bioRxiv (Cold Spring Harbor Laboratory), Jul 19, 2020
The evolutionary mechanisms underlying the emergence of new cell types are still unclear. Here, w... more The evolutionary mechanisms underlying the emergence of new cell types are still unclear. Here, we address the origin and diversification of muscle cells in the diploblastic sea anemone Nematostella vectensis. We discern two fast and two slow-contracting muscle cell populations in Nematostella differing by extensive sets of paralogous genes. The regulatory gene set of the slow cnidarian muscles and the bilaterian cardiac muscle are remarkably similar. By contrast, the two fast muscles differ substantially from each other, while driving the same set of paralogous structural protein genes. Our data suggest that extensive gene duplications and co-option of individual effector modules may have played an important role in cell type diversification during metazoan evolution.
Cephalopods provide an unprecedented opportunity for comparative studies of the developmental gen... more Cephalopods provide an unprecedented opportunity for comparative studies of the developmental genetics of organ systems that are convergent with analogous vertebrate structures. The Sox-family of transcription factors is an important class of DNA-binding proteins that are known to be involved in many aspects of differentiation, but have been largely unstudied in lophotrochozoan systems. Using a degenerate primer strategy we have isolated coding sequence for three members of the Sox family of transcription factors from a cephalopod mollusk, the European cuttlefish Sepia officinalis: Sof-SoxE, Sof-SoxB1, and Sof-SoxB2. Analyses of their expression patterns during organogenesis reveals distinct spatial and temporal expression domains. Sof-SoxB1 shows early ectodermal expression throughout the developing epithelium, which is gradually restricted to presumptive sensory epithelia. Expression within the nervous system appears by mid-embryogenesis. Sof-SoxB2 expression is similar to Sof-SoxB1 within the developing epithelia in early embryogenesis, however appears in largely non-overlapping expression domains within the central nervous system and is not expressed in the maturing sensory epithelium. In contrast, Sof-SoxE is expressed throughout the presumptive mesodermal territories at the onset of organogenesis. As development proceeds, Sof-SoxE expression is elevated throughout the developing peripheral circulatory system. This expression disappears as the circulatory system matures, but expression is maintained within undifferentiated connective tissues throughout the animal, and appears within the nervous system near the end of embryogenesis. SoxB proteins are widely known for their role in neural specification in numerous phylogenetic lineages. Our data suggests that Sof-SoxB genes play similar roles in cephalopods. In contrast, Sof-SoxE appears to be involved in the early stages of vasculogenesis of the cephalopod closed circulatory system, a novel role for a member of this gene family.
Animals are grouped into ~35 'phyla' based upon the notion of distinct body plans 1-4. Morphologi... more Animals are grouped into ~35 'phyla' based upon the notion of distinct body plans 1-4. Morphological and molecular analyses have revealed that a stage the middle of developmentknown as the phylotypic period-is conserved among species within some phyla 5-9. While these analyses provide evidence for their existence, phyla have also been criticized as lacking an objective definition, and consequently based on arbitrary groupings of animals 10. Here, we compare the developmental transcriptomes of ten species, each annotated to a different phylum, with a wide range of life histories and embryonic forms. We find that, in all ten species, Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Cephalopod mollusks possess a number of anatomical traits that often parallel vertebrates in morp... more Cephalopod mollusks possess a number of anatomical traits that often parallel vertebrates in morphological complexity, including a centralized nervous system with sophisticated cognitive functionality. Very little is known about the genetic mechanisms underlying patterning of the cephalopod embryo to arrive at this anatomical structure. Homeodomain (HD) genes are transcription factors that regulate transcription of downstream genes through DNA binding, and as such are integral parts of gene regulatory networks controlling the specification and patterning of body parts across lineages. We have used a degenerate primer strategy to isolate homeobox genes active during late-organogenesis from the European cuttlefish Sepia officinalis. With this approach we have isolated fourteen HD gene fragments and examine the expression profiles of five of these genes during late stage (E24-28) embryonic development (Sof-Gbx, Sof-Hox3, Sof-Arx, Sof-Lhx3/4, Sof-Vsx). All five genes are expressed within the developing central nervous system in spatially restricted and largely nonoverlapping domains. Our data provide a first glimpse into the diversity of HD genes in one of the largest, yet least studied, metazoan clades and illustrate how HD gene expression patterns reflect the functional partitioning of the cephalopod brain.
