Search

Kruppel-like factor 9 (Klf9) is a feedforward regulator of glucocorticoid receptor (GR) signaling. We asked if klf9 interacts with fkbp5, a negative feedback regulator of the GR implicated in stress-induced psychiatric and metabolic disorders. We show that in zebrafish fkbp5 and klf9 are expressed with highly correlated, GR-dependent oscillatory dynamics. Expression of fkbp5 is basally elevated in klf9-/- mutants, and Klf9 binds sequences in the fkbp5 promoter, suggesting that Klf9 is an incoherent feedforward regulator of the GR-fkbp5 feedback circuit that governs GR activity. Given that both the GR and Fkbp5 regulate metabolism, we asked how loss of Klf9 function affects metabolic rate and gene expression. We found that klf9-/- mutants have a decreased rate of oxygen consumption and overexpress glycolytic genes, the promoters of which are enriched for Klf9 binding motifs. Our results suggest that Klf9 functions as a transcriptional repressor to dynamically regulate cellular glucocorticoid responsivity and metabolic homeostasis.

The zebrafish has recently emerged as a model system for investigating the developmental roles of glucocorticoid signaling and the mechanisms underlying glucocorticoid-induced developmental programming. To assess the role of the Glucocorticoid Receptor (GR) in such programming, we used CRISPR-Cas9 to produce a new frameshift mutation, GR369-, which eliminates all potential in-frame initiation codons upstream of the DNA binding domain. Using RNA-seq to ask how this mutation affects the larval transcriptome under both normal conditions and with chronic cortisol treatment, we find that GR mediates most of the effects of the treatment, and paradoxically, that the transcriptome of cortisol-treated larvae is more like that of larvae lacking a GR than that of larvae with a GR, suggesting that the cortisol-treated larvae develop GR resistance. The one transcriptional regulator that was both underexpressed in GR369- larvae and consistently overexpressed in cortisol-treated larvae was klf9. We therefore used CRISPR-Cas9-mediated mutation of klf9 and RNA-seq to assess Klf9-dependent gene expression in both normal and cortisol-treated larvae. Our results indicate that Klf9 contributes significantly to the transcriptomic response to chronic cortisol exposure, mediating the upregulation of proinflammatory genes that we reported previously.

Objective Chronic early life stress can affect development of the neuroendocrine stress system, leading to its persistent dysregulation and consequently increased disease risk in adulthood. One contributing factor is thought to be epigenetic programming in response to chronic cortisol exposure during early development. We have previously shown that zebrafish embryos treated chronically with cortisol develop into adults with constitutively elevated whole-body cortisol and aberrant immune gene expression. Here we further characterize that phenotype by assessing persistent effects of the treatment on cortisol tissue distribution and dynamics, chromatin accessibility, and activities of glucocorticoid-responsive regulatory genes klf9 and fkbp5. To that end cortisol levels in different tissues of fed and fasted adults were measured using ELISA, open chromatin in adult blood cells was mapped using ATAC-seq, and gene activity in adult blood and brain cells was measured using qRT-PCR. Results Adults derived from cortisol-treated embryos have elevated whole-body cortisol with aberrantly regulated tissue distribution and dynamics that correlate with differential activity of klf9 and fkbp5 in blood and brain.

Oxygen is essential to contemporary life, providing the major electron sink underlying cellular energy metabolism. In addition to providing energy, largely involving redox reactions within mitochondria, oxidative metabolism produces reactive byproducts that are damaging to cellular components. Eukaryotic organisms have evolved multiple physiological mechanisms and signaling pathways to deal with fluctuating levels of oxygen and reactive oxygen species (ROS), and many of these are used in animals to regulate developmental processes. Here we review recent findings showing how mitochondria, ROS and hypoxia signaling contribute to the regulation of early axial patterning in embryos, to nervous system development, and to the regulation of cell proliferation and differentiation during development and regeneration.

Life has many defining characteristics, not the least of which is that it exists in a self-perpetuating homeostatic state that is far from thermodynamic equilibrium. However, since the universe contains many abiotic dissipative structures that are far from thermodynamic equilibrium, that alone cannot be taken to be definitive of life, at least not life on earth as we know it. What distinguishes the latter from nonlife, and hence biology as a science distinct from chemistry and physics, is genomic information, i.e., the information encoded in DNA. Living systems do what they do only by virtue of that information. As Robert Rosen showed by mathematical abstraction, life is a complex self-entailing system of metabolism, repair, and replication, and organisms embody modeling relations with their environment. In all known organisms, metabolism, repair, and replication require a genome, which informs the entailments through which the organism relates and adapts to its environment. Biologists came to recognize this in the latter part of the twentieth century, and hence the cutting edge of biology is now focused almost entirely on genomics (and its derivatives such as epigenomics). With genomic information as its central concern, biology is a historical as much as a physical science. Life cannot be reduced to chemistry and physics because genomically informed modeling relations are physically indeterminate and context-dependent, with historical contingency being an irreducible part of the relevant context. As a result, the science of paleontology is essential for reconstructing the evolutionary development of life on earth. This then constitutes the hard problem for the origin-of-life discourse: the sequence of contingencies that produced the first functional genome is not well preserved in the fossil record. Therefore, the problem of abiogenesis will probably remain unsolved and a matter of speculation until we learn how to create a true (i.e., genomically informed) organism from scratch.

Life is inherently regenerative. However, in humans and many other animal species development imposes epigenetic constraints on cellular plasticity and proliferative potency that limit somatic regenerative capacity. Such constraints can be surmised to have evolved as adaptations or tradeoffs that increased organismal fitness. A major goal of regenerative biology is to identify the limiting constraints that can be clinically targeted by regenerative medicine. Here I review the fundamental problems and processes of tissue repair and regeneration that pertain to all eukaryotic organisms, then discuss the evolution of somatic regenerative potential in animals in relation to constraints imposed by ecology, development of structural and functional complexity, epigenetics, and metabolism. Within this context I address the question of why regenerative capacity is generally more limited in amniotes than in fish and amphibians. This survey identifies some key constraints limiting mammalian regenerative capacity that might be productively targeted by regenerative medicine.

Elk proteins are Ets family transcription factors that regulate cell proliferation, survival, and differentiation in response to ERK (extracellular-signal regulated kinase)-mediated phosphorylation. Here we report the embryonic expression and function of Sp-Elk, the single Elk gene of the sea urchin Strongylocentrotus purpuratus. Sp-Elk is zygotically expressed throughout the embryo beginning at late cleavage stage, with peak expression occurring at blastula stage. Morpholino antisense-mediated knockdown of Sp-Elk causes blastula-stage developmental arrest and embryo disintegration due to apoptosis, a phenotype that is rescued by wild-type Elk mRNA. Development is also rescued by Elk mRNA encoding a serine to aspartic acid substitution (S402D) that mimics ERK-mediated phosphorylation of a conserved site that enhances DNA binding, but not by Elk mRNA encoding an alanine substitution at the same site (S402A). This demonstrates both that the apoptotic phenotype of the morphants is specifically caused by Elk depletion, and that phosphorylation of serine 402 of Sp-Elk is critical for its anti-apoptotic function. Knockdown of Sp-Elk results in under-expression of several regulatory genes involved in cell fate specification, cell cycle control, and survival signaling, including the transcriptional regulator Sp-Runt-1 and its target Sp-PKC1, both of which were shown previously to be required for cell survival during embryogenesis. Both Sp-Runt-1 and Sp-PKC1 have sequences upstream of their transcription start sites that specifically bind Sp-Elk. These results indicate that Sp-Elk is the signal-dependent activator of a feed-forward gene regulatory circuit, consisting also of Sp-Runt-1 and Sp-PKC1, which actively suppresses apoptosis in the early embryo.

Chronic early-life stress increases adult susceptibility to numerous health problems linked to chronic inflammation. One way that this may occur is via glucocorticoid-induced developmental programming. To gain insight into such programming we treated zebrafish embryos with cortisol and examined the effects on both larvae and adults. Treated larvae had elevated whole-body cortisol and glucocorticoid signaling, and upregulated genes associated with defense response and immune system processes. In adulthood the treated fish maintained elevated basal cortisol levels in the absence of exogenous cortisol, and constitutively mis-expressed genes involved in defense response and its regulation. Adults derived from cortisol-treated embryos displayed defective tailfin regeneration, heightened basal expression of pro-inflammatory genes, and failure to appropriately regulate those genes following injury or immunological challenge. These results support the hypothesis that chronically elevated glucocorticoid signaling early in life directs development of a pro-inflammatory adult phenotype, at the expense of immunoregulation and somatic regenerative capacity., Summary: Chronically elevated glucocorticoid stress signaling during early zebrafish development has long-term consequences that affect immune gene expression and regulation in adulthood.

