12A variability is sensitive to genetic background and temperature.

<p>(<b>A-B</b>) Proportions of unsplit hemilineages and ectopic branching in various genotypes at 25°C. (<b>A</b>) T/CS proportions were significantly different from all other groups (p < 0.05), but only the comparison with T/OR is shown. (<b>B</b>) T/OR had the largest proportion of ectopic branches, followed by T/CS. (<b>C</b>) Proportion of hemilineages with the typical morphology. (<b>D-E</b>) Effect of temperature on bundle splitting and ectopic branching. Data at 25°C are from 7A and 7B. p values are for comparisons between 16°C and 29°C for each genotype. (<b>F</b>) Effect of temperature on typical morphology. For all panels, vertical error bars represent 95% confidence intervals. Chi-squared tests were used to obtain p values and 95% CI. Holm correction for multiple comparisons (21 pairwise comparisons) was applied to p values in Fig 7A and 7B. T/CS: progeny of tester strains crossed to Canton S; T/OR: tester/Oregon R; T/yw: tester/<i>yw</i>; HI: Recently derived strain from Hawaii; CT: Recently derived strain from Connecticut.</p

12A in T2 is a variability “hot-spot.”

<p>(<b>A</b>) Two examples of transverse projections through the T2 neuromere showing the neurite bundles of hemilineages 11A and 11B (magenta) relative to those of 12A (green). The right 12A hemilineages failed to split but the lineage 11 projections were invariant. Lineages from the genotype: <i>w; R26B05-LexA(attP40)/LexAop2-myr</i>::<i>tdTomato-P10; R24B02-GAL4(attP2)/UAS-myr</i>::<i>GFP-P10</i>. (<b>B</b>) Single examples of S3, T1, and T2, showing the effects of expressing UAS-Ubx.Ia. The immature neurite bundles in S3 are consistently transformed toward the unsplit T2 morphology. The 12A neurons in T1 become T2-like with respect to variability in the failure to split and in forming ectopic branches. Percentages shown are for the fraction of hemilineages that split into a dorsal and intermediate bundle and the fraction of split hemilineages that have an ectopic branch. (<b>C</b>) Quantification of anti-Ubx staining for four individuals that showed bilateral asymmetry. A difference in staining intensity was only observed in one of the four individuals. (<b>D</b>) An image stack from one of the four individuals (Sample1), with GFP in green and anti-Ubx in magenta. (<b>E</b>) A single optical section of the sample in D (showing area in the dashed box). Note the Ubx expression in T2 and lack of expression in T1. (<b>F</b>) When Ubx was expressed in 12A neurons in T1 using R24B02-GAL4 with UAS-Ubx.1a, Ubx was detectable (arrows) in the T1 neurons but still very low when compared to the endogenous levels in T2.</p

Developmental variability in hemilineage 12A.

<p>(<b>A</b>) Transverse optical sections showing examples of variation in T2 and A1. Note in T2 the unsplit bundle of the hemilineage on the right and the ectopic branch on the left. (<b>B-C</b>) Two examples of T2 at 18h APF. Upper panels are transverse optical sections, lower panels are partial z-projections corresponding to the depth indicated by black bars in the upper panels. Arrowheads show the location of ectopic branches in C and the corresponding location in B. (<b>D</b>) Transverse optical sections of two examples of T2 at 72h APF. The upper panel shows an animal with typical branch morphology, whereas the bottom panel shows an unsplit bundle in the right hemisegment. (<b>E</b>) comparison between number of cells in split hemilineages (n = 44 hemilineages) and unsplit hemilineages (n = 36 hemilineages) in late wandering third instar larvae. We did not observe a significant difference in cell number (p = 0.36 Welch’s t-test), indicating the difference between the split and unsplit phenotype is not due to differences in neuronal proliferation or death.</p

Other examples of 12A variants.

