High resolution AKT signaling in individual cells

Cells sense and respond to their environment by activating distinct intracellular signaling pathways, however an individual cell’s ability to faithfully transmit and discriminate environmental signals is thought to be limited. To assess the fidelity of signal transmission in the PI3K-AKT signaling pathway, we first developed an optimized genetically encoded sensor that had an increased dynamic range and reduced variation under basal conditions. We then used this reporter to track responses to varying doses of IGF-I in live cells and found that signaling responses from individual cells overlapped across a wide range of IGF-I doses, suggesting limited transmission accuracy. However, further analysis of individual cell traces revealed that responses were constant over time without stochastic fluctuations. We devised a new information theoretic approach to calculate the channel capacity using variance of the single cell time course data--rather than population-level variance as has been previously used—and predicted that cells were capable of discriminating multiple growth factor doses. We validated these predictions by tracking individual cell responses to multiple IGF-I doses and found that cells can accurately distinguish at least four different IGF-I concentrations, as demonstrated by their distinct responses. Furthermore, we found a similar discriminatory ability to pathway inhibition, as assessed by responses to the PI3K inhibitor alpelisib. Our studies indicate that cells can faithfully transmit an IGF-I input into a down-stream signaling response and that heterogeneous responses result from variation in the input-output relation across the population. These observations reveal the importance of viewing each cell as having its own communication channel and underscore the importance of understanding responses at the single cell level. in in , it differences in basal signaling , cell shape , , or phosphatase activity. Assessing each of these mechanisms independently is challenging, but our work suggests that it may be possible to tease apart these different mechanisms using a single-cell multi-treatment approach. studies indicate that signaling responses to molecules that activate or inhibit the IGF-I-AKT signaling pathway are not necessarily noisy. This is important as it rules out one mechanism for in that cells have large and variable signaling fluctuations that allow to overcome This conclusion also suggests a role for microenvironmental For may whereby dose fully inhibit these cells 19, . These experiments open the door to more in depth studies on how various pathway inhibitors alter signaling activity and how treatments regimens could be optimized for use in the clinic, particularly in the metastatic setting where cancer cells have to distinct tissue sites enriched with unique complements of signaling molecules.

Abstract
Cells sense and respond to their environment by activating distinct intracellular signaling pathways, however an individual cell's ability to faithfully transmit and discriminate environmental signals is thought to be limited. To assess the fidelity of signal transmission in the PI3K-AKT signaling pathway, we first developed an optimized genetically encoded sensor that had an increased dynamic range and reduced variation under basal conditions. We then used this reporter to track responses to varying doses of IGF-I in live cells and found that signaling responses from individual cells overlapped across a wide range of IGF-I doses, suggesting limited transmission accuracy. However, further analysis of individual cell traces revealed that responses were constant over time without stochastic fluctuations. We devised a new information theoretic approach to calculate the channel capacity using variance of the single cell time course data--rather than population-level variance as has been previously used-and predicted that cells were capable of discriminating multiple growth factor doses. We validated these predictions by tracking individual cell responses to multiple IGF-I doses and found that cells can accurately distinguish at least four different IGF-I concentrations, as demonstrated by their distinct responses. Furthermore, we found a similar discriminatory ability to pathway inhibition, as assessed by responses to the PI3K inhibitor alpelisib. Our studies indicate that cells can faithfully transmit an IGF-I input into a down-stream signaling response and that heterogeneous responses result from variation in the input-output relation across the population.
These observations reveal the importance of viewing each cell as having its own communication channel and underscore the importance of understanding responses at the single cell level.

Introduction
Cells sense and respond to their local environment by activating distinct intracellular signaling pathways. These pathways are composed of multi-step relays that use second messengers, protein translocations, and kinase/phosphatase cycles to encode, relay, and decode the extracellular signal into an intracellular response 1 . Given the multi-step nature of cell signaling in both space and time, much remains unknown about exactly how individual cells process extracellular signals into accurate intracellular responses [2][3][4][5] . Previous studies have observed that cell signaling is inherently noisy and that individual cells are limited in their detection of extracellular stimuli [2][3][4] , which suggests that ensemble averaging is required to decode extracellular signals into accurate signaling responses. However, these prior studies assessed signaling pathways with predominantly pulsatile--rather than sustained--responses, which hinders the ability to quantify and compare individual cell responses across time and may also obscure alternate explanations for heterogeneity.
The IGF-I -PI3K -AKT signaling pathway provides an excellent model to study cell signaling: it uses multiple modalities to encode, relay, and decode extracellular signals and also produces sustained responses. In brief, IGF-I binds to the receptor tyrosine kinase IGF1R to stimulate intracellular kinase activity 6 . This leads to recruitment of protein binding partners and translation of the protein signal into a lipid second messenger by PI3-Kinase and the subsequent phosphorylation and activation of AKT. The protein kinase AKT can then phosphorylate a range of proteins including the transcription factor FoxO1 [6][7][8][9] . Previously, we developed a fluorescent fusion protein using FoxO1, which can be used as a readout for IGF-I signaling activity 10 , and found that responses to IGF-I were sustained across time but heterogeneous across the population. We surmised that this heterogeneity could arise from various sources, including: noise in the signaling pathway, variability in the reporter and its quantification, non-specificity in the reporter, or cell-to-cell variation in the encoding of the signal.
Transitions from healthy to diseased states frequently involve changes to cell signaling, therefore a deep understanding of the fidelity of signaling in individual cells can provide insights into disease progression and treatment. For example, cancers arise from genetic alterations that drive activation of oncogenic pathways such as the PI3K-AKT pathway [11][12][13] . Moreover, cancers are frequently marked by substantial intratumoral heterogeneity 14 , indicating the need to quantify signaling at the single cell level. Several small molecule inhibitors have been developed to inhibit PI3K-AKT pathway activity and block cancer growth 15 . While promising, responses to these compounds are variable and resistance inevitably develops 16,17 . One possible explanation for incomplete therapeutic responses is variation in the signaling capability of individual cells [18][19][20] . Thus, a more complete understanding of responses to pathway-targeted activators and inhibitors at the single cell level will influence how clinical responses are interpreted and could inform mechanisms of drug resistance.
Here we report the development of an improved FoxO1 reporter to monitor AKT activity in individual cells and use it to assess growth factor signaling responses. In genetically transformed HeLa cells, we observed heterogeneous responses to IGF-I that were consistent with individual cells having different input-output relationships. To account for differences in the input-output relation across the population, we calculated the channel capacity-which provides an assessment of signaling fidelity-by measuring variance in individual cells over a time window when signaling responses had stabilized. This new approach nearly doubled the predicted number of IGF-I doses a cell could accurately discriminate. We then experimentally validated these predictions by treating cells with multiple IGF-I doses and showed that individual cells could accurately discriminate at least four IGF-I concentrations. Furthermore, we found that signaling fidelity extends to pathway inhibition such that the level of activation is largely correlated with the level of inhibition. We conclude that single cell responses to submaximal doses of IGF-I vary across a population because individual cells have different input-output relationships, and further that accounting for these differences reveals high signaling fidelity in individual cells.

