Neeru Gandotra

The developmental dynamics of the maize leaf transcriptome

We have analyzed the maize leaf transcriptome using Illumina sequencing. We mapped more than 120 million reads to define gene structure and alternative splicing events and to quantify transcript abundance along a leaf developmental gradient and in mature bundle sheath and mesophyll cells. We detected differential mRNA processing events for most maize genes. We found that 64% and 21% of genes were differentially expressed along the developmental gradient and between bundle sheath and mesophyll cells, respectively. We implemented Gbrowse, an electronic fluorescent pictograph browser, and created a two-cell biochemical pathway viewer to visualize datasets. Cluster analysis of the data revealed a dynamic transcriptome, with transcripts for primary cell wall and basic cellular metabolism at the leaf base transitioning to transcripts for secondary cell wall biosynthesis and C4 photosynthetic development toward the tip. This dataset will serve as the foundation for a systems biology approach to the understanding of photosynthetic development.

A transcriptome atlas of rice cell types uncovers cellular, functional and developmental hierarchies

The functions of the plant body rely on interactions among distinct and nonequivalent cell types. The comparison of transcriptomes from different cell types should expose the transcriptional networks that underlie cellular attributes and contributions. Using laser microdissection and microarray profiling, we have produced a cell type transcriptome atlas that includes 40 cell types from rice (Oryza sativa) shoot, root and germinating seed at several developmental stages, providing patterns of cell specificity for individual genes and gene classes. Cell type comparisons uncovered previously unrecognized properties, including cell-specific promoter motifs and coexpressed cognate binding factor candidates, interaction partner candidates and hormone response centers. We inferred developmental regulatory hierarchies of gene expression in specific cell types by comparison of several stages within root, shoot and embryo.

VH1/BRL2 receptor-like kinase interacts with vascular-specific adaptor proteins VIT and VIK to influence leaf venation.

VH1/BRL2 is a receptor-like kinase of the BRI1 family with a role in vascular development. In developing Arabidopsis leaves it is expressed first in ground cells and then becomes restricted to provascular and procambial cells as venation forms. We isolated proteins interacting with the activated (phosphorylated) cytoplasmic domain of VH1/BRL2, and found that most belong to three processes: proteasome activity, vesicle traffic and intracellular signal transduction. Two adaptor proteins are included that we named VIT [VH1-interacting tetratricopeptide repeat (TPR)-containing protein] and VIK (VH1-interacting kinase), which are co-expressed in the same cells as VH1/BRL2 at two distinct time points in vein differentiation. Mutation of either adaptor or of VH1 results in vein pattern defects and in alterations in response to auxin and brassinosteroids. We propose that these two adaptors facilitate the diversification and amplification of a ligand signal perceived by VH1/BRL2 in multiple downstream pathways affecting venation.

Plant cell types: reporting and sampling with new technologies

Plants have relatively few cell types, but their specialized functions and their interactions are essential for physiology, development, and defense. The contributions of individual cells have been distinguished by methods including in situ reporting, cell sampling, and cell separation, thus far mostly limited to measurement of single transcripts, proteins, or metabolites. Advances in transcriptomics, proteomics, metabolomics, and activity assays with small samples and in the modeling of these data into networks of expression, regulation, interaction, and metabolism make it possible to evaluate the roles of cell types at system levels. Recent analyses include cell types of developing roots, bundle sheath and mesophyll cells of C4-type leaves, xylem and phloem cells of vascular systems, and specialized regions of embryos and shoot apices.

Developmental dynamics of Kranz cell transcriptional specificity in maize leaf reveals early onset of C4-related processes

Summary The measured differential expression of genes between bundle sheath and mesophyll cells at successive developmental stages of the maize leaf is used to identify C4-photosynthesis-related candidates.

Basic peptide-morpholino oligomer conjugate that is very effective in killing bacteria by gene-specific and nonspecific modes

Basic peptides covalently linked to nucleic acids, or chemically modified nucleic acids, enable the insertion of such a conjugate into bacteria grown in liquid medium and mammalian cells in tissue culture. A unique peptide, derived from human T cells, has been employed in a chemical synthesis to make a conjugate with a morpholino oligonucleotide. This new conjugate is at least 10- to 100-fold more effective than previous peptides used in altering the phenotype of host bacteria if the external guide sequence methodology is employed in these experiments. Bacteria with target genes expressing chloramphenicol resistance, penicillin resistance, or gyrase A function can effectively be reduced in their expression and the host cells killed. Several bacteria are susceptible to this treatment, which has a broad range of potency. The loss in viability of bacteria is not due only to complementarity with a target RNA and the action of RNase P, but also to a non-gene-specific tight binding of the complexed nontargeted RNA to the basic polypeptide-morpholino oligonucleotide.

