The germline coordinates mitokine signaling.

The ability of mitochondria to coordinate stress responses across tissues is critical for health. In C. elegans, neurons experiencing mitochondrial stress elicit an inter-tissue signaling pathway through the release of mitokine signals, such as serotonin or the Wnt ligand EGL-20, which activate the mitochondrial unfolded protein response (UPRMT) in the periphery to promote organismal health and lifespan. We find that germline mitochondria play a surprising role in neuron-to-periphery UPRMT signaling. Specifically, we find that germline mitochondria signal downstream of neuronal mitokines, Wnt and serotonin, and upstream of lipid metabolic pathways in the periphery to regulate UPRMT activation. We also find that the germline tissue itself is essential for UPRMT signaling. We propose that the germline has a central signaling role in coordinating mitochondrial stress responses across tissues, and germline mitochondria play a defining role in this coordination because of their inherent roles in germline integrity and inter-tissue signaling.

Cell non-autonomous control of autophagy and metabolism by glial cells.

Glia are the protectors of the nervous system, providing neurons with support and protection from cytotoxic insults. We previously discovered that four astrocyte-like glia can regulate organismal proteostasis and longevity in C. elegans. Expression of the UPRER transcription factor, XBP-1s, in these glia increases stress resistance, and longevity, and activates the UPRER in intestinal cells via neuropeptides. Autophagy, a key regulator of metabolism and aging, has been described as a cell autonomous process. Surprisingly, we find that glial XBP-1s enhances proteostasis and longevity by cell non-autonomously reprogramming organismal lipid metabolism and activating autophagy. Glial XBP-1s regulates the activation of another transcription factor, HLH-30/TFEB, in the intestine. HLH-30 activates intestinal autophagy, increases intestinal lipid catabolism, and upregulates a robust transcriptional program. Our study reveals a novel role for glia in regulating peripheral lipid metabolism, autophagy, and organellar health through peripheral activation of HLH-30 and autophagy.

The germline coordinates mitokine signaling.

The ability of mitochondria to coordinate stress responses across tissues is critical for health. In C. elegans , neurons experiencing mitochondrial stress elicit an inter-tissue signaling pathway through the release of mitokine signals, such as serotonin or the WNT ligand EGL-20, which activate the mitochondrial unfolded protein response (UPR MT ) in the periphery to promote organismal health and lifespan. We find that germline mitochondria play a surprising role in neuron-to-peripheral UPR MT signaling. Specifically, we find that germline mitochondria signal downstream of neuronal mitokines, like WNT and serotonin, and upstream of lipid metabolic pathways in the periphery to regulate UPR MT activation. We also find that the germline tissue itself is essential in UPR MT signaling. We propose that the germline has a central signaling role in coordinating mitochondrial stress responses across tissues, and germline mitochondria play a defining role in this coordination because of their inherent roles in germline integrity and inter-tissue signaling.

Widespread priming of transcriptional regulatory elements by incipient accessibility or RNA polymerase II pause in early embryos of the sea urchin Strongylocentrotus purpuratus.

Transcriptional regulatory elements (TREs) are the primary nodes that control developmental gene regulatory networks. In embryo stages, larvae, and adult differentiated red spherule cells of the sea urchin Strongylocentrotus purpuratus, transcriptionally engaged TREs are detected by Precision Run-On Sequencing (PRO-seq), which maps genome-wide at base pair resolution the location of paused or elongating RNA polymerase II (Pol II). In parallel, TRE accessibility is estimated by the Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-seq). Our analysis identifies surprisingly early and widespread TRE accessibility in 4-cell cleavage embryos that is not necessarily followed by concurrent or subsequent transcription. TRE transcriptional differences identified by PRO-seq provide more contrast among embryonic stages than ATAC-seq accessibility differences, in agreement with the apparent excess of accessible but inactive TREs during embryogenesis. Global TRE accessibility reaches a maximum around the 20-hour late blastula stage, which coincides with the consolidation of major embryo regionalizations and peak histone variant H2A.Z expression. A transcriptional potency model based on labile nucleosome TRE occupancy driven by DNA sequences and the prevalence of histone variants is proposed in order to explain the basal accessibility of transcriptionally inactive TREs during embryogenesis. However, our results would not reconcile well with labile nucleosome models based on simple A/T sequence enrichment. In addition, a large number of distal TREs become transcriptionally disengaged during developmental progression, in support of an early Pol II paused model for developmental gene regulation that eventually resolves in transcriptional activation or silencing. Thus, developmental potency in early embryos may be facilitated by incipient accessibility and transcriptional pause at TREs.

