAi animals, Axons traveling through the VNC neuropil regularly exit to form orthogonal commissures that extend from the VNC to the edge of the body.

An anti-Synapsin antibody labels large clusters of synapses within the neuropil and in nerve plexuses in the grid-like network of commissural axon bundles called the Orthogon Adell et al. In the ventral nerve cords, synapses accumulated into discrete, regularly spaced structures that strongly resembled synaptic glomeruli described in insect species Boeckh and Tolbert, To further study the morphology of these cells, we performed protein-retention expansion microscopy Tillberg et al. This video cannot be played in place because your browser does support HTML5 video.

You may still download the video for offline viewing. Immunofluorescence for IF-1 magenta and Synapsin green followed by protein-retention expansion microscopy. Anterior up, ventral side shown.

Hedgehog signaling is required at multiple stages of zebrafish tooth development

Inhibition of hh resulted in complete ablation of IF-1 immunofluorescence signal and no change in expression or localization of Synapsin protein, whereas inhibition of ptc caused an increase in IF-1 protein presence in cellular processes observed throughout the animal Figure 4L. Normally Conversely, if inhibition of Hh signaling affected transcription of if-1 but did not impact survival of these cells, then we would expect IF-1 protein to perdure for some time after if-1 mRNA is lost.

We performed a shortened RNAi treatment 3 feedings at 4-day intervals because IF-1 protein is completely eliminated by full treatment Figure 4L. Statistical significance indicated by labels n. Anterior up, ventral side shown for A — B. To further investigate the role of Hh signaling in glia biology we examined the expression of gs, gat, eaat , and eaat in uninjured hh RNAi and ptc RNAi animals.

Unlike the case for if-1 and cali, expression of the other glia markers was still observed throughout the neuropil and was indistinguishable from control animals. Similarly, inhibition of ptc had no effect on the expression or localization of gs, gat, eaat , or eaat Figure 5B. Given that planarian glia outside the neuropil also expressed ptc , these data suggest that if-1 and cali were induced in these cells when Hh signaling was activated by ptc inhibition.

We also examined transcript abundance of glia markers in our RNA-Seq data and found no statistically significant differential expression for eaat, eaat, gs, gat, glut , and trpm following hh or ptc RNAi Figure 5—figure supplement 1. Anterior blastemas of control RNAi animals after six days of regeneration contained cells expressing planarian glia markers, both inside the forming neuropil and outside.

Similar results were observed in ptc RNAi animals, despite defective head formation Figure 5—figure supplement 2. In hh RNAi animals, expression of if-1 and cali was eliminated, but cells expressing gat, eaat , and eaat were observed throughout the blastema Figure 5—figure supplement 2. The presence of these markers in newly formed cells of the blastema suggests that the animal is capable of regenerating glia in the absence of Hh signaling.

To distinguish between these two possibilities, we examined whether ectopic if-1 and cali expression in ptc RNAi animals required new cell production. After irradiation, animals can survive for a short time but are unable to produce new cells Reddien et al.

We exposed animals to rads of ionizing radiation and subsequently began RNAi. Yellow boxed area indicates region detailed in B. Control RNAi animals had Previous electron microscopy studies had identified candidate planarian glia based on their localization and appearance but did not provide any molecular evidence for their identity Golubev, ; Morita and Best, We have described here the first molecular and morphological evidence for neuronal support cells in planarians.

Second, the cells have branched processes that are closely associated with neurons. These processes extend through the synapse-rich regions of the neuropil, travel along orthogonal commissures of the peripheral nervous system, and encapsulate synaptic glomeruli. Third, these cells express three neurotransmitter transporters.

Orthologs of the proteins encoded by planarian eaat and eaat have known roles in the transport of glutamate from the extracellular environment into the cytoplasm where it is metabolized by orthologs of the enzyme encoded by glutamine synthetase Anderson and Swanson, , another gene expressed in these planarian cells.

Glutamate released from the pre-synaptic neuron, if not removed from the synaptic cleft, can continue to activate glutamate receptors on the post-synaptic neuron, resulting in high intracellular levels of calcium and activation of pathways that lead to cellular damage Manev et al.

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Additionally, because of the expression of gat , which encodes an ortholog of a GABA transporter, we predict that this cell type is also involved in GABA reuptake. Based upon these data, we propose that these planarian cells uptake the excitotoxic neurotransmitter glutamate from areas near synapses to prevent damage to the nervous system, similar to the function of astrocytes in other animals Schousboe, Taking these data together, we propose that these planarian cells are glia.

Continued study of the function, morphology, and molecular characteristics of these cells will allow further comparison of similarities and differences between these cells and glia in other organisms.

These glia markers are also co-expressed in cells outside the neuropil region, indicating the presence of glia in the nervous system beyond the neuropil of the cephalic ganglia and ventral nerve cords. One hypothesis is that they are specialized to modulate environments of extremely high synaptic density, particularly around synaptic glomeruli that are characteristic of this region of the CNS.

