Biology subjects

Miller, A.

Publications and source records attributed to Miller, A..

8 recordsLinked to original sources

bioRxiv2018

The Heterotaxy Gene, CCDC11, is Essential for Cytokinesis and Cell-Cell Adhesion Via RhoA Regulation

Mutations in CCDC11 have been identified in multiple patients with heterotaxy (Htx), a disorder of left-right (LR) patterning of the internal organs. In Xenopus, depletion of Ccdc11 causes defects in LR patterning, recapitulating the patient phenotype. Upon Ccdc11 depletion, normally monociliated cells of the Left-Right Organizer (LRO) exhibit multiple cilia per cell. Unexpectedly, we found that Ccdc11 is necessary for successful cytokinesis, and the multiciliation observed in Ccdc11-depleted cells was due to failed cytokinesis. Furthermore, CCDC11 depletion alters cell-cell adhesion with reduction in junctional localization of adhesion molecules. The small GTPase RhoA is critical for cytokinesis and cell-cell adhesion. Because the CCDC11 depletion phenotypes are reminiscent of RhoA loss of function, we investigated a possible connection to regulation of RhoA signaling. We demonstrate that CCDC11 is localized to the cytokinetic contractile ring overlapping with RhoA during cytokinesis and regulates total RhoA protein levels. Our results suggest that CCDC11 connects cytokinesis and LR patterning via RhoA regulation, providing a potential mechanism for heterotaxy disease pathogenesis.

cell biology↗

bioRxiv2018

Pregnancy associated plasma protein-aa (Pappaa) regulates photoreceptor synaptic development to mediate visually guided behavior

To guide behavior, sensory systems detect the onset and offset of stimuli and process these distinct inputs via parallel pathways. In the retina, this strategy is implemented by splitting neural signals for light onset and offset via synapses connecting photoreceptors to ON and OFF bipolar cells, respectively. It remains poorly understood which molecular cues establish the architecture of this synaptic configuration to split light onset and offset signals. A mutant with reduced synapses between photoreceptors and one bipolar cell type, but not the other, could reveal a critical cue. From this approach, we report a novel synaptic role pregnancy associated plasma protein aa (pappaa) in promoting the structure and function of cone synapses that transmit light offset information. Electrophysiological and behavioral analyses indicated pappaa mutant zebrafish have dysfunctional cone to OFF bipolar cell synapses and impaired responses to light offset, but intact cone to ON bipolar cell synapses and light onset responses. Ultrastructural analyses of pappaa mutant cones showed a lack of presynaptic domains at synapses with OFF bipolar cells. pappaa is expressed postsynaptically to the cones during retinal synaptogenesis and encodes a secreted metalloprotease known to stimulate insulin-like growth factor 1 (IGF1) signaling. Induction of dominant negative IGF1 receptor expression during synaptogenesis reduced light offset responses. Conversely, stimulating IGF1 signaling at this time improved pappaa mutants light offset responses and cone presynaptic structures. Together, our results indicate Pappaa-regulated IGF1 signaling as a novel pathway that establishes how cone synapses convey light offset signals to guide behavior.\n\nSignificance StatementDistinct sensory inputs, like stimulus onset and offset, are often split at distinct synapses into parallel circuits for processing. In the retina, photoreceptors and ON and OFF bipolar cells form discrete synapses to split neural signals coding light onset and offset, respectively. The molecular cues that establish this synaptic configuration to specifically convey light onset or offset remain unclear. Our work reveals a novel cue: pregnancy associated plasma protein aa (pappaa), which regulates photoreceptor synaptic structure and function to specifically transmit light offset information. Pappaa is a metalloprotease that stimulates local insulin-like growth factor 1 (IGF1) signaling. IGF1 promotes various aspects of synaptic development and function and is broadly expressed; thus requiring local regulators, like Pappaa, to govern its specificity.

neuroscience↗

bioRxiv2017

Transcriptional control by Sall4 in blastocysts facilitates lineage commitment of inner cell mass cells

The enhancer-binding zinc finger transcription factor Sall4 is essential for early mammalian postimplantation development and plays important roles in lineage commitment of embryonic stem cells. Enhancer binding by Sall4 results in transcriptional activation of some genes, but repression of others. Exactly how cells in preimplantation stage embryos use this transcriptional modulatory activity of Sall4 during early developmental transitions has not been determined. Using single cell gene expression analyses we show that Sall4 is required to maintain the gene regulatory network in inner cell mass (ICM) cells prior to lineage commitment. Although Sall4 is not required for ICM cells to adopt a correct epiblast or primitive endoderm gene expression profile, in the absence of Sall4 early ICM cells commit to either lineage at reduced frequency. We propose a model whereby Sall4 activity sets the stage for efficient progression from the uncommitted ICM progenitor state by modulating the gene regulatory network in early ICM cells.

developmental biology↗

bioRxiv2017

Nanopore sequencing of full-length BRCA1 mRNA transcripts reveals co-occurrence of known exon skipping events