Pattern formation originates during embryogenesis by a series of symmetry-breaking steps througho... more Pattern formation originates during embryogenesis by a series of symmetry-breaking steps throughout an expanding cell lineage. InDrosophila, classic work has shown that the embryo segmentation is established by morphogens within a syncytium, and the subsequent action of the gap, pair-rule, and segment polarity genes. This classic model however does not translate directly to species that lack a syncytium – such asC. elegans– where cell fate is specified by cell-autonomous cell lineage programs and their inter-signaling. Previous single-cell RNA-Seq studies inC. eleganshave analyzed cells from a mixed suspension of cells from many embryos to study late differentiation stages, or individual early stage embryos to study early gene expression in the embryo. To study the intermediate stages of early and late gastrulation (28- to 102- cells stages) missed by these approaches, here we determine the transcriptomes of the 1- to 102-cell stage to identify 119 embryonic cell-states during cell-...
bioRxiv (Cold Spring Harbor Laboratory), Feb 5, 2023
Pattern formation originates during embryogenesis by a series of symmetry-breaking steps througho... more Pattern formation originates during embryogenesis by a series of symmetry-breaking steps throughout an expanding cell lineage. In Drosophila, classic work has shown that the embryo segmentation is established by morphogens within a syncytium, and the subsequent action of the gap, pair-rule, and segment polarity genes. This classic model however does not translate directly to species that lack a syncytium-such as C. elegans-where cell fate is specified by cell-autonomous cell lineage programs and their inter-signaling. Previous single-cell RNA-Seq studies in C. elegans have analyzed cells from a mixed suspension of cells from many embryos to study late differentiation stages, or individual early stage embryos to study early gene expression in the embryo. To study the intermediate stages of early and late gastrulation (28-to 102-cells stages) missed by these approaches, here we determine the transcriptomes of the 1to 102-cell stage to identify 119 embryonic cell-states during cell-fate specification, including 'equivalence-group' cell identities. We find that gene expression programs are modular according to the sub-cell lineages, each establishing a set of stripes by combinations of transcription factor gene expression across the anAterior-posterior axis. In particular, expression of the homeodomain genes establishes a comprehensive lineage-specific positioning system throughout the embryo beginning at the 28-cell stage. Moreover, we find that genes that segment the entire embryo in Drosophila have orthologs in C. elegans that exhibit sub-lineage specific expression. These results suggest that the C. elegans embryo is patterned by a juxtaposition of distinct lineage-specific gene regulatory programs each with a unique encoding of cell location and fate. This use of homologous gene regulatory patterning codes suggests a deep homology of cell fate specification programs across diverse modes of development.
Animals are typically composed of hundreds of different cell types, yet mechanisms underlying the... more Animals are typically composed of hundreds of different cell types, yet mechanisms underlying the emergence of new cell types remain unclear. Here we address the origin and diversification of muscle cells in the non-bilaterian, diploblastic sea anemone Nematostella vectensis. We discern two fast and two slow-contracting muscle cell populations, which differ by extensive sets of paralogous structural protein genes. We find that the regulatory gene set of the slow cnidarian muscles is remarkably similar to the bilaterian cardiac muscle, while the two fast muscles differ substantially from each other in terms of transcription factor profiles, though driving the same set of structural protein genes and having similar physiological characteristics. We show that anthozoan-specific paralogs of Paraxis/Twist/Hand-related bHLH transcription factors are involved in the formation of fast and slow muscles. Our data suggest that the subsequent recruitment of an entire effector gene set from the ...
Highlights d Development and homeostasis of a sea anemone is captured at single-cell resolution d... more Highlights d Development and homeostasis of a sea anemone is captured at single-cell resolution d Developmental differentiation trajectories are maintained throughout the life cycle d Neurons, gland, and stinging cells derive from a common pool of putative stem cells d SoxC is an upstream regulator of neural progenitor cell specification
<p>Expression territories are extrapolated from analysis of serially sectioned embryos from... more <p>Expression territories are extrapolated from analysis of serially sectioned embryos from stages E24–28 (this paper) and previously published data<sup>*</sup> °. Dorsal view of the head is shown on the left, and lateral view on the right as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109627#pone-0109627-g001" target="_blank">Figure 1</a>. Territories with potentially overlapping expression domains are multi-coloured. A) ANTP-class Hox genes <i>Sof-Hox3</i> (dark green), <i>Sof-Gbx</i> (orange), and <i>Sof-Mnx*</i> (pink). B) PRD-class genes <i>Sof-Arx</i> (red), <i>Sof-Vsx</i> (dark blue), <i>Sof-Phox2*</i> (light green), and <i>Sof-otx</i>° (yellow). C) LIM-class gene <i>Sof-Lhx3/4</i> (light blue). Note that ANTP-class genes are largely restricted to the subesophageal masses, whereas PRD-class genes are more expansive within the supraesophageal mass. Both <i>Sof-Hox3</i> and <i>Sof-Lhx3/4</i> show extensive expression throughout the nervous system, and five of the eight genes represented here are found within the stellate ganglion. Abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109627#pone-0109627-g001" target="_blank">Figure 1</a>; * data extrapolated from Nomaksteinsky <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109627#pone.0109627-Nomaksteinsky1" target="_blank">[26]</a> for stage E29 embryos; ° data extrapolated from Buresi <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109627#pone.0109627-Buresi2" target="_blank">[25]</a> for embryos at stages E25–26.</p
