Search

Three central cell populations play roles in morphogen action, the cells that produce, the cells that distribute, and the cells that respond to the morphogen. Taking advantage of the properties of embryonic stem cell to aggregate and readily differentiate into neural progenitor tissue, we describe an approach using genetically modified murine stem cell lines to individually address the contribution of these cells in the establishment and response to a morphogenetic gradient in mosaic spinal cord organoids.

BackgroundSonic Hedgehog (Shh) has a catalytic cleft characteristic for zinc metallopeptidases and has significant sequence similarities with some bacterial peptidoglycan metallopeptidases defining a subgroup within the M15A family that, besides having the characteristic zinc coordination motif, can bind two calcium ions. Extracellular matrix (ECM) components in animals include heparan-sulfate proteoglycans, which are analogs of bacterial peptidoglycan and are involved in the extracellular distribution of Shh.ResultsWe found that the zinc-coordination center of Shh is required for its association to the ECM as well as for non-cell autonomous signaling. Association with the ECM requires the presence of at least 0.1 μM zinc and is prevented by mutations affecting critical conserved catalytical residues. Consistent with the presence of a conserved calcium binding domain, we find that extracellular calcium inhibits ECM association of Shh.ConclusionsOur results indicate that the putative intrinsic peptidase activity of Shh is required for non-cell autonomous signaling, possibly by enzymatically altering ECM characteristics.

As a morphogen, Sonic Hedgehog (Shh) mediates signaling at a distance from its sites of synthesis. After secretion, Shh must traverse a distance through the extracellular matrix (ECM) to reach the target cells and activate the Hh response. ECM proteins, in particular, the heparan sulfate proteoglycans (HSPGs) of the glypican family, have both negative and positive effects on Shh signaling, all attributed to their ability to bind Shh. Using mouse embryonic stem cell-derived mosaic tissues with compartments that lack the glycosyltransferases Exostosin1 and Exostosin2, or the HSPG core protein Glypican5, we show that Shh accumulates around its source cells when they are surrounded by cells that have a mutated ECM. This accumulation of Shh is correlated with an increased noncell autonomous Shh response. Our results support a model in which Shh presented on the cell surface accumulates at or near ECM that lacks HSPGs, possibly due to the absence of these Shh sequestering molecules. Stem Cells 2019;37:899-909.

Sonic Hedgehog (Shh) signaling is characterized by non-cell autonomy; cells expressing Shh do not respond to the ligand. Here, we identify several Shh mutations that can activate the Hedgehog (Hh) pathway cell-autonomously. Cell-autonomous pathway activation requires the extracellular cysteine rich domain of Smoothened, but is otherwise independent of the Shh receptors Patched1 and -2. Many of the Shh mutants that gain activity fail to undergo auto processing resulting in the perdurance of the Shh pro-peptide, a form of Shh that is sufficient to activate the Hh response cell-autonomously. Our results demonstrate that Shh is capable of activating the Hh pathway via Smoothened, independently of Patched1/2, and that it harbors an intrinsic mechanism that prevents cell-autonomous activation of the Shh response.

Sonic Hedgehog (Shh) coordinates Zn2+ in a manner that resembles that of peptidases. The ability of Shh to undergo autoproteolytic processing is impaired in mutants that affect the Zn2+ coordination, while mutating residues essential for catalytic activity results in more stable forms of Shh. The residues involved in Zn2+ coordination in Shh are found to be mutated in some individuals with the congenital birth defect holoprosencephaly, demonstrating their importance in development. Highly conserved Shh domains are found in parts of some bacterial proteins that are members of the larger family of DD-peptidases, supporting the notion that Shh acts as a peptidase. Whereas this Hh/DD-peptidase motif is present in Hedgehog (Hh) proteins of nearly all animals, it is not present in Drosophila Hh, indicating that Hh signaling in fruit flies is derived, and perhaps not a good model for vertebrate Shh signaling. A sequence analysis of Hh proteins and their possible evolutionary precursors suggests that the evolution of modern Hh might have involved horizontal transfer of a bacterial gene coding of a Hh/DD-peptidase into a Cnidarian ancestor, recombining to give rise to modern Hh.

UVR in sunlight causes mutations that drive basal cell carcinomas. However, the incidence of these tumors plateaus with prolonged exposure, but the incidence of other skin cancers increases. Makarova et al. now show that vitamin D3 produced in the skin by UVR protects against its oncogenic effects by inhibiting Hedgehog signaling, whereas dietary vitamin D3 does not.

