BMP signaling is required cell autonomously in NCPCs for their specification.

<p>(A) Z-projection of confocal sections showing that wild-type donor cells transplanted into wild-type hosts readily express the neural crest marker Foxd3 at the 3-somite stage. (B,C,D) Single confocal section of the embryo in A. Foxd3 (B) and lineage tracer rhodamine dextran (C) are found in the same cells (D, arrowheads). (E) Z-projection of confocal sections showing that <i>smad5</i> morphant donor cells within the neural crest region of a wild-type host do not express the neural crest marker Foxd3. (F, G, H, I, J, K) Single confocal sections of the cells indicated by arrows in E. Foxd3 (F, I) and lineage tracer (G, J) do not colocalize (H, K). (L, M, N) Single confocal section of a different host embryo containing <i>smad5</i> morphant cells (Rho) within the neural crest region that do not express Foxd3. (O, P, Q) Z-projection of confocal sections of chimera in which donor cells were induced as ectopic neural crest. Foxd3 (O) and lineage tracer (P) colocalize (Q) in a patch of cells (arrowhead) located ventrally from the normal neural crest domain (asterisk).</p

Reduction of BMP signaling in wild-type embryos causes expansion or loss of NCPC in dosage-sensitive manner.

<p><i>foxd3</i> expression in <i>chd</i> mRNA injected embryos (A) and <i>smad5</i> MO injected embryos (B) at the end of gastrulation. (A) Injection of a low dose of <i>chordin</i> mRNA (50 pg) generates weaker NCPC phenotypes (WT = normal or very mild expansion, “<i>snh</i>”  =  moderate expansion), whereas a high dose (200 pg) leads to strong phenotypes (“<i>sbn</i>” = large expansion, or “<i>swr</i>”  = loss). (B) Injection of a low 2.5 ng dose of <i>smad5</i> MO leads to “<i>snh</i>” and “<i>sbn</i>” phenotypes. Injection of a high 4 ng dose of <i>smad5</i> MO leads to “<i>sbn</i>” and “<i>swr</i>” phenotypes exclusively.</p

Levels of P-Smad1/5 correlate with the strength of NCPC phenotype in <i>smad5</i> morphant embryos.

<p>(A) P-Smad1/5 levels in <i>smad5</i> morphant embryos. Embryos injected with increasing higher concentrations of <i>smad5</i> MOs show decreasing P-Smad1/5 levels relative to uninjected controls. Actin was used as a loading control. (B, C) Expression of <i>foxd3</i> in embryos injected with increasing doses of <i>smad5</i> MOs. Injection of a low dose of <i>smad5</i> MOs (3 ng) leads to WT, “<i>snh</i>” and “<i>sbn</i>” phenotypes, whereas injection of higher doses of <i>smad5</i> MOs (5 ng, 6 ng) lead to “<i>sbn</i>” and “<i>swr</i>” phenotypes. The embryos used for in situ hybridization of <i>foxd3</i> in Fig. 4B are from the same batch of injected embryos used for Western blotting in Fig. 4A.</p

NCPC domains when BMP signaling is reduced in <i>somitabun</i> and <i>snailhouse</i> and increased in <i>swirl</i> embryos.

<p><i>foxd3</i> expression at the end of gastrulation in <i>tBR</i> mRNA injected embryos of <i>sbn</i> (A) and of <i>snh</i> (B), and <i>smad5</i> mRNA injected into <i>swr</i> embryos (C). (A) Injection of <i>tBR</i> mRNA into <i>sbn</i> mutants leads to the majority of embryos displaying a “<i>swr</i>” phenotype. (B) Injection of a low 15 pg dose of <i>tBR</i> mRNA into <i>snh</i> mutants leads to nearly equal numbers of “<i>snh</i>” and “<i>sbn</i>” phenotypes. Injection of a higher 100 pg dose leads to the majority of embryos displaying the “<i>sbn</i>” phenotype and also a percentage displaying a stronger “<i>swr</i>” phenotype. (C) Injection of a low 30 pg dose of murine <i>smad5</i> mRNA results in nearly half of embryos displaying a “<i>sbn</i>” phenotype. Injection of a higher 150 pg dose results in a small percentage of embryos displaying the “<i>swr</i>” phenotype, and the rest of the embryos divided between “<i>sbn</i>”, “<i>snh</i>”, and WT phenotypes.</p

BMP gradient model for NCPC specification.

