Gross morphological deformations

Water accumulated fractions (WAF) of the Macondo crude oil were vortex mixed in embryo medium at a 1:10 dilution in accordance with conventional methods [40]. Analysis of different mixing procedures and serial dilution experiments were performed and are described in Additional files 1 and 2. Based on our dose response analysis we conducted all embryo treatments at the 100% concentration of the vortex-mixed, 1:10 WAF stock solution, which will from here on be referred to as WAF. Only during our analysis of locomotor behavior was 50% WAF solution used. These WAF preparations serve to model the portions of the Macondo crude oil sampled from the riser insertion tube during the Deepwater Horizon oil spill that are capable of dissolving in embryo medium. It is important to acknowledge that according to communications with BP during the period of 2 and 3 May 2010 when this sample was being collected Nalco EC9323A defoamer was being applied topside and methanol with VX9831 oxygen scavenger/catalysts solution injected subsea. While we cannot rule out the possibility that these additional agents in the vicinity of the spill site could be present in this sample, it is highly unlikely due to the method of direct sampling through the riser tube.

Embryos exposed to WAF starting at 3.5 hours post-fertilization (hpf) until a maximum of 5 days post-fertilization (dpf) caused an array of morphological phenotypes that included dorsal tail curvature, cardiac edema, cyst formation, reduced head structures and brain hemorrhages (Figure 1). More specifically, WAF-treated embryos showed moderate (Figure 1F) to severe cardiac edema (Figure 1C-E, arrows) that, in some cases, also included yolk sac edema (Figure 1C, right most arrow). In addition, a dorsal curling of the tail and caudal cyst formation were variably observed within a treated clutch of embryos but were consistently present across treatments (Figure 1E, F, double arrows; Figure 1C, D, F, arrowhead). Qualitatively, the overall size of the brain and eyes were often reduced, which was most apparent in the ventral jaw structures (Figure 1G, H, brackets). Equally obvious was the average 28% of embryos with brain hemorrhages present in the forebrain, midbrain, or hindbrain regions (Control 0.0%, n = 452; WAF, n = 441; Figure 1H-J, arrowheads). While the eyes of some WAF-treated embryos were noticeably smaller, the mean measurements of the area and perimeter of the retina and lens showed only subtle, but statistically significant, reductions in size (Figure 1K-M). These varied, but relatively specific, morphological deformations suggest that certain embryonic processes may be affected by exposure to Macondo crude oil.

Figure 1 Exposure to Macondo crude oil-derived WAFs induced diverse gross morphological deformations in zebrafish embryos. (A-F) Lateral and ventral views of live untreated control (A, B) and WAF-treated embryos (C-F) at 5 dpf. Severe cardiac and yolk edema (C, D, E, arrows), dorsal tail curvature (E, F, double arrows), and cysts at the tip of the tail (C, D, F, arrowhead) were visible. (G, H) WAF-treated embryos (H) had reduced jaws compared to controls (G, brackets). (G-J) At 3 dpf cardiac edema was evident in WAF-treated embryos (arrows), and 28% of embryos had hemorrhaging in the forebrain, midbrain and hindbrain (arrowheads). Lateral (G, H, I, J) and dorsal views (H', I', J'). (K-M) Retinal architecture appeared normal in control and WAF-treated embryos (K, L) but there was a slight reduction in the area and perimeter of WAF-treated retinas (M). Except for lens area, the size reductions were statistically significant (M, asterisks; t-tests: lens area, P = 0.015; lens perimeter, P = 0.007; retina area P < 0.0005; retina perimeter, P < 0.0005). Scale bars: 200 μm, F, J'; 50 μm, L. Abbreviations: gcl, ganglion cell layer; ipl, inner plexiform layer; inl, inner nuclear layer; opl, outer plexiform layer; onl. outer nuclear layer. Full size image

Chemical analysis of water accumulated fraction

To determine what components of the crude oil were released into the WAF mixture and thus exposed to the embryos during experimental treatments, WAF samples were analyzed within one hour of being made using solid phase microextraction (SPME, 100 mm polydimethylsiloxane) and gas chromatography mass spectrometry (GCMS, Agilent 7890A GC/5975C MSD). Chemical analysis confirmed the presence of n-hexane, toluene, xylene, benzene, naphthalene and ethylbenzene that were reported by BP to be present in this source oil B. This analysis also revealed a variety of additional components, of which the most prominent were aromatics and alkanes (Figure 2A), as observed in chemical analyses of Gulf of Mexico waters after the Deepwater Horizon blowout [41–43]. Aromatic concentrations were similar over the four WAF solutions sampled (Figure 2A). We interpret the large variation in heavier alkane concentrations (Figure 2A) to the inclusion of a non-aqueous phase, into which these poorly soluble components fractionate. Such partitioning of these less-soluble components into deep-plume oil droplets [42] and into the surface slick [43] was observed in the Gulf of Mexico after the BP Oil Spill. For a full detailed report of the chemical analysis obtained see Additional file 2.

