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Phenotypic and Genetic Analysis of det2, a New Mutant That Affects Light-Regulated Seedling Development in Arabidopsis.

The greening phenotypes produced by recessive mutations in a gene designated de-etiolated-2 (DET2) are described. Recessive mutations in the DET2 gene uncouple light signals from a number of light-dependent processes. det2 mutations result in dark-grown Arabidopsis thaliana seedlings with many characteristics of light-grown plants, including hypocotyl growth inhibition, cotyledon expansion, primary leaf initiation, anthocyanin accumulation, and derepression of light-regulated gene expression. In contrast to these morphological and gene expression changes, however, the chloroplast development program is not initiated in the dark in det2 mutants, suggesting that light-regulated gene expression precedes the differentiation of etioplasts to chloroplasts. det2 mutations thus reveal at least two classes of downstream light-regulated responses that differ in their timing and control mechanisms. Homozygous det2 mutations also affect photoperiodic responses in light-grown plants, including timing of flowering, dark adaptation of gene expression, and onset of leaf senescence. The phenotype of det1 det2 double mutants is additive, implying that DET1 and DET2 function in distinct pathways that affect downstream light-regulated genes. Furthermore, these pathways are not utilized solely during early seedling development but must also be required to regulate different aspects of the light developmental program during later stages of vegetative growth.

Journal Article↗

Characterization of a fertilization-induced and developmentally regulated plasma-membrane aquaporin expressed in reproductive tissues, in the wild potato Solanum chacoense Bitt.

Fertilization triggers a unique and complex developmental program leading to embryogenesis and seed set. Growth and differentiation are accompanied by rapid metabolic changes as well as massive cellular reorganization inside the fertilized ovule. Using differential display to isolate genes involved in pollen-pistil interactions and early fertilization events, we isolated from Solanum chacoense Bitt. a fertilization-induced plasma-membrane intrinsic protein of the PIP2 family that is predominantly expressed in pistil and anthers tissues. Major intrinsic proteins (MIPs) consist of a large family of highly conserved membrane-spanning proteins that are mainly represented in plants by the aquaporins. Aquaporins, mainly of the PIP and TIP type, have been shown in many species to serve as water channels. In S. chacoense the ScPIP2a mRNA is developmentally regulated during anthesis, with mRNA levels gradually decreasing as the pistil reaches maturity. In flowers, strongest expression was observed in elongating styles, in stamens, and transiently in ovules following fertilization. In styles, maximal expression levels correlated with phases of rapid style elongation and with the formation of epidermal papillae. ScPIP2a mRNA was also strongly expressed in developing fruit, consistent with a role in cell expansion during maturation and development.

Amino Acid Sequence↗

Molecular characterization of a Dictyostelium G-protein alpha-subunit required for development.

Dictyostelium discoideum utilizes G-protein-regulated transmembrane signaling systems to implement its developmental program. This report describes the Dictyostelium G-protein alpha-subunit, G alpha3, and demonstrates that it is required for normal development. G alpha3 is the largest of the four completely sequenced Dictyostelium G-protein alpha-subunits. The difference in size is due to variability in the N-terminal regions. The regions which are affected by the increased size of G alpha3 are the Gbeta gamma binding region and the helical domain that protects the guanine nucleotide cleft. g alpha3- mutants created by gene disruption fail to aggregate. However, when treated with exogenous pulses of cAMP that mimic the endogenous cAMP oscillations, they are able to aggregate, but development arrests at the tipped mound stage. This conditional developmental phenotype suggests that G alpha3 is required for production of the cAMP signal.

Amino Acid Sequence↗

Molecular mechanisms in the developmental regulation of the maize Suppressor-mutator transposable element.

