Abstract Neuroblastoma (NB) is the most common extracranial solid tumor in infancy and originates in the peripheral sympathetic nervous system (PSNS). High-risk NB is fatal in the majority of patients, despite intensive myeloablative chemotherapy. The MYCN oncogene is amplified in over 20% of NB, particularly in those with highest risk treatment failure. We have developed a zebrafish NB model by overexpressing human MYCN under the control of the dopamine-beta-hydroxylase (D) promoter specific for noradrenergic cells in the PSNS. Fish from this stable transgenic line developed tumors as early as 4 months of age with approximately 20% penetrance at 8 months of age. The tumors resemble human NB histologically, immunohistochemically, and ultrastructurally. The expression of MYCN suppressed the normal development of PSNS neurons and chromaffin cells in the head kidney during embryogenesis and in young adult fish. In some fish, tumor cells began to repopulate the interrenal gland of the head kidney by 2 months of age. Germline and somatic activating mutations have been identified in the ALK gene, which encodes a receptor tyrosine kinase, in human NB, including those with MYCN amplification. To assess cooperativity between amplified MYCN and mutant ALK genes in transformation, we generated a zebrafish stable transgenic line in which the activated mutant form of ALK (F1174L) was expressed under the control of the DßH promoter. These transgenic animals did not display an abnormal phenotype nor did they develop tumors during the first 6 months of life. In contrast, mutant ALK expression accelerated the onset of MYCN-induced neuroblastoma when the 2 genes were co-expressed in double transgenic fish, indicating that MYCN over-expression and activating ALK mutations can cooperate in tumorigenesis. Although co-expression of mutant ALK accelerated the onset of tumors, it did not rescue the MYCN-induced suppression of PSNS development, suggesting that an as-yet-unidentified tumor suppressor gene, possibly located on distal 1p in the human genome, may be lost to fulfill this role in NB tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-162.
Abstract Background Short tandem repeats (STRs) are widely distributed across the human genome and are associated with numerous neurological disorders. However, the extent that STRs contribute to disease is likely under-estimated because of the challenges calling these variants in short read next generation sequencing data. Several computational tools have been developed for STR variant calling, but none fully address all of the complexities associated with this variant class. Results Here we introduce LUSTR which is designed to address some of the challenges associated with STR variant calling by enabling more flexibility in defining STR loci, allowing for customizable modules to tailor analyses, and expanding the capability to call somatic and multiallelic STR variants. LUSTR is a user-friendly and easily customizable tool for targeted or unbiased genome-wide STR variant screening that can use either predefined or novel genome builds. Using both simulated and real data sets, we demonstrated that LUSTR accurately infers germline and somatic STR expansions in individuals with and without diseases. Conclusions LUSTR offers a powerful and user-friendly approach that allows for the identification of STR variants and can facilitate more comprehensive studies evaluating the role of pathogenic STR variants across human diseases.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Chemokines are secreted proteins that regulate a range of processes in eukaryotic organisms. Interestingly, different chemokine receptors control distinct biological processes, and the same receptor can direct different cellular responses, but the basis for this phenomenon is not known. To understand this property of chemokine signaling, we examined the function of the chemokine receptors Cxcr4a, Cxcr4b, Ccr7, Ccr9 in the context of diverse processes in embryonic development in zebrafish. Our results reveal that the specific response to chemokine signaling is dictated by cell-type-specific chemokine receptor signal interpretation modules (CRIM) rather than by chemokine-receptor-specific signals. Thus, a generic signal provided by different receptors leads to discrete responses that depend on the specific identity of the cell that receives the signal. We present the implications of employing generic signals in different contexts such as gastrulation, axis specification and single-cell migration. https://doi.org/10.7554/eLife.33574.001 eLife digest Every process in the body is regulated by a complex network of interactions between