Figure 2 Compound composition of Macondo crude oil-derived WAFs. A) Concentrations of selected components in samples taken from four different WAF experiments. The most prominent components were aromatics and alkanes. See Supplementary Information for details. B) Selected aromatic (blue) and alkane (red) WAF components decreased in concentration over a 10-hour test period. Full size image

To determine how component concentrations changed with time, samples from a WAF solution were taken every five hours from petri dishes maintained under the same conditions as the treated zebrafish embryos. As demonstrated by a few selected representative components, there is a dramatic drop-off in concentrations with time (Figure 2B). For aromatics (blue), the drop-off is less extreme for the heavier napthalenes (75 to 80% drop in five hours), compared to the lighter alkylated benzenes (93 to 95% drop in five hours). The initial concentrations for the heavy alkanes (red) are beyond reported hydrocarbon solubilities, such that the WAF solution is likely to have been initially fully saturated with these poorly soluble components (as discussed further in Additional files 1 and 2). Assuming initial saturation, the drop-off for tetradecane is similar to the heavier napthalenes, at 75% within five hours. These results suggest that while embryos are initially exposed to high levels of hydrocarbon compounds, within the 5- to 10-hour window, they are experiencing hydrocarbon concentrations that approach levels observed in underwater plumes in the Gulf of Mexico [41, 42] and those along the Louisiana marshes capable of altering gene expression in adult killifish [44]. For example, within 10 hours, our concentrations of toluene, ethylbenzene and total xylenes fall below ranges observed in underwater plumes [42].

Cell proliferation and cell death

We next wanted to characterize the gross morphological phenotypes we observed at higher resolution to glean better insight into whether exposure to Macondo oil affected specific developmental processes during zebrafish embryogenesis. WAF-treated embryos exhibit subtle reductions in brain and eye size and changes to the symmetric elongation of the tail (Figure 1), all of which could indicate reductions in cell numbers. No obvious greying of tissue, indicative of cell necrosis, was ever observed in WAF treated embryos; reduction in tissue size could be attributed to either reduced cell proliferation or increased programmed cell death.

Embryos treated with WAF from 3.5 hpf to 30 hpf were immunolabeled for Phosphorylated Histone H3 (PH3) to visualize all cells undergoing mitosis [45]. Pooled averaging of three replicate WAF treatments did not show significant changes in the number of PH3-positive cells as compared to untreated controls (Figure 3A, B; control, 79.9; WAF, 76.2; t-test, P = 0.18). To control for the presence of outlying values, the lack of statistical significance was confirmed with a non-parametric test (Mann-Whitney, P = 0.0511). If mitotic rates are not altered in WAF-treated embryos, then reductions in tissue size may be due to increased apoptosis or programmed cell death.

Figure 3 Macondo crude oil exposure did not affect cell proliferation but did induce programmed cell death. (A, B) Phospho-Histone H3 labeling of cells in mitosis were unaffected in 30 hpf WAF-treated embryos (B). (C) Quantification of anti-Activated Caspase 3-positive cells in 30 hpf control and WAF-treated embryos over 4 replicates and a WAF-refreshing procedure. The number of apoptotic cells decreased with each successive replicate, but increased following application of freshly-mixed WAF. (D-F) Activated caspase-3 labeled 30 hpf control (D, arrowheads) and WAF-treated embryos (E) from experiments in January 2011, and a WAF-treated embryo from an experiment in March 2011 (F, arrowheads). There was a significant decrease in the number of apoptotic cells in WAF-treated embryos between January (E) and March (F). (G-I) Representative images from the refreshed WAF experiments (arrowheads denote positive anti-Caspase 3 cells). Embryos were either untreated (G), exposed to the same WAF from 3.5 hpf to 30 hpf (H), or exposed to WAF from 3.5 hpf to 15 hpf and then exposed to a fresh WAF solution from 15 hpf to 30 hpf (I). Embryos in the refreshed WAF group (I) partially recovered the cell death phenotype of earlier replicates (E). (A, B, D-I) Lateral trunk views centered on somites 14 to 21. Scale bar 50 μm, A, B, D-I. Full size image

