Multi-omics characterization of NIST seafood reference materials and alternative matrix preparations.

The National Institute of Standards and Technology (NIST) has prepared four seafood reference materials (RMs) for use in food safety and nutrition studies: wild-caught and aquacultured salmon (RM 8256 and RM 8257) and wild-caught and aquacultured shrimp (RM 8258 and RM 8259). These materials were characterized using genetic, metabolomic (1H-NMR, nuclear magnetic resonance and LC-HRMS/MS, liquid chromatography high-resolution tandem mass spectrometry), lipidomic, and proteomic methods to explore their use as matrix-matched, multi-omic differential materials for method development towards identifying product source and/or as quality control in untargeted omics studies. The results from experimental replicates were reproducible for each reference material and analytical method, with the most abundant features reported. Additionally, differences between the materials could be detected, where wild-caught and aquacultured seafood could be distinguished using untargeted metabolite, lipid, and protein analyses. Further processing of the fresh-frozen RMs by freeze-drying revealed the freeze-dried seafoods could still be reliably discerned. These results demonstrate the usefulness of these reference materials as tools for omics instrument validation and measurement harmonization in seafood-related studies. Furthermore, their use as differential quality control (QC) materials, regardless of preparation method, may also provide a tool for laboratories to demonstrate proficiency at discriminating between products based on source/species.

Spiking and homogenization of biological matrices for production of reference materials using cryogenic processes.

Biological reference materials (RMs) are essential for quality assurance, traceability of measurement results and for method validation. When addressing new measurement questions or emerging regulatory issues, rigorous large-scale CRM production may not be time efficient or economically practical using current production methods. By amending a relatively small matrix batch with a compound(s) of interest at the homogenization step, the National Institute of Standards and Technology (NIST) can create a custom material on an "as-needed" basis and circumvent the time delay inherent in large-batch production, thereby generating a fit-for-purpose, rapid-response RM. Here, Coho salmon (Oncorhynchus kisutch) was cryohomogenized and spiked with an aquaculture antibiotic and antibiotic metabolite. The resultant material was analyzed using liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) to determine the effectiveness of the amendment technique in a fresh-frozen matrix by assessing homogeneity and accuracy to the target concentration (e.g. mass fraction). Target mass fractions were achieved for both spike components, with RSDs below 5% in replicate measurements of each compound (n = 8). The stability of the spiked compounds was assessed one year post-production and mass fractions were stable, within 1-6% of the initial measurement results, indicating minimal change to the amended analyte concentrations over time. The results support this method as a promising new technique for custom, small-batch RM generation.

Dynamic temporal requirement of Wnt1 in midbrain dopamine neuron development.

Wnt1-expressing progenitors generate midbrain dopamine (MbDA) and cerebellum (Cb) neurons in distinct temporal windows and from spatially discrete progenitor domains. It has been shown that Wnt1 and Lmx1a participate in a cross-regulatory loop that is utilized during MbDA neuron development. However, Wnt1 expression dynamically changes over time and precedes that of Lmx1a. The spatial and temporal requirements of Wnt1 in development and specifically its requirement for MbDA neurons remain to be determined. To address these issues, we generated a conditional Wnt1 allele and temporally deleted Wnt1 coupled with genetic lineage analysis. Using this approach, we show that patterning of the midbrain (Mb) and Cb by Wnt1 occurs between the one-somite and the six- to eight-somite stages and is solely dependent on Wnt1 function in the Mb, but not in the Cb. Interestingly, an En1-derived domain persists after the early deletion of Wnt1 and mutant cells express OTX2. However, the En1-derived Wnt1-mutant domain does not contain LMX1a-expressing progenitors, and MbDA neurons are depleted. Thus, we demonstrate an early requirement of Wnt1 for all MbDA neurons. Subsequently, we deleted Wnt1 in the ventral Mb and show a continued late requirement for Wnt1 in MbDA neuron development, but not in LMX1a-expressing progenitors. Specifically, Wnt1 deletion disrupts the birthdating of MbDA neurons and causes a depletion of MbDA neurons positioned medially and a concomitant expansion of MbDA neurons positioned laterally during embryogenesis. Collectively, our analyses resolve the spatial and temporal function of Wnt1 in Mb and Cb patterning and in MbDA neuron development in vivo.

Genetic dissection of midbrain dopamine neuron development in vivo.