Smoothened (Smo) inhibition by Patched (Ptch) is central to Hedgehog (Hh) signaling. Ptch, a proton driven antiporter, is required for Smo inhibition via an unknown mechanism. Hh ligand binding to Ptch reverses this inhibition and activated Smo initiates the Hh response. To determine whether Ptch inhibits Smo strictly in the same cell or also mediates non-cell-autonomous Smo inhibition, we generated genetically mosaic neuralized embryoid bodies (nEBs) from mouse embryonic stem cells (mESCs). These experiments utilized novel mESC lines in which Ptch1, Ptch2, Smo, Shh and 7dhcr were inactivated via gene editing in multiple combinations, allowing us to measure non-cell autonomous interactions between cells with differing Ptch1/2 status. In several independent assays, the Hh response was repressed by Ptch1/2 in nearby cells. When 7dhcr was targeted, cells displayed elevated non-cell autonomous inhibition. These findings support a model in which Ptch1/2 mediate secretion of a Smo-inhibitory cholesterol precursor.

The distribution of Sonic Hedgehog (Shh) is a highly regulated and critical process for development. Several negative feedback mechanisms are in place, including the Shh-induced upregulation of Hedgehog-interacting protein (Hhip). Hhip sequesters Shh, leading to a non-cell autonomous inhibition of the pathway. Hhip overexpression has a severe effect on neural tube development, raising the question why normal sites of Hhip expression have a seemingly unimpaired response to Shh. Here we show that although Hhip is able to leave its sites of synthesis to inhibit Shh non-cell autonomously, activation of Smoothened (Smo) drastically increases Hhip internalization and degradation cell autonomously. Although Hhip is unable to cell autonomously inhibit the consequences of Smo activation, it can inhibit the Shh response non-cell autonomously. Our data provide a mechanism by which the Shh ligand can activate the response and negate cell autonomous effects of Hhip, while Hhip can still induce non-cell autonomous inhibition.

Emerging evidence points to an unexpected diversification of core promoter recognition complexes that serve as important regulators of cell-type specific gene transcription. Here, we report that the orphan TBP-associated factor TAF9B is selectively up-regulated upon in vitro motor neuron differentiation, and is required for the transcriptional induction of specific neuronal genes, while dispensable for global gene expression in murine ES cells. TAF9B binds to both promoters and distal enhancers of neuronal genes, partially co-localizing at binding sites of OLIG2, a key activator of motor neuron differentiation. Surprisingly, in this neuronal context TAF9B becomes preferentially associated with PCAF rather than the canonical TFIID complex. Analysis of dissected spinal column from Taf9b KO mice confirmed that TAF9B also regulates neuronal gene transcription in vivo. Our findings suggest that alternative core promoter complexes may provide a key mechanism to lock in and maintain specific transcriptional programs in terminally differentiated cell types.DOI: http://dx.doi.org/10.7554/eLife.02559.001.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. It is typically detected at an advanced stage, at which the therapeutic options are very limited. One remarkable feature of PDAC that contributes to its resilience to treatment is the extreme stromal activation seen in these tumors. Often, the vast majority of tumor bulk consists of non-tumor cells that together provide a tumor-promoting environment. One of the signals that maintains and activates the stroma is the developmental protein Sonic Hedgehog (SHH). As the disease progresses, tumor cells produce increasing amounts of SHH, which activates the surrounding stroma to aid in tumor progression. To better understand this response and identify targets for inhibition, we aimed to elucidate the proteins that mediate the SHH-driven stromal response in PDAC. For this a novel mixed-species coculture model was set up in which the cancer cells are human, and the stroma is modeled by mouse fibroblasts. In conjunction with next-generation sequencing we were able to use the sequence difference between these species to genetically distinguish between the epithelial and stromal responses to SHH. The stromal SHH-dependent genes from this analysis were validated and their relevance for human disease was subsequently determined in two independent patient cohorts. In non-microdissected tissue from PDAC patients, in which a large amount of stroma is present, the targets were confirmed to associate with tumor stroma versus normal pancreatic tissue. Patient survival analysis and immunohistochemistry identified CDA, EDIL3, ITGB4, PLAUR and SPOCK1 as SHH-dependent stromal factors that are associated with poor prognosis in PDAC patients. Summarizing, the presented data provide insight into the role of the activated stroma in PDAC, and how SHH acts to mediate this response. In addition, the study has yielded several candidates that are interesting therapeutic targets for a disease for which treatment options are still inadequate.

