characterizing-epithelial-wound-healing-vivo-using-cnidarian-model

Characterizing Epithelial Wound Healing <em>In Vivo</em> Using the Cnidarian Model Organism <em>Clytia hemisphaerica</em>

All animal organs, from the skin to eyes to intestines, are covered with sheets of epithelial cells that allow them to maintain homeostasis while ...

The University of Chicago

Although root system architecture is known to be highly plastic and strongly affected by environmental conditions, we have little understanding of the underlying mechanisms controlling root system development. Here we demonstrate that the formation of a lateral root from a lateral root primordium is repressed as water availability is reduced. This osmotic-responsive regulatory mechanism requires abscisic acid (ABA) and a newly identified gene, LRD2. Mutant analysis also revealed interactions of ABA and LRD2 with auxin signaling. Surprisingly, further examination revealed that both ABA and LRD2 control root system architecture even in the absence of osmotic stress. This suggests that the same molecules that mediate responses to environmental cues can also be regulators of intrinsic developmental programs in the root system.

Osmotic regulation of root system architecture.

Root system size (RSS) is a complex trait that is greatly influenced by environmental cues. Hence, both intrinsic developmental pathways and environmental-response pathways contribute to RSS. To assess the natural variation in both types of pathways, we examined the root systems of the closely related Arabidopsis ecotypes Landsberg erecta (Ler) and Columbia (Col) grown under mild osmotic stress conditions. We found that Ler initiates more lateral root primordia, produces lateral roots from a higher percentage of these primordia, and has an overall larger root system than Col under these conditions. Furthermore, although each of these parameters is reduced by osmotic stress in both ecotypes, Ler shows a decreased sensitivity to osmotica. To understand the genetic basis for these differences, QTL for RSS under mild osmotic stress were mapped in a Ler x Col recombinant inbred population. Two robust quantitative trait loci (QTL) were identified and confirmed in near-isogenic lines (NILs). The NILs also allowed us to define distinct physiological roles for the gene(s) at each locus. This study provides insight into the genetic and physiological complexity that determines RSS and begins to dissect the molecular basis for naturally occurring differences in morphology and developmental plasticity in the root system.

Identification of quantitative trait loci that regulate Arabidopsis root system size and plasticity.

We have identified a gene, Lateral Root Development 3 (LRD3), that is important for maintaining a balance between primary and lateral root growth. The lrd3 mutant has decreased primary root growth and increased lateral root growth. We determined that the LRD3 gene encodes a LIM-domain protein of unknown function. LRD3 is expressed only in the phloem companion cells, which suggested a role in phloem function. Indeed, while phloem loading and export from the shoot appear to be normal, delivery of phloem to the primary root tip is limited severely in young seedlings. Abnormalities in phloem morphology in these seedlings indicate that LRD3 is essential for correct early phloem development. There is a subsequent spontaneous recovery of normal phloem morphology, which is correlated tightly with increased phloem delivery and growth of the primary root. The LRD3 gene is one of very few genes described to affect phloem development, and the only one that is specific to early phloem development. Continuous growth on auxin also leads to recovery of phloem development and function in lrd3, which demonstrates that auxin plays a key role in early phloem development. The root system architecture and the pattern of phloem allocation in the lrd3 root system suggested that there may be regulated mechanisms for selectively supporting certain lateral roots when the primary root is compromised. Therefore, this study provides new insights into phloem-mediated resource allocation and its effects on plant root system architecture.

Arabidopsis Lateral Root Development 3 is essential for early phloem development and function, and hence for normal root system development.

Plants adapt to their unique soil environments by altering the number and placement of lateral roots post-embryonic. Mutants were identified in Arabidopsis thaliana that exhibit increased lateral root formation. Eight mutants were characterized in detail and were found to have increased lateral root formation due to at least three distinct mechanisms. The causal mutation in one of these mutants was found in the XEG113 gene, recently shown to be involved in plant cell wall biosynthesis. Lateral root primordia initiation is unaltered in this mutant. In contrast, synchronization of lateral root initiation demonstrated that mutation of XEG113 increases the rate at which lateral root primordia develop and emerge to form lateral roots. The effect of the XEG113 mutation was specific to the root system and had no apparent effect on shoot growth. Screening of 17 additional cell wall mutants, altering a myriad of cell wall components, revealed that many (but not all) types of cell wall defects promote lateral root formation. These results suggest that proper cell wall biosynthesis is necessary to constrain lateral root primordia emergence. While previous reports have shown that lateral root emergence is accompanied by active remodelling of cell walls overlying the primordia, this study is the first to demonstrate that alteration of the cell wall is sufficient to promote lateral root formation. Therefore, inherent cell wall properties may play a previously unappreciated role in regulation of root system architecture.

Cell wall properties play an important role in the emergence of lateral root primordia from the parent root.

All animals have mechanisms for healing damage to the epithelial sheets that cover the body and line internal cavities. Epithelial wounds heal either by cells crawling over the wound gap, by contraction of a super-cellular actin cable ("purse string") that surrounds the wound, or some combination of the two mechanisms. Both cell crawling and purse string closure of epithelial wounds are widely observed across vertebrates and invertebrates, suggesting early evolution of these mechanisms. Cnidarians evolved ~600 million years ago and are considered a sister group to the Bilateria. They have been much studied for their tremendous regenerative potential, but epithelial wound healing has not been characterized in detail. Conserved elements of wound healing in bilaterians and cnidarians would suggest an evolutionary origin in a common ancestor. Here we test this idea by characterizing epithelial wound healing in live medusae of Clytia hemisphaerica.

In vivo imaging of epithelial wound healing in the cnidarian Clytia hemisphaerica demonstrates early evolution of purse string and cell crawling closure mechanisms.

Epithelial cell dynamics can be difficult to study in intact animals or tissues. Here we use the medusa form of the hydrozoan Clytia hemisphaerica, which is covered with a monolayer of epithelial cells, to test the efficacy of an orientation-independent differential interference contrast microscope for in vivo imaging of wound healing. Orientation-independent differential interference contrast provides an unprecedented resolution phase image of epithelial cells closing a wound in a live, nontransgenic animal model. In particular, the orientation-independent differential interference contrast microscope equipped with a 40x/0.75NA objective lens and using the illumination light with wavelength 546 nm demonstrated a resolution of 460 nm. The repair of individual cells, the adhesion of cells to close a gap, and the concomitant contraction of these cells during closure is clearly visualized.

Jocelyn Malamy

Department of Molecular Genetics and Cell Biology

The University of Chicago

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica