Micromeres are required for normal vegetal plate specification in sea urchin embryos

Vegetal plate specification was assessed in S. purpuratus embryos after micromere deletions at the 4th, 5th and 6th cleavages, by assaying expression of the early vegetal plate marker Endo 16, using whole-mount in situ hybridization. After 4th cleavage micromere deletions, the embryos typically displayed weak Endo16 expression in relatively few cells of the lineages that normally constitute the vegetal plate, while after 5th and 6th cleavage micromere deletions the embryos exhibited strong Endo16 expression in larger fractions of cells belonging to those lineages. When all four micromeres were deleted, the embryos were severely delayed in initiating gastrulation and sometimes failed to complete gastrulation. However, if only one micromere was allowed to remain in situ throughout development, the embryos exhibited strong Endo16 expression and gastrulation occurred normally, on schedule with controls. Additional measurements showed that these microsurgical manipulations do not alter cleavage rates or generally disrupt embryo organization. These results constitute direct evidence that the micromeres provide signals required by the macromere lineages for initiation of vegetal plate specification. The specification of the vegetal plate is completed in a normal manner only if micromere signaling is allowed to continue at least to the 6th cleavage stage

Specification of cell fate in the sea urchin embryo: summary and some proposed mechanisms

An early set of blastomere specifications occurs during cleavage in the sea urchin embryo, the result of both conditional and autonomous processes, as proposed in the model for this embryo set forth in 1989. Recant experimental results have greatly illuminated the mechanisms of specification in some early embryonic territories, though others remain obscure. We review the progressive process of specification within given lineage elements, and with reference to the early axial organization of the embryo. Evidence for the conditional specification of the veg(2) lineage subelement of the endoderm and other potential interblastomere signaling interactions in the cleavage-stage embryo are summarized. Definitive boundaries between mesoderm and endoderm territories of complex. the vegetal plate, and between endoderm and overlying ectoderm, are not established until later in development. These processes have been clarified by numerous observations on spatial expression of various genes, and by elegant lineage labeling studies. The early specification events depend on regional mobilization of regulatory factors resulting at once in the zygotic expression of genes encoding transcription factors, as well as downstream genes encoding proteins characteristic of the cell types that will much later arise from the progeny of the specified blastomeres. This embryo displays a maximal form of indirect development. The gene regulatory network underlying the embryonic development reflects the relative simplicity of the completed larva and of the processes required for its formation. The requirements for postembryonic adult body plan formation in the larval rudiment include engagement of a new level of genetic regulatory apparatus, exemplified by the Hox gene complex

The relationship between cell size and cell fate in Volvox carteri

In Volvox carteri development, visibly asymmetric cleavage divisions set apart large embryonic cells that will become asexual reproductive cells (gonidia) from smaller cells that will produce terminally differentiated somatic cells. Three mechanisms have been proposed to explain how asymmetric division leads to cell specification in Volvox: (a) by a direct effect of cell size (or a property derived from it) on cell specification, (b) by segregation of a cytoplasmic factor resembling germ plasm into large cells, and (c) by a combined effect of differences in cytoplasmic quality and cytoplasmic quantity. In this study a variety of V. carteri embryos with genetically and experimentally altered patterns of development were examined in an attempt to distinguish among these hypotheses. No evidence was found for regionally specialized cytoplasm that is essential for gonidial specification. In all cases studied, cells with a diameter > approximately 8 microns at the end of cleavage--no matter where or how these cells had been produced in the embryo--developed as gonidia. Instructive observations in this regard were obtained by three different experimental interventions. (a) When heat shock was used to interrupt cleavage prematurely, so that presumptive somatic cells were left much larger than they normally would be at the end of cleavage, most cells differentiated as gonidia. This result was obtained both with wild-type embryos that had already divided asymmetrically (and should have segregated any cytoplasmic determinants involved in cell specification) and with embryos of a mutant that normally produces only somatic cells. (b) When individual wild-type blastomeres were isolated at the 16-cell stage, both the anterior blastomeres that normally produce two gonidia each and the posterior blastomeres that normally produce no gonidia underwent modified cleavage patterns and each produced an average of one large cell that developed as a gonidium. (c) When large cells were created microsurgically in a region of the embryo that normally makes only somatic cells, these large cells became gonidia. These data argue strongly for a central role of cell size in germ/soma specification in Volvox carteri, but leave open the question of how differences in cell size are actually transduced into differences in gene expression