different molecules and cells. Chemokines, for example, are tiny molecules produced by a cell that are involved in a range of processes, from development to immune responses and cancer. When chemokines bind to a specific protein on another cell, called the chemokine receptor, it stimulates different signaling pathways inside the cell. Consequently, chemokine receptors are equally important for regulating processes as diverse as the movement of cells during development and growth, or activating immune responses. Mammals have over 20 different chemokine receptors, and the same receptor can have different roles depending in which cell type it is found in. For example, in one cell type it may stimulate an action such as cell growth, but in another, it may block this process. Until now, it was unclear how chemokine receptors can achieve such different effects. One theory was that chemokine receptors initiate a distinct signaling cascade, a phenomenon termed 'signaling bias', depending on the type of chemokine or receptor. Here, Malhotra et al. used zebrafish embryos to investigate how four specific chemokine receptors regulate different events during early development. They found that the same chemokine receptor could direct different reactions in distinct cell types, while different receptors could also cause the same response in a specific cell type. In other words, the effect of a chemokine receptor depends on the cell type rather than the type of receptor. Since each of these receptors was able to control processes that it normally does not regulate in other cells, Malhotra et al. suggest that different chemokine receptors provide the same generic signal when activated, which the specific cell types then interpret accordingly. A next step will be to test how other chemokine receptors behave in different contexts, for example during an immune response. If the receptors work on the same principle regardless of the process, it could help to explain why faulty expression of chemokine receptors play such an important role during development and in disease. It could further highlight why blocking one receptor may not have any consequences, as they are dispensable and can be replaced by other receptors in the cell. https://doi.org/10.7554/eLife.33574.002 Introduction Chemokines are small proteins that signal upon binding seven-pass-transmembrane G protein-coupled receptors (GPCRs) (Zlotnik and Yoshie, 2000). Chemokine receptors are classified into four categories namely, CXCR, CCR, XCR and CX3CR (Nomiyama et al., 2011). Chemokines were originally shown to function in the context of immune response, but were thereafter implicated in a range of developmental processes such as angiogenesis (Strieter et al., 1995), neural development (Zou et al., 1998) and migration of non-immune cells. Following binding to their ligands, chemokine receptors activate a wide range of effectors, including adenylyl cyclases, phospholipase isoforms, protein tyrosine kinases, ion channels, and mitogen-activated protein kinases (MAPKs). These responses can result from the activation of G proteins, as well as from other second messengers to initiate G-protein-independent signaling (Steen et al., 2014). For example, in addition to signaling through the G protein Gαi and Gβγ, chemokine receptors can initiate JAK/STAT signaling and signal through β-arrestin, in the context of chemotaxis of hematopoietic progenitor cells and in the context of activation and release of granules in neutrophils, respectively (Barlic et al., 2000; Zhang et al., 2001). Thus, chemokine receptor signaling through a range of second messenger molecules potentially expands an individual chemokine receptor's ability to control qualitatively different cellular responses. In many cases, the same chemokine receptor is expressed in different cell types, where it initiates very different types of biological responses. For example, Cxcr4 expression in hematopoietic progenitors is important for these cells' mobilization (Möhle et al., 1998), yet, the same receptor, when expressed in neuronal progenitor cells inhibits their proliferation (Krathwohl and Kaiser, 2004). Similarly, CCR7 is expressed by T lymphocytes to facilitate their homing to secondary lymphoid organs (Sallusto et al., 1999), but it is also expressed in and important for the development of the human placenta (Drake et al., 2004). On the other hand, in early zebrafish embryos Ccr7 is expressed broadly and is involved in proper dorsoventral axis formation (Wu et al., 2012). These observations raise the question of how different chemokine receptors control such a wide range of different processes. Several models have been suggested