Midbrain dopamine (MbDA) neurons are partitioned into medial and lateral cohorts that control complex functions. However, the genetic underpinnings of MbDA neuron heterogeneity are unclear. While it is known that Wnt1-expressing progenitors contribute to MbDA neurons, the role of Wnt1 in MbDA neuron development in vivo is unresolved. We show that mice with a spontaneous point mutation in Wnt1 have a unique phenotype characterized by the loss of medial MbDA neurons concomitant with a severe depletion of Wnt1-expressing progenitors and diminished LMX1a-expressing progenitors. Wnt1 mutant embryos also have alterations in a hierarchical gene regulatory loop suggesting multiple gene involvement in the Wnt1 mutant MbDA neuron phenotype. To investigate this possibility, we conditionally deleted Gbx2, Fgf8, and En1/2 after their early role in patterning and asked whether these genetic manipulations phenocopied the depletion of MbDA neurons in Wnt1 mutants. The conditional deletion of Gbx2 did not result in re-positioning or distribution of MbDA neurons. The temporal deletion of Fgf8 did not result in the loss of either LMX1a-expressing progenitors nor the initial population of differentiated MbDA neurons, but did result in a complete loss of MbDA neurons at later stages. The temporal deletion and species specific manipulation of En1/2 demonstrated a continued and species specific role of Engrailed genes in MbDA neuron development. Notably, our conditional deletion experiments revealed phenotypes dissimilar to Wnt1 mutants indicating the unique role of Wnt1 in MbDA neuron development. By placing Wnt1, Fgf8, and En1/2 in the context of their temporal requirement for MbDA neuron development, we further deciphered the developmental program underpinning MbDA neuron progenitors.

Proteomics as a Metrological Tool to Evaluate Genome Annotation Accuracy Following De Novo Genome Assembly: A Case Study Using the Atlantic Bottlenose Dolphin (Tursiops truncatus).

The last decade has witnessed dramatic improvements in whole-genome sequencing capabilities coupled to drastically decreased costs, leading to an inundation of high-quality de novo genomes. For this reason, the continued development of genome quality metrics is imperative. Using the 2016 Atlantic bottlenose dolphin NCBI RefSeq annotation and mass spectrometry-based proteomic analysis of six tissues, we confirmed 10,402 proteins from 4711 protein groups, constituting nearly one-third of the possible predicted proteins. Since the identification of larger proteins with more identified peptides implies reduced database fragmentation and improved gene annotation accuracy, we propose the metric NP10, which attempts to capture this quality improvement. The NP10 metric is calculated by first stratifying proteomic results by identifying the top decile (or 10th 10-quantile) of identified proteins based on the number of peptides per protein and then returns the median molecular weight of the resulting proteins. When using the 2016 versus 2012 Tursiops truncatus genome annotation to search this proteomic data set, there was a 21% improvement in NP10. This metric was further demonstrated by using a publicly available proteomic data set to compare human genome annotations from 2004, 2013 and 2016, which showed a 33% improvement in NP10. These results demonstrate that proteomics may be a useful metrological tool to benchmark genome accuracy, though there is a need for reference proteomic datasets across species to facilitate the evaluation of new de novo and existing genome.

Tamoxifen dose response and conditional cell marking: is there control?

Conditional marking and gene inactivation experiments are valuable approaches used to understand developmental and molecular mechanisms. CreER(T) is a fundamental component in recombinase-based conditional strategies and is used to gain temporal control subsequent to tamoxifen administration. We tested the hypothesis that tamoxifen dose linearly correlates with recombination efficiency in vivo. Wnt1-CreER(T) and tamoxifen administration were used to mark progenitors that contributed to the trigeminal ganglia. We executed a dose response study to determine the number of neurons that had undergone recombination in response to tamoxifen administered at doses ranging from 50 to 500 mg/kg. Our findings show a substantial variability in the amount of recombination within and between dose groups with no clear correlation between tamoxifen dose and the number of marked cells. This is the first study that we are aware of in which cell counts, robust quantitative data, and statistical analyses were performed on sections obtained from embryos marked in response to a wide range of tamoxifen dose in vivo. We provide an important quantitative and statistical framework for designing CreER(T)-based experiments and choosing tamoxifen dosing paradigms.

Characterization of a human liver reference material fit for proteomics applications.

The National Institute of Standards and Technology (NIST) is creating new, economical, qualitative reference materials and data for proteomics comparisons, benchmarking and harmonization. Here we describe a large dataset from shotgun proteomic analysis of RM 8461 Human Liver for Proteomics, a reference material being developed. Consensus identifications using multiple search engines and sample preparations demonstrate a homogeneous and fit-for-purpose material that can be incorporated into automated or manual sample preparation workflows, with the resulting data used to directly assess complete sample-to-data workflows and provide harmonization and benchmarking between laboratories and techniques. Data are available via PRIDE with identifier PXD013608.