Summary Aging in many animals is characterized by a failure to maintain tissue homeostasis and the loss of regenerative capacity. In this study, the ability to maintain tissue homeostasis and regenerative potential was investigated in sea urchins, a novel model to study longevity and negligible senescence. Sea urchins grow indeterminately, regenerate damaged appendages and reproduce throughout their lifespan and yet different species are reported to have very different life expectancies (ranging from 4 to more than 100 years). Quantitative analyses of cell proliferation and apoptosis indicated a low level of cell turnover in tissues of young and old sea urchins of species with different lifespans (Lytechinus variegatus, Strongylocentrotus purpuratus and Mesocentrotus franciscanus). The ability to regenerate damaged tissue was maintained with age as assessed by the regrowth of amputated spines and tube feet (motor and sensory appendages). Expression of genes involved in cell proliferation (pcna), telomere maintenance (tert) and multipotency (seawi and vasa) was maintained with age in somatic tissues. Immunolocalization of the Vasa protein to areas of the tube feet, spines, radial nerve, esophagus and a sub‐population of circulating coelomocytes suggests the presence of multipotent cells that may play a role in normal tissue homeostasis and the regenerative potential of external appendages. The results indicate that regenerative potential was maintained with age regardless of lifespan, contrary to the expectation that shorter lived species would invest less in maintenance and repair.

James A Coffman speaks to Adam Price-Evans, Commissioning Editor: James A Coffman is a scientist at the MDI Biological Laboratory in Bar Harbor, Maine, and Director of the Maine INBRE, a NIH-sponsored program to promote biomedical research. He holds a doctorate in zoology from Duke University and a bachelor's in biology from Carleton College. He is currently studying the mechanisms by which chronic early-life stress increases adult disease risk, using zebrafish as a model organism to ask how such stress affects immune system development and regulation.

Chronic stress undermines physical and mental health, in part via dysregulation of the neuroendocrine stress system. Key to understand this dysregulation is recognizing that the problem is not stress per se, but rather its chronicity. The optimally functioning stress system is highly dynamic, and negative feedback regulation enforces transient responses to acute stressors. Chronic stress overrides this, and adaptation to the chronicity can result in persistent dysregulation by altering sensitivity thresholds critical for control of system dynamics. Such adaptation involves plasticity within the central nervous system (CNS) as well as epigenetic regulation. When it occurs during development, it can have persistent effects on neuroendocrine regulation. Understanding how chronic stress programs development of the neuroendocrine stress system requires elucidation of stress-responsive gene regulatory networks that control CNS plasticity and development.

It is proposed that the evolution of complex animals required repressive genetic mechanisms for controlling the transcriptional and proliferative potency of cells. Unicellular organisms are transcriptionally potent, able to express their full genetic complement as the need arises through their life cycle, whereas differentiated cells of multicellular organisms can only express a fraction of their genomic potential. Likewise, whereas cell proliferation in unicellular organisms is primarily limited by nutrient availability, cell proliferation in multicellular organisms is developmentally regulated. Repressive genetic controls limiting the potency of cells at the end of ontogeny would have stabilized the gene expression states of differentiated cells and prevented disruptive proliferation, allowing the emergence of diverse cell types and functional shapes. We propose that distal cis-regulatory elements represent the primary innovations that set the stage for the evolution of developmental gene regulatory networks and the repressive control of key multipotency and cell-cycle control genes. The testable prediction of this model is that the genomes of extant animals, unlike those of our unicellular relatives, encode gene regulatory circuits dedicated to the developmental control of transcriptional and proliferative potency.

The oral–aboral axis of the sea urchin embryo is specified conditionally via a regulated feedback circuit involving the signaling gene nodal and its antagonist lefty. In normal development nodal activity becomes localized to the prospective oral side of the blastula stage embryo, a process that requires lefty. In embryos of Strongylocentrotus purpuratus, a redox gradient established by asymmetrically distributed mitochondria provides an initial spatial input that positions the localized domain of nodal expression. This expression is perturbed by hypoxia, leading to development of radialized embryos lacking an oral–aboral axis. Here we show that this radialization is not caused by a failure to express nodal, but rather by a failure to localize nodal activity to one side of the embryo. This occurs even when embryos are removed from hypoxia at late cleavage stage when nodal is first expressed, indicating that the effect involves the initiation phase of nodal activity, rather than its positive feedback-driven amplification and maintenance. Quantitative fluorescence microscopy of MitoTracker Orange-labeled embryos expressing nodal-GFP reporter gene revealed that hypoxia abolishes the spatial correlation between mitochondrial distribution and nodal expression, suggesting that hypoxia eliminates the initial spatial bias in nodal activity normally established by the redox gradient. We propose that absent this bias, the initiation phase of nodal expression is spatially uniform, such that the ensuing Nodal-mediated community effect is not localized, and hence refractory to Lefty-mediated enforcement of localization.