To test whether cells were dying by apoptosis we immunolabeled embryos for Activated Caspase 3, which is a marker for cells undergoing programmed cell death [46]. Pooled averaging of four replicate experiments did show a statistically significant increase in the number of dying cells positive for Activated Caspase 3 along the trunk in WAF-treated embryos as compared to controls (Figure 3C; control, 1.94; WAF, 12.9; t-test, P = 0.0001). Apoptotic cells were present both inside and outside the spinal cord (Figure 3D-F). Interestingly, despite using the same crude oil source for each WAF preparation, the number of apoptotic cells in WAF-treated embryos decreased with each successive replicate experiment (Figure 3C). In the first treatment an average of 38.25 Activated Caspase 3-positive cells were seen in WAF-treated embryos (Control, 3.9; t-test P = 0.0001), and in the second treatment this average dropped to eight cells (Control, 2.05; t-test P = 0.0062). Furthermore, in the third and fourth replicate experiments the number of apoptotic cells in WAF-treated embryos was no longer statistically different from their respective controls (Rep3: control, 1.1; WAF, 3.05 t-test P = 0.0966; Rep4: control, 0.7; WAF, 2.15; t-test P = 0.1151). A total of 20 embryos were examined for each condition in each separate replicate experiment.

The prevalence of WAF-induced cell death during the initial experiments suggests our crude oil sample was changing and becoming less potent over time. Chemical analysis did show much of the aromatics and alkanes in the WAF drop off dramatically within five hours after exposure to the embryos (Figure 2B), thus it is possible that our crude oil source sample experienced an incremental loss of some compounds over the course of storage. To test this hypothesis directly, we treated embryos with WAF from 3.5 hpf to 18.5 hpf and then replaced this solution with a freshly mixed WAF solution and continued the experiment until 30 hpf. This WAF refreshing protocol did show a statistically significant increase in the number of Activated Caspase 3 cells (36.74 cells; n = 42) as compared to untreated (1.75 cells; n = 40) or non-refreshed WAF treated controls (11.97 cells; n = 38) (Kruskal-Wallis Test P < 0.0005) (Figure 3G-I). In fact, the number of apoptotic cells in the refreshed WAF experiment was similar to the initial WAF experiment (Rep1, 38.25 cells; P = 0.2276) (Figure 3C). This result suggests refreshing the WAF solution returned the cell death-inducing properties of the crude oil.

Circulation and vasculogenesis

We have demonstrated that embryos treated with WAF derived from the Macondo Oil exhibit cardiac edema and hemorrhaging in the brain (Figure 1). We next wanted to determine whether the Macondo Oil WAF likewise interferes with the proper development and physiology of the circulatory system that has been demonstrated previously for other crude oil types and components [9, 10, 36–39, 47, 48]. Closer examination for the presence of blood through whole-mount hemoglobin stained control and WAF-treated embryos showed a qualitative reduction in the amount of blood cells in treated embryos (Figure 4A-D). This reduction was particularly obvious in the vasculature of the pharyngeal arches (Figure 4A, B, brackets; C, arrowheads, D). Importantly, significant hemoglobin staining was still present in the common cardinal vein distal to and including the heart, but abruptly ended in the heart or bulbus artery prior to filling the aortic arches (Figure 4A-D, arrow).

Figure 4 Defects in head and trunk vascular development result in reduced circulatory function. (A-D) Hemoglobin staining revealed a reduction in the amount of blood cells in 3 dpf WAF-treated embryos, notably in the vasculature of the pharyngeal arches (A, B, brackets, lateral view; C, D, arrowheads, ventral view), with staining abruptly ending in the heart or bulbus artery prior to filling the aortic arches (arrow). (E-H') Microangiography analysis with QTracker 655 fluorescent quantum dots (red) injected into 3 dpf tg[fli:eGfp] transgenic larvae to visualize endothelial cells associated with the vasculature (green). Endothelial vasculature in moderately affected WAF-treated embryos (G) was comparable to controls (E), however in severe cases posterior arch vasculature was lost and circulation was reduced (H, brackets, arrowhead, H', arrowheads). (E', F', G', H') Arrowheads and arrows denote the specific blood vessels associated with the pharyngeal arches. Accumulation of quantum dots in the heart atrium suggests reduced flow into the ventricle (H', dashed line). (I-K) Real time analysis of the flow speed of individual blood cells (I-J', arrowheads) over a 7-somite distance in the dorsal aorta. WAF-treated embryos have reduced blood circulation (K, right half of graph). (L-O') Intersegmental blood vessels had reduced circulation of quantum dots as demonstrated by either a complete absence of flow (M-O, arrowheads) or truncated flow (M-O arrows). Ectopic branching and vascular remodeling was evident in some segments devoid of circulation (N', O'). Abbreviations: BuA, bulbus arteriosus; H, heart; HA, hypobranchial artery; ORA, opercular artery; PHS, primary head sinus. Numbers and affiliated arrowheads in E' and F' represent the first through sixth aortic arch. Scale bars = 200 μm, A-D; 100 μm, E-H'; 50 μm, I, J, N', O'; 20 μm, I', J'; 50 μm, L-O. Full size image