During homeostasis, constitutive expression of hh is required for expression of if-1 and cali in planarian glia in the neuropil. Upon inhibition of hh , these cells cease transcription of if-1 and cali. Inhibition of ptc results in ectopic if-1 and cali transcription in cells distributed broadly in the animal, likely as a consequence of derepression of the Gli-1 transcription factor.

This indicates that cells competent to respond to Hh ligand normally exist outside of the medial CNS. We currently do not have evidence supporting or rejecting the presence of multiple planarian glia cell types. It will be of interest to further investigate these possibilities by examining the function of planarian glia responsive to Hh signaling as well as planarian glia outside of the neuropil that do not express if-1 or cali in ptc RNAi animals. Planarian glia are distributed throughout the nervous system and concentrated in the neuropil.

Pathway Description:

Upon global inhibition of hh by RNAi, Ptc remains high in all cells and if-1 and cali are repressed in glia. Upon global inhibition of ptc by RNAi, expression of if-1 and cali is derepressed in glia. The ability of Hh signaling to modulate function in glia has been described in vertebrates. Increased levels of GFAP protein result in an increase in cell size, which is necessary for the formation of an astrocytic scar at the wound site Wilhelmsson et al.

Although the function of this regulation has differences, the regulation of intermediate filament proteins by Hh signaling in glia is another striking commonality between planarians and vertebrates. Additionally, SHH secreted from neurons has been reported to regulate distinct subpopulations of mammalian astrocytes Farmer et al. Although the genes regulated by Hh signaling in these glial populations is different, the ability for neurons to instruct astrocyte expression profiles in mammals is a strikingly similar feature to what we observe in planarians Farmer et al.

SHH expression in the vertebrate floor plate establishes distinct domains of transcription factor expression in the ventral neural tube. These domains first give rise to neurons and then, at later stages of development, glia Dessaud et al. The dorsal-ventral distribution of transcription factors in the developing CNS of Drosophila and the Lophotrochozoan Platynereis dumerilii bear a resemblance to the distribution of orthologous transcription factors in the vertebrate neural tube Cornell and Ohlen, ; Denes et al.

Hh, however, appears to play a role in the anterior-posterior patterning of Drosophila neuroblasts rather than dorsal-ventral patterning Bhat, Similarly, in Platynereis , a role for Hh in segment patterning has been identified, but no effect of pathway perturbation on the dorsal-ventral medial-lateral arrangement of CNS progenitor domains was described Denes et al. Here, we also find that regionalized expression in the cephalic ganglia of several orthologs of Hh-responsive vertebrate neural tube transcription factors appear unaffected by Hh signaling in uninjured planarians.

This is consistent with the possibility that Hh signaling was co-opted into a dorsal-ventral patterning role in the nervous system in the deuterostome lineage. The lack of head formation in ptc RNAi animals is a challenge for investigating head patterning in regenerating planarians, and further research into the neuronal progenitor pool in head blastemas following Hh pathway perturbation will be of interest for continuing to assess whether any role of Hh signaling exists in planarian nervous system patterning.

The floor plate, which is induced by SHH secreted from the notochord, serves as a mediator of axonal midline crossing through the secretion of axon guidance cues Colamarino and Tessier-Lavigne, SHH continues its involvement in neural patterning by acting as a chemoattractant and by mediating cellular responses to other guidance cues Parra and Zou, The Drosophila midline glia are considered to be an analogous structure to the vertebrate floor plate because of similar gene expression and roles in controlling midline crossing Evans and Bashaw, Hh in Drosophila is required for the decision to form posterior midline glia, the function for which is still not fully understood, instead of anterior midline glia, which develop into ensheathing glia in the Drosophila neuropil Watson et al.

A shared function of Hh signaling among Deuterostomes, Ecdysozoans, and Lophotrochozoans therefore appears to be in the control of glia near the midline. Planarians are an ideal model for the study of regeneration because of their nearly unrivaled regenerative ability, their ease of culture, and the molecular tools developed for rapid study of gene function. The role of glia in regeneration has been investigated in vertebrates, where glia proliferate in response to brain injury, and in insects, where surface glia can reform the blood-brain barrier Sofroniew, ; Treherne et al.

Interestingly, astrocytic scars appear to counteract neural regeneration by blocking the extension of axons into the damaged region Silver and Miller, Whether planarian glia actively participate in repatterning the nervous system after injury is an interesting topic to explore, possibly leading to studies on both mechanisms of glia-neuron interaction and glial roles in neural network connectivity. If, on the other hand, planarian glia passively extend their processes into existing neural architecture, then the mechanisms that guide glial cell development and migration could be studied instead.

The work we present here opens the field to a number of opportunities for continued research. Glia are now gaining recognition as an active player in nervous system development, function, and regeneration Freeman and Rowitch, ; Perea and Araque, ; Robel et al.

Planarians are a tractable model organism that will be amenable to the study of glia in a highly regenerative member of the understudied Lophotrochozoan superphylum.