<p>(<b>A</b>) An example of the ectopic branch phenotype in T1 (compare with the typical T1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g002" target="_blank">Fig 2B</a> and the ectopic branch phenotype of T2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g003" target="_blank">Fig 3A</a>). All examples were bilaterally symmetrical with a fully formed commissure (arrowhead), in contrast to 12A in T2, which often exhibits bilateral asymmetry. (<b>B</b>) Example of ventral arch routing of the late-born 12A neurons in T2. Compare the branch on the left (arrowhead) with the unoccupied Neuroglian-stained tract on the right (arrow). (<b>C</b>) Transverse optical section of S3 in which 12A shows the T2 morphology (with unsplit bundles). Compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g006" target="_blank">Fig 6B</a> and the variants of 12A that are missing the intermediate bundle as shown in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g003" target="_blank">3A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g006" target="_blank">6A</a>. (<b>D</b>) Transverse optical section of T1 in which the left bundle fails to split. (<b>E</b>) Two examples of animals with duplicated hemilineages. (<b>F</b>) One of two cases in which one hemilineage ectopically projected all of its neurites to the contralateral side, leaving one T1 hemisegment completely uninnervated by 12A and the other double innervated.</p

Correlation between ectopic branch phenotype and delayed flight initiation.

<p>(<b>A</b>) Plot of the time it takes for an individual fly to spontaneously initiate flight after being released onto a small pedestal. The kernel density estimate for each condition was calculated in R using the base density function with the default bandwidth; this is shown as a gray violin plot behind the data points. The spread of data in the T/OR and T/CS groups is much broader than in the CT and HI groups, and bimodality is suggested in the CT (29°C), T/OR (16°C), and T/CS groups. (<b>B</b>) Combined data from 8A. Using the kernel density estimate of the combined data, a cutoff was established at the local minimum between the two modes: 19.1 s. This cutoff was then applied to the data in 8A to sort individual flies into early or late groups. (<b>C</b>) Pearson correlation comparing the proportion of flies taking off in the late mode to the expected proportion of flies that should harbor the ectopic branch phenotype in either 12A hemilineage in T2. Gray diagonal line indicates the expected 1:1 correlation. (<b>D</b>) Pearson correlation comparing the proportion of flies taking off in the late mode to the expected proportion of flies that should harbor the unsplit phenotype (<b>E</b>) Ability of flies to recover from free fall. Each point represents the height at which a falling fly hits the side of the test cylinder. Flies that fly poorly (CyO) mostly reach the bottom of the cylinder. Note that the distributions of T/OR (~50% of animals have ectopic branch phenotype) as compared to HI (~8% of animals have ectopic branch phenotype) are not suggestive of a mixed population as with spontaneous flight initation in 8A. Strains as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g007" target="_blank">Fig 7</a>.</p

Genetic and Environmental Control of Neurodevelopmental Robustness in <i>Drosophila</i>

<div><p>Interindividual differences in neuronal wiring may contribute to behavioral individuality and affect susceptibility to neurological disorders. To investigate the causes and potential consequences of wiring variation in <i>Drosophila melanogaster</i>, we focused on a hemilineage of ventral nerve cord interneurons that exhibits morphological variability. We find that late-born subclasses of the 12A hemilineage are highly sensitive to genetic and environmental variation. Neurons in the second thoracic segment are particularly variable with regard to two developmental decisions, whereas its segmental homologs are more robust. This variability “hotspot” depends on Ultrabithorax expression in the 12A neurons, indicating variability is cell-intrinsic and under genetic control. 12A development is more variable and sensitive to temperature in long-established laboratory strains than in strains recently derived from the wild. Strains with a high frequency of one of the 12A variants also showed a high frequency of animals with delayed spontaneous flight initiation, whereas other wing-related behaviors did not show such a correlation and were thus not overtly affected by 12A variation. These results show that neurodevelopmental robustness is variable and under genetic control in Drosophila and suggest that the fly may serve as a model for identifying conserved gene pathways that stabilize wiring in stressful developmental environments. Moreover, some neuronal lineages are variation hotspots and thus may be more amenable to evolutionary change.</p></div

Summary of neuroblast clones in T2 examined at 24 hours APF.

<p>Summary of neuroblast clones in T2 examined at 24 hours APF.</p

Single-neuron clones reveal variable development of late-born 12A neurons in T2.

