Biomedical subjects
Publications and source records attributed to L Dolan.
The radial pattern of the plant root is determined by the action of two transcription factors, SHORT ROOT and SCARECROW, which are produced in different cell types. The SHORT ROOT protein has now been shown to move from cell to cell, regulating transcription of the downstream SCARECROW gene in the target cells.
Multicellular organisms are composed of groups of different cell types. The importance in the evolution of cellular diversity of regulatory changes in genes that control cell specification has been revealed by a recent study of developmental genes in Arabidopsis.
The cell wall is an important determinant of plant cell form. Here we define a class of Arabidopsis root hair mutants with defective cell walls. Plants homozygous for kojak (kjk) mutations initiate root hairs that rupture at their tip soon after initiation. The KJK gene was isolated by positional cloning, and its identity was confirmed by the molecular complementation of the Kjk(-) phenotype and the sequence of three kjk mutant alleles. KOJAK encodes a cellulose synthase-like protein, AtCSLD3. KOJAK/AtCSLD3 is the first member of this subfamily of proteins to be shown to have a function in cell growth. Subcellular localization of the KOJAK/AtCSLD3 protein using a GFP fusion shows that KOJAK/AtCSLD3 is located on the endoplasmic reticulum, indicating that KOJAK/AtCSLD3 is required for the synthesis of a noncellulosic wall polysaccharide. Consistent with the cell specific defect in the roots of kjk mutants, KOJAK/AtCSDL3 is preferentially expressed in hair cells of the epidermis. The Kjk(-) phenotype and the pattern of KOJAK/AtCSLD3 expression suggest that this gene acts early in the process of root hair outgrowth. These results suggest that KOJAK/AtCSLD3 is involved in the biosynthesis of beta-glucan-containing polysaccharides that are required during root hair elongation.
In this review we integrate the information available on the cell biology of root hair formation with recent findings from the analysis of root hair mutants of Arabidopsis thaliana. The mature Arabidopsis root epidermis consists of root-hair-producing cells and non-root-hair-producing cells. Root hair growth begins with a swelling of the outer epidermal wall. It has been postulated that this is due to a pH-mediated localised cell wall loosening. From the bulge a single root hair emerges which grows by tip growth. The root hair tip consists of a vesicle-rich zone and an organelle-rich subapical zone. The vesicles supply new plasma membrane and cell wall material for elongation. The cytoskeleton and its associated regulatory proteins such as profilin and spectrin are proposed to be involved in the targeting of vesicles. Ca2+ influxes and gradients are present in hair tips, but their function is still unclear. Mutants have been isolated with lesions in various parts of the root hair developmental pathway from bulge identity and initiation to control of tip diameter and extent and polarity of elongation.
The root hair of Arabidopsis has become a model system for investigations of the patterning and morphogenesis of cells in plants. A cascade of transcriptional regulators controls the pattern of cellular differentiation. Recently, one of the genes that plays a specific role in cellular differentiation in roots, WEREWOLF, has been shown to be functionally equivalent to GLABRA1, which functions only in the shoot. The cloning of genes defined by mutants with defective root-hair growth has provided insights into the roles of the cell wall, ion transport and the cytoskeleton during hair growth. Genetic analyses continue to identify mutants that will be instructive in furthering our understanding of the growth and development of root-hair cells.
Root hair pattern develops in a number of different ways in angiosperm. Cells in the epidermis of some species undergo asymmetric cell divisions to form a smaller daughter cell from which a hair grows, and a larger cell that forms a non-hair epidermal cell. In other species any cell in the epidermis can form a root hair. Hair cells are arranged in files along the Arabidopsis root, located in the gaps between underlying cortical cell files. Epidermal cells overlying a single cortical cell file develop as non-hair epidermal cells. Genetic analysis has identified a transcription factor cascade required for the formation of this pattern. WEREWOLF (WER) and GLABRA2 (GL2) are required for the formation of non-hair epidermal cells while CAPRICE (CPC) is required for hair cell development. Recent analyses of the pattern of epidermal cells among the angiosperms indicate that this striped pattern of cell organization evolved from non-striped ancestors independently in a number of diverse evolutionary lineages. The genetic basis for the evolution of epidermal pattern in angiosperms may now be examined.
