Major Developmental Transitions Research Articles

Antagonistic Actions of Ecdysone and Insulins Determine Final Size in Drosophila

All animals coordinate growth and maturation to reach their final size and shape. In insects, insulin family molecules control growth and metabolism, whereas pulses of the steroid 20-hydroxyecdysone (20E) initiate major developmental transitions. We show that 20E signaling also negatively controls animal growth rates by impeding general insulin signaling involving localization of the transcription factor dFOXO and transcription of the translation inhibitor 4E-BP. We also demonstrate that the larval fat body, equivalent to the vertebrate liver, is a key relay element for ecdysone-dependent growth inhibition. Hence, ecdysone counteracts the growth-promoting action of insulins, thus forming a humoral regulatory loop that determines organismal size.

The genomic response to 20-hydroxyecdysone at the onset of Drosophila metamorphosis

The genome-wide transcriptional response to 20-hydroxyecdisone at the onset of Drosophila metamorphosis, as well as its dependency on one of the ecdysone receptors is described.

The evolution of plant development

The last decade has witnessed a resurgence in the study of the evolution of plant development, combining investigations in systematics, developmental morphology, molecular developmental genetics, and molecular evolution. The integration of phylogenetic studies, structural analyses of fossil and extant taxa, and molecular developmental genetic information allows the formulation of explicit and testable hypotheses for the evolution of morphological characters. These comprehensive approaches provide opportunities to dissect the evolution of major developmental transitions among land plants, including those associated with apical meristems, the origins of the root/shoot dichotomy, diversification of leaves, and origin and subsequent modification of flower structure. The evolution of these major developmental innovations is discussed within both phylogenetic and molecular genetic contexts. We conclude that it is the combination of these approaches that will lead to the greatest understanding of the evolution of plant development.

Expression profiling using random genomic DNA microarrays identifies differentially expressed genes associated with three major developmental stages of the protozoan parasite Leishmania major.

To complete its life cycle, protozoan parasites of the genus Leishmania undergo at least three major developmental transitions. However, previous efforts to identify genes showing stage regulated changes in transcript abundance have yielded relatively few. Here we used expression profiling to assess changes in transcript abundance in three stages: replicating promastigotes and infective non-replicating metacyclics, which occur in the sand fly vector, and in the amastigote stage residing with macrophage phagolysosomes in mammals. Microarrays were developed containing 11,484 PCR products that included a number of known genes and 10,464 random 1 kb genomic DNA fragments. Arrays were hybridized in triplicate and genes showing two-fold or greater changes in 2/3 experiments were scored as differentially expressed. Remarkably, only about one percent of the DNAs expression varied by this criteria, in either stage comparison. Northern blot analysis confirmed the predicted change in mRNA abundance for most of these (68%). This set of genes included most of those previously identified in the literature as differentially regulated as well as a number of novel genes. Notably, Leishmania maxicircle transcripts showed strong up-regulation in metacyclic and amastigote parasites, probably associated with changes in parasite energy metabolism. However, current data suggest that expression profiling using shotgun DNA libraries significantly underestimates the extent of regulated transcripts.

Multiple pathways in the decision to flower: enabling, promoting, and resetting.

At a certain point in their life cycle, annual plants undergo a major developmental transition and switch from vegetative to reproductive development. This process is rarely reversible, and ensuring that the timing of this transition is optimal for pollination and seed development is a major factor

Induction of flowering by seasonal changes in photoperiod.

In many plants, major developmental transitions such as the initiation of flowering are synchronized to the changing seasons. Day length provides one of the environmental cues used to achieve this. We describe the molecular mechanisms that measure day length and control flowering in Arabidopsis. Also, we compare these mechanisms with those that control flowering time in rice. This comparison suggests that components of the Arabidopsis regulatory network are conserved in other species, but that their regulation can be altered to generate different phenotypic responses.