Evolutionary plasticity of developmental gene regulatory network architecture

Sea stars and sea urchins evolved from a last common ancestor that lived at the end of the Cambrian, approximately half a billion years ago. In a previous comparative study of the gene regulatory networks (GRNs) that embody the genomic program for embryogenesis in these animals, we discovered an almost perfectly conserved five-gene network subcircuit required for endoderm specification. We show here that the GRN structure upstream and downstream of the conserved network kernel has, by contrast, diverged extensively. Mesoderm specification is accomplished quite differently; the Delta–Notch signaling system is used in radically distinct ways; and various regulatory genes have been coopted to different functions. The conservation of the conserved kernel is thus the more remarkable. The results indicate types of network linkage subject to evolutionary change. An emergent theme is that subcircuit design may be preserved even while the identity of genes performing given roles changes because of alteration in their cis-regulatory control systems

Development of Protacs to Target Cancer-promoting Proteins for Ubiquitination and Degradation

The proteome contains hundreds of proteins that in theory could be excellent therapeutic targets for the treatment of human diseases. However, many of these proteins are from functional classes that have never been validated as viable candidates for the development of small molecule inhibitors. Thus, to exploit fully the potential of the Human Genome Project to advance human medicine, there is a need to develop generic methods of inhibiting protein activity that do not rely on the target protein’s function. We previously demonstrated that a normally stable protein, methionine aminopeptidase-2 or MetAP-2, could be artificially targeted to an Skp1-Cullin-F-box (SCF) ubiquitin ligase complex for ubiquitination and degradation through a chimeric bridging molecule or Protac (proteolysis targeting chimeric molecule). This Protac consisted of an SCFß-TRCP-binding phosphopeptide derived from I{kappa}B{alpha} linked to ovalicin, which covalently binds MetAP-2. In this study, we employed this approach to target two different proteins, the estrogen (ER) and androgen (AR) receptors, which have been implicated in the progression of breast and prostate cancer, respectively. We show here that an estradiol-based Protac can enforce the ubiquitination and degradation of the {alpha} isoform of ER in vitro, and a dihydroxytestosterone-based Protac introduced into cells promotes the rapid disappearance of AR in a proteasome-dependent manner. Future improvements to this technology may yield a general approach to treat a number of human diseases, including cancer

Gene regulatory network subcircuit controlling a dynamic spatial pattern of signaling in the sea urchin embryo

We dissect the transcriptional regulatory relationships coordinating the dynamic expression patterns of two signaling genes, wnt8 and delta, which are central to specification of the sea urchin embryo endomesoderm. cis-Regulatory analysis shows that transcription of the gene encoding the Notch ligand Delta is activated by the widely expressed Runx transcription factor, but spatially restricted by HesC-mediated repression through a site in the delta 5′UTR. Spatial transcription of the hesC gene, however, is controlled by Blimp1 repression. Blimp1 thus represses the repressor of delta, thereby permitting its transcription. The blimp1 gene is itself linked into a feedback circuit that includes the wnt8 signaling ligand gene, and we showed earlier that this circuit generates an expanding torus of blimp1 and wnt8 expression. The finding that delta expression is also controlled at the cis-regulatory level by the blimp1-wnt8 torus-generating subcircuit now explains the progression of Notch signaling from the mesoderm to the endoderm of the developing embryo. Thus the specific cis-regulatory linkages of the gene regulatory network encode the coordinated spatial expression of Wnt and Notch signaling as they sweep outward across the vegetal plate of the embryo

Whose urn is it anyway? Discussions on decolonisation & repatriation efforts in the cultural heritage sector in Portugal

