Genetic dissection of cytokinesis

Higher plants have evolved specific mechanisms for partitioning the cytoplasm of dividing cells. In the predominant mode of phragmoplast-assisted cytokinesis, a cell wall and flanking plasma membranes are made de novo from a transient membrane compartment, the cell plate, which in turn forms by vesicle fusion from the centre to the periphery of the dividing cell. Other modes of cytokinesis appear to occur in meiotic cells and developing gametophytes. Here we review recent progress in the analysis of plant cytokinesis, focusing on genetic studies in Arabidopsis which are beginning to identify structural and regulatory components of phragmoplast-assisted cytokinesis. Two classes of mutations have been described. In one class, the defects appear to be confined to cell plate formation, suggesting that the execution of cytokinesis is specifically affected. Mutations in the other class display more general defects in cell division. We also discuss possible roles of proteins that have been localised in cytokinetic cells but not characterised genetically. Finally, mutations affecting meiotic or gametophytic cell divisions suggest that mechanistically different modes of cytokinesis occur in higher plants.     

The classic cadherins in synaptic specificity

During brain development, billions of neurons organize into highly specific circuits. To form specific circuits, neurons must build the appropriate types of synapses with appropriate types of synaptic partners while avoiding incorrect partners in a dense cellular environment. Defining the cellular and molecular rules that govern specific circuit formation has significant scientific and clinical relevance because fine scale connectivity defects are thought to underlie many cognitive and psychiatric disorders. Organizing specific neural circuits is an enormously complicated developmental process that requires the concerted action of many molecules, neural activity, and temporal events. This review focuses on one class of molecules postulated to play an important role in target selection and specific synapse formation: the classic cadherins. Cadherins have a well-established role in epithelial cell adhesion, and although it has long been appreciated that most cadherins are expressed in the brain, their role in synaptic specificity is just beginning to be unraveled. Here, we review past and present studies implicating cadherins as active participants in the formation, function, and dysfunction of specific neural circuits and pose some of the major remaining questions.     

The classic cadherins in synaptic specificity

During brain development, billions of neurons organize into highly specific circuits. To form specific circuits, neurons must build the appropriate types of synapses with appropriate types of synaptic partners while avoiding incorrect partners in a dense cellular environment. Defining the cellular and molecular rules that govern specific circuit formation has significant scientific and clinical relevance because fine scale connectivity defects are thought to underlie many cognitive and psychiatric disorders. Organizing specific neural circuits is an enormously complicated developmental process that requires the concerted action of many molecules, neural activity, and temporal events. This review focuses on one class of molecules postulated to play an important role in target selection and specific synapse formation: the classic cadherins. Cadherins have a well-established role in epithelial cell adhesion, and although it has long been appreciated that most cadherins are expressed in the brain, their role in synaptic specificity is just beginning to be unraveled. Here, we review past and present studies implicating cadherins as active participants in the formation, function, and dysfunction of specific neural circuits and pose some of the major remaining questions.     

Genetic dissection of retinoid dehydrogenases

Biochemical studies indicate that alcohol dehydrogenase (ADH) metabolizes retinol to retinal, and that aldehyde dehydrogenase (ALDH) metabolizes retinal to retinoic acid, a molecule essential for growth and development. Summarized herein are several genetic studies supporting in vivo functions for ADH and ALDH in retinoic acid synthesis. Gene targeting was used to create knockout mice for either Adh1 or Adh4. Both knockout mice were viable and fertile without obvious defects. However, when wild-type and Adh4 knockout mice were subjected to vitamin A deficiency during gestation, the survival rate at birth was 3.3-fold lower for Adh4 knockout mice. When adult mice were examined for production of retinoic acid following retinol administration, Adh1 knockout mice exhibited 10-fold lower retinoic acid levels in liver compared with wild-type, whereas Adh4 knockout mice differed from wild-type by less than 2-fold. Thus, Adh1 plays a major role in the metabolism of a large dose of retinol to retinoic acid in adults, whereas Adh4 plays a role in maintaining sufficient retinol metabolism for development during retinol deficiency. ALDHs were examined by overexpression studies in frog embryos. Injection of mRNAs for either mouse Raldh1 or Raldh2 stimulated retinoic acid synthesis in frog embryos at the blastula stage when retinoic acid is normally undetectable. Overexpression of human ALDH2, human ALDH3, and mouse Aldh-pb did not stimulate retinoic acid production. In addition, Raldh2 knockout mice exhibit embryonic lethality with defects in retinoid-dependent tissues. Overall, these studies provide genetic evidence that Adh1, Adh4, Raldh1, and Raldh2 encode retinoid dehydrogenases involved in retinoic acid synthesis in vivo.     