to explain this phenomenon and these are collectively referred to as 'signaling bias' (Steen et al., 2014). Related to that, it has been recently demonstrated that the extracellular and membrane-spanning domains of chemokine receptors are not responsible for signaling specificity (Xu et al., 2014). In this work, the extracellular and transmembrane domains of rhodopsin were combined with the intracellular domains of Cxcr4. In this case, in response to light (the ligand of rhodopsin), the chimeric protein elicited Cxcr4 signaling, such that it could direct the migration of T-cells. A receptor could show signaling bias by preferentially activating different signaling cascades depending on the specific ligand or agonist it binds. For example, when chemokine receptor Ccr7 is bound by Ccl19, it induces β-arrestin recruitment more potently than when it is bound by Ccl21 (Kohout et al., 2004). Another type of signaling bias, which could increase the range of processes that chemokine receptors control involves the initiation of different signaling cascades upon binding of the same ligand. For example, Cxcr4 interaction with its ligand Cxcl12 triggers both Gαi and β-arrestin signaling (Dumstrei et al., 2004; Sun et al., 2002; Thomsen et al., 2016), while Cxcr7 receptor interaction with Cxcl12 was reported to activate β-arrestin but not G-protein signaling (Rajagopal et al., 2010). Overall, the above-mentioned studies help explain how chemokine receptor signaling bias occurs, as these receptors and their ligands can differentially activate certain second messengers. However, previous studies have not thoroughly examined the relevance of specific differences in second messenger activation with respect to the resulting biological responses in vivo. Here, we demonstrate that distinct responses to chemokine receptor signaling depend on the responding cell type rather than on the specific receptor activated by its ligand. We demonstrate this principle via chemokine receptor signaling in zebrafish embryos by examining the function of four different chemokine receptors: Cxcr4a, Cxcr4b, Ccr7, and Ccr9. We gathered several lines of evidence to show that these chemokine receptors initiate specific biological processes in a way that depends on the cell types they are expressed in. These results present chemokine receptor signal interpretation module (CRIM) as a new mechanism for biasing the biological response resulting from chemokine receptor signaling. Specifically, we showed that each of those receptors is capable of controlling biological processes that it normally does not regulate, indicating that different chemokine receptors provide the same signal when activated. We thus suggest that different cell types express specific response modules or CRIM that interpret generic signals produced by different types of chemokine receptors. Results The signaling cascades initiated by Cxcr4a and Cxcr4b can direct similar biological processes As a result of an additional genome duplication in teleosts relative to other vertebrates (Lu et al., 2012; Meyer and Schartl, 1999), the zebrafish genome encodes two cxcr4 genes, specifically cxcr4a and cxcr4b (Chong et al., 2001). These two genes were shown to regulate very different biological events and respond to different ligands; Cxcr4a is activated by the chemokine Cxcl12b and Cxcr4b is activated by the chemokine Cxcl12a (Boldajipour et al., 2011). Cxcr4a plays a central role in vascular system patterning by guiding multicellular vessel growth (Siekmann et al., 2009). Additionally, Cxcr4a controls endodermal cell-matrix adhesion, thereby ensuring proper gastrulation movements (Nair and Schilling, 2008). At the same time, Cxcr4b is involved in different processes such as the guided migration of primordial germ cells (PGCs) (Doitsidou et al., 2002; Knaut et al., 2003). To examine whether qualitative differences between Cxcr4a and Cxcr4b signaling exist, we investigated this issue in the contexts of gastrulation (Cxcr4a-controlled endodermal cell adhesion) and directional migration (Cxcr4b-controlled single-cell migration). First, we expressed Cxcr4a instead of the Cxcr4b receptor in PGCs and examined whether the foreign receptor (Cxcr4a) could function in the context of guided single-cell migration, which is normally directed by Cxcr4b. We assayed the function of the receptor by monitoring the position of the PGCs in 12 hours post-fertilization (hpf) old embryos, a stage when cxcl12b (encoding for the Cxcr4a ligand) and cxcl12a (encoding for the Cxcr4b ligand) exhibit distinct expression patterns (Boldajipour et al., 2011), allowing us to determine the response of the cells towards each of the