The Arabidopsis leaf provascular cell transcriptome is enriched in genes with roles in vein patterning

Several classes of genes have been associated, by mutant phenotypes or cell biology, with the formation of vein patterns during early leaf development, including genes for certain transcription factors, auxin transport and response factors, endomembrane traffic components and other signaling pathway components. The majority of these are expressed with spatial and temporal specificity that includes expression in the precursors of vascular cells - provascular (PV) and procambial (PC) cells - suggesting that other PV/PC-specific genes might have roles in vein patterning. We inventoried the PV/PC transcriptome of Arabidopsis leaves using a combination of laser microdissection and microarray expression profiling, and determined the phenotypes of knock-outs of previously uncharacterized PV/PC-specific genes. As examples, we observed vein pattern defects in knock-out lines of KEG and a CCCH zinc finger protein. This strategy of gene discovery, based on the identification of a gene set co-expressed in the same cells during the targeted developmental event, appears to be an efficient means of identifying genes functionally relevant to the event. In the case of vein patterning, this strategy would have identified many or most of the genes previously obtained by labor-intensive screening for pattern-defective mutants.

A peptide-morpholino oligomer conjugate targeting Staphylococcus aureus gyrA mRNA improves healing in an infected mouse cutaneous wound model

Management of skin wound infections presents a serious problem in the clinic, in the community, and in both civilian and military clinical treatment centers. Staphylococcus aureus is one of the most common microbial pathogens in cutaneous wounds. Peptide-morpholino oligomer (PMO) conjugates targeted to S. aureus gyrase A mRNA have shown the ability to reduce bacterial viability by direct site-specific mRNA cleavage via RNase P. As a treatment, these conjugates have the added advantages of not being susceptible to resistance due to genetic mutations and are effective against drug resistant strains. While this strategy has proven effective in liquid culture, it has yet to be evaluated in an animal model of infected surface wounds. In the present study, we combined PMO conjugates with a thermoresponsive gel delivery system to treat full-thickness mouse cutaneous wounds infected with S. aureus. Wounds treated with a single dose of PMO conjugate displayed improved healing that was associated with increased epithelialization, reduced bacterial load, and increased matrix deposition. Taken together, our findings demonstrate the efficacy and flexibility of the PMO conjugate drug delivery system and make it an attractive and novel topical antimicrobial agent.

Distribution of muscarinic acetylcholine receptor subtypes in the murine small intestine

Aims: Serotonin stimulates enterocyte turnover in the small intestine and studies suggest this is mediated by neuronal signaling via a cholinergic pathway. Distribution of the five known muscarinic receptor subtypes (mAChRs) in the small intestine has not been fully studied, and their role in intestinal growth is unknown. We hypothesized that mAChRs have distinct anatomic distributions within the bowel, and that mAChRs present within intestinal crypts mediate the effects of acetylcholine on the small intestinal mucosa. Main methods: Small intestine from male C57BL/6 mice ages 2, 4, 6, and 8 weeks were harvested. RNA was isolated and cDNA synthesized for PCR‐amplification of subtype specific mAChRs. Ileum was fixed with Nakane, embedded in epon, and immunofluorescence microscopy performed using polyclonal antibodies specific to each mAChR1‐5. Key findings: All five mAChR subtypes were present in the mouse duodenum, jejunum, and ileum at all ages by RT‐PCR. Immunofluorescence microscopy suggested the presence of mAChR1‐5 in association with mature enterocytes along the villus and within the myenteric plexus. Only mAChR2 clearly localized to the crypt stem cell compartment, specifically co‐localizing with Paneth cells at crypt bases. Significance: Muscarinic receptors are widely distributed along the entire alimentary tract. mAChR2 appears to localize to the crypt stem cell compartment, suggesting it is a plausible regulator of stem cell activity. The location of mAChR2 to the crypt makes it a potential therapeutic target for treatment of intestinal disease such as short bowel syndrome. The exact cellular location and action of each mAChR requires further study.

Enhanced serotonin signaling increases intestinal neuroplasticity

BACKGROUND The intestinal mucosa recovers from injury by accelerating enterocyte proliferation resulting in villus growth. A similar phenomenon is seen after massive bowel resection. Serotonin (5-HT) has been implicated as an important regulator of mucosal homeostasis by promoting growth in the epithelium. The impact of 5-HT on other components of growing villi is not known. We hypothesized that 5-HT-stimulated growth in the intestinal epithelium would be associated with growth in other components of the villus such as enteric neural axonal processes. MATERIALS AND METHODS Enteric serotonergic signaling is inactivated by the serotonin reuptake transporter, or SERT, molecule. Enhanced serotonin signaling was achieved via SERT knockout (SERTKO) and administration of selective serotonin reuptake inhibitors (SSRI) to wild-type mice (WT-SSRI). 5-HT synthesis inhibition was achieved with administration of 4-chloro-L-phenylalanine (PCPA). Intestinal segments from age-matched WT, SERTKO, WT-SSRI, and corresponding PCPA-treated animals were assessed via villus height, crypt depth, and crypt proliferation. Gap 43, a marker of neuroplasticity, was assessed via immunofluorescence and Western blot. RESULTS SERTKO and WT-SSRI mice had taller villi, deeper crypts, and increased enterocyte proliferation compared with WT mice. Gap 43 expression via immunofluorescence was significantly increased in SERTKO and WT-SSRI samples, as well as in Western blot analysis. PCPA-treated SERTKO and WT-SSRI animals demonstrated reversal of 5-HT-induced growth and Gap 43 expression. CONCLUSIONS Enhanced 5-HT signaling results in intestinal mucosal growth in both the epithelial cell compartment and the enteric nervous system. Furthermore, 5-HT synthesis inhibition resulted in reversal of effects, suggesting that 5-HT is a critically important regulator of intestinal mucosal growth and neuronal plasticity.