Construction of a novel FoxO1 reporter with increased specificity
Signaling responses in the AKT, Erk, NF-kB, TRAIL and Ca 2+ pathways have been shown to be heterogeneous across the population [2][3][4][5]21 . This heterogeneity is frequently interpreted as arising from noise in signal transmission [2][3][4] . In previous work, we developed a live-cell reporter to study IGF-I responses at the single cell level: a fluorescently tagged FoxO1 protein 10 , which contains three canonical AKT phosphorylation sites that regulate protein localization to the nucleus or cytoplasm depending on their phosphorylation status 9,22 (Fig. 1A). We found that the relative fraction of the reporter in the nucleus was variable across a population of HeLa cells cultured in serum free conditions 21 and speculated that this variation resulted from phosphorylation on other non-AKT phosphorylation sites. In order to assess signaling noise in the IGF-PI3K-AKT pathway, we first sought to reduce basal variation in our readout for pathway activity.
To construct a new reporter with increased specificity, we started from the template of the previously developed reporter, termed Fox Tracer 1 (FT1) and made alanine substitutions to 10 serine and threonine sites previously identified as phosphorylation sites of other kinases [23][24][25][26][27] (Fig.   1A and Supplementary Fig. 1A, B). To reduce the protein size and eliminate other potential sources of non-specificity, we deleted a 234 amino acid segment from the C-terminus. Lastly, to increase the ease and efficiency of creating stable cell lines, we transferred the reporter from a lentiviral to a transposase-based genetic delivery system 28 (Sup. Fig. 1C).
The first construct tested, FT2-LAP, had a significantly reduced variance and increased nuclear localization compared to FT1 under serum free conditions, which suggested an increase in specificity from the alanine mutations. However, this came at the cost of a reduced export rate following IGF-I treatment, which limits the ability to rapidly assess responses. We next tested reporter variants containing a series of mutations near S326, which is located in an AKT phosphorylation domain thought to regulate the rate of FoxO1 nuclear export by creation of a negatively charged region when phosphorylated 26,29 (Fig. 1B). The single mutation of alanine 326 back to serine (FT2-LSP) increased the rate of nuclear export, but also added back a DYRK1/Erk phosphorylation site 26,29 (Fig. 1D, Sup. Fig. 1D). To eliminate this unwanted phosphorylation site, we mutated the peptide segment using the phosphomimetic amino acid aspartic acid. Furthermore, based on the concept that multiple negative charges better mimic the effect from phosphorylation 30, 31 , we tested four reporter variants that differed in the number of aspartic acid substitutions in this peptide segment. A comparison of these variants revealed that response rates increased with peptide charge, leading to a maximum rate of export for the FT2-DDD reporter (Fig. 1E, Movies 1-3).
As a control for the FT2-DDD reporter, we created a mutant with the three canonical AKT phosphorylation sites Thr24, Ser253 and Ser316 all mutated to alanine. As expected, this reporter (FT2-DDD 3A) does not translocate in response to IGF-I, which indicates that FT2-DDD responses to IGF-I were dependent on the canonical AKT phosphorylation sites (Fig. 1D). In summary, comparing the FT1 to the FT2-DDD reporter, we found that FT2-DDD is the optimal reporter for tracking the fidelity of IGF-I signaling responses based on its: increased specificity ( Fig. 1A, B), reduced population variance in both serum free conditions and following IGF-I treatment (Sup. Fig. 1E), and increased measurement range (Fig. 1F).

Responses to IGF-I stimulation are heterogeneous across the population
We next used the optimized FT2-DDD reporter to quantify responses of individual cells to IGF-I.
As expected, we found a progressive increase in the average population response with increasing IGF-I dose, which was quantified as a change in the relative nuclear intensity of the fluorescent reporter in individual HeLa cells ( Fig. 2A and B). Analysis of individual cells revealed heterogeneous responses to single doses of IGF-I and overlapping responses across multiple IGF-I doses, which has been interpreted as an inability of individual cells to discriminate different ligand doses 2 (Fig. 2C). Importantly, this heterogeneity was not due to differences in the relative expression of the reporter between cells, nor differences in the basal level of activation in individual cells (Sup. Fig. 2). Thus, despite reducing possible effects of nonspecificity in the readout for pathway activity, we find that responses to IGF-I remain heterogeneous across a population of individual cells.
Integration of multiple time points to quantify a cell's response has been shown to reduce the level of heterogeneity by reducing measurement noise 3 . To measure the effect of varying the number of measurements for each response, we compared dose-response relationships using either one image, which corresponds to the fluorescence at 90 min (Raw), two images, which allows cell responses at 90 min to be normalized based on their starting value (T0 normalized), or 15 images, which incorporates the signal change over a longer time window (Summed). We found that the EC 50 values remained similar between measures (Fig. 2D), which indicates that the quantification method does not distort the relationship between doses. Yet, a comparison of the cell responses to 0, 15, 25, and 125 pM showed that overlap between the populations was reduced--but not eliminated--as the measurement window increased (Fig. 2D). These observations indicate that single cell responses to IGF-I are heterogeneous, even with use of analytical approaches that reduce variance.

Responses of individual cells are stable across time
The Shannon-Hartley formula has been extensively used to quantify the signaling accuracy of biological systems [2][3][4] . A central assumption of the Shannon-Hartley channel capacity measurement, when applied to a population, is that the input-output relationship is identical for all cells in the population and observed variation is therefore attributed to noise in signal interpretation, transmission, and encoding. Based on this assumption, a noisy pathway would lead to varying responses in single cells over time (Fig. 3A, upper). Alternatively, there could be variation across the population in the input-output relationships of individual cells. In this case, we would expect to observe individual cells with minimal variation in signal transmission over time as well as heterogeneous responses across the population (Fig. 3A, lower). To separate these two scenarios, we quantified variation using the standard deviation of responses from each dose of IGF-I in the first 90-minute response window compared to the second 90-minute window when responses had reached a steady state (Fig. 3B). We found that the standard deviation during the first 90-minute response window increased as the dose of IGF-I increased  Based on this interpretation, we hypothesized that variance quantified from stabilized responses provides a more robust calculation of noise for channel capacity measurements. To test this, we quantified channel capacity in various ways. Channel capacity calculated from a single time point yielded 1 bit (Fig. 3D), which is similar to prior measures of other signaling pathways 2 .
This measure was increased to 1.2 bits when we computed channel capacity from the T0 normalized responses (Fig. 3D), and was further increased to 1.4 bits by using the summed responses drawn from 15 images. Finally, we quantified noise using the stable response window and found a channel capacity of 2.38 bits, which indicates that cells can discriminate at least 5 distinct IGF-I doses. While higher than previously reported, this is likely still an underestimate due to limitations in measuring individual cell responses. To test this idea, we treated cells with the nuclear export inhibitor Leptomycin B (LMB), which results in complete and All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/373993 doi: bioRxiv preprint sustained nuclear localization of the reporter. As a consequence, any variation in nuclear intensity after reaching steady state can be attributed to measurement noise rather than signaling noise. Comparison of how the signal varied across time for cells treated with LMB compared to cells treated with a submaximal dose of 17.5 pM IGF-I revealed nearly equivalent variances, which indicates that the true channel capacity may actually be much higher (Sup. Fig.   3B, C).

Individual cells can recapitulate population-derived dose response curves to reveal high signaling fidelity
To test the capacity of a single cell to discriminate different signal intensities, we treated cells showed measurable responses to even the lowest dose tested. Comparison of traces from cells centered at the 25 th and 75 th percentiles revealed similar responses within the groupings but distinct differences between the groupings (Fig. 5B). Likewise, the average dose response relationships from these cell subpopulations and a subpopulation of cells centered at the 50 th percentile revealed consistent responses within the groupings (Fig. 5C). Moreover, a cell's response to the first dose can be used to predict its response to the second dose (Sup. Fig. 4A).
Finally, we find that responses of multi-dose treated cells are significantly different from timeand dose-matched cells maintained in the same dose (Sup. Fig. 4B). These studies indicate that individual cells can discriminate a minimum of four distinct IGF-I concentrations, and the inputoutput relationship is consistent in individual cells but varies across the population. All rights reserved. No reuse allowed without permission.

Single cell responses to PI3Kα inhibition are influenced by IGF-I activation level
Our multi-dose single cell data indicate that cells can discriminate multiple concentrations of IGF-I, and that a continuum of responses exists across the population due to different inputoutput relations. This raised the question of whether responses to pathway inhibition similarly vary across a population of cells. To test this, we treated cells with 25 pM IGF-I, a submaximal dose that leads to a wide variation in single cell responses (see Fig. 2B), followed by dose escalation of the PI3Kα inhibitor alpelisib (0-500 nM). At the population level, we observed the expected dose-response relationship to alpelisib treatment (Fig. 6A). To understand single cell responses to alpelisib, we compared cells from the upper (blue traces) and lower (red traces) quantiles after treatment with 25 pM IGF-I. We found that these two populations remain separable after inhibitor treatment ( Fig. 6 B, Movie 6), and that responses to PI3Kα inhibition were correlated with the level of activation from IGF-I (Fig. 6C). Comparison of all drug doses revealed similar separations between quantiles, up to 500 nM alpelisib, the dose at which signaling from IGF-I is nearly completely blocked (Fig. 6D). Together, these observations indicate that a cell's response to inhibition is determined by its response to IGF-I, and as a consequence, responses to pathway inhibition are in line with a model in which there is variation in how cells encode a signaling input. All rights reserved. No reuse allowed without permission.

