Kn specifically mediates Hiw regulation of dendritic growth.

<p>(A) <i>hiw</i> and <i>kn</i> interact genetically. Shown are representative dendrites of the following genotypes: (1) <i>hiw^ΔN</i> heterozygote (<i>hiw^ΔN/+</i>); (2) <i>kn ^KN4</i> heterozygote (<i>kn^KN4/+</i>); (3) <i>hiw^ΔN</i> and <i>kn^KN4</i> trans-heterozygote (<i>hiw^ΔN/+</i>; <i>kn^KN4/+</i>). Scale bar, 50 µm. (B) Quantification of total dendrite length of denoted genotypes. <i>wt</i> samples used for statistical analysis are the same as those in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001572#pbio-1001572-g001" target="_blank">Figure 1</a>. (C and C′) Overexpressing Kn partially rescues dendritic defects in <i>hiw^ΔN</i> mutants. Representative dendrites (C) and tracings (C′) of ddaC MARCM clones of following genotypes: (1) <i>wt</i>; (2) <i>hiw^ΔN</i>; (3) overexpressing Kn with MARCM (OE Kn); (4) overexpressing Knot in <i>hiw^ΔN</i> genetic background with MARCM (<i>hiw^ΔN</i>+OE Kn). Scale bar, 50 µm. (D) Quantification of total dendrite length (left) and number of dendrite termini (right). (E) Overexpressing Kn does not alter axon terminal morphology in <i>hiw^ΔN</i> mutants. Shown are representative axon terminals of ddaC MARCM clones of the indicated genotypes. Scale bar, 10 µm. (F) Quantification of the length of axon terminals.</p

Wnd kinase inhibits dendrite growth in C1da neurons expressing ectopic Kn.

<p>(A) Wnd overexpression does not alter dendrite morphology in wild-type C1da neurons, but restrains the dendritic overgrowth caused by ectopic Kn in these neurons. Shown are representative dendrites of C1da neurons ddaD (left) and ddaE (right), labeled by <i>Gal4^2-21</i>/<i>UAS-mCD8::GFP</i>, of the following genotypes: (1) <i>wt</i>; (2) overexpressing Kn by <i>Gal4^2-21</i>(OE Kn); (3) <i>hiw^ΔN</i> homozygotes (<i>hiw</i>); (4) overexpressing Wnd by <i>Gal4^2-21</i> (OE Wnd); (5) overexpressing Kn and Wnd by <i>Gal4^2-21</i> (OE Kn+Wnd); (6) overexpressing Kn and a kinase-dead form of Wnd by <i>Gal4^2-21</i>(OE Kn+Wnd^KD). Scale bar, 50 ⋯µm. Magnified views of the boxed areas are shown on the right for each genotype. (B) Quantification of total dendrite length (left) and number of dendrite termini (right) of ddaEs of denoted genotypes. (C) Wnd kinase specifically down-regulates the expression of <i>UAS-Kn</i>, but not <i>UAS-RedStinger</i> (a nuclear red fluorescent protein) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001572#pbio.1001572-Barolo1" target="_blank">[66]</a> in a posttranscriptional manner. Representative images of ddaEs labeled with antibodies against Kn (top) and RedStinger (bottom) in “OE Kn+Wnd” and “OE Kn+Wnd^KD” using <i>Gal4^2-21</i>. Scale bar, 5 µm. (D) Quantification of immunofluorescence intensity of nuclear Kn normalized to that of RedStinger. Two different <i>Gal4</i> lines, <i>Gal4^2-21</i> (left) and <i>Gal4^21-7</i> (right), were tested in this experiment.</p

Bimodal Control of Dendritic and Axonal Growth by the Dual Leucine Zipper Kinase Pathway

<div><p>Knowledge of the molecular and genetic mechanisms underlying the separation of dendritic and axonal compartments is not only crucial for understanding the assembly of neural circuits, but also for developing strategies to correct defective dendrites or axons in diseases with subcellular precision. Previous studies have uncovered regulators dedicated to either dendritic or axonal growth. Here we investigate a novel regulatory mechanism that differentially directs dendritic and axonal growth within the same neuron in vivo. We find that the dual leucine zipper kinase (DLK) signaling pathway in <i>Drosophila</i>, which consists of Highwire and Wallenda and controls axonal growth, regeneration, and degeneration, is also involved in dendritic growth in vivo. Highwire, an evolutionarily conserved E3 ubiquitin ligase, restrains axonal growth but acts as a positive regulator for dendritic growth in class IV dendritic arborization neurons in the larva. While both the axonal and dendritic functions of <i>highwire</i> require the DLK kinase Wallenda, these two functions diverge through two downstream transcription factors, Fos and Knot, which mediate the axonal and dendritic regulation, respectively. This study not only reveals a previously unknown function of the conserved DLK pathway in controlling dendrite development, but also provides a novel paradigm for understanding how neuronal compartmentalization and the diversity of neuronal morphology are achieved.</p></div

Wnd mediates the functions of Hiw on dendritic growth.