Mammalian cartilage is a complex and developmentally important tissue type. Outside the mammalian... more Mammalian cartilage is a complex and developmentally important tissue type. Outside the mammalian lineage, cartilage may persist as an adult tissue, which shows a much wider diversity of histological structure. Tissues similar to vertebrate cartilage are also found within multiple invertebrate lineages, including mollusks, arthropods, and polychaetes, however the relationship of these tissues to vertebrate cartilage is unknown. Detailed molecular analysis of these invertebrate tissues is necessary to assess the degree of homology, if any, of cartilage throughout the metazoans. The purpose of the following review is to introduce readers to this diversity of cartilage and to synthesize the known genetic interactions that give rise to vertebrate cartilage into the format of a gene regulatory network (GRN). This chondrogenesis GRN highlights a large number of transcription factors known to be expressed during chondrogenesis, whose role in this process has yet to be elucidated. Verification and expansion of this initial GRN will assist in the identification of the core set of the genetic interactions necessary for the specification of the vertebrate chondrocyte. This is the necessary first step in allowing detailed comparison of the molecular signature of vertebrate chondrocytes with that of invertebrates with the ultimate goal of understanding the evolutionary origin of this important skeletal cell type.
SUMMARYCommunication in bilaterian nervous systems is mediated by electrical and secreted signals... more SUMMARYCommunication in bilaterian nervous systems is mediated by electrical and secreted signals, however, the evolutionary origin and relation of neurons to other secretory cell types has not been elucidated. Here we use developmental single cell RNA-sequencing in the cnidarian Nematostella vectensis, representing an early evolutionary lineage with a simple nervous system. Validated by transgenics, we demonstrate that neurons, stinging cells, and gland cells arise from a common multipotent progenitor population. We identify the conserved transcription factor gene SoxC as a key upstream regulator of all neurosecretory lineages and demonstrate that SoxC knockdown eliminates both neuronal and secretory cell types. While in vertebrates and many other bilaterians neurogenesis is largely restricted to early developmental stages, we show that in the sea anemone differentiation of neurosecretory cells is maintained throughout all life stages, and follows the same molecular trajectories fr...
<p>Column 1 shows a schematic illustration of stage-specific gene expression. <b>a) &... more <p>Column 1 shows a schematic illustration of stage-specific gene expression. <b>a) <i>Sof-SoxB1</i></b> Expression is seen throughout the presumptive ectoderm, including expression within the developing eye (arrow); far right: higher magnification illustrating the absence of expression within the statocysts (st) and gill buds (g). <b>b) <i>Sof-SoxE</i>:</b> (1) Expression is found throughout presumptive mesoderm. (2) Higher magnification of a different embryo illustrating a ring of expression within the mesoderm underlying the forming mantle (m) and within the gill buds (g). The dash line indicates the area where hearts form in the pygmy squid [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157821#pone.0157821.ref064" target="_blank">64</a>] (3) A distinct ring of expression is also seen within the stomodeum. (4) Higher magnification illustrating medial expression within the arms and ring of underlying mesoderm (arrow). <b>c)</b> representative control embryo, sense probe. Abbreviations: *: stomodeum; ey: eye; f: funnel; g: gills; m: mantle; st: statocyst; i-v: arms. Scale bar in b3: 125μm; other scale bars: 500μm. body axis: D/V: dorsal/ventral. L/R: left/right.</p
<p>Column 1 shows a schematic illustration of stage-specific gene expression. Examples of c... more <p>Column 1 shows a schematic illustration of stage-specific gene expression. Examples of control embryos (inset) show variation of non-specific colouration found within the eyes and developing cuttlebone. <b>a) stage E24</b>. (2) dorsal, (3) ventral and (4) lateral views of a stage E24 embryo showing extensive expression at the level of the developing circulatory system. The ophthalmic vessels are particularly evident (arrowheads); (5–9) higher magnification to illustrate details. (5) anterior right side of the head showing the ophthalmic vessels (arrowhead) and specific expression within the eyes (arrows). (6) Anterior-medial view of the head showing expression at the level of the developing beak (b). Notice also the fine capillary network of the head in this region. (7) Anterior view of developing fins highlighting the expression with the network of