The Temporal Contribution of the Gbx2 Lineage to Cerebellar Neurons.

The cerebellum (Cb) is an exquisite structure that controls elaborate motor behaviors and is essential for sensory-motor learning. During development, the Cb is derived from rhombomere 1 (r1). Within this embryonic compartment, precursors in r1 are patterned by signaling cues originating from the isthmus organizer (IsO) and subsequently undergo complex morphogenic movements to establish their final position in the mature Cb. The transcription factor Gbx2 is expressed in the developing Cb and is intimately involved in organizing and patterning the Cb. Nevertheless, how precursors expressing Gbx2 at specific embryonic time points contribute to distinct cell types in the adult Cb is unresolved. In this study, we used Genetic Inducible Fate Mapping (GIFM) to mark Gbx2-expressing precursors with fine temporal resolution and to subsequently track this lineage through embryogenesis. We then determined the terminal neuronal fate of the Gbx2 lineage in the adult Cb. Our analysis demonstrates that the Gbx2 lineage contributes to the Cb with marking over the course of five stages: Embryonic day 7.5 (E7.5) through E11.5. The Gbx2 lineage gives rise to Purkinje cells, granule neurons, and deep cerebellar neurons across these marking stages. Notably, the contribution of the Gbx2 lineage shifts as development proceeds with each marking stage producing a distinct profile of mature neurons in the adult Cb. These findings demonstrate the relationship between the temporal expression of Gbx2 and the terminal cell fate of neurons in the Cb. Based on these results, Gbx2 is critical to Cb development, not only for its well-defined role in positioning and maintaining the IsO, but also for guiding the development of Cb precursors and determining the identity of Cb neurons.

The distribution and stratification of persistent organic pollutants and fatty acids in bottlenose dolphin (Tursiops truncatus) blubber.

Blubber has been used for decades to monitor exposure of marine mammals to persistent organic pollutants (POPs). However, little is known about POP variability as a function of blubber depth and across the body of the animal. Remote blubber biopsy sampling (e.g, projectile biopsy) is the most common technique used to acquire samples from free-swimming animals, yet such techniques may result in variable sampling. It is important to understand whether blubber stratification or body location affects POP concentration or the concentration of other important blubber constituents such as fatty acids (FA). To investigate the influence of sampling depth and location on POP concentration, full depth blubber samples were taken from one stranded bottlenose dolphin (Tursiops truncatus) at six different body sites to assess variation in FA distribution and contaminant storage with body location. Three of the samples from different body locations were separated into histologically distinct layers to examine the effect of blubber depth and body location on POPs and FAs. In this individual, both POPs and FAs were heterogeneous with blubber depth and body location. POP concentrations were significantly greater in ventral (average ΣPBDEs 1350 ng/g lipid) and anterior (average ΣPCBs 28,700 ng/g lipid) body locations and greater in the superficial blubber layer (average ΣPCBs 35,500 ng/g lipid) when compared to the deep (8390 ng/g lipid) and middle (23,700 ng/g lipid) layers. Proportionally more dietary FAs were found in dorsal blubber and in middle and deep layers relative to other locations while the reverse was true for biosynthesized FAs. Stratification was further examined in blubber from the same body location in five additional stranded bottlenose dolphins. Although FAs were stratified with blubber depth, lipid-normalized POPs were not significantly different with depth, indicating that POP concentrations can vary in an individual with blubber depth though the direction of POP stratification is not consistent among individuals.

Christianson syndrome protein NHE6 modulates TrkB endosomal signaling required for neuronal circuit development.

Neuronal arborization is regulated by cell-autonomous and nonautonomous mechanisms including endosomal signaling via BDNF/TrkB. The endosomal Na⁺/H⁺ exchanger 6 (NHE6) is mutated in a new autism-related disorder. NHE6 functions to permit proton leak from endosomes, yet the mechanisms causing disease are unknown. We demonstrate that loss of NHE6 results in overacidification of the endosomal compartment and attenuated TrkB signaling. Mouse brains with disrupted NHE6 display reduced axonal and dendritic branching, synapse number, and circuit strength. Site-directed mutagenesis shows that the proton leak function of NHE6 is required for neuronal arborization. We find that TrkB receptor colocalizes to NHE6-associated endosomes. TrkB protein and phosphorylation are reduced in NHE6 mutant neurons in response to BDNF signaling. Finally, exogenous BDNF rescues defects in neuronal arborization. We propose that NHE6 mutation leads to circuit defects that are in part due to impoverished neuronal arborization that may be treatable by enhanced TrkB signaling.

