PlexinA3 restricts spinal exit points and branching of trunk motor nerves in embryonic zebrafish

The pioneering primary motor axons in the zebrafish trunk are guided by multiple cues along their pathways. Plexins are receptor components for semaphorins that influence motor axon growth and path finding. We cloned plexinA3 in zebrafish and localized plexinA3 mRNA in primary motor neurons during axon outgrowth. Antisense morpholino knock-down led to substantial errors in motor axon growth. Errors comprised aberrant branching of primary motor nerves as well as additional exit points of axons from the spinal cord. Excessively branched and supernumerary nerves were found in both ventral and dorsal pathways of motor axons. The trunk environment and several other types of axons, including trigeminal axons, were not detectably affected by plexinA3 knock-down. RNA overexpression rescued all morpholino effects. Synergistic effects of combined morpholino injections indicate interactions of plexinA3 with semaphorin3A homologs. Thus, plexinA3 is a crucial receptor for axon guidance cues in primary motor neurons

Zebrafish Pou5f1-dependent transcriptional networks in temporal control of early development

Time-resolved transcriptome analysis of early pou5f1 mutant zebrafish embryos identified groups of developmental regulators, including SoxB1 genes, that depend on Pou5f1 activity, and a large cluster of differentiation genes which are prematurely expressed.Pou5f1 represses differentiation genes indirectly via activation of germlayer-specific transcriptional repressor genes, including her3, which may mediate in part Pou5f1-dependent repression of neural genes.A dynamic mathematical model is established for Pou5f1 and SoxB1 activity-dependent temporal behaviour of downstream transcriptional regulatory networks. The model predicts that Pou5f1-dependent increase in SoxB1 activity significantly contributes to developmental timing in the early gastrula.Comparison to mouse Pou5f1/Oct4 reveals evolutionary conserved targets. We show that Pou5f1 developmental function is also conserved by demonstrating rescue of Pou5f1 mutant zebrafish embryos by mouse POU5F1/OCT4

Pou5f1/Oct4 Promotes Cell Survival via Direct Activation of <i>mych</i> Expression during Zebrafish Gastrulation

<div><p>Myc proteins control cell proliferation, cell cycle progression, and apoptosis, and play important roles in cancer as well in establishment of pluripotency. Here we investigated the control of <i>myc</i> gene expression by the Pou5f1/Oct4 pluripotency factor in the early zebrafish embryo. We analyzed the expression of all known zebrafish Myc family members, <i>myca</i>, <i>mycb</i>, <i>mych</i>, <i>mycl1a, mycl1b</i>, and <i>mycn</i>, by whole mount <i>in situ</i> hybridization during blastula and gastrula stages in wildtype and maternal plus zygotic <i>pou5f1</i> mutant (MZ<i>spg</i>) embryos, as well as by quantitative PCR and in time series microarray data. We found that the broad blastula and gastrula stage <i>mych</i> expression, as well as late gastrula stage <i>mycl1b</i> expression, both depend on Pou5f1 activity. We analyzed ChIP-Seq data and found that both Pou5f1 and Sox2 bind to <i>mych</i> and <i>mycl1b</i> control regions. The regulation of <i>mych</i> by Pou5f1 appears to be direct transcriptional activation, as overexpression of a Pou5f1 activator fusion protein in MZ<i>spg</i> embryos induced strong <i>mych</i> expression even when translation of zygotically expressed mRNAs was suppressed. We further showed that MZ<i>spg</i> embryos develop enhanced apoptosis already during early gastrula stages, when apoptosis was not be detected in wildtype embryos. However, Mych knockdown alone did not induce early apoptosis, suggesting potentially redundant action of several early expressed <i>myc</i> genes, or combination of several pathways affected in MZ<i>spg.</i> Experimental <i>mych</i> overexpression in MZ<i>spg</i> embryos did significantly, but not completely suppress the apoptosis phenotype. Similarly, p53 knockdown only partially suppressed apoptosis in MZ<i>spg</i> gastrula embryos. However, combined knockdown of p53 and overexpression of Mych completely rescued the MZ<i>spg</i> apoptosis phenotype. These results reveal that Mych has anti-apoptotic activity in the early zebrafish embryo, and that p53-dependent and Myc pathways are likely to act in parallel to control apoptosis at these stages.</p></div

Mych overexpression and p53 knockdown suppress cell death in MZ<i>spg</i> gastrulae.

<p>TUNEL staining to detect apoptosis at bud stage (A-J) and subsequent computational image analysis (K) for quantification of the number of apoptotic cells. The images show maximum intensity projections of z-stacks taken from single embryos with dorsal to the right. WT embryos display almost no apoptosis, whereas MZ<i>spg</i> mutants show an increase in cell death throughout the embryo (A-D). This mutant apoptosis phenotype was partially repressed either by <i>mych</i> overexpression (E,F) or p53 morpholino knockdown (G,H). The co-injection of <i>mych</i> mRNA and p53-morpholino could completely suppress cell death in MZ<i>spg</i> mutants, but did not rescue the delay in epiboly movement (I-J; arrowheads). The quantification of cell death (K) revealed that the number of apoptotic cells is decreased by a factor of six in MZ<i>spg</i> embryos after <i>mych</i> mRNA injection. By combined knockdown of p53 and Mych overexpression, apoptosis in MZ<i>spg</i> embryos was reduced to WT levels.</p

MZ<i>spg</i> mutants have enhanced apoptosis during gastrulation.