Subdivision of the neuroectoderm into three rows of cells along the dorsal-ventral axis by neural identity genes is a highly conserved developmental process. While neural identity genes are expressed in remarkably similar patterns in vertebrates and invertebrates, previous work suggests that these patterns may be regulated by distinct upstream genetic pathways. Here we ask whether a potential conserved source of positional information provided by the BMP signaling contributes to patterning the neuroectoderm. We have addressed this question in two ways: First, we asked whether BMPs can act as bona fide morphogens to pattern the Drosophila neuroectoderm in a dose-dependent fashion, and second, we examined whether BMPs might act in a similar fashion in patterning the vertebrate neuroectoderm. In this study, we show that graded BMP signaling participates in organizing the neural axis in Drosophila by repressing expression of neural identity genes in a threshold-dependent fashion. We also provide evidence for a similar organizing activity of BMP signaling in chick neural plate explants, which may operate by the same double negative mechanism that acts earlier during neural induction. We propose that BMPs played an ancestral role in patterning the metazoan neuroectoderm by threshold-dependent repression of neural identity genes.

A concentration gradient of Shh is thought to pattern the ventral neural tube, and these ventral cell types are absent in [shh.sup.-/-] mice. Based on in vitro and genetic studies, the zinc finger-containing transcription factors Gli 1, 2, and 3 are mediators of the Shh intracellular response. The floorplate and adjacent cell types are absent in gli[1.sup.-/-];gli[2.sup.-/-] mice, but part of the [Shh.sup.-/-] phenotype in the neural tube is alleviated in the [Shh.sup.-/-];gli[3.sup.-/-] double mutant. This is consistent with the predicted role of Gli3 as a repressor of the Shh response. Gli3 repressor activity is blocked by Shh. In order to test the role of the repressor form of Gli3 in the neural tube, a truncated version of Gli3 (Gli3R*) was designed to mimic a Pallister Hall allele. Gli3R* acts as a constitutive repressor independent of Shh signaling. Misexpression of Gli3R* in the chick neural tube caused a ventral expansion of class-I, dorsal progenitor proteins and a loss of class-II, ventral progenitor proteins consistent with expected activity as a repressor of the Shh response. Activation of the BMP response is sufficient to maintain gli3 expression in neural plate explants, which might be a mechanism by which BMPs antagonize the Shh response. Keywords: gli3; shh; BMP; Neural: Chick; Spinal cord; Patterning; Pallister Hall Syndrome

Sonic Hedgehog (SHH) signaling is required for the short-range induction of floor plate cells by the notochord and separately, for the induction of motor neurons by both the notochord and midline neural cells. Two periods of SSH signaling are crucial for motor neuron generation, namely, an early period where naive neural plate cells are converted into ventralized progenitors and a late period that even reaches the S phase of the final progenitor cell division. During this late period, SHH drives the differentiation of ventralized progenitors into motor neurons.

Near the floor plate of the embryonic neural tube there is a group of neuroepithelial precursor cells that are specialized for production of the oligodendrocyte lineage. We performed experiments to test whether specification of these neuroepithelial oligodendrocyte precursors, like other ventral neural cell types, depends on signals from the notochord and/or floor plate. We analyzed heterozygous Danforth's short tail (Sd/+) mutant mice, which lack a notochord and floor plate in caudal regions of the neural tube, and found that oligodendrocyte precursors did not appear at the ventricular surface where there was no floor plate. Moreover, oligodendrocytes did not develop in explant cultures of Sd/+ spinal cord in the absence of a floor plate. When a second notochord was grafted into an ectopic position dorsolateral to the endogenous notochord of a chicken embryo, an additional floor plate was induced along with an ectopic focus of oligodendrocyte precursors at the ventricular surface. Oligodendrocytes developed in explants of intermediate neural tube only when they were cocultured with fragments of notochord or in the presence of purified Sonic hedgehog (Shh) protein. Thus, signals from the notochord/floor plate, possibly involving Shh, are necessary and sufficient to induce the development of ventrally derived oligodendroglia. These signals appear to act by specifying the future fate(s) of neuroepithelial cells at the ventricular surface rather than by influencing the proliferation or differentiation of prespecified progenitor cells in the parenchyma of the cord.

The differentiation of cells into dorsal cell type starts at the neural plate stage of development and is controlled by the dorsalizing signal of the bone morphogenetic protein genes (BMP4 and BMP7) and the ventralizing signal of the sonic hedgehog (SHH) gene. BMP4 and BMP7 are produced by the ectoderm of the epidermis and SHH by the notochord. Although the dorsal positioning of the genes present in the dorsal neural plate is controlled by the SHH gene signal, definitive dorsal cell fate is controlled by the signal from epidermal ectoderm which is due to the BMP genes.

