Distinct Developmental Programs Research Articles

The Medicago species A2-type cyclin is auxin regulated and involved in meristem formation but dispensable for endoreduplication-associated developmental programs.

Phytohormones as well as temporal and spatial regulation of the cell cycle play a key role in plant development. Here, we investigated the function and regulation of an alfalfa (Medicago sativa) A2-type cyclin in three distinct root developmental programs: in primary and secondary root development, nodule development, and nematode-elicited gall formation. Using transgenic plants carrying the Medsa;cycA2;2 promoter-beta-glucuronidase gene fusion, in combination with other techniques, cycA2;2 expression was localized in meristems and proliferating cells in the lateral root and nodule primordia. Rapid induction of cycA2;2 by Nod factors demonstrated that this gene is implicated in cell cycle activation of differentiated cells developing to nodule primordia. Surprisingly, cycA2;2 was repressed in the endoreduplicating, division-arrested cells both during nodule development and formation of giant cells in nematode-induced galls, indicating that CycA2;2 was dispensable for S-phase in endoreduplication cycles. Overexpression of cycA2;2 in transgenic plants corresponded to wild type protein levels and had no apparent phenotype. In contrast, antisense expression of cycA2;2 halted regeneration of somatic embryos, suggesting a role for CycA2;2 in the formation or activity of apical meristems. Expression of cycA2;2 was up-regulated by auxins, as expected from the presence of auxin response elements in the promoter. Moreover, auxin also affected the spatial expression pattern of this cyclin by shifting the cycA2;2 expression from the phloem to the xylem poles.

Distinct roles for c-Myb and core binding factor/polyoma enhancer-binding protein 2 in the assembly and function of a multiprotein complex on the TCR delta enhancer in vivo.

Enhancers and promoters within TCR loci functionally collaborate to modify chromatin structure and to confer accessibility to the transcription and V(D)J recombination machineries during T cell development in the thymus. Two enhancers at the TCRalphadelta locus, the TCR alpha enhancer and the TCR delta enhancer (Edelta), are responsible for orchestrating the distinct developmental programs for V(D)J recombination and transcription of the TCR alpha and delta genes, respectively. Edelta function depends critically on transcription factors core binding factor (CBF)/polyoma enhancer-binding protein 2 (PEBP2) and c-Myb as measured by transcriptional activation of transiently transfected substrates in Jurkat cells, and by activation of V(D)J recombination within chromatin-integrated substrates in transgenic mice. To understand the molecular mechanisms for synergy between these transcription factors in the context of chromatin, we used in vivo footprinting to study the requirements for protein binding to Edelta within wild-type and mutant versions of a human TCR delta minilocus in stably transfected Jurkat cells. Our data indicate that CBF/PEBP2 plays primarily a structural role as it induces a conformational change in the enhanceosome that is associated with augmented binding of c-Myb. In contrast, c-Myb has no apparent affect on CBF/PEBP2 binding, but is critical for transcriptional activation. Thus, our data reveal distinct functions for c-Myb and CBF/PEBP2 in the assembly and function of an Edelta enhanceosome in the context of chromatin in vivo.

Developmental Differences Between Cranial Bones in the Zebrafish ( Danio rerio ): Some Preliminary Light and TEM Observations

To test whether cartilage bones and dermal bones, which belong to two different units of the vertebrate skeleton, have distinct developmental programs possibly reflected in a different molecular control of their ossification process, we currently investigate the development of some selected cranial bones in the zebrafish, Danio rerio. Here we present some light microscopical and ultrastructural findings with respect to the maxillary bone (a dermal bone that is edentulous in the zebrafish) and the basioccipital bone (a cartilage bone, i.e., with a perichondral phase followed by endochondral invasion). The two bones differ in (a) the area where matrix is first deposited--an unstructured extracellular domain in the former versus intermingling of bone matrix elements with cartilage matrix in the latter--and (b) the progression of ossification--continuously from an initium in the former versus through fusion of separate anlagen in the latter. These findings seem to support the hypothesis that the two types of bone have at least some distinctive features in their developmental programs.

Assessing a role for enhancer-blocking activity in gene regulation within the murine T-cell receptor alpha/delta locus.