Rigor mortisencodes a novel nuclear receptor interacting protein required for ecdysone signaling duringDrosophilalarval development

Pulses of the steroid hormone ecdysone trigger the major developmental transitions in Drosophila, including molting and puparium formation. The ecdysone signal is transduced by the EcR/USP nuclear receptor heterodimer that binds to specific response elements in the genome and directly regulates target gene transcription. We describe a novel nuclear receptor interacting protein encoded by rigor mortis (rig) that is required for ecdysone responses during larval development. rig mutants display defects in molting, delayed larval development, larval lethality, duplicated mouth parts, and defects in puparium formation--phenotypes that resemble those seen in EcR, usp, E75A and betaFTZ-F1 mutants. Although the expression of these nuclear receptor genes is essentially normal in rig mutant larvae, the ecdysone-triggered switch in E74 isoform expression is defective. rig encodes a protein with multiple WD-40 repeats and an LXXLL motif, sequences that act as specific protein-protein interaction domains. Consistent with the presence of these elements and the lethal phenotypes of rig mutants, Rig protein interacts with several Drosophila nuclear receptors in GST pull-down experiments, including EcR, USP, DHR3, SVP and betaFTZ-F1. The ligand binding domain of betaFTZ-F1 is sufficient for this interaction, which can occur in an AF-2-independent manner. Antibody stains reveal that Rig protein is present in the brain and imaginal discs of second and third instar larvae, where it is restricted to the cytoplasm. In larval salivary gland and midgut cells, however, Rig shuttles between the cytoplasm and nucleus in a spatially and temporally regulated manner, at times that correlate with the major lethal phase of rig mutants and major switches in ecdysone-regulated gene expression. Taken together, these data indicate that rig exerts essential functions during larval development through gene-specific effects on ecdysone-regulated transcription, most likely as a cofactor for one or more nuclear receptors. Furthermore, the dynamic intracellular redistribution of Rig protein suggests that it may act to refine spatial and temporal responses to ecdysone during development.

Temporal profiles of nuclear receptor gene expression reveal coordinate transcriptional responses during Drosophila development.

The recent completion of the Drosophila genome sequence revealed 21 members of the nuclear receptor superfamily. Many of these genes are transcriptionally regulated by the steroid hormone ecdysone and play a role during the onset of metamorphosis, including the EcR/USP ecdysone receptor heterodimer. As a first step toward a genomic analysis of this gene family, we have characterized the temporal patterns of expression for all detectable nuclear receptor transcripts throughout major ecdysone-regulated developmental transitions in the life cycle: embryogenesis, a larval molt, puparium formation, and the prepupal-pupal transition. We find an unexpected close temporal relationship between DHR3, E75B, and betaFTZ-F1 expression after each major ecdysone pulse examined, reflecting the known cross-regulatory interactions of these genes in prepupae and suggesting that they act together at other stages in the life cycle. In addition, E75A, E78B, and DHR4 are expressed in a reproducible manner with DHR3, E75B, and betaFTZ-F1, suggesting that they intersect with this regulatory cascade. Finally, we find that known ecdysone-inducible primary-response transcripts are coordinately induced at times when the ecdysteroid titer is low, implying the existence of novel, as yet uncharacterized, temporal signals in Drosophila.

The Drosophila Orphan Nuclear Receptor DHR38 Mediates an Atypical Ecdysteroid Signaling Pathway

Ecdysteroid pulses trigger the major developmental transitions during the Drosophila life cycle. These hormonal responses are thought to be mediated by the ecdysteroid receptor (EcR) and its heterodimeric partner Ultraspiracle (USP). We provide evidence for a second ecdysteroid signaling pathway mediated by DHR38, the Drosophila ortholog of the mammalian NGFI-B subfamily of orphan nuclear receptors. DHR38 also heterodimerizes with USP, and this complex responds to a distinct class of ecdysteroids in a manner that is independent of EcR. This response is unusual in that it does not involve direct binding of ecdysteroids to either DHR38 or USP. X-ray crystallographic analysis of DHR38 reveals the absence of both a classic ligand binding pocket and coactivator binding site, features that seem to be common to all NGFI-B subfamily members. Taken together, these data reveal the existence of a separate structural class of nuclear receptors that is conserved from fly to humans.