The nested expression of transcription factors in the prosomeres of the developing forebrain suggests that transverse subdivisions are important for anterior-posterior patterning of the future telencephalon and diencephalon. At the zona limitans intrathalamica (zli) in the prospective diencephalon, marking the border between the future dorsal and ventral thalamus, there is a conspicuous anterior-posterior restriction of gene expression. At chick HH stage 14, Dlx-1 and Dlx-2 are expressed anterior to the zli, while Gbx-2 is expressed posterior to the zli. Superimposed on this transcription factor expression pattern, the known morphogens Shh, Wnt-3, and Wnt-3a are expressed in a ring that posteriorly abuts the zli. We hypothesize that this ring of tissue acts as a regional organizer and that the Shh, Wnt-3, and Wnt-3a signals emanating from this tissue are critical for the proposed organizer function. Our results suggest that the zli tissue is capable of inducing prosomere-specific gene expression in naive forebrain explants. Moreover, there appears to be a competency difference between neural tissue taken anterior to the zli vs posterior to the zli in its ability to respond to zli-derived cues. Coculture of HH stage 14 posterior zli with naive stage 8 cephalic neural plate explants from anterior to the zli results in the upregulation of Dlx-2 expression. Naive explants taken from posterior to the zli show no Dlx-2 expression after coculture. The addition of CKI7, a pharmacological inhibitor of the transforming Wnt pathway, to coculture media can reverse this Dlx-2 expression pattern, causing some posterior cocultured explants to upregulate Dlx-2 expression. These results suggest that a Wnt signal is involved in, but not solely responsible for, the induction and/or maintenance of Dlx-2 expression in the forebrain. In support of a Wnt-mediated induction event, soluble Wnt-3a is sufficient to induce Gbx-2 expression in naive posterior forebrain explants in vitro. Furthermore, injection of CKI7 into stage 10 embryos in ovo results in a reduction of Gbx-2 expression in p2. Experiments are ongoing to assess the role of Shh in zli-mediated inductive events. Through this series of experiments we hope to elucidate the role of the zli in the proper patterning of the developing forebrain.

Sonic hedgehog (Shh), secreted from the notochord and floor plate, is required for the induction of a variety of cell types in the early neural tube. There is evidence that Shh acts as a morphogen, but its wide-ranging effects are not accomplished by Shh alone, but rather by Shh in combination with other signaling factors. One antagonistic force on Shh signaling, PKA, is active in the presence of cAMP. In certain biological situations, another cyclic nucleotide, cGMP can result in effects that are opposite those of cAMP. We tested whether cGMP can affect Shh signaling using chick neural plate explants, which are induced by Shh to produce proteins characteristic of motor neurons and floor plate cells. In the presence of stabilized, soluble cGMP analogs, more motor neuron and floor plate cells were induced than in the presence of Shh alone. More motor neurons and floor plate cells were also produced when neural plate explants were exposed to chick natriuretic peptide (chNP), a ligand that stimulates membrane-bound guanylate cyclase (GC) to produce cGMP. ChNP and cGMP did not induce ventral cell types in the absence of Shh. cGMP also had no effect on the induction of a Slug-expressing neural crest cells by the BMP family member Dorsalin. Another protein that produces cGMP, soluble GC, is stimulated by nitric oxide (NO). When chick embryos were exposed to an NO donor, cGMP staining was seen in ventral and dorsal neural regions, indicating that these cyclases may active at the time and place of DV neural ;patterning. Taken together, these data indicate that cGMP can enhance the patterning effects of Shh and that core components of cGMP signaling are present and functional in the embryo at appropriate locations and times to influence Shh patterning, cGMP provides an interesting addition to the complexity of signals that interact with Shh during development, bringing us closer to an understanding of how a diversity of cells arises in an embryo.

Cyclopamine is a teratogenic steroidal alkaloid that causes cyclopia by blocking Sonic hedgehog (Shh) signal transduction. We have tested whether this activity of cyclopamine is related to disruption of cellular cholesterol transport and putative secondary effects on the Shh receptor, Patched (Ptc). First, we report that the potent antagonism of Shh signaling by cyclopamine is not a general property of steroidal alkaloids with similar structure. The structural features of steroidal alkaloids previously associated with the induction of holoprosencephaly in whole animals are also associated with inhibition of Shh signaling in vitro. Second, by comparing the effects of cyclopamine on Shh signaling with those of compounds known to block cholesterol transport, we show that the action of cyclopamine cannot be explained by inhibition of intracellular cholesterol transport. However, compounds that block cholesterol transport by affecting the vesicular trafficking of the Niemann-Pick C1 protein (NPC1), which is structurally similar to Ptc, are weak Shh antagonists. Rather than supporting a direct link between cholesterol homeostasis and Shh signaling, our findings suggest that the functions of both NPC1 and Ptc involve a common vesicular transport pathway. Consistent with this model, we find that Ptc and NPC1 colocalize extensively in a vesicular compartment in cotransfected cells. [C] 2000 Academic Press Key Words: neural development; holoprosencephaly; teratogen; U18666A; progesterone; membrane trafficking.