CSB340 Midterm Notes.doc

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Cell and Systems Biology
Thomas Berleth

Lecture 5: Mutations in the FASS gene uncouple pattern formation and morphogenesis in Arabidopsis Development Torres et al. Fass gene recessive mutations in this gene alter the pattern of cell division from the zygote without interfering with pattern formation o Affects cell elongation and orientation of cell walls but dont interfere with polarity Evidence: unequal division of zygote still occurs as in wild type Stele (vasculature) derived signals radial patterning, root meristem formation, cotyledon positioning depend on vasculature derived signals Forward genetic screen define indicative phenotypes, collect mutants for this phenotype, derive complementation units (genes), and confirm saturation of screen Pattern defect defined structures (tissues and organs) misplaced in space and time, deletion, duplication, incorrectly polarized Morphogenesis defect abnormal appearance, cell shape, physiological condition, viability and health Fass mutants organ and tissue positions intact, continuous vascular strands, functional apical meristem, and clear separation of vascular strands Weak alleles are useful to show that the phenotype should move in the same direction of severity and the strong allele phenotype. Demonstrates that when you have residual function you should see a gradient of the same phenotype ,i.e. will show you that this is what the gene does o Trans-heterozygote demonstrates that the phenotype is due to loss-of-function or reduced function of the fass gene In order for a mutant to be a patterning mutant, it has to be established in the pre- heart stage, but morphogenesis mutants can be established in any stage due to abnormal cell shapes and abnormal rates of cell division Longitudinal cross-sections of fass mutants epidermal layer clearly recognizable, stomatal guard cells are well-differentiated, narrow cells in the center correspond to vascular cells o Apical-basal patterns: SAM, RAM, hypocotyls, cotyledons, root hair, root o Radial patterns: epidermal layer, vascular strands Fass mutants exhibit abnormal arrangements of cell walls and look abnormal before the heart stage Mutant phenotype was expressed at the cellular level in the leaf epidermis (no pattern defect in seedlings): o Epidermal cells: Interdigitated in wild type and round in mutant o Leaf Trichomes: Three branches in wild type and unbranched or abolished in fass mutant These results suggest that the fass gene is required during post- embryonic development in a similar manner as embryogenesis Fass mutants look highly abnormal at the heart stage but not all mutants defective at the heart stage are patterning mutants, but all patterning mutants are defective at the heart stage Polarity is important for pattern formation Fass gene is required for position-dependent cell shape + correct orientation of cell walls + oriented cell divisions o Fass gene is not required for polarity which is essential for pattern formation Fass gene acts in a quantitative manner amount of functional product directly determines biological effect o The weak alleles make less product, rather than less active product; consistent with partial lack of complementation between weak alleles o The strong alleles probably lack gene activity The fass gene is required throughout embryonic development and post-embryonic development in all cell types All changes in morphogenesis (orderly division, elongation) can be brought by reorientation of cortical microtubules + cellulose microfibrils Pattern formation genes = MP, GNOM o Fass mutant suggests that oriented cell divisions are not associated with pattern formation o Pattern formation is not dependent on morphogenesis Additive phenotype of the fass gnom double mutant suggests that the two processes are independent Conclusion Although the division of the zygote produces a variably oriented cell wall and subsequent divisions of both the daughter cells are abnormal, cell polarity is not affected since the apical and basal cell differ in staining intensity as they do in wild type Cell polarity as set up in the zygote might be essential for cells to acquire information relative to their positions which may underlie pattern formation Although cell division is distorted in the fass mutants, the arrangement of internal patterns, layers, and tissues are intact Fass dont undergo regular or stereotypic divisions, but patterns are still established. Its not a cell by cell mechanism to lineage doesnt seem to matter In the wild type plant development, pattern formation and morphogenesis are coupled. Mutations in the fass gene affected morphogenesis but not pattern formation, suggesting that the regularity of cell division is not associated with pattern formation in the embryos Cell divisions look so regular in plants that they suggest the importance of lineage, but when distorted as in fass mutants, it turns out that its position (cell to cell communication), not lineage that determines a cells fate Lecture 6: Regulation of Polar Auxin Transport by Pin1 in Arabidopsis Vascular Tissue Galweiler et al. Polar auxin transport controls vascular tissue formation. PIN1 mutants have diminished polar auxin transport. Polar auxin transport provides directional information influencing: vascular tissue differentiation, apical development, organ regeneration, and cell elongation Conclusion of stem incision experiment: IAA is required and sufficient, response is polar, response is semi-linear, and response generates continuity Chemiosmotic model: the driving the driving force for polar auxin transport is provided by the transmembrane proton motive force and the cellular efflux of auxin anions is mediated by saturable, auxin-specific carriers in located at the basal end of transport-competent cells Immunocytochemical work with monoclonal antibodies indicated that auxin efflux carriers are located at the basal end of cells Canalization of auxin flow auxin acts to polarize its own transport. The initial diffusion of auxin away from a source positively reinforces its own transport, which ultimately leads to the distribution of auxin into narrow canals, and this canalization underlies coordinated tissue polarization Model: Influx carrier (AUX1) + Efflux carrier + Efflux carrier polarly mobilized to the basal side of the cell Polar auxin transport could is essential for cell polarity and embryo axis formation (tissue patterning) o If this is the case, inhibition of auxin before the heart stage should distort cell polarity o Using a range of concentrations of NPA/PCIB, apical-basal and radial axes are distorted, therefore auxin causes patterning in the embryo Chemical inhibition mimics pin1 phenotype due to inhibition of polar auxin transport Chemical inhibition of auxin results in pin-shaped, flowerless inflorescences Isolation of AtPIN1 Use of autonomous transposable elements to generate mutants allowed isolation of AtPIN1 gene locus Analyze function of AtPIN1 proteins: raise polyclonal antibodies to a portion of recombinant AtPIN1 Northern Blots with AtPIN1-specific probe showed that the gene was transcribed in all wild-type organs and was absent in the homozygous PIN1 mutants but expressed in heterozygotes, indicating a recessive mutant. Distribution of transported radioactive IAA: Endogenous amount of free IAA is much lower in pin1 mutants than in wild type. This is evidence that an auxin efflux carrier might exist and that there might be a defect in it
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