Root hair initiation involves the formation of a bulge at the basal end of the trichoblast by localized diffuse growth. Tip growth occurs subsequently at this initiation site and is accompanied by the establishment of a polarized cytoplasmic organization. Arabidopsis plants homozygous for a complete loss-of-function tiny root hair 1 (trh1) mutation were generated by means of the T-DNA-tagging method. Trichoblasts of trh1 plants form initiation sites but fail to undergo tip growth. A predicted primary structure of TRH1 indicates that it belongs to the AtKT/AtKUP/HAK K(+) transporter family. The proposed function of TRH1 as a K(+) transporter was confirmed in (86)Rb uptake experiments, which demonstrated that trh1 plants are partially impaired in K(+) transport. In line with these results, TRH1 was able to complement the trk1 potassium transporter mutant of Saccharomyces, which is defective in high-affinity K(+) uptake. Surprisingly, the trh1 phenotype was not restored when mutant seedlings were grown at high external potassium concentrations. These data demonstrate that TRH1 mediates K(+) transport in Arabidopsis roots and is responsible for specific K(+) translocation, which is essential for root hair elongation.
OBJECTIVE: The purpose of this study was to investigate the impact of knowing chronologic age on the variability of pediatric bone age determination using the method of Greulich and Pyle. MATERIALS AND METHODS: Radiographs of the left hand of 107 patients were interpreted by four radiologists on two separate occasions, once with and once without knowledge of the patient's chronologic age at time of interpretation. Twenty-five radiographs were randomly selected and reevaluated twice by each radiologist. Interobserver and intraobserver variability were calculated and compared for the two conditions. The distribution of studies with normal and abnormal findings was then compared across knowledge conditions for all observers and by individual observer, using two standard deviations above and below chronologic age as the range of "normal". RESULTS: When the chronologic age was known, the interobserver reliability coefficient for knowledge of chronologic age was 0.954 and when not known, 0.952. The intraobserver reliability coefficients when chronologic age was known ranged from 0.944 to 0.967, and when not known from 0.938 to 0.980. Observers interpreted 58% (248/428) of the radiographs as having normal findings when chronologic age was known and 48% (205/428) when chronologic age was not known. CONCLUSION: Knowing chronologic age before assessing bone age radiographs does not affect the reproducibility of assessment. However, observers are more likely to interpret the radiograph as showing normal findings when chronologic age is known than if the interpretation is performed with the observer unaware of chronologic age.
The cellular organization of the Arabidopsis thaliana (L.) Heynh. root meristem suggests that a regular pattern of cell divisions occurs in the root tip. Deviations from this pattern of division might be expected to disrupt the organization of cells and tissues in the root. A clonal analysis of the 3-d-old primary root meristem was carried out to determine if there is variability in division patterns, and if so to discover their effect on cellular organization in the root. Clones induced in the seedling meristem largely confirmed the predicted pattern of cell divisions. However, the cellular initials that normally give rise to the different cell files in the root were shown to exhibit some instability. For example, it was calculated that a lateral root cap/epidermal initial is displaced every 13 d. Furthermore, the existence of large marked clones that included more than two adjacent cell layers suggests that intrusive growth followed by cell division may occur at low frequency, perhaps in response to local cell deaths in the meristem. These findings support the view that even in plant organs with stereotypical cell division patterns, positional information is still the key determinant of cell fate.
The root forms at the basal end of an axis that is set up early in embryogenesis, and recent genetic analysis has indicated that auxin transport is required for the formation of the root pole. Drug studies show that auxin transport is also required for the maintenance of the tissue organisation in the seedling root. These studies support existing models for tissue patterning that involve canalised auxin flow. Molecular insights into the mechanism of cell-type specification and patterning has come from the epidermis where positionally controlled cell specification is regulated by a cascade of transcription factors.