vessels (arrows). (8) Posterior view at the level of the funnel. Expression within the vasculature of the funnel (f), arms (a) and capillaries of the head (arrow) is evident. (9) Posterior view of the mantle (m) showing the intricate network of vessels, and signal within the gills (g). <b>b) stage E25</b>. (2) anterior and (3) posterior views illustrating a reduction of expression at the level of the circulatory system. (4–9) higher magnification to illustrate details. Lower levels of expression with respect to stage E24 are retained within the circulatory system of the fins (4), within the eyes (5), and within the mantle (6). (7–9) higher magnification illustrating expression details within the arms and tentacles. (7) Expression is evident both anteriorly and posteriororly within the tentacles, as well as within the ventral most suckers (s) on the posterior side. (8) An optical cross section though the arms at this stage reveals expression corresponding to the three posterior vessels (arrows) in addition to the sucker staining. (9) Posterior view of the expression within the suckers illustrates strong signal within the posterior vessel and tissues surrounding the developing suckers, as well as the formation of the lateral blood vessels (arrow). Abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157821#pone.0157821.g005" target="_blank">Fig 5</a>. Scale bars: 500μm.</p
<p>The phylogenetic tree on the left depicts the consensus relationships from analysis of t... more <p>The phylogenetic tree on the left depicts the consensus relationships from analysis of the gene fragments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157821#sec002" target="_blank">methods</a>). The alignment used for the analysis is shown on right; the consensus HMG sequence is shown above, with highly conserved residues underlined. Family specific amino acid positions are shaded grey. The first sequence from each family is presented in full, and only amino acids that differ from this sequence are shown in subsequent lines. The three isolated <i>Sepia officinalis</i> gene fragments are evidenced in bold typeface. The HMG domains of known SOX families fall out together in the tree as expected. Members of the SoxA family are known to be mammalian specific and are excluded from the analysis. We find no clear separation of the SoxB1 and B2 groups in our analysis, however residues specific to these two sub-families are present within the two isolated SoxB genes–SoxB2 residues are highlighted in black. Hs: <i>Homo sapiens</i>; Ce: <i>Ceanorhabditis elegans</i>; Pv: <i>Patella vulgata;</i> Sk: Saccoglossus kowalevskii; Dm: <i>Drosophila malangaster</i>; Ci: <i>Ciona instestinalis</i>; So: <i>Sepia officinalis</i>; Nv: <i>Nematostella;</i> Pd: <i>Platyneries dumerilii</i>; (“.”): conserved amino acid residues.</p
<p>Figure layout as in <a href="http://www.plosone.org/article/info:doi/10.1371/jou... more <p>Figure layout as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109627#pone-0109627-g004" target="_blank">Figure 4</a>. Clockwise from upper left: Medial section through the head detecting moderate expression levels within all lobes of the CNS and the posterior mesenchyme of the buccal mass; A more lateral section of the same embryo, highlighting the strong expression domain within the middle and anterior subesophageal masses, and expression within the nerve cords of the arms; Lateral section showing expression within the optic lobes and the presumptive olfactory pit epithelium (*); Expression within the stellate ganglion is absent in the ventral-anterior most portion; Medial section at the level of the buccal mass showing expression within the buccal ganglion and neural cord of the arms; Section at the level of the optic lobes demonstrating expression throughout. Expression is also evident in the nerve cords of the arms but absent in the dorsal-lateral portion of the supraesophageal mass; Medial section through the supraesophageal mass showing expression domain in the dorsal-anterior most portion; Medial section through the subesophageal mass illustrating expression domains within the anterior-most portion of the anterior and middle masses, as well as the posterior-most portion of the posterior mass and underlying statocyst. Abbreviations: bg: buccal ganglia; nc: nerve cord; all others as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109627#pone-0109627-g003" target="_blank">Figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109627#pone-0109627-g004" target="_blank">4</a>.</p
Animals are grouped into ~35 'phyla' based upon the notion of distinct body plans. Morpho... more Animals are grouped into ~35 'phyla' based upon the notion of distinct body plans. Morphological and molecular analyses have revealed that a stage in the middle of development-known as the phylotypic period-is conserved among species within some phyla. Although these analyses provide evidence for their existence, phyla have also been criticized as lacking an objective definition, and consequently based on arbitrary groupings of animals. Here we compare the developmental transcriptomes of ten species, each annotated to a different phylum, with a wide range of life histories and embryonic forms. We find that in all ten species, development comprises the coupling of early and late phases of conserved gene expression. These phases are linked by a divergent 'mid-developmental transition' that uses species-specific suites of signalling pathways and transcription factors. This mid-developmental transition overlaps with the phylotypic period that has been defined previously ...