The reduced blood volume specifically beyond the heart could be due to diminished hematopoiesis, improper vessel formation or altered heart function. To better discern between these possibilities, we conducted angiography with fluorescent quantum dots in 72 hpf live embryos treated with WAF starting at 3.5 hpf. This procedure was done in tg[fli:eGfp] transgenic larvae, which allowed for visualization of green endothelial cells as the red quantum dots flowed throughout the circulatory system, illuminating both the extent of circulatory function and blood vessel anatomy [49]. Imaging of the aortic arches revealed that in some cases the endothelial vasculature was present and capable of supporting circulation (Figure 4E, G, arrows and arrowheads), while in more severe cases the more posterior arch vasculature was lost and circulation was significantly reduced in the remaining vasculature (Figure 4F, H). In addition, there was often a build-up of the quantum dots in the heart atrium paired with a reduced flow of these quantum dots into the ventricle (H', dashed line). These results suggest that the endothelial vasculature and proper heart development and function are all variably compromised in WAF-treated embryos. Further supporting these results, real time imaging of blood flow through the dorsal aorta showed that individual blood cells from 60 different WAF-treated embryos (3 replicates) took, on average, 2.68 times longer to travel the distance of 7 somites (Figure 4I-K; Control, 1.44s; WAF, 3.85s; t-test, P = 0.0001).

Continued analysis of quantum dot circulation and endothelial vasculature in the trunk revealed additional phenotypes involving intersegmental blood vessel development. The most prevalent phenotype was varied frequency of reduced circulation of fluorescent quantum dots through the intersegmental blood vessels, such that vessels showed either a complete absence of flow (Figure 4L-O, arrowheads), truncated flow (Figure 4L-O, arrows), or normal circulation. Interestingly some vessels devoid of any quantum dots often showed ectopic branching and an overall improper vessel pattern (Figure 4N', O'). These phenotypes were consistent over three separate experimental replicates with at least 10 embryos imaged per replicate. As a whole, these results suggest the Macondo crude oil is capable of causing specific deformations in vasculogenesis in both the trunk and head of zebrafish that leads to reduced circulatory function.

Craniofacial development

Due to the vascular defects associated with pharyngeal arches, we next examined whether exposure to Macondo crude oil similarly causes defects in craniofacial development, a phenotype known to occur with specific PAH exposure [11, 36]. We treated embryos from 3.5 hpf to 4 dpf and then performed Alcian Blue staining to visualize cartilage [50]. Consistent with head vasculature phenotypes, WAF-treated larvae showed a range of head and jaw cartilage phenotypes (Figure 5A-F). WAF-treated embryos had a general size reduction in all cartilage elements with a significant lack of anterior extension of the most prominent jaw elements (Meckel's and ceratohyal cartilage) (Figure 5B, C, E, F). The most severely affected embryos showed dramatic reduction in all of the pharyngeal arch cartilage elements (ceratobranchial) with a complete loss of the most posterior three arches as compared to untreated control larvae (Figure 5A, B, E). In addition, we report here for the first time, in response to any crude oil treatment, a lack of anterior midline fusion of the basihyal cartilage (Figure 5E, arrowhead). Less severely affected embryos, however, did not display this basihyal phenotype (Figure 5F).