Exploiting genomics in evolutionary developmental biology

Genomics is providing a new stimulus to research in developmental biology. Here we describe how genomic approaches are giving new insight into the genetic basis for two major developmental transitions in evolution: the origin of vertebrates and the origin of multicellular animals. Our first approach uses complete genome sequences. We compared orthologue group complexity (an estimate of gene family size) between human, fruitfly, nematode and yeast genomes. These analyses suggest extensive gene duplication somewhere on the deuterostome lineage leading to humans. We show that similar events did not occur on the lineages leading to flies or to nematodes. To determine the timing of this event, we then incorporate gene sampling data from a cephalochordate, amphioxus. Molecular phylogenetic analysis of amphioxus genes reveals that gene duplications occurred close to vertebrate origins, coincident with a major developmental transition. By contrast, whole genome comparison between invertebrates and yeast did not reveal a similar event during the origin of multicellular animals. In this case, an EST survey of choanoflagellates is being used to identify genetic changes coincident with animal origins.

Lipid dynamics in the embryos of Patiriella species (Asteroidea) with divergent modes of development.

Evolution of lecithotrophic development in sea stars involved a modification in maternal provisioning from the production of yolk-dominated to lipid-dominated eggs. The dynamics of lipid reserves in the embryos of four Patiriella species differing in their lipid provisions were examined. Patiriella regularis had small yolk protein-dominated eggs (150 microm in diameter) and an ancestral mode of development through planktotrophic larvae. Patiriella calcar, Patiriella exigua and Patiriella pseudoexigua had large eggs (390-440 microm in diameter) and lecithotrophic planktonic, benthic and intragonadal larvae, respectively. Patiriella exigua deposited negatively buoyant eggs containing substantial yolk protein and lipid reserves onto the substratum. In contrast, the planktonic eggs of P. calcar and the intragonadal eggs of P. pseudoexigua were dominated by lipid and were neutrally and positively buoyant, respectively. By the blastula stage there was little trace of lipid in P. regularis embryos. Blastulae of the lecithotrophic developers, by contrast, had conspicuous lipid droplets distributed through their cells. In parallel with the change from cuboidal to columnar epithelium during the blastula to gastrula transition, lipid reserves became redistributed into the basal cytoplasm. The extent of lipid transport reflected the amount of lipid reserves. In P. pseudoexigua embryos with the greatest lipid load, basal shunting was followed by secretion of lipid into the blastocoele where it was stored for the perimetamorphic period. Evolution of lecithotrophy in Patiriella appears to reflect selection to provide metamorphic stages with nutrients normally accrued by feeding larvae with the consequence that early development is burdened by voluminous, potentially inert nutritive stores. Lipid redistribution coincident with a major developmental stage transition may be required to facilitate unimpeded morphogenesis. This phenomenon may be characteristic of lecithotrophic development in echinoderms and appears pre-adaptive for extrusion of lipid in species like P. pseudoexigua with particularly extensive lipid reserves.

The RXR homolog ultraspiracle is an essential component of the Drosophila ecdysone receptor.

Pulses of the steroid hormone ecdysone function as key temporal signals during insect development, coordinating the major postembryonic developmental transitions, including molting and metamorphosis. In vitro studies have demonstrated that the EcR ecdysone receptor requires an RXR heterodimer partner for its activity, encoded by the ultraspiracle (usp) locus. We show here that usp exerts no apparent function in mid-third instar larvae, when a regulatory hierarchy prepares the animal for the onset of metamorphosis. Rather, usp is required in late third instar larvae for appropriate developmental and transcriptional responses to the ecdysone pulse that triggers puparium formation. The imaginal discs in usp mutants begin to evert but do not elongate or differentiate, the larval midgut and salivary glands fail to undergo programmed cell death and the adult midgut fails to form. Consistent with these developmental phenotypes, usp mutants show pleiotropic defects in ecdysone-regulated gene expression at the larval-prepupal transition. usp mutants also recapitulate aspects of a larval molt at puparium formation, forming a supernumerary cuticle. These observations indicate that usp is required for ecdysone receptor activity in vivo, demonstrate that the EcR/USP heterodimer functions in a stage-specific manner during the onset of metamorphosis and implicate a role for usp in the decision to molt or pupariate in response to ecdysone pulses during larval development.

