The formation of perinucleolar bodies is important for normal leaf development and requires the zinc‐finger DNA‐binding motif in Arabidopsis ASYMMETRIC LEAVES2

In Arabidopsis, the ASYMMETRIC LEAVES2 (AS2) protein plays a key role in the formation of flat symmetric leaves via direct repression of the abaxial gene ETT/ARF3. AS2 encodes a plant‐specific nuclear protein that contains the AS2/LOB domain, which includes a z inc‐f inger (ZF) motif that is conserved in the AS2/LOB family. We have shown that AS2 binds to the coding DNA of ETT/ARF3, which requires the ZF motif. AS2 is co‐localized with AS1 in perinucleolar bodies (AS2 bodies). To identify the amino acid signals in AS2 required for formation of AS2 bodies and function(s) in leaf formation, we constructed recombinant DNAs that encoded mutant AS2 proteins fused to yellow fluorescent protein. We examined the subcellular localization of these proteins in cells of cotyledons and leaf primordia of transgenic plants and cultured cells. The amino acid signals essential for formation of AS2 bodies were located within and adjacent to the ZF motif. Mutant AS2 that failed to form AS2 bodies also failed to rescue the as2‐1 mutation. Our results suggest the importance of the formation of AS2 bodies and the nature of interactions of AS2 with its target DNA and nucleolar factors including NUCLEOLIN1. The partial overlap of AS2 bodies with perinucleolar chromocenters with condensed ribosomal RNA genes implies a correlation between AS2 bodies and the chromatin state. Patterns of AS2 bodies in cells during interphase and mitosis in leaf primordia were distinct from those in cultured cells, suggesting that the formation and distribution of AS2 bodies are developmentally modulated in plants.     

Subcellular localizations of AS1 and AS2 suggest their common and distinct roles in plant development

During leaf organogenesis, a critical step for normal leaf primordium initiation is the repression of the class 1 KNOTTED1-like homeobox (KNOX) genes. After leaf primordia are formed, they must establish polarity for normal leaf morphogenesis.Recent studies have led to the identification of a number of genes that participate in the class 1 KNOX gene repression and/or the leaf polarity establishment. ASTMMETRIC LEAVES1 and 2 (AS1 and AS2) are two of these genes, which are critical for both of these two processes. As a first step towards understanding the molecular genetic basis of the AS1-AS2 action, we determined the subcellular Iocalizations of the two proteins in both tobacco BY2 cells and Arabidopsis plants,by fusing them to yellow/cyan fluorescent protein (YFPICFP). Our data showed that AS1 and AS2 alone were predominantly localized in the nucleolus and the nucleoplasm, respectively. The presence of both AS1 and AS2 proteins in the same interphase cell demonstrated their co-localization in both nucleolus and nucleoplasm. In addition, AS1 alone was able to associate with the condensed chromosome in the metaphase cell. Our data suggest that AS1, AS2 and the AS1-AS2 protein complex may have distinct functions, which are all required for normal plant development.     

EARLY HISTOGENESIS AND SEMIQUANTITATIVE HISTOCHEMISTRY OF LEAF INITIATION IN TRITICUM AESTIVUM

The shoot apex of Triticum aestivum cv. Ramona 50 was investigated histologically to describe cell lineages and events during leaf initiation. During histogenesis three periclinal divisions occurred in the first apical layer, with one or two divisions in the second apical layer. This sequence of cell divisions initially occurred in one region and spread laterally in both directions to encircle the meristem. Cells of the third apical layer were not involved in leaf histogenesis. Initially, young leaf primordia were produced from daughter cells of periclinal divisions in the two outer apical layers. Nuclear contents of protein, histone, and RNA in the shoot apex were evaluated as ratios to DNA by means of semiquantitative histochemistry. Daughter cells of periclinal divisions in the outer apical layer which produced the leaf primordia had higher histone/DNA ratios than cells of the remaining meristem. However, protein/DNA and RNA/DNA ratios were similar in both regions. Leaf initial cells had a higher ³H-thymidine labeling index, a higher RNA synthesis rate, and smaller nuclear volumes than cells of the residual apical meristem.     