Although situated close together within the T-cell receptor (TCR) alpha/delta locus, TCR delta and TCR alpha gene segments are controlled by two developmental stage-specific enhancers and are activated according to distinct developmental programmes. We previously used a stable transfection colony assay to identify an enhancer-blocking element, blocking element alpha/delta-1 (BEAD-1), between the TCR delta and alpha gene segments of the human TCR alpha/delta locus. We hypothesized that enhancer-blocking by BEAD-1 might be required to prevent the TCR delta enhancer from activating TCR alpha gene segment transcription and rearrangement at the double negative stage of thymocyte development. Here, we used a transfection approach to define partial enhancer-blocking activity in an analogous position of the murine TCR alpha/delta locus. To test the functional significance of this activity in vivo, we used gene targeting to delete the region from the endogenous locus. We found no perturbation of TCR delta and TCR alpha gene expression and rearrangement on targeted alleles, indicating that enhancer-blocking activity in this region is not required to maintain the developmentally distinct activation profiles of the two genes. We suggest that appropriate regulation may be achieved as a result of intrinsic biases in enhancer-promoter interactions or a developmental stage specificity to promoter function that is distinct from any additional specificity imposed by the enhancers themselves.

Repression of GCN4 mRNA Translation by Nitrogen Starvation in Saccharomyces cerevisiae

Saccharomyces cerevisiae activates a regulatory network called "general control" that provides the cell with sufficient amounts of protein precursors during amino acid starvation. We investigated how starvation for nitrogen affects the general control regulatory system, because amino acid biosynthesis is part of nitrogen metabolism. Amino acid limitation results in the synthesis of the central transcription factor Gcn4p, which binds to specific DNA-binding motif sequences called Gcn4-protein-responsive elements (GCREs) that are present in the promoter regions of its target genes. Nitrogen starvation increases GCN4 transcription but efficiently represses expression of both a synthetic GCRE6::lacZ reporter gene and the natural amino acid biosynthetic gene ARO4. Repression of Gcn4p-regulated transcription by nitrogen starvation is independent of the ammonium sensing systems that include Mep2p and Gpa2p or Ure2p and Gln3p but depends on the four upstream open reading frames in the GCN4 mRNA leader sequence. Efficient translation of GCN4 mRNA is completely blocked by nitrogen starvation, even when cells are simultaneously starved for amino acids and eukaryotic initiation factor-2 alpha is fully phosphorylated by Gcn2p. Our data suggest that nitrogen starvation regulates translation of GCN4 by a novel mechanism that involves the four upstream open reading frames but that still acts independently of eukaryotic initiation factor-2 alpha phosphorylation by Gcn2p.

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.

The Unique Developmental Program of the Acoel Flatworm, Neochildia fusca

Acoel embryos exhibit a unique form of development that some investigators argue is related to that found in polyclad turbellarians and coelomate spiralians, which display typical quartet spiral cleavage. We generated the first cell-lineage fate map for an acoel flatworm, Neochildia fusca, using modern intracellular lineage tracers to assess the degree of similarity between these distinct developmental programs. N. fusca develops via a “duet” cleavage pattern in which second cleavage occurs in a leiotropically oblique plane relative to the animal–vegetal axis. At the four-cell stage, the plane of first cleavage corresponds to the plane of bilateral symmetry. All remaining cleavages are symmetrical across the sagittal plane. No ectomesoderm is formed; the first three micromere duets generate only ectodermal derivatives. Endomesoderm, including the complex assemblage of circular, longitudinal, and oblique muscle fibers, as well as the peripheral and central parenchyma, is generated by both third duet macromeres. The cleavage pattern, fate map, and origins of mesoderm in N. fusca share little similarity to that exhibited by other spiralians, including the Platyhelminthes (e.g., polyclad turbellarians). These findings are considered in light of the possible evolutionary origins of the acoel duet cleavage program versus the more typical quartet spiral cleavage program. Finally, an understanding of the cell-lineage fate map allows us to interpret the results of earlier cell deletion studies examining the specification of cell fates within these embryos and reveals the existence of cell–cell inductive interactions in these embryos.

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.

Chromatin silencing and Arabidopsis development: A role for polycomb proteins.

All organisms undergo dramatic shifts in gene expression patterns during early development, particularly at fertilization when the zygotic program is initiated. In flowering plants, fertilization launches four distinct developmental programs that lead to the formation of the embryo, endosperm, seed

Common role for each of the cGATA-4/5/6 genes in the regulation of cardiac morphogenesis.

