Key aspects of the cycling of refractory dissolved organic carbon (DOC) remain unknown, making it challenging to predict how this large carbon reservoir will respond to climate change. Herein we investigate mechanisms that remove refractory DOC using bioassay experiments with DOC isolated from surface, mesopelagic and deep waters of the Atlantic Ocean. The isolated DOC was refractory to degradation by native microbial communities, even at elevated concentrations. However, when the refractory DOC was introduced to a series of novel environmental conditions, including addition of a labile substrate, a microbial community from coastal waters and exposure to solar radiation, a substantial fraction (7–13%) was removed within 1.5 years. Our results suggest that while refractory molecules can persist in the ocean for millennia, removal is rapid when they encounter their fate. The observed and projected climate-induced slowdown of global overturning circulation could reduce the exposure of refractory molecules to disparate removal processes. Assuming a constant rate of production, the reservoir size of refractory DOC could increase as overturning circulation slows, providing a negative feedback to rising atmospheric CO Investigations of the chemical nature of DOC reveal an incredibly diverse and complex mixture of predominantly small molecules (<1 kDa) with an average radiocarbon age of about five thousand years5. This long-lived DOC reservoir sequesters carbon dioxide from the atmosphere and likely plays an important role in shaping global climate, but little is known about the stability of the reservoir. Seawater bioassay experiments provide insight about the microbial utilization of DOC, and overall they indicate most marine DOC is very resistant to degradation and is considered to be refractory7. The long-term persistence of DOC in the ocean is widely recognized, but the mechanisms responsible for its removal and the processes regulating the size of this large carbon reservoir are not well understood8.A vast reservoir (~660 Pg) of dissolved organic carbon (DOC) resides in the ocean, and the cycling of this carbon remains an enigma after decades of inquiry10, the intrinsic composition and structure of molecules12 and low concentrations of molecules that are below a threshold for microbial utilization14. Diverse and versatile microorganisms distributed throughout the ocean are the primary consumers of DOC, and it appears abiotic processes also play an important role in the removal of refractory molecules. Photochemical processes degrade and alter molecular structures of DOC in ocean surface waters15, and elevated temperatures in hydrothermal vent systems pyrolyze DOC in deep waters17. Thus, the removal of DOC from the ocean appears to occur through disparate processes that are heterogeneously distributed in space and time and connected by physical transport processes.The persistence of marine DOC has been related to environmental conditions and microbial communitiesBuilding on insights from prior studies, we examined the removal of refractory DOC under varying environmental conditions using experimental approaches. Solid phase extraction C-18) was used to isolate refractory molecules from surface (50 m), mesopelagic (500 m), and deep (1200 m) waters of the Atlantic Ocean. Bioassay experiments with concentrated C-18 extracts were used to verify the refractory nature of the isolated DOC, followed by a series of manipulations to investigate the removal of refractory DOC under different scenarios, including substrate priming and exposure to irradiation and distinct microbial communities  and potentially labile DOC in the deep Atlantic Ocean18. Sinking particles formed in productive surface waters are likely sources of labile DOC in mesopelagic and deep waters where solubilization processes release bioavailable dissolved organic matter (DOM)19. The bulk (≥90%) of the DOC throughout the water column remained at the end of the incubations, confirming the current view that the majority of natural DOC is very resistant to microbial degradation.Bioassay experiments with seawater collected from three depths in the Atlantic Ocean revealed a wide range of DOC reactivity Fig. . About 120. Carboxyl-rich alicyclic molecules (CRAM) are a diverse mixture of carboxylated molecules that comprise about 8% of the DOC in seawater11. The CRAM components of DOC appear to be old and refractory components of the deep ocean DOC reservoir21. The C-18 extraction process recovered 29–30% of the DOC from surface, mesopelagic and deep waters. The average C/N of the isolated DOM (36.6 ± 1.3) was similar to that of humic substances extracted from seawater using XAD-2 resin20 and is indicative of CRAM and other N-depleted aliphatic molecules.Solid phase extraction (C-18) preferentially isolates nonpolar compounds commonly found in marine humic substances, such as carboxylated aliphatic moleculesin situ concentrations. The concentrations of the isolated DOC showed minor changes throughout the incubations at all depths , which were ~10-fold greater than ths Fig. , in cont13 examining the microbial utilization of high concentrations of DOC extracted from deep waters using a modified styrene-divinylbenzene polymer (PPL), which isolates more polar molecules than C-18. Microbial growth was observed to increase with increasing concentrations (up to 10-fold) of PPL extracted DOC13, thereby providing evidence that the concentrations of some components of DOC were below threshold concentrations required for microbial utilization and growth14. The PPL extraction used by Arrieta et al.13 recovers a greater fraction of seawater DOC (≥40%) than the C-18 extraction (≤30%) and includes more N-containing molecules compared to the C-18 extraction22. As demonstrated in the present bioassay experiments with unamended seawater, ~3% of the DOC in deep water was labile and rapidly utilized by microbes  was added to replicate experiments to investigate the priming effect on refractory DOC removal. Glucose utilization occurred during the initial stage of the experiments  in ultraviolet light absorption (a350) and an increase (>50%) in the spectral slope coefficient (S275-295)  to unfiltered seawater collected from five depths (50–1500 m) at the Bermuda Atlantic Time-Series Study (BATS) site  in concentrations of C-18 DOC at three of the five depths , indicating potential removal of refractory DOC when exposed to different microbes and environmental conditions.The removal of refractory DOC was further investigated using a different experimental design. The C-18 isolated DOC from deep water (1200 m) was added  and the collective metabolic activities of various microbial specialists48.Marine heterotrophic microbes have dual roles as consumers and producers of DOC in the ocean carbon cycle. Most of the DOC they consume is respired to carbon dioxide, but a small fraction is transformed into refractory DOC that sequesters carbon in the ocean for ages, a process known as the microbial carbon pump39. Mixing exposes refractory molecules to varying environmental conditions and microbial communities, and it facilitates their degradation and removal , mesopelagic (500 m), and deep waters (1200 m) using Niskin bottles mounted on a CTD rosette sampler. At each depth, 80 L of seawater was collected in acid-cleaned high-density polyethylene carboys for solid-phase extraction, 250 ml of seawater was collected in clean (400 °C for 4 h) Kimax glass bottles (3 replicates per depth) for microbial degradation experiments, and 120 ml of seawater was collected in clean glass vials for a microbial inoculum.Sampling was conducted in the North Atlantic Ocean  on the R/V −1 sulfuric acid. Acidified samples from each depth were passed through C-18 cartridges  at a flow rate of 50 ml min−1 and 20 L per C-18 cartridge. Cartridges were pre-conditioned with methanol (HPLC grade) and Milli Q water (pH 2.5) immediately prior to use. The extracted DOC was eluted from each cartridge in 30 ml of methanol and dried under a gentle stream of N2. The C-18 extraction recovered 29 ± 1% of the bulk DOC and 53 ± 6% of the CDOM absorption coefficient at 350 nm (a350).Water samples for solid-phase extraction were filtered onboard through 0.2 µm Nuclepore polycarbonate cartridge filters (pre-rinsed with Milli Q water and seawater) and acidified to pH 2.5 with 6 mol LMicrobial utilization of bulk DOC was determined using bioassay experiments with unfiltered seawater. Water samples (~250 ml) were incubated in glass bottles at room temperature (21–24 °C) in the dark immediately after collection. Subsamples for DOC analysis were taken on days 0, 20, 40, 90, and 190. All experiments in this study were conducted with 3 replicates.et al.43. Six replicate clean Kimax glass bottles, each containing 300 ml of ASW, were used for each depth. Dried C-18 DOC from 50 m, 500 m, and 1200 m was re-dissolved in ASW, passed through a glass fiber filter , and mixed into the bottles at final concentrations of 707, 508, and 535 µmol C L−1, respectively. Inorganic nutrients (NaNO3 and KH2PO4) were added at final concentrations of 48.5 µmol N L−1 and 3.0 µmol P L−1. Natural microbial inocula collected from these depths were added to the corresponding bottles at a 1:50 (v/v) dilution within a week of collection.Biological and photochemical degradation of refractory DOC were determined in a series of laboratory experiments  and priming groups (P) as in Eq.  of glucose is transformed into refractory DOC during microbial utilization and production41. Therefore, the net change (%) calculated using Eq. (The six replicates at each depth were divided into two groups: a control group and a priming group amended with ~120 µmol C LThe experiments were ended after 146 days by passing the samples through Supor membranes (0.2 µm pore-size) to remove microbial populations. Filtered seawater from the priming group received a freshly collected coastal microbial assemblage , and was incubated in the dark at room temperature (22–24 °C) for another 346 days . The irradiation was conducted in an Atlas Suntest XPS+ solar simulator at 750 W mI) from values in the dark controls  for 346 days  were negligible throughout the measurements56. Deep-sea reference standards were injected every 6th sample to monitor the accuracy and consistency of the analysis. The coefficient of variation among injections of a given sample was typically within ±1%.Samples for DOC and CDOM analyses were filtered through GF/F filters (400 °C for 4 h) immediately after collection. Concentrations of DOC were measured by high temperature combustion using a Shimadzu total organic carbon TOC-V analyzer equipped with an autosamplerAλ) at a wavelength of λ was blank corrected (minus the average absorbance between 690–700 nm) and converted to a Napierian absorption coefficient, aλ (m−1): aλ = 2.303 Aλ/0.01. The spectral slope coefficient (S) was calculated as the slope of the linear regression of log-linearized aλ: aλ1 = aλ2 exp [−S (λ1 − λ2)], where aλ1 and aλ2 are absorption coefficients at wavelengths of λ1 and λ2. The spectral slope coefficient between 275 and 295 nm is reported as S275–295 with a unit of µm−1.Absorbance spectra (250–800 nm) were measured using a Shimadzu ultraviolet-visible 1601 dual beam spectrophotometer and 1-cm path length quartz cuvettes. The absorbance .Supplementary material