Letter to the Editor

Letter to the Editor Racelessness a Difficult Characterization Dear Editor: I would appreciate an opportunity to respond to a recent article in the American Annals of the Deaf, entitled, "The Association Between Racelessness and Achievement Among African American Deaf Adolescents" (Mosley-Hall, 1998). My initial reaction to the construct on which the study was based—"racelessness" per se—was disdain. I have read Fordham's (1990) original study of Black hearing teens and am familiar with Ogbu's theory (Ogbu & Simons, 1994) of minority status, the model on which racelessness is built. I agree with his assertions that African American students experience social pressures that inhibit their ability to engage in pro-school attitudes and behaviors because they (the attitudes and behaviors) are associated with the oppressor. However, it does not matter from which angle I view it, I find racelessness a difficult characterization to make of young people who are still undergoing the developmental process of racial identity formation (Tatum, 1998). The students being referred to may not have a solid racial identity and may not be aware of the full impact race has in this society, and to label them raceless is a rather strong indictment, particularly when the scale used to measure racelessness reflects such a narrow and stereotypical view of African American culture that its face validity is rather questionable. To accept such a view is feeding into the same negative stereotype which equates being smart with acting White. If we do not broaden our view of Black culture, then how do we expect our children to be able to do so? And to label children as "modern-day Uncle Toms" implies they are undermining the community simply by succeeding in school. What about the students who are racially aware enough to know that their history is filled with examples of Black high achievement so that succeeding in school is seen as their legacy and something they feel expected to do? Where do these students fit into the model—being neither raceless nor low achieving? Such a dichotomous line of thinking does almost as much damage as those in the school systems who devalue our children, disproportionately place them in lower academic or vocational tracks, administer culturally biased tests to our students, and expect low achievement from them. According to the racelessness model, none of these teacher variables is considered as contributing to the low academic performance of Black Deaf students. African American students, as measured by the raceless scale, are being judged by how closely they subscribe to stereotypic views of Black people. However, the teachers are not being judged on the extent to which they make judgments against the students who fit the stereotype. Are they seating them in the back of the room, calling on them less frequently, allowing them shorter time to answer questions , or providing less constructive feedback to their work? These teacher variables have a more direct causal relationship to student success (Hilliard, 1989). Although I subscribe to Delpit's (1993) view of recognizing the "power" of achievement and, in effect, teaching our children to adopt those behaviors necessary to achieve success (access power), we as professionals would be doing a disservice to the generation succeeding us if we do not fight to change the mind-set of others responsible for educating our children so that African American students expressing their cultural competence (in the way they dress, gesture, etc.) are not viewed as threatening or menacing by teachers (namely those of the dominant culture). We need to challenge the notions and behaviors they (teachers) bring into the classroom and how these notions and behaviors perpetuate the self-fulfilling prophecy instead of "blaming the victim"—i.e., Black Deaf adolescents who are in the midst of a major developmental transition period. Educators and parents are the ones who have to effect change. It is the responsibility of the adults to accommodate the needs of students, not the other way around. In addition, what would have been helpful to me in reading the article is a little more discussion on the development of a Deaf cultural identity and the effect that has on ethnic identity. The Aramburo (1989) study that was...

