Occurrence of sdh Mutations in German Alternaria solani Isolates and Potential Impact on Boscalid Sensitivity In Vitro, in the Greenhouse, and in the Field.

The fungus Alternaria solani is the main pathogen causing early blight on potatoes (Solanum tuberosum L.). An increase in the development of resistance to the succinate dehydrogenase inhibitor (SDHI) boscalid, one of the main active ingredients for the control of early blight, has been reported. For this study, monitoring data from Germany were collected between 2013 and 2016 and an increase in the occurrence of A. solani succinate dehydrogenase (SDH) mutant isolates was observed. In addition to the known point mutations in sdh complex II, a new mutation in subunit C was found in German isolates (SdhC-H134Q). SDHI fungicide sensitivity testing was performed in the laboratory, greenhouse, and field. Reduced boscalid sensitivity was shown for mutant isolates (SdhB-H278Y and SdhC-H134R) both in vitro and in vivo. In addition, field trials with artificial inoculation were performed in 2016 and 2017. In both years, fungicide efficacy was significantly reduced after mutant inoculation compared with wild-type inoculation.

Identification of neural progenitor pools by E(Spl) factors in the embryonic and adult brain.

The maintenance of progenitor cells is a crucial aspect of central nervous system development and maturation, and bHLH transcription factors of the E(Spl) subfamily are involved in this process in all vertebrates studied to date. In the zebrafish embryonic neural plate, a large number of E(Spl) genes (her genes) are at play. We review recent data on this point, and propose a model where distinct subsets of these genes define different progenitor subtypes. Analysis of her genes expression in the adult zebrafish brain suggests that part of the embryonic her cascade might also be reused to define progenitors during adulthood. Further, available evidence on orthologous genes in the mouse (Hes genes) point to different modes of Hes regulation depending on cell location within the embryonic neural tube, perhaps associated with distinct progenitor types in this species as well. Out of these comparisons emerges a simple model of neural stem cell maintenance applicable from embryonic development until adulthood as well as across species. This working model suggests the directions for future experiments.

atoh1.2 and beta3.1 are two new bHLH-encoding genes expressed in selective precursor cells of the zebrafish anterior hindbrain.

Transcription factors of the bHLH class play crucial roles in neurogenesis by controlling the location and timing of neuronal commitment and differentiation, as well as influencing neuronal identity. Proneural bHLH factors belong to the Olig, Neurogenin, NeuroD, Achaete-scute and Atonal subfamilies, and are expressed in partially overlapping or complementary patterns within the vertebrate embryonic neural tube. The combinatorial expression of these factors likely drives the generic and cell type-specific properties of neurogenesis throughout the nervous system. As an approach towards identifying a complete set of vertebrate proneural factors, we report here the isolation of two new zebrafish neural bHLH gene members, atoh1.2 and beta3.1. Among other sites, both are expressed in the late embryonic and early larval anterior hindbrain. In this territory we demonstrate that atoh1.2 and beta3.1 are transcribed in distinct precursors, further highlighting the subdivision of anterior zebrafish hindbrain into subdomains of bHLH expression.

Expression of hairy/enhancer of split genes in neural progenitors and neurogenesis domains of the adult zebrafish brain.

All subdivisions of the adult zebrafish brain maintain niches of constitutive neurogenesis, sustained by quiescent and multipotent progenitor populations. In the telencephalon, the latter potential neural stem cells take the shape of radial glia aligned along the ventricle and are controlled by Notch signalling. With the aim of identifying new markers of this cell type and of comparing the effectors of embryonic and adult neurogenesis, we focused on the family of hairy/enhancer of split [E(spl)] genes. We report the expression of seven hairy/E(spl) (her) genes and the new helt gene in three neurogenic areas of the adult zebrafish brain (telencephalon, hypothalamus, and midbrain) in relation to radial glia, proliferation, and neurogenesis. We show that the expression of most her genes in the adult brain characterizes quiescent radial glia, whereas only few are expressed in progenitor domains engaged in active proliferation or neurogenesis. The low proliferation status of most her-positive progenitors contrasts with the embryonic nervous system, in which her genes are expressed in actively dividing progenitors. Likewise, we demonstrate largely overlapping expression domains of a set of her genes in the adult brain, which is in striking contrast to their distinct embryonic expression profiles. Overall, our data provide a consolidated map of her expression, quiescent glia, proliferation, and neurogenesis in these various subdivisions of the adult brain and suggest distinct regulation and function of Her factors in the embryonic and adult contexts.