Recently, coherent control of the optical response of thin films in standing waves has attracted considerable attention, ranging from applications in excitation-selective spectroscopy and nonlinear optics to all-optical image processing. Here, we show that integration of metamaterial and optical fibre technologies allows the use of coherently controlled absorption in a fully fiberized and packaged switching metadevice. With this metadevice, which controls light with light in a nanoscale plasmonic metamaterial film on an optical fibre tip, we provide proof-of-principle demonstrations of logical functions XOR, NOT and AND that are performed within a coherent fibre network at wavelengths between 1530 and 1565 nm. The metadevice has been tested at up to 40 gigabits per second and sub-milliwatt power levels. Since coherent absorption can operate at the single-photon level and with 100 THz bandwidth, we argue that the demonstrated all-optical switch concept has potential applications in coherent and quantum information networks. Here, the authors show that integration of metamaterial and optical fibre technologies enables all-optical XOR, NOT and AND logical functions that are performed at up to 40 gigabits per second with few femtojoules per bit energy consumption within a coherent fully fiberized network. However, recently it was shown that an effective nonlinear response may be derived from coherent interaction of light with light on linear materials of substantially sub-wavelength thickness7. In contrast to conventional optical nonlinearities, the effect has been shown to allow intensity-independent control over absorption of light, from almost 0% to almost 100%8, with 100 THz bandwidth10 and even for single-photon signals11. The concept has enabled all-optical control of luminescence13, redirection of light15 as well as control of nonlinear16, polarization17 and quantum19 effects in films of nanoscale thickness and excitation-selective spectroscopy20. In particular, it has been predicted that coherent interaction of light waves on lossy ultrathin films could perform signal processing functions7 and proof-of-concept experiments in the static regime have been reported22.All-optical signal processing fundamentally relies on modulation of one optical signal with another. Therefore all-optical logical functions have long been perceived as the exclusive domain of nonlinear opticsHere, we report the realization of a switching network using a fully packaged, fiberized metamaterial device based on this concept. It uses a plasmonic nanostructure of substantially sub-wavelength thickness as a switchable absorber that allows the absorption of one optical pulse to be controlled by another coherent optical pulse. We demonstrate nonlinear input–output characteristics and all-optical operations analogous to logical functions NOT, AND and XOR at both kHz and GHz bitrates, in a fiberized configuration assembled from standard telecoms components, at wavelengths between 1530 and 1565 nm.23, this implies that a planar thin film placed at a node will be perfectly transparent, while the same thin film will be strongly excited if placed at an anti-node. With respect to absorption, which is limited to 50% in planar materials illuminated by a travelling plane wave24, standing wave illumination allows absorption to be controlled from 0 to 100% in the ideal case7. Such performance in the optical part of the spectrum, as is relevant to optical fibre technology, may be approximated with materials that are thin compared to the optical wavelength and exhibit equal levels of transmission and reflection in addition to around 50% travelling wave absorption. This combination of parameters may be achieved for example in nanostructured plasmonic metamaterials8 and 30-layer graphene25.As illustrated in Fig. 8. The dependence of this structure’s transmission, reflection and absorption characteristics on aperture size and geometry26 is well understood, allowing for easy optimization throughout the optical telecommunications bands. Our switch operates at wavelengths around λ = 1550 nm, where its 70 nm thickness corresponds to λ/22. The metamaterial structure was fabricated by thermal evaporation of gold and subsequent focused ion beam milling on a 25 × 25 μm2 area covering the core of a cleaved polarization-maintaining single-mode silica fibre  and equal reflection and transmission for both directions of propagation, both output intensities would be identical and modulated from complete absorption to perfect transmission. Differences between the measured output channels arise in the present case from the asymmetric construction of our metadevice that contains a metasurface fabricated on the glass/air interface of one of the optical fibres. The whole metadevice exhibits about 24% single-beam transmission, 18% (8%) reflection and 58% (68%) losses for a single input signal α (β). However, only part of these losses correspond to metasurface absorption that can be coherently controlled, while other sources of loss include the fibre connections of the metadevice, scattering and unwanted reflections within the microcollimator and fabrication imperfections such as imperfect alignment of the metasurface orientation with the slow axis of the fibres.Figure Iδ depends on the input intensity Iβ while Iα remains constant for various phase differences between the input beams. The measured output intensity Iδ as a function of input Iβ is nonlinear and generally follows the behaviour predicted by Fang et al.7. For input phase differences of less than π/2 it is also nonmonotonic—counterintuitively the output intensity decreases with increasing input intensity and reaches a minimum before increasing when Iβ becomes large. For an input phase difference of π/2, changes in the measured output intensity Iδ are approximately proportional to changes in the input intensity Iβ. For larger input phase differences, Iδ flattens with increasing Iβ, but is steep for small Iβ suggesting possible applications in small signal amplification. Thus, the results presented in Fig. The nonlinear functionality of the switch is illustrated by Fig. θext = π to one of the input signals. Furthermore, a fixed input signal β of + or − could be used to map the phase-modulated signal α to an intensity-modulated signal with (NOT α) or without (IDENTITY α) inversion.All-optical signal processing operations with input/output relations analogous to logical functions may now be realized in the network by exploiting coherent transparency and/or coherent absorption in the metadevice. In what follows, binary logical states encoded in beams of equal intensity but opposite phase in the metasurface plane are denoted '+' and '−', while opposing states encoded as low/high intensity are denoted '0'/'1'. Consider, in the first instance, the case of mutually coherent, binary, phase-modulated input signals + and −: constructive interference of identical bits α and β will lead to coherent absorption on the metasurface, while destructive interference of opposing bits will lead to coherent transparency, producing an intensity-modulated (0/1) output α XOR β. The behaviour of an ideal switch is summarized in Table θext = 0). Input pulses α  will be coherently absorbed resulting in low output . On the other hand, low input signals α  will allow light from input β to reach the outputs . For an ideal metadevice, the expected output intensities are 0 and 25% of the input intensity, respectively; our experimental device achieves about 5 and 23% at a modulation frequency of 10 kHz, which is more than sufficient to distinguish the logical states . For an ideal device, this would lead to 100% output intensity for interaction of two pulses on the metasurface and at least 4× lower output intensity for any other combination of input bits , while the AND function suffers from limited contrast of 6 dB (though in principle without insertion loss) and the NOT function suffers from insertion loss of 6 dB (in principle with unlimited contrast).All-optical signal processing applications based on coherently controlling the absorption of light with light promise extremely high bandwidth and extremely low energy requirements, but they will require mutually coherent signals and phase stability. Mutual coherence is most easily achieved in local systems, where multiple signals are derived from the same seed laser. Indeed, as locally coherent networks become part of the mainstream telecommunications agenda31. Devices based on coherent perfect absorption (as considered here) and lossless devices are limiting cases among a much wider range of possibilities. Beyond all-optical data processing, potential applications of coherent metadevices include small signal amplification and coherence filtering8.It is notable that metasurfaces can be engineered to enable a broad range of switching and control functions, which may be used to implement various signal processing functionalities34.The switching network demonstrated here is one example among many opportunities arising from fibre-integration of metasurfaces, which could also be used to control, for example, focusing, polarization, spectral characteristics, propagation direction and angular momentum of lightIn summary, we report a metamaterial-based fiberized switching network for all-optical signal processing that is compatible with optical telecommunications fibre components. The multi-functional switch can perform effectively-nonlinear signal processing functions including input/output relations analogous to XOR, AND and NOT operations, and the underlying mechanism of coherent transparency and coherent absorption is compatible with single-photon signals and 100 THz bandwidth. We therefore anticipate that such fiberized metadevices may provide solutions for quantum information networks as well as orders-of-magnitude improvements in speed and energy consumption over existing nonlinear approaches to all-optical signal processing in coherent information networks.For the realization of the metadevice, the metamaterial was fabricated on the cleaved end face of a polarization-maintaining single-mode fibre as described above. The metamaterial nanostructure was fabricated with its symmetry axis aligned with the slow axis of the Panda-style fibre. In order to allow metamaterial illumination from both sides, the metamaterial-covered fibre was interfaced with a second polarization-maintaining fibre using a microcollimator arrangement consisting of a pair of microlenses. Specifically, the metamaterial-covered fibre end was inserted into a fibre ferrule and positioned at the focal point of an anti-reflection-coated microlens to fabricate a compact microcollimator that was stabilized by a glass capillary shell. The second cleaved fibre tip was mounted in the same way. Using kinematic mounts, the two microcollimators were aligned with each other by maximizing optical coupling efficiency and polarization contrast. The aligned microcollimator arrangement was then fixed with UV-cured adhesive, stabilized with an outer glass capillary shell and placed in an anodized aluminum enclosure for additional protection Fig. .Iα, Iβ, Iγ and Iδ in Figs. Iα = Iβ was 10 μW. The input signal modulators used were low-loss electro-optical 10 Gbit s−1 phase and intensity modulators (EOspace) driven by a waveform generator (AM300 by Rohde & Schwarz).Experimental characterization was performed using the interferometer arrangement presented in Fig. Iα = Iβ is ~100 μW in the case of Fig. −1 phase and intensity modulators (EOspace) driven by an arbitrary waveform generator (Tektronix AWG7122C) and a radio frequency amplifier (LA Techniques) in the case of Fig. Measurements at GHz frequencies used a fibre-coupled tuneable CW laser (ID Photonics CoBrite-DX4). Here the EDFA-amplified output power was detected by an oscilloscope (Agilent Infiniium DCA-J 86100C) as described in the main text. The peak input power at the metadevice input connector The data from this paper is available from the University of Southampton ePrints research repository: 10.5258/SOTON/D0172.Supplementary Information


