Publications by authors named "Charles A Ettensohn"

43 Publications

Classifying domain-specific text documents containing ambiguous keywords.

Database (Oxford) 2021 Sep 29;2021. Epub 2021 Sep 29.

Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada.

A keyword-based search of comprehensive databases such as PubMed may return irrelevant papers, especially if the keywords are used in multiple fields of study. In such cases, domain experts (curators) need to verify the results and remove the irrelevant articles. Automating this filtering process will save time, but it has to be done well enough to ensure few relevant papers are rejected and few irrelevant papers are accepted. A good solution would be fast, work with the limited amount of data freely available (full paper body may be missing), handle ambiguous keywords and be as domain-neutral as possible. In this paper, we evaluate a number of classification algorithms for identifying a domain-specific set of papers about echinoderm species and show that the resulting tool satisfies most of the abovementioned requirements. Echinoderms consist of a number of very different organisms, including brittle stars, sea stars (starfish), sea urchins and sea cucumbers. While their taxonomic identifiers are specific, the common names are used in many other contexts, creating ambiguity and making a keyword search prone to error. We try classifiers using Linear, Naïve Bayes, Nearest Neighbor, Tree, SVM, Bagging, AdaBoost and Neural Network learning models and compare their performance. We show how effective the resulting classifiers are in filtering irrelevant articles returned from PubMed. The methodology used is more dependent on the good selection of training data and is a practical solution that can be applied to other fields of study facing similar challenges. Database URL: The code and date reported in this paper are freely available at http://xenbaseturbofrog.org/pub/Text-Topic-Classifier/.
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http://dx.doi.org/10.1093/database/baab062DOI Listing
September 2021

A nomenclature for echinoderm genes.

Database (Oxford) 2021 08;2021

Department of Biological Sciences, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, PA 15213, USA.

Echinoderm embryos and larvae are prominent experimental model systems for studying developmental mechanisms. High-quality, assembled, annotated genome sequences are now available for several echinoderm species, including representatives from most classes. The increased availability of these data necessitates the development of a nomenclature that assigns universally interpretable gene symbols to echinoderm genes to facilitate cross-species comparisons of gene functions, both within echinoderms and across other phyla. This paper describes the implementation of an improved set of echinoderm gene nomenclature guidelines that both communicates meaningful orthology information in protein-coding gene symbols and names and establishes continuity with nomenclatures developed for major vertebrate model organisms, including humans. Differences between the echinoderm gene nomenclature guidelines and vertebrate guidelines are examined and explained. This nomenclature incorporates novel solutions to allow for several types of orthologous relationships, including the single echinoderm genes with multiple vertebrate co-orthologs that result from whole-genome-duplication events. The current version of the Echinoderm Gene Nomenclature Guidelines can be found at https://www.echinobase.org/gene/static/geneNomenclature.jsp Database URL https://www.echinobase.org/.
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http://dx.doi.org/10.1093/database/baab052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8361234PMC
August 2021

Global patterns of enhancer activity during sea urchin embryogenesis assessed by eRNA profiling.

Genome Res 2021 Sep 30;31(9):1680-1692. Epub 2021 Jul 30.

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.

We used capped analysis of gene expression with sequencing (CAGE-seq) to profile eRNA expression and enhancer activity during embryogenesis of a model echinoderm: the sea urchin, We identified more than 18,000 enhancers that were active in mature oocytes and developing embryos and documented a burst of enhancer activation during cleavage and early blastula stages. We found that a large fraction (73.8%) of all enhancers active during the first 48 h of embryogenesis were hyperaccessible no later than the 128-cell stage and possibly even earlier. Most enhancers were located near gene bodies, and temporal patterns of eRNA expression tended to parallel those of nearby genes. Furthermore, enhancers near lineage-specific genes contained signatures of inputs from developmental gene regulatory networks deployed in those lineages. A large fraction (60%) of sea urchin enhancers previously shown to be active in transgenic reporter assays was associated with eRNA expression. Moreover, a large fraction (50%) of a representative subset of enhancers identified by eRNA profiling drove tissue-specific gene expression in isolation when tested by reporter assays. Our findings provide an atlas of developmental enhancers in a model sea urchin and support the utility of eRNA profiling as a tool for enhancer discovery and regulatory biology. The data generated in this study are available at Echinobase, the public database of information related to echinoderm genomics.
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http://dx.doi.org/10.1101/gr.275684.121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8415375PMC
September 2021

Analysis of the DNA-binding properties of Alx1, an evolutionarily conserved regulator of skeletogenesis in echinoderms.

J Biol Chem 2021 07 19;297(1):100901. Epub 2021 Jun 19.