Figure 5 Craniofacial defects induced by Macondo crude oil exposure were correlated with defects in neural crest development. (A-F) Alcian blue staining of head and jaw cartilage in 4 dpf control (A, D) and severely (B, E) or moderately (C, F) affected WAF-treated embryos. WAF-treated embryos had a variable reduction in the size of all cartilage components, notably a lack of anterior extension of jaw elements and a dramatic reduction in posterior pharyngeal arches (B, C, E, F). (G-L) Whole mount in situ hybridization of crestin expression in neural crest cells. crestin expression is normal in the trunks of control and WAF-treated embryos (G, I, K, bracket; circles in G, I, K represent magnified view in H, J, L). However, crestin expression was variably reduced specifically in the anterior migratory streams, an area of cells that will populate the pharyngeal arches (H, J, L, arrowheads and bracket). (M, N) Cranial neural crest forming pharyngeal arches (p1-5) at 31 hpf as visualized by fli driven expression of GFP. One of the posterior-most pharyngeal arches is missing in WAF treated embryos (N, arrows) as compared to controls (M, 3, 4, 5). (O, P) dlx2 expression in the region of pharyngeal arches is reduced in 26 hpf WAF-treated embryos (P, p1, arrow, bracket) as compared to controls (O). dlx2 expression is nearly lost in the most posterior regions of the presumptive pharyngeal arches (P, bracket) despite robust expression still seen in the forebrain. Abbreviations: bh, basihyal cartilage; cb1-5, ceratobranchial branches; ch, ceratohyal; m, Meckel's; pq, palatoquadrate. Scale bars = 200 μm, A-F; 100 μm, G-L. Full size image

Because of the corresponding deformations in both endothelial and cartilage cell development in the head, we next hypothesized that these phenotypes may be derived from an earlier defect in the differentiation or migration of neural crest cells, which are a common progenitor cell contributing to pharyngeal arch cartilage, smooth muscle of the pharyngeal arch arteries as well as portions of the heart such as the arterial pole and endocardial cushions [51–56]. To test this we conducted whole-mount in situ hybridizations for crestin transcripts in WAF-treated and control embryos at 27 hpf. crestin is a known marker of neural crest cells during their early specification, delamination from the dorsal neural tube, and subsequent migration into the characterized pathways of the trunk and head [57]. Interestingly, embryos treated with WAF from 3.5 hpf to 27 hpf of development showed qualitative reductions in crestin-labeled cells, specifically in the anterior migratory streams known to populate the pharyngeal arches (Figure 5G-L; H, J, L, arrowheads and bracket). In contrast, the amount and position of crestin-positive cells in the trunk qualitatively appeared normal (Figure 5G, I, K, insets).

To confirm that deformations in pharyngeal arch development were associated with defects in early cranial neural crest populations, we examined the presence of rostral migratory streams of neural crest cells in tg(fli:GFP) transgenic embryos and whether they appropriately expressed the cell specification marker dlx2 [58, 59]. A total of 76% of WAF treated embryos (n = 83) showed a specific loss of one of the more posterior pharyngeal arches (Figure 5M, N, arrows) (control = 8.6%; n = 83; P < 0.0005). Importantly, despite normal dlx2 expression in the forebrain, 89.4% of WAF-treated embryos (n = 94) showed a significant reduction of dlx2 expression in all of the rostral migratory streams with a near complete absence in the presumptive posterior-most arch locations (Figure 5O, P, bracket) (control = 15.6%; n = 122; P < 0.0005). These findings suggest that Macondo crude oil is compromising the early development of cranial neural crest cell differentiation, which results in reduced posterior pharyngeal arches and the more visible defects in the heart, arch vasculature and craniofacial development.

Locomotor behavior

During our many WAF treatments it was clearly evident embryos displayed irregular swimming behaviors. The locomotor escape response to stimuli is an important survival behavior that develops later in embryogenesis. Interestingly, previous studies examining the effect of PAHs on zebrafish swimming behavior did not reveal any significant phenotypes [36]. Therefore, we systematically tested whether exposure to Macondo crude oil WAF impacted swimming patterns and escape responses. To do this, we recorded the swimming behavior of individual 48 hpf larvae with a high-speed video camera (1,000 frames/second) following the administration of a specific touch stimulus [60]. WAF-treated embryos demonstrated abnormal swimming behavior and a failure to escape based on multiple criteria. WAF-treated embryos showed reduced sensitivity to touch stimuli, as demonstrated by 70% response rate for WAF-treated embryos as compared to a 99% response rate for untreated control embryos (n = 100 trials from 10 embryos each). When a response was produced in WAF-treated embryos they showed a significantly reduced frequency of body bends (Control, 39.10Hz; WAF, 18.82Hz; n = 10 each; t-test, P < 0.01) and swam for less time than untreated control embryos (Control, 875.8mS; WAF, 282mS; n = 10 each; t-test, P = 0.01) (Figure 6). The presence of locomotor behavior phenotypes suggests that there could either be a problem with neural transmission or a developmental problem resulting from an anatomical deformation in the nervous or musculature systems.