Injectable Magnetic Resonance Imaging (MRI) contrast agents have been widely used to provide critical assessments of disease for both clinical and basic science imaging research studies. The scope of available MRI contrast agents has expanded over the years with the emergence of molecular imaging contrast agents specifically targeted to biological markers. Unfortunately, synergistic application of more than a single molecular contrast agent has been limited by MRI’s ability to only dynamically measure a single agent at a time. In this study, a new Dual Contrast - Magnetic Resonance Fingerprinting (DC – MRF) methodology is described that can detect and independently quantify the local concentration of multiple MRI contrast agents following simultaneous administration. This “multi-color” MRI methodology provides the opportunity to monitor multiple molecular species simultaneously and provides a practical, quantitative imaging framework for the eventual clinical translation of molecular imaging contrast agents. Clinical use of these contrast-enhanced MRI scans has further expanded as multiple contrast agents have been approved for specific clinical imaging applications .Over the past 3 decades, Magnetic Resonance Imaging (MRI) has become an essential medical imaging modality due to its exceptional soft tissue contrast and lack of ionizing radiation. Along with a wide variety of endogenous tissue contrast mechanisms, many MRI applications utilize an intravenous injection of an MRI contrast agent  to enable sensitive identification of numerous pathologies such as tumors8, cell receptors10, and other molecular species12. In addition, a number of activatable agents have been described that have different relaxivities based on the local tissue environment in vivo14. In a typical preclinical molecular MRI study, the T1 or T2 relaxation time  is measured dynamically before and after contrast agent administration allowing tracking of the agent’s distribution. These studies often incorporate non-targeted contrast agents  as controls to verify the in vivo molecular specificity of the targeting moiety. Due to the difficulty of previously-developed contrast enhanced MRI strategies to uniquely identify two MRI contrast agents administered simultaneously, the targeted and untargeted contrast agents must be studied in separate imaging sessions and likely in separate animal cohorts17. This significant limitation can result in experimental bias due to phenotypic variation. As such, the development of a “multi-color” MRI methodology to independently monitor simultaneously-administered targeted and control MRI contrast agents, and potentially multiple targeted MRI contrast agents, would significantly improve preclinical molecular MRI studies. This “multi-color” MRI capability would also provide a robust pathway for clinical translation of molecular MRI contrast agents by providing the capability to simultaneously compare the biodistribution of a molecular imaging agent with a conventional clinical MRI agent.With the emergence of the field of molecular imaging, there has been a dramatic increase in the number of MRI contrast agents targeted to proteins1 and r2) shown in equations , each paramagnetic MRI contrast agent still impacts both the T1 and T2 relaxation times. This important factor limits the capability of MRI to independently assess simultaneously-administered contrast agents .The primary limiting factor in the simultaneous assessment of multiple paramagnetic MRI contrast agents is that the unique identification of each agent is challenging. MRI contrast agents directly impact both the T1 and T2 relaxation times according to well-established concentration-dependent linear relationships to their magnetic relaxivities (rquations  and 1b)1 and r2)21 and animal models23. MRF uses a unique acquisition and quantification strategy that combines a priori acquisition parameter variation with a dictionary-based pattern matching algorithm to obtain quantitative assessments of multiple imaging parameters simultaneously. Importantly, MRF has been shown to provide quantitative T1 and T2 maps in 10–50 seconds per imaging slice providing the opportunity to dynamically generate quantitative maps of these two important MRI parameters simultaneously27. In this study, we demonstrate that the framework for simultaneous T1 and T2 assessments provided by MRF can be used to analytically quantify the local concentration of two different MRI contrast agents present at the same time. Herein, we describe a straightforward multiple contrast agent relaxation model (equations (METHODS) that can be used in combination with the rapid, multi-parametric MRF strategy to independently calculate inherently co-registered concentration maps for two MRI contrast agents. These initial in vitro results represent a proof-of-concept study to: (1) validate the multiple contrast agent relaxation model using a 60 MHz magnetic relaxometer; and (2) demonstrate the application of the rapid MRF method to enable simultaneous calculation of concentration maps for two different paramagnetic MRI contrast agents on a clinical 3 T MRI scanner. Overall, these in vitro studies suggest an imaging framework for future in vivo MRI studies to simultaneously quantify multiple contrast agents.The Magnetic Resonance Fingerprinting (MRF) methodology has recently been developed to simultaneously generate inherently co-registered T1 and T2 relaxation time maps in both patientsquations  and 2b)21 and anin vitro phantoms and their respective concentrations of contrast agents is shown in Supplementary Table 2 > 0.993, p < 0.0001). The 60 MHz magnetic relaxivities at 37 °C for the gadolinium agent (slope of the linear correlation lines) were 0.0040 mM−1ms−1 (r1) and 0.0048 mM−1ms−1 (r2). The corresponding magnetic relaxivities for the manganese agent were 0.0054 mM−1ms−1 (r1) and 0.0652 mM−1ms−1 (r2). The magnetic relaxivity values for the 60 MHz relaxometer, as well as the relaxivities measured at 3 T, are shown in Table 0) and 2760 ms (T20), respectively. NMR relaxivity measurements are in reasonable agreement with previously reported results for both the Gd28 and Mn30 contrast agents used here.Figure METHODS) were then used to calculate the gadolinium and manganese concentrations for all of the phantoms (n = 17) using the measured T1 and T2 values for each in vitro sample, the calculated magnetic relaxivities for each contrast agent, and the “non-contrast” T10 and T20 values for the deionized water samples. Plots of the relaxometry-based concentration estimates against the known concentration in each phantom for both gadolinium and manganese chloride are shown in Fig. 2 > 0.998, two-tailed probability p < 0.0001) for both the gadolinium and manganese contrast agents. Importantly, the slopes of these correlations are both near unity (1.003 (Gd) and 0.975 (Mn), respectively) suggesting that the concentration estimates obtained from the multiple contrast agent model results in good agreement with the known concentrations. The results from only the six samples with both agents (Supplementary Table Equations  and 3b)3b)  and the manganese agent . These MRF-based relaxivity values compared favorably to relaxivity values obtained from conventional inversion recovery and single-echo spin echo MRI experiments for both gadolinium  and manganese . Relaxivity results at 60 MHz and 3 T are summarized in Table 31. DC-MRF was also used to obtain T1 and T2 relaxation times for the deionized water sample with no contrast agent .Similar to the relaxometry results above, T1 and T2 relaxation time assessments for gadolinium and manganese containing phantoms (n = 17) were obtained using the MRF method on a clinical 3 T MRI scanner at room temperature. Phantoms scanned contained the same contrast agent concentrations as for the NMR relaxometer experiments described above. This data was acquired using a FISP-MRF acquisition repeated 12 times following repositioning to measure average T1 and T2 values for each phantom. In contrast to the relaxometer studies above, the MRF-based T1 and T2 measurements were obtained simultaneously for all phantoms (n = 17). Figure 2 = 0.9987, two-tailed probability p < 0.0001; Mn: R2 = 0.998, p < 0.0001). The mean DC-MRF concentrations were also in reasonable agreement with actual values as evidenced by the slopes of the correlation lines equal to 0.988 (Gd) and 0.980 (Mn), respectively. Results from the phantoms containing mixtures of both contrast agents are presented separately 3b) are sMean and standard deviations of the gadolinium and manganese concentrations were calculated from the MRF-based concentration maps for the 6 phantoms containing both agents using an ROI analysis. The DC-MRF method resulted in significant differences among all samples for both the gadolinium concentration estimates  and the manganese concentration estimates . Overall, these results suggest that the DC-MRF methodology provides accurate and precise assessments of two paramagnetic MRI contrast agents at the same time.in vitro relaxometry measurements at 60 MHz to evaluate the proposed multiple contrast agent relaxation model for two paramagnetic MRI contrast agents -MRF method to simultaneously measure the concentration of two paramagnetic MRI contrast agents. Herein, we present initial quations  and 2b)in vitro 2 > 0.998, Fig. 2 > 0.998, Figs. ®, gadobenate dimeglumine and MnCl2) used in this study. These results also demonstrate the ability of the model to accurately measure contrast agent concentrations at multiple field strengths. This straightforward linear relaxation model assumes that the two contrast agents impact the overall T1 and T2 relaxation times independently with minimal interactions between the two contrast agents. While this model may be expected to be valid for moderate agent concentrations, high local concentrations of one or both of the contrast agents could result in deviations from the linear model as the agents compete for interactions with the surrounding water molecules. Implicit in this model also is the assumption that the relaxivities (r1 and r2) are distinctly different for the two MRI contrast agents. Therefore, this relaxation model may have limitations if used to detect two MRI contrast agents with similar r1 and r2 values ). Furthermore, multiple follow-on studies (in vitro and in vivo) will be needed to thoroughly explore the limitations of the proposed relaxation model. Regardless, these initial results demonstrate that the multiple contrast agent relaxation model can provide an analytical basis to determine the concentration of two different paramagnetic MRI contrast agents.We first validated the multiple contrast agent relaxation model described in equations  and 2b)2b). The 32, ratiometric methods to detect the presence of activatable MRI agents14, and machine learning33. Other groups have utilized chemical exchange saturation transfer (CEST) MRI techniques which can have reduced sensitivity on low-field (≤ 3 T) MRI scanners36. It is important to note that these prior studies primarily incorporated sequential MRI assessments to separately assess the different MRI contrast agents. Sequential T1 and T2 measurements (or any other method that results in both T1 and T2 maps) could be used with equations 32, ratioin vitro results suggest that DC-MRF can provide accurate assessments of two paramagnetic MRI contrast agents, in vivo imaging studies will be required to more fully evaluate the DC-MRF methodology. A schematic for an eventual in vivo DC-MRF implementation is shown in Supplementary Fig. in vivo DC-MRF experiments could be conducted similarly to conventional dynamic contrast enhanced MRI studies where images are collected before, during, and after administration of a contrast agent42. For in vivo DC-MRF, a pre-contrast MRF scan is performed to obtain baseline T1 and T2 maps in vitro quations  during dquations  and 3b)in vitro in vivo experiments. First, MRF