Cross-talking among Drosophila nuclear receptors at the promiscuous response element of the ng-1 and ng-2 intermolt genes

In Drosophila, peaks of the titer of the steroid hormone ecdysone act as molecular signals that trigger all the major developmental transitions occurring along the life cycle. The EcR/USP heterodimer, known to constitute the functional ecdysone receptor, binds with high affinity to specific target sequences, the ecdysone response elements (EcREs), whose repertoire still remains to be fully characterized at both the molecular and functional levels. In order to investigate the properties of EcREs composed of directly repeated half-sites (DRs), we have analysed the binding properties of the ng-EcRE, a DR element located within the coding region of ng-1 and ng-2, two highly homologous genes mapping at the ecdysone-regulated 3C intermolt puff. We report here that the ng-EcRE contacts the ecdysone receptor through its directly repeated half-sites spaced by 12 bp, and that this element may interact efficiently with at least three Drosophila orphan receptors, namely DHR38, DHR39 and βFTZ-F1. Interestingly, DHR38 is bound alone or in combination with USP, providing the first evidence that the EcR-USP and DHR38-USP may directly compete for binding to a common response element. These results suggest that EcREs composed of widely spaced DRs may contribute to the establishment of extensive nuclear receptors cross-talking along the development, a mechanism that might play a relevant role in determining the temporal and spatial specificty of the ecdysone response. Finally, we show that the ng-EcRE can promote functional interactions in vitro as well as in vivo, acting as a transcriptional enhancer able to confer a specific developmental expression profile to a minimal promoter in transgenic flies.

Friendships and adaptation in the life course.

To consider friendships and their significance through the life course requires, first, differentiation of deep structure (i.e., reciprocity) from surface structure (i.e., the social exchange) and, second, assessment within a multifaceted framework that simultaneously emphasizes having friends, the identity of one's friends, and relationship quality. Having friends is correlated with a sense of well being across the life span, but developmental outcome also depends on the identity of one's friends as well as the quality of one's relationships with them. Greater attention needs to be given to the manner in which friendships differ from one another, continuities and changes across major developmental transitions, and differentiation of developmental pathways through which friendship experience contributes to individual outcome.

Contrasting roles for c-Myc and L-Myc in the regulation of cellular growth and differentiation in vivo.

Although myc family genes are differentially expressed during development, their expression frequently overlaps, suggesting that they may serve both distinct and common biological functions. In addition, alterations in their expression occur at major developmental transitions in many cell lineages. For example, during mouse lens maturation, the growth arrest and differentiation of epithelial cells into lens fiber cells is associated with a decrease in L- and c-myc expression and a reciprocal rise in N-myc levels. To determine whether the down-regulation of L- and c-myc are required for mitotic arrest and/or completion of differentiation and whether these genes have distinct or similar activities in the same cell type, we have studied the consequences of forced L- and c-myc expression in the lens fiber cell compartment using the alpha A-crystallin promoter in transgenic mice (alpha A/L-myc and alpha A/c-myc mice). With respect to morphological and molecular differentiation, alpha A/L-myc lenses were characterized by a severely disorganized lens fiber cell compartment and a significant decrease in the expression of a late-stage differentiation marker (MIP26); in contrast, differentiation appeared to be unaffected in alpha A/c-myc mice. Furthermore, an analysis of proliferation indicated that while alpha A/L-myc fiber cells withdrew properly from the cell cycle, inappropriate cell cycle progression occurred in the lens fiber cell compartment of alpha A/c-myc mice. These observations indicate that continued late-stage expression of L-myc affected differentiation processes directly, rather than indirectly through deregulated growth control, whereas constitutive c-myc expression inhibited proliferative arrest, but did not appear to disturb differentiation. As a direct corollary, our data indicate that L-Myc and c-Myc are involved in distinct physiological processes in the same cell type.

Sensitivity Thresholds and Variable Time Scales in Plant Hormone Action.