Figure 6 Macondo crude Oil exposure impaired escape behavior by 48 hpf. (A) Individual frames from high-speed video recordings are shown for control larvae. The images are overlaid in 20 mS intervals and the duration of the response captured within the field is indicated. (B) Kinematics traces are shown for 10 escape responses each for control larvae. 0° indicates a strait body and positive and negative angles represent body bends in opposite directions. The time is indicated in seconds. (C) Image overlays for a WAF-treated larva escape response illustrates the failure to clear the field that was frequently observed. (D) Kinematic traces for WAF-treated larvae reveal reduced, abnormal body bend frequencies. (E) Quantification of body bend frequencies. (F) Quantification of the duration of escape responses reveals that WAF-treated larvae respond for shorter periods of time. Asterisks in E and F indicate statistically significant differences (n = 10, P < 0.01). Full size image

Neuronal development in the central and peripheral nervous system

Development of locomotor movement in response to touch stimuli requires a complex neural network that begins with an elaborate meshwork of Rohon Beard sensory axons at the periphery that originate from the dorsal neural tube. These bipolar sensory neurons make connections with a variety of interneurons that function to relay signals to Mauthner neurons, which serve as the major motor control center in the hindbrain. Mauthner neurons then send signals back down the spinal cord to stimulate coordinated motor neuron activation to achieve the alternating contraction of skeletal muscle required for swimming behaviors [61–63]. In order to determine what underlying developmental processes may be contributing to the visible defects in locomotor behavior, we used specific antibody markers to visualize each cellular step in the locomotor neural circuit.

Anti-Acetylated Tubulin (AT) labels all axonal pathways in the zebrafish as well as specifically marks the somas of Rohon Beard sensory neurons [64]. Anti-Islet-1 labels both the nuclei of Rohon Beard Sensory neurons and motorneurons, anti-Gaba labels a series of interneurons, and the 3A10 antibody marks Mauthner neurons in the hindbrain [65–67]. Surprisingly, using this spectrum of neuronal markers we found only one consistent and specific anatomical deformation that was restricted to the peripheral projections of sensory axons. The Mauthner neurons that serve as the central mediators for startle-reflex behavior in the hindbrain, showed absolutely no difference in number or axonal projections between WAF-treated and untreated control embryos (Figure 7A, B; n = 60) [67–69]. Likewise, no statistical difference was seen in the average number or position of Gaba-positive DoLa, CoSA, VeLD or KA interneurons (Figure 7C, D; Control, 41.1 cells, n = 60; WAF, 41.3 cells, n = 59; t-test, P = 0.855).

Figure 7 Macondo crude oil exposure caused specific deformations in the peripheral but not central nervous system. (A-L) 30 hpf control and WAF-treated embryos labeled by immunohistochemistry. (A, B) 3A10 labeled Mauthner neurons in the hindbrain were normal. (C, D) Distribution of Gaba-positive interneurons in the spinal cord were not impacted by WAF treatment (d, DoLa; c, CoSa; v, VeLD; k, KA). (E, F) Anti-Acetylated tubulin (AT) labeling of primary motor axons (arrows) or Rohon Beard sensory neuronal somas (asterisk) were correctly positioned in WAF-treated embryos (lateral view of trunk and spinal cord). (G, H) Qualitatively, Islet1 labeling for primary and secondary motor neurons (lower bracket) and Rohon Beard sensory neurons (upper bracket) were positioned normally. (I, J) The branching pattern of AT-labeled sensory axonal projections along the trunk epidermis was significantly reduced (I', J', magnified views of boxed area in I, J). (K, L) Anti-Gfap labeled radial glia somas in WAF-treated embryo spinal cords (L) were correctly positioned in the ventricular zone (arrowheads) and similar in number to controls (K). (A, B) Dorsal views of the hindbrain. (C-L) Lateral views of the spinal cord (C, D, E, F, G, H, K, L) and trunk (I, J). Scale bars = 50 μm, A-L. Full size image