Genetic dissection of neural circuits

Understanding the principles of information processing in neural circuits requires systematic characterization of the participating cell types and their connections, and the ability to measure and perturb their activity. Genetic approaches promise to bring experimental access to complex neural systems, including genetic stalwarts such as the fly and mouse, but also to nongenetic systems such as primates. Together with anatomical and physiological methods, cell-type-specific expression of protein markers and sensors and transducers will be critical to construct circuit diagrams and to measure the activity of genetically defined neurons. Inactivation and activation of genetically defined cell types will establish causal relationships between activity in specific groups of neurons, circuit function, and animal behavior. Genetic analysis thus promises to reveal the logic of the neural circuits in complex brains that guide behaviors. Here we review progress in the genetic analysis of neural circuits and discuss directions for future research and development.     

Calorimetric dissection of colicin DNase--immunity protein complex specificity

We explore the thermodynamic strategies used to achieve specific, high-affinity binding within a family of conserved protein-protein complexes. Protein-protein interactions are often stabilized by a conserved interfacial hotspot that serves as the anchor for the complex, with neighboring variable residues providing specificity. A key question for such complexes is the thermodynamic basis for specificity given the dominance of the hotspot. We address this question using, as our model, colicin endonuclease (DNase)-immunity (Im) protein complexes. In this system, cognate and noncognate complexes alike share the same mechanism of association and binding hotspot, but cognate complexes (K(d) approximately 10(-)(14) M) are orders of magnitude more stable than noncognate complexes (10(6)-10(10)-fold discrimination), largely because of a much slower rate of dissociation. Using isothermal titration calorimetry (ITC), we investigated the changes in enthalpy (DeltaH), entropy (-TDeltaS), and heat capacity (DeltaC(p)) accompanying binding of each Im protein (Im2, Im7, Im8, and Im9) to the DNase domains of colicins E2, E7, E8, and E9, in the context of both cognate and noncognate complexes. The data show that specific binding to the E2, E7, and E8 DNases is enthalpically driven but entropically driven for the E9 DNase. Analysis of DeltaC(p), a measure of the change in structural fluctuation upon complexation, indicates that E2, E7, and E8 DNase specificity is coupled to structural changes within cognate complexes that are consistent with a reduction in the conformational dynamics of these complexes. In contrast, E9 DNase specificity appears coupled to the exclusion of water molecules, consistent with the nonpolar nature of the interface of this complex. The work highlights that although protein-protein interactions may be centered on conserved structural epitopes the thermodynamic mechanism underpinning binding specificity can vary considerably.     

Genetic dissection of centromere function.

A system to detect a minimal function of Saccharomyces cerevisiae centromeres in vivo has been developed. Centromere DNA mutants have been examined and found to be active in a plasmid copy number control assay in the absence of segregation. The experiments allow the identification of a minimal centromere unit, CDE III, independently of its ability to mediate chromosome segregation. Centromere-mediated plasmid copy number control correlates with the ability of CDE III to assemble a DNA-protein complex. Cells forced to maintain excess copies of CDE III exhibit increased loss of a nonessential artificial chromosome. Thus, segregationally impaired centromeres can have negative effects in trans on chromosome segregation. The use of a plasmid copy number control assay has allowed assembly steps preceding chromosome segregation to be defined.     