has been previously shown to be sensitive to inhomogeneities in both the B1 and B0 fields. As variation in B1 and B0 are expected to significantly increase for in vivo experiments, accurate B0 shimming and/or B1 corrections will be needed to avoid significant errors in T1 and T2 measurements43 as well as the calculated contrast agent concentrations. Additionally, in vivo relaxivities will likely be different from the in vitro relaxivities shown here due to complex molecular interactions experienced by the contrast agents in vivo. This may be particularly complicated as these interactions can vary considerably between normal tissues and pathologies. Addressing this important limitation will require careful in vivo relaxivity assessments and potentially validation using elemental analysis of excised tissues. The pre-contrast T1 and T2 relaxation times of the tissues and pathologies of interest may also pose specific challenges. For example, if the tissue of interest has a low T2 relaxation time before contrast administration , resolving the concentration of the two agents may be problematic due to excessive T2 decay. In addition, in vivo studies may be impacted by multiple factors including partial volume effects, flow effect, and magnetization transfer and/or chemical exchange that may benefit from multi-exponential relaxation models instead of the simple mono-exponential models used here45. Despite these challenges, DC-MRF offers a unique opportunity to expand the portfolio of in vivo contrast-enhanced MRI applications.Importantly, there are several challenges that must be overcome to utilize the DC-MRF methodology for in vivo studies, the DC-MRF results presented in this initial report have multiple areas for future exploration. As described above, the multiple contrast agent relaxation model described herein provides an analytical solution to independently quantify two MRI contrast agents. It is conceivable that DC-MRF could be applied for three or more contrast agents. However, in that case, equations in vivo s46 labeled with gadolinium, and a second agent targeted to apoptosis47 labeled with dysprosium). Importantly, the DC-MRF methodology may also directly aid in the clinical translation of molecular imaging agents by allowing for the direct comparison between a conventional, non-targeted clinical MRI contrast agent  with a molecular imaging agent in a single patient. In this way, DC-MRF would provide the opportunity to efficiently establish the molecular specificity of the molecular contrast agent in heterogeneous human diseases.Although DC-MRF may have numerous applications, this new methodology is particularly well suited to support the development and eventual clinical translation of molecular MRI contrast agents. As described above, DC-MRF would provide the opportunity to detect both a molecularly-targeted contrast agent and a control non-targeted contrast agent at the same time in the same subject. The only constraint in achieving these simultaneous assessments using DC-MRF is that the two contrast agents must have different relaxivities. This constraint may require that the targeted and control contrast agents incorporate different lanthanides  in order to produce differential relaxivities while also retaining similar pharmacokinetic properties. A similar approach could also be used to allow multiple molecular imaging targets to be assessed simultaneously. For example, two targeted MRI contrast agents could be used to simultaneously track drug delivery and therapeutic impact in cancer treatment INTRODUCTquations  and 2b)INTRODUCTin vitro phantoms containing varying concentrations of gadolinium  and manganese  contrast agents either as single agents  or as mixtures of the two agents  . A table explicitly listing the concentrations of the phantoms is provided online as Supplementary Table In this initial study, we tested the validity of this model using 48. All relaxometry experiments were conducted at 37 °C. The relaxometric measurements were repeated twice for T1 measurements and three times for T2 measurements to ensure accurate assessments. These repeated measures were averaged to obtain single T1 and T2 values for each in vitro phantom.To test the multiple contrast agent relaxation model in equations  and 2b)2b), each0 and T20). Second, the phantoms containing the individual agents were analyzed to calculate the magnetic relaxivities of both agents  through a linear least-squares fit to the plot of R1  alone were measured and established as “pre-contrast” relaxation times (T1Equation ) and R2 Equation ) as a fuquations  and 3b)0 and T20quations  and 2b)0 and T20a priori variation in both flip angle (FA) and repetition time  to generate MRF signal evolution profiles sensitive to both T1 and T2 relaxation times25. The MRF method acquired 3000 images with time-varying contrast generated by the FA and TR variation. This FISP-MRF implementation included a non-selective inversion preparation (inversion time = 21 ms) immediately prior to the FISP-MRF image acquisitions to increase the sensitivity of the MRF signal evolution profiles to T1 relaxation times22. The MRF acquisition also incorporated undersampled spiral trajectories as described previously25. The acquisition time of one slice was 47 seconds with a FOV of 380 × 380 mm, an image matrix of 352 × 352, and a slice thickness of 5 mm. Measurement of the excitation (B1) field was incorporated to mitigate the effects of inhomogeneous B1 field on the T1 and T2 relaxation time estimates43.To determine the capability of the DC-MRF technique to provide quantitative imaging based assessments of contrast agent concentration, we obtained MRF-based T1 and T2 maps of the gadolinium-containing and manganese-containing phantoms evaluated in the relaxometry studies described above. The solutions in these phantoms were taken from the same source solutions as the NMR studies and placed into 15 mL centrifuge tubes . All MRF studies were conducted on a Siemens Skyra 3 T MRI scanner . The MRF acquisition (Siemens Work-In-Progress #881v23) utilized a FISP acquisition kernel designed with 24 assuming a mono-exponential relaxation model. MRF image reconstruction as well as subsequent dictionary matching were performed on the Siemens Skyra 3 T MRI scanner. Generation of the quantitative MRF-based T1 and T2 relaxation time maps was attained by matching the acquired MRF profiles on a pixel-by-pixel basis to the MRF dictionary of simulated profiles from all logical combinations of T1  and T2 . The T1 and T2 maps were exported for further offline processing in MATLAB .A fundamental component of the MRF reconstruction is the development of a large dictionary of signal evolution profiles that are subsequently “matched” to the acquired MRF signal evolution profile for each imaging voxel using vector-based inner product comparisons. The MRF dictionary was created as described previously0 and T20, respectively. The mean T1 and T2 relaxation times from the phantoms containing only a single agent were used to estimate the magnetic relaxivities for the gadolinium and manganese contrast agents. The T1 and T2 maps from the MRF acquisition were then used to calculate gadolinium and manganese concentration maps using the calculated 3 T relaxivities and equations  analysis. Similar to the relaxometric studies above, mean MRF-based T1 and T2 values for the sample with no contrast agent (deionized water only) was used as a measure of T1quations  and 3b)0 and T201 and r2 for the gadolinium and manganese contrast agents, respectively.Magnetic relaxivities were also obtained using conventional MRI assessments for comparison with the MRF-based relaxivity assessments. T1 relaxation time assessments were obtained with an inversion recovery spin echo acquisition (8 inversion times), and T2 relaxation times were obtained with a single-echo spin echo acquisition (8 echo times). Fitting was performed with the appropriate mono-exponential model generating T1 and T2 maps. Equations  and 1b)1b) were Data were compared using Pearson correlation and unpaired, two-tailed Student’s t-tests when appropriate. In this study, p < 0.05 was used to establish statistical significance and the p-values are reported as less than the largest reasonable, round number. Repeated studies were performed on the same days after repositioning of the specimen and performing scanner adjustments including magnetic field shimming, radiofrequency excitation calibration, and receiver gain adjustments.The data and protocols used in the current study are available from the corresponding author on reasonable request.Supplementary Materials


We show, across a range of different incident light regimes, that such radiative energy budgets are highly dominated by heat dissipation constituting up to 99.5% of the absorbed light energy. Highest photosynthetic energy conservation efficiency was found in the coral sediment under low light conditions and amounted to 18.1% of the absorbed light energy. Additionally, the effect of light directionality, i.e., diffuse or collimated light, on energy conversion efficiency was tested on the two surface-associated systems. The effects of light directionality on the radiative energy budgets of these phototrophic communities were not unanimous but, resulted in local spatial differences in heat-transfer, gross photosynthesis, and light distribution. The light acclimation index, Ek, i.e., the irradiance at the onset of saturation of photosynthesis, was >2 times higher in the coral sediment compared to the biofilm and changed the pattern of photosynthetic energy conservation under light-limiting conditions. At moderate to high incident irradiances, the photosynthetic conservation of absorbed energy was highest in collimated light; a tendency that changed in the biofilm under sub-saturating incident irradiances, where higher photosynthetic efficiencies were observed under diffuse light. The aim was to investigate how the physical structure and light propagation affected energy budgets and light utilization efficiencies in loosely organized vs. compact phototrophic sediment under diffuse and collimated light. Our results suggest that the optical properties and the structural organization of phytoelements are important traits affecting the photosynthetic efficiency of biofilms and sediments.We investigated the radiative energy budgets of a heterogeneous photosynthetic coral reef sediment and a compact uniform cyanobacterial biofilm on top of coastal sediment. By combining electrochemical, thermocouple and fiber-optic microsensor measurements of O Sediment cores were then mounted in a custom-made flow-chamber flushed with aerated seawater (26°C and S = 35) for another 24 h prior to measurements. The flow-chamber (interior dimensions: 25 × 8 × 8 cm) had a honeycomb baffle between the water inlet and the sample, ensuring a stable laminar flow . Before measurement at each experimental irradiance, the coral sediment core was illuminated for at least 45 min to ensure steady state O2 and temperature conditions; as confirmed from repeated microprofile measurements. Throughout measurements, the flow-chamber was flushed with a stable laminar flow (~0.5 cm s−1) of filtered aerated seawater over the sediment surface as generated by a Fluval U1 pump submerged in a 20 L thermostated aquarium (26°C and S = 35) and connected with tubing to the flow-chamber.The CS samples were collected with Perspex corers (inner diameter 5.3 cm), and were maintained under a continuous flow of aerated seawater at ambient temperature and salinity (26°C and −2 s−1) in a thermostated room (16–18°C). The biofilm surface appeared dark green–brownish due to predominance of dense communities of cyanobacteria and diatoms  under a downwelling photon irradiance of ~500 μmol photons m−2 s−1. During