ligands. In this experimental setup, embryos homozygous for the cxcr4b odysseus nonsense mutation, inactivating the gene (Knaut et al., 2003) were used. In odysseus mutant embryos injected with control RNA encoding for the human CD14 the PGCs were randomly distributed, whereas injecting RNA encoding for Cxcr4b in the PGCs reversed the phenotype, such that the cells clustered at regions where the ligand Cxcl12a was expressed (Figure 1A). Intriguingly, PGCs expressing Cxcr4a were located closer to the midline at the region where the RNA encoding for the Cxcr4a ligand Cxcl12b is normally expressed (see green label in Figure 1A right panel, n = 60 embryos in three experimental repeats). Thus, the mis-expressed chemokine receptor Cxcr4a is capable of directing PGC migration toward sites where its ligand Cxcl12b is expressed, despite the fact that it is normally not involved in this process. This result is consistent with the idea that the signals provided by the two receptors are qualitatively similar, allowing the cells to respond in a similar way to the signals generated by either of the receptors. Figure 1 with 1 supplement see all Download asset Open asset The signaling cascades initiated by Cxcr4a and Cxcr4b are functionally equivalent. (A) The position of PGCs (detected by nanos3 RNA expression, yellow) relative to tissues expressing cxcl12a (magenta) and cxcl12b (green) RNAs in 12 hpf cxcr4b-/- embryos. PGCs express control RNA (cntl, left panel) or RNAs encoding for Cxcr4b (middle panel) or Cxcr4a (right panel). 20 pg of control RNA and RNA encoding for Cxcr4a and Cxcr4b were injected. (B) Epifluorescence image of transgenic sox17::gfp embryos at 8 hpf. The embryos were injected with control (cntl) morpholino and control RNA (left panel). Experimental embryos were knocked down for Cxcr4a and Cxcl12b and the effect of control RNA, cxcr4a and Cxcl12b or cxcr4b along with cxcl12a mRNAs was examined. The quantitation of the endoderm displacement with respect to the forerunner cells is presented in the graph on the right. See also Figure 1—figure supplement 1. 0.8 pmol of each morpholino was used. 100 pg of receptor and 50 pg of ligand encoding mRNA was used, as well as equimolar amounts of control RNA. https://doi.org/10.7554/eLife.33574.003 Figure 1—source data 1 Source data file contains the results of the measured displacement of endoderm from forerunner cells under different experimental conditions. The data shows that the expression of Cxcr4b together with Cxcl12a can reverse the phenotype of displaced endoderm in Cxcr4a morphants. Three biological replicates are presented. https://doi.org/10.7554/eLife.33574.005 Download elife-33574-fig1-data1-v1.xlsx To further investigate if the signals the two receptors elicited were indeed equivalent, we examined the ability of Cxcr4b to support a process normally controlled by Cxcr4a and its ligand Cxcl12b, namely the proper adhesion and positioning of endodermal cells during gastrulation. In this experiment, we made use of a transgenic fish line (sox17::GFP, [Mizoguchi et al., 2008]) in which all the endodermal cells and the dorsal forerunner cells are labeled with GFP. Inhibiting the translation of cxcr4a along with cxcl12b RNAs using antisense morpholino oligonucleotides elicited the previously described abnormal displacement of endoderm from the dorsal forerunner cells, as seen in Figure 1B (Nair and Schilling, 2008). Unlike the case of chemokine-guided migration, in the context of controlling the interaction of the endoderm with the mesoderm, the distribution of the ligand is not critical. Accordingly, global expression of the chemokine acting in a paracrine or autocrine manner is expected to effectively control the process. Indeed, the cxcr4a/cxcl12b morpholino–induced phenotype was effectively rescued by co-expressing the morpholino-resistant cxcr4a and cxcl12b mRNAs in the embryos (Figure 1B). Interestingly, consistent with the idea that the intracellular signals generated by the two receptors are equivalent, the expression of cxcr4b and cxcl12a RNAs in embryos knocked down for cxcr4a and cxcl12b reversed the phenotype as well. Thus, Cxcr4b signaling in endodermal cells could effectively replace that of Cxcr4a as determined by the reduction in the displacement between endodermal cells and forerunner cells. CC chemokine receptors can control processes regulated by CXC receptors The results presented above show that distinct CXC receptors can control processes they are not normally involved in. Nevertheless, Cxcr4a and Cxcr4b show relatively high similarity in their protein sequence (Figure 2—figure supplement 1). Therefore, to examine the equivalence of chemokine receptor