Genetic dissection of plant cell-wall biosynthesis

The plant cell wall is a complex structure consisting of a variety of polymers including cellulose, xyloglucan, xylan and polygalacturonan. Biochemical and genetic analysis has made it possible to clone genes encoding cellulose synthases (CesA). A comparison of the predicted protein sequences in the Arabidopsis genome indicates that 30 divergent genes with similarity to CesAs exist. It is possible that these cellulose synthase-like (Csl) proteins do not contribute to cellulose synthesis, but rather to the synthesis of other wall polymers. A major challenge is, therefore, to assign biological function to these genes. In an effort to address this issue we have systematically identified T-DNA or transposon insertions in 17 Arabidopsis Csls. Phenotypic characterization of "knock-out" mutants includes the determination of spectroscopic profile differences in mutant cell walls from wild-type plants by Fourier-transform IR microscopy. A more precise characterization includes cell wall fractionation followed by neutral sugar composition analysis by anionic exchange chromatography.     

Molecular mechanisms of synaptic specificity in developing neural circuits

The function of the brain depends on highly specific patterns of connections between populations of neurons. The establishment of these connections requires the targeting of axons and dendrites to defined zones or laminae, the recognition of individual target cells, the formation of synapses on particular regions of the dendritic tree, and the differentiation of pre- and postsynaptic specializations. Recent studies provide compelling evidence that transmembrane adhesion proteins of the immunoglobulin, cadherin, and leucine-rich repeat protein families, as well as secreted proteins such as semaphorins and FGFs, regulate distinct aspects of neuronal connectivity. These observations suggest that the coordinated actions of a number of molecular signals contribute to the specification and differentiation of synaptic connections in the developing brain.     

Genetic dissection of cadherin function during nephrogenesis

The distinct expression of R-cadherin in the induced aggregating metanephric mesenchyme suggests that it may regulate the mesenchymal-epithelial transition during kidney development. To address whether R-cadherin is required for kidney ontogeny, R-cadherin-deficient mice were generated. These mice appeared to be healthy and were fertile, demonstrating that R-cadherin is not essential for embryogenesis. The only kidney phenotype of adult mutant animals was the appearance of dilated proximal tubules, which was associated with an accumulation of large intracellular vacuoles. Morphological analysis of nephrogenesis in R-cadherin(-/-) mice in vivo and in vitro revealed defects in the development of both ureteric bud-derived cells and metanephric mesenchyme-derived cells. First, the morphology and organization of the proximal parts of the ureteric bud epithelium were altered. Interestingly, these morphological changes correlated with an increased rate of apoptosis and were further supported by perturbed branching and patterning of the ureteric bud epithelium during in vitro differentiation. Second, during in vitro studies of mesenchymal-epithelial conversion, significantly fewer epithelial structures developed from R-cadherin(-/-) kidneys than from wild-type kidneys. These data suggest that R-cadherin is functionally involved in the differentiation of both mesenchymal and epithelial components during metanephric kidney development. Finally, to investigate whether the redundant expression of other classic cadherins expressed in the kidney could explain the rather mild kidney defects in R-cadherin-deficient mice, we intercrossed R-cadherin(-/-) mice with cadherin-6(-/-), P-cadherin(-/-), and N-cadherin(+/-) mice. Surprisingly, however, in none of the compound knockout strains was kidney development affected to a greater extent than within the individual cadherin knockout strains.     

Genetic dissection of barley morphology and development

Since the early 20th century, barley (Hordeum vulgare) has been a model for investigating the effects of physical and chemical mutagens and for exploring the potential of mutation breeding in crop improvement. As a consequence, extensive and well-characterized collections of morphological and developmental mutants have been assembled that represent a valuable resource for exploring a wide range of complex and fundamental biological processes. We constructed a collection of 881 backcrossed lines containing mutant alleles that induce a majority of the morphological and developmental variation described in this species. After genotyping these lines with up to 3,072 single nucleotide polymorphisms, comparison to their recurrent parent defined the genetic location of 426 mutant alleles to chromosomal segments, each representing on average <3% of the barley genetic map. We show how the gene content in these segments can be predicted through conservation of synteny with model cereal genomes, providing a route to rapid gene identification.     