The maize Suppressor-mutator (Spm) element can exist in one of three heritable forms: (1) a stably active form, (2) a stably inactive form, termed cryptic, and (3) a labile form, here termed programmable, in which the element exhibits one of a variety of heritable developmental programs of expression. Active elements are transcribed and are hypomethylated at sites upstream of the transcription start site, whereas inactive elements are transcriptionally silent and largely methylated at the upstream sites. Active (both stable and programmable), inactive programmable, and cryptic elements are unmethylated, partially methylated, and fully methylated, respectively, at sites within an 0.35-kb 80% G + C region just downstream from the transcription start site. An active Spm element in a genome with a cryptic element promotes its partial demethylation but not its transcriptional activation. In contrast, a trans-acting Spm promotes extensive demethylation and transcriptional activation of an inactive programmable element, as well as its heritable reactivation. These observations define the molecular components of the Spm element's developmental regulatory mechanism. We discuss their general relevance to the developmental regulation of gene expression.

DNA Probes↗

The cell lineage of the polyplacophoran, Chaetopleura apiculata: variation in the spiralian program and implications for molluscan evolution.

Polyplacophorans, or chitons, are an important group of molluscs, which are argued to have retained many plesiomorphic features of the molluscan body plan. Polyplacophoran trochophore larvae posses several features that are distinctly different from those of their sister trochozoan taxa, including modifications of the ciliated prototrochal cells, the postrochal position of the larval eyes or ocelli, epidermal calcareous spicules, and a collection of serially reiterated epidermal shell plates. Despite these differences, chitons demonstrate a canonical pattern of equal spiral cleavage shared by other spiralian phyla that permits the identification of homologous cells across this animal clade. Cell lineage analysis using intracellular labeling on one chiton species, Chaetopleura apiculata, shows that the ocelli are generated from different lineal precursors (second-quartet micromeres: 2a, 2c) compared to those in all other spiralians studied to date (first-quartet micromeres: 1a, 1c). This situation implies that significant changes have also occurred in terms of the inductive interactions that control eye development in the spiralians. Although radical departures from the spiralian developmental program are seen in some molluscs (i.e., cephalopods), the findings presented here indicate that important changes can occur even within the highly constrained framework of the spiral cleavage program. Among spiralians, variation has been reported for the origin of the anterior, sensory, apical organ, which arises from the 1c and 1d micromeres in C. apiculata. The prototroch of C. apiculata consists of two to three irregular rows of ciliated cells but arise from 1q and 2q daughters, similar to that of Ischnochiton rissoi, as well as the gastropod, Patella vulgata. Despite certain early claims, there is no supporting evidence that any of the shell plates arise pretrochally in C. apiculata. The first seven of eight definitive shell plates that arise in the larva originate from shell secreting grooves in the postrochal region (derived from 2c, 2d, 3d). Earlier descriptions indicate that the eighth plate arises later at metamorphosis, and as this is formed posteriorly, it too forms in the postrochal region. On the other hand, epidermal spicules originate from both pretrochal and postrochal cells (1a,1d, 2a, 2c, 3c, 3d). The significance of these observations is discussed in light of various hypotheses concerning the origin of the conchiferan shell. This study reveals conservation, as well as evolutionary novelty, in the assignment of specific cell fates in the spiralians.

Animals↗

Single-cell analysis of normal and FOXP3-mutant human T cells: FOXP3 expression without regulatory T cell development.

Forkhead winged-helix transcription factor Foxp3 serves as the dedicated mediator of the genetic program governing CD25+CD4+ regulatory T cell (T(R)) development and function in mice. In humans, its role in mediating T(R) development has been controversial. Furthermore, the fate of T(R) precursors in FOXP3 deficiency has yet to be described. Making use of flow cytometric detection of human FOXP3, we have addressed the relationship between FOXP3 expression and human T(R) development. Unlike murine Foxp3- T cells, a small subset of human CD4+ and CD8+ T cells transiently up-regulated FOXP3 upon in vitro stimulation. Induced FOXP3, however, did not alter cell-surface phenotype or suppress T helper 1 cytokine expression. Furthermore, only ex vivo FOXP3+ T(R) cells persisted after prolonged culture, suggesting that induced FOXP3 did not activate a T(r) developmental program in a significant number of cells. FOXP3 flow cytometry was also used to further characterize several patients exhibiting symptoms of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) with or without FOXP3 mutations. Most patients lacked FOXP3-expressing cells, further solidifying the association between FOXP3 deficiency and immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome. Interestingly, one patient bearing a FOXP3 mutation enabling expression of stable FOXP3(mut) protein exhibited FOXP3(mut)-expressing cells among a subset of highly activated CD4+ T cells. This observation raises the possibility that the severe autoimmunity in FOXP3 deficiency can be attributed, in part, to aggressive T helper cells that have developed from T(R) precursors.