Evolution of promoter-proximal pausing enabled a new layer of transcription control.

Promoter-proximal pausing of RNA polymerase II (Pol II) is a key regulatory step during transcription. Despite the central role of pausing in gene regulation, we do not understand the evolutionary processes that led to the emergence of Pol II pausing or its transition to a rate-limiting step actively controlled by transcription factors. Here we analyzed transcription in species across the tree of life. We found that unicellular eukaryotes display a slow acceleration of Pol II near transcription start sites. This proto-paused-like state transitioned to a longer, focused pause in derived metazoans which coincided with the evolution of new subunits in the NELF and 7SK complexes. Depletion of NELF reverts the mammalian focal pause to a proto-pause-like state and compromises transcriptional activation for a set of heat shock genes. Collectively, this work details the evolutionary history of Pol II pausing and sheds light on how new transcriptional regulatory mechanisms evolve.

Identification and prediction of developmental enhancers in sea urchin embryos.

BACKGROUND: The transcription of developmental regulatory genes is often controlled by multiple cis-regulatory elements. The identification and functional characterization of distal regulatory elements remains challenging, even in tractable model organisms like sea urchins. RESULTS: We evaluate the use of chromatin accessibility, transcription and RNA Polymerase II for their ability to predict enhancer activity of genomic regions in sea urchin embryos. ATAC-seq, PRO-seq, and Pol II ChIP-seq from early and late blastula embryos are manually contrasted with experimental cis-regulatory analyses available in sea urchin embryos, with particular attention to common developmental regulatory elements known to have enhancer and silencer functions differentially deployed among embryonic territories. Using the three functional genomic data types, machine learning models are trained and tested to classify and quantitatively predict the enhancer activity of several hundred genomic regions previously validated with reporter constructs in vivo. CONCLUSIONS: Overall, chromatin accessibility and transcription have substantial power for predicting enhancer activity. For promoter-overlapping cis-regulatory elements in particular, the distribution of Pol II is the best predictor of enhancer activity in blastula embryos. Furthermore, ATAC- and PRO-seq predictive value is stage dependent for the promoter-overlapping subset. This suggests that the sequence of regulatory mechanisms leading to transcriptional activation have distinct relevance at different levels of the developmental gene regulatory hierarchy deployed during embryogenesis.

Electron microscopic characterization of nuclear egress in the sea urchin gastrula.

Nuclear egress, also referred to as nuclear envelope (NE) budding, is a process of transport in which vesicles containing molecular complexes or viral particles leave the nucleus through budding from the inner nuclear membrane (INM) to enter the perinuclear space. Following this event, the perinuclear vesicles (PNVs) fuse with the outer nuclear membrane (ONM), where they release their contents into the cytoplasm. Nuclear egress is thought to participate in many functions such as viral replication, cellular differentiation, and synaptic development. The molecular basis for nuclear egress is now beginning to be elucidated. Here, we observe in the sea urchin gastrula, using serial section transmission electron microscopy, strikingly abundant PNVs containing as yet unidentified granules that resemble the ribonucleoprotein complexes (RNPs) previously observed in similar types of PNVs. Some PNVs were observed in the process of fusion with the ONM where they appeared to release their contents into the cytoplasm. These vesicles were abundantly observed in all three presumptive germ layers. These findings indicate that nuclear egress is likely to be an important mechanism for nucleocytoplasmic transfer during sea urchin development. The sea urchin may be a useful model to characterize further and gain a better understanding of the process of nuclear egress.

The origins of developmental gene regulation.

The leap from simple unicellularity to complex multicellularity remains one of life's major enigmas. The origins of metazoan developmental gene regulatory mechanisms are sought by analyzing gene regulation in extant eumetazoans, sponges, and unicellular organisms. The main hypothesis of this manuscript is that, developmental enhancers evolved from unicellular inducible promoters that diversified the expression of regulatory genes during metazoan evolution. Promoters and enhancers are functionally similar; both can regulate the transcription of distal promoters and both direct local transcription. Additionally, enhancers have experimentally characterized structural features that reveal their origin from inducible promoters. The distal co-operative regulation among promoters identified in unicellular opisthokonts possibly represents the precursor of distal regulation of promoters by enhancers. During metazoan evolution, constitutive-type promoters of regulatory genes would have acquired novel receptivity to distal regulatory inputs from promoters of inducible genes that eventually specialized as enhancers. The novel regulatory interactions would have caused constitutively expressed genes controlling differential gene expression in unicellular organisms to become themselves differentially expressed. The consequence of the novel regulatory interactions was that regulatory pathways of unicellular organisms became interlaced and ultimately evolved into the intricate developmental gene regulatory networks (GRNs) of extant metazoans.