Discussion
Understanding how individual cells respond to diverse stimuli is a fundamental issue in cell biology [32][33][34] . To that end, we describe here the development of a FoxO1 reporter with a higher specificity for IGF-I signaling compared to our previous FoxO1 reporter 10 . With this improved reporter, we observed that individual cell responses to IGF-I still overlapped between doses, suggesting low signaling fidelity. Further analysis showed that this overlap was from differences in how cells encode the signal, rather than from noise in the signaling pathway. Multi-dose treatment protocols validated these findings and showed that cells could accurately discriminate at least four different IGF-I doses. These studies demonstrate that individual cells can accurately process extracellular growth factors into downstream signaling responses despite the observation that population responses overlap between doses.

Reporter optimization for live cell imaging studies
Genetically encoded fluorescent reporters provide a powerful tool for understanding and quantifying dynamic signaling responses 35 . Various types of signaling reporters have been developed, including FRET sensors 36,37 , synthetic translocation reporters 38 , and native proteins that translocate between the nucleus and cytoplasm (FoxO1 10, 39 , p53 40 , SMAD2 41 , NFkB 42 , Erk 43 ). Native proteins are advantageous in that they are biologically tuned to respond under physiological conditions and the function of critical protein regions are typically well-described.
Here we used knowledge of the FoxO1 sequence to optimize an existing FoxO1 reporter.
Specifically, we increased specificity by both targeting a large peptide region for deletion that is not involved in nuclear translocation and by mutating multiple phosphorylation sites that are not phosphorylated by AKT. Additionally, we optimized nuclear export through the modification of a short peptide sequence. These modifications would have been difficult to predict in the absence of previous studies. For example, nuclear export was improved by substitution of a phosphomimetic amino acid rather than an alanine at serine 326, and export was further aided by mutating the surrounding amino acids to better match the charge from phosphorylation 30,31 . All together these modifications significantly increased the specificity of the reporter for IGF-I signaling in HeLa cells, and allowed for increased accuracy in tracking individual cell responses.
Our approach to optimize the FoxO1 reporter, which considers the molecular structure and function of FoxO1, could be used to efficiently develop reporters of other signaling pathways.

Channel capacity quantification
Previous studies of the Erk, NFkB, and Ca 2+ signaling pathways have found channel capacities from 1-1.7 bits 2, 3, 5, 44 . Here, we assessed the channel capacity of the PI3K-AKT pathway using IGF-I. Importantly, this growth factor produces sustained Akt signaling responses 45 , making it All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.

Multi-treatment experiments reveal variation in single cell responses
The ability to quantify responses to multiple treatments in individual cells provides a powerful technique for understanding mechanisms of population variation 46,47 . This is in stark contrast to standard experiments in which single measures on individual cells are drawn from different populations, or the response to only a single stimulus is tracked across time. We used this multitreatment approach to achieve several means: first we used LMB normalization to quantify the measurement range of the reporter and to show that individual cell responses are not biased by the level of reporter expression, second we used multi-dose IGF-I treatments to validate that cells can accurately discriminate multiple doses of IGF-I and to identify variability in IGF-I encoding across the population, and third we used activation-inhibition experiments to determine that the level of signaling activity in an individual cell biases the extent of inhibition.
Without multiple measurements on the same cells, it would not be possible to identify these patterns nor accurately quantify potential sources of biological variation.

Cells exist in different states with different input-output relationships
Our data indicate that cells can accurately interpret multiple growth factor signals in their extracellular environment and that across the population there is variation in how cells encode this signal. As a consequence, it is not possible to accurately decode the input based on a single response. It is only through knowledge about how a cell responds to a second perturbation-either activation or inhibition-that the input signal could be decoded. This raises the question of what cellular parameter varies to create different input-output relationships All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/373993 doi: bioRxiv preprint across the population. It is reasonable to assume that variation in the input-output relationships between cells occurs because they differ in the content of the IGF-I receptor or another pathway component 48 , but it could also be from differences in basal signaling 49 , cell shape 50 , nuclear import/export machinery 38 , or phosphatase activity. Assessing each of these mechanisms independently is challenging, but our work suggests that it may be possible to tease apart these different mechanisms using a single-cell multi-treatment approach.
Our studies indicate that signaling responses to molecules that activate or inhibit the IGF-I-AKT signaling pathway are not necessarily noisy. This is important as it rules out one mechanism for drug resistance in cancer: that cells have large and variable signaling fluctuations that allow them to overcome inhibition. This conclusion also suggests a role for microenvironmental signals to modulate therapeutic response. For instance, subpopulations of cells may be subject to signals that lead to pathway activation, whereby a larger drug dose may be required to fully inhibit these cells 19,51 . These experiments open the door to more in depth studies on how various pathway inhibitors alter signaling activity and how treatments regimens could be optimized for use in the clinic, particularly in the metastatic setting where cancer cells have disseminated to distinct tissue sites enriched with unique complements of signaling molecules.

Summary
A significant question in cell biology is how much noise is present in a signaling pathway, and as a consequence how accurately a signal can be interpreted and processed. Along with the development of an improved reporter, we found that individual cells can faithfully interpret multiple IGF-I concentrations, although each cell encodes the signal differently. This conclusion has significant impacts on how we think about the regulation of biochemical pathways and cellcell heterogeneity.
All rights reserved. No reuse allowed without permission.

Cloning
The new FoxO1-Clover reporter was constructed from a modified pSBbi-RP plasmid 28   The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/373993 doi: bioRxiv preprint that migrated over the course of the experiment such that the T0 location no longer aligned with later time points were excluded from analysis. Additionally, cells that divided, died, were multinucleated, or overlapped with another cell were excluded from analysis.

Transfection and selection of stable cell lines
Three measures were used to quantify individual cell responses: the 'nuclear localization', the 'relative nuclear intensity', and the 'scaled relative nuclear intensity'. The 'nuclear localization' for each cell was calculated by normalizing fluorescence intensities to the nuclear fluorescence intensity 90 min after leptomycin (LMB) treatment. The 'relative nuclear intensity' for each cell was calculated by normalizing fluorescence intensities to the nuclear fluorescence intensity at T0 following 90 min in serum free media. The 'scaled relative nuclear intensity' was calculated by scaling each cell's response to the population average, where 0 pM was set to 0 and 125 pM IGF-I was set to 100.

Reporter Characterization
To characterize each HeLa reporter cell line, cells were serum starved for 90 minutes in Fluorobrite media (ThermoFisher) supplemented with 2 mM L-glutamine, 0.1% bovine serum albumin, penicillin and streptomycin. Cells were then treated with 250 pM R3-IGF-I for 90 minutes followed by the combined addition of the dual PI3K and mTOR inhibitor PI-103 (1 µM) and LMB (100 nM) for 90 minutes. From these experiments, we quantified: the average rate of reporter nuclear exclusion following IGF-I treatment, the average rate of nuclear localization following PI-103 and LMB, and the average measurement range, which was defined as the difference between the percent nuclear localization after serum starvation and the percent nuclear localization 90 min after IGF-I treatment. Export and import rates were calculated using the population average response and fit via non-linear regression (Graph Pad Prism 7). All cell lines were imaged using the same 75 millisecond imaging duration. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/373993 doi: bioRxiv preprint 0 min value. 'Summed responses' were calculating by summing the relative change of the T0 normalized data using 15 equally spaced images (every 6 min). Individual cell responses to multiple doses of IGF-I were sorted using the summed responses across the 300-minute experiment. Heat maps were created in R (https://www.R-project.org/) using the gplots heatmap package version 3.0.1.