Recent initiatives and reports, such as the Savoy–Sarr report in France, show some positive response among Western countries to the increasingly frequent calls for repatriating artefacts from museums, often as part of larger decolonisation efforts, although many of these calls still go unanswered. As this movement gains traction, the modern states of former empires and present-day hegemons, including Portugal, will face more legal, cultural, and ideological battles over the objects in their care. Thus, establishing guidelines for handling repatriation requests would be an acknowledgement of the power and importance of culture and increase Portugal’s soft power to make peace with the past and foster stronger international ties. This thesis analyses actors and arguments involved in public decolonisation and repatriation debates and efforts in the Portuguese cultural heritage sector. First, a brief history and contextualisation of decolonisation and repatriation efforts in museums situates Portugal within a larger movement to include previously excluded voices and perspectives. Then, the Portuguese context is specifically examined, considering the legacy of lusotropicalism and the ways Portugal has begun to reckon with its historical role in constructing and perpetuating the idea of race. Recent public debates reveal two outspoken camps in such polarising debates, with a notable absence of in-between voices, including rural and less formally educated Portuguese. Finally, a shared piece of cultural patrimony, the mortal remains of Dom Pedro I of Brazil (IV of Portugal), shows the potential difficulty in determining ownership and dominance in the relationship between empires and their former colonies.Iniciativas e relatórios recentes mostram algumas respostas positivas por parte dos países Ocidentais aos crescentes pedidos de repatriação de artefactos de museu, que vêm associados a esforços de descolonização mais abrangentes. Enquanto este movimento cresce, os estados modernos de antigos impérios e hegemons atuais, incluindo Portugal, irão enfrentar mais batalhas legais, culturais e ideológicas acerca dos objetos que estão debaixo do seu cuidado. Assim, o estabelecimento de orientações para lidar com pedidos de repatriação representaria um reconhecimento da importância da cultura e aumentaria o soft power de Portugal, mostrando a sua capacidade de fazer paz com o passado e de nutrir laços internacionais mais fortes. Esta tese analisa os atores e argumentos envolvidos nos debates e esforços públicos de descolonização e de repatriação dentro do setor português de herança cultural. Uma contextualização dos esforços de descolonização e repatriação nos museus situa Portugal dentro de um movimento maior para incluir vozes anteriormente excluídas. Esta tese analisa especificamente o contexto português, considerando o legado do luso-tropicalismo e as formas como Portugal tem começado a lidar com o seu papel histórico na construção e perpetuação da ideia de raça. Debates públicos, recentes, e polarizantes revelam dois campos proeminentes, com uma ausência notável de vozes intermédias, incluindo de portugueses com menor educação formal e provenientes de zonas rurais. Finalmente, um pedaço partilhado de património cultural, os restos mortais de Dom Pedro I do Brasil, mostra a potencial dificuldade em determinar a pertença e a dominância na relação entre impérios e as antigas colónias

Regulative recovery in the sea urchin embryo and the stabilizing role of fail-safe gene network wiring

Design features that ensure reproducible and invariant embryonic processes are major characteristics of current gene regulatory network models. New cis-regulatory studies on a gene regulatory network subcircuit activated early in the development of the sea urchin embryo reveal a sequence of encoded “fail-safe” regulatory devices. These ensure the maintenance of fate separation between skeletogenic and nonskeletogenic mesoderm lineages. An unexpected consequence of the network design revealed in the course of these experiments is that it enables the embryo to “recover” from regulatory interference that has catastrophic effects if this feature is disarmed. A reengineered regulatory system inserted into the embryo was used to prove how this system operates in vivo. Genomically encoded backup control circuitry thus provides the mechanism underlying a specific example of the regulative development for which the sea urchin embryo has long been famous

The spatial and temporal distribution of imp clusters in sea urchin embryos

Sea urchin embryos of Strongylocentrotus purpuratus, Lytechinus pictus and Lytechinus variegatus were investigated with respect to appearance, disappearance, and distribution of clusters of larger than average intramembraneous particles and associated cytoplasmic vesicles using thin section and freeze-fracture TEM as well as experimental procedures. This study identified a stable pattern of IMP clusters that becomes established during the eight-cell stage in S. purpuratus embryos. The pattern essentially distinguishes apical surface and lateral surface compartments in blastomere P-face plasma membranes and is spatially the same as the distribution of cytoplasmic (pigment) vesicles which occupy the apical cortices of the blastomeres. Analysis of post 16-cell stages revealed the IMP pattern persists through the early stages of blastulation. Largely due to a process called the vegetal cortical rearrangement, the micromeres are formed without the typical IMP pattern and without the pigment vesicles. Thus, the micromeres have a significantly different plasma membrane organization, and probably, also, a significantly different degree of stability in their cortex at the time of their formation. The implications of these findings are discussed in relation to the development of radial polarity and micromere determination. (See more in text.)California State University, Northridge. Department of Biology.Includes bibliographical references (pages 75-83