Investigation of motorneuron development did not reveal major differences in their cell differentiation. Specifically, anti-AT labeling of primary motor axons did not show consistent defects over the length of the trunk (Figure 7E, F, arrows). It should be noted that while occasional errors in motor axon pathfinding were detected, they were always associated with an affiliated muscle patterning defect that we describe in detail later. Quantification of three separate replicate experiments of Islet1 labeling for primary and secondary motorneurons at 30 hpf showed only minor reductions in the number of motorneurons, with only one of the three replicates actually exhibiting statistical significance (Figure 7G, H, lower bracket; Rep 1: 16% reduction (control n = 11, WAF n = 20), t-test, P = 0.0001; Rep 2: 4.6% reduction (control n = 18, WAF n = 18), Mann-Whitney test, P = 0.456; Rep3: 6.6% reduction (control n = 20, WAF n = 19), Mann-Whitney test, P = 0.043). These results suggest that in some limited way motorneuron number may be impacted by exposure to Macondo oil WAF. However, based on the relatively normal pattern of primary motor axons, very modest overall reductions in number, variable significance and time period of analysis correlating with the middle of secondary motorneuron birth [65], we attribute these minor decreases in motorneuron populations more likely to a mild developmental delay in treated embryos rather than any biologically relevant effect from the oil.

Similarly, no qualitative differences were seen in the position or morphology of Rohon Beard sensory neurons as detected with anti-AT labeling (Figure 7E, F asterisks). Quantification of Rohon Beard sensory neuron numbers with anti-Islet1 showed little to no effect (Figure 7G, H, upper bracket). Similar to motorneurons, only one of the three replicates revealed a statistically significant difference (Rep 1: 2% increase (control n = 11, WAF n = 20), t-test, P = 0.72; Rep 2: 6.3% reduction (control n = 18, WAF n = 18), t-test, P = 0.184; Rep 3: 15.6% reduction (control n = 20, WAF n = 19), t-test, P = 0.0001). Again, based on the normal morphology and position of Rohon Beard cells, with no statistical difference present in the first two replicates, and only a small effect observed in the third replicate, we conclude Rohon Beard sensory neuronal number to be unaffected by Macondo oil. However, analysis of the meshwork of sensory axonal projections in the periphery was consistently both visually and quantitatively significantly reduced (Figure 7I, J, Control axonal labeling intensity = 1274.1 pixels, n = 55; WAF axonal labeling intensity = 883.9 pixels, n = 59; t-test, P = 0.0001). These results suggest most of the anatomical development of the locomotor circuitry is unaffected by Macondo crude oil WAF with the exception of subtle but consistent deformations in the amount and/or branching dynamics of sensory axonal projections.

While the overall number of many key neuronal cell types was normal in WAF-treated embryos, we wanted to confirm that radial glial cells, which serve as the neural stem cell population in the developing spinal cord, were also unaffected [70–72]. Using an antibody to Glial fibrillary acidic protein (Gfap) to mark radial glia we show that radial glial cell staining throughout the spinal cord appeared normal (data not shown). Most relevant was the normal number of Gfap-positive cell somas located at the ventricular surface of the neural tube (Figure 7K, L, arrowheads; Control, 5.99 cells, n = 40; WAF, 5.67 cells, n = 40; t-test, P = 0.582). Anti-Gfap labeling fills radial glial cell bodies when they are undergoing mitosis at the ventricular zone (Johnson and Barresi, in preparation; [73]). Normal numbers of such dividing radial glia indicates that exposure to Macondo crude oil WAF does not affect neural stem cell proliferation during embryogenesis.

Somitogenesis and muscle fiber type development

Because our analysis of the nervous system showed specific deformation restricted to the axonal projections of the peripheral nervous system, it was possible that these defects were secondary to deformations in the paraxial mesodermal environment through which these axonal projections need to navigate. During somite development, radially migrating slow muscle precursor cells provide key guidance cues to the simultaneously outgrowing motor axons [74]. Therefore, any defects in the proper specification, migration and/or position and integrity of slow muscle fibers could influence the development of the peripheral nervous system. We exposed embryos from 3.5 hpf to 48 hpf in Macondo crude oil WAF and assayed for F59 labeling, which preferentially recognizes slow muscle myosin in zebrafish [75, 76]. As with most assays, we discovered embryos displaying a range of slow muscle development phenotypes from no observable defects to the loss of muscle fibers and segment boundary errors. Interestingly, similar to what was observed for our cell death analysis over time, embryos displaying slow muscle phenotypes were extremely severe during initial treatments but these phenotypes decreased significantly over the course of several months of repeated experiments using the same crude oil source for WAF preparation (Figure 8).