Leptomycin B experiments
To quantify the relationship between reporter expression and sub-maximal IGF-I responses,

Quantification of channel capacity
Channel capacity was calculated using the Shannon-Hartley formula: CC = ½ log 2 (S 2 /N 2 ), where CC is the channel capacity in bits, S is the signal magnitude quantified by taking the variance of the average response across six relatively evenly spaced IGF-I doses representing the full dose range (0, 10, 15, 25, 40, 125 pM R3-IGF-I), and N is the noise magnitude quantified by taking the average variance within a dose and averaging across all doses 3 .

Data Availability
Source data are available from the corresponding author upon reasonable request.  The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/373993 doi: bioRxiv preprint   The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/373993 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.  The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.

Channel Capacity (bits)
A All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/373993 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.  All rights reserved. No reuse allowed without permission.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Impact of maternal western diet-induced obesity on offspring mortality and peripheral endocannabinoid system in mice

Over two-thirds of adults in the United States are obese or overweight, which is largely due to chronic overconsumption of diets high in fats and sugars (i.e., Western diet). Recent studies reveal that maternal obesity may predispose offspring to development of obesity and other metabolic diseases; however, the molecular underpinnings of these outcomes are largely unknown. The endocannabinoid system is an important signaling pathway that controls feeding behavior and energy homeostasis, and its activity becomes upregulated in the upper small intestinal epithelium of Western diet-induced obese mice, which drives overeating. In the current investigation, we examined the impact of chronic maternal consumption of Western diet on the expression and function of the endocannabinoid system in several peripheral organs important for food intake and energy homeostasis in offspring. Female C57BL/6Tac mice were fed a Western diet or low-fat/no-sucrose control chow for 10 weeks, then males were introduced for mating. Dams were maintained on their respective diets through weaning of pups, at which time pups were maintained on low-fat/no-sucrose chow for 10 weeks. Neonates born from dams fed Western diet, when compared to those born from mice fed control chow, unexpectedly displayed increases in mortality that occurred exclusively within six days following birth (greater than 50% mortality). Males comprised a larger fraction of surviving offspring from obese dams. Furthermore, surviving offspring displayed transient increases in body mass for first two days post weaning, and no marked changes in feeding patterns and endocannabinoid levels in upper small intestinal epithelium, pancreas, and plasma, or in expression of key endocannabinoid system genes in the upper small intestinal epithelium and pancreas at 10 weeks post-weaning. Collectively, these results suggest that maternal diet composition greatly influences survival of neonate C57BL/6Tac mice, and that surviving offspring from dams chronically fed a Western diet do not display marked changes in body mass, eating patterns, or expression and function of the endocannabinoid system in several peripheral organs important for feeding behavior and energy homeostasis.

Introduction important for food intake and energy balance, which include pancreas, small-intestinal epithelium, and plasma in C57BL/6Tac mice. Neonate mortality was also evaluated.

Animals
Six-week old male and female C57BL/6Tac (Taconic, Oxnard, CA, USA) were grouped housed according to sex with free access to water and food and maintained on a 12-hour light/dark cycle (lights off at 1800 hours). Test diets composed of low-fat/no-sucrose standard lab rodent chow [(SD) Teklad 2020x Global Soy Protein-Free Extruded Rodent Diet; 16% kcal from fat, 60% kcals from carbohydrate, no sucrose], or Western diet [(WD) Research Diets D127098, New Brunswick, NJ, USA; 40% kcal from fat, 43% carbohydrates, mostly sucrose) ( Table 1). All procedures met the U.S. National Institute of Health guidelines for care and use of laboratory animals, and were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, Riverside.

Breeding parameters
Female mice were fed either SD or WD during pre-gestation for 10 weeks, gestation, and lactation. Male mice were only given access to SD. Following 10 weeks, male mice were harem bred with female mice for mating at 1000 hr and separated at 1600 hr (see Fig 1 for experimental design). Food was removed during mating times and returned immediately after to avoid male consumption of WD diet. When a vaginal plug was observed during mating, females were single housed with their respective diets to give birth. Birth dates, litter sizes, neonate survival, and overall health of all animals were monitored daily. Body weights of offspring were recorded twice weekly. At postnatal day 21, pups were weaned, ear tagged, grouped housed with mice of same condition (i.e., those born from the same maternal test diet), and given access to only SD for the duration of the 10-week study. Pregnancies all occurred approximately at the same date. Thus, the outcome from all litters occurred at approximately the same date.
Notably, increased mortality of neonates was not an expected outcome or anticipated endpoint of our experimental design, which was aimed at evaluating feeding patterns and endocannabinoid system expression and function in peripheral organs of offspring born from dams maintained on WD or SD. Thus, despite daily monitoring of health and adherence to well-defined IACUC-approved humane endpoints for determining when animals will be removed from studies, treated, or euthanized, their implementation was not possible for mice in this study given that no signs of suffering or distress were observed for non-surviving neonates and that mortality of neonates occurred unexpectedly and exclusively within the first six days following birth. The cause of increased mortality is unknown but is associated with chronic maternal consumption of WD. Out of a total of 70 pups, 44 failed to survive by six days post-birth, at which time the remaining pups survived throughout the entirety of the study. All dams born from WD fed mothers suffered from at least one dead pup, with several

Feeding behavior
Age-matched females (to those used in maternal studies) and surviving offspring were separated into single house feeding chambers (TSE Systems) and acclimated for 5 days prior to feeding behavior testing. Food and water intakes were obtained every 60 seconds. Baseline feeding behavior was monitored for 24 hours to assess daily intake patterns. In addition, a preference test between SD and WD was conducted after baseline reading. Animals had free access to either diet for 24 hours and their feeding behaviors were recorded. Feeding parameters included total caloric intake, average meal size, average rate of intake, average number of meals, average meal duration, average post-meal interval, and percentage of total meals between SD and WD (preference test).
Tissue processing Tissue collection. Isoflurane was used to anesthetize animals at time of tissue harvest (0900 to 1100 hours). Blood was collected by cardiac puncture and stored in EDTA-lined tubes on ice. Tubes were centrifuged (1500g for 10 minutes at 4˚C) to obtain plasma. Pancreas was rapidly collected, washed with ice-chilled phosphate-buffer solution (PBS), and flash frozen in liquid nitrogen. Jejunum was rapidly collected, washed with ice-chilled PBS, sliced open longitudinally on a stainless steel plate kept on ice, scraped with a glass slide to separate mucosal layer, then mucosa was flash frozen in liquid nitrogen. All samples were stored at -80˚C until time of processing.
Lipid extraction. Tissues were weighed and homogenized in 1.0 mL of methanol solution containing internal standards: [ 2 H 5 ]-2-arachidonoyl-sn-glycerol (2-AG), [ 2 H 4 ]-arachidonoylethanolamide (AEA), and [ 2 H 4 ]-oleoylethanolamide (OEA) (Cayman Chemical, Ann Arbor, MI, USA). Lipids were extracted using chloroform (2.0 mL) and washed with ultra-pure (0.2 μm filtered) water. Lipids were extracted from plasma using sterile 0.9% saline solution in lieu of water (0.1 mL plasma at expense of saline). Organic phases were collected and separated using silica gel column chromatography as previously described [40]. Eluate was gently dried under N 2 stream Female mice were fed a SD or WD for 10 weeks during the pregestation period, then males were introduced for mating. Females were then mainained on respective diets throughout the entirety of the experiment. Surviving offspring were weaned from dams at day 21 and placed on SD for 10 weeks of body mass monitoring. Behavioral analysis of feeding patterns were made after a 5 day acclimation period. At the conclusion of 10 weeks, tissues in offspring were collected and processed for analysis of endocannabinoid levels and expression of genes for components of the endocannabinoid system. Tissues from dams were collected during the pregestation phase and after 10 weeks on corresponding diet. Behavioral analysis in dams also occurred at this timepoint. (99.998% pure) and re-suspended in LCMS grade methanol:chloroform (9:1) solution [100 μL for plasma, 200 μL for tissue]. 1 μL was injected for analysis by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) as described [40,49].