We describe a real-time (rt) PCR-based method of quantifying DNA damage, adapted from the long-run rtPCR method of DNA damage quantification (LORD-Q) developed by Lehle et al. (Nucleic Acids Res 42(6):e41, 2014). We show that semi-long run rtPCR, which generates amplicons half the length of those generated in LORD-Q, provides equivalent sensitivity for detecting low lesion frequencies, and better sensitivity for detecting high frequencies. The smaller amplicon size greatly facilitates PCR optimization and allows greater flexibility in the use of detection dyes, and a modified data analysis method simplifies the calculation of lesion frequency. The method was used to measure DNA damage in the nuclear and mitochondrial genomes of different tissues in zebrafish of different ages. We find that nuclear DNA damage generally increases with age, and that the amount of mitochondrial DNA damage varies substantially between tissues, increasing with age in liver and brain but not in heart or skeletal muscle, the latter having the highest levels of damage irrespective of age. Electronic supplementary material The online version of this article (doi:10.1186/s13104-017-2593-x) contains supplementary material, which is available to authorized users.

Summary In animal development following the initial cleavage stage of embryogenesis, the cell cycle becomes dependent on intercellular signaling and controlled by the genomically encoded ontogenetic program. Runx transcription factors are critical regulators of metazoan developmental signaling, and we have shown that the sea urchin Runx gene runt-1, which is globally expressed during early embryogenesis, functions in support of blastula stage cell proliferation and expression of the mitogenic genes pkc1, cyclinD, and several wnts. To obtain a more comprehensive list of early runt-1 regulatory targets, we screened a Strongylocentrotus purpuratus microarray to identify genes mis-expressed in mid-blastula stage runt-1 morphants. This analysis showed that loss of Runx function perturbs the expression of multiple genes involved in cell division, including the pro-growth and survival kinase Akt (PKB), which is significantly underexpressed in runt-1 morphants. Further genomic analysis revealed that Akt is encoded by two genes in the S. purpuratus genome, akt-1 and akt-2, both of which contain numerous canonical Runx target sequences. The transcripts of both genes accumulate several fold during blastula stage, contingent on runt-1 expression. Inhibiting Akt expression or activity causes blastula stage cell cycle arrest, whereas overexpression of akt-1 mRNA rescues cell proliferation in runt-1 morphants. These results indicate that post-cleavage stage cell division requires Runx-dependent expression of akt.

The Symposium on the Comparative Biology of Tissue Repair, Regeneration and Aging, held 26 June to 28 June 2015 at the MDI Biological Laboratory in Salisbury Cove, Maine, brought together a diverse group of biologists with a common interest in understanding why regenerative capacity varies among animal species, why it is progressively lost in senescence, and how answers obtained from studies that address those questions might be applied in regenerative medicine.

Nodal proteins are diffusible morphogens that drive pattern formation via short-range feedback activation coupled to long-range Lefty-mediated inhibition. In the sea urchin embryo, specification of the secondary (oral-aboral) axis occurs via zygotic expression of nodal, which is localized to the prospective oral ectoderm at early blastula stage. In mid-blastula stage embryos treated with low micromolar nickel or zinc, nodal expression expands progressively beyond the confines of this localized domain to encompass the entire equatorial circumference of the embryo, producing radialized embryos lacking an oral-aboral axis. RNAseq analysis of embryos treated with nickel, zinc, or cadmium (which does not radialize embryos) showed that several genes involved in endocytosis were similarly perturbed by nickel and zinc but not cadmium. Inhibiting dynamin, a GTPase required for receptor-mediated endocytosis, phenocopies the effects of nickel and zinc, suggesting that dynamin-mediated endocytosis is required as a sink to limit the range of Nodal signaling. Developmental Dynamics 240:704–711, 2011. © 2011 Wiley-Liss, Inc.

In sea urchin embryos, specification of the secondary (oral–aboral) axis occurs via nodal, expression of which is entirely zygotic and localized to prospective oral ectoderm at blastula stage. The initial source of this spatial anisotropy is not known. Previous studies have shown that oral–aboral (OA) polarity correlates with a mitochondrial gradient, and that nodal activity is dependent both on mitochondrial respiration and p38 stress-activated protein kinase. Here we show that the spatial pattern of nodal activity also correlates with the mitochondrial gradient, and that the latter correlates with inhomogeneous levels of intracellular reactive oxygen species. To test whether mitochondrial H2O2 functions as a redox signal to activate nodal, zygotes were injected with mRNA encoding either mitochondrially-targeted catalase, which quenches mitochondrial H2O2 and down-regulates p38, or superoxide dismutase, which augments mitochondrial H2O2 and up-regulates p38. Whereas the former treatment inhibits the initial activation of nodal and entrains OA polarity toward aboral when confined to half of the embryo via 2-cell stage blastomere injections, the latter does not produce the opposite effects. We conclude that mitochondrial H2O2 is rate-limiting for the initial activation of nodal, but that additional rate-limiting factors, likely also involving mitochondria, contribute to the asymmetry in nodal expression.