Animals↗

Neural crest survival and differentiation in zebrafish depends on mont blanc/tfap2a gene function.

Neural crest progenitor cells are the main contributors to craniofacial cartilage and connective tissue of the vertebrate head. These progenitor cells also give rise to the pigment, neuronal and glial cell lineages. To study the molecular basis of neural crest differentiation, we have cloned the gene disrupted in the mont blanc (mob(m610)) mutation, which affects all neural crest derivatives. Using a positional candidate cloning approach we identified an A to G transition within the 3' splice site of the sixth intron of the tfap2a gene that abolishes the last exon encoding the crucial protein dimerization and DNA-binding domains. Neural crest induction and specification are not hindered in mob(m610) mutant embryos, as revealed by normal expression of early neural crest specific genes such as snail2, foxd3 and sox10. In addition, the initial stages of cranial neural crest migration appear undisturbed, while at a later phase the craniofacial primordia in pharyngeal arches two to seven fail to express their typical set of genes (sox9a, wnt5a, dlx2, hoxa2/b2). In mob(m610) mutant embryos, the cell number of neuronal and glial derivatives of neural crest is greatly reduced, suggesting that tfap2a is required for their normal development. By tracing the fate of neural crest progenitors in live mont blanc (mob(m610)) embryos, we found that at 24 hpf neural crest cells migrate normally in the first pharyngeal arch while the preotic and postotic neural crest cells begin migration but fail to descend to the pharyngeal region of the head. TUNEL assay and Acridine Orange staining revealed that in the absence of tfap2a a subset of neural crest cells are unable to undergo terminal differentiation and die by apoptosis. Furthermore, surviving neural crest cells in tfap2a/mob(m610) mutant embryos proliferate normally and later differentiate to individual derivatives. Our results indicate that tfap2a is essential to turn on the normal developmental program in arches 2-7 and in trunk neural crest. Thus, tfap2a does not appear to be involved in early specification and cell proliferation of neural crest, but it is a key regulator of an early differentiation phase and is required for cell survival in neural crest derived cell lineages.

Amino Acid Sequence↗

Molecular mechanisms of natural de novo shoot organogenesis and their applications.

Natural de novo shoot organogenesis (DNSO) is the spontaneous regeneration of shoots from wound sites outside the shoot apical region through endogenous developmental programs. This regenerative capacity enables plants to recover from severe tissue damage by re-establishing the shoot-root axis. Here, we review current knowledge about the molecular mechanisms of natural DNSO, focusing on transcriptomic and physiological studies in model plants. Accumulating evidence suggests that natural DNSO proceeds through three sequential phases: (i) early wound responses, characterized by the activation of the WIND1-ESR1 module and the establishment of apical-basal auxin asymmetry; (ii) cellular proliferation driven by metabolic and cell-cycle reprogramming; and (iii) cytokinin-mediated establishment of shoot apical meristem identity. We also discuss how these mechanistic insights have been harnessed for practical applications, including tissue culture-free transformation systems such as the cut-dip-budding (CDB) method, and developmental reprogramming strategies that employ ectopic expression of developmental regulator (DR) genes to induce DNSO in otherwise recalcitrant species. Together, these advances illustrate how understanding natural regeneration can guide the development of simplified, broadly applicable plant transformation technologies.

Plant Shoots↗

Differential gene expression in the ookinete stage of the malaria parasite Plasmodium berghei.