signaling more rigorously, we performed analogous experiments where we exchanged CC and CXC receptors in different processes. Here, Ccr9 and Ccr7 (and their ligands Ccl25 and Ccl19, respectively), which do not share high-sequence similarity with Cxcr4a, Cxcr4b (Figure 2—figure supplement 2) were tested in the context of Cxcr4-controlled PGC directional migration and endoderm cell adhesion. To examine the general nature of chemokine receptor signals, we tested the potency of Ccr7 and Ccr9 in regulating endodermal cell movement. We expressed these receptors with their cognate ligands in early embryos and observed their effects on endodermal cell positioning in the embryos knocked down for Cxcr4a, the receptor that normally regulates this process. The ubiquitous expression of chemokine receptors and their ligand in the early embryos by way of injecting the RNA results in a uniform expression pattern, which in the context of this process is similar to that of the endogenous receptor-ligand pair (Cxcr4a and Cxcl12b). Remarkably, both Ccr9 and Ccr7 reversed the Cxcr4a phenotype concerning the displacement between endoderm cells and dorsal forerunner cells, effectively controlling endodermal cell positioning (Figure 2A,B) Figure 2 with 2 supplements see all Download asset Open asset CC receptors can control a process regulated by CXC receptors. (A) Epifluorescence image of sox17::gfp transgenic control embryos (left panel) and embryos knock down for Cxcr4a. The Cxcr4a knocked down embryos were injected with control RNA (cntl) or with cxcr4a RNA, or co-injected with ccr9 RNA and its ligand ccl25 RNA. The quantitation of the endoderm displacement with respect to the forerunner cells is presented in the graph on the right. See also Figure 1—figure supplement 1. (B) Epifluorescence image of sox17::gfp transgenic control embryos (left panel) and of embryos knocked down for Cxcr4a. The embryos were injected with control RNA, with cxcr4a RNA, or co-injected with ccr7 RNA and its ligand ccl19 RNA. The quantitation of the endoderm displacement with respect to the forerunner cells is presented in the graph on the right. 0.8 pmol of each morpholino was used. 100 pg of receptor and 50 pg of ligand encoding mRNA was used, as well as equimolar amounts of control RNA. https://doi.org/10.7554/eLife.33574.006 Figure 2—source data 1 Cxcr4b can initiate signaling cascade functionally equivalent to Cxcr4a in endoderm cells. Source data contains two files, Figure 2—source data 1A contains the results of the measured displacement of endoderm from forerunner cells under different experimental conditions. The data shows that the expression of Ccr9 together with Ccl25 can reverse the phenotype of displaced endoderm in Cxcr4a morphants. Figure 2—source data 1B contains data showing that expression of Ccr7 together with Ccl19 can reverse the phenotype of displaced endoderm in Cxcr4a morphants. Three biological replicates are presented for both the experiments. https://doi.org/10.7554/eLife.33574.009 Download elife-33574-fig2-data1-v1.xlsx To further test the capability of receptors to direct cell migration, we expressed receptors in PGCs and their cognate ligands in one half of the embryo in the absence of the regular endogenous signals guiding the cells (i.e. the Cxcl12a in the environment and Cxcr4b in the PGCs). In this experimental setup, we thus generated spatially restricted source of chemokine, simulating the uneven distribution of the endogenous guidance cue within the embryo (see Figure 3 for a schematic representation of the experimental setup). If the receptor can direct cell migration, it would lead to PGC accumulation within the part of the embryo expressing the ligand, as compared with a random distribution in control (Doitsidou et al., 2002). Interestingly, in contrast with PGCs expressing control RNA that were randomly distributed throughout the embryo, under conditions where the endogenous Cxcl12a signals were knocked down, PGCs expressing Ccr9 were preferentially present on the part of the embryo engineered to express Ccl25 (Figure 3A,C). Similar results were observed when testing the activity of Ccr7 and its ligand Ccl19 (Figure 3A,D) and Cxcr4a and its ligand Cxcl12b (Figure 3A,B), demonstrating that receptors from the same and from different families can control directional PGC migration. Figure 3 with 1 supplement see all Download asset Open asset CC and CXC receptors can direct the migration of primordial cells. The experimental scheme is provided at the top. (A) Epifluorescence image of 10 hpf embryos expressing the control RNA or RNA encoding for the indicated ligand (Cxcl12a, Cxcl12b, Ccl25 and Ccl19) in one half of the embryo and control RNA or Receptor-encoding RNA (Cxcr4b, Cxcr4a, Ccr9 or Ccr7) in PGCs. Merged images show the position of PGCs with respect to control or ligand-expressing domains (red). (B–D) Graphs show the quantitation of the migration of PGCs as the percentage of GFP-labeled cells located within the ligand-expressing part of the embryos. 