Advances in single cell analysis techniques have demonstrated cell-to-cell variability in both homogeneous and heterogeneous cell populations strengthening our understanding of multicellular organisms and individual cell behaviour. However, additional tools are needed for non-targeted metabolic analysis of live single cells in their native environment. Here, we combine capillary microsampling with electrospray ionization (ESI) mass spectrometry (MS) and ion mobility separation (IMS) for the analysis of various single A. thaliana epidermal cell types, including pavement and basal cells, and trichomes. To achieve microsampling of different cell types with distinct morphology, custom-tailored microcapillaries were used to extract the cell contents. To eliminate the isobaric interferences and enhance the ion coverage in single cell analysis, a rapid separation technique, IMS, was introduced that retained ions based on their collision cross sections. For each cell type, the extracted cell material was directly electrosprayed resulting in ∼200 peaks in ESI-MS and ∼400 different ions in ESI-IMS-MS, the latter representing a significantly enhanced coverage. Based on their accurate masses and tandem MS, 23 metabolites and lipids were tentatively identified. Our results indicated that profound metabolic differences existed between the trichome and the other two cell types but differences between pavement and basal cells were hard to discern. The spectra indicated that in all three A. thaliana cell types the phenylpropanoid metabolism pathway had high coverage. In addition, metabolites from the subpathway, sinapic acid ester biosynthesis, were more abundant in single pavement and basal cells, whereas compounds from the kaempferol glycoside biosynthesis pathway were present at significantly higher level in trichomes. Our results demonstrate that capillary microsampling coupled with ESI-IMS-MS captures metabolic differences between A. thaliana epidermal cell types, paving the way for the non-targeted analysis of single plant cells and subcellular compartments.

Background: Many animals utilize maternal mRNAs to pre‐pattern the embryo before the onset of zygotic transcription. In Xenopus laevis, vegetal factors specify the germ line, endoderm, and dorsal axis, but there are few studies demonstrating roles for animal‐enriched maternal mRNAs. Therefore, we carried out a microarray analysis to identify novel maternal transcripts enriched in 8‐cell‐stage animal blastomeres. Results: We identified 39 mRNAs isolated from 8‐cell animal blastomeres that are >4‐fold enriched compared to vegetal pole mRNAs. We characterized 14 of these that are of unknown function. We validated the microarray results for 8/14 genes by qRT‐PCR and for 14/14 genes by in situ hybridization assays. Because no developmental functions are reported yet, we provide the expression patterns for each of the 14 genes. Each is expressed in the animal hemisphere of unfertilized eggs, 8‐cell animal blastomeres, and diffusely in blastula animal cap ectoderm, gastrula ectoderm and neural ectoderm, neural crest (and derivatives) and cranial placodes (and derivatives). They have varying later expression in some mesodermal and endodermal tissues in tail bud through larval stages. Conclusions: Novel animal‐enriched maternal mRNAs are preferentially expressed in ectodermal derivatives, particularly neural ectoderm. However, they are later expressed in derivatives of other germ layers. Developmental Dynamics 243:478–496, 2014. © 2013 Wiley Periodicals, Inc.

Genes that code for proteins involved in organelle biogenesis and intracellular trafficking produce products that are critical in normal cell function . Conserved orthologs of these are present in most or all eukaryotes, including Drosophila melanogaster. Some of these genes were originally identified as eye color mutants with decreases in both types of pigments found in the fly eye. These criteria were used for identification of such genes, four eye color mutations that are not annotated in the genome sequence: chocolate, maroon, mahogany, and red Malpighian tubules were molecularly mapped and their genome sequences have been evaluated. Mapping was performed using deletion analysis and complementation tests. chocolate is an allele of the VhaAC39-1 gene, which is an ortholog of the Vacuolar H+ ATPase AC39 subunit 1. maroon corresponds to the Vps16A gene and its product is part of the HOPS complex, which participates in transport and organelle fusion. red Malpighian tubule is the CG12207 gene, which encodes a protein of unknown function that includes a LysM domain. mahogany is the CG13646 gene, which is predicted to be an amino acid transporter. The strategy of identifying eye color genes based on perturbations in quantities of both types of eye color pigments has proven useful in identifying proteins involved in trafficking and biogenesis of lysosome-related organelles. Mutants of these genes can form the basis of valuable in vivo models to understand these processes.

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.