<p>The Y axis shows BMP signaling levels. The X axis indicates position along the dorsoventral axis. The threshold range of BMP signaling that specifies NCPC is shown in yellow. The intersection of the gradient with the threshold range for NCPC specification leads to NCPC formation in a lateral region in the size domain shown. In WT, the gradient of BMP signaling reaches a high level ventrally and NCPCs are located in a lateral region of the embryo where BMP signaling levels are low. The region of NCPCs specified in WT is shown with black stripes over the yellow area. In <i>snh</i>, the BMP signaling gradient is lower than WT. Therefore, the NCPCs in <i>snh</i> (blue striped area) are slightly expanded compared to wild type and are located in a more ventral region than WT. In <i>sbn</i>, the BMP signaling gradient is lower than <i>snh</i>. The NCPCs are located in a more ventral region than <i>snh</i> and the NCPCs in <i>sbn</i> (red striped area) are greatly expanded compared to wild type. In the <i>swirl/bmp2b</i> mutant, BMP signaling level is absent or very low, leading to the great reduction or absence of NCPCs.</p

SLO BK Potassium Channels Couple Gap Junctions to Inhibition of Calcium Signaling in Olfactory Neuron Diversification

<div><p>The <i>C</i>. <i>elegans</i> AWC olfactory neuron pair communicates to specify asymmetric subtypes AWC^OFF and AWC^ON in a stochastic manner. Intercellular communication between AWC and other neurons in a transient NSY-5 gap junction network antagonizes voltage-activated calcium channels, UNC-2 (CaV2) and EGL-19 (CaV1), in the AWC^ON cell, but how calcium signaling is downregulated by NSY-5 is only partly understood. Here, we show that voltage- and calcium-activated SLO BK potassium channels mediate gap junction signaling to inhibit calcium pathways for asymmetric AWC differentiation. Activation of vertebrate SLO-1 channels causes transient membrane hyperpolarization, which makes it an important negative feedback system for calcium entry through voltage-activated calcium channels. Consistent with the physiological roles of SLO-1, our genetic results suggest that <i>slo-1</i> BK channels act downstream of NSY-5 gap junctions to inhibit calcium channel-mediated signaling in the specification of AWC^ON. We also show for the first time that <i>slo-2</i> BK channels are important for AWC asymmetry and act redundantly with <i>slo-1</i> to inhibit calcium signaling. In addition, <i>nsy-5</i>-dependent asymmetric expression of <i>slo-1</i> and <i>slo-2</i> in the AWC^ON neuron is necessary and sufficient for AWC asymmetry. SLO-1 and SLO-2 localize close to UNC-2 and EGL-19 in AWC, suggesting a role of possible functional coupling between SLO BK channels and voltage-activated calcium channels in AWC asymmetry. Furthermore, <i>slo-1</i> and <i>slo-2</i> regulate the localization of synaptic markers, UNC-2 and RAB-3, in AWC neurons to control AWC asymmetry. We also identify the requirement of <i>bkip-1</i>, which encodes a previously identified auxiliary subunit of SLO-1, for <i>slo-1</i> and <i>slo-2</i> function in AWC asymmetry. Together, these results provide an unprecedented molecular link between gap junctions and calcium pathways for terminal differentiation of olfactory neurons.</p></div

<i>slo-1</i> and <i>slo-2</i> regulate the subcellular localization of synaptic markers in AWC neurons.

<p><b>(A</b>) Left panels: Images of wild type, <i>slo-1(ky399gf)</i>, and <i>slo-1(eg142lf); slo-1(ok2214lf)</i> mutants expressing the single copy insertion transgene <i>odr-3p</i>::<i>GFP</i>::<i>unc-2</i> (the same transgene as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005654#pgen.1005654.g005" target="_blank">Fig 5A and 5B</a>) in AWC cell bodies (arrows) and axons (arrowheads) in L1. Right panel: Quantification of GFP::UNC-2 fluorescence intensity in AWC axons and cell bodies. <i>slo-1(eg142lf); slo-1(ok2214lf)</i> mutants displayed a significant decrease in GFP::UNC-2 intensity in AWC axons and cell bodies. (<b>B</b>) Left panels: Images of wild-type, <i>slo-1(ky399gf)</i>, and <i>slo-1(eg142lf); slo-1(ok2214lf)</i> mutants expressing the single copy insertion transgene <i>odr-3p</i>::<i>YFP</i>::<i>rab-3</i> in AWC cell bodies (arrows) and axons (arrowheads) in L1. Right panel: Quantification of YFP::RAB-3 fluorescence intensity in AWC axons and cell bodies. <i>slo-1(eg142lf); slo-1(ok2214lf)</i> mutants had a significant decrease in YFP::RAB-3 intensity in AWC axons and cell bodies. (<b>A</b>, <b>B</b>) Anterior is at left and ventral is at bottom. Scale bar, 5 μm. Student’s <i>t-</i>test was used for statistical analysis. ns, not significant. Error bars, standard error of the mean. AU, arbitrary unit.</p

<i>slo-1</i> and <i>slo-2</i> act downstream of <i>nsy-5</i> to antagonize the function of voltage-gated calcium channel-activated kinase cascade in promoting AWC^ON.

