Kepler-16: A Transiting Circumbinary Planet

We report the detection of a planet whose orbit surrounds a pair of low-mass stars. Data from the Kepler spacecraft reveal transits of the planet across both stars, in addition to the mutual eclipses of the stars, giving precise constraints on the absolute dimensions of all three bodies. The planet is comparable to Saturn in mass and size, and is on a nearly circular 229-day orbit around its two parent stars. The eclipsing stars are 20% and 69% as massive as the sun, and have an eccentric 41-day orbit. The motions of all three bodies are confined to within 0.5 degree of a single plane, suggesting that the planet formed within a circumbinary disk.Comment: Science, in press; for supplemental material see http://www.sciencemag.org/content/suppl/2011/09/14/333.6049.1602.DC1/1210923.Doyle.SOM.pd

Coordination of olfactory receptor choice with guidance receptor expression and function in olfactory sensory neurons.

Olfactory sensory neurons choose to express a single odorant receptor (OR) from a large gene repertoire and extend axons to reproducible, OR-specific locations within the olfactory bulb. This developmental process produces a topographically organized map of odorant experience in the brain. The axon guidance mechanisms that generate this pattern of connectivity, as well as those that coordinate OR choice and axonal guidance receptor expression, are incompletely understood. We applied the powerful approach of single-cell RNA-seq on newly born olfactory sensory neurons (OSNs) in young zebrafish larvae to address these issues. Expression profiles were generated for 56 individual Olfactory Marker Protein (OMP) positive sensory neurons by single-cell (SC) RNA-seq. We show that just as in mouse OSNs, mature zebrafish OSNs typically express a single predominant OR transcript. Our previous work suggests that OSN targeting is related to the OR clade from which a sensory neuron chooses to express its odorant receptor. We categorized each of the mature cells based on the clade of their predominantly expressed OR. Transcripts expressed at higher levels in each of three clade-related categories were identified using Penalized Linear Discriminant Analysis (PLDA). A genome-wide approach was used to identify membrane-associated proteins that are most likely to have guidance-related activity. We found that OSNs that choose to express an OR from a particular clade also express specific subsets of potential axon guidance genes and transcription factors. We validated our identification of candidate axon guidance genes for one clade of OSNs using bulk RNA-seq from a subset of transgene-labeled neurons that project to a single protoglomerulus. The differential expression patterns of selected candidate guidance genes were confirmed using fluorescent in situ hybridization. Most importantly, we observed axonal mistargeting in knockouts of three candidate axonal guidance genes identified in this analysis: nrp1a, nrp1b, and robo2. In each case, targeting errors were detected in the subset of axons that normally express these transcripts at high levels, and not in the axons that express them at low levels. Our findings demonstrate that specific, functional, axonal guidance related genes are expressed in subsets of OSNs that that can be categorized by their patterns of OR expression

Confirmation of differential gene expression using dual fluorescent <i>in situ</i> hybridization.

<p>(A) Dual-FISH of <i>nrp1a</i> with an <i>or111</i> sub-family probe cocktail. Two cells have been highlighted to show that <i>nrp1a</i> signal (red) is localized in cells with <i>or111</i> subfamily labelling (green). (B) Summary of dual-FISH expression for four candidate guidance receptors. The percentage of OSNs expressing the indicated OR subfamily that also expressed the indicated candidate guidance transcript are noted. The total number of OR subfamily expressing cells counted are shown in parentheses. Pink shading indicates transcripts that showed higher overlap with the <i>or111</i> sub-family probe-mix and green shading indicates transcripts that showed higher overlap with <i>or133</i> sub-family probe mix. <i>nrp1a</i> shows greater overlap with <i>or111</i> subfamily expression than with <i>or133</i> subfamily expression. <i>nrp1b</i>, <i>pcdh11</i>, and <i>robo2</i> show greater overlap with <i>or133</i> subfamily expression than with <i>or111</i> subfamily expression.</p

OR clade choice is related to the expression of distinct guidance gene transcripts.

<p>(A) PLDA using a ‘membrane-associated’ subset of transcripts selected using TargetP. OSNs can be discriminated into three distinct clusters along the two extracted determinants. (B, C) Distribution of the V1 and V2 loading values for 343 genes shows the top candidate plasma membrane associated or secreted transcripts (red dots) that contribute towards OSN discrimination. Top genes that have not been shaded in red localize to intracellular compartments like the endoplasmic reticulum, Golgi complex or lysosomes and hence are not likely to place a direct role in axon guidance. (D) Heatmap of normalized expression values of top 47 candidate guidance related transcripts. Red, blue, and green boxes indicate higher expression of genes in single OSNs expressing ORs from clade A, B or C, respectively. Genes at the bottom are expressed in more than one clade of OSNs and are not boxed.</p

Isolation of single OSNs and identification of mature cells expressing a single predominant OR.