Transcriptional and post-transcriptional regulation of histone variant H2A.Z during sea urchin development.

Histone variant H2A.Z promotes chromatin accessibility at transcriptional regulatory elements and is developmentally regulated in metazoans. We characterize the transcriptional and post-transcriptional regulation of H2A.Z in the purple sea urchin Strongylocentrotus purpuratus. H2A.Z depletion by antisense translation-blocking morpholino oligonucleotides during early development causes developmental collapse, in agreement with its previously demonstrated general role in transcriptional multipotency. During H2A.Z peak expression in 24-h embryos, endogenous H2A.Z 3' UTR sequences stabilize GFP mRNAs relative to those with SV40 3' UTR sequences, although the 3' UTR of H2A.Z does not determine the spatial distribution of H2A.Z transcripts during embryonic and postembryonic development. We elaborated an H2A.Z::GFP BAC reporter that reproduces embryonic H2A.Z expression. Genome-wide chromatin accessibility analysis using ATAC-seq revealed a cis-regulatory module (CRM) that, when deleted, causes a significant decline of the H2A.Z reporter expression. In addition, the mutation of a Sox transcription factor binding site motif and, more strongly, of a Myb motif cause significant decline of reporter gene expression. Our results suggest that an undetermined Myb-family transcription factor controls the transcriptional regulation of H2A.Z.

Expression of GATA and POU transcription factors during the development of the planktotrophic trochophore of the polychaete serpulid Hydroides elegans.

The expression of transcription factors with endodermal and mesodermal roles in bilaterians is characterized during the development of Hydroides elegans, a serpulid polychaete with planktotrophic trochophore. GATA 4/5/6 is expressed in endodermal and mesodermal precursors during embryogenesis and in the midgut of trochophore larvae. HeGATA1/2/3a is expressed in animal hemisphere blastomeres 1d121 and 1d122, in dorsal ectoderm and in 4d endomesodermal derivatives that maintain their expression in trochophore larvae. HeGATA1/2/3b is not expressed during embryogenesis, but in several regions of the larva during postembryonic development. During very early gastrulation, Brn1/2/4 is first expressed in cells associated with the prospective oral/foregut side of the blastopore, and during larval development in 4d blastomere descendants. Comparison with orthologs in other metazoans suggests ancestral expression of GATA4/5/6 in the midgut of the last common ancestor of protostomes and deuterostomes. The conserved expression of Brn1/2/4 in the foregut precursors of Hydroides and sea urchins suggests an ancestral role in patterning the tripartite gut of planktotrophic larvae. Broader analysis of these and other regulatory genes reveals variability of developmental gene expression among polychaetes with lecithotrophic larvae, suggesting that they are evolutionarily derived from polychaetes with planktotrophic larvae.

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.

Development of a feeding trochophore in the polychaete Hydroides elegans.

Hydroides elegans is an indirectly developing polychaete with equal spiral cleavage, gastrulation by invagination, and a feeding trochophore. Expression of several transcription factors and differentiation genes has been characterized. Comparative analysis reveals evolutionarily conserved roles. For example, the synexpression of transcription factors FoxA and Brachyury suggests homology of primary and secondary gut openings in protostomes and deuterostomes, and the expression of Sall suggests similar regulatory controls in the posterior growth zone of bilaterians. Differences in gene expression suggest regulatory differences control gastrulation by invagination in polychaetes with a feeding trochophore and gastrulation by epiboly in polychaetes without a feeding trochophore. Association of histone variant H2A.Z with transcriptional potency and its expression suggest a developmental role during both embryogenesis and the larva-to-adult transformation. Methods are being developed for experimental exploration of the gene regulatory networks involved in trochophore development in Hydroides. It is unknown if polychaete feeding trochophores evolved from a larval stage already present in the life cycle of the last common ancestor of protostomes and deuterostomes. Previous evolutionary scenarios about larval origins overemphasize the discontinuity between larval and adult development and require the early evolution of undifferentiated and transcriptionally potent "set aside" cells. Indirect development may proceed by developmental remodeling of differentiated cells and could have evolved after gradual transformation of juveniles into larvae; undifferentiated and transcriptionally potent cells would have evolved secondarily. Comprehensive characterization of gene regulatory networks for feeding trochophore development may help resolve these major evolutionary questions.

Brachyury, Tbx2/3 and sall expression during embryogenesis of the indirectly developing polychaete Hydroides elegans.

