Androgenesis: Affecting the fate of the male gametophyte

Natural leaf senescence proceeds through an orderly program of events referred to, generally, as the 'senescence syndrome'. Leaf senescence consists of primarily, but not exclusively, a set of degradative and remobilization activities that salvage valuable nutrients by reallocation to the seeds or other viable parts of the plant. The program requires changes in gene expression and eventually culminates in death of the leaf or whole plant. Leaf/whole plant senescence has now been scrutinized extensively using molecular genetic approaches and a clearer picture of the events that comprise the developmental program is beginning to emerge. However, while understandings of the phenomenological aspects of the program have become apparent, the mechanistic aspects, particularly with regard to the processes required for induction and regulation of the program, are still far from clear. Molecular evidence suggests the process is complex in terms of the wide array of genes and activities expressed, and in terms of the overall regulation of progression of the events of the syndrome. This article attempts to review our current understanding of leaf senescence and includes a brief discussion of aspects of the process that require clarification if we are to more fully understand this complex developmental program.     

AtDEK1 is essential for specification of embryonic epidermal cell fate

The specification of epidermal (L1) identity occurs early during plant embryogenesis. Here we show that, in Arabidopsis, AtDEK1 encodes a key component of the embryonic L1 cell-layer specification pathway. Loss of AtDEK1 function leads to early embryo lethality characterized by a severe loss of cell organization in the embryo proper and abnormal cell divisions within the suspensor. Markers for L1 identity, ACR4 and ATML1, are not expressed in homozygous mutant embryos. In order to clarify the function of AtDEK1 further, an RNAi knockdown approach was used. This allowed embryos to partially complete embryogenesis before losing AtDEK1 activity. Resulting seedlings showed a specific loss of epidermal cell identity within large portions of the cotyledons. In addition, meristem structure and function was systematically either reduced or entirely lost. AtDEK1 expression is not restricted to the L1 epidermal cell layer at any stage in development. This is consistent with AtDEK1 playing an upstream role in the continuous generation or interpretation of positional information required for epidermal specification. Our results not only identify a specific role for AtDEK1 during embryogenesis, but underline the potential key importance of L1 specification at the globular stage for subsequent progression through embryogenesis.     

Retinoic acid signalling is required for specification of pronephric cell fate

The mechanisms by which a subset of mesodermal cells are committed to a nephrogenic fate are largely unknown. In this study, we have investigated the role of retinoic acid (RA) signalling in this process using Xenopus laevis as a model system and Raldh2 knockout mice. Pronephros formation in Xenopus embryo is severely impaired when RA signalling is inhibited either through expression of a dominant-negative RA receptor, or by expressing the RA-catabolizing enzyme XCyp26 or through treatment with chemical inhibitors. Conversely, ectopic RA signalling expands the size of the pronephros. Using a transplantation assay that inhibits RA signalling specifically in pronephric precursors, we demonstrate that this signalling is required within this cell population. Timed antagonist treatments show that RA signalling is required during gastrulation for expression of Xlim-1 and XPax-8 in pronephric precursors. Moreover, experiments conducted with a protein synthesis inhibitor indicate that RA may directly regulate Xlim-1. Raldh2 knockout mouse embryos fail to initiate the expression of early kidney-specific genes, suggesting that implication of RA signalling in the early steps of kidney formation is evolutionary conserved in vertebrates.     

FGF signaling regulates mesoderm cell fate specification and morphogenetic movement at the primitive streak.

Although FGF signaling plays an integral role in the migration and patterning of mesoderm at gastrulation, the mechanism and downstream targets of FGF activity have remained elusive. Here, we demonstrate that FGFR1 orchestrates the epithelial to mesenchymal transition and morphogenesis of mesoderm at the primitive streak by controlling Snail and E-cadherin expression. Furthermore, we show that FGFR1 functions in mesoderm cell fate specification by positively regulating Brachyury and Tbx6 expression. Finally, we provide evidence that the attenuation of Wnt3a signaling observed in Fgfr1 -/- embryos can be rescued by lowering E-cadherin levels. We propose that modulation of cytoplasmic beta-catenin levels, associated with FGF-induced downregulation of E-cadherin, provides a molecular link between FGF and Wnt signaling pathways at the streak.     

Satb2 is a postmitotic determinant for upper-layer neuron specification in the neocortex

Pyramidal neurons of the neocortex can be subdivided into two major groups: deep- (DL) and upper-layer (UL) neurons. Here we report that the expression of the AT-rich DNA-binding protein Satb2 defines two subclasses of UL neurons: UL1 (Satb2 positive) and UL2 (Satb2 negative). In the absence of Satb2, UL1 neurons lose their identity and activate DL- and UL2-specific genetic programs. UL1 neurons in Satb2 mutants fail to migrate to superficial layers and do not contribute to the corpus callosum but to the corticospinal tract, which is normally populated by DL axons. Ctip2, a gene required for the formation of the corticospinal tract, is ectopically expressed in all UL1 neurons in the absence of Satb2. Satb2 protein interacts with the Ctip2 genomic region and controls chromatin remodeling at this locus. Satb2 therefore is required for the initiation of the UL1-specific genetic program and for the inactivation of DL- and UL2-specific genes.     