The Runt domain (Runx) is a 128 amino acid sequence motif that defines a metazoan family of sequence-specific DNA binding proteins, which appears to have originated in concert with the intercellular signaling systems that coordinate multicellular development in animals. In the model organisms where they have been studied (fruit fly, mouse, sea urchin, and nematode) Runx genes are essential for normal development, and in humans they are causally associated with a variety of cancers, manifesting both oncogenic and tumor suppressive attributes. During development Runx proteins support both cell proliferation and differentiation, and function in both transcriptional activation and repression. Runx function is thus context-dependent, with the context provided genetically by cis-regulatory sequence architecture and epigenetically by development. This context dependency makes it difficult to formulate reductionistic generalizations concerning Runx function in normal and carcinogenic development. However, a growing body of literature links Runx function to each of the major intercellular signaling systems in animals, suggesting that the general function of Runx transcription factors may be to potentiate and govern genomic responsiveness to developmental signaling.

Planktotrophic sea urchin larvae are developmentally plastic: in response to food scarcity, development of the juvenile rudiment is suspended and larvae instead develop elongated arms, thus increasing feeding capacity and extending larval life. Here, data are presented on the effect of different feeding regimes on gene expression in larvae of the green sea urchin Strongylocentrotus droebachiensis. These data indicate that during periods of starvation, larvae down-regulate genes involved in growth and metabolic activity while up-regulating genes involved in lipid transport, environmental sensing, and defense. Additionally, we show that starvation increases FoxO activity and that in well-fed larvae rapamycin treatment impedes rudiment growth, indicating that the latter requires TOR activity. These results suggest that the developmental plasticity of echinoplutei is regulated by genes known to control aging and longevity in other animals.

Chance has somewhat different meanings in different contexts, and can be taken to be either ontological (as in quantum indeterminacy) or epistemological (as in stochastic uncertainty). Here I argue that, whether or not it stems from physical indeterminacy, chance is a fundamental biological reality that is meaningless outside the context of knowledge. To say that something happened by chance means that it did not happen by design. This of course is a cornerstone of Darwin's theory of evolution: random undirected variation is the creative wellspring upon which natural selection acts to sculpt the functional form (and hence apparent design) of organisms. In his essay Chance & Necessity, Jacques Monod argued that an intellectually honest commitment to objectivity requires that we accord chance a central role in an otherwise mechanistic biology, and suggested that doing so may well place the origin of life outside the realm of scientific tractability. While that may be true, ongoing research on the origin of life problem suggests that abiogenesis may have been possible, and perhaps even probable, under the conditions that existed on primordial earth. Following others, I argue that the world should be viewed as causally open, i.e. primordially indeterminate or vague. Accordingly, chance ought to be the default scientific explanation for origination, a universal 'null hypothesis' to be assumed until disproven. In this framework, creation of anything new manifests freedom (allowing for chance), and causation manifests constraint, the developmental emergence of which establishes the space of possibilities that may by chance be realized.

This paper reports a preliminary in silico analysis of the sea urchin kinome. The predicted protein kinases in the sea urchin genome were identified, annotated and classified, according to both function and kinase domain taxonomy. The results show that the sea urchin kinome, consisting of 353 protein kinases, is closer to the Drosophila kinome (239) than the human kinome (518) with respect to total kinase number. However, the diversity of sea urchin kinases is surprisingly similar to humans, since the urchin kinome is missing only 4 of 186 human subfamilies, while Drosophila lacks 24. Thus, the sea urchin kinome combines the simplicity of a non-duplicated genome with the diversity of function and signaling previously considered to be vertebrate-specific. More than half of the sea urchin kinases are involved with signal transduction, and approximately 88% of the signaling kinases are expressed in the developing embryo. These results support the strength of this nonchordate deuterostome as a pivotal developmental and evolutionary model organism.