Expression of the transcription factor genes brachyury, Tbx2/3 and sall is characterized in detail for the first time in an indirectly developing spiralian with a feeding trochophore. In Hydroides elegans, gut formation proceeds by invagination during embryogenesis and is followed by feeding-dependent posterior growth during larval stages. Posterior growth gives rise to the reproductive and segmented portion of the adult and derives primarily from multipotent dorsal blastomeres. Dorsal fate becomes morphologically evident at the 60-cell stage during spiral cleavage, although the timing of dorsal specification remains uncertain. Expression of brachyury anticipates the morphogenetic events associated with both gastrulation by invagination in the endoderm and ventral midline convergent extension in the ectoderm. The absence of brachyury expression in endoderm precursors previously reported in annelids that do not have feeding larvae suggests evolutionarily conserved roles associated with morphogenesis rather than endoderm specification. Synexpression of brachyury and FoxA in the blastopore of eumetazoans as well as in the secondarily formed anus of some protostomes and the mouth of deuterostomes suggests shared regulatory circuits during the formation of both oral and anal openings in protostomes and deuterostomes. Expression of sall during gastrulation, in the protonephridium, and in posterior growth zone precursors, also suggests evolutionarily conserved roles. The dorsal sides of the Hydroides and sea urchin embryos express Tbx2/3 in all three germ layer precursors, suggesting evolutionarily conserved dorsal regionalization functions. The results suggest specific gene usage during tubular gut formation, endoderm specification, dorsoventral specification and anteroposterior body elongation in the context of development by feeding larva.

Indirect development, transdifferentiation and the macroregulatory evolution of metazoans.

It is proposed here that a biphasic life cycle with partial dedifferentiation of intermediate juvenile or larval stages represents the mainstream developmental mode of metazoans. Developmental plasticity of differentiated cells is considered the essential characteristic of indirect development, rather than the exclusive development of the adult from 'set-aside' cells. Many differentiated larval cells of indirect developers resume proliferation, partially dedifferentiate and contribute to adult tissues. Transcriptional pluripotency of differentiated states has premetazoan origins and seems to be facilitated by histone variant H2A.Z. Developmental plasticity of differentiated states also facilitates the evolution of polyphenism. Uncertainty remains about whether the most recent common ancestor of protostomes and deuterostomes was a direct or an indirect developer, and how the feeding larvae of bilaterians are related to non-feeding larvae of sponges and cnidarians. Feeding ciliated larvae of bilaterians form their primary gut opening by invagination, which seems related to invagination in cnidarians. Formation of the secondary gut opening proceeds by protostomy or deuterostomy, and gene usage suggests serial homology of the mouth and anus. Indirect developers do not use the Hox vector to build their ciliated larvae, but the Hox vector is associated with the construction of the reproductive portion of the animal during feeding-dependent posterior growth. It is further proposed that the original function of the Hox cluster was in gonad formation rather than in anteroposterior diversification.

The transcription factors HeBlimp and HeT-brain of an indirectly developing polychaete suggest ancestral endodermal, gastrulation, and sensory cell-type specification roles.

The expression of Blimp and T-brain was characterized during embryogenesis of the indirectly developing polychaete Hydroides elegans. The expression of both genes in the vegetal blastomeres of this lophotrochozoan is restricted to the lateral and oral regions of the blastopore. Both transcription factors also have expression patterns consistent with ancestral neural functions. HeBlimp is expressed in a couple of animal cap cells and exhibits a marked left bias that correlates with earlier development of the left eye in H. elegans. HeT-brain is expressed in animal cap blastomeres and eventually becomes restricted to apical tuft cells of early trochophore stages. The expression of Blimp and T-brain among directly and indirectly developing bilaterians suggests ancestral sensory cell-type specification, gastrulation by invagination, endoderm specification, mesoderm specification, and/or tripartite gut subdivision. These characterizations add to the long-term goal of understanding the regulatory evolution functions that underlies complex life cycles in metazoans.

Sinistral equal-size spiral cleavage of the indirectly developing polychaete Hydroides elegans.

Two major variants of the stereotypic spiral cleavage correlate with distinct developmental modes in polychaetes. Indirect development through a feeding trochophore larva correlates with development from four equal-sized blastomeres, whereas direct development correlates with unequal cleavage characterized by a large dorsal blastomere precursor maternally predetermined. The equal-size spiral cleavage of the indirectly developing serpulid Hydroides elegans has been reconstructed from serial sections of nuclei-stained embryos. The order of cell divisions has been determined from the 2-cell stage to the 80-cell stage, when gastrulation cell movements start to overlap with late spiral-cleavage divisions. In contrast to related species, the third cleavage in Hydroides elegans is invariably sinistral. The four quadrants remain indistinct until the 60-cell stage, when the small 2d22 and large 2d21 cells are generated. The developmental significance of the invariant spiral cleavage relates to the spatial distribution of gene functions that it partitions and their relation to blastomere fate commitments. The conservation and divergence of the cleavage pattern among spiralians is well suited to study the developmental control of the cell-cleavage machinery and its evolution.