bioRxiv (Cold Spring Harbor Laboratory), Jul 19, 2020
The evolutionary mechanisms underlying the emergence of new cell types are still unclear. Here, w... more The evolutionary mechanisms underlying the emergence of new cell types are still unclear. Here, we address the origin and diversification of muscle cells in the diploblastic sea anemone Nematostella vectensis. We discern two fast and two slow-contracting muscle cell populations in Nematostella differing by extensive sets of paralogous genes. The regulatory gene set of the slow cnidarian muscles and the bilaterian cardiac muscle are remarkably similar. By contrast, the two fast muscles differ substantially from each other, while driving the same set of paralogous structural protein genes. Our data suggest that extensive gene duplications and co-option of individual effector modules may have played an important role in cell type diversification during metazoan evolution.
Cephalopods provide an unprecedented opportunity for comparative studies of the developmental gen... more Cephalopods provide an unprecedented opportunity for comparative studies of the developmental genetics of organ systems that are convergent with analogous vertebrate structures. The Sox-family of transcription factors is an important class of DNA-binding proteins that are known to be involved in many aspects of differentiation, but have been largely unstudied in lophotrochozoan systems. Using a degenerate primer strategy we have isolated coding sequence for three members of the Sox family of transcription factors from a cephalopod mollusk, the European cuttlefish Sepia officinalis: Sof-SoxE, Sof-SoxB1, and Sof-SoxB2. Analyses of their expression patterns during organogenesis reveals distinct spatial and temporal expression domains. Sof-SoxB1 shows early ectodermal expression throughout the developing epithelium, which is gradually restricted to presumptive sensory epithelia. Expression within the nervous system appears by mid-embryogenesis. Sof-SoxB2 expression is similar to Sof-SoxB1 within the developing epithelia in early embryogenesis, however appears in largely non-overlapping expression domains within the central nervous system and is not expressed in the maturing sensory epithelium. In contrast, Sof-SoxE is expressed throughout the presumptive mesodermal territories at the onset of organogenesis. As development proceeds, Sof-SoxE expression is elevated throughout the developing peripheral circulatory system. This expression disappears as the circulatory system matures, but expression is maintained within undifferentiated connective tissues throughout the animal, and appears within the nervous system near the end of embryogenesis. SoxB proteins are widely known for their role in neural specification in numerous phylogenetic lineages. Our data suggests that Sof-SoxB genes play similar roles in cephalopods. In contrast, Sof-SoxE appears to be involved in the early stages of vasculogenesis of the cephalopod closed circulatory system, a novel role for a member of this gene family.
Animals are grouped into ~35 'phyla' based upon the notion of distinct body plans 1-4. Morphologi... more Animals are grouped into ~35 'phyla' based upon the notion of distinct body plans 1-4. Morphological and molecular analyses have revealed that a stage the middle of developmentknown as the phylotypic period-is conserved among species within some phyla 5-9. While these analyses provide evidence for their existence, phyla have also been criticized as lacking an objective definition, and consequently based on arbitrary groupings of animals 10. Here, we compare the developmental transcriptomes of ten species, each annotated to a different phylum, with a wide range of life histories and embryonic forms. We find that, in all ten species, Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Cephalopod mollusks possess a number of anatomical traits that often parallel vertebrates in morp... more Cephalopod mollusks possess a number of anatomical traits that often parallel vertebrates in morphological complexity, including a centralized nervous system with sophisticated cognitive functionality. Very little is known about the genetic mechanisms underlying patterning of the cephalopod embryo to arrive at this anatomical structure. Homeodomain (HD) genes are transcription factors that regulate transcription of downstream genes through DNA binding, and as such are integral parts of gene regulatory networks controlling the specification and patterning of body parts across lineages. We have used a degenerate primer strategy to isolate homeobox genes active during late-organogenesis from the European cuttlefish Sepia officinalis. With this approach we have isolated fourteen HD gene fragments and examine the expression profiles of five of these genes during late stage (E24-28) embryonic development (Sof-Gbx, Sof-Hox3, Sof-Arx, Sof-Lhx3/4, Sof-Vsx). All five genes are expressed within the developing central nervous system in spatially restricted and largely nonoverlapping domains. Our data provide a first glimpse into the diversity of HD genes in one of the largest, yet least studied, metazoan clades and illustrate how HD gene expression patterns reflect the functional partitioning of the cephalopod brain.
