Population-Specific Regulation of Chmp2b by <em>Lbx1</em> during Onset of Synaptogenesis in Lateral Association Interneurons

<div><p>Chmp2b is closely related to Vps2, a key component of the yeast protein complex that creates the intralumenal vesicles of multivesicular bodies. Dominant negative mutations in Chmp2b cause autophagosome accumulation and neurodegenerative disease. Loss of Chmp2b causes failure of dendritic spine maturation in cultured neurons. The homeobox gene <em>Lbx1</em> plays an essential role in specifying postmitotic dorsal interneuron populations during late pattern formation in the neural tube. We have discovered that Chmp2b is one of the most highly regulated cell-autonomous targets of <em>Lbx1</em> in the embryonic mouse neural tube. Chmp2b was expressed and depended on <em>Lbx1</em> in only two of the five nascent, Lbx1-expressing, postmitotic, dorsal interneuron populations. It was also expressed in neural tube cell populations that lacked Lbx1 protein. The observed population-specific expression of Chmp2b indicated that only certain population-specific combinations of sequence specific transcription factors allow Chmp2b expression. The cell populations that expressed Chmp2b corresponded, in time and location, to neurons that make the first synapses of the spinal cord. Chmp2b protein was transported into neurites within the motor- and association-neuropils, where the first synapses are known to form between E11.5 and E12.5 in mouse neural tubes. Selective, developmentally-specified gene expression of Chmp2b may therefore be used to endow particular neuronal populations with the ability to mature dendritic spines. Such a mechanism could explain how mammalian embryos reproducibly establish the disynaptic cutaneous reflex only between particular cell populations.</p> </div

Chmp2b Colocalization with MAP2a in DLF and VLF.

<p>(A–I) Comparison of Chmp2b/MAP2a colabeling with Chmp2b/ß-tubulin colabeling in the VLF in adjacent sections (J–R) Comparison of Chmp2b/MAP2a colabeling with Chmp2b/ß-tubulin colabeling in the DLF in adjacent sections. Note that Chmp2b and MAP2a colocalize, while Chmp2b and ß-tubulin are only in close apposition.</p

Lbx1-Independent Domain Absent at Cervical Levels.

<p>(A–D) Dorsolateral neural tube at cervical levels. A lateral column (bracketed by arrowheads) shows cells that are colabeled by Lbx1(GFP) and Lhx1/5. A subset of these cells are also labeled by Chmp2b in heterozygotes but not in mutants. The size of the lateral column is also reduced, possibly reflecting the loss of dI4 cells, as observed at thoracic levels. The <i>Lbx1</i>-independent expression domain in the circumferential trajectory, representing dI1B cells, is absent in both genotypes. (E–H). Dorsolateral neural tube at thoracic levels. A lateral column (bracketed by arrowheads) shows dI4 cells that are colabeled by Lbx1(GFP) and Lhx1/5. Almost all of the cells in this column are labeled by Chmp2b in heterozygotes. The column is absent in mutants. A large <i>Lbx1</i>-independent expression domain in the circumferential trajectory, representing dI1B cells, is present in both genotypes. It is adjacent to the dI4 column and obscured the loss of Chmp2b RNA in the dI4 column at thoracic levels (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048573#s3" target="_blank">results</a>). (I, J) Quantification of cells in GFP/Lhx1/5/Chmp2b labeled heterozygote sections at cervical (n = 4) and thoracic (n = 4) levels, respectively. Primary antibodies against GFP, Lhx1/5, and Chmp2b were detected using appropriate Cy2 (green), Cy3 (red), and Cy5 (infrared)secondary antibodies, respectively. Colors were electronically switched, for clarity, to those indicated by the labels.</p

Lbx1-Dependent and Lbx1-Independent Chmp2b Expression Domains.

<p>(A,B) Chmp2b RNA at mid-forelimb levels in the dorsal and ventral portions of the mantle zone is <i>Lbx1</i>-dependent and <i>Lbx1</i>-independent, respectively. RNA expression in the motor columns (black arrow) varied considerably in adjacent sections, but was not generally affected when series of sections along corresponding parts of the body axis were compared. Arrowheads indicate the region of the intermediate zone (IZ), where Chmp2b RNA signal remains intense in mutants. The IZ lies lateral and adjacent to the ventricular zone (VZ). Chmp2b signal can be compared in the circumferential trajectory at the level of the sulcus limitans (SL), in the circled region (white arrow). (C, D) Chmp2b protein at E12.5 at mid-forelimb levels in the dorsal and ventral portions of the mantle zone (MZ) is Lbx1-dependent and <i>Lbx1</i>-independent, respectively. The dorsolateral funiculus (DLF; *) and ventrolateral funiculus (VLF; **) are indicated. (E) Only a subset of the Lbx(GFP)⁺ cells within the lateral dorsal horn express Chmp2b (arrowheads). Green cells lacking Chmp2b protein are intermingled (arrows). (F) Quantitative evaluation of the fraction of Chmp2b⁺ area in the neural tube that was colabeled by GFP. All images of the neural tube in this report will be oriented medial to lateral (left to right) and dorsal to ventral (top to bottom).</p

Funicular Localization of Chmp2b Protein.