<p>(A) Method of isolation of single OSNs. Transgenic zebrafish embryos expressing OMP:RFP were raised to 48hpf. Olfactory epithelia were dissected and dissociated to obtain single-cell suspensions. OMP:RFP expressing neurons were enriched using FACS and loaded onto a Fluidigm C1 microfluidics chip to isolate single cells in singe wells. cDNA synthesis and amplification were carried out directly on the chip. Samples with high yields of cDNA and smooth distributions of RNA lengths were processed though Nextera library prep and Illumina sequencing. (B) A heatmap of normalized marker gene values for 47 single OSN transcriptomes. 5 cells were enriched in markers of progenitor and/or early mature stages (shaded in black on top bar). 42 cells showed strong expression of mature OSN marker genes and low expression of progenitor/earlier mature genes. High OR expression levels (second bar:cyan) were detected in 29 of these mature cells (shaded in magenta on top bar). Three color keys are shown at the bottom: the first pertains to cell age inferred in the topmost bar, the second pertains to log10 values of normalized (predominant) OR expression, and the third relates to log10 values of normalized developmental gene expression.</p

Clade specific co-expression of selected transcription factors and guidance related transcripts.

<p>Unsupervised clustering using average linkage of the Pearson’s correlation coefficients relating expression levels of 71 guidance related genes and transcription factors. Three distinct clusters are seen and each cluster is comprised of genes that show higher expression in the same clade as compared to the others. Transcripts expressed at higher levels in clades A, B, and C are indicated using red, blue, and green circles, respectively.</p

OR transcripts detected in single OSN transcriptomes.

<p>OR transcripts detected in single OSN transcriptomes.</p

<i>Nrp1b</i> and <i>robo2</i>, but not <i>nrp1a</i>, are required for axonal targeting to the DZ.

<p><i>Tg(BACor111</i>-7:<i>IRES</i>:<i>GAL4; UAS</i>:<i>citrine</i>) (‘<i>or111-7’)</i> expressing OSNs project axons to the CZ in wild-type olfactory bulbs (A, D, G) while <i>Tg(BACor130-1</i>:<i>IRES</i>:<i>GAL4; UAS</i>:<i>citrine</i>) (‘<i>or130-1’)</i> expressing OSNs project axons to the DZ in wild type olfactory bulbs at 72 hpf (J, M, P). In <i>nrp1a-/-</i> larvae, <i>or111</i>-<i>7</i> axons misproject to the DZ (B, C). Mistargeting of <i>or111-7</i> axons is not observed in <i>nrp1b-/-</i> (E,F) or <i>robo2-/-</i> larvae. (H, I). However, in <i>nrp1b-/-</i> larvae, <i>or130-1</i> axons misproject to the CZ (N, O). Similarly, <i>robo2-/-</i> larvae show misprojections of <i>or130-1</i> axons to the MG (Q, R). Mistargeting of <i>or130-1</i> axons is not observed in <i>nrp1a-/-</i> larvae (K,L). Fisher’s exact test (two-tailed) * = p<0.05, ** = p< = 0.005. CZ: central zone, DZ: dorsal zone, MG: medial glomerulus, LG2: lateral glomerulus 2, LG3: lateral glomerulus 3. OE: olfactory epithelium, OB: olfactory bulb.</p

Confirmation of differential gene expression using dual fluorescent <i>in situ</i> hybridization.

<p>(A) Dual-FISH of <i>nrp1a</i> with an <i>or111</i> sub-family probe cocktail. Two cells have been highlighted to show that <i>nrp1a</i> signal (red) is localized in cells with <i>or111</i> subfamily labelling (green). (B) Summary of dual-FISH expression for four candidate guidance receptors. The percentage of OSNs expressing the indicated OR subfamily that also expressed the indicated candidate guidance transcript are noted. The total number of OR subfamily expressing cells counted are shown in parentheses. Pink shading indicates transcripts that showed higher overlap with the <i>or111</i> sub-family probe-mix and green shading indicates transcripts that showed higher overlap with <i>or133</i> sub-family probe mix. <i>nrp1a</i> shows greater overlap with <i>or111</i> subfamily expression than with <i>or133</i> subfamily expression. <i>nrp1b</i>, <i>pcdh11</i>, and <i>robo2</i> show greater overlap with <i>or133</i> subfamily expression than with <i>or111</i> subfamily expression.</p