Figure 8 The severity of deformations in slow-twitch skeletal muscle development decreased with each experimental replicate. (A-M) Lateral views of F59 labeled Myosin heavy chains in slow-twitch muscle fibers (red) and anti-Acetylated tubulin labeled motor axons (green) in the embryonic trunk at 72 hpf (A-D) and 48 hpf (E-M). (A-D) Initial Macondo crude oil WAF treatments beginning at either 3.5 hpf (B) or 8 hpf (C, D) and ending at 72 hpf showed severely defective neuromuscular phenotypes, such as improper somite boundary formation (B, C, arrowhead) or slow muscle loss and disorganization (D). Somite boundary defects associated with the middle or ventral portion of the somite correlated with motor axon pathfinding errors (B, asterisks, arrow; D). (E-M) Subsequent WAF treatments beginning at 3.5 hpf and ending at 48 hpf induced somitogenesis (F, G; F, G', arrowheads), slow muscle (I-M', arrowheads), and motor axon pathfinding (F, G; F", G", arrows) defects, however the severity of these defects decreased over time with each experimental replicate from November to March. (A-M) Primed letters represent single channel images of slow muscle (single prime) or axon (double prime) labeling for the whole image (A-D) or just the boxed regions (F, G, I, J, L, M). Scale bars = 50 μm, A-M". Full size image

Initial experiments in November 2010 showed clear defects that represented several distinguishable phenotypes in the treated embryos (Figure 8A-D). The first was the sporadic instance of defects in proper somite boundary formation, such that a segment boundary in a portion of one somite would be absent as represented by slow muscle fibers extending from the anterior boundary of one somite to the posterior boundary of the neighboring somite (Figure 8B, C, arrowhead). Most interestingly, double labeling for motor axons showed pathfinding errors in the affected somite that were directly correlated to the somite/slow muscle defect (Figure 8B, asterisk, arrow). These results suggest that Macondo crude oil may not only be affecting slow muscle fiber development but also somitogenesis, the process by which segment boundaries are established in the paraxial mesoderm [77–79]. The second distinguishable phenotype was the sporadic loss and disorganization of slow muscle fibers in the superficial monolayer (Figure 8D), which suggests impairments in slow muscle differentiation and subsequent migration in WAF-treated embryos.

Later experiments revealed somitogenesis defects with corresponding motor axon pathfinding errors, but these deformations were not as dramatic as compared to our first experiments (Figure 8E-G) and subsequently absent from the final replicates (Figure 8H-M). Later experimentation still showed extremely sporadic loss and disorganization of the slow muscle monolayer (Figure 8H-M). Interestingly, high resolution imaging of regions showing reductions in slow muscle fibers revealed muscle phenotypes resembling muscle degeneration [80–85]. Variable sized fragments of slow muscle myosin were found present in locations devoid of morphologically normal slow-twitch fibers (Figure 8I, J, red). In contrast to the somite boundary defects, motor axon pathfinding appeared normal in areas showing these missing or degenerating muscle fibers (Figure 8I, J, green). Lastly, these phenotypes continued to diminish in severity upon our last round of treatment (Figure 8K-M).

The sporadic nature of muscle phenotypes both in a clutch of treated embryos and within an affected embryo paired with the gradual loss of these phenotypes over time suggests that the causative agent in the crude oil is not particularly potent and somehow loses its activity over time. Therefore, in an attempt to reproduce the severity of the muscle and somitogenesis defects observed initially, we exposed embryos from 3.5 hpf to 48 hpf in WAF that was refreshed every 15 h. Similar to our results of increased apoptosis in refreshed WAF treatments, more severe muscle deformations were present in refreshed WAF-treated embryos as compared to untreated controls and non-refreshed WAF-treated embryos (Figure 9). The sporadic nature of these deformations was consistent, but the severity was now qualitatively similar to our initial experiments. Remarkably, somitogenesis defects were detected again, showing slow muscle fibers crossing in locations that would normally have a somitic boundary (Figure 9D, D', arrowheads) as well as the presence of irregularly shaped somites (Figure 9D', arrows). In addition, significant slow muscle loss and potential degeneration was also observed following this WAF refreshing protocol. Specifically, slow muscle myosin fibrils were seen in varying stages of degeneration (Figure 9E). This type of dose-response approach confirms that some component(s) within the Macondo crude oil WAF do directly disrupt somitogenesis and slow muscle development.