Plasmodium, the malaria parasite, undergoes a complex developmental program in its mosquito vector. The ookinete is the parasite form which invades the mosquito midgut and is an important stage for genetic mixing. To identify genes expressed during ookinete development and mosquito midgut invasion, purified zygotes and ookinetes of the rodent parasite Plasmodium berghei were used to construct a suppression subtractive hybridization cDNA library, enriched in sequences expressed in the ookinete stage. In addition to four genes coding for previously described major ookinete-secreted proteins, we isolated ookinete-expressed sequences representing 18 predicted genes. Their gene products include proteins involved in signal transduction and regulatory processes. For six of these genes our analysis provides the first evidence for expression in the ookinete stage. A majority of the genes are not expressed in the zygote, the preceding developmental stage. Furthermore, four of the genes are also transcribed in sporozoites, and one of these in merozoites, suggesting that they code for proteins with a function common to Plasmodium invasive stages.

Animals↗

Regulation of mammary epithelial cell phenotypes by the helix-loop-helix protein, Id-1.

Mammary epithelial cells undergo cycles of proliferation, invasion, differentiation and apoptotic cell-death throughout adult life. The molecular mechanisms that regulate these complex and co-ordinated developmental programs are poorly understood. We have identified Id-1 protein, a negative regulator of basic helix-loop-helix transcription factors, as a critical regulator of these normal mammary epithelial cell phenotypes. We also found that Id-1 is an important regulator of the aggressive and invasive phenotype, as well as mediator of the effects of sex steroid hormones, in human breast cancer cells.

Animals↗

Hemoglobin switching across vertebrate classes: exchange of developmental signals by cell fusion.

Our aim is to obtain evidence for trans-acting factors that regulate developmental hemoglobin (Hb) switching. Our approach is to fuse erythroid cells that have different developmental programs, allowing the trans-acting factors from the adult cell to have access to the nucleus of the fetal or embryonic cell and vice-versa. After cell fusion, the heteropolykaryons are cultured for six hours, and globin gene expression is assayed at two levels: (1) at the level of mRNAs on dot blots hybridized with globin-specific cDNA probes, and (2) at the level of fully-formed Hb tetramers separated by native polyacrylamide gel electrophoresis (PAGE). Since the donor erythroid cells are from different species, it is easy to determine which globin gene products are from which nucleus. And since there is no nuclear fusion for at least twelve hours, the Hb switching that occurs is due to regulation in trans. Our results show that developmental Hb switching occurs in mouse-frog erythroid cell polykaryons. When DMSO-induced murine erythroleukemia (MEL) cells (which express only adult mouse Hbs) are fused with Rana tadpole RBCs (which express only embryonic and fetal-like Hbs), the resultant heteropolykaryons express adult frog globin mRNA and adult frog Hbs. We conclude that there are developmental stage-specific trans-acting factors for Hb switching, that trans-acting factors from adult mouse erythroid cells can induce expression of adult frog globin genes in a tadpole RBC nucleus, and that Hb switching mechanisms are conserved across vertebrate classes.

Animals↗

The abaA homologue of Penicillium marneffei participates in two developmental programmes: conidiation and dimorphic growth.

Penicillium marneffei is the only known species of its genus that is dimorphic. At 25 degrees C, P. marneffei exhibits true filamentous growth and undergoes asexual development producing spores borne on complex structures called conidiophores. At 37 degrees C, P. marneffei undergoes a dimorphic transition to produce uninucleate yeast cells that divide by fission. We have cloned a homologue of the Aspergillus nidulans abaA gene encoding an ATTS/TEA DNA-binding domain transcriptional regulator and shown that it is involved in both these developmental programs. Targeted deletion of abaA blocks asexual development at 25 degrees C before spore production, resulting in aberrant conidiophores with reiterated terminal cells. At 37 degrees C, the abaA deletion strain fails to switch correctly from multinucleate filamentous to uninucleate yeast cells. Both the transitional hyphal cells, which produce the yeast cells, and the yeast cells themselves contain multiple nuclei. Expression of the abaA gene is activated during both conidiation and the hyphal-yeast switch. Interestingly, the abaA gene of the filamentous monomorphic fungus A. nidulans can complement both conidiation and dimorphic switching defects in the P. marneffei abaA mutant. In addition, ectopic overexpression of abaA results in anucleate yeast cells and multinucleate vegetative filamentous cells. These data suggest that abaA regulates cell cycle events and morphogenesis in two distinct developmental programmes.