measurements, a stable laminar flow (~0.5 cm s−1) over the biofilm surface was maintained by a water pump  immersed in a 20 L aquarium with filtered aerated seawater  and connected with tubing to the flow-chamber.The BF samples were collected and contained in small rectangular plastic trays (7 × 2 × 5 cm) with the upper surface exposed and flush with the upper edge of the tray wall. After collection, the samples were kept humid and under a 12:12 h light-dark regime (~100 μmol photons md(PAR), (see definitions of abbreviations in Appendix) was measured with a calibrated irradiance meter  equipped with a cosine collector . Defined experimental irradiances  were achieved by adjusting the aperture on the fiber-optic lamp. The downwelling spectral irradiance at the above-mentioned levels was also measured in radiometric energy units (in W m−2 nm−1) with a calibrated spectroradiometer .Illumination was provided by a fiber-optic tungsten halogen lamp equipped with a collimating lens  positioned vertically above the flow-chamber. A spectrum of the used halogen lamp can be found in the Suppl. Info. in Lichtenberg et al.  and is cCollimated light was achieved by attaching a collimating lens to the fiber cable of the lamp. Diffuse light was achieved by inserting a TRIMMS diffuser  with a fast response time (<0.5 s) and a low stirring sensitivity , which was connected to a PC. All microsensors were mounted in a PC-interfaced motorized micromanipulator  controlled by dedicated data acquisition and positioning software . The micromanipulator was oriented in a 45° angle relative to the vertically incident light to avoid self-shading, especially in the light measurements. Depth profiles of temperature and O2 concentration were measured in vertical steps of 100 μm. Before profiling, the microsensor tips were manually positioned on the sample surface to define the z = 0 position, determined from visual detection through a stereo microscope. The precisions of this approach is about ± the average grain size of the sediments, i.e., 125–500 μm.Temperature measurements were performed with a thermocouple microsensor  connected to a thermocouple meter . The temperature microsensors were linearly calibrated against readings of a high precision thermometer  in seawater at different temperatures. Analogue outputs from the temperature and OG(z); in units of nmol O2 cm−3 s−1) were measured with O2 microsensors using the light-dark shift method  darkening equalled the local rate of photosynthesis just prior to darkening; while no response in the O2 signal upon darkening indicated a zero rate of photosynthesis. Areal rates of gross photosynthesis (in nmol O2 cm−2 s−1) were calculated by depth integration over the euphotic zone with respect to the measuring interval used in the depth profile measurement of PG(z), similar to Al-Najjar et al.  from measured steady-state O2 concentration profiles using Fick's first law of diffusion:The net upward flux of O0 is the diffusion coefficient of O2 in seawater at experimental temperature and salinity and 2 concentration gradient in the DBL.where D2 flux from the photic zone of the sediments to the aphotic part of the sediment/biofilm was calculated in a similar manner as:The downward O2 out of the photic zone, i.e., the total net photosynthesis in the photic zone (NPP), was subsequently calculated as the difference between the upward and downward O2 flux  we multiplied the areal gross photosynthesis, GPP, with the Gibbs free energy formed in the light-dependent reactions, where O2 is formed by splitting water, which gains (including the formation of ATP) a Gibbs free energy of EG = 482.9 kJ (mol O2)−1  from the sediment/biofilm into the water column was calculated by Fourier's law of conduction:The amount of the absorbed light energy that was not photochemically conserved was dissipated as heat resulting in a local increase of the sediment/biofilm temperature relatively to the ambient seawater and thereby leading to the establishment of a TBL. The heat dissipation, i.e., the heat flux  and dT/dz is the measured linear temperature gradient in the TBL  but with the thermal conductivity constant of the sediment, k(b), which was estimated as:where ks is the carbonate thermal conductivity , (in units of counts nm−1) with the tip of the scalar irradiance microsensor positioned in the light path at the same distance from the light source as the sediment surface. Using identical light settings, the absolute downwelling irradiance, EABS(λ) (in W m−2) was also quantified with a calibrated spectroradiometer .Spectral photon scalar irradiance was measured in units of counts nmE0(λ), was measured in vertical steps of 0.1–0.2 mm in the sediment and was calculated as the fraction of the incident downwelling irradiance, i.e., E0(λ)/Ed(λ), and plotted as transmittance spectra in % of Ed(λ). The relative measurements of scalar irradiance in different depths in the biofilm/sediment were converted to absolute scalar irradiance spectra in units of W m−2 nm−1 as EABS(λ)×E0(λ)/Ed(λ). Absolute scalar irradiance spectra were converted to photon scalar irradiance spectra (in units of μmol photons m−2 s−1 nm−1) by using Planck's equation:The spectral scalar irradiance, Eλ is the energy of a photon with wavelength, λ, h is Planck's constant (6.626 × 10−34 W s2), and c is the speed of light in vacuum (in m s−1).where 0(λ), were calculated as 1 and E0(λ)2 are the spectral scalar irradiances measured at depth z1 and z2, respectively.where E0(PAR), in μmol photons m−2 s−1), i.e., the light energy available for oxygenic photosynthesis at each measurement depth. The diffuse attenuation coefficient of E0(PAR), K0(PAR), was obtained by fitting the measured E0(PAR) vs. depth profiles with an exponential model:Light attenuation was also calculated by integrating the spectral quantum irradiance over PAR (420–700 nm) yielding the PAR scalar irradiance (The PAR irradiance reflectance (R) of the sediment/biofilm surface was calculated asEu(λ) is the upwelling irradiance at the sediment surface, here estimated as the diffuse backscattered spectral radiance measured at the sediment surface  is the downwelling irradiance estimated as the backscattered spectral radiance measured over a white reflectance standard ; both measured with a fiber-optic field radiance microprobe  in the sediment/biofilm was estimated by subtracting the downwelling and upwelling irradiance at the surface:The absorbed light energy (JEd(λ) and R(λ) are the downwelling spectral irradiance and irradiance reflectance, respectively. This parameter is equivalent to the so-called vector irradiance, which is a measure of the net downwelling radiative energy flux.where A balanced radiative energy budget of the sediment/biofilm was calculated according to  (in J m−2 s−1):Areal gross photosynthesis rates as a function of JPS,max. The respective efficiencies under light-limiting conditions, i.e., for JABS→0, were then calculated as:This yielded an estimate of the maximum photochemically conserved energy flux Ek is the photochemical light acclimation index, i.e., the irradiance at the onset of photosynthetic saturation, calculated as Ek = JPS,max/α, where α is the initial slope of the fitted photosynthesis vs. JABS curve.where 0(PAR), decreased with increasing sediment depth , the exponential attenuation of collimated light within the biofilm was observed below 0.2 mm, whereas diffuse light was attenuated exponentially from the biofilm surface under all investigated irradiance levels . The top layer of the biofilm showed ~10 times stronger light attenuation than the coral sediment with average PAR attenuation coefficient of α = 9.52 and α = 10.54 mm−1 for diffuse and collimated light, respectively, compared to α = 1.18 mm−1 in the coral sediment (both light types).At all incident irradiances, the photon scalar irradiance, Eh Figure . Light ay Figure . Surfacey Figure . The proy Figure  in the uy Figure . At the  Figure − s−1, the Figure − s−1, the Figure − s−1, thea (440 and 670 nm) and phycocyanin , where the strongest attenuation occurred over the 0.3–0.6 mm zone  while the lowest attenuation was found in the upper 0–1 mm, consistent with the scalar irradiance profiles , while saturation of photosynthesis was reached at a higher downwelling photon irradiance of ~200 μmol photons m−2 s−1  developed between the biofilm and the surrounding water Figures , 4. In dy Figure . At incie Figure . The O2 −2 s−1 . At the highest irradiance , the biofilm surface was 0.51 ± 0.036 and 0.41 ± 0.008°C warmer than the overlaying water, while the coral sediment surface was 0.53 ± 0.031 and 0.48 ± 0.040°C warmer than the surrounding water for diffuse and collimated light, respectively. Similar temperature profiles were observed between collimated and diffuse light, although a slightly enhanced surface heating and thus a higher efflux of heat was observed under diffuse light  under low irradiance (50–200 μmol photons m−2 s−1), while rates decreased at photon irradiances of >200 μmol photons m−2 s−1  was higher under diffusive illumination, while PG under diffuse and collimated illumination were similar at higher irradiances  curve of the coral sediment measured in diffuse light increased with increasing light intensity with an initial slope of 0.05 ± 0.01, until reaching an asymptotic saturation level at J1 Figure . In cont1 Figure . In the t Figure .diff = 4.33·10−3 °C (J m−2 s−1)−1 and CSαcoll = 2.14·10−3 °C (J m−2 s−1)−1 in the coral sediment, as compared to BFαdiff = 2.77·10−3 °C (J m−2 s−1)−1 and BFαcoll = 2.0·10−3 °C (J m−2 s−1)−1 in the biofilm  serves as a control to determine the potential deviations between absorbed and dissipated energy , photosynthetic energy conservation in the coral sediment illuminated with diffuse light decreased from 6.7 to 2.0% of the incident light energy, favoring heat dissipation (which increased from 63.1 to 67.8%), and from 9.3 to 2.1% of the incident light energy under collimated light  of the incident light energy was conserved, whereas 96.3 and 96.0% of the incident light energy was dissipated as heat, respectively , whereas the biofilm had maximum energy conservation through photosynthesis (14.7% of the absorbed light energy) at the lowest measured incident irradiance , εPS,max, were calculated from the initial slope of the areal PG vs. vector irradiance curve to 26.2% of the absorbed light energy  compared to 16 and 9.0% of the absorbed light energy .The photosynthetic efficiencies of biofilm and coral sediment under light-limiting conditions  dark sandy sediment, whereas the photosynthetic microorganisms exhibited a more patchy distribution within the large-grained (100–500 μm) bright and highly scattering coral sediment. This structural difference between the two systems led to a ~15 times higher surface reflection and a decreased energy absorption in the coral sediment compared to the biofilm that displayed >8 times higher light attenuation coefficients. As previously shown . Thus, the dense biofilm appeared acclimated to low irradiances as previously shown  favoring effective light utilization under diffuse light  photosynthetic energy conservation was higher (3–4% of the absorbed light energy) in collimated light as compared to diffuse light; a tendency that dramatically changed in the photosynthetic biofilm at low and light-limiting incident irradiances (≤100 μmol photons m−2 s−1) favoring effective light utilization under diffuse light (up to a ~2-fold increase). However, cyanobacterial and diatom dominated mats have been shown to migrate vertically employing photo- and/or chemo-tactic responses  diffuse light enhanced dissipation of heat (~30%) in the upper sediment layers as compared to collimated light; (ii) at low incident irradiance (200 μmol photons mML, KB, and MK designed the experiments; ML and KB performed experiments; ML, KB, and MK analyzed data; ML wrote the paper with editorial inputs from KB and MK.