The nucleus in plants and animals is a highly structured organelle containing several well-defined subregions or suborganelles. These include the nucleolus, interphase chromosome territories and coiled bodies. We have visualized transcription sites in plants at both light- and electron-microscopy level by the incorporation of BrUTP. In the nucleolus many dispersed foci are revealed within the dense fibrillar component, each of which probably corresponds to a single gene copy. In the nucleoplasm there are also many dispersed foci of transcription, but not enough to correspond to one site per transcribed gene. We have shown that in wheat, and probably many other plant species, interphase chromosome territories are organized in a very regular way, with all the chromosomes in the Rabl configuration, all the centromeres clustered at the nuclear membrane and all the telomeres located at the nuclear membrane on the opposite side of the nucleus. However, despite this regular, polarized structure, there is no sign of polarization of transcription sites, or of any preferred location for them with respect to chromosome territorial boundaries. The nucleus is also highly dynamic. As an example, we have shown by the use of a green fluorescent protein fusion to the spliceosomal protein U2B" that coiled bodies move and coalesce within the nucleus, and may act as transport structures within the nucleus and nucleolus.
Root hairs are a major site for the uptake of water and nutrients into plants and form an increasingly important model system for studies of development of higher plants and cell biology. We have identified loss-of-function mutations in eight new genes required for hair growth in Arabidopsis: SHAVEN1 (SHV1), SHV2, and SHV3; CENTIPEDE1 (CEN1), CEN2, and CEN3; BRISTLED1 (BST1); and SUPERCENTIPEDE1 (SCN1). We combined mutations in 79 pairs of genes to determine the stages at which these and six previously known genes contribute to root hair formation. Double mutant phenotypes revealed roles for several genes that could not have been predicted from the single mutant phenotypes. For example, we show that TIP1 and RHD3 are required much earlier in hair formation than previous studies have suggested. We present a genetic model for root hair morphogenesis that defines the roles of each gene, and we suggest hypotheses about functional relationships between genes.
The cell layers of the Arabidopsis primary root are arranged in a simple radial pattern. The outermost layer is the lateral root cap and lies outside the epidermis that surrounds the ground tissue. The files of epidermal and lateral root cap cells converge on a ring of initials (lateral root cap/epidermis initial) from which the epidermal and lateral root cap tissues of the seedling are derived, once root growth is initiated after germination. Each initial gives rise to a clone of epidermal cells and a clone of lateral root cap cells. These initial divisions in the epidermal/lateral root cap initial are defective in tornado1 (trn1) and trn2 plants indicating a requirement for TRN1 and TRN2 for initial cell function. Furthermore, lateral root cap cells develop in the epidermal position in trn1 and trn2 roots indicating that TRN1 and TRN2 are required for the maintenance of the radial pattern of cell specification in the root. The death of these ectopic lateral root cap cells in the elongation zone (where lateral root cap cells normally die) results in the development of gaps in the epidermis. These observations indicate that TRN1 and TRN2 are required to maintain the distinction between the lateral root cap and epidermis and suggest that lateral root cap fate is the default state. It also suggests that TRN1 and TRN2 repress lateral root cap fate in cells in the epidermal location. Furthermore, the position-dependent pattern of root hair and non-root hair cell differentiation in the epidermis is defective in trn1 and trn2 mutants. Together these results indicate that TRN1 and TRN2 are required for the maintenance of both the radial pattern of tissue differentiation in the root and for the subsequent circumferential pattern within the epidermis.
Positional information is an important determinant in the establishment of cellular identity in plants. It is established during pattern formation and is maintained in growing organs. Cells maintain the ability to respond to changes in positional information during development indicating that the mechanism for perceiving such information must remain intact until relatively late in development. Once positional cues are perceived they set in motion a number of cascades resulting in the differentiation of particular cell types in defined locations. The circuitry underpinning these later events is being teased out using genetics. Evidence is emerging for the existence of an array of both positive and negative genetic regulators from studies in a number of diverse plant model systems
Root hairs of Arabidopsis roots develop on trichoblasts located over the anticlinal (radial) walls of underlying cortical cells. Non-hair cells, on the other hand, develop on atrichoblasts overlying the periclinal (tangential) walls of cortical cells. Dark-grown wild-type seedlings, which produce little ethylene, are largely root hairless. Exogenous treatment of dark-grown plants with either ethylene or 1-aminocyclopropane-1-carboxylic acid (ACC) restores the development of root hairs in cells overlying the anticlinal cortical cell walls, indicating that cells in this position are more sensitive to ethylene than atrichoblasts. We used mutations in genes that overproduce ethylene (eto1, eto2, eto3 and eto4) to illustrate the positive regulatory role of ethylene. The preferential development of root hairs on epidermal cells overlying the cortical anticlinal cell walls in these mutants also illustrates that trichoblasts are more sensitive to ethylene than atrichoblasts. CTR1 is a negative regulator of the ethylene response and might, therefore, be a candidate regulator of differential sensitivity. CTR1 mRNA is expressed in all cell types in the root, suggesting that its transcriptional pattern alone cannot account for the differential sensitivity of epidermal cells to ethylene. Cellular mapping of wild-type and mutant roots supports previous findings indicating that ethylene acts after, and perhaps independently, of TTG during the establishment of cell fate in the root epidermis.