<p>TUNEL staining of WT (left embryo in each panel) and MZ<i>spg</i> (right embryo in each panel) embryos at distinct embryonic stages between 32 cells and 90%-epiboly. Embryos are shown in animal (left column) and lateral (right column) view. Pou5f1 deficient embryos show enhanced apoptosis, starting at 60%-epiboly, compared to WT (G-J).</p

Analysis of the Mych contribution to the morphology of the MZ<i>spg</i> mutant phenotype.

<p>Knockdown of Mych function in WT by injecting splice-blocking (A-D) or translation-blocking (E-H) morpholinos. Morphological analysis of injected (A,C,E,G) and non-injected (B,D,F,H) embryos at 90%-epiboly and 25 hpf. Morphants show neither a delay in epiboly (A, E) nor obvious morphological defects by 25 hours development (C, G). Rescue of Mych activity in MZ<i>spg</i> mutants by injecting <i>mych</i> mRNA into 1-cell stage embryos (I-L). Mych alone is not able to rescue the strong defects developed by Pou5f1 deficient embryos during gastrulation (I) and after 25 hpf (K).</p

Spatial expression pattern of zebrafish <i>myc</i> genes in WT and MZ<i>spg</i> embryos at 60% epiboly.

<p>Whole mount <i>in situ</i> hybridization (WISH) analysis of <i>myca</i>, <i>mycb</i>, <i>mych</i>, <i>mycl1a</i> and <i>mycl1b</i> expression in WT (right embryo in each panel) and MZ<i>spg</i> (left embryo in each panel). All embryos are shown in lateral (left column) and animal (right column) views with dorsal oriented to the right. All analyzed <i>myc</i> genes are broadly expressed in mid-gastrula embryos, except for c-<i>myc</i> homologues, <i>myca</i> and <i>mycb</i>. <i>mycb</i> is specifically expressed in the shield, whereas <i>myca</i> was not detectable at this stage (A-D). <i>mych</i> and <i>mycl1a</i> in addition have a strong expression domain in the involuting axial mesoderm (E-H). Only <i>mych</i> (E-F) and <i>mycl1b</i> (I-J) depend on the function of Pou5f1 and their expression is strongly decreased in MZ<i>spg</i> mutants. However, the <i>mych</i> expression domain in the involuting axial mesoderm is less affected in Pou5f1 deficient embryos (E-F; arrows). We used <i>notail</i> as control, because its expression is not altered in MZ<i>spg</i> mutants compared to WT (K-L).</p

<i>mych</i> and potentially also <i>mycl1b</i> are directly regulated by Pou5f1.

<p>Pou5f1 overexpression in MZ<i>spg</i> mutants by <i>pou5f1-VP16</i> mRNA injection into one-cell stage embryos (A-F; left embryos in each panel) and non-injected controls (A, C and E; MZ<i>spg</i> middle and WT right embryos in each panel; B, D and F; MZ<i>spg</i> right embryo in each panel). The embryos in B, D and F are in addition treated with CHX starting at 1.5 hpf. The <i>mych</i> expression can be rescued by Pou5f1-VP16 in both, CHX treated and untreated embryos, and therefore the regulatory influence of Pou5f1 should be direct (A-B). The expression of <i>mycl1b</i> can also be rescued by Pou5f1-VP16 overexpression in CHX untreated MZ<i>spg</i> embryos, but is also strongly upregulated in CHX treated MZ<i>spg</i> embryos, even without the injection of <i>pou5f1-VP16</i> mRNA (C-D). Thus the experiment cannot proove whether activation of <i>mycl1b</i> by Pou5f1 is direct or not. We used <i>notail</i> as negative control, because its expression is independent of Pou5f1 function (E), and depends on zygotic gene products.</p

Pou5f1 binds to the promoters and regulatory regions of <i>mych</i> and <i>mycl1b</i>.

<p>ChIP-seq reads for Sox2 (red) and Pou5f1 (blue) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092356#pone.0092356-Leichsenring1" target="_blank">[50]</a> mapped to the <i>mycl1b</i> (A) and <i>mych</i> (B) genetic loci are shown. Colocalisation of Pou5f1 (blue) and Sox2 (red) at the regulatory regions of <i>mycl1b</i> and <i>mych</i> were detected. Y-axis: ChIP-Seq reads per base, y-axis floor is set to 2.</p

Implementing liquid biopsies into clinical decision making for cancer immunotherapy

During the last decade, novel immunotherapeutic strategies, in particular antibodies directed against immune checkpoint inhibitors, have revolutionized the treatment of different malignancies leading to an improved survival of patients. Identification of immune-related biomarkers for diagnosis, prognosis, monitoring of immune responses and selection of patients for specific cancer immunotherapies is urgently required and therefore areas of intensive research. Easily accessible samples in particular liquid biopsies (body fluids), such as blood, saliva or urine, are preferred for serial tumor biopsies.Although monitoring of immune and tumor responses prior, during and post immunotherapy has led to significant advances of patients' outcome, valid and stable prognostic biomarkers are still missing. This might be due to the limited capacity of the technologies employed, reproducibility of results as well as assay stability and validation of results. Therefore solid approaches to assess immune regulation and modulation as well as to follow up the nature of the tumor in liquid biopsies are urgently required to discover valuable and relevant biomarkers including sample preparation, timing of the collection and the type of liquid samples. This article summarizes our knowledge of the well-known liquid material in a new context as liquid biopsy and focuses on collection and assay requirements for the analysis and the technical developments that allow the implementation of different high-throughput assays to detect alterations at the genetic and immunologic level, which could be used for monitoring treatment efficiency, acquired therapy resistance mechanisms and the prognostic value of the liquid biopsies.status: publishe