<p>(<b>A</b>) Proportions of single-neuron clones taking either a dorsal or intermediate path in segments T1 or T2. Each bar shows the proportion of clones recovered from heat shocks at the time point on the x-axis. Numbers of clones are indicated above each bar. (<b>B</b>) Transverse optical section of T1 showing a twin-spot MARCM clone at 0 hours APF. Green and magenta show a single-neuron and neuroblast clone from the same neuroblast. The magenta neurons are all born after the green neuron. Neuroglian-labeled tracts are shown in grayscale. (<b>C-H</b>) Single-neuron clones of 12A in T2 at 24h APF. Transverse optical sections are shown on the left, followed by a z-projection of a dorsal substack, then a tracing of the dorsal view. In tracings, green indicates the clone and gray indicates landmarks as seen by anti-Neuroglian staining (magenta). Green asterisks indicate the point most proximal to the cell soma at which the neurite enters the substack. Dashed boxes indicate the core region of the mesothoracic triangle. (<b>C</b>) Example of a 12Ama clone induced at 72h AEL. (<b>D</b>) Example of a 12Amb clone, induced at 72h AEL. Arrow points to arbor originating from a T1 clone that is not part of the traced clone. (<b>E</b>) Example of a 12Amc clone, induced at 72h AEL. Arrows point to unrepressed signal that is not part of the clone. <b>(F</b>) Three examples of 12Ala clones. Note that the top clone routes along the dorsal path while the other two route along the intermediate path. While all three clones produce contralateral projections at the level of the intermediate commissure, only the bottom clone does so at the location of the ectopic branch of the neurite bundle (arrowheads). The top two clones produce this branch at a more anterior location (long arrows). (<b>G</b>) Two examples of 12Alb clones, induced at 96h AEL. Note the upper clone takes the dorsal path, whereas the lower example takes the intermediate path but otherwise looks identical. Arrows point to unrepressed signal from the right hemilineage and is not part of the clone. (<b>H</b>) Four examples of 12Alc clones induced at either 96h or 108h AEL. Projections into the T2 spur (sp) are indicated with brackets. The top two clones take either the intermediate (top) or dorsal (second from the top) path to an intermediate target, after which they look nearly identical and remain ipsilateral. In the third clone, the longitudinal projection jumps across the midline and continues projecting anteriorly. The bottom example shows two 12Alc clones, one on each side. The clone on the right sends a branch across the midline at the same point as the ectopic commissure (arrowheads); compare this example with the full 18h APF patterns in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g003" target="_blank">Fig 3C</a> and the 0h APF example in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g003" target="_blank">Fig 3A</a>.</p

Neuroblast clones further demonstrate 12A variability.

<p>(<b>A</b>) Full 24B02-GAL4 pattern in T2 at 24h APF. A transverse optical section is shown on the left, with two dorsal views of partial z-projections on the right, representing two levels along the D-V axis (indicated by black bars). The core region is indicated by dashed boxes and the T2 spur (Sp) is indicated with a bracket. (<b>B-C</b>) Two neuroblast clones induced at 72h AEL and examined at 24h APF. Note the presence of the contralateral longitudinal projections in C (arrow), but not B. <b>(D-F)</b> Schematic of the hemilineage 12A neurons in T2 at 24 hours APF. Neurons are colored according to the key at the bottom of the Fig, with landmarks (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g002" target="_blank">Fig 2B</a>) shown in gray. Transverse views are shown on the left, dorsal views on the right. Some features of the neurons are slightly shifted or exaggerated as a visual aid. Only one neuron of each class is shown, but each hemilineage contains multiple neurons of each neuron class. This schematic does not capture all possible neuronal classes produced by this hemilineage, only those that we recovered this study. <b>(D)</b> Typical development of 12A in T2. <b>(E)</b> Atypical unsplit 12A phenotype in which the late-born neurons all take the dorsal path (arrow), although they still reach the appropriate target zone. <b>(F)</b> Atypical ectopic branch 12A phenotype in which 12Ala and 12Alc neurons produce branches that split off from the intermediate bundle (arrowhead). 12Alc can also ectopically cross the midline at more anterior locations, but these variants can only be seen with clones and were not quantified in multiple genotypes/temperatures. (<b>G-J</b>) Neuroblast clones induced at 96h AEL and examined at 24h APF. z-projections of the full dorsal neuropil are shown. The clones contain only 12Alc neurons. Clones vary with respect to neurons taking either the dorsal (G) or intermediate route (H and I) or both (J). They also vary with respect to contralateral projections (arrows). (<b>I</b>) Note the ectopic contralateral projections run long the ectopic branch route (arrowhead). Compare this clone with Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g003" target="_blank">3A, 3C</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g004" target="_blank">4H</a>.</p

Expected and observed frequencies of bilateral asymmetry.

<p>Expected and observed frequencies of bilateral asymmetry.</p