<p>(A) Loss of <i>wnd</i> blocks dendrite reduction in <i>hiw</i> mutants, and ectopic Wnd restrains dendritic growth. Shown are representative dendrites of ddaC neurons, labeled by <i>ppk-CD4::tdTomato</i>, of the following genotypes: (1) <i>wt</i>; (2) <i>hiw^ΔN</i> homozygotes (<i>hiw</i>); (3) <i>wnd¹/wnd³</i>(<i>wnd</i>); (4) <i>hiw^ΔN</i>; <i>wnd¹/wnd³</i> double mutants (<i>hiw</i>; <i>wnd</i>); (5) overexpressing Wnd by <i>ppkGal4</i> (OE Wnd); (6) overexpressing a kinase dead form (K188A) of Wnd by <i>ppkGal4</i> (OE Wnd^KD). Scale bar, 50 µm. (B) Bar charts showing the quantification of total dendrite length (left) and number of dendrite termini (right). Samples of <i>wt</i> and <i>hiw^ΔN</i> that are used for statistical analysis are the same as those in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001572#pbio-1001572-g001" target="_blank">Figure 1</a>.</p

Regulatory mechanisms underlying dendritic and axonal growth.

<p>(A) Three distinct mechanisms regulating dendritic and axonal growth. Shared mechanisms control dendrite and axon co-growth. Dedicated mechanisms direct compartment-specific growth. Bimodal mechanisms differentially regulate dendritic and axonal growth. (B) A model that postulates the differential control of dendritic and axonal growth by the DLK pathway, which is based on the present study. In this model, DLK plays a dual role in neuron morphogenesis. Up-regulated DLK, caused either by <i>PHR</i> mutations or DLK overactivation, promotes the growth of axon terminals but restricts that of high-order dendritic branches. Such a dichotomous function is the result of signaling divergence into two transcriptional programs that are each dedicated to either dendritic or axonal growth. Fos serves a permissive role in the axonal regulation by DLK, whereas Kn specifically mediates the dendritic regulation by DLK.</p

Transcription factor Fos specifically mediates axonal overgrowth induced by Wnd.

<p>(A) Loss of the <i>Drosophila fos</i>, <i>kay</i>, blocks axonal overgrowth caused by Wnd overexpression. Shown are representative axon terminals of ddaC MARCM clones of following genotypes: (1) <i>wt</i>; (2) overexpressing Wnd with MARCM (OE Wnd); (3) <i>kay¹</i>; (4) overexpressing Wnd in <i>kay¹</i> genetic background with MARCM (OE Wnd+<i>kay¹</i>). Scale bar, 10 µm. (B–B′) <i>kay¹</i> impairs dendritic growth in <i>wt</i> genetic background and exacerbates the dendritic reduction caused by Wnd overexpression. Shown are representative dendrites (B) and tracings (B′) of ddaC MARCM clones of indicated genotypes. Scale bar, 50 µm. (C) Bar charts showing the quantification of axon terminal length (left), total dendrite length (middle), and number of dendrite termini (right).</p

Hiw differentially regulates dendrite and axon growth in C4da neurons.

<p>(A) Dendrites of the C4da neuron ddaC in <i>hiw^ΔN</i> homozygous mutant larvae are reduced, as compared to <i>wild-type</i> (<i>wt</i>). C4da neurons were labeled by the C4da marker <i>ppk-CD4::tdTomato</i>. Scale bar, 100 µm. (B) Bar charts showing the quantification of total dendrite length (top), number of dendrite termini (bottom) of ddaC in <i>wt</i>, <i>hiw^ΔN</i>, and <i>hiw^ND8</i> larvae. Sample numbers are shown in the bars of the bar charts throughout this article. (C–D) <i>hiw</i> mutant MARCM clones exhibit impaired dendritic growth and overgrowth of axon terminals. (C) Representative dendrites of <i>wt</i> and <i>hiw^ΔN</i> mutant ddaC neurons. Scale bar, 50 µm. (D) Representative axon terminals of a single <i>wt</i> ddaC and a single <i>hiw^ΔN</i> mutant ddaC. The axon terminals of wild-type ddaC clones (green) extend within one segment length of the C4da neuropil (magenta) labeled by <i>ppk-CD4::tdTomato</i>. The axon terminals of <i>hiw^ΔN</i> mutant clones (green) expand over multiple segment lengths of the C4da neuropil (magenta). Scale bar, 10 µm. (E) Quantification of total dendrite length (left) and number of dendrite termini (right) of <i>wt</i> and <i>hiw^ΔN</i> MARCM clones. (F) Quantification of axon terminal length of <i>wt</i> and <i>hiw^ΔN</i> MARCM clones.</p

Clinical Characteristics of Study Subjects according to Smoking Status.

<p>Data are presented as means ± SD.</p>*<p>P value <0.05,</p>†<p>P value<0.001.</p

Adjusted Odds Ratio of Visceral Obesity and Others according to the Smoking Amounts during a Lifetime.

<p>BMI = Body Mass Index, BF% = Body Fat Percentage, WC = Waist Circumference.</p

Comparing Mean Values of Anthropometric Measures with Smoking Amounts during a Lifetime.

<p>BMI = Body Mass Index, BF% = Body Fat Percentage, WC = Waist circumference, SCF = Abdominal Subcutaneous Fat.</p><p>Means in the same row with different superscript letters are significantly different, P<0.05 (ANOVA with Duncan’s post hoc test).</p