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA. Electronic address:

Alx1, a homeodomain-containing transcription factor, is a highly conserved regulator of skeletogenesis in echinoderms. In sea urchins, Alx1 plays a central role in the differentiation of embryonic primary mesenchyme cells (PMCs) and positively regulates the transcription of most biomineralization genes expressed by these cells. The alx1 gene arose via duplication and acquired a skeletogenic function distinct from its paralog (alx4) through the exonization of a 41-amino acid motif (the D2 domain). Alx1 and Alx4 contain glutamine-50 paired-type homeodomains, which interact preferentially with palindromic binding sites in vitro. Chromatin immunoprecipitation sequencing (ChIP-seq) studies have shown, however, that Alx1 binds both to palindromic and half sites in vivo. To address this apparent discrepancy and explore the function of the D2 domain, we used an endogenous cis-regulatory module associated with Sp-mtmmpb, a gene that encodes a PMC-specific metalloprotease, to analyze the DNA-binding properties of Alx1. We find that Alx1 forms dimeric complexes on TAAT-containing half sites by a mechanism distinct from the well-known mechanism of dimerization on palindromic sites. We used transgenic reporter assays to analyze the functional roles of half sites in vivo and demonstrate that two sites with partially redundant functions are essential for the PMC-specific activity of the Sp-mtmmpb cis-regulatory module. Finally, we show that the D2 domain influences the DNA-binding properties of Alx1 in vitro, suggesting that the exonization of this motif may have facilitated the acquisition of new transcriptional targets and consequently a novel developmental function.
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http://dx.doi.org/10.1016/j.jbc.2021.100901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8319359PMC
July 2021

Conditional gene knockdowns in sea urchins using caged morpholinos.

Dev Biol 2021 07 5;475:21-29. Epub 2021 Mar 5.

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA. Electronic address:

Echinoderms are important experimental models for analyzing embryonic development, but a lack of spatial and temporal control over gene perturbations has hindered developmental studies using these animals. Morpholino antisense oligonucleotides (MOs) have been used successfully by the echinoderm research community for almost two decades, and MOs remain the most widely used tool for acute gene knockdowns in these organisms. Echinoderm embryos develop externally and are optically transparent, making them ideally-suited to many light-based approaches for analyzing and manipulating development. Studies using zebrafish embryos have demonstrated the effectiveness of photoactivatable (caged) MOs for conditional gene knockdowns. Here we show that caged MOs, synthesized using nucleobase-caged monomers, provide light-regulated control over gene expression in sea urchin embryos. Our work provides the first robust approach for conditional gene silencing in this prominent model system.
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http://dx.doi.org/10.1016/j.ydbio.2021.02.014DOI Listing
July 2021

Transcription Factors of the Alx Family: Evolutionarily Conserved Regulators of Deuterostome Skeletogenesis.

Front Genet 2020 23;11:569314. Epub 2020 Nov 23.

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, United States.

Members of the gene family encode transcription factors that contain a highly conserved Paired-class, DNA-binding homeodomain, and a C-terminal OAR/Aristaless domain. Phylogenetic and comparative genomic studies have revealed complex patterns of gene duplications during deuterostome evolution. Remarkably, genes have been implicated in skeletogenesis in both echinoderms and vertebrates. In this review, we provide an overview of current knowledge concerning genes in deuterostomes. We highlight their evolutionarily conserved role in skeletogenesis and draw parallels and distinctions between the skeletogenic gene regulatory circuitries of diverse groups within the superphylum.
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http://dx.doi.org/10.3389/fgene.2020.569314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7719703PMC
November 2020

The gene regulatory control of sea urchin gastrulation.

Mech Dev 2020 06 28;162:103599. Epub 2020 Feb 28.

Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Ave., Pittsburgh, PA 15213, USA. Electronic address:

The cell behaviors associated with gastrulation in sea urchins have been well described. More recently, considerable progress has been made in elucidating gene regulatory networks (GRNs) that underlie the specification of early embryonic territories in this experimental model. This review integrates information from these two avenues of work. I discuss the principal cell movements that take place during sea urchin gastrulation, with an emphasis on molecular effectors of the movements, and summarize our current understanding of the gene regulatory circuitry upstream of those effectors. A case is made that GRN biology can provide a causal explanation of gastrulation, although additional analysis is needed at several levels of biological organization in order to provide a deeper understanding of this complex morphogenetic process.
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http://dx.doi.org/10.1016/j.mod.2020.103599DOI Listing
June 2020

The evolution of a new cell type was associated with competition for a signaling ligand.

PLoS Biol 2019 09 18;17(9):e3000460. Epub 2019 Sep 18.

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of America.