<p>(<b>A</b>) Double and triple mutant analysis of <i>slo-1(ky389gf)</i>, <i>slo-1(ky399gf)</i>, and <i>slo-1(eg142lf); slo-1(ok2214lf)</i> animals with mutants of known genes involved in establishment of AWC asymmetry. 2AWC^ON, both AWC cells express <i>str-2</i>; 1AWC^OFF/AWC^ON, only one of the two AWC cells expresses <i>str-2</i>; 2AWC^OFF, neither AWC cell expresses <i>str-2</i>. (<b>B</b>) The genetic pathway that demonstrates possible relationships between <i>slo-1</i>, <i>slo-2</i> and other genes required for AWC asymmetry. Genes in green represent AWC^OFF promoting, genes in red represent AWC^ON promoting, and those in grey represent less active or inactive genes.</p

Model of <i>slo-1</i> and <i>slo-2</i> function in AWC asymmetry.

<p>AWC asymmetry is stochastic, and this figure illustrates the case when AWC^ON is on the left side of the head. Molecules in green represent AWC^OFF promoting, molecules in red represent AWC^ON promoting, and those in grey represent less active or inactive molecules. In the AWC^OFF neuron (right), calcium enters the cell through voltage-gated calcium channels (UNC-2/UNC-36 and EGL-19/UNC-36) and stimulates a MAP kinase cascade consisting of UNC-43 (CaMKII), TIR-1 (Sarm1) adaptor protein, and NSY-1 (MAPKKK). This leads to expression of the AWC^OFF marker <i>srsx-3</i> and suppression of the AWC^ON marker <i>str-2</i>. In the AWC^ON cell (left), NSY-5 gap junctions activate SLO-1 and SLO-2 voltage- and calcium-activated potassium channels, which antagonize the function of UNC-2/UNC-36 and EGL-19/UNC-36 calcium channels by suppressing the calcium-activated CaMKII-MAP kinase cascade. NSY-4 (claudin) acts in parallel with NSY-5, SLO-1, and SLO-2 to inhibit calcium channel-mediated signaling, resulting in de-repression of <i>str-2</i> expression.</p

SLO-1 and SLO-2 BK potassium channels are localized in the vicinity of UNC-2 voltage-gated calcium channels in AWC axons.

<p>(<b>A-C</b>) Images of wild-type L1 animals expressing single copy insertion transgenes <i>odr-3p</i>::<i>slo-1</i>::<i>TagRFP</i> and <i>odr-3p</i>::<i>GFP</i>::<i>unc-2</i> (A), <i>odr-3p</i>::<i>slo-2</i>::<i>TagRFP</i> and <i>odr-3p</i>::<i>GFP</i>::<i>unc-2</i> (B), as well as <i>odr-3p</i>::<i>slo-2</i>::<i>TagRFP</i> and <i>odr-3p</i>::<i>slo-1</i>::<i>GFP</i> (C) in AWC neurons. SLO-1::TagRFP (A), SLO-1::GFP (C), SLO-2::TagRFP (B, C), and GFP::UNC-2 (A, B) were localized in AWC cell bodies (arrows) and in a punctate pattern along AWC axons (arrowheads). In AWC axons, SLO-1::TagRFP was localized next to GFP::UNC-2 (A); SLO-2::TagRFP was adjacent to GFP::UNC-2 (B); and SLO-2::TagRFP was localized near SLO-1::GFP (C). Insets show higher magnification of the outlined areas that exemplify localization of two translational reporters in close proximity. Scale bar, 5 μm. Anterior is at left and ventral is at bottom. (D) Quantification of mean correlation coefficient between SLO-1 and UNC-2, SLO-2 and UNC-2, as well as SLO-1 and SLO-2 using three algorithms of the Coloc 2 plugin in Fiji: Pearson’s correlation coefficient, Spearman’s rank correlation coefficient, and Li’s ICQ. For each colocalization class, images of three animals were used for quantification. Positive values of each coefficient indicate positive correlation, values close to zero indicate no correlation, and negative values indicate anti-correlation. Pearson's correlation coefficient ranges from -1 to +1; Spearman’s rank correlation coefficient ranges from -1 to +1; Li's ICQ value ranges from -0.5 to +0.5. A schematic diagram of the AWC cell body, axon, dendrite, and cilia that represents the approximate region of images in A-C is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005654#pgen.1005654.s002" target="_blank">S2D Fig</a>.</p