International audience; A search of the Strongylocentrotus purpuratus genome for genes associated with cell cycle control and DNA metabolism shows that the known repertoire of these genes is conserved in the sea urchin, although with fewer family members represented than in vertebrates, and with some cases of echinoderm-specific gene diversifications. For example, while homologues of the known cyclins are mostly encoded by single genes in S. purpuratus (unlike vertebrates, which have multiple isoforms), there are additional genes encoding novel cyclins of the B and K/L types. Almost all known cyclin-dependent kinases (CDKs) or CDK-like proteins have an orthologue in S. purpuratus; CDK3 is one exception, whereas CDK4 and 6 are represented by a single homologue, referred to as CDK4. While the complexity of the two families of mitotic kinases, Polo and Aurora, is close to that found in the nematode, the diversity of the NIMA-related kinases (NEK proteins) approaches that of vertebrates. Among the nine NEK proteins found in S. purpuratus, eight could be assigned orthologues in vertebrates, whereas the ninth is unique to sea urchins. Most known DNA replication, DNA repair and mitotic checkpoint genes are also present, as are homologues of the pRB (two) and p53 (one) tumor suppressors. Interestingly, the p21/p27 family of CDK inhibitors is represented by one homologue, whereas the INK4 and ARF families of tumor suppressors appear to be absent, suggesting that these evolved only in vertebrates. Our results suggest that, while the cell cycle control mechanisms known from other animals are generally conserved in sea urchin, parts of the machinery have diversified within the echinoderm lineage. The set of genes uncovered in this analysis of the S. purpuratus genome should enhance future research on cell cycle control and developmental regulation in this model.

Development is a process whereby a relatively unspecified system comprised of loosely connected lower level parts becomes organized into a coherent, higher-level agency. Its temporal corollaries are growth, increasingly deterministic behavior, and a progressive reduction of developmental potential. During immature stages with relatively low specification and high potential, development is largely controlled by local interactions from the “bottom-up,” whereas during more highly specified stages with reduced potential, emergent autocatalytic processes exert “top-down” control. Robert Ulanowicz has shown that this phenomenology of ascendency follows thermodynamic principles and can be described quantitatively using information theory, providing a general theory of development. However, the theory has not found a wide audience among developmental biologists, as genetic determinism encourages the popular reductionistic perception that ontogeny is controlled entirely by molecular mechanisms that exert efficient causality from the bottom-up. Nonetheless, measurements of metabolic rates and mRNA complexity in developing embryos, as well as functional analyses of gene regulatory systems, indicate that ontogeny fits the paradigm of developmental ascendency. Beyond informing biomedical research and the interpretation of large datasets obtained by systems-biological approaches, developmental ascendency helps explain the origin of life, and, being independent of scale, provides an overarching explanation for phylogenetic change that contextualizes Darwinian evolution.

Perhaps no piece of office equipment is more taken for granted than the common business telephone. The technology behind this basic communication device, however, is in the midst of a major transformation. Businesses are now converging their voice and data networks in order to simplify their network operations and take advantage of the new functional benefits and capabilities that a converged network delivers from greater productivity and cost savings to enhanced mobility.

The initial asymmetry that specifies the oral–aboral (OA) axis of the sea urchin embryo has long been a mystery. It was shown previously that OA polarity can be entrained in embryos by imposing a respiratory asymmetry, with the most oxidizing side of the embryo tending to develop as the oral pole. This suggests that one of the earliest observable asymmetries along the incipient OA axis, a redox gradient established by a higher density and/or activity of mitochondria on the prospective oral side of the embryo, might play a causal role in establishing the axis. Here, we examine the origin and functional significance of this early redox gradient. Using MitoTracker Green, we show that mitochondria are asymmetrically distributed in the unfertilized egg of Strongylocentrotus purpuratus, and that the polarity of the maternal asymmetry is maintained in the zygote. Vital staining indicates that the side of the embryo that inherits the highest density of mitochondria tends to develop into the oral pole. This correlation holds when mitochondria are redistributed by centrifugation of eggs or by transfer of purified mitochondria into zygotes, indicating that an asymmetric mitochondrial distribution can entrain OA polarity, possibly through effects on intracellular redox state. In support of this possibility, we find that specification of oral ectoderm is suppressed when embryos are cultured under hypoxic conditions that enforce a relatively reducing redox state. This effect is reversed by overexpression of nodal, an early zygotic marker of oral specification whose localized expression suffices to organize the entire OA axis, indicating that redox state is upstream of nodal expression. We therefore propose that a threshold level of intracellular oxidation is required to effectively activate nodal, and that precocious attainment of this threshold within the blastomeres containing the highest density of mitochondria results in asymmetric nodal activity and consequent specification of the OA axis.

The runt box (runx) is a highly conserved DNA binding and protein-protein interaction domain that defines a family of heterodimeric transcription factors that regulate development in metazoans. The three mammalian runx genes are oncogenes with essential functions in normal development: Runx1 is required for hematopoiesis and is frequently mutated in human and murine leukemias; Runx2 is required for bone development and is associated with human cleidocranial dysplasia and murine leukemias; and Runx3 (the evolutionarily basal member of the mammalian family) regulates growth of the gut and functions as a tumor suppressor in the gastric epithelium (Westendorf and Hiebert, 1999; Li et al., 2002). The sea urchin Strongylocentrotus purpuratus contains a single runx gene, SpRunt. We present here the initial structural characterization of SpRunt, and its pattern of expression during embryogenesis. SpRunt contains two introns, the locations of which are identical to those of the second and third introns from promoter P2 of the mammalian runx genes. A approximately 6 kb transcript begins to accumulate during cleavage. At mesenchyme blastula stage, SpRunt transcripts are found throughout the embryo, but specifically enriched in the vegetal plate, skeletogenic mesenchyme, and part of the ectoderm. By late gastrula stage expression is localized to the endomesoderm and oral ectoderm. In the pluteus larva SpRunt transcripts remain confined to the endomesoderm and oral ectoderm, with highest levels of accumulation in the foregut and in the ciliary band. These data suggest that SpRunt expression is enhanced in proliferating cells.