The SCARECROW gene's role in asymmetric cell divisions in rice plants

Asymmetric cell division is one of the most important mechanisms in the diversification of cell function and fate. In Arabidopsis, SCARECROW (SCR) is essential for the asymmetric division of the cortex/endodermis progenitor cell in the root. To learn more about how SCR is involved in asymmetric division, we analyzed the rice SCR (OsSCR) expression. In the root tip, OsSCR expression was observed in the endodermal cell layer and downregulated in the daughter cortex cell after asymmetric division, just as with Arabidopsis SCR. In leaf primordia, expression of OsSCR was observed in stomatal and ligule formation. In stomatal development, OsSCR was specifically expressed in the stomatal cell files before formation of guard mother cells (GMCs), and then, its expression was localized in GMCs, when the first asymmetric division occurred to generate the GMCs. Before the second asymmetric division of subsidiary mother cells (SMCs), localized OsSCR expression was observed in SMCs in the area close to the GMCs. Before these asymmetric divisions, the localization of OsSCR mRNA in GMC-forming cells and SMCs was observed in the area of the daughter GMC and subsidiary cells. OsSCR expression was also observed in the initiation area of ligule formation, and its downregulation occurred in the inner L2 cells generated by asymmetric division. Based on these observations, we proposed that OsSCR is involved not only in the asymmetric division of the cortex/endodermis progenitor cell but also during stomata and ligule formation by establishing the polarization of cytoplasm.     

How to keep proliferative neural stem/progenitor cells: A critical role of asymmetric inheritance of cyclin D2

It has long been argued that cell cycle regulators such as cyclins, cyclin-dependent kinases and their inhibitors affect the fate of neuronal progenitor cells. Recently, we identified that cyclin D2, which localizes at the basal tip of the radial glial cell (i.e., the neural progenitor in the developing neocortex), functions to give differential cell fates to its daughter cells just after cell division. This basally biased localization is due to transportation of cyclin D2 mRNA via its unique cis-regulatory sequence and local translation into cyclin D2 protein at the basal endfoot. During division of the neural progenitor cells, cyclin D2 protein is inherited by the daughter cell that retain the basal process, resulting in asymmetric distribution of cyclin D2 protein between the two daughter cells. Cyclin D2 is similarly localized in the human fetal cortical primordium, suggesting a common mechanism for the maintenance of neural progenitors and a possible scenario in evolution of primate brains. Here we introduce our recent findings and discuss how cyclin D2 functions in mammalian brain development and evolution.     

The induced sector Arabidopsis apical embryonic fate map

Creation of an embryonic fate map may provide insight into the patterns of cell division and specification contributing to the apical region of the early Arabidopsis embryo. A fate map has been constructed by inducing genetic chimerism during the two-apical-cell stage of embryogenesis to determine if the orientation of the first anticlinal cell division correlates with later developmental axes. Chimeras were also used to map the relative locations of precursors of the cotyledon and leaf primordia. Genetic chimeras were induced in embryos doubly heterozygous for a heat shock regulated Cre recombinase and a constitutively expressed beta-glucuronidase (GUS) gene flanked by the loxP binding sites for Cre. Individual cells in the two-apical-cell stage embryo responding to heat shock produce GUS-negative daughter cells. Mature plants grown from seed derived from treated embryos were scored for GUS-negative sector extent in the cotyledons and leaves. The GUS-negative daughters of apical cells had a strong tendency to contribute primarily to one cotyledon or the other and to physically adjacent true leaf margins. This result indicated that patterns of early cell division correlate with later axes of symmetry in the embryo and that these patterns partially limit the fates available for adoption by daughter cells. However, GUS-negative sectors were shared between all regions of the mature plant, suggesting that there is no strict fate restriction imposed on the daughters of the first apical cells.     