<p>(A) Chmp2b staining between the cellular region and basal lamina was observed in a dorsolateral zone (between arrowheads) and a ventrolateral zone (between arrows)in heterozygotes. (B) The dorsolateral zone is lost in mutants while the ventrolateral zone is retained. (C–E) ß-tubulin Stains longitudinal axons in the lateral white matter as blue puncta. (C) The dorsolateral zone of Chmp2b expression is located between the tubulin stained DLF (*) and lateral funiculus (LF; †) in heterozygotes. It appears smaller in this section than in panel A. (D) Tubulin staining at the sulcus limitans (horizontal line) and in the VLF is severely reduced in mutants. The LF appears to be absent and Chmp2b staining in the VLF has an altered appearance. (E) Chmp2b stained projections enter the heterozygote DLF at E12.5. Afferent projections in this funiculus project longitudinally and have not entered the grey matter at this stage. They are labeled with tubulin (blue). Single channel images of this panel are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048573#pone.0048573.s003" target="_blank">Fig. S3</a> A–C. GFP/Chmp2b double labels of the insets are enlarged in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048573#pone.0048573.s003" target="_blank">Fig. S3D</a>–F. They show coincident labeling of GFP and Chmp2b in the white matter (F–K) The red channel of the color images in F, H, J were converted to greyscale images in G, I, and K, respectively. This allows Chmp2b staining in the circumferential trajectory (arrows) to be seen at thoracic levels at E11.5 (G) and E12.5 (I). No such staining can be seen at cervical levels at E12.5 (K). Note that Chmp2b staining is only seen in GFP stained areas at cervical levels (J), whereas it is observed outside of the GFP stained areas at thoracic levels (F, H).</p

Chmp2b Protein in Soma and Dendrites of Motor Neurons.

<p>Chmp2b and Isl1 are co-expressed in motor columns of the ventral horn. (A, C, E) Isl1 primary antibody was detected with Cy3 (red). Chmp2b antibody was detected with Cy5 (infrared) and is shown in green. (B, D, F) Greyscale images of the green channel of images shown in A, C, and E, respectively. Chmp2b staining within soma can be distinguished from staining of projections between soma. The most sensitive secondary antibody (Cy3) and relatively long image acquisition times are required to detect Chmp2b signal in the motor columns. (G, H) Comparison of the VLF of heterozygotes and mutants stained with Chmp2b (red; Cy3) and tubulin (blue; Cy5). Radial breaks in the tubulin stains of heterozygotes (indicated by pairs of arrowheads) may represent the endfeet of radial glial cells (see inset I) that dendrites of motor neurons have been shown to follow into the VLF during early synaptogenesis (diagrammed by flow arrow). Note the loss of tubulin staining in the LF (†) and in the VLF below the sulcus limitans (horizontal line). Chmp2b staining in the VLF is present but distributed differently in mutants. (I) High magnification image of a putative radial glial endfoot that can be seen by background stain in the green channel. Note that Chmp2b and tubulin staining do not colocalize but appear to associate closely on the surface of the endfoot.</p

Non-SSTF, Cell-Autonomous, Neural Tube, Genetic Target Genes.

<p>Neural tubes were dissected from heterozygote Lbx1^GFP/+(h) and mutant Lbx1^GFP/GFP (m) E12.5 embryos. Pools of neural tubes from each genotype were rapidly dissociated, flow sorted into GFP⁺ (green; G) and GFP⁻ (white; W) cells, and total RNA prepared. Three replicates of the flow sort experiment were used to produce triplicate array data for each of the four conditions, namely heterozygous green (hG), mutant green (mG), heterozygous white (hW), and mutant white (mW). Error bars in each direction indicate the standard deviation in three biological replicates. Only 41,500 of the array's probe sets, corresponding to 22,300 non-sequence specific transcription factor (non-SSTF) genes are shown. (A) Cell autonomous changes in gene expression due to loss of <i>Lbx1</i> function were measured in cells that normally express Lbx1 (green cells). Chmp2b probe sets are circled. Significant (P<0.05; <i>t</i>-test) changes in 3400 probes sets, corresponding to 2200 genes were observed. Similar numbers of probe sets had significantly higher (1660) or lower (1730) signals in mG, respectively. At least 1000 and 1200 genes were genetically repressed and activated by <i>Lbx1</i>, respectively. (B) Non-cell autonomous changes in gene expression due to loss of <i>Lbx1</i> function were measured in cells that normally lack Lbx1 expression (white cells). Significant (P<0.05; <i>t</i>-test) changes in 1600 probe sets, corresponding to 930 genes were observed. Similar numbers of probe sets had significantly higher (880) or lower (690) signals in mW, respectively. At least 550 and 700 genes were genetically repressed and activated by <i>Lbx1</i>, respectively. The magnitude of cell autonomous changes (shown in A) was generally far greater than the magnitude of non-cell autonomous changes (shown in B). (C–E) Array data for two different Chmp2b probe sets (E) shown as bar graphs for green cells (C) and white cells (D).</p

Lack of Chmp2b in dI4L^B Late Population.

