Origins of the brain tumor, medulloblastoma, from stem cells or restricted progenitor cells are unclear. To investigate this, we activated oncogenic Hedgehog (Hh) signaling in multipotent and lineage-restricted CNS progenitors. We observed that normal unipotent cerebellar granule neuron precursors (CGNP) derive from hGFAP+ and Olig2+ RL progenitors. Hh activation in a spectrum of early and late stage CNS progenitors generated similar medulloblastomas, but not other brain cancers, indicating that acquisition of CGNP identity is essential for tumorigenesis. We show in human and mouse medulloblastoma that cells expressing the glia-associated markers Gfap and Olig2 are neoplastic and that they retain features of embryonic-type granule lineage progenitors. Thus, oncogenic Hh signaling promotes medulloblastoma from lineage-restricted granule cell progenitors.

Glutamatergic and GABAergic neurons mediate much of the excitatory and inhibitory neurotransmission, respectively, in the vertebrate nervous system. The process by which developing neurons select between these two cell fates is poorly understood. Here we show that the homeobox genes Tlx3 and Tlx1 determine excitatory over inhibitory cell fates in the mouse dorsal spinal cord. First, we found that Tlx3 was required for specification of, and expressed in, glutamatergic neurons. Both generic and region-specific glutamatergic markers, including VGLUT2 and the AMPA receptor Gria2, were absent in Tlx mutant dorsal horn. Second, spinal GABAergic markers were derepressed in Tlx mutants, including Pax2 that is necessary for GABAergic differentiation, Gad1/2 and Viaat that regulate GABA synthesis and transport, and the kainate receptors Grik2/3. Third, ectopic expression of Tlx3 was sufficient to suppress GABAergic differentiation and induce formation of glutamatergic neurons. Finally, excess GABA-mediated inhibition caused dysfunction of central respiratory circuits in Tlx3 mutant mice.

Correct removal of RNA primers of Okazaki fragments during lagging-strand DNA synthesis is a critical process for the maintenance of genome integrity. Disturbance of this process has severe mutagenic consequences and could contribute to the development of cancer. The role of the mammalian nucleases RNase HI and FEN-1 in RNA primer removal has been substantiated by several studies. Recently, RNase H(35), the Saccharomyces cerevisiae homologue of mammalian RNase HI, was identified and its possible role in DNA replication was proposed (P. Frank, C. Braunshofer-Reiter, and U. Wintersberger, FEBS Lett. 421:23-26, 1998). This led to the possibility of moving to the genetically powerful yeast system for studying the homologues of RNase HI and FEN-1, i.e., RNase H(35) and Rad27p, respectively. In this study, we have biochemically defined the substrate specificities and the cooperative as well as independent cleavage mechanisms of S. cerevisiae RNase H(35) and Rad27 nuclease by using Okazaki fragment model substrates. We have also determined the additive and compensatory pathological effects of gene deletion and overexpression of these two enzymes. Furthermore, the mutagenic consequences of the nuclease deficiencies have been analyzed. Based on our findings, we suggest that three alternative RNA primer removal pathways of different efficiencies involve RNase H(35) and Rad27 nucleases in yeast.Replication of double-stranded DNA is an asymmetric process. While leading-strand synthesis proceeds continuously, lagging-strand synthesis takes place by synthesis, processing, and ligation of Okazaki fragments (39). These fragments, measuring about 200 nucleotides (nt) in eukaryotes, are primed by DNA polymerase alpha/primase with a short oligoribonucleotide of 7 to 14 residues. Before the nascent Okazaki fragments are ligated to form a continuous lagging strand, the short RNA primers must be removed by an enzyme exhibiting RNase H activity. Although several such enzymes from eukaryotes are known, the process of RNA primer hydrolysis is as yet not fully understood.An RNase H was first detected in calf thymus extracts (57). Subsequently, RNase H enzymatic activity was detected in all prokaryotes and eukaryotes examined as well as in a bacteriophage and in retroviruses as a part of reverse transcriptases (for reviews, see references 12 and 66). Generally, RNases H are defined as ribonucleotide-specific endonucleases, cleaving the RNA portion of RNA-DNA/DNA or RNA/DNA duplexes. Several RNases H implicated in RNA primer removal have been purified and/or cloned from diverse organisms ranging from bacteriophages to human cells (see, e.g., references 8, 9, 13, 14, 24, 26, 37, 51, and 52). Nevertheless, conclusive evidence of the involvement of these enzymes in primer removal is still lacking. In the budding yeast, Saccharomyces cerevisiae, three different RNases H were identified and partially characterized as RNase H(70), RNase H1, and RNase H(35) (17,32,34). These enzymes are evolutionarily related to prokaryotic as well as to mammalia...