The C terminus of p53 family proteins is a cell fate determinant

The p53 tumor suppressor is the most commonly mutated gene in human cancers. The ability of p53 to induce cell cycle arrest, apoptosis, DNA repair, and other p53-dependent activities is well known; however, the mechanism by which p53 induces a specific activity over another is unclear. Here, we showed that stringent regulation of and by p53 family isoforms facilitates differential target gene expression and thus determines cell fate. Through the use of engineered deletion mutants, we found that activation domain 2 is required for induction of the proapoptotic target gene insulin-like growth factor binding protein 3 (IGFBP3) by p53 and that the basic domain inhibits induction of this gene by p53. Thus, for the first time we provide evidence that the basic domain of p53 is inhibitory in vivo as has been determined in vitro. We also showed that the in vivo inhibitory activity of the basic domain depends upon activation domain 1, such that combined deletion of activation domain 1 and the basic domain was required to alleviate the inhibition by the basic domain. Importantly, deletion of the inhibitory functional domains, namely N-terminal activation domain 1 and the C-terminal basic domain, is paralleled in nature. We found that the IGFBP3 promoter was activated by p53(DeltaNDeltaBD), which mimics a naturally occurring N- and C-terminally truncated human p53 isoform, and by p53AS, a C-terminally truncated murine p53 isoform generated through alternative splicing, but not by full-length human or murine p53. In addition, we found that the C termini of p63 and p73 inhibit the induction of IGFBP3, such that C-terminally truncated p63 and p73 isoforms induce the expression of IGFBP3, whereas full-length ones cannot. We also demonstrated that IGFBP3 is an important effector of the apoptosis induced by N- and C-terminally truncated p53, such that knockdown of IGFBP3 by using an IGFBP3 neutralizing antibody or IGFBP3 small interfering RNA partially rescues the cell death induced by N- and C-terminally truncated p53. In addition, we identified that histone deacetylase activity, not p53 DNA binding ability, governs the regulation of IGFBP3 by full-length p53 family proteins, as inhibition of histone deacetylases restores the induction of IGFBP3 by exogenous full-length p53, p63, and p73 proteins. Furthermore, we found that activation of p53 or inhibition of histone deacetylases alone was not sufficient to induce IGFBP3; however, combined treatment endowed endogenous p53 with this activity. To better understand the significance of this regulation, we performed a microarray study and identified several target genes differentially regulated by full-length p53 and p53 lacking the N-terminal activation domain 1 and the C-terminal basic domain. Taken together, our data suggest a novel mechanism by which p53 family proteins differentially regulate gene expression and provide an insight for designing a combined therapy for cancer treatment.     

Nuclear behavior, cell polarity, and cell specification in the female gametophyte

In flowering plants, the haploid gamete-forming generation comprises only a few cells and develops within the reproductive organs of the flower. The female gametophyte has become an attractive model system to study the genetic and molecular mechanisms involved in pattern formation and gamete specification. It originates from a single haploid spore through three free nuclear division cycles, giving rise to four different cell types. Research over recent years has allowed to catch a glimpse of the mechanisms that establish the distinct cell identities and suggests dynamic cell–cell communication to orchestrate not only development among the cells of the female gametophyte but also the interaction between male and female gametophytes. Additionally, cytological observations and mutant studies have highlighted the importance of nuclei migration- and positioning for patterning the female gametophyte. Here we review current knowledge on the mechanisms of cell specification in the female gametophyte, emphasizing the importance of positional cues for the establishment of distinct molecular profiles.     

Revisiting cell fate specification in the inner ear

Generating the diversity of cell types in the inner ear may require an interplay between regional compartmentalization and local cellular interactions. Recent evidence has come from gene targeting, lineage analysis, fate mapping and gene expression studies. Notch signaling and neurogenic gene regulation are involved in patterning or specification of sensory organs, ganglion cells and hair cell mechanoreceptors.     