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Previous work in the zebrafish embryo has shown that laminin γ-3 (lamc3) is enriched in endothelial cells marked by expression offli1a, but the role of Lamc3 has been unknown.Methods: We use antisense morpholino oligonucleotides, and CRISPR/Cas9 mutagenesis of F0 embryos, to create zebrafish embryos in whichlamc3 expression is compromised. Transgenic imaging, immunofluorescence, andin situ hybridisation reveal that Lamc3 loss-of-function affects the development of muscle pioneers, endothelial cells, and motoneurons.Results: Lamc3 is enriched in endothelial cells during zebrafish development, but it is also expressed by other tissues. Depletion of Lamc3 by use of antisense morpholino oligonucleotides perturbs formation of the parachordal chain and subsequently the thoracic duct, but Lamc3 is not required for sprouting of the cardinal vein. F0 embryos in whichlamc3 expression is perturbed by a CRISPR/Cas9 approach also fail to form a parachordal chain, but we were unable to establish a stablelamc3 null line. Lamc3 is dispensable for muscle pioneer specification and for the expression ofnetrin-1a in these cells. Lamc3 knockdown causesnetrin-1a up-regulation in the neural tube and there is increased Netrin-1 protein throughout the trunk of the embryo. Axonal guidance of rostral primary motoneurons is defective in Lamc3 knockdown embryos.Conclusions: We suggest that knockdown of Lamc3 perturbs migration of rostral primary motoneurons at the level of the horizontal myoseptum, indicating that laminin γ3 plays a role in motoneuron guidance. Danio rerio) is a powerful model for studying the formation of the vascular system. It is well suited to live imaging due to its transparency and external fertilisation. Development is rapid, with functional circulation established within a day of fertilisation (The zebrafish (isation . Zebrafiisation . For thefli1a:egfp) has frequently been used in this approach, coupled with both microarray analysis and high throughput RNA sequencing  cells compared with gfp negative cells (fli1a expressing cells between 26 and 28 hours post fertilisation (hpf), compared with the rest of the embryo (lamc3 (ENSDARG00000060396) gene, which encodes the zebrafish laminin γ3 protein.Several attempts have been made to define the transcriptomes of haematopoietic and vascular precursors in the zebrafish, including the comparison of wild type and vascular deficient cloche mutants by microarray (uencing . Such woHomo sapiens) have four laminin β chain isoforms (LAMB1-4), mice (Mus musculus) have three β chain isoforms (LAMB1-3), and zebrafish have six laminin β genes:lamb1,lamb1b,lamb2,lamb2l,lamb3, andlamb4. In both zebrafish and mammals there are three γ chain isoforms (LAMC1-3) ,grumpy (gup), andsleepy (sly), which encode the laminin α1, β1, and γ1 proteins respectively  vasculature and that loss of the PAC is an indirect effect, caused by the failure of rostral primary motoneurons to extend at the level of the horizontal myoseptum  Act 1986, the Animal Welfare Act (2006) and the Welfare of Animals in Transport Order. Care was taken to minimize the number of animals used in these experiments, in accordance with theHomo sapiens), mouse (Mus musculus), and zebrafish (Danio rerio) were aligned using MUSCLE. Full length alignments were represented as a barcode with RasMol colouring using Geneious software  embryos was a kind gift from Elsie Place , extracted as previously described  are located in Supplementary data repository,cDNA from 26 hpf FACS sorted Tg(fli1a:egfp) GFP+ and GFP- cells. One-way ANOVA test for multiple comparisons were performed using Prism software to generate p-values between control, uninjected, Lamc3 SBMO, and TBMO PAC quantifications. Two-way ANOVA tests were performed using Prism software to compare the number of arterial and venous intersegmental vessels between control and Lamc3 SBMO embryos. Fisher’s exact Two-tailed tests (2x2 contingency table) were performed using GraphPad software to compare the number of CRISPR/Cas9 F0 embryos with normal PAC development or PAC defects to control embryos. All raw quantified data can be found in Supplementary data repository,Tukey box blots and bar graphs were generated using R and annotated in Adobe Illustrator. Four statistical analyses were performed in this work: Unpaired Student’s t test was performed using Prism software to compare quantitative Lamc3 SBMO data to standard control embryos, and to generate p-values for qPCR of Lamc3 in Tg(Danio rerio) adults were maintained and bred under standard conditions  “Tg(fli1a:egfp)” was a gift from Dr Tim Chico  (flt1:yfp)hu4624Tg “Tg(flt1:yfp)” and Tg(kdrl:Hsa.HRAS-mCherry)s896 “Tg(kdrl:mCherry)” lines were gifts from Dr Roger Patient  nim5 “Tg(prox1a:rfp)” line was a gift from Dr Elke Ober  (gfap:gfp)mi2001“Tg(gfap:gfp)” transgenic line was a gift from Dr David Wilkinson  (Zebrafish (ditions . Embryosffield) . The Tg .netrin-1a (ntn1a) plasmid  was digested with EcoRV  according to the manufacturer’s instructions. DIG-labelled mRNA was synthesised using 1 μg of linearised plasmid DNA and T7 RNA polymerase  in 1× transcription buffer and DIG RNA Labeling Mix . Probes were purified using Illustra Microspin G-50 columns  according to the manufacturer’s instructions.In situ hybridisation forlamc3 andntn1a were performed as described previously , and SP6 RNA polymerase  in 1× transcription buffer and DIG RNA Labeling Mix . The zebrafishviously . After fFor immunostaining, all steps were performed at room temperature unless otherwise stated. Control and knockdown zebrafish embryos were fixed in 4% paraformaldehyde for 1 hour before being washed 3× in phosphate buffered saline pH7.4 (PBS) for 5 minutes each. Embryos were digested in 10 μg/ml proteinase K  in blocking solution  for 30 minutes and refixed in 4% paraformaldehyde for 30 minutes. Next, embryos were washed 3× in PBS 0.1% Tween-20 (PBST) for 5 minutes each. Embryos were then incubated in blocking solution for 1 hour before being incubated in blocking solution with primary antibody overnight at 4°C. The next day embryos were washed 3× in PBS with 0.2% Triton-X for 10 minutes each before being incubated in blocking solution containing secondary antibody for at least 1 hour. Embryos were then washed 3× in PBS with 0.1% Triton-X for 1 hour each before a single 10 minute wash in PBST. For imaging, embryos were transferred to 75% glycerol overnight and mounted for imaging on glass cover slides. For transverse sections, embryos were cut by hand using a clean scalpel at the level of the yolk extension and mounted on glass slides with coverslips.Drosophila engrailed monoclonal primary antibody  was used at a concentration of 1:4, followed by rabbit anti-mouse HRP secondary  at 1:100 and Cyanine 5 tyramide reaction  according to the manufacturer’s instructions. In all cases, samples containing no primary antibody were used as negative controls. Whole embryos or sections were imaged on a Zeiss LSM710 confocal microscope using ZEN software. Z-stacks were compiled using FIJI (Fiji Is Just Image-J) software using a Max Intensity projection.The following primary antibodies were used at a concentration of 1:200: rabbit anti-human LAMC3 C-terminal polyclonal primary antibody ; rabbit anti-human NETRIN-1 (H104) polyclonal primary antibody ; and mouse anti-chicken MNR/HB9/MNX1 monoclonal primary antibody . Each of these antibodies was visualised using Alexa Fluor 488-conjugated goat anti-rabbit secondary  used at 1:1000. The mouse anti-Where necessary for clarity, contrast was increased using Adobe Photoshop “Brightness/contrast” or “Curves” tools. All images were positioned to have anterior to the left and posterior to the right. Examples of such manipulated images compared to original images are shown inOne-cell stage embryos were injected with 1 nl solution containing up to 10 ng of antisense morpholino oligonucleotides, from Gene Tools . Morpholino sequences and concentrations are shown inLamc3 vector  was linearised with NotI  according to manufacturer’s recommendations. Column purified plasmid was used as a template forin vitro mRNA synthesis using mMESSAGE mMACHINE SP6 transcription kit  according to manufacturer’s instructions. The resulting mRNA was then lithium chloride precipitated, dissolved in RNase-free water and stored in aliquots at -80°C. Either 1 nl of 200 ng/μl or 400 ng/μl of mLamc3 mRNA was injected into single embryos at the one-cell stage.For overexpression of mouse Lamc3 mRNA, the mLive embryos were transferred to a glass cover slip bottomed dish in embryo medium and 2.1 ml of stock tricaine solution  was added per 50 ml of embryo medium. Anaesthetised animals were imaged on the Zeiss LSM710 confocal microscope using ZEN software. All images of the trunk were taken at the level of the horizontal myoseptum. 8–12 Z-stacks for images of the whole trunk, or 4–6 Z-stacks for images from the embryo midline to the distal surface of the embryo were collected at approximately 8 μm intervals. Z-stacks were compiled using Fiji (Fiji Is Just Image-J) software using “Max Intensity” projections.lamc3 were designed using CRISPR Design tool (crispr.mit.edu) to span an endonuclease target site and constructed using the oligonucleotides shown infli1a:egfp) zebrafish embryos were injected at the one-cell stage with 1 nl of solution containing ~12–15 ng/μl of each Lamc3 sgRNA and ~300 ng/μl of Cas9 mRNA. In addition, sgRNA alone or Cas9 mRNA alone were injected as negative controls. Embryos were imaged at 2 dpf on a Leica M165FC fluorescent microscope using Leica Application Suite 3.4.1 software. Embryos that lacked a parachordal chain in more than 5 segments of the trunk at the level of the yolk extension were recorded as having parachordal chain defects.Custom CRISPR short guide RNAs (sgRNAs) targeting exon 1 ofTo extract genomic DNA, single 48 hpf embryos or tail fin clips of individual adult fish were incubated for up to 3 hours at 55°C in 20–50 μl of genomic DNA extraction buffer  with fresh 0.08 μg/ml Proteinase K  added before use. Samples were then heated to 95°C for 10 minutes to inactivate proteinase K and debris was spun down using micro centrifugation. The sgRNA target site was amplified from extracted genomic DNA using the following primers: Fw: 5′-TTCTGCTTTTTGCCAGCGTC-3′; Rv: 5′-GCAATACCAGCACTGCTCTAC-3′. Mutation of the target site was identified by digestion with SphI-HF  or TaqαI  for sgRNA1 and sgRNA2 respectively, overnight at 37°C according to the manufacturer’s instructions. After digestion, samples were run on a 2% agarose gel for 30 minutes at 120V and compared to a 100 bp ladder . For SphI digests, complete digestion of wild type product produces three bands of 84, 164 and 263 bp. Some animals were later identified to contain a single nucleotide polymorphism (SNP) in one of the SphI sites. Complete digestion of wild type genomic DNA in such animals produces two bands of 164 and 346 bp. Digestion of mutant product yields bands of 83 and 427 bp, or 510 bp if the SNP is present. For TaqαI digests, complete digestion of wild type PCR product produces two bands of 205 and 305 bp. Digestion of mutant products yields a single band of 510 bp. For presentation, agarose gel images were inverted and cropped but were not otherwise manipulated.48 hpf zebrafish embryos were dechorionated in batches of around 100 and transferred to ice cold Calcium-free Ringer’s solution. Embryos were rinsed 3× in Ringer’s solution. Samples were then transferred to ice cold Ringer’s solution containing 1mM EDTA and 0.3 mM phenylmethylsulfonyl fluoride (PMSF). Embryos were de-yolked by pipetting using a 200 μl pipette tip. De-yolked