<p>Lmx1b and Brn 3a mark both the dI4L^B and dI5 populations of interneurons at E12.5 in mid-forelimb cross sections. These populations express Lbx1(GFP) (not shown), but lack Chmp2b. (A–C) Nuclei stained with the Brn3a antibody are located within the region stained by Chmp2b antibodies. Dorsal and ventral clusters of these nuclei correspond to the dI4L^B and dI5 populations, respectively. Brn3a stained nuclei (arrowheads) in these regions are not associated with Chmp2b staining. Other cell bodies in the immediate vicinity lack Brn3a and are stained for Chmp2b (arrows). (D) Loss of Chmp2b and severe reduction of Brn3a in dorsal horn of mutants. (E–G) Nuclei stained with the Lmx1b antibody are located within the region stained by Chmp2b antibodies. Dorsal and ventral clusters of these nuclei correspond to the dI4L^B and dI5 populations, respectively. Lmx1b stained nuclei (arrowheads) in these regions are not associated with Chmp2b staining. Other cell bodies in the immediate vicinity lack Lmx1b and are stained for Chmp2b (arrows). (H) Loss of Chmp2b and severe reduction of Lmx1b in dorsal horn of mutants. Primary antibodies against GFP (rat), Brn3a (guinea pig) or Lmx1b (guinea pig), and Chmp2b (rabbit) were detected using appropriate Cy2(green), Cy3(red), and Cy5 (infrared) secondary antibodies. The Lbx1(GFP) channel was omitted and the colors of the remaining channels switched for clarity to those indicated by the labels. (I, J) Blind quantification of cells in Brn3a/GFP/Chmp2b stained sections for heterozygote (n = 7) and mutants (n = 4), and Lmx1b/GFP/Chmp2b stained sections for heterozygote (n = 5) and mutants (n = 6). <i>VZ, ventricular zone; IZ, intermediate zone; MZ, mantle zone; SL, sulcus limitans; DLF (*), dorsolateral funiculus</i>.</p

The Genome of <i>Tolypocladium inflatum</i>: Evolution, Organization, and Expression of the Cyclosporin Biosynthetic Gene Cluster

<div><p>The ascomycete fungus <i>Tolypocladium inflatum</i>, a pathogen of beetle larvae, is best known as the producer of the immunosuppressant drug cyclosporin. The draft genome of <i>T. inflatum</i> strain NRRL 8044 (ATCC 34921), the isolate from which cyclosporin was first isolated, is presented along with comparative analyses of the biosynthesis of cyclosporin and other secondary metabolites in <i>T. inflatum</i> and related taxa. Phylogenomic analyses reveal previously undetected and complex patterns of homology between the nonribosomal peptide synthetase (NRPS) that encodes for cyclosporin synthetase (<i>simA</i>) and those of other secondary metabolites with activities against insects (e.g., beauvericin, destruxins, etc.), and demonstrate the roles of module duplication and gene fusion in diversification of NRPSs. The secondary metabolite gene cluster responsible for cyclosporin biosynthesis is described. In addition to genes necessary for cyclosporin biosynthesis, it harbors a gene for a cyclophilin, which is a member of a family of immunophilins known to bind cyclosporin. Comparative analyses support a lineage specific origin of the cyclosporin gene cluster rather than horizontal gene transfer from bacteria or other fungi. RNA-Seq transcriptome analyses in a cyclosporin-inducing medium delineate the boundaries of the cyclosporin cluster and reveal high levels of expression of the gene cluster cyclophilin. In medium containing insect hemolymph, weaker but significant upregulation of several genes within the cyclosporin cluster, including the highly expressed cyclophilin gene, was observed. <i>T. inflatum</i> also represents the first reference draft genome of Ophiocordycipitaceae, a third family of insect pathogenic fungi within the fungal order Hypocreales, and supports parallel and qualitatively distinct radiations of insect pathogens. The <i>T. inflatum</i> genome provides additional insight into the evolution and biosynthesis of cyclosporin and lays a foundation for further investigations of the role of secondary metabolite gene clusters and their metabolites in fungal biology.</p></div

Modular domain structure and A-domain specificities of NRPSs grouping within the <i>simA</i> clade.

<p>Color coding of NRPS modules denotes clade assignment of A-domains in phylogeny (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003496#pgen-1003496-g004" target="_blank">Figure 4</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003496#pgen.1003496.s003" target="_blank">Figure S3</a>): light blue = groups within cyclosporin (<i>simA</i>) clade, red = groups within enniatin (<i>esyn1</i>) module 1 clade, white = groups with <i>perA</i>-like outside both <i>simA</i> and enniatin module 1 clade (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003496#pgen.1003496.s003" target="_blank">Figure S3</a>). Abbreviations for unusual amino acid substrates: Bmt = (4<i>R</i>)-4-[(<i>E</i>)-2-butenyl]-4-methyl-threonine, Abu = Aminobutyric acid, Hiv = D-2-hydroxyvaleric acid, Hmp = D-Hmp, D-2-hydroxy-3-methylpentanoic acid.</p