The GATA-4/5/6 genes encode transcription factors implicated previously in the regulation of cardiac-specific differentiation programs. However, recent analyses of mouse GATA-4 null mutations found evidence for function in endoderm development (in vitro) and embryonic morphogenesis (in vivo). Whether each of the three cardiac-associated GATA factors function within distinct or common developmental programs was previously untested; past studies defined specific and distinct roles for each of the GATA-1/2/3 genes in embryonic hematopoiesis. In this study, we compare the transcript patterns of cGATA-4/5/6 during chick embryogenesis. Each of the three GATA factors is expressed in a similar pattern within gastrulating cells of the primitive streak, prior to determination of the cardiomyocyte progenitors, and later within the lateral plate mesoderm and associated endoderm layer. The patterns overlap but extend beyond the presumptive cardiomyocyte population expressing cNkx-2.5. Later in development, cGATA-4/5/6 are all transcribed throughout the differentiating heart, in similar but not identical patterns, within the endocardium, myocardium, and great vessels. In order to test the function of GATA factors during chick cardiogenesis, embryos were cultured in vitro in the presence of antisense oligomers designed to deplete specifically transcripts encoding cGATA-4/5/6, beginning around stage 7. When oligomers are used to target transcripts for all three genes, a high percentage of the embryos develop abnormal hearts related to the failure to form a normal primitive heart tube. In the most severe phenotype, cardiac bifida results in two bilateral beating hearts. In some embryos, the paired heart primordia undergo partial fusion but fail to form a single looping heart tube. In all cases, cellular differentiation is not obviously affected, as the abnormal hearts form beating tissue. Depletion of transcripts encoding any single GATA factor, or any combination of two GATA factors, does not affect development. The partial depletion of all three genes in chick results in a remarkably similar phenotype compared to the null GATA-4 mutation in mouse. Therefore, in the chick, each of the GATA-4/5/6 genes functions in a common pathway, at the time of cardiac crescent formation, for regulating early embryonic cardiac morphogenesis, apparently associated with embryonic folding or the migration of primordia to form a primitive tube.

Reciprocal signaling between Drosophila epidermal muscle attachment cells and their corresponding muscles.

Directed intercellular interactions between distinct cell types underlie the basis for organogenesis during embryonic development. This paper focuses on the establishment of the final somatic muscle pattern in Drosophila, and on the possible cross-talk between the myotubes and the epidermal muscle attachment cells, occurring while both cell types undergo distinct developmental programs. Our findings suggest that the stripe gene is necessary and sufficient to initiate the developmental program of epidermal muscle attachment cells. In stripe mutant embryos, these cells do not differentiate correctly. Ectopic expression of Stripe in various epidermal cells transforms these cells into muscle-attachment cells expressing an array of epidermal muscle attachment cell-specific markers. Moreover, these ectopic epidermal muscle attachment cells are capable of attracting somatic myotubes from a limited distance, providing that the myotube has not yet been attached to or been influenced by a closer wild-type attachment cell. Analysis of the relationships between muscle binding and differentiation of the epidermal muscle attachment cell was performed in mutant embryos in which loss of muscles, or ectopic muscles were induced. This analysis indicated that, although the initial expression of epidermal muscle-attachment cell-specific genes including stripe and groovin is muscle independent, their continuous expression is maintained only in epidermal muscle attachment cells that are connected to muscles. These results suggest that the binding of a somatic muscle to an epidermal muscle attachment cell triggers a signal affecting gene expression in the attachment cell. Taken together, our results suggest the presence of a reciprocal signaling mechanism between the approaching muscles and the epidermal muscle attachment cells. First the epidermal muscle attachment cells signal the myotubes and induce myotube attraction and adhesion to their target cells. Following this binding, the muscle cells send a reciprocal signal to the epidermal muscle attachment cells inducing their terminal differentiation into tendon-like cells.

The WH11 gene of Candida albicans is regulated in two distinct developmental programs through the same transcription activation sequences.