Pattern formation originates during embryogenesis by a series of symmetry-breaking steps througho... more Pattern formation originates during embryogenesis by a series of symmetry-breaking steps throughout an expanding cell lineage. InDrosophila, classic work has shown that the embryo segmentation is established by morphogens within a syncytium, and the subsequent action of the gap, pair-rule, and segment polarity genes. This classic model however does not translate directly to species that lack a syncytium – such asC. elegans– where cell fate is specified by cell-autonomous cell lineage programs and their inter-signaling. Previous single-cell RNA-Seq studies inC. eleganshave analyzed cells from a mixed suspension of cells from many embryos to study late differentiation stages, or individual early stage embryos to study early gene expression in the embryo. To study the intermediate stages of early and late gastrulation (28- to 102- cells stages) missed by these approaches, here we determine the transcriptomes of the 1- to 102-cell stage to identify 119 embryonic cell-states during cell-...
bioRxiv (Cold Spring Harbor Laboratory), Feb 5, 2023
Pattern formation originates during embryogenesis by a series of symmetry-breaking steps througho... more Pattern formation originates during embryogenesis by a series of symmetry-breaking steps throughout an expanding cell lineage. In Drosophila, classic work has shown that the embryo segmentation is established by morphogens within a syncytium, and the subsequent action of the gap, pair-rule, and segment polarity genes. This classic model however does not translate directly to species that lack a syncytium-such as C. elegans-where cell fate is specified by cell-autonomous cell lineage programs and their inter-signaling. Previous single-cell RNA-Seq studies in C. elegans have analyzed cells from a mixed suspension of cells from many embryos to study late differentiation stages, or individual early stage embryos to study early gene expression in the embryo. To study the intermediate stages of early and late gastrulation (28-to 102-cells stages) missed by these approaches, here we determine the transcriptomes of the 1to 102-cell stage to identify 119 embryonic cell-states during cell-fate specification, including 'equivalence-group' cell identities. We find that gene expression programs are modular according to the sub-cell lineages, each establishing a set of stripes by combinations of transcription factor gene expression across the anAterior-posterior axis. In particular, expression of the homeodomain genes establishes a comprehensive lineage-specific positioning system throughout the embryo beginning at the 28-cell stage. Moreover, we find that genes that segment the entire embryo in Drosophila have orthologs in C. elegans that exhibit sub-lineage specific expression. These results suggest that the C. elegans embryo is patterned by a juxtaposition of distinct lineage-specific gene regulatory programs each with a unique encoding of cell location and fate. This use of homologous gene regulatory patterning codes suggests a deep homology of cell fate specification programs across diverse modes of development.
Animals are typically composed of hundreds of different cell types, yet mechanisms underlying the... more Animals are typically composed of hundreds of different cell types, yet mechanisms underlying the emergence of new cell types remain unclear. Here we address the origin and diversification of muscle cells in the non-bilaterian, diploblastic sea anemone Nematostella vectensis. We discern two fast and two slow-contracting muscle cell populations, which differ by extensive sets of paralogous structural protein genes. We find that the regulatory gene set of the slow cnidarian muscles is remarkably similar to the bilaterian cardiac muscle, while the two fast muscles differ substantially from each other in terms of transcription factor profiles, though driving the same set of structural protein genes and having similar physiological characteristics. We show that anthozoan-specific paralogs of Paraxis/Twist/Hand-related bHLH transcription factors are involved in the formation of fast and slow muscles. Our data suggest that the subsequent recruitment of an entire effector gene set from the ...
Highlights d Development and homeostasis of a sea anemone is captured at single-cell resolution d... more Highlights d Development and homeostasis of a sea anemone is captured at single-cell resolution d Developmental differentiation trajectories are maintained throughout the life cycle d Neurons, gland, and stinging cells derive from a common pool of putative stem cells d SoxC is an upstream regulator of neural progenitor cell specification
<p>Expression territories are extrapolated from analysis of serially sectioned embryos from... more <p>Expression territories are extrapolated from analysis of serially sectioned embryos from stages E24–28 (this paper) and previously published data<sup>*</sup> °. Dorsal view of the head is shown on the left, and lateral view on the right as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109627#pone-0109627-g001" target="_blank">Figure 1</a>. Territories with potentially overlapping expression domains are multi-coloured. A) ANTP-class Hox genes <i>Sof-Hox3</i> (dark green), <i>Sof-Gbx</i> (orange), and <i>Sof-Mnx*</i> (pink). B) PRD-class genes <i>Sof-Arx</i> (red), <i>Sof-Vsx</i> (dark blue), <i>Sof-Phox2*</i> (light green), and <i>Sof-otx</i>° (yellow). C) LIM-class gene <i>Sof-Lhx3/4</i> (light blue). Note that ANTP-class genes are largely restricted to the subesophageal masses, whereas PRD-class genes are more expansive within the supraesophageal mass. Both <i>Sof-Hox3</i> and <i>Sof-Lhx3/4</i> show extensive expression throughout the nervous system, and five of the eight genes represented here are found within the stellate ganglion. Abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109627#pone-0109627-g001" target="_blank">Figure 1</a>; * data extrapolated from Nomaksteinsky <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109627#pone.0109627-Nomaksteinsky1" target="_blank">[26]</a> for stage E29 embryos; ° data extrapolated from Buresi <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109627#pone.0109627-Buresi2" target="_blank">[25]</a> for embryos at stages E25–26.</p