Gene expression
Tissues were chosen at random to reflect a sample from each distinct litter in each of the two groups (SD vs. WD). Total RNA was extracted from jejunum mucosa and pancreas using QIAzol (Qiagen, Maryland, USA) and RNeasy (Qiagen, Germany) combined method, and generated first strand complementary DNA using M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA). Surfaces for tissue collection and processing were sanitized using 70% EtOH solution followed by RNAse inhibitor (RNAse out, G-Biosciences, St. Louis, MO, USA) to maintain integrity of isolated RNA. Reverse transcription of RNA was performed with random hexamers (Invitrogen, Carlsbad, CA, USA) for 50 minutes at 37˚C, RT-PCR was carried out using PrimePCR assays (BioRad, Irvine, CA, USA) with primers for cannabinoid receptor 1 and 2 (Cnr1, Cnr2), diacylglycerol α and β (DAGLα, DAGLβ), monoacylglycerol lipase (MGLL), N-acyl phosphatidylethanolaminespecific phospholipase D (NAPE-PLD), and fatty acid amide hydrolase (FAAH) using preconfigured SYBR green assay (BioRad, Irvine, CA, USA). Reactions were run in triplicate. Hprt was selected as a housekeeping gene for jejunum mucosa studies; β-actin was chosen as a housekeeping gene for pancreas studies. No changes in expression were found in conditions tested between respective housekeeping genes [Cq values for conditions, n = 5; hprt: SD born offspring, 24.51 ±0.84, WD born offspring, 24.47±0.77, not significant; β-actin: SD born offspring, 25.59±0.56, WD born offspring, 25.12±0.32, not significant].

Statistical analyses
Data was analyzed using Graphpad Prism 7.0 software. Results are expressed as the mean ±S.E. M. Significant differences between groups were assessed using Student's two-tailed t-test or two-way analysis of variance (ANOVA) with repeated or non-repeated measures with Sidak or Newman-Keuls post hoc analysis, respectively. Survival curves were analyzed using Mentel-Cox and Gehan-Breslow-Wilcoxon tests. Differences were considered significant if p<0.05. Grubbs' tests for statistical outliers were performed on data from biochemical analyses. Statistical significance of any parameter was not affected by inclusion of outliers.

Female mice fed Western diet display altered feeding behavior during pregestation phase
Female mice fed Western diet (WD) ad-libitum over a time course of 10 weeks (age-matched to mice used in maternal experiments below), when compared to mice maintained on standard rodent chow (SD), gained weight at a higher rate based on change in body weight [ Total daily caloric consumption trended towards an increase but did not reach statistical significance (Fig 2G), which is in contrast to reports of significant increases in body weight and daily caloric intake in male mice fed a WD versus SD under similar conditions (see our [40]).
These results highlight important sex differences in feeding patterns in mice chronically maintained on WD and may represent differential expression of the eCB system and function among sexes in organs important for food intake and energy balance, including the upper small-intestinal epithelium [35,52] and pancreas [21]. Indeed, when compared to male mice fed WD for 60 days that display increases in levels of the eCBs anandamide and 2-AG in the upper small intestinal epithelium and plasma [40], female mice fed WD displayed decreases and increases in levels of plasma 2-AG ( Table 2, t = 2.654, p = 0.025) and anandamide (Table 2, t = 3.025, p = 0.0128) respectively, and no changes in levels of these eCBs in upper small intestinal epithelium when compared to mice fed SD ( Table 2). Levels of the eCB-related molecule oleoylethanolamide (OEA) was increased in plasma of WD mice (

Female mice fed WD display changes in endocannabinoid and related lipid profiles in plasma and jejunal epithelium during pre-gestation phase
Lipids were extracted from blood plasma, epithelium of the proximal small intestine (jejunum), and pancreas of female mice maintained on WD or SD for 10 weeks (age-matched to mice used in maternal experiments below). Female WD mice, when compared to those fed SD for 10 weeks, had significantly higher levels of plasma AEA (t = 3.025, p = 0.0128) and OEA (t = 3.223, p = 0.01), and reductions in 2-AG (t = 2.654, p = 0.024), but did not display differences in jejunal epithelium or pancreas ( Table 2). Levels of the less characterized DHEA Chronic consumption of western diet results in altered feeding behavior in female mice. Female mice maintained for ten weeks (a, cumulative change in body weight; b, gross body weight) on a western diet (Western) become obese and display increases in 24 h meal size and rate of intake paired with a decrease in meal duration (c-e) when compared to mice maintained on a standard chow diet (Stand). Meal frequency, total caloric intake, dark cycle intake, light cycle intake, and post meal interval do not significantly differ between diets (f-j). Repeated measures two-way ANOVA, with Sidak's multiple comparison post hoc test, ÃÃÃ = p<0.001, ÃÃ = p<0.01, Ã = p<0.05 (a,b); unpaired Student's t-test (two-tailed), ÃÃÃ = p<0.001, Ã = p<0.05 between Stand and Western. Results are expressed as means ± SEM; n = 6 per condition. https://doi.org/10.1371/journal.pone.0205021.g002 Impact of maternal obesity on offspring mortality and peripheral endocannabinoid system PLOS ONE | https://doi.org/10.1371/journal.pone.0205021 October 1, 2018 (t = 3.605, p = 0.005) and DHG (t = 2.963, p = 0.014) were also decreased and increased, respectively, in jejunal epithelium. These results are in contrast to male C57BL/6 mice similarly fed WD for 60 days, which were reported to have increases in plasma and jejunal epithelial 2-AG and AEA (see our [40]). This data highlights important sex differences in the impact of DIO on the peripheral eCB system, which may underlie differential feeding patterns (i.e., total caloric intake) in females (Fig 2) versus males (see our [40]).

Offspring born from DIO mothers have lower rates of survival
Female mice were fed a SD or WD during pre-gestation (10 weeks) and lactation phases (see Fig 1). Neonates born from DIO dams had lower rates of survival when compared to mice born to lean dams (Fig 3A, HR 0.2309; 95% CI, 0.1032-0.5166, p = 0.0004). All mortality occurred within the first six days following birth. Average litter sizes of the total offspring born between DIO and lean mothers did not differ at time of birth (Fig 3B, t = 0.691, p = 0.528). However, the average litter sizes of surviving offspring born from DIO mothers decreased by post-natal day 21 (Fig 3C, t = 3.06, p = 0.012)The remaining offspring that survived the lactation period born from DIO mice had a higher male fraction than surviving pups born from lean mothers (Fig 3D, 60% vs. 45%, respectively).

Surviving offspring display similar feeding behaviors between groups
Male ( Fig 4A) and female (Fig 5A) offspring born from dams fed WD had higher body mass at time of wean when compared to those born from dams maintained on SD; however, body weights rapidly normalized over the monitoring period of WD mice to levels found in offspring born from SD dams. Male offspring did not display a difference in meal size (Fig 4B, t = 1.16, p = 0.244), rate

Surviving offspring display similar endocannabinoid and related lipid profiles irrespective of dam's diet
Lipids were extracted from blood plasma, epithelium of the proximal small intestine (jejunum), and pancreas from male and female offspring from dams maintained on WD or SD for 10 weeks prior to mating and through to weaning ( Table 2). Irrespective of dams' diet, no changes were found in levels of 2-AG, AEA, or OEA in plasma, jejunal epithelium, or pancreas; however, plasma levels of DHG (t = 2.727, p = 0.017) in male offspring born from WD dams, and DHEA (t = 2.955, p = 0.018) in female offspring born from WD dams, were moderately decreased.