LACHESIS restricts gametic cell fate in the female gametophyte of Arabidopsis

In flowering plants, the egg and sperm cells form within haploid gametophytes. The female gametophyte of Arabidopsis consists of two gametic cells, the egg cell and the central cell, which are flanked by five accessory cells. Both gametic and accessory cells are vital for fertilization; however, the mechanisms that underlie the formation of accessory versus gametic cell fate are unknown. In a screen for regulators of egg cell fate, we isolated the lachesis (lis) mutant which forms supernumerary egg cells. In lis mutants, accessory cells differentiate gametic cell fate, indicating that LIS is involved in a mechanism that prevents accessory cells from adopting gametic cell fate. The temporal and spatial pattern of LIS expression suggests that this mechanism is generated in gametic cells. LIS is homologous to the yeast splicing factor PRP4, indicating that components of the splice apparatus participate in cell fate decisions.     

beta-Catenin is required for specification of proximal/distal cell fate during lung morphogenesis

The lungs are divided, both structurally and functionally, into two distinct components, the proximal airways, which conduct air, and the peripheral airways, which mediate gas exchange. The mechanisms that control the specification of these two structures during lung development are currently unknown. Here we show that beta-catenin signaling is required for the formation of the distal, but not the proximal, airways. When the gene for beta-catenin was conditionally excised in epithelial cells of the developing mouse lung prior to embryonic day 14.5, the proximal lung tubules grew and differentiated appropriately. The mice, however, died at birth because of respiratory failure. Analysis of the lungs by in situ hybridization and immunohistochemistry, using molecular markers of the epithelial and mesenchymal components of both proximal and peripheral airways, showed that the lungs were composed primarily of proximal airways. These observations establish, for the first time, both the sites and timing of specification of the proximal and peripheral airways in the developing lung, and that beta-catenin is one of the essential components of this specification.     

Dcas is required for importin-alpha3 nuclear export and mechano-sensory organ cell fate specification in Drosophila

We have studied the in vivo function and tissue specificity of Dcas, the Drosophila ortholog of CAS, the importin beta-like export receptor for importin alpha. While dcas mRNA is specifically expressed in the embryonic central nervous system, Dcas protein is maternally supplied to all embryonic cells and its nuclear/cytoplasmic distribution varies in different tissues and times in development. Unexpectedly, hypomorphic alleles of dcas show specific transformations in mechano-sensory organ cell identity, characteristic of mutations that increase Notch signaling. Dcas is essential for efficient importin-alpha3 nuclear export in mechano-sensory cells and the surrounding epidermal cells and is indirectly required for the import of one component of the Notch pathway, but not others tested. We interpret the specificity of the dcas phenotype as indicating that one or more Notch signaling components are particularly sensitive to a disruption in nuclear protein import. We propose that mutations in house keeping genes often cause specific developmental phenotypes, such as those observed in many human genetic disorders.     

Solute analysis and water relations of gametophyte mutants tolerant to NaCl in the fern Ceratopteris richardii

The stl1 and stl2 mutations confer low and high levels of NaCl tolerance to gametophytes of the fern Ceratopteris richardii, respectively. As an initial characterization of these mutations, the levels of various organic solutes, tissue ion content and water relations were examined in the wild-type and mutant strains in the absence and presence of 60 mol m^-3 NaCl stress (a level which results in a 20, 15 and 0% reduction in gametophyte growth in the wild-type, stl1 mutant and stl2 mutant, respectively). All strains exhibited major changes in organic and inorganic solute levels and water relations in response to 60 mol m^-3 NaCl stress. Differences in organic solute levels and water relations between the wild-type and mutant strains in the absence and in response to 60 mol m^-3 NaCl stress were minimal. Analysis of tissue ion content showed that stl1 was associated with a slight reduction in Na⁺ accumulation during 60 mol m^-3 NaCl stress. stl2 was associated with (1) higher constitutive levels of K⁺ and (2) continued selective accumulation of K⁺ and reduced accumulation of Na⁺ during 60 mol m^-3 NaCl stress. A K⁺/Na⁺ ratio close to 1 was observed in the wild-type during 60 mol m^-3 NaCl stress, while higher ratios were detected in stl1 and stl2 (1·7 and 4·0, respectively). The findings of this study suggest that the tolerance imparted by stl1 and stl2 is associated with altered ion accumulation during NaCl stress, rather than an enhanced ability to accumulate organic solutes to be used for osmotic adjustment of the cytoplasm.     

Effects of water stress on male gametophyte development in plants

 Male reproductive development in plants is highly sensitive to water deficit during meiosis in the microspore mother cells. Water deficit during this stage inhibits further development of microspores or pollen grains, causing male sterility. Female fertility, in contrast, is quite immune to stress. The injury is apparently not caused by desiccation of the reproductive tissue, but is an indirect consequence of water deficit in the vegetative organs, such as leaves. The mechanism underlying this stress response probably involves a long-distance signaling molecule, originating in the organs that undergo water loss, and affecting fertility in the reproductive tissue, which conserves its water status. Much research has been focused on the involvement of abscisic acid in this regard, but the most recent evidence tends to reject a role for this hormone in the induction of male sterility. Stress-induced arrest of male gametophyte development is preceded by disturbances in carbohydrate metabolism and distribution within anthers, and an inhibition of the key sugar-cleaving enzyme, acid invertase. Since invertase gene expression can be modulated by sugar concentration, it is possible that decreased sugar delivery to reproductive tissue upon inhibition of photosynthesis by stress is the signal that triggers metabolic lesions leading to failure of male gametophyte development. Received: 31 October 1996 / Revision accepted: 18 February 1997