Fgf signaling in the zebrafish adult brain: association of Fgf activity with ventricular zones but not cell proliferation.

The zebrafish adult brain contains numerous neural progenitors and is a good model to approach the general mechanisms of adult neural stem cell maintenance and neurogenesis. Here we use this model to test for a correlation between Fgf signaling and cell proliferation in adult progenitor zones. We report expression of Fgf signals (fgf3,4,8a,8b,17b), receptors (fgfr1-4), and targets (erm, pea3, dusp6, spry1,2,4, and P-ERK) and document that genes of the embryonic fgf8 synexpression group acquire strikingly divergent patterns in the adult brain. We further document the specific expression of fgf3, fgfr1-3, dusp6, and P-ERK in ventricular zones, which contain neural progenitors. In these locations, however, a comparison at the single-cell level of fgfr/P-ERK expression with bromo-deoxy-uridine (BrdU) incorporation and the proliferation marker MCM5 indicates that Fgf signaling is not specifically associated with proliferating progenitors. Rather, it correlates with the ventricular radial glia state, some of which only are progenitors. Together these results stress the importance of Fgf signaling in the adult brain and establish the basis to study its function in zebrafish, in particular in relation to adult neurogenesis.

Genomic regulatory blocks encompass multiple neighboring genes and maintain conserved synteny in vertebrates.

We report evidence for a mechanism for the maintenance of long-range conserved synteny across vertebrate genomes. We found the largest mammal-teleost conserved chromosomal segments to be spanned by highly conserved noncoding elements (HCNEs), their developmental regulatory target genes, and phylogenetically and functionally unrelated "bystander" genes. Bystander genes are not specifically under the control of the regulatory elements that drive the target genes and are expressed in patterns that are different from those of the target genes. Reporter insertions distal to zebrafish developmental regulatory genes pax6.1/2, rx3, id1, and fgf8 and miRNA genes mirn9-1 and mirn9-5 recapitulate the expression patterns of these genes even if located inside or beyond bystander genes, suggesting that the regulatory domain of a developmental regulatory gene can extend into and beyond adjacent transcriptional units. We termed these chromosomal segments genomic regulatory blocks (GRBs). After whole genome duplication in teleosts, GRBs, including HCNEs and target genes, were often maintained in both copies, while bystander genes were typically lost from one GRB, strongly suggesting that evolutionary pressure acts to keep the single-copy GRBs of higher vertebrates intact. We show that loss of bystander genes and other mutational events suffered by duplicated GRBs in teleost genomes permits target gene identification and HCNE/target gene assignment. These findings explain the absence of evolutionary breakpoints from large vertebrate chromosomal segments and will aid in the recognition of position effect mutations within human GRBs.

her5 expression reveals a pool of neural stem cells in the adult zebrafish midbrain.