Mammalian cartilage is a complex and developmentally important tissue type. Outside the mammalian... more Mammalian cartilage is a complex and developmentally important tissue type. Outside the mammalian lineage, cartilage may persist as an adult tissue, which shows a much wider diversity of histological structure. Tissues similar to vertebrate cartilage are also found within multiple invertebrate lineages, including mollusks, arthropods, and polychaetes, however the relationship of these tissues to vertebrate cartilage is unknown. Detailed molecular analysis of these invertebrate tissues is necessary to assess the degree of homology, if any, of cartilage throughout the metazoans. The purpose of the following review is to introduce readers to this diversity of cartilage and to synthesize the known genetic interactions that give rise to vertebrate cartilage into the format of a gene regulatory network (GRN). This chondrogenesis GRN highlights a large number of transcription factors known to be expressed during chondrogenesis, whose role in this process has yet to be elucidated. Verification and expansion of this initial GRN will assist in the identification of the core set of the genetic interactions necessary for the specification of the vertebrate chondrocyte. This is the necessary first step in allowing detailed comparison of the molecular signature of vertebrate chondrocytes with that of invertebrates with the ultimate goal of understanding the evolutionary origin of this important skeletal cell type.
SUMMARYCommunication in bilaterian nervous systems is mediated by electrical and secreted signals... more SUMMARYCommunication in bilaterian nervous systems is mediated by electrical and secreted signals, however, the evolutionary origin and relation of neurons to other secretory cell types has not been elucidated. Here we use developmental single cell RNA-sequencing in the cnidarian Nematostella vectensis, representing an early evolutionary lineage with a simple nervous system. Validated by transgenics, we demonstrate that neurons, stinging cells, and gland cells arise from a common multipotent progenitor population. We identify the conserved transcription factor gene SoxC as a key upstream regulator of all neurosecretory lineages and demonstrate that SoxC knockdown eliminates both neuronal and secretory cell types. While in vertebrates and many other bilaterians neurogenesis is largely restricted to early developmental stages, we show that in the sea anemone differentiation of neurosecretory cells is maintained throughout all life stages, and follows the same molecular trajectories fr...
<p>Column 1 shows a schematic illustration of stage-specific gene expression. <b>a) &... more <p>Column 1 shows a schematic illustration of stage-specific gene expression. <b>a) <i>Sof-SoxB1</i></b> Expression is seen throughout the presumptive ectoderm, including expression within the developing eye (arrow); far right: higher magnification illustrating the absence of expression within the statocysts (st) and gill buds (g). <b>b) <i>Sof-SoxE</i>:</b> (1) Expression is found throughout presumptive mesoderm. (2) Higher magnification of a different embryo illustrating a ring of expression within the mesoderm underlying the forming mantle (m) and within the gill buds (g). The dash line indicates the area where hearts form in the pygmy squid [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157821#pone.0157821.ref064" target="_blank">64</a>] (3) A distinct ring of expression is also seen within the stomodeum. (4) Higher magnification illustrating medial expression within the arms and ring of underlying mesoderm (arrow). <b>c)</b> representative control embryo, sense probe. Abbreviations: *: stomodeum; ey: eye; f: funnel; g: gills; m: mantle; st: statocyst; i-v: arms. Scale bar in b3: 125μm; other scale bars: 500μm. body axis: D/V: dorsal/ventral. L/R: left/right.</p
<p>Column 1 shows a schematic illustration of stage-specific gene expression. Examples of c... more <p>Column 1 shows a schematic illustration of stage-specific gene expression. Examples of control embryos (inset) show variation of non-specific colouration found within the eyes and developing cuttlebone. <b>a) stage E24</b>. (2) dorsal, (3) ventral and (4) lateral views of a stage E24 embryo showing extensive expression at the level of the developing circulatory system. The ophthalmic vessels are particularly evident (arrowheads); (5–9) higher magnification to illustrate details. (5) anterior right side of the head showing the ophthalmic vessels (arrowhead) and specific expression within the eyes (arrows). (6) Anterior-medial view of the head showing expression at the level of the developing beak (b). Notice also the fine capillary network of the head in this region. (7) Anterior view of developing fins highlighting the expression with the network of vessels (arrows). (8) Posterior view at the level