Male offspring display changes in expression of mRNA for select endocannabinoid system components in jejunal epithelium
We next evaluated expression of mRNA for components of the eCB system in jejunal epithelium and pancreas from male ( Fig 6) and female (Fig 7) offspring from dams maintained on WD or SD for 10 weeks prior to mating and through to weaning. Expression of mRNA for the monoacylglyerol (e.g., 2-AG, DHG) degradative enzyme, monoacylglycerol lipase (MGL), was decreased in jejunal epithelium of male mice born from WD dams (Fig 6A), when compared to those born from SD dam, (t = 3.1, p = 0.017). In contrast, no changes in mRNA expression Surviving male mice born from DIO or lean dams do not display differences in feeding behavior. Male offspring weaned from dams fed a standard chow diet (SD) or a Western diet (WD) were given ad libitum access to standard chow for 8 weeks. Body weights were monitored and feeding behavior was tested. WD offspring had higher body mass at time of wean but body weights were rapidly normalized over the monitoring period (a). There were no differences meal size, rate of intake, meal duration, meal frequency, total caloric intake, dark cycle intake, light cycle intake, or in post meal interval (b-i). The offspring also displayed no difference in preference when given the choice between consuming a standard chow and highly palatable western diet (j). Data analyzed using repeated measures two-way ANOVA, with Sidak's multiple comparison post hoc test, ÃÃ = p<0.01, ns = p>0.05 (a); unpaired Student's t-test (two-tailed), ns = p>0.05 between SD and WD (b-j). Results are expressed as means ± SEM; n = 8/9 (SD/WD). Surviving female mice born from DIO or lean dams display a small decrease in rate of intake. Female offspring weaned from dams fed a standard chow (SD) or a Western diet (WD) were given ad libitum access to standard chow for 8 weeks. Body weights were monitored and feeding behavior was tested. WD offspring had higher body mass at time of wean but body weights were rapidly normalized over the monitoring period (a). There were no differences meal size (a) but slight decrease in rate of intake between groups (b). Further, there were no differences in meal duration, meal frequency, total caloric intake, dark cycle intake, light cycle intake, or in post meal interval (d-i). The offspring also displayed no difference in preference when given the choice between consuming a standard chow and highly palatable western diet (j). Data analyzed using repeated measures two-way ANOVA, with Sidak's multiple comparison post hoc test, ÃÃ = p<0.01, ns = p>0.05 (a); unpaired Student's t-test (two-tailed), ns = p>0.05 between SD and WD (b-j). Results are expressed as means ± SEM; n = 8/4 (SD/WD). https://doi.org/10.1371/journal.pone.0205021.g005 Impact of maternal obesity on offspring mortality and peripheral endocannabinoid system (LE). In female jejunal epithelium, (Fig 7A), no changes were found in Cnr1 (t = 0.331, p = 0.

Discussion
These studies suggest that maternal diet is an important predictor of survival in neonate C57BL/6Tac mice, and maternal WD is not associated with disruptions in feeding patterns or marked abnormalities in eCB system expression or function in jejunal epithelium, pancreas, or plasma in surviving offspring maintained on a SD for 10 weeks. DIO is associated with a host of metabolic abnormalities that include type-2 diabetes [5,6]. Obesity rates have increased over the past several decades in human females of child-bearing age, and obesity during gestation is associated with abnormal metabolic profiles in offspring, which may include an epigenetic component in these outcomes [53]. Thus, research aimed at exploring the impact of maternal diet on feeding behavior and glucose homeostasis is critical. In the present study, chronic exposure to WD greatly influenced offspring survival within the first 6 days following birth. Female mice were maintained on a WD for 10 weeks, at which time they displayed significantly increased body weight gain and altered feeding behaviors.
Maternal obesity led to high rates of neonate mortality, with a higher male fraction in surviving pups. Our results are consistent with reports of increased mortality in rat pups born from mothers that were exposed to a highly-palatable "cafeteria diet" (i.e., chocolate candy bars) for eight weeks prior to mating and through to weaning [54]. Importantly, however, we found a much higher rate of mortality in our studies (above 50% mortality by 6 days post-birth in pups born from WD dams) when compared to studies by Ramirez-Lopez and colleagues (4.3% pups died at birth and about 10% during lactation by 21 days) [54]. It is plausible that dietary composition plays a large role in these effects and WD in our studies, which is high in milk fat (40% total diet kcals) and sucrose (29% total diet kcals), leads to considerably higher neonate deaths. A comprehensive analysis of the impact of specific dietary components (i.e., various types of fats and carbohydrates) on neonate survival in rodents remains.
Surviving offspring were then monitored for 10 weeks and feeding behaviors were assessed. Surviving offspring did not display notable differences in body weights or feeding parameters at 10 weeks of age (i.e., total caloric intake, meal size, meal duration, meal frequency, rate of intake, or preference tests). Other groups report increased body weights and food intake in mice or rats born from dams maintained on high-fat diets versus standard diets, but these effects were mostly apparent after 8-10 weeks post-weaning [13,17,55]. Thus, changes in feeding behaviors and body weight change under our conditions may occur at later timepoints from those included in our analysis (i.e., 10 weeks post-weaning). Furthermore, in contrast to our findings in mice that offspring born from dams maintained on WD or SD displayed no differences in preference for WD during a 24-h test, rats born from dams maintained on a "cafeteria diet" were reported to display "an exacerbated preference for fatty, sugary and salty foods at the expense of protein-rich foods" [56]. These results may reflect important species differences and differential impact of specific nutrients on palatable food preference. Future studies should include tracking the impact of maternal DIO on feeding patterns of offspring born from WD or SD dams over extended periods of time (i.e., greater than 10 weeks).
We next analyzed levels of eCBs (i.e., the fatty acid ethanolamide, AEA, and the monoacylglycerol, 2-AG) and related fatty acid ethanolamides (i.e., OEA and DHEA) and monoacylglycerols (i.e., DHG) in plasma, upper small intestinal epithelium, and pancreas. We found no changes in the eCBs in organs tested between groups born from dams chronically fed WD or SD, and modest reductions in levels of DHG in male plasma and DHEA in female plasma of those born from WD dams. DHG and DHEA are synthesized from the omega-3 fatty acid, docosahexaenoic acid (DHA, 22:6n-3). Their physiological relevance, however, is not wellestablished but may include roles in inflammation and neural development [57,58]. It is plausible that lower levels of these molecules in plasma may play a role in systemic inflammation associated with DIO; however, a direct test of this hypothesis remains. Furthermore, we found no appreciable changes in expression of eCB biosynthetic and degradative enzymes in small intestinal epithelium and pancreas of males and females, with the exception of a small but significant reduction in expression of the 2-AG degradative enzyme, MGL, in males. The latter effect suggests a possible remodeling of the eCB metabolic machinery in the small intestinal epithelium in male mice born from mothers maintained on WD; however, no changes in levels of select monoacylglycerols (i.e., 2-AG and DHG) were found, which raises the question of the physiological significance of this change in expression of MGL. A complete analysis of the expression in tissue of a wider variety of monoacylglycerols under our conditions is warranted.
Notably, we analyzed eCB expression in organs of pups 10 weeks after weaning and maintenance on SD. Thus, we cannot rule out that changes in expression or function of the eCB system in pancreas and small intestinal epithelium occur in pups born from WD mothers at earlier time points and is transient, or at later time points from our analysis of eCB system expression and feeding behavior in pups (i.e., 10 weeks after weaning). Indeed, other groups reported sex-specific changes in eCB system expression in white and brown adipose depots in rat pups born from mothers maintained on a high-fat diet versus standard; however, in contrast to our experiments, their analysis occurred immediately after weaning [15]. Similarly, Ramirez-Lopez and colleagues reported reductions at birth in levels of the eCBs, AEA and 2-AG, in the hypothalamus of male rats born from mothers maintained on a high-calorie test diet versus standard chow during the pregestational and gestational periods [54]. Furthermore, rats born to mothers maintained on either a palatable chocolate diet in combination with standard chow (ad-libitum access to both) or control rats given access to only standard chow for 8 weeks displayed very small decreases in body weight gain over a period of nineteen weeks [16]. The authors then analyzed expression of eCB system components in brain, liver, and adipose tissue in these rats at nineteen weeks postnatal and found a variety of sex-specific changes in expression of mRNA for several components of the eCB system (see for details [16]). Collectively, these studies reveal possible changes in eCB system function or expression in several organs immediately after birth [54] or weaning [15], and at much later timepoints (i.e., nineteen weeks postnatal [16]) in offspring born from dams consuming high-energy diets. Furthermore, under our conditions, similar expression and function of the eCB system between groups in the upper small intestinal epithelium-particularly given its critical role in feeding behavior [45][46][47]49]-may underlie a lack of change in feeding patterns or palatable food preferences observed between mice born from WD or SD dams at the time point analyzed in our experiments.

