Loss of Krüppel-like factor 9 deregulates both physiological gene expression and development.

Krüppel-like factor 9 (Klf9) is a ubiquitously expressed transcription factor that is a feedforward regulator of multiple stress-responsive and endocrine signaling pathways. We previously described how loss of Klf9 function affects the transcriptome of zebrafish larvae sampled at a single time point 5 days post-fertilization (dpf). However, klf9 expression oscillates diurnally, and the sampled time point corresponded to its expression nadir. To determine if the transcriptomic effects of the klf9-/- mutation vary with time of day, we performed bulk RNA-seq on 5 dpf zebrafish embryos sampled at three timepoints encompassing the predawn peak and midmorning nadir of klf9 expression. We found that while the major effects of the klf9-/- mutation that we reported previously are robust to time of day, the mutation has additional effects that manifest only at the predawn time point. We used a published single-cell atlas of zebrafish development to associate the effects of the klf9-/- mutation with different cell types and found that the mutation increased mRNA associated with digestive organs (liver, pancreas, and intestine) and decreased mRNA associated with differentiating neurons and blood. Measurements from confocally-imaged larvae suggest that overrepresentation of liver mRNA in klf9-/- mutants is due to development of enlarged livers.

Glucocorticoid-Responsive Transcription Factor Krüppel-Like Factor 9 Regulates fkbp5 and Metabolism.

Krüppel-like factor 9 (Klf9) is a feedforward regulator of glucocorticoid receptor (GR) signaling. Here we show that in zebrafish klf9 is expressed with GR-dependent oscillatory dynamics in synchrony with fkbp5, a GR target that encodes a negative feedback regulator of GR signaling. We found that fkbp5 transcript levels are elevated in klf9 -/- mutants and that Klf9 associates with chromatin at the fkbp5 promoter, which becomes hyperacetylated in klf9 -/ - mutants, suggesting that the GR regulates fkbp5 via an incoherent feedforward loop with klf9. As both the GR and Fkbp5 are known to regulate metabolism, we asked how loss of Klf9 affects metabolic rate and gene expression. We found that klf9 -/- mutants have a decreased oxygen consumption rate (OCR) and upregulate glycolytic genes, the promoter regions of which are enriched for potential Klf9 binding motifs. Our results suggest that Klf9 functions downstream of the GR to regulate cellular glucocorticoid responsivity and metabolic homeostasis.

Glucocorticoid-Mediated Developmental Programming of Vertebrate Stress Responsivity.

Glucocorticoids, vertebrate steroid hormones produced by cells of the adrenal cortex or interrenal tissue, function dynamically to maintain homeostasis under constantly changing and occasionally stressful environmental conditions. They do so by binding and thereby activating nuclear receptor transcription factors, the Glucocorticoid and Mineralocorticoid Receptors (MR and GR, respectively). The GR, by virtue of its lower affinity for endogenous glucocorticoids (cortisol or corticosterone), is primarily responsible for transducing the dynamic signals conveyed by circadian and ultradian glucocorticoid oscillations as well as transient pulses produced in response to acute stress. These dynamics are important determinants of stress responsivity, and at the systemic level are produced by feedforward and feedback signaling along the hypothalamus-pituitary-adrenal/interrenal axis. Within receiving cells, GR signaling dynamics are controlled by the GR target gene and negative feedback regulator fkpb5. Chronic stress can alter signaling dynamics via imperfect physiological adaptation that changes systemic and/or cellular set points, resulting in chronically elevated cortisol levels and increased allostatic load, which undermines health and promotes development of disease. When this occurs during early development it can "program" the responsivity of the stress system, with persistent effects on allostatic load and disease susceptibility. An important question concerns the glucocorticoid-responsive gene regulatory network that contributes to such programming. Recent studies show that klf9, a ubiquitously expressed GR target gene that encodes a Krüppel-like transcription factor important for metabolic plasticity and neuronal differentiation, is a feedforward regulator of GR signaling impacting cellular glucocorticoid responsivity, suggesting that it may be a critical node in that regulatory network.

Chronic cortisol exposure in early development leads to neuroendocrine dysregulation in adulthood.

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.

Klf9 is a key feedforward regulator of the transcriptomic response to glucocorticoid receptor activity.

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.

Redox regulation of development and regeneration.

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.

Simple and fast quantification of DNA damage by real-time PCR, and its application to nuclear and mitochondrial DNA from multiple tissues of aging zebrafish.

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.

Cortisol-treated zebrafish embryos develop into pro-inflammatory adults with aberrant immune gene regulation.

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.

An Elk transcription factor is required for Runx-dependent survival signaling in the sea urchin embryo.

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.

Maintenance of somatic tissue regeneration with age in short- and long-lived species of sea urchins.

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.

Gene Expression Changes Associated With the Developmental Plasticity of Sea Urchin Larvae in Response to Food Availability.

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.

Developmental control of transcriptional and proliferative potency during the evolutionary emergence of animals.

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.

Oral-aboral axis specification in the sea urchin embryo, IV: hypoxia radializes embryos by preventing the initial spatialization of nodal activity.

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.

On the meaning of chance in biology.

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.

Developmental cis-regulatory analysis of the cyclin D gene in the sea urchin Strongylocentrotus purpuratus.

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.

Sea urchin akt activity is Runx-dependent and required for post-cleavage stage cell division.

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.

Nodal-mediated epigenesis requires dynamin-mediated endocytosis.

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.