embryos were then rinsed twice in Ringer’s solution with EDTA and PMSF, and snap frozen on dry ice. Frozen embryos were thawed and homogenised in 100 μl of protein extraction buffer  with 1× cOmplete Mini Protease Inhibitor Cocktail  overnight at 4°C with rotation. Samples were pelleted and 150 μl of 1× Laemmli sample buffer was added . Pellets were homogenised in sample buffer using a microfuge pestle in a round-bottom 1.5 ml tube. Before loading, samples were boiled at 99°C for 5 minutes.20 μl of protein extract was loaded into a NuSEP 4–20% glycine pre-cast gel with Spectra multicolour broad range protein ladder  and run in SDS running buffer for 1 hour at 100V. Proteins were transferred to methanol-activated Immobilon-P PVDF membranes (Millipore) in Tris-Glycine transfer buffer. Transfer was performed for approximately 1 hour at 400 amps with ice cooling. Membranes were blocked in 5% milk powder in PBST  for 1 hour at room temperature. Membranes were incubated with rabbit anti-human NETRIN-1 (H104) polyclonal antibody  was in 5% milk powder blocking solution. For data presented in this work, the antibody was used at a concentration of 1:100, although bands of identical molecular weights can also be detected up to a concentration of 1:1000. Membranes were rinsed with 5 washes in PBST before being incubated with goat anti-rabbit HRP-conjugated antibody  1:2000 in 5% milk powder blocking solution for 2 hours at room temperature. Membranes were rinsed 3× in PBST at room temperature. Protein bands were visualised using chemifluorescence, SuperSignal West Dura Extended Duration Substrate  according to manufacturer’s instructions and exposed using the ChemiDoc gel imaging system (BioRad). An image of the gel prior to cropping and brightness/contrast manipulation can be found in the Supplementary data repository,Amino acid sequences of human, mouse, and zebrafish laminin γ1, γ2, and γ3 chains were aligned using MUSCLE . Zebrafifli1a:egfp) zebrafish embryos had suggested thatlamc3 is enriched 4-fold infli1a:gfp expressing (GFP+) endothelial cells (fli1a:egfp) embryos. Our results revealed only a 3-fold enrichment that was not statistically significant  in GFP+ cells compared with the rest of the embryo. This observation confirms thatlamc3 is expressed by GFP+ cells, but suggests that other cells also express the gene   . In addi (n=10) . These dlamc3 expression: protein is located at the horizontal myoseptum, the dorsal plate of the neural tube, and axial vessels in the trunk  is found adjacent to regions of gene expression . At 26 he trunk . To showe trunk . We beliTo validate Lamc3 knockdown, embryos injected with standard control morpholino or Lamc3 SBMO were immunostained using the human anti-LAMC3 C-terminal antibody. Control embryos (n=11/12) showed γ3 protein in the dorsal plate of the neural tube, horizontal myoseptum and ventral vasculature as seen previously . In Lamcfli1a:egfp) embryos and observed the developing vasculature over the following 3 days. A minority of MO injected embryos exhibited phenotypes associated with MO toxicity, including cell death and oedema in the head  and splice-blocking morpholino oligonucleotides (SBMO) targeting Lamc3 mRNA into Tg(he head  (Eisen he head . For thiOur results show that in uninjected (n=19) or standard control morpholino injected embryos (n=16) the parachordal chain  develops between the intersegmental vessels at the level of the horizontal myoseptum . In embrflt1:yfp;kdrl:mCherry) embryos were injected with Lamc3 SBMO and venous intersegmental vessels were identified by their lack of flt1 expression, which is specific to arterial endothelium (flt1 expression (white outline), which form the PAC and venous intersegmental vessels  embryos were injected with Lamc3 SBMO (n=12) or standard control MO (n=12) and were observed at 5 dpf for the presence or absence of the thoracic duct, identified by expression ofprox1a. Despite increasing in size, γ3-deficient embryos were severely morphologically abnormal compared with controls  embryos together with Cas9 mRNA, and Cas9 mRNA alone was also injected as a negative control. At 2 dpf embryos were assessed for formation of the PAC. Of embryos injected with Cas9 mRNA alone (n=70), only 1 had developmental defects  embryos with Cas9 mRNA and sgRNA1. However, by 5 dpf almost half (n=31/72) of the injected embryos presented severe oedema, with swelling around the eyes (red lines), heart, gut and yolk  line. In control embryos (n=6)prox1a:rfp was expressed in the neural tube, caudal motoneurons and slow-twitch muscle  in Lamc3 morphants  compared with control embryos (n=12). To our surprise however, the expression domain ofnetrin-1a appeared to be broader in γ3-deficient embryos, with increased expression in the neural tube  embryos using a human anti-NETRIN-1 polyclonal antibody. This antibody was chosen because its epitope is highly conserved between human and zebrafish Netrin-1a and Netrin-1b proteins. In control embryos, we observed Netrin-1 protein in the neural tube and in the middle of somites (white outline) in the trunk  and Lamc3 SBMO (n=8) injected Tg zebrafish line to ask whether primary motoneurons are affected by Lamc3 knockdown at 48 hpf.The development of the PAC depends on the correct migration of rostral primary motoneurons, which extend axons along the horizontal myoseptum in response to Netrin-1a signalling (In 48 hpf control embryos (n=5) HB9 positive cells are elongated along the anterior-posterior axis and are concentrated at the dorsal neural tube and horizontal myoseptum . The motgfap:gfp) embryos. In control embryos (n=12) the caudal motoneuron (CaP) extends axons ventrally through the somite (red outline). Rostral motoneurons (RoPs) extend axons ventrally to the level of the horizontal myoseptum and then move anteriorly  it remains possible that this is a nonspecific effect rather than a phenotype of γ3-deficiency, because no oedema was observed in Lamc3 SBMO embryos at 5 dpf. Although further attempts to generate alamc3 genetic line were not possible within the scope of this work, we would encourage attempts to generate alamc3 loss-of-function mutant in any further study of Lamc3, as suggested by the zebrafish community  proteins fromNematostella vectensis,Mus musculus,Danio rerio andHomo sapiens.File 2 - Trimmed alignments of LAMC1, LAMC2 and LAMC3 proteins for phylogenetic tree building. Raw data used for generating phylogenetic trees, gaps and areas of low conservation of the aligned amino acid sequences fromFile 3 - Raw quantitative RT-PCR data . Duplicate raw Cp-values from qPCR analysis of cDNA synthesised from GFP FACS-sorted Tg(fli1a:egfp) transgenic zebrafish. GFP-ve, cells that did not expressGFP; GFP+ve,GFP expressing cells; No RTase, No reverse transcription of RNA control. Dilution curve and negative (water) controls as labelled.File 4 - Number of intersegmental vessels and parachordal chains at 48 hpf. Counting of vessels in uninjected, standard control morpholino injected, and Lamc3 splice-blocking morpholino (SBMO) and translation-blocking morpholino (TBMO) injected embryos. Hpf, hours post fertilisation.File 5 - Number of intersegmental vessels and parachordal chains at 72 hpf. Counting of vessels in uninjected, standard control morpholino injected and Lamc3 splice-blocking morpholino (SBMO) and translation-blocking morpholino (TBMO) injected Tg(fli1a:egfp) embryos. Hpf, hours post fertilisation.File 6 - Number of hemisegments with thoracic duct at 5 dpf. Counting of thoracic duct vessels in standard control morpholino injected and Lamc3 splice-blocking morpholino (SBMO) injected Tg embryos. Dpf, days post fertilisation.File 7 - Number of HB9 cells at 2 dpf. Counting of HB9 immunostained cells in standard control morpholino injected and Lamc3 splice-blocking morpholino (SBMO) injected embryos. Dpf, days post fertilisation.File 8 - Number of caudal and rostral motoneurons at 2 dpf. Counting of caudal and rostral motoneurons in standard control morpholino injected and Lamc3 splice-blocking morpholino (SBMO) injected Tg(gfap:gfp) embryos. Dpf, days post fertilisation.File 9 - Raw data of Western blot gel used in This is a well constructed and clearly written manuscript.  The authors present a straightforward story of a knockdown of laminin 3 (Lamc3) and the consequence of this on parachordal chain and motorneuron development.  The knockdown of Lamc3 was achieved using morpholino oligonucleotide injection that was validated by transient, mosaic genetic lesions in F0 embryos using using CRISPR/Cas9.  They indicate that upon reduced lamc3 expression, the parachordal chain is lost and that motorneuron morphology is perturbed; the former defect is suggested to be the consequence of the latter, consistent with earlier findings.   Abnormal midline development evidenced by altered netrin expression and changes in prox1a transgene expression as a consequence of the loss of lamc3 is cited as a probable cause of the defects. The experimental procedures admirably well presented: they are thoroughly described, and they seem to have been carried out carefully.  The data presented are of a high quality.  The authors are sensibly cautious and measured in the language employed to describe their results and to conclude from them.  Their findings do throw up several further questions and many of these are discussed by the authors.  I might normally ask for more experiments to develop the study more but I recognise that the reviewer guidelines are limited to an analysis of the quality of the work presented; discussion of the further questions would seem to suffice in this context.  Nevertheless, I do have a few reservations about the study, as it is presented, these can probably be addressed through adjustment of the manuscript.  The main issues are listed below, followed by some smaller items that would improve/correct the manuscript prior to formal publication. Larger issuesBecause the study heavily relies upon morpholino use, it is imperative to carefully validate the efficacy of the reagents employed.  The authors employ several approaches to this problem, including the use of both splice- and translation-blocking morpholinos, immunohistochemistry against the target protein as well as the replication of the findings using transient CRIPSR/Cas9 injection. This is good and refreshing when compared with other published data employing morpholinos.  I believe the specificity and efficacy of the morpholino employed is reasonably sure.  However, I was a bit surprised to see that the authors gave up attempting the generation of a full mutant line after apparently only attempting to raise 72 F0 embryos/larvae with only 12 making it to adulthood for genomic testing.  .  With the apparently reliable restriction digest assay that the authors have already generated I would have thought it were feasible to either titrate down the dose of the Cas9/sgRNA injections sufficiently, or use the less efficient sgRNA so that there was less mutation load to allow more fish to survive to adulthood for germ line genomic lesion screening.  This is certainly a time-consuming undertaking because of generation time, and that may be the problem here but the authors should be honest about that.  The morpholino data is certainly convincing as it stands but, as acknowledged by the authors in the discussion, consensus in the community is that bona fide genetic mutants are a ‘gold standard’ for examination of gene function, particularly also in concert with morpholino use. I feel that the presented reasons for abandoning efforts to obtain a lamc3 mutant are not really sufficient.  I recommend a change to this section to acknowledge it may be possible in the future to generate a mutant using the same reagents employed for ‘morpholino validation’ here.It is not clear whether the authors were able to correlate more severe genetic lesions using CRISPR/Cas9 and sgRNAs with more severe PAC defects.  