Conclusions
Our studies reveal large increases in mortality in neonates born from dams maintained on a WD for 10 weeks before mating, during gestation, and through to weaning of pups. Mortality was restricted to the first six days after birth. Furthermore, changes in eCB system function and expression in these offspring, when compared to dams maintained on a control SD, were largely absent at 10 weeks post-weaning in small-intestinal epithelium, pancreas, and plasma.
Future studies under our conditions should include a comprehensive temporal evaluation of eCB system expression and function in small intestinal epithelium, pancreas, and plasma of pups born from mothers maintained on WD and SD, from immediately after weaning through to time points after 10 weeks post-weaning. Furthermore, despite a lack of marked changes in eCB system profile or feeding behavior in offspring born from dams maintained on WD, it is important to consider that behavioral and biochemical analysis of the animals tested under our conditions were performed on surviving pups, which may be considered "extraordinary". It is possible that the neonates that died within six days following their birth had significant changes in eCB system and other regulatory factors affecting feeding and glucose homeostasis that led to their failure to thrive. A test of this hypothesis, however, remains, but is difficult given the inability to predict when mice will die, and which mice will survive.


extfieldsofstudy:
Materials Science
Medicine

text:
Simultaneously Measuring Image Features and Resolution in Live-Cell STED Images

Reliable interpretation and quantification of cellular features in fluorescence microscopy requires an accurate estimate of microscope resolution. This is typically obtained by measuring the image of a nonbiological proxy for a point-like object, such as a fluorescent bead. Although appropriate for confocal microscopy, bead-based measurements are problematic for stimulated emission depletion microscopy and similar techniques where the resolution depends critically on the choice of fluorophore and acquisition parameters. In this article, we demonstrate that for a known geometry (e.g., tubules), the resolution can be measured in situ by fitting a model that accounts for both the point spread function (PSF) and the fluorophore distribution. To address the problem of coupling between tubule diameter and PSF width, we developed a technique called nested-loop ensemble PSF fitting. This approach enables extraction of the size of cellular features and the PSF width in fixed-cell and live-cell images without relying on beads or precalibration. Nested-loop ensemble PSF fitting accurately recapitulates microtubule diameter from stimulated emission depletion images and can measure the diameter of endoplasmic reticulum tubules in live COS-7 cells. Our algorithm has been implemented as a plugin for the PYthon Microscopy Environment, a freely available and open-source software.

INTRODUCTION
Fluorescence microscopy images never represent the underlying object perfectly, failing to discern details smaller than a certain size. This imperfection is described by the system's point-spread function (PSF). Knowledge of the PSF is essential when interpreting the images produced and in ensuring that quantitative measurements are accurate. For many purposes, it is sufficient to summarize the effects of the PSF in a simple resolution metric (e.g., the full-width at half maximum (FWHM)). A popular method for obtaining the PSF FWHM is extracting an intensity line profile from a fluorescent bead image and either directly measuring the FWHM or estimating it more accurately by fitting a Gaussian or Lorentzian (in the case of stimulated emission depletion (STED)) model to the profile. For diffractionlimited microscopes, beads can be regarded as point-sources because they are significantly smaller than the FWHM of the PSF (beads are typically 20-100 nm compared to the $250 nm PSF FWHM), and the fit FWHM is taken to be that of the PSF.
When considering STED microscopy, where the PSF FWHM is typically 25-70 nm, the assumption that the PSF is much larger than the bead size is no longer valid, as beads whose size is similar to that of the PSF are often needed to achieve reasonable signal levels. The resolution in STED microscopy is also strongly affected by laser powers, sample-induced aberrations, and the depletion cross-section of the dye, which is additionally dependent on the local environment, making beads a poor proxy for the true resolution achieved when imaging cellular samples.
Measuring STED resolution on a target in the same cellular environment labeled with the same dye(s) and imaged with the same laser powers avoids these issues. Microtubules labeled with the same fluorescent dye as the final target structure are an attractive candidate that can be readily prepared. The simplest and most common labeling protocol that usually results in bright stainings is indirect immunofluorescence. Labeling the 25-nm outer diameter of a microtubule with primary and secondary antibodies results in a structure that is 60 nm in diameter as observed using electron microscopy (1). However, this is within the size range of a STED PSF, so as with beads the thickness of the structure is nonnegligible when quantifying the resolution of a microtubule image. To determine the impact finite object size has on resolution quantification using the popular Gaussian-and Lorentzian-fitting techniques, we simulated intensity line profiles perpendicular to the long axis of antibody-labeled microtubules imaged at various resolutions and fit them with Gaussian and Lorentzian functions.
We modeled the fluorophore distribution for the primary and secondary antibody labeled microtubule as an annulus of 25-nm inner diameter and 60-nm outer diameter, as measured for densely-labeled microtubules (1) (Fig. 1 A,  top). For most STED microscopes, the axial (z) PSF FWHM is considerably larger (500-700 nm) than the FWHM along the lateral (xy) directions. This means that the entire cross-section of a microtubule is effectively summed along the axial dimension during imaging, producing the red dash-dot curve in Fig. 1 A. The imaging process is simulated by convolving the fluorophore distribution with the PSF model ( Fig. 1 A, teal dashed curve). For these simulations, we modeled the PSF as a Lorentzian, a common STED PSF approximation (2,3) that represents our experimental PSF better than a Gaussian (see Fig. S1 in the Supporting Material). The resulting cross section of a microtubule imaged with a 50-nm FWHM PSF is shown in Fig. 1 A (green solid curve).
We simulated line profiles across microtubules imaged with STED resolutions ranging from 20 to 100 nm (PSF FWHM) with added shot noise and fit them with simple Lorentzian and Gaussian functions. The FWHM of the Lorentzian fits, and in particular the Gaussian fits, were sub-stantially larger than the PSF FWHM they were simulated with ( Fig. 1 B), confirming that simple fitting of these models does not result in an accurate resolution measure. The accuracy deteriorates, as expected, at higher resolutions (smaller PSF FWHM), making it particularly problematic for STED microscopy, in which systematic errors of 100% can easily occur.
Similar Gaussian or Lorentzian fits in which the width is interpreted as the size of an imaged structure rather than resolution are popular in various types of fluorescence (superresolution) microscopy. Interpreting Fig. 1 B this way shows that this approach only yields reasonable results when the PSF is much smaller than the imaged structure. We conclude that using the FWHM of Gaussian or Lorentzian fits is an unreliable measure for both resolution and feature size quantification.
As an alternative to fitting of line profiles, Fourier ring correlation, a powerful model-free method of measuring relative resolution in images of similar objects (4,5), has been used to quantify STED resolution (6). Unfortunately, Fourier ring correlation results are highly dependent on the structure being imaged, subject to the same limitations relating to object dimensions as simple profile fits, and are not directly translatable into FWHM values.
In order to accurately determine microscope resolution or feature size from line profile cross-sections, both the microscope PSF and the geometric distribution of fluorophores on the labeled structure must be modeled in the function used for fitting. Previous efforts have held-fixed one of these parameters, assuming that either the PSF or the structure size is already known and enforcing this assumption either during fitting (7) or in a simulation to quantify the FWHM bias of the fit (8). However, these approaches are limited because in biological STED microscopy, both structure size and resolution are typically unknown. Fitting both of these parameters simultaneously, on the other hand, is difficult; increases in either parameter give rise to an increased profile width, albeit with subtly different effects on profile shape. At the signal-to-noise (SNR) level typical of a single profile, it is difficult to separate the effects of the two parameters. This coupling can result in inaccurate estimates for both values. Here, we present a tool that overcomes this challenge and allows simultaneous fitting of structure size and PSF width.