of the funnel. Expression within the vasculature of the funnel (f), arms (a) and capillaries of the head (arrow) is evident. (9) Posterior view of the mantle (m) showing the intricate network of vessels, and signal within the gills (g). <b>b) stage E25</b>. (2) anterior and (3) posterior views illustrating a reduction of expression at the level of the circulatory system. (4–9) higher magnification to illustrate details. Lower levels of expression with respect to stage E24 are retained within the circulatory system of the fins (4), within the eyes (5), and within the mantle (6). (7–9) higher magnification illustrating expression details within the arms and tentacles. (7) Expression is evident both anteriorly and posteriororly within the tentacles, as well as within the ventral most suckers (s) on the posterior side. (8) An optical cross section though the arms at this stage reveals expression corresponding to the three posterior vessels (arrows) in addition to the sucker staining. (9) Posterior view of the expression within the suckers illustrates strong signal within the posterior vessel and tissues surrounding the developing suckers, as well as the formation of the lateral blood vessels (arrow). Abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157821#pone.0157821.g005" target="_blank">Fig 5</a>. Scale bars: 500μm.</p
<p>The phylogenetic tree on the left depicts the consensus relationships from analysis of t... more <p>The phylogenetic tree on the left depicts the consensus relationships from analysis of the gene fragments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157821#sec002" target="_blank">methods</a>). The alignment used for the analysis is shown on right; the consensus HMG sequence is shown above, with highly conserved residues underlined. Family specific amino acid positions are shaded grey. The first sequence from each family is presented in full, and only amino acids that differ from this sequence are shown in subsequent lines. The three isolated <i>Sepia officinalis</i> gene fragments are evidenced in bold typeface. The HMG domains of known SOX families fall out together in the tree as expected. Members of the SoxA family are known to be mammalian specific and are excluded from the analysis. We find no clear separation of the SoxB1 and B2 groups in our analysis, however residues specific to these two sub-families are present within the two isolated SoxB genes–SoxB2 residues are highlighted in black. Hs: <i>Homo sapiens</i>; Ce: <i>Ceanorhabditis elegans</i>; Pv: <i>Patella vulgata;</i> Sk: Saccoglossus kowalevskii; Dm: <i>Drosophila malangaster</i>; Ci: <i>Ciona instestinalis</i>; So: <i>Sepia officinalis</i>; Nv: <i>Nematostella;</i> Pd: <i>Platyneries dumerilii</i>; (“.”): conserved amino acid residues.</p
<p>Figure layout as in <a href="http://www.plosone.org/article/info:doi/10.1371/jou... more <p>Figure layout as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109627#pone-0109627-g004" target="_blank">Figure 4</a>. Clockwise from upper left: Medial section through the head detecting moderate expression levels within all lobes of the CNS and the posterior mesenchyme of the buccal mass; A more lateral section of the same embryo, highlighting the strong expression domain within the middle and anterior subesophageal masses, and expression within the nerve cords of the arms; Lateral section showing expression within the optic lobes and the presumptive olfactory pit epithelium (*); Expression within the stellate ganglion is absent in the ventral-anterior most portion; Medial section at the level of the buccal mass showing expression within the buccal ganglion and neural cord of the arms; Section at the level of the optic lobes demonstrating expression throughout. Expression is also evident in the nerve cords of the arms but absent in the dorsal-lateral portion of the supraesophageal mass; Medial section through the supraesophageal mass showing expression domain in the dorsal-anterior most portion; Medial section through the subesophageal mass illustrating expression domains within the anterior-most portion of the anterior and middle masses, as well as the posterior-most portion of the posterior mass and underlying statocyst. Abbreviations: bg: buccal ganglia; nc: nerve cord; all others as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109627#pone-0109627-g003" target="_blank">Figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109627#pone-0109627-g004" target="_blank">4</a>.</p
Animals are grouped into ~35 'phyla' based upon the notion of distinct body plans. Morpho... more Animals are grouped into ~35 'phyla' based upon the notion of distinct body plans. Morphological and molecular analyses have revealed that a stage in the middle of development-known as the phylotypic period-is conserved among species within some phyla. Although these analyses provide evidence for their existence, phyla have also been criticized as lacking an objective definition, and consequently based on arbitrary groupings of animals. Here we compare the developmental transcriptomes of ten species, each annotated to a different phylum, with a wide range of life histories and embryonic forms. We find that in all ten species, development comprises the coupling of early and late phases of conserved gene expression. These phases are linked by a divergent 'mid-developmental transition' that uses species-specific suites of signalling pathways and transcription factors. This mid-developmental transition overlaps with the phylotypic period that has been defined previously ...
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Papers by Alison Cole