Veterinary medical education is undergoing rapid change in terms of pedagogy, the demographics of the student body, and, in turn, the membership of the profession. Central to the value of the traditional curriculum and the total student experience is the small-group environment, both in client service and in clinical rounds. It is one of the few Socratic learning experiences in higher education today. Similarly, experience in private practice is of inestimable value in terms of developing people skills and a lasting sense of service and accountability. In a generation, the student body has transformed from vanishingly small numbers of women to a predominance of female students. However, the profession still is very white, in a world becoming more and more diverse. With a predominantly white faculty and student body today, this circumstance shows little promise of rapid enough change to maintain relevance to a workforce that, a generation from now, likely will be dominated numerically by people of color. The incorporation of various worldviews and the impact of stereotyping on performance are central to issues of success and failure of minorities and, in somewhat different ways, women in the veterinary medical profession. These issues must become better understood and addressed. And to accomplish this, and to address a host of other culturally important issues, a greater diversity of worldviews must be engaged in the work and planning of veterinary medical education and the profession at large. Addressing these issues in an environment in which the values of faculty and administrators are intensely focused on the science of veterinary medicine, and in which the participants hold dear a system that places value only on teaching, research, and clinical service, is a formidable undertaking and will require substantial reconsideration of faculty role and reward systems.

In embryos of indirectly developing echinoids, the secondary (oral–aboral) larval axis is established after fertilization by an as yet undiscovered process. One of the earliest manifestations of this axis is an asymmetry in mitochondrial respiration, with the prospective oral side of the embryo exhibiting a higher rate of respiration than the prospective aboral side. We show here that respiratory asymmetry can be experimentally induced within embryos by immobilizing them in tight clusters of four (“rosettes”). Within such clusters a redox gradient is established from the inside to the outside of the rosette. Vital staining of clustered embryos demonstrates that the side of the embryo facing the outside of the rosette (i.e., the most oxidizing) tends to become the oral side, while the side facing the inside tends to become the aboral side. Effective entrainment of the oral–aboral axis requires that the embryos remain immobilized in rosettes until the hatching blastula stage. To begin to investigate the molecular mechanisms underlying this effect we made use of P3A2, a transcriptional regulatory protein whose activity is spatially modulated along the oral–aboral axis. When synthetic mRNA encoding P3A2 fused to the VP16 activation domain is injected into eggs, it activates embryonic expression of a green fluorescent protein reporter gene containing a basal promoter and a single strong P3A2 target site. In embryo rosettes, such activation occurs predominantly on the outside of the rosette, suggesting that the activity of the P3A2 protein is spatially regulated by the respiratory asymmetry established by clustering the embryos. These findings are discussed with reference to earlier work on both oral–aboral axis specification and P3A2 and used to develop a testable model of the mechanism of oral–aboral axis specification in the sea urchin embryo.

The Cyllla actin gene of Strongylocentrotus purpuratus is transcribed exclusively in the embryonic aboral ectoderm, under the control of 2.3 kb cis-regulatory domain that contains a proximal module that controls expression in early embryogenesis, and a middle module that controls expression in later embryogenesis. Previous studies demonstrated that the SpRunt-1 target site within the middle module is required for the sharp increase in Cyllla transcription which accompanies differentiation of the aboral ectoderm, and that a negative regulatory region near the SpRunt-1 target site is required to prevent ectopic transcription in the oral ectoderm and skeletogenic mesenchyme. This negative regulatory region contains a consensus binding site for the myb family of transcription factors. In vitro DNA-binding experiments reveal that a protein in blastula-stage nuclei interacts specifically with the myb target site. Gene transfer experiments utilizing Cyllla reporter constructs containing oligonucleotide substitutions indicate that this site is both necessary and sufficient to prevent ectopic expression of Cyllla. Synthetic oligonucleotides containing the myb target site were used to purify a protein from sea urchin embryo nuclear extracts by affinity chromatography. This protein is immunoprecipitated by antibodies specific to the evolutionarily conserved myb domain, and amino acid sequences obtained from the purified protein were found to be identical to sequences within the myb domain. Sequence information was used to obtain cDNA clones of SpMyb, the S. purpura-tus member of the myb family of transcription factors. Through interactions within the middle module, SpMyb functions to repress activation of Cyllla in the oral ectoderm and skeletogenic mesenchyme.