Brainstem visceral sensory and (nor)adrenergic neurons play crucial roles in modulating cardiovascular and respiratory functions. The origins and formation of these neurons are poorly understood. Here we show that these two classes of neurons are derived from Mash1-positive precursor cells, and can be prospectively identified by combinatorial expression of two homeobox genes, Rnx and Phox2 (Phox2a or Phox2b). It was previously shown that Rnx-deficient mice die from respiratory failure. Here we show that Rnx function is required for formation of first-order relay visceral sensory neurons in the brainstem. In addition, as in Phox2b-deficient mice, the development of most (nor)adrenergic centers is compromised in Rnx mutants. We also provide genetic evidence to show that Rnx and Phox2 proteins may function independently to specify the (nor)adrenergic phenotype. Our studies reveal a surprising ontogenetic relationship between relay visceral sensory and (nor)adrenergic neurons, and suggest that it may be a common theme in the developing nervous system that the same set of transcriptional regulators is associated with formation of multiple components within a neuronal network.

Trigeminal nuclei and the dorsal spinal cord are first-order relay stations for processing somatic sensory information such as touch, pain, and temperature. The origins and development of these neurons are poorly understood. Here we show that relay somatic sensory neurons and D2/D4 dorsal interneurons likely derive from Mash1-positive neural precursors, and depend on two related homeobox genes, Rnx and Tlx-1, for proper formation. Rnx and Tlx-1 maintain expression of Drg11, a homeobox gene critical for the development of pain circuitry, and are essential for the ingrowth of trkA+ nociceptive/thermoceptive sensory afferents to their central targets. We showed previously that Rnx is necessary for proper formation of the nucleus of solitary tract, the target for visceral sensory afferents. Together, our studies demonstrate a central role for Rnx and Tlx-1 in the development of two major classes of relay sensory neurons, somatic and visceral.

The dorsal spinal cord synthesizes a variety of neuropeptides that modulate the transmission of nociceptive sensory information. Here, we used genetic fate mapping to show that Tlx3 ϩ spinal cord neurons and their derivatives represent a heterogeneous population of neurons, marked by partially overlapping expression of a set of neuropeptide genes, including those encoding the anti-opioid peptide cholecystokinin, pronociceptive Substance P (SP), Neurokinin B, and a late wave of somatostatin. Mutations of Tlx3 and Tlx1 result in a loss of expression of these peptide genes. Brn3a, a homeobox transcription factor, the expression of which is partly dependent on Tlx3, is required specifically for the early wave of SP expression. These studies suggest that Tlx1 and Tlx3 operate high in the regulatory hierarchy that coordinates specification of dorsal horn pain-modulatory peptidergic neurons.

qnr and aac(6')-Ib-cr genes were detected in 8.0% and 9.9% of ESBL-producing E. coli and K. pneumoniae, respectively. The plasmids carrying the qnr gene could be transferred by conjugation together with ESBL-encoding genes and aac(6')-Ib-cr.

Metarhizium robertsii has been used as a model to study fungal pathogenesis in insects, and its pathogenicity has many parallels with plant and mammal pathogenic fungi. MAPK (Mitogen-activated protein kinase) cascades play pivotal roles in cellular regulation in fungi, but their functions have not been characterized in M. robertsii. In this study, we identified the full complement of MAPK cascade components in M. robertsii and dissected their regulatory roles in pathogenesis, conidiation and stress tolerance. The nine components of the Fus3, Hog1 and Slt2-MAPK cascades are all involved in conidiation. The Fus3- and Hog1-MAPK cascades are necessary for tolerance to hyperosmotic stress, and the Slt2- and Fus3-MAPK cascades both mediate cell wall integrity. The Hog1 and Slt2-MAPK cascades contribute to pathogenicity; the Fus3-MAPK cascade is indispensable for fungal pathogenesis. During its life cycle, M. robertsii experiences multiple microenvironments as it transverses the cuticle into the haemocoel. RNA-seq analysis revealed that MAPK cascades collectively play a major role in regulating the adaptation of M. robertsii to the microenvironmental change from the cuticle to the haemolymph. The three MAPKs each regulate their own distinctive subset of genes during penetration of the cuticle and haemocoel colonization, but they function redundantly to regulate adaptation to microenvironmental change.