Laboratory assays evaluating the effect of DNA sequence variants on BRCA1 mRNA splicing may contribute to classification by providing molecular evidence. However, our knowledge of normal and aberrant BRCA1 splicing events to date has been limited to data derived from assays targeting partial transcript sequences. For the first time, we resolve the exon structure of whole BRCA1 transcripts using MinION nanopore sequencing of long-range PCR amplicons. Our study identified 32 BRCA1 isoforms, including 18 novel isoforms which comprised skipping of multiple contiguous and/or non-contiguous exons. Furthermore, we show that known BRCA1 exon skipping events, such as {Delta}(9,10) and {Delta}21, can co-occur in a single transcript, with some isoforms containing four or more alternative splice junctions. Our results highlight complexity in BRCA1 transcript structure that has not previously been described. This finding has key implications for predicting translation frame of splicing transcripts, important for interpreting the clinical significance of spliceogenic variants. Future research is warranted to quantitatively assess full length BRCA1 transcript levels, and to assess the application of nanopore sequencing for routine evaluation of potential spliceogenic variants.

genomics↗

bioRxiv2017

Disequilibrium of fire-prone forests sets the stage for a rapid decline in conifer dominance during the 21st century

As trees are long-lived organisms, the impacts of climate change on forest communities may not be apparent on the time scale of years to decades. While lagged responses to environmental change are common in forested systems, potential for abrupt transitions under climate change may occur in environments where alternative vegetation states are influenced by disturbances, such as fire. The Klamath mountains (northern California and southwest Oregon, USA) are currently dominated by carbon rich and hyper-diverse temperate conifer forests, but climate change could disrupt the mechanisms promoting forest stability- regeneration and fire tolerance-- via shifts in the fire regime in conjunction with lower fitness of conifers under a hotter climate. Understanding how this landscape will respond to near-term climate change (before 2100) is critical for predicting potential climate change feedbacks and to developing sound forest conservation and management plans. Using a landscape simulation model, we estimate that 1/3 of the Klamath could transition from conifer forest to shrub/hardwood chaparral, triggered by an enhanced fire activity coupled with lower post-fire conifer establishment. Such shifts were more prevalent under higher climate change forcing (RCP 8.5) but were also simulated under the climate of 1950-2000, reflecting the joint influences of early warming trends and historical forest legacies. Our results demonstrate that there is a large potential for loss of conifer forest dominance--and associated carbon stocks and biodiversity- in the Klamath before the end of the century, and that some losses would likely occur even without the influence of climate change. Thus, in the Klamath and other forested landscapes subject to similar feedback dynamics, major ecosystem shifts should be expected when climate change disrupts key stabilizing feedbacks that maintain the dominance of long-lived, slowly regenerating trees.

ecology↗

bioRxiv2017

Persistent homology demarcates a leaf morphospace

Current morphometric methods that comprehensively measure shape cannot compare the disparate leaf shapes found in seed plants and are sensitive to processing artifacts. We explore the use of persistent homology, a topological method applied across the scales of a function, to overcome these limitations. The described method isolates subsets of shape features and measures the spatial relationship of neighboring pixel densities in a shape. We apply the method to the analysis of 182,707 leaves, both published and unpublished, representing 141 plant families collected from 75 sites throughout the world. By measuring leaves from throughout the seed plants using persistent homology, a defined morphospace comparing all leaves is demarcated. Clear differences in shape between major phylogenetic groups are detected and estimates of leaf shape diversity within plant families are made. This approach does not only predict plant family, but also the collection site, confirming phylogenetically invariant morphological features that characterize leaves from specific locations. The application of a persistent homology method to measure leaf shape allows for a unified morphometric framework to measure plant form, including shape and branching architectures.

plant biology↗

bioRxiv2017

Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, while less is known about human intestinal stem cells due to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas, and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC) associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from 4 adenoma organoid lines and were subjected to RNA-sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA-seq data from adenoma and normal organoids, we found correlations between LGR5 and CRC- specific genes, including DKK4 (dickkopf WNT signaling pathway inhibitor 4) and SMOC2 (SPARC related modular calcium binding 2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.

cancer biology↗

bioRxiv2017

Integrated growth factor signaling promotes lung epithelial progenitor cell expansion and maintenance in mice and humans

The bud tip epithelium of the branching mouse and human lung contains multipotent progenitors that are able to self-renew and give rise to all mature lung epithelial cell types. The current study aimed to understand the developmental signaling cues that regulate bud tip progenitor cells in the human fetal lung, which are present during branching morphogenesis, and to use this information to induce a bud tip progenitor-like population from human pluripotent stem cells (hPSCs) in vitro. We identified that FGF7, CHIR-99021 and RA maintained isolated human fetal lung epithelial bud tip progenitor cells in an undifferentiated state in vitro, and led to the induction of a 3-dimensional lung-like epithelium from hPSCs. 3-dimensional hPSC-derived lung tissue was initially patterned, with airway-like interior domains and bud tip-like progenitor domains at the periphery. Epithelial bud tip-like domains could be isolated, expanded and maintained as a nearly homogeneous population by serial passaging. Comparisons between human fetal lung epithelial bud tip cells and hPSC-derived bud tip-like cells were carried out using immunostaining, in situ hybridization and transcriptome-wide analysis, and revealed that in vitro derived tissue was highly similar to native lung. hPSC-derived epithelial bud tip-like structures survived in vitro for over 16 weeks, could be easily frozen and thawed and maintained multi-lineage potential. Furthermore, hPSC-derived epithelial bud tip progenitors successfully engrafted in the proximal airways of injured immunocompromised NSG mouse lungs, where they persisted for up to 6 weeks and gave rise to several lung epithelial lineages.

developmental biology↗