Centrin-2 is required for centriole duplication in mammalian cells

BACKGROUND: Centrosomes are the favored microtubule-organizing framework of eukaryotic cells. Centrosomes contain a pair of centrioles that normally duplicate once during the cell cycle to give rise to two mitotic spindle poles, each containing one old and one new centriole. However, aside from their role as an anchor point for pericentriolar material and as basal bodies of flagella and cilia, the functional attributes of centrioles remain enigmatic. RESULTS: Here, using RNA interference, we demonstrate that "knockdown" of centrin-2, a protein of centrioles, results in failure of centriole duplication during the cell cycle in HeLa cells. Following inhibition of centrin-2 synthesis, the preexisting pair of centrioles separate, and functional bipolar spindles form with only one centriole at each spindle pole. Centriole dilution results from the ensuing cell division, and daughter cells are "born" with only a single centriole. Remarkably, these unicentriolar daughter cells may complete a second and even third bipolar mitosis in which spindle microtubules converge onto unusually broad spindle poles and in which cell division results in daughter cells containing either one or no centrioles at all. Cells thus denuded of the mature or both centrioles fail to undergo cytokinesis in subsequent cell cycles, give rise to multinucleate products, and finally die. CONCLUSIONS: These results demonstrate a requirement for centrin in centriole duplication and demonstrate that centrioles play a role in organizing spindle pole morphology and in the completion of cytokinesis.     

The influence of fission line expression on the number and positioning of oral primordia in the cdaA1 mutant of Tetrahymena thermophila.

During cytokinesis, furrowing creates new boundaries for daughter cells. Following a shift to a restrictive temperature, cells of the temperature-sensitive cell-division-arrest (cdaA1) mutant of Tetrahymena thermophila complete development of the oral apparatus for the prospective posterior daughter cell before becoming arrested in cytokinesis. When maintained under weak restrictive conditions (35 degrees C), some of the chains were arrested prior to the start of fission line formation (D-shaped chains), whereas others manifested rudimentary unilateral furrowing on the ventral side (B-shaped chains). In their second cell cycle following the temperature shift, the D-shaped chains usually formed only one oral primordium, at a position highly correlated with the length of the entire chain. The B-shaped chains always produced two separate oral primordia, located at irregular positions anterior and posterior to the division furrow, often close to the posterior oral apparatus produced during the first cycle. These results suggest that the formation of the fission line sets a reference boundary to assess the number of oral primordia and influence their position, that appear during subsequent morphogenetic episodes. They also indicate that, during cell division cycles, pre-existing oral apparatuses do not strongly inhibit the formation of new oral apparatuses in their close vicinity.     

Unraveling cell division mechanisms with small-molecule inhibitors

Cell division is the process by which a cell creates two genetically identical daughter cells. To maintain genomic integrity, a complex and highly regulated sequence of events ensures that the replicated chromosomes are equally partitioned between the daughter cells. For more than 50 years, strategies designed around small-molecule inhibitors have been critical in advancing our understanding of this essential process. Here we introduce a series of questions on the biology of cell division and illustrate how small molecules have been used to design experiments to address these questions. Because of the highly dynamic nature of cell division, the temporal control over protein function that is possible with small molecules has been particularly valuable in dissecting biological mechanisms.     

Novel as1 and as2 defects in leaf adaxial-abaxial polarity reveal the requirement for ASYMMETRIC LEAVES1 and 2 and ERECTA functions in specifying leaf adaxial identity

The shoot apical meristem (SAM) of seed plants is the site at which lateral organs are formed. Once organ primordia initiate from the SAM, they establish polarity along the adaxial-abaxial, proximodistal and mediolateral axes. Among these three axes, the adaxial-abaxial polarity is of primary importance in leaf patterning. In leaf development, once the adaxial-abaxial axis is established within leaf primordia, it provides cues for proper lamina growth and asymmetric development. It was reported previously that the Arabidopsis ASYMMETRIC LEAVES1 (AS1) and ASYMMETRIC LEAVES2 (AS2) genes are two key regulators of leaf polarity. In this work, we demonstrate a new function of the AS1 and AS2 genes in the establishment of adaxial-abaxial polarity by analyzing as1 and as2 alleles in the Landsberg erecta (Ler) genetic background. We provide genetic evidence that the Arabidopsis ERECTA (ER) gene is involved in the AS1-AS2 pathway to promote leaf adaxial fate. In addition, we show that AS1 and AS2 bind to each other, suggesting that AS1 and AS2 may form a complex that regulates the establishment of leaf polarity. We also report the effects on leaf polarity of overexpression of the AS1 or AS2 genes under the control of the cauliflower mosaic virus (CAMV) 35S promoter. Although plants with as1 and as2 mutations have very similar phenotypes, 35S::AS1/Ler and 35S::AS2/Ler transgenic plants showed dramatically different morphologies. A possible model of the AS1, AS2 and ER action in leaf polarity formation is discussed.     