It seems logical to me that this would be done but the two results are presented separately.  Was this carried out?  Also, in other studies employing F0 mutagenesis, experimenters have multiplexed their sgRNAs to increase the chance of lesion.  Did the authors try this approach?In Fig.4G’ there is an abrupt drop off in the NTN1 staining on the left hand side of the sectioned embryo, when passing from dorsal to ventral there is almost a line apparent, below which the expression is much lower compared to the right hand side.  What is the cause of this? Was it consistently seen? Perhaps it is a sectioning artifact? Despite this it is clear that the morphant (Panel H’) staining is more extensive, the difference between sides in the Std control section is something that stands out. Please comment on this.     Smaller issuesA little more detail on the qRT-PCR experiment would be good eg. How was the FACs undertaken?  How was the RNA isolated? Were the primer efficiencies determined? Are the n numbers in the panel Fig1D replications of the whole experiment (FACs sorting and qRT-PCR) or just the qRT-PCR? I realise it is a negative result but the detail helps with interpretation.in situ hybridisation heading, the manscript states that the T7 promoter sequence is underlined and SP6 sequence is in italics, neither can be seen in the primer sequences as presented in the version I see online and in PDF.In the methods section, under the probe synthesis and embryos …” where the legend statesadult fish; c) The ordering of the panel labelling in fig. 3 is rather odd: Panels A-G are labelled left to right by row and H-J are by column, it makes it hard to read; d) The picture of the three embryos dorsally mounted in the bottom right of figure 3 does not have a panel label or a corresponding legend; e) In my opinion, the “U” in Panel G that denotes undigested would be better described in the section of the legend about this panel rather than at the end of the legend; f) There is no “U” sample in panel H, why? This is a different assay and it would be useful for interpretation.Figure 3 issues: a) The n-numbers for the data in panel E would be useful to present in the panel or at least in the legend.  I can see they are in the body but it is much easier for the reader to see them either in legend or panel itself; b) In Fig 3J the blue box above the gel image says “Surviving F0 SG RNAI believe that the results section entitled “Lamc3 is required for parachordal chain (PAC) formation” was intended to go after the “Lamc3 is required for parachordal chain (PAC) formation”.  It seems like the more logical order.  This is especially true regarding the p53 MO use.  The order it is written currently it is ambiguous whether the experiments described in the earlier section employed co-injection of p53 MO or not.  I suspect an editing error but, either way, the authors should make clear which experiments employed p53 MO in addition to the various other morpholinos (including Std control).     If the authors are able to address the points above I would be happy for the paper to be formally published.  As I hope is clear, the changes are mainly possible to be dealt with through changes in the text.  Some of the points I raise are open to debate and I’m happy to engage in this, if necessary.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. In this paper, the Authors used a morpholino and CRISPR/Cas9 approach and they find a  role of laminin γ3 in motoneuron guidance. The experiments are technically sound and the results are clear. The images are of high quality and the quantification data are well-described, including numbers of animals and numbers of experiments.  The manuscript is well written and may be recommended for indexing. For revision, the following changes are recommended: Fig1 (Panel D) Were sufficient animals analysed to conclude that no difference exists? It would be interesting if the authors presented a double IF to show if there is a colocalization of Lamc3 (red) and fli1a  (anti-GFP) in some vessels. Fig2. To assess the morpholino efficacy, lam3c specific RT-PCR reactions could be performed on RNA populations or check the decrease of protein level by western blot? Did you try also a mismatch MO instead of std control? Fig3 (Panel A) Did you check for sgRNA1 the decrease of mRNA or protein level for Lam3c in the mutant with the phenotype?1. In this way you can identify some mutation that could not be identified by digestion. To try to obtained a stable line in the CRISPR/Cas9 approach, you can try to decrease the concentration and nanoliters inject of sgRNA1 due to its toxicity or too high efficiency. A suggestion could be the genotyping of mutations in F0 using heteroduplex mobility assay (HMA)I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In the results section, the authors discribed that LAMC3 is most closely related to LAMC1. Whether LAMC1 were closer to the clade of LAMC2 based on Fig.1B. ?kdrl, using immunostaining or the transgenic lines which express florescent proteins in endothelial cells.In Fig.1G-H, it will be more convincing to colocalize the expression of LAMC3 and endothelial cell specific gene, such asIn the Figure1, Is the legend (I) indicating the panel (H’)?The content of figure S4 was similar with figure3B-D. What is the difference between them?The authors showed that lamc3 loss-of-function impaired the formation of parachordal chain (PAC) and thoracic duct. To confirm the phenotype was caused specifically by loss of lamc3, I suggest the authors to examine whether lamc3 mRNA could rescue the phenotypes in morphants and mutants.Since the impaired blood flow affects the formation of parachordal chain (PAC) and thoracic duct in zebrafish. Whether the phenotype of lamc3 deficient embryos was caused by blood flow defect as a secondary effect needs to be seriously checked.Tg(gfap:gfp) line to study motoneuron development instead ofTg(mnx1:EGFP)?line is often employed in the study of astrocytes. Why did the authors chose theMotoneurons are essential for vascular pathfinding. Were the parachordal chain (PAC) defects were caused by primary motoneurons malformation?In figure 4F, expression domain of netrin-1a was increased in the neural tube, the netrin1a signals in horizontal myoseptum and the whole embryo were stronger too. In contrast, the authors found that the netrin-1 protein was reduced in all Lamc3 knockdown embryos at the level of the horizontal myoseptum compared with controls, the authors should give some interpretation.Results that netrin expression was affected by LAMC3 knockdown are interesting. I suggest the authors add the relevant discussion, for example the potential molecular mechanism.The authors provided with evidences that lamc3 was enriched in endothelial cells as a regulator of retinal neuronal guidance. However, it has not been investigated on zebrafish model. The authors performed Lamc3 knockdown and knockout using Mo and CRISPR/Cas9 approaches during zebrafish development. It was revealed that Lamc3 deficiency disturbed the formation of the PAC, the thoracic duct, and axonal guidance of rostral primary moto neuron which might be caused by up-regulation of netrin-1a in the neural tube, but not sprouting of the cardinal vein. The manuscript was well written and the figures were well presented. However, I have some questions that need to be addressed:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. SUMMARY I enjoyed reading this paper, which was conducted technically well and presented in a very accessible and thorough manner. The authors are sensibly cautious about the conclusions that they draw. I  wholeheartedly believe that the huge amount of unpublished morpholino data, were it available to the community, would be of great value even with the difficulty in being certain that all the described effects are specific so am very keen that this paper, and the hundreds like it that I suspect could be written, be disseminated. I make some general comments below, but the main point that I think should be kept in mind when interpreting this paper is that non-specific effects on cardiac output induced by the morpholino or the CRISPR gRNA would be expected to induce the same reduction in lymphatic formation; unless the authors revise the manuscript to include an assessment of cardiac output and blood flow then I would be a little uncomfortable assuming that these effects have much to do with LAMC3 directly. It would be reasonable however, to acknowledge this and other potential confounders a little more clearly in the results and discussion if it is not possible at this stage to examine the effects of the genetic manipulations on blood flow. The immunostaining against Laminin 3  is nice but without more validation that it binds selectively to zebrafish LAMC3 (such as a western blot) it is hard to be certain that the immunostaining is specific to this isoform of laminin, especially with a cross-species antibody. The overexpression study in the supplemental data does show that there is increased antibody binding to mouse LAMC3 when overexpressed but does not rule out it binding to other laminins or non-specifically, nor does it confirm it binds to zebrafish LAMC3. I would therefore consider the protein expression data to be likely correct but not completely validated. However, the reduction of staining in the LAMC3 morphants makes it likely that the antibody binds to the MO target; it just cannot be concluded that it doesn't bind to anything else! Figure 2 shows nicely that the LAMC3 morpholino prevents lymphatic formation. However, a similar effect is seen if blood flow is prevented in embryos, for example by troponin knockdown. To be certain that this does not explain the observed phenotype, it would be necessary to show that the morpholino does not alter cardiac output and blood flow in the dose used. The CRISPR/Cas9 methods indicate that some embryos were injected with gRNAs alone (which is an appropriate control) but the results don't show these data; they only show Cas9 alone as a control. My own group has found that some gRNAs at high doses do induce toxicity and these data should be shown or discussed. I'm not convinced that the different effects of the second guide RNA exclude off target toxicity from a different guide; we see varying effects with different guide sequences. Although the LAMC3 crispants show similar defects in lymphatic development as the morphants, again this could be secondary to alterations in blood flow which would need to be examined to exclude such a confounding effect. The authors do seem to have encountered a problem with the CRISPR/Cas9 system, in that they have found a really efficient guideRNA that cuts bi-allelically and so induces a lethal phenotype that means a heterozygous founder cannot be identified. It would have made more sense to raise the embryos injected with the second, less efficient guide as this does seem to induce mosaic/heterozygous mutations more compatible with finding a founder adult. The alteration of netrin expression induced by LAMC3 knockdown is interesting, but would be strengthened by quantification . For example, if the expression domains are quantified blindedly, is there a statistically significant alteration? It seems this would be very achievable and would make the conclusions far more solid. In passing, no mention of blinding during analysis is made in the methods, and it would be important to know if when the quantification was performed in for example Figure 2 that the observer was blinded to group allocation. Minor points Table 1 refers to concentrations of morpholinos but these are doses per embryo.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.