Cyclin D genes regulate the cell cycle, growth and differentiation in response to intercellular signaling. While the promoters of vertebrate cyclin D genes have been analyzed, the cis-regulatory sequences across an entire cyclin D locus have not. Doing so would increase understanding of how cyclin D genes respond to the regulatory states established by developmental gene regulatory networks, linking cell cycle and growth control to the ontogenetic program. Therefore, we conducted a cis-regulatory analysis on the cyclin D gene, SpcycD, of the sea urchin, Strongylocentrotus purpuratus, during embryogenesis, identifying upstream and intronic sequences, located within six defined regions bearing one or more cis-regulatory modules each.

The role of a 150-kD SR-cyclophilin (NK-TR1) in monocyte differentiation was investigated. Using an antipeptide monoclonal antibody, we have detected NK-TR1 in human peripheral blood monocytes and HL-60 cells. Unstimulated monocytes showed a low intracellular level of NK-TR1 protein that increased over 3 days of lipopolysaccharide + interferon-gamma treatment, consistent with the kinetics of monocyte differentiation. Normal HL-60 cells also had a low level of NK-TR1 protein, and exposure to 1.25% dimethyl sulfoxide (DMSO) resulted in a marked transient increase in expression that returned to basal levels before the development of granulocyte differentiation-associated biochemical changes. Phorbol myristate acetate, a promoter of monocytic differentiation in HL-60 cells, also caused a significant increase in NK-TR1 over basal levels. Transfection of a vector expressing NK-TR1 antisense RNA into HL-60 cells suppressed DMSO-mediated growth arrest. In addition, the development of a more mature phenotype, as measured by expression of CD16, and the ability to reduce nitroblue tetrazoleum dye was inhibited in transfectants when compared with controls. These results are consistent with the hypothesis that the NK-TR1 gene product is required for the progression towards a mature differentiated phenotype.

In this paper we present a structural and functional characterization of a new sea urchin embryo transcription factor, SpRunt-1. This factor was isolated by means of its specific interaction with a cis-regulatory target site of the CyIIIa gene. Here we show that this target site, the P7I site, is required for normal embryonic activation of CyIIIa x CAT reporter gene constructs. An oligonucleotide affinity column bearing the P7I target site purifies a 21-kDa polypeptide from blastula-stage nuclear extracts, and the amino acid sequence obtained from this polypeptide was used to generate a nucleic acid probe with which the corresponding cDNA was cloned. The cDNA encodes an approximately 60-kDa protein, SpRunt-1, which includes a "runt domain" that is closely homologous to those of Drosophila and mammalian runt domain transcription factors. RNA and genomic blots show that SpRunt-1 is represented by a single embryonic transcript, encoded by one of possibly two runt-domain-containing genes. By RNA probe protection we found that transcripts of SpRunt-1 increase in concentration dramatically after the blastula stage of development, suggesting that the up-regulation of CyIIIa that occurs after blastula stage is a function of zygotically transcribed SpRunt-1. These results are discussed with reference to known features of the runt domain family of transcription factors.

In the undisturbed sea urchin embryo, cleavage of the blastomeres is invariant and gives rise to five polyclonal territories that are each defined by the larval structures to which they give rise, and by unique programmes of gene expression. These territories are the aboral ectoderm, the oral ectoderm, the vegetal plate, the skeletogenic mesenchyme, and the small micromeres. Structural gene markers for four of these territories (all except the small micromeres, which participate in later development) have been cloned and characterized, and the regulatory domains of several of these genes have been mapped to the level of specific protein-binding sites.

Publisher Summary This chapter describes developmentalism that views all non-random causality as a product of development at some level. Developmentalism helps resolve a number of long-standing dialectics concerned with causality, including reductionism/holism, orthogenesis/adaptation, and stasis/ change. In biological sciences, developmentalism engenders a discourse that overcomes barriers imposed by the still-dominant paradigms of molecular reductionism on the one hand and Darwinian evolution on the other. With regard to the former, it provides a better interpretive framework for the new science of “systems-biology,” which seeks to elucidate regulatory networks that control ontogeny, stem cell biology, and the etiology of disease. With regard to the latter, it provides an intelligible bridge between chemistry and biology, and hence an explanation for the natural origin of life. Finally, developmentalism, being an inherently ecological perspective, is well suited as a paradigm for addressing problems of environmental management and sustainability.