How to keep proliferative neural stem/progenitor cells

It has long been argued that cell cycle regulators such as cyclins, cyclin-dependent kinases and their inhibitors affect the fate of neuronal progenitor cells. Recently, we identified that cyclin D2, which localizes at the basal tip of the radial glial cell (i.e., the neural progenitor in the developing neocortex), functions to give differential cell fates to its daughter cells just after cell division. This basally biased localization is due to transportation of cyclin D2 mRNA via its unique cis-regulatory sequence and local translation into cyclin D2 protein at the basal endfoot. During division of the neural progenitor cells, cyclin D2 protein is inherited by the daughter cell that retain the basal process, resulting in asymmetric distribution of cyclin D2 protein between the two daughter cells. Cyclin D2 is similarly localized in the human fetal cortical primordium, suggesting a common mechanism for the maintenance of neural progenitors and a possible scenario in evolution of primate brains. Here we introduce our recent findings and discuss how cyclin D2 functions in mammalian brain development and evolution.     

Asymmetric inheritance of Cyclin D2 maintains proliferative neural stem/progenitor cells: A critical event in brain development and evolution

Asymmetric cell division and cell cycle regulation are fundamental mechanisms of mammalian brain development and evolution. Cyclin D2, a positive regulator of G1 progression, shows a unique localization within radial glial (RG) cells (i.e., the neural progenitor in the developing neocortex). Cyclin D2 accumulates at the very basal tip of the RG cell (i.e., the basal endfoot) via a unique cis‐regulatory sequence found in the 3′ untranslated region (3′UTR) of its mRNA. During RG division, Cyclin D2 protein is asymmetrically distributed to two daughter cells following mitosis. The daughter cell that inherits Cyclin D2 mRNA maintains its self‐renewal capability, while its sibling undergoes differentiation. A similar localization pattern of Cyclin D2 protein has been observed in the human fetal cortical primordium, suggesting a common mechanism of maintenance of neural progenitors that may be evolutionarily conserved across higher mammals such as primates. Here, we discuss our findings and the Cyclin D2 function in mammalian brain development and evolution.     

Cyclin D2 in the basal process of neural progenitors is linked to non-equivalent cell fates

Asymmetric cell division plays an indispensable role during corticogenesis for producing new neurons while maintaining a self-renewing pool of apical progenitors. The cellular and molecular determinants favouring asymmetric division are not completely understood. Here, we identify a novel mechanism for generating cellular asymmetry through the active transportation and local translation of Cyclin D2 mRNA in the basal process. This process is regulated by a unique cis-regulatory sequence found in the 3' untranslated region (3'UTR) of the mRNA. Unequal inheritance of Cyclin D2 protein to the basally positioned daughter cell with the basal process confers renewal of the apical progenitor after asymmetric division. Conversely, depletion of Cyclin D2 in the apically positioned daughter cell results in terminal neuronal differentiation. We demonstrate that Cyclin D2 is also expressed in the developing human cortex within similar domains, thus indicating that its role as a fate determinant is ancient and conserved.     

The influence of fission line expression on the number and positioning of oral primordia in the cdaA1 mutant of Tetrahymena thermophila

During cytokinesis, furrowing creates new boundaries for daughter cells. Following a shift to a restrictive temperature, cells of the temperature-sensitive cell-division-arrest (cdaA1) mutant of Tetrahymena thermophila complete development of the oral apparatus for the prospective posterior daughter cell before becoming arrested in cytokinesis. When maintained under weak restrictive conditions (35°C), some of the chains were arrested prior to the start of fission line formation (D-shaped chains), whereas others manifested rudimentary unilateral furrowing on the ventral side (B-shaped chains). In their second cell cycle following the temperature shift, the D-shaped chains usually formed only one oral primordium, at a position highly correlated with the length of the entire chain. The B-shaped chains always produced two separate oral primordia, located at irregular positions anterior and posterior to the division furrow, often close to the posterior oral apparatus produced during the first cycle. These results suggest that the formation of the fission line sets a reference boundary to assess the number of oral primordia and influence their position, that appear during subsequent morphogenetic episodes. They also indicate that, during cell division cycles, pre-existing oral apparatuses do not strongly inhibit the formation of new oral apparatuses in their close vicinity. © 1992 Wiley-Liss, Inc.