Histone H2A.Z expression in two indirectly developing marine invertebrates correlates with undifferentiated and multipotent cells.

The embryos of indirect developers generate an intermediate larval stage that nourishes the proliferation of undifferentiated multipotent cell precursors in charge of postembryonic adult formation. Multipotency affects the regulation of many genes and seems to be mediated in part by chromatin modification. Chromatin transcriptional properties are regulated by histone modification and by incorporation of peculiar histone variants. The histone variant H2A.Z is associated with transcriptionally competent chromatin and silent genes primed for activation or permanent repression. However, despite the extensive mechanistic characterizations in unicellular eukaryotes, the essential role of the highly conserved H2A.Z variant during animal embryogenesis remains obscure. We show that the expression of H2A.Z in the larvae of two distant indirectly developing marine invertebrates, a polychaete and a sea urchin, remains high in all their embryonic and postembryonic developmentally competent cell precursors, and declines during their differentiation. In particular, the expression in undifferentiated multipotent adult precursors during feeding larval stages in both organisms provides unique insight about its general association with developmental potential. Our experiments confirm previous reports indicating that the expression of H2A.Z is proliferation (DNA synthesis) independent, in contrast with the DNA synthesis dependence of "mainstream" histones. We suggest that similar H2A.Z transcriptional functions previously identified in unicellular organisms also help to maintain an open chromatin state competent for transcriptional-regulatory transactions during metazoan development.

HeOtx expression in an indirectly developing polychaete correlates with gastrulation by invagination.

The expression of an Otx homolog in the indirectly developing polychaete Hydroides elegans was characterized during embryo, trochophore, and feeding-larva stages. In the animal hemisphere, HeOtx is first expressed in 1q(12) blastomeres and their immediate descendants. Such discrete embryonic animal hemisphere Otx expression perhaps relates to cell-type specification functions of the larva. During feeding stages, transcripts are detected in adult cerebral ganglia precursors and putative adult eye precursors, where it may have adult brain regionalization functions. HeOtx is not expressed in primary trochoblast precursors, but it is expressed in cells adjacent to the ciliary band. HeOtx is also expressed in a group of cells in the dorsal midline of the early trochophore larva in putative posterior sensory organ precursors. The vegetal hemisphere expression starts in oral and lateral sides of the blastopore and later expands to central blastomeres that lead the gastrulation movements. During late gastrulation stages, the expression declines in foregut precursors, but it is maintained in midgut precursors, suggesting its involvement in tripartite gut subdivision functions. HeOtx broader and earlier endoderm expression correlates with gastrulation by invagination associated with the formation of the feeding trochophore, in contrast with a later and orally restricted Otx expression found in a polychaete that gastrulates by epiboly and forms a non-feeding trochophore. The endoderm expression and functional roles in other bilaterians suggest an ancestral role of Otx related to gastrulation by invagination.

Ciliary band gene expression patterns in the embryo and trochophore larva of an indirectly developing polychaete.

The trochophore larvae of indirectly developing spiralians have ciliary bands with motor and feeding functions. The preoral prototroch ciliary band is the first differentiating organ in annelid and mollusk embryos. Here we report the expression of several ciliary band markers during embryogenesis and early larval stages of the indirectly developing polychaete Hydroides elegans. Genes with similarity to caveolin, beta-tubulin, alpha-tubulin, and tektin are expressed in the eight primary prototroch precursors, 1q(221) and 1q(212). Blastomeres 1q(221) and 1q(212) locate at the same equatorial latitude after the complementary asymmetric division of their 1q(22) and 1q(21) precursors. In addition, caveolin and alpha-tubulin are expressed in the metatroch and adoral ciliary zone. Caveolin is expressed in foregut ciliated cells, and alpha-tubulin is expressed in apical tuft ciliated cells. The expression of a beta-thymosin homolog is restricted to 1q(122) and 1q(121) blastomeres, which locate just above and in close association with the eight primary prototroch cells 1q(221) and 1q(212). In addition, the beta-thymosin homolog has a transient expression in the hindgut and apical zone. The expression of all these genes provides a landmark for the early specification of ciliary bands and other ciliated organs.