60 pg of mGFP-nanos was used to label PGCs in green and 40 pg of m-cherry mRNA was used for labeling the ligand expressing half of the embryo. 20 pg of receptor-encoding RNA was used and 30 pg of ligand-encoding RNA was used. 0.2 pmol of Cxcl12a morpholino was used. Equimolar amounts of control RNA were used. For raw data see Figure 3—source data 1. https://doi.org/10.7554/eLife.33574.010 Figure 3—source data 1 The data presents the percentage of PGCs located on the ligand expressing embryo half under conditions where the Cxcr4b and Cxcl12a proteins are not expressed. Figure 3—source data 1B contains data showing that Cxcr4a can direct PGCs towards the Cxcl12b-expressing half. Figure 3—source data 1C contains data showing that Ccr9 can direct PGCs toward the Ccl25 expressing half. Figure 3—source data 1D contains data showing that Ccr7 can direct PGCs toward Ccl19 expressing half. Three biological replicates are presented for each experiment. https://doi.org/10.7554/eLife.33574.013 Download elife-33574-fig3-data1-v1.xlsx Different chemokine receptors can control dorsoventral axis specification Since the biological contexts studied above are based on cell migration, we further tested the equivalency of chemokine receptor signals in the context of dorsoventral fate specification in the early zebrafish embryo. Ccr7 was previously shown to be important for dorsoventral axis specification in early zebrafish embryos (Wu et al., 2012). Here, the activated Ccr7 limits β-catenin-induced dorsalization throughout the early embryo, thereby controlling the relative size of dorsal and ventral domains. Embryos lacking Ccr7 function (maternal zygotic ccr7stl7/stl7, MZccr7 mutants) do not appear morphologically dorsalized as do ccr7 morpholino-treated embryos (Wu et al., 2012), a finding that could be attributed to off-target effects of the morpholinos on genes in addition to ccr7. Nevertheless, MZccr7 mutant embryos do exhibit increased sensitivity to β-catenin-induced dorsalization. Consequently, a very low dose of RNA encoding for Δβ-catenin (2.5 pg) caused expansion of the dorsal region in MZccr7 homozygous mutants, whereas in wild-type embryos the same dose of Δβ-catenin has no effect (Figure 4—figure supplement 1). To quantify the effect of chemokine signaling on the size of the dorsal tissue induced by Δβ-Catenin, we used MZccr7 mutant embryos expressing GFP under the control of the goosecoid promoter (Doitsidou et al., 2002). We counted the number of pixels showing GFP expression above the auto threshold in those embryos following different experimental manipulations. MZccr7 mutants Δβ-catenin RNA-sensitized embryos co-injected with control RNA showed high level of GFP expression at 5 hpf as compared with non-injected embryos (Figure 4A–D). Interestingly, goosecoid promoter-driven GFP expression was reduced in Δβ-catenin RNA-sensitized MZccr7 embryos in which different chemokine receptors were activated by expression of cxcr4a, cxcr4b and ccr9 RNAs along with their cognate ligands (Figure 4A–D). These results show that Ccr7's effect on the extent of dorsalization (Wu et al., 2012) can be directed by other chemokine receptors from different families. Figure 4 with 1 supplement see all Download asset Open asset Control of dorsoventral axis specification by different chemokine receptors. (A) Epifluorescence image of 5 hpf ccr7-/- mutant embryos carrying the gsc::gfp transgene. Uninjected embryo (left panel) and embryos sensitized by injection of Δβ-Catenin encoding RNA (right panels) are presented. The embryos were also injected with control RNA (cntl) or with RNA encoding for different chemokine receptors (Cxcr4a, Cxcr4b or Ccr9) and their cognate ligands (Cxcl12b, Cxcl12a or Ccl25). (B–D) Graphs showing the area of the goosecoid expression domain determined by quantifying the number of pixels with GFP signal above the auto threshold in the different treatments. 100 pg of mRNA encoding for the receptors was injected and 60 pg of RNA encoding for the ligands. 