Amino Acid Sequence↗

Tribolium mae expression suggests roles in terminal and midline patterning and in the specification of mesoderm.

In Drosophila, the Mae protein ("modulator of the activity of Ets") regulates receptor tyrosine kinase (RTK)-dependent mitogen-activated protein kinase (MAPK) signaling. Mae has been shown to bind the Yan and Pointed-P2 transcription factors, thereby changing their ability to activate or repress target genes. In this work we show that the mae ortholog of the red flour beetle Tribolium castaneum (Tc'mae) is active at the posterior, but not the anterior pole of the blastoderm. Since MAPK signaling is known to be active at both poles, Tc'Mae could function to modulate terminal MAPK signaling to differentiate the developmental programs at the anterior and posterior poles of the Tribolium blastoderm embryo. Tc'mae is also expressed along the midline of the germband, similar as in Drosophila, where it is involved in the patterning of midline cells. Before gastrulation and in the growth zone, Tc'mae is active in mesoderm precursor cells. This suggests that in short germ embryos MAPK signaling may also be involved in the specification of mesoderm.

Amino Acid Sequence↗

Differential modulation of BMP signaling promotes the elaboration of cerebral cortical GABAergic neurons or oligodendrocytes from a common sonic hedgehog-responsive ventral forebrain progenitor species.

During cerebral cortical development, excitatory glutamatergic projection neurons are generated from neural stem cells intrinsic to the early embryonic cortical ventricular zone by a process of radial migration, whereas most inhibitory gamma-aminobutyric acid (GABA)ergic interneurons and oligodendrocytes (OLs) appear to be elaborated from ventral forebrain stem cells that initially undergo tangential cortical migration before terminal lineage maturation. In contrast to the more compartmentalized developmental organization of the spinal cord, the generation of neurons and OLs from a common ventral forebrain stem cell would expose these cells to the sequential actions of ventral and dorsal gradient morphogens [sonic hedgehog (Shh) and bone morphogenetic proteins (BMPs)] that normally mediate opposing developmental programs. Here we report that Shh promotes GABAergic neuronalOL lineage restriction of forebrain stem cells, in part, by activation of the basic helix-loop-helix transcription factors, Olig2 and Mash1. In mutant mice with a generalized defect in tangential cortical migration (Dlx12--), there is a profound and selective reduction in the elaboration of both cortical GABAergic neurons and OLs. Our studies further demonstrate that the sequential elaboration of cortical GABAergic neurons and OLs from common Shh-responsive ventral forebrain progenitors requires the spatial and temporal modulation of cortical BMP signaling by BMP ligands and the BMP antagonist, noggin, respectively. These findings suggest an integrative model for cerebral cortical GABAergic neuronal and OL lineage maturation that would incorporate the sequential contributions of the ventral and dorsal forebrain, and the potential role of regional developmental cues in modulating transcriptional codes within evolving neural lineage species.

Animals↗

Polycomb group proteins ESC and E(Z) are present in multiple distinct complexes that undergo dynamic changes during development.

The Polycomb Group proteins are required for stable long-term maintenance of transcriptionally repressed states. Two distinct Polycomb Group complexes have been identified, a 2-MDa PRC1 complex and a 600-kDa complex containing the ESC and E(Z) proteins together with the histone deacetylase RPD3 and the histone-binding protein p55. We report here that there are at least two embryonic ESC/E(Z) complexes that undergo dynamic changes during development and a third larval E(Z) complex that forms after disappearance of ESC. We have identified a larger embryonic ESC complex containing RPD3 and p55, along with E(Z), that is present only until mid-embryogenesis, while the previously identified 600-kDa ESC/E(Z) complex persists until the end of embryogenesis. Constitutive overexpression of ESC does not promote abnormal persistence of the larger or smaller embryonic complexes and does not delay a dissociation of E(Z) from the smaller ESC complex or delay appearance of the larval E(Z) complex, indicating that these changes are developmentally programmed and not regulated by the temporal profile of ESC itself. Genetic removal of ESC prevents appearance of E(Z) in the smaller embryonic complex, but does not appear to affect formation of the large embryonic ESC complex or the PRC1 complex. We also show that the ESC complex is already bound to chromosomes in preblastoderm embryos and present genetic evidence that ESC is required during this very early period.