Candida albicans strain WO-1 undergoes two developmental programs, the bud-hypha transition and high-frequency phenotypic switching in the form of the white-opaque transition. The WH11 gene is expressed in the white budding phase but is inactive in the white hyphal phase and in the opaque budding phase. WH11 expression, therefore, is regulated in the two developmental programs. Through fusions between deletion derivatives of the WH11 promoter and the newly developed Renilla reniformis luciferase, the WH11 promoter has been characterized in the two developmental programs. Three transcription activation sequences, two strong and one weak, are necessary for the full expression of WH11 in the white budding phase, but no negative regulatory sequences were revealed as playing a role in either the white hyphal phase or the opaque budding phase. These results suggest that regulation is solely through activation in the white budding phase and the same mechanism, therefore, is involved in regulating the differential expression of WH11 in the alternative white and opaque phases of switching and the budding and hyphal phases of dimorphism.

The Dictyostelium MAP kinase ERK2 regulates multiple, independent developmental pathways.

We showed previously that the MAP kinase ERK2 is essential for aggregation. erk2 null cells lack cAMP stimulation of adenylyl cyclase and thus cannot relay the cAMP chemotactic signal, although the cells chemotax to cAMP (Segall et al. 1995). In this paper we have examined the role of ERK2 in controlling developmental gene expression and morphogenesis during the multicellular stages, making use of a temperature-sensitive ERK2 mutation. Using suspension assays, we show that ERK2 is not essential for aggregation-stage, cAMP pulse-induced gene expression, or for the expression of postaggregative genes, which are induced at the onset of mound formation in response to cAMP in wild-type cells. In contrast, the prespore-specific gene SP60 is not induced and the prestalk-specific gene ecmA is induced but at a significantly reduced level. Chimeric organisms, comprised of wild-type and erk2 null cells expressing the prestalk-specific ecmA/lacZ reporter, show an abnormal spatial patterning, in which Erk2ts/erk2 cells are excluded from the very anterior prestalk A region. To further examine the function of ERK2 during the multicellular stages, we bypassed the requirement of ERK2 for aggregation by creating an ERK2 temperature-sensitive mutant. erk2 null cells expressing the ERK2ts mutant develop normally at 20 degrees C and express cell-type-specific genes but do not aggregate at temperatures above 25 degrees C. Using temperature shift experiments, we showed that ERK2 is essential for proper morphogenesis and for the induction and maintenance of prespore but not prestalk gene expression. Our results indicate that ERK2 functions at independent stages during Dictyostelium development to control distinct developmental programs: during aggregation, ERK2 is required for the activation of adenylyl cyclase and during multicellular development, ERK2 is essential for morphogenesis and cell-type-specific gene expression. Analysis of these results and other supports the conclusion that the requirement of ERK2 for cell-type differentiation is independent of its role in the activation of adenylyl cyclase.

Establishment of the apical-basal axis in multicellular plant embryos

Apical-basal polarity of multicellular plant embryos is defined originally in the zygote before the first cell division and the apical and basal daughter cells follow distinct developmental programs. Mechanisms controlling these initial embryonic steps are still poorly understood but advances both in the flowering plant Arabidopsis and the alga Fucus have lead to various models and hypotheses. These point to the positional information as a major source for patterning the early embryo. Hypotheses concerning the possible location of this positional information are reviewed and discussed.

Overexpression of Arabidopsis COP1 results in partial suppression of light-mediated development: evidence for a light-inactivable repressor of photomorphogenesis.

Arabidopsis seedlings are genetically endowed with the capability to follow two distinct developmental programs: photomorphogenesis in the light and skotomorphogenesis in darkness. The regulatory protein CONSTITUTIVE PHOTO-MORPHOGENIC1 (COP1) has been postulated to act as a repressor of photomorphogenesis in the dark because loss-of-function mutations of COP1 result in dark-grown seedlings phenocopying the light-grown wild-type seedlings. In this study, we tested this working model by overexpressing COP1 in the plant and examining its inhibitory effects on photomorphogenic development. Stable transgenic Arabidopsis lines overexpressing COP1 were generated through Agrobacterium-mediated transformation. Overexpression was achieved using either the strong cauliflower mosaic virus 35S RNA promoter or additional copies of the wild-type gene. Analysis of these transgenic lines demonstrated that higher levels of COP1 can inhibit aspects of photomorphogenic seedling development mediated by either phytochromes or a blue light receptor, and the extent of inhibition correlated quantitatively with the vivo COP1 levels. This result provides direct evidence that COP1 acts as a molecular repressor of photomorphogenic development and that multiple photoreceptors can independently mediate the light inactivation of COP1. It also suggests that a controlled inactivation of COP1 may provide a basis for the ability of plants to respond quantitatively to changing light signals, such as fluence rate and photoperiod.