The lineage contribution and role of Gbx2 in spinal cord development.

BACKGROUND: Forging a relationship between progenitors with dynamically changing gene expression and their terminal fate is instructive for understanding the logic of how cell-type diversity is established. The mouse spinal cord is an ideal system to study these mechanisms in the context of developmental genetics and nervous system development. Here we focus on the Gastrulation homeobox 2 (Gbx2) transcription factor, which has not been explored in spinal cord development. METHODOLOGY/PRINCIPAL FINDINGS: We determined the molecular identity of Gbx2-expressing spinal cord progenitors. We also utilized genetic inducible fate mapping to mark the Gbx2 lineage at different embryonic stages in vivo in mouse. Collectively, we uncover cell behaviors, cytoarchitectonic organization, and the terminal cell fate of the Gbx2 lineage. Notably, both ventral motor neurons and interneurons are derived from the Gbx2 lineage, but only during a short developmental period. Short-term fate mapping during mouse spinal cord development shows that Gbx2 expression is transient and is extinguished ventrally in a rostral to caudal gradient. Concomitantly, a permanent lineage restriction boundary ensures that spinal cord neurons derived from the Gbx2 lineage are confined to a dorsal compartment that is maintained in the adult and that this lineage generates inhibitory interneurons of the spinal cord. Using lineage tracing and molecular markers to follow Gbx2-mutant cells, we show that the loss of Gbx2 globally affects spinal cord patterning including the organization of interneuron progenitors. Finally, long-term lineage analysis reveals that the presence and timing of Gbx2 expression in interneuron progenitors results in the differential contribution to subtypes of terminally differentiated interneurons in the adult spinal cord. CONCLUSIONS/SIGNIFICANCE: We illustrate the complex cellular nature of Gbx2 expression and lineage contribution to the mouse spinal cord. In a broader context, this study provides a direct link between spinal cord progenitors undergoing dynamic changes in molecular identity and terminal neuronal fate.

A practical approach to genetic inducible fate mapping: a visual guide to mark and track cells in vivo.

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage. GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreER(T)). CreER(T) contains a modified estrogen receptor ligand binding domain which renders CreER(T) sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreER(T), which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP. Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreER(T) release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreER(T). We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders. This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreER(T);mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

Comparative analysis of conditional reporter alleles in the developing embryo and embryonic nervous system.

A long-standing problem in development is understanding how progenitor cells transiently expressing genes contribute to complex anatomical and functional structures. In the developing nervous system an additional level of complexity arises when considering how cells of distinct lineages relate to newly established neural circuits. To address these problems, we used both cumulative marking with Cre/loxP and Genetic Inducible Fate Mapping (GIFM), which permanently and heritably marks small populations of progenitors and their descendants with fine temporal control using CreER/loxP. A key component used in both approaches is a conditional phenotyping allele that has the potential to be expressed in all cell types, but is quiescent because of a loxP flanked Stop sequence, which precedes a reporter allele. Upon recombination, the resulting phenotyping allele is 'turned on' and then constitutively expressed. Thus, the reporter functions as a high fidelity genetic lineage tracer in vivo. Currently there is an array of reporter alleles that can be used in marking strategies, but their recombination efficiency and applicability to a wide array of tissues has not been thoroughly described. To assess the recombination/marking potential of the reporters, we utilized CreER(T) under the control of a Wnt1 transgene (Wnt1-CreER(T)) as well as a cumulative, non-inducible En1(Cre) knock-in line in combination with three different reporters: R26R (LacZ reporter), Z/EG (EGFP reporter), and Tau-Lox-STOP-Lox-mGFP-IRES-NLS-LacZ (membrane-targeted GFP/nuclear LacZ reporter). We marked the Wnt1 lineage using each of the three reporters at embryonic day (E) 8.5 followed by analysis at E10.0, E12.5, and in the adult. We also compared cumulative marking of cells with a history of En1 expression at the same stages. We evaluated the reporters by whole-mount and section analysis and ascertained the strengths and weaknesses of each of the reporters. Comparative analysis with the reporters elucidated complexities of how the Wnt1 and En1 lineages contribute to developing embryos and to axonal projection patterns of neurons derived from these lineages.