Ensemble PSF Fitting
We enable robust simultaneous determination of PSF width and structure size by fitting multiple line profiles as an ensemble, exploiting prior knowledge that the PSF width should be the same for each profile. We accomplish this by performing a two-layer nested fit, such that in the inner fit, all tubules are fit with the same PSF FWHM, g, and the mean squared error (MSE) for each tubule is reported.
The MSE averaged over all tubule fits as a function of g has a propensity to be well behaved and smooth ( Fig. 1 C). The outer fit is then responsible for finding the value of g which minimizes the mean MSE. This technique, which we refer to as nested-loop ensemble PSF (NEP) fitting, constrains the fit enough that accurate PSF widths and microtubule diameters can be determined, as shown in Fig. 1, B and D. NEP fitting using the antibody-coated tubule model yielded significantly better results than fitting with plain Gaussian or Lorentzian functions, and the PSF widths calculated by the fit are in close agreement with the ground truth ( Fig. 1 B). Simultaneously, it yielded accurate measures of the simulated microtubule diameter, 25 nm, for all simulated PSFs with FWHM equal to or less than 60 nm, which is the value at which the PSF FWHM becomes larger than the outer diameter of the antibody coat (Fig. 1 D). For structures whose size does not vary in a cell (e.g., microtubule diameters), the structure size can additionally be constrained as an ensemble parameter during the fit, although we did not find this to be a necessary step.
We carried out additional simulations to determine the robustness of NEP fitting on low SNR data as well as data obtained with aberrated PSFs. Not surprisingly, the variability in fit tubule diameters for individual profiles is increased at lower SNRs (Fig. S2), yet NEP fitting estimates for tubule diameter and PSF width do not suffer from systematic errors. Systematic changes in initial fit parameter estimates led to negligible differences in the fit outcomes (see Fig. S3), demonstrating the robustness of the nested fit approach. We then simulated STED PSFs with various aberrations and found that sizes of the convolved structures were still accurately determined (Fig. S4). Additionally, the PSF width estimates from NEP fitting were in good agreement with the FWHM of the simulated (aberrated) PSFs (Fig. S5).

Software and Validation
We implemented NEP fitting for STED images of labelfilled or surface-labeled cylindrical structures in the PYthon Microscopy Environment (PYME). Line profiles of a userdefined width are extracted from images loaded into PYME, after which they can be fit using a variety of model functions. Alternatively, they can be saved or appended to two file formats (Hierarchical Data Format, JavaScript Object Notation) for later analysis or ensemble fitting with profiles from multiple images. Fitting performed through the graphical user interface (GUI) saves results to an Hierarchical Data Format table and generates an HTML report (see Fig. S6 for an example report). The line profile extraction GUI is shown in Fig. 2 A. STED PSF width is dependent on the STED laser power with a scaling of a= ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 þ I=I s p , where I is the depletion intensity and a and I s are constants (9). To test the efficacy of ensemble fitting on real data, we imaged primary and secondary antibody-labeled microtubules using a Leica SP8 STED 3X microscope (Leica Microsystems, Wetzlar, Germany) at different STED laser powers. Fig. 2 C shows that our fit is responsive to changes in PSF size and can reproduce the expected scaling of PSF width with respect to the STED laser power.
To determine if our NEP fitting approach in which the PSF width is constrained to a single, global value for all profiles is beneficial, we compared its results with those obtained when both PSF width and tubule diameter were optimized on a per-profile basis. We performed this comparison on images recorded with 111-mW depletion power. Although fits performed without an ensemble PSF have more degrees of freedom and therefore usually yield smaller residuals, they come at the expense of accuracy in the measured values. This can be seen in Fig. 2 D, where the standard least squares fit results in an average microtubule diameter of 30 5 13 nm (mean 5 standard deviation (SD)), compared to the NEP-fitted value of 28 5 8 nm (mean 5 SD). NEP fitting, with its global PSF constraint, improves the measurement of the microtubule diameter, as evident from the reduced spread in tubule diameters and average value closer to the expected 25 nm. The PSF FWHM of standard least squares fitting was 45 5 10 nm (mean 5 SD), compared to the NEP-fitted value of 44 nm.
We note that fitting a single line profile would not provide a reliable measure of either PSF FWHM or tubule diameter and could lead to relative errors of 100% for both values. The minimum number of profiles necessary for robust NEP fitting depends on the fluorophore distribution and the relative PSF size. However, even for cases of low SNR, we found 100 profiles to be sufficient for the fluorophore distributions tested (see Fig. S2). To improve performance in low SNR images, we recommend averaging the line profiles over several pixels width during extraction (as can be done by entering, for example, 10 pixels for the ''line width'' in the GUI as described in the Supporting Material). This measure, which averages signal along a segment of the tubule, is also effective at reducing any discrepancies between the modeled and true fluorophore distribution that might occur as a result of sparse labeling.

Application to live-cell images
Although the robust PSF measurement in fixed cells by NEP fitting is a substantial improvement over bead calibrations, a large advantage of NEP fitting is that it can be performed on live-cell data for in situ resolution calibration in the most biologically relevant state. We applied ensemble PSF fitting in live-cell STED images of label-filled or membrane-labeled endoplasmic reticulum (ER) tubules using SNAP-KDEL or SNAP-Sec61b, respectively (Fig. 3, A-D). In order to fit the label-filled tubules, we modeled the fluorophore distribution perpendicular to the long axis of the tubule as a filled circle, which projects as 2 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi , where R is the radius. We then convolved this tubule profile with a Lorentzian to account for the imaging process. For membrane-labeled tubules, we modeled the fluorophore distribution as an annulus, like the antibodylabeled microtubules only with a thinner coat. SNAP-tag (10) is $4 nm in diameter, and the organic dye itself can be estimated to have a radius of 0.5 nm, assuming they are both globular (11), resulting in a 4.5-nm thick annulus. Fits of ER tubule diameter for various test PSF widths show stronger coupling between the tubule diameter and PSF width for label-filled tubules than membrane-labeled tubules (Fig. 3, E and F). The PSF width results from the NEP fits were, however, very similar for label-filled and membrane-labeled profiles (45.8 and 43.7 nm FWHM, respectively) as shown in Fig. 3, G and H.
Notably, the SD for both the label-filled and membranelabeled ER tubule diameters is fairly large: 30 and 15 nm, respectively (with mean values of 132 and 101 nm). To test whether this variability in tubule diameter is primarily biological in nature or dominated by the SNR-limited fit precision, we simulated tubule profiles of known diameter with similar SNRs, convolved with 50-nm FWHM Lorentzians. The distributions of fitted diameters were narrower than those observed in the live-cell images, with SDs of only 5 nm for the membrane-labeled tubules and 12 nm for the label-filled tubules (Fig. S7). The larger range of label-filled tubule diameters is expected because the fluorophore distribution orthogonal to the long axis of the tubule looks more similar to the PSF than a surface-labeled fluorophore distribution. This is reflected in the live-cell data, where the tubule diameter is more strongly coupled to the PSF width for the label-filled tubules, as shown in the heat-maps of tubule diameter histograms when fit with various fixed PSF widths (Fig. 3, E and F). Since the standard deviations add in quadrature, we expect roughly 80% of the spread in tubule diameter to be biological in origin.

Discussion
Traditional methods of resolution calibration in STED microscopy are problematic for biological quantification. The NEP fitting method introduced in this article provides a robust and practical alternative to both quantify the performance of a microscope as well as improve feature measurements within the image. Its implementation in a freely downloadable, open source, cross-platform software package allows for rapid adoption by others without requiring mathematical or programming expertise. The principle of ensemble fitting can be readily extended to other fluorescence microscopy modalities (e.g., confocal) by substituting a different functional representation of the PSF when  producing the model function for fitting; the only requirement is that the labeling geometry of the structure is known. This known geometry is not limited to tubules and can be extended to fit objects like beads or vesicles, which would be useful for cell-trafficking studies. The accurate measure of PSF width afforded by NEP fitting can be used to quantify microscope performance under various conditions, refine models of organelle morphology, and remove uncertainty in parameter selection for deconvolution or other image enhancement algorithms.

Software availability
All line profiles were drawn, extracted, and fit using the open source PYME and the NEP fitting plug-in, which are both freely available (12,13). Supplemental data are provided alongside this article for readers to test the program.