Plant growth regulators are involved in major developmental transitions, such as flowering, embryogenesis, or dormancy, and in real-time responses to environmental conditions, such as adjustments in growth rates or stomatal conductance. In addition, it is generally the case that only specific cells or tissues in the plant are responsive to a particular regulatory factor. For example, only certain cells at or near the abscission zone express cellulase when exposed to ethylene (del Campillo et al., 1990); aleurone cells of cereal grains respond specifically to GA by synthesizing a-amylase (Jacobsen and Knox, 1973); stomatal guard cells are dramatically responsive to ABA (MacRobbie, 1991); and transcriptional responses to auxin vary markedly among different cell and tissue types (Gee et al., 1991). The wide range of hormonal responses evident among individual cell types and the different developmental and physiological time scales on which hormones act raise two questions that will be addressed here. (a) How is the variation in hormonal sensitivity or content among cells or tissues integrated into developmental patterns? (b) What is the relationship of hormonal sensitivity or content to the timing of developmental or physiological events? Recently, new insights into these fundamental questions have begun to form into a general model of plant hormone action. Our objective here is to outline a novel conceptual framework that integrates variation in both hormonal sensitivity and content with an expanded view of biological time.

Structure of the murine homeobox gene cdx-2. Expression in embryonic and adult intestinal epithelium.

To gain insight into the mechanisms which govern cellular identity in the intestinal epithelium we have begun a detailed study of the murine cdx-2 homeobox gene. We isolated and sequenced both cDNA and genomic clones in order to define the open reading frame and mature transcript. A detailed analysis of cdx-2 transcript levels late in embryogenesis showed that they increased 6-7-fold at a time when the gut undergoes a major developmental transition. In the adult, cdx-2 was expressed in colon in a region-specific manner with transcripts some 5-fold more abundant in the cecum as compared with the rectum. In situ hybridization and immunohistochemical experiments showed cdx-2 transcripts and protein were present in all epithelial cells in the proximal colon irrespective of their degree of differentiation. In distal colon, however, transcripts were most abundant in undifferentiated cells at the bottom of crypts, whereas the highest protein levels were present in mature cells in the upper half of crypts. We conclude that cdx-2 is expressed specifically in gut epithelium where it is not restricted to a particular cell lineage. Rather, the rostrocaudal expression gradient suggests that it may play a role in specifying positional identity.

Temporal coordination of regulatory gene expression by the steroid hormone ecdysone.

In Drosophila, pulses of the steroid hormone ecdysone function as temporal signals that trigger the major postembryonic developmental transitions. The best characterized of these pulses activates a series of puffs in the polytene chromosomes as it triggers metamorphosis. A small set of early puffs is induced as a primary response to the hormone. These puffs encode regulatory proteins that both repress their own expression and activate a large set of late secondary response genes. We have used Northern blot analysis of RNA isolated from staged animals and cultured organs to study the transcription of three primary response regulatory genes, E75, BR-C and EcR. Remarkably, their patterns of transcription in late larvae can be defined in terms of two responses to different ecdysone concentrations. The class I transcripts (E74B and EcR) are induced in mid-third instar larvae in response to the low, but increasing, titer of ecdysone. As the hormone concentration peaks in late third instar larvae, these transcripts are repressed and the class II RNAs (E74A, E75A and E75B) are induced. The BR-C RNAs appear to have both class I and class II characteristics. These data demonstrate that the relatively simple profile of a hormone pulse contains critical temporal information that is transduced into waves of primary response regulatory gene activity.

Effect of incest on self and social functioning: A developmental psychopathology perspective.

The effects of child sexual abuse have become a leading concern of mental health service providers. Despite an explosion of studies, one major difficulty in this research is the lack of a developmentally sensitive model for conceptualizing short- and long-term effects and continuity and discontinuity of effects over time. This article proposes a model based in the perspective of developmental psychopathology. It is argued that incest has its unique negative effects in the domains of self and social functioning, specifically in jeopardizing self-definition and integration, self-regulatory processes, and a sense of security and trust in relationships. Studies with clinical samples indicate that diagnostic conditions associated uniquely with a history of incest reflect serious self- and social impairments. A review of the developmental literature on self and social development summarizes each major developmental transition from infancy to middle adulthood, and the implications for the negative effects of incest on development are discussed. Finally, implications for developmentally sensitive research are discussed.