Animals↗

Transcriptional control of B cell activation.

The developmental program that commits a hematopoietic stem cell to the B lymphocyte lineage employs transcriptional regulators to enable the assembly of an antigen receptor complex with a useful specificity and with signalling competence. Once a naive IgM+ B cell is generated, it must correctly integrate signals from the antigen receptor with those from cytokine receptors and co-receptors delivering T cell help. The B cell responds through the regulated expression of genes that implement specific cell expansion and differentiation, secretion of high levels of high-affinity antibody, and generation of long-term memory. The transcriptional regulators highlighted in this chapter are those for which genetic evidence of function in IgM+ B cells in vivo has been provided, often in the form of mutant mice generated by conventional or conditional gene targeting. A critical developmental step is the maturation of bone marrow emigrant "transitional" B cells into the mature, long-lived cells of the periphery, and a number of the transcription factors discussed here impact on this process, yielding B cells with poor mitogenic responses in vitro. For mature B cells, it is clear that not only the nature, but the duration and amplitude of an activating signal are major determinants of the transcription factor activities enlisted, and so the ultimate outcome. The current challenge is the identification of the target genes that are activated to implement the correct response, so that we may more precisely and safely manipulate B cell behavior to predictably and positively influence humoral immune responses.

Animals↗

Evolution of cis-acting elements in 5' flanking regions of vertebrate actin genes.

Regulation of the vertebrate actin multigene family involves the recognition of various regulatory sequences (cis-acting elements) that specify the distinct tissue type and developmental program of expression for each actin paralogue, which implies that the distribution of cis-acting elements may be unique for each paralogue gene. To elucidate the evolution of these unique distribution patterns, we improved a method to scan for cis-acting elements in the 5' flanking regulatory region of genes and used it to analyze five cis-acting elements (SRE, MyoD binding site, Elk-1 binding site, positive and negative YY1 binding sites) of six actin paralogue genes (beta and gamma cytoplasmic actins, alpha and gamma smooth muscle actins, and alpha skeletal and alpha cardiac actins) among various vertebrates. It was shown that although an element(s) may exist in all paralogue genes of the same species, its numbers, compositions, and distribution patterns or even sequences vary remarkably among paralogues, which contributes to their different tissue- and developmental-specific expression. However, each pair of coexpressed paralogues has some certain similarity in distribution patterns. Furthermore, among various orthologues of actin genes derived from diverse vertebrates, the sequences, numbers, and distribution patterns of these cis-acting elements are highly conserved or even identical in the long run of phylogeny of vertebrates. Taken together, the results described above strongly indicate that not only the structures of actins but also their expression patterns are essential in both the phylogeny and the physiology of vertebrates. The distribution patterns of cis-acting elements of various actin genes can be regarded as indicators of both horizontal (paralogues) and vertical (orthologous) evolution of actins.

Actins↗

Regulatory elements in the promoter of the vitelline membrane gene VM32E of Drosophila melanogaster direct gene expression in distinct domains of the follicular epithelium.

The Drosophila vitelline membrane protein gene VM32E is expressed according to a precise temporal and spatial program in the follicle cells. Results from germ line transformation experiments using different fragments of the -465/-39 VM32E region fused to the hsp/lacZ reporter gene revealed that the region -348/-39 is sufficient to confer the wild-type expression pattern. Within this segment, distinct cis-regulatory elements control VM32E expression in ventral and dorsal follicle cells. The region between -135/-113 is essential for expression of the VM32E gene in the ventral columnar follicle cells. Expression in the dorsal domain requires the two regions -348/-254 and -118/-39. Furthermore, the region -253/-119 appears to contain a negative element that represses gene activity in anterior centripetal cells. We suggest that the expression of the VM32E gene throughout the follicular epithelium is controlled by specific cis-regulatory elements acting in distinct spatial domains and following a precise developmental program.

Animals↗