Current models of vertebrate adult neural stem cells are largely restricted to the rodent forebrain. To extract the general mechanisms of neural stem cell biology, we sought to identify new adult stem cell populations, in other model systems and/or brain areas. The teleost zebrafish appears to be an ideal system, as cell proliferation in the adult zebrafish brain is found in many more niches than in the mammalian brain. As a starting point towards identifying stem cell populations in this system, we used an embryonic neural stem cell marker, the E(spl) bHLH transcription factor Her5. We demonstrate that her5 expression is not restricted to embryonic neural progenitors, but also defines in the adult zebrafish brain a new proliferation zone at the junction between the mid- and hindbrain. We show that adult her5-expressing cells proliferate slowly, self-renew and express neural stem cell markers. Finally, using in vivo lineage tracing in her5:gfp transgenic animals, we demonstrate that the her5-positive population is multipotent, giving rise in situ to differentiated neurons and glia that populate the basal midbrain. Our findings conclusively identify a new population of adult neural stem cells, as well as their fate and their endogenous environment, in the intact vertebrate brain. This cell population, located outside the forebrain, provides a powerful model to assess the general mechanisms of vertebrate neural stem cell biology. In addition, the first transcription factor characteristic of this cell population, Her5, points to the E(Spl) as a promising family of candidate adult neural stem cell regulators.

Conserved and acquired features of adult neurogenesis in the zebrafish telencephalon.

Our understanding of the cellular and molecular mechanisms underlying the adult neural stem cell state remains fragmentary. To provide new models on this issue, we searched for stem cells in the adult brain of the zebrafish. Using BrdU tracing and immunodetection of cell-type-specific markers, we demonstrate that the adult zebrafish telencephalon contains self-renewing progenitors, which show features of adult mammalian neural stem cells but distribute along the entire dorso-ventral extent of the telencephalic ventricular zone. These progenitors give rise to newborn neurons settling close to the ventricular zone within the telencephalon proper. They have no equivalent in mammals and therefore constitute a new model of adult telencephalic neural stem cells. In addition, progenitors from the ventral subpallium generate rapidly dividing progenitors and neuroblasts that reach the olfactory bulb (OB) via a rostral migratory stream and differentiate into GABAergic and TH-positive neurons. These ventral progenitors are comparable to the mammalian neural stem cells of the subependymal zone. Interestingly, dorsal and ventral progenitors in the adult telencephalon express a different combination of transcription factors than their embryonic counterparts. In the case of neurogenin1, this is due to the usage of different enhancer elements. Together, our results highlight the conserved and unique phylogenic and ontogenic features of adult neurogenesis in the zebrafish telencephalon and open the way to the identification of adult neural stem cell characters in cross-species comparative studies.

Selective control of neuronal cluster size at the forebrain/midbrain boundary by signaling from the prechordal plate.

Within the vertebrate embryonic neural plate, the first neuronal clusters often differentiate at the border of patterning identities. Whether the information inherent in the intersection of patterning identities alone controls all aspects of neuronal cluster development (location, identity, and size) is unknown. Here, we focus on the cluster of the medial longitudinal fascicle (nMLF) and posterior commissure (nPC), located at the forebrain/midbrain (fore/mid) boundary, to address this issue. We first identify expression of the transcription factor Six3 as a common and distinct molecular signature of nMLF and nPC neurons in zebrafish, and we use this marker to monitor mechanisms controlling the location and number of nMLF/nPC neurons. We demonstrate that six3 expression is induced at the fore/mid boundary in pax2.1/no-isthmus and smoothened/slow muscle omitted mutants, where identities adjacent to the six3 cluster are altered; however, in these mutants, the subpopulation of six3-positive cells located within the mispatterned territory is reduced. These results show that induction of the six3 cluster is triggered by the information derived from the intersection in patterning identities alone, whereas correct cluster size depends, in a modular manner, on the identities themselves. The size of the six3 cluster is also controlled independently of neural tube patterning: we demonstrate that the prechordal plate (PCP) is impaired in mixer/bonnie and clyde mutants and that this phenotype secondarily results in an increased production of six3-positive cells at the fore/mid boundary, without correlatively affecting patterning in this area. Thus, a signaling process originating from the PCP distinguishes between neural patterning and the control of six3 cluster size at the fore/mid junction in vivo. Together, our results suggest that a combination of patterning-related and -unrelated mechanisms specifically controls the size of individual early neuronal clusters within the anterior neural plate.