2.5 pg of Δβ-Catenin-encoding RNA was inject to sensitize the embryos. Equimolar amounts of control RNA were used. For raw data see Figure 4—source data 1. https://doi.org/10.7554/eLife.33574.014 Figure 4—source data 1 The data presents the number of pixels showing GFP expression above the threshold (Area of goosecoid RNA expression) in embryos under different experimental conditions. Figure 4—source data 1B shows that expression of Cxcr4a together with Cxcl12b lead to a reduction in the area of goosecoid expression. Figure 4—source data 1C shows that expression of Cxcr4b together with Cxcl12a lead to a reduction in the area of goosecoid expression. Figure 4—source data 1D shows that expression of Ccr9 together with Ccl25 lead to a reduction in the area of goosecoid expression. A minimum of three biological replicates are presented for each experiment. https://doi.org/10.7554/eLife.33574.017 Download elife-33574-fig4-data1-v1.xlsx Different chemokine receptors activate the same downstream signaling pathway to direct cell migration In contrast to the notion that different receptors initiate different signaling cascades to mediate various biological processes, our results suggest that different chemokine receptors initiate similar signaling. Accordingly, the specific response to receptor signaling might depend on its interpretation by the cell type within which the receptor was activated. To test this idea, we examined the signaling downstream of chemokine receptors in the context of directional cell migration. Cxcr4 was shown to signal through Gαi in response to Cxcl12 binding (Moepps et al., 1997). Accordingly, Gαi was shown to be important for Cxcr4b-mediated directed PGC migration in zebrafish (Dumstrei et al., 2004). To examine if the guidance signals that receptors other than Cxcr4b transmitted are Gαi dependent as well, we expressed pertussis toxin (PTX) in the PGCs. PTX catalyzes ADP ribosylation of Gαi impairing its interaction with the receptor thereby inhibiting G-protein-dependent signaling (Casey et al., 1989; Mangmool and Kurose, 2011). We examined if this treatment that inhibits Gαi signaling affected the activity of different chemokine receptors in steering PGCs toward ligand-expressing domains within the embryo (Figure 5A). Indeed, the guidance of PGCs mediated by the four receptor-ligand pairs (Cxcr4b-Cxcl12a, Cxcr4a-Cxcl12b, Ccr9-Ccl25 and Ccr7-Ccl19) was abrogated by inhibiting Gαi function (Figure 5A–E). These results are consistent with the idea that Gαi is essential for the directional cues the four chemokine receptors provide to the motile PGCs. Figure 5 with 1 supplement see all Download asset Open asset CC and CXC receptors can regulate the migration of primordial cells employing the same downstream signaling pathway. (A) Epifluorescence image of 10 hpf embryos expressing different ligands (Cxcl12a, Cxcl12b, Ccl25 and Ccl19) in one half of the embryo (mCherry-expressing cells) and PGCs (mGFP) expressing different chemokine receptors (Cxcr4b, Cxcr4a, Ccr9 and Ccr7) along with PTX. Merged images show the position of PGCs with respect to control (cntl), or ligand-expressing cells. (B–E) Graphs showing the quantitation of directed cell migration, by presenting the percentage of PGCs located within the ligand-expressing domains. 60 pg of mGFP-nanos was used to label PGCs in green and 40 pg of m-cherry-globin mRNA was used for labeling the ligand-expressing half of the embryo. 10 pg of PTX-encoding RNA were injected to inhibit the Gi protein. 20 pg of receptor-encoding RNA was used and 30 pg of ligand-encoding RNA was used. 0.2 pmol of cxcl12a morpholino was used. Equimolar amounts of cntl RNA were used. For raw data see Figure 5—source data 1 . https://doi.org/10.7554/eLife.33574.018 Figure 5—source data 1 The data presents the percentage of PGCs expressing pertussis toxin present on ligand expressing embryo half. Figure 5—source data 1B shows that Cxcr4b cannot direct PGCs expressing PTX towards the Cxcl12a expressing half. Figure 5—source data 1C shows that Cxcr4a cannot direct PGCs expressing ptx toward the Cxcl12b expressing embryo half. Figure 5—source data 1D shows that Ccr9 cannot direct PGCs expressing PTX toward the Ccl25 expressing embryo half. Figure 5—source data 1E shows that Ccr7 cannot direct PGCs expressing ptx toward Ccl19 expressing embryo half. Minimum of three biological replicates are presented for each experiment. https://doi.org/10.7554/eLife.33574.021 Download elife-33574-fig5-data1-v1.xlsx The finding that different types of chemokine receptors depend on the same signaling cascade to control the same process highlights the importance of tight regulation over their expression. This would ensure th