Coiled bodies are nuclear organelles that contain components of at least three RNA-processing pathways: pre-mRNA splicing, histone mRNA 3'- maturation, and pre-rRNA processing. Their function remains unknown. However, it has been speculated that coiled bodies may be sites of splicing factor assembly and/or recycling, play a role in histone mRNA 3'-processing, or act as nuclear transport or sorting structures. To study the dynamics of coiled bodies in living cells, we have stably expressed a U2B"-green fluorescent protein fusion in tobacco BY-2 cells and in Arabidopsis plants. Time-lapse confocal microscopy has shown that coiled bodies are mobile organelles in plant cells. We have observed movements of coiled bodies in the nucleolus, in the nucleoplasm, and from the periphery of the nucleus into the nucleolus, which suggests a transport function for coiled bodies. Furthermore, we have observed coalescence of coiled bodies, which suggests a mechanism for the decrease in coiled body number during the cell cycle. Deletion analysis of the U2B" gene construct has shown that the first RNP-80 motif is sufficient for localization to the coiled body.
BACKGROUND: Researchers and clinicians commonly use the classification system of Stulberg et al. as a basis for treatment decisions during the active phase of Legg-Calvé-Perthes disease because of its putative utility as a predictor of long-term outcome. It is generally assumed that this system has an acceptable degree of reliability. This assumption, however, is not convincingly supported by the literature. METHODS: The purpose of the present study was to assess the inter-rater and intra-rater reliability of the classification system of Stulberg et al. with use of a pre-test, post-test design. During the pre-test phase, nine raters independently used the system to evaluate the radiographs of skeletally mature patients who had been managed for Legg-Calvé-Perthes disease. The intervention between the pre-test and post-test phases consisted of a consensus-building session during which all raters jointly arrived at standardized definitions of the various joint structures that are assessed with use of the classification system. The effect of these definitions on reliability then was assessed by reevaluating the radiographs during the post-test phase. RESULTS: The pre-test intra-rater reliability coefficients ranged from 0.709 to 0.915, and the post-test coefficients ranged from 0.568 to 0.874. The pre-test inter-rater reliability coefficients ranged from 0.603 to 0.732, and the post-test coefficients ranged from 0.648 to 0.744. Contributing to the variance was a lack of agreement concerning the assessment of joint structures and the way in which the raters translated these evaluations into a classification according to the system of Stulberg et al. CONCLUSIONS: Although intra-rater reliability was marginally acceptable, the degree of variability between the classifications assigned by different raters even after the intervention - calls into question the reliability of the system of Stulberg et al.; consequently, the validity of any treatment decisions, outcome evaluations, or epidemiological studies based on this system is also in question.
We have used whole mount immunofluorescence labelling with the antibody 4G3, raised against the human snRNP-specific protein U2B", and whole mount in situ hybridization with an anti-sense probe to a conserved region of U2 snRNA, in combination with confocal microscopy, to examine the organization of spliceosomal components throughout the development of the Arabidopsis thaliana root epidermis. We show that the number of coiled bodies, nuclear organelles in which splicing snRNPs and snRNAs concentrate, is developmentally regulated in the Arabidopsis root epidermis. Firstly, there is a progression from a small number of coiled bodies in the quiescent centre and initial cells, to a larger number in the cell division zone, returning to a lower number in the cell elongation and differentiation zone. Secondly, trichoblasts (root-hair forming epidermal cells) have on average 1.5 times more and often smaller coiled bodies than atrichoblasts (hairless epidermal cells). Moreover, we have shown that these differences in coiled body numbers are related to differences in cell cycle stage, cell type and developmental stage, but are not due to differences in nucleolar or general metabolic activity per se. We discuss possible explanations, including a model in which coiled bodies coalesce during interphase, for the developmental dynamics of coiled bodies.