Genetic dissection of the Drosophila circadian system

Genetic experiments involving selected strains as well as single gene mutations have provided information concerning the organization of the Drosophila circadian system. The phase of the emergence rhythm of D. pseudoobscura can be altered by genetic selection without significantly affecting the phase and period of the light-sensitive pacemaker. The period of the D. melanogaster pacemaker, over the range 19 hours to 29 hours, can be encoded in the DNA sequence of a single genetic locus. The short-period and long-period mutations do not eliminate the pacemaker's temperature compensation mechanism. The short-period mutation alters the resetting behavior of the pacemaker from weak (type 1) in wild-type to strong (type 0) in the mutant. Five aperiodic mutations isolated in D. pseudoobscura belong to two complementation groups. In complements bearing one mutation from each group, the periodicity of the pacemaker is wild-type, but the phase of the emergence rhythm is 5 hours later than wild-type. Thus mutations in particular genetic loci have dramatic effects on the basic properties of circadian pacemakers and rhythms.     

Genetic dissection of parallel sister-chromatid cohesion pathways

Sister-chromatid cohesion, the process of pairing replicated chromosomes during mitosis and meiosis, is mediated through the essential cohesin complex and a number of nonessential cohesion genes, but the specific roles of these nonessential genes in sister-chromatid cohesion remain to be clarified. We analyzed sister-chromatid cohesion in double mutants of mrc1Delta, tof1Delta, and csm3Delta and identified additive cohesion defects that indicated the existence of at least two pathways that contribute to sister-chromatid cohesion. To understand the relationship of other nonessential cohesion genes with respect to these two pathways, pairwise combinations of deletion and temperature-sensitive alleles were tested for cohesion defects. These data defined two cohesion pathways, one containing CSM3, TOF1, CTF4, and CHL1, and the second containing MRC1, CTF18, CTF8, and DCC1. Furthermore, we found that the nonessential genes are not important for the maintenance of cohesion at G(2)/M. Thus, our data suggest that nonessential cohesion genes make critical redundant contributions to the establishment of sister-chromatid cohesion and define two cohesion pathways, thereby establishing a framework for understanding the role of nonessential genes in sister-chromatid cohesion.     

Genetic dissection of histidine biosynthesis in Arabidopsis

The biosynthesis of histidine (His) in microorganisms, long studied through the isolation and characterization of auxotrophic mutants, has emerged as a paradigm for the regulation of metabolism and gene expression. Much less is known about His biosynthesis in flowering plants. One limiting factor has been the absence of large collections of informative auxotrophs. We describe here the results of a systematic screen for His auxotrophs of Arabidopsis (Arabidopsis thaliana). Ten insertion mutants disrupted in four different biosynthetic genes (HISN2, HISN3, HISN4, HISN6A) were identified through a combination of forward and reverse genetics and were shown to exhibit an embryo-defective phenotype that could be rescued by watering heterozygous plants with His. Male transmission of the mutant allele was in several cases reduced. Knockouts of two redundant genes (HISN1B and HISN5A) had no visible phenotype. Another mutant blocked in the final step of His biosynthesis (hisn8) and a double mutant altered in the redundant first step of the pathway (hisn1a hisn1b) exhibited a combination of gametophytic and embryonic lethality in heterozygotes. Homozygous mutant seedlings and callus tissue produced from rescued seeds appeared normal when grown in the presence of His but typically senesced after continued growth in the absence of His. These knockout mutants document the importance of His biosynthesis for plant growth and development, provide valuable insights into amino acid transport and source-sink relationships during seed development, and represent a significant addition to the limited collection of well-characterized auxotrophs in flowering plants.