Abstract Genetic factors predictive of severe adolescent idiopathic scoliosis (AIS) are largely unknown. To identify genetic variation associated with severe AIS, we performed an exome-wide association study of 457 severe AIS cases and 987 controls. We find a missense SNP in SLC39A8 (p.Ala391Thr, rs13107325) associated with severe AIS ( P = 1.60 × 10 −7 , OR = 2.01, CI = 1.54–2.62). This pleiotropic SNP was previously associated with BMI, blood pressure, cholesterol, and blood manganese level. We replicate the association in a second cohort (841 cases and 1095 controls) resulting in a combined P = 7.02 × 10 −14 , OR = 1.94, CI = 1.63–2.34. Clinically, the minor allele of rs13107325 is associated with greater spinal curvature, decreased height, increased BMI and lower plasma manganese in our AIS cohort. Functional studies demonstrate reduced manganese influx mediated by the SLC39A8 p.Ala391Thr variant and vertebral abnormalities, impaired growth, and decreased motor activity in slc39a8 mutant zebrafish. Our results suggest the possibility that scoliosis may be amenable to dietary intervention.
Diphthamide is a post-translationally modified histidine essential for messenger RNA translation and ribosomal protein synthesis. We present evidence for DPH5 as a novel cause of embryonic lethality and profound neurodevelopmental delays (NDDs).Molecular testing was performed using exome or genome sequencing. A targeted Dph5 knockin mouse (C57BL/6Ncrl-Dph5em1Mbp/Mmucd) was created for a DPH5 p.His260Arg homozygous variant identified in 1 family. Adenosine diphosphate-ribosylation assays in DPH5-knockout human and yeast cells and in silico modeling were performed for the identified DPH5 potential pathogenic variants.DPH5 variants p.His260Arg (homozygous), p.Asn110Ser and p.Arg207Ter (heterozygous), and p.Asn174LysfsTer10 (homozygous) were identified in 3 unrelated families with distinct overlapping craniofacial features, profound NDDs, multisystem abnormalities, and miscarriages. Dph5 p.His260Arg homozygous knockin was embryonically lethal with only 1 subviable mouse exhibiting impaired growth, craniofacial dysmorphology, and multisystem dysfunction recapitulating the human phenotype. Adenosine diphosphate-ribosylation assays showed absent to decreased function in DPH5-knockout human and yeast cells. In silico modeling of the variants showed altered DPH5 structure and disruption of its interaction with eEF2.We provide strong clinical, biochemical, and functional evidence for DPH5 as a novel cause of embryonic lethality or profound NDDs with multisystem involvement and expand diphthamide-deficiency syndromes and ribosomopathies.
Cytoplasmic dynein provides the main motor force for minus-end-directed transport of cargo on microtubules. Within the vertebrate central nervous system (CNS), proliferation, neuronal migration, and retrograde axon transport are among the cellular functions known to require dynein. Accordingly, mutations of DYNC1H1, which encodes the heavy chain subunit of cytoplasmic dynein, have been linked to developmental brain malformations and axonal pathologies. Oligodendrocytes, the myelinating glial cell type of the CNS, migrate from their origins to their target axons and subsequently extend multiple long processes that ensheath axons with specialized insulating membrane. These processes are filled with microtubules, which facilitate molecular transport of myelin components. However, whether oligodendrocytes require cytoplasmic dynein to ensheath axons with myelin is not known.We identified a mutation of zebrafish dync1h1 in a forward genetic screen that caused a deficit of oligodendrocytes. Using in vivo imaging and gene expression analyses, we additionally found evidence that dync1h1 promotes axon ensheathment and myelin gene expression.In addition to its well known roles in axon transport and neuronal migration, cytoplasmic dynein contributes to neural development by promoting myelination.
Chemokines are secreted proteins that regulate a range of processes in eukaryotic organisms. Interestingly, different chemokine receptors control distinct biological processes, and the same receptor can direct different cellular responses, but the basis for this phenomenon is not known. To understand this property of chemokine signaling, we examined the function of the chemokine receptors Cxcr4a, Cxcr4b, Ccr7, Ccr9 in the context of diverse processes in embryonic development in zebrafish. Our results reveal that the specific response to chemokine signaling is dictated by cell-type-specific chemokine receptor signal interpretation modules (CRIM) rather than by chemokine-receptor-specific signals. Thus, a generic signal provided by different receptors leads to discrete responses that depend on the specific identity of the cell that receives the signal. We present the implications of employing generic signals in different contexts such as gastrulation, axis specification and single-cell migration.
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