There is presently a very limited understanding of the mechanisms that underlie the evolution of new cell types. The skeleton-forming primary mesenchyme cells (PMCs) of euechinoid sea urchins, derived from the micromeres of the 16-cell embryo, are an example of a recently evolved cell type. All adult echinoderms have a calcite-based endoskeleton, a synapomorphy of the Ambulacraria. Only euechinoids have a micromere-PMC lineage, however, which evolved through the co-option of the adult skeletogenic program into the embryo. During normal development, PMCs alone secrete the embryonic skeleton. Other mesoderm cells, known as blastocoelar cells (BCs), have the potential to produce a skeleton, but a PMC-derived signal ordinarily prevents these cells from expressing a skeletogenic fate and directs them into an alternative developmental pathway. Recently, it was shown that vascular endothelial growth factor (VEGF) signaling plays an important role in PMC differentiation and is part of a conserved program of skeletogenesis among echinoderms. Here, we report that VEGF signaling, acting through ectoderm-derived VEGF3 and its cognate receptor, VEGF receptor (VEGFR)-10-Ig, is also essential for the deployment of the skeletogenic program in BCs. This VEGF-dependent program includes the activation of aristaless-like homeobox 1 (alx1), a conserved transcriptional regulator of skeletogenic specification across echinoderms and an example of a "terminal selector" gene that controls cell identity. We show that PMCs control BC fate by sequestering VEGF3, thereby preventing activation of alx1 and the downstream skeletogenic network in BCs. Our findings provide an example of the regulation of early embryonic cell fates by direct competition for a secreted signaling ligand, a developmental mechanism that has not been widely recognized. Moreover, they reveal that a novel cell type evolved by outcompeting other embryonic cell lineages for an essential signaling ligand that regulates the expression of a gene controlling cell identity.
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http://dx.doi.org/10.1371/journal.pbio.3000460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6768484PMC
September 2019

Genome-wide identification of binding sites and gene targets of Alx1, a pivotal regulator of echinoderm skeletogenesis.

Development 2019 08 19;146(16). Epub 2019 Aug 19.

Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA

Alx1 is a conserved regulator of skeletogenesis in echinoderms and evolutionary changes in Alx1 sequence and expression have played a pivotal role in modifying programs of skeletogenesis within the phylum. Alx1 regulates a large suite of effector genes that control the morphogenetic behaviors and biomineral-forming activities of skeletogenic cells. To better understand the gene regulatory control of skeletogenesis by Alx1, we used genome-wide ChIP-seq to identify Alx1-binding sites and direct gene targets. Our analysis revealed that many terminal differentiation genes receive direct transcriptional inputs from Alx1. In addition, we found that intermediate transcription factors previously shown to be downstream of Alx1 all receive direct inputs from Alx1. Thus, Alx1 appears to regulate effector genes by indirect, as well as direct, mechanisms. We tested 23 high-confidence ChIP-seq peaks using GFP reporters and identified 18 active cis-regulatory modules (CRMs); this represents a high success rate for CRM discovery. Detailed analysis of a representative CRM confirmed that a conserved, palindromic Alx1-binding site was essential for expression. Our work significantly advances our understanding of the gene regulatory circuitry that controls skeletogenesis in sea urchins and provides a framework for evolutionary studies.
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http://dx.doi.org/10.1242/dev.180653DOI Listing
August 2019

Genome-wide analysis of chromatin accessibility using ATAC-seq.

Methods Cell Biol 2019 21;151:219-235. Epub 2018 Dec 21.

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, United States. Electronic address:

Programs of gene transcription are controlled by cis-acting DNA elements, including enhancers, silencers, and promoters. Local accessibility of chromatin has proven to be a highly informative structural feature for identifying such regulatory elements, which tend to be relatively open due to their interactions with proteins. Recently, ATAC-seq (assay for transposase-accessible chromatin using sequencing) has emerged as one of the most powerful approaches for genome-wide chromatin accessibility profiling. This method assesses DNA accessibility using hyperactive Tn5 transposase, which simultaneously cuts DNA and inserts sequencing adaptors, preferentially in regions of open chromatin. ATAC-seq is a relatively simple procedure which can be applied to only a few thousand cells. It is well-suited to developing embryos of sea urchins and other echinoderms, which are a prominent experimental model for understanding the genomic control of animal development. In this chapter, we present a protocol for applying ATAC-seq to embryonic cells of sea urchins.
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http://dx.doi.org/10.1016/bs.mcb.2018.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259819PMC
July 2019

Techniques for analyzing gene expression using BAC-based reporter constructs.

Methods Cell Biol 2019 23;151:197-218. Epub 2019 Feb 23.

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, United States.

To characterize the complex regulatory control of gene expression using fluorescent protein reporters, it is often necessary to analyze large genomic regions. Bacteria artificial chromosome (BAC) vectors, which are able to support DNA fragments of up to 300kb, provide stable platforms for experimental manipulation. Using phage-based systems of homologous recombination, BACs can be efficiently engineered for a variety of aims. These include expressing fluorescent proteins to delineate gene expression boundaries using high-resolution, in vivo microscopy, tracing cell lineages using stable fluorescent proteins, perturbing endogenous protein function by expressing dominant negative forms, interfering with development by mis-expressing transcription factors, and identifying regulatory regions through deletion analysis. Here, we present a series of protocols for identifying BAC clones that contain genes of interest, modifying BACs for use as reporter constructs, and preparing BAC DNA for microinjection into fertilized eggs. Although the protocols here are tailored for use in echinoderm embryonic and larval stages, these methods are easily adaptable for use in other transgenic systems. As fluorescent protein technology continues to expand, so do the potential applications for recombinant BACs.
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http://dx.doi.org/10.1016/bs.mcb.2019.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7215881PMC
July 2019

From genome to anatomy: The architecture and evolution of the skeletogenic gene regulatory network of sea urchins and other echinoderms.

Genesis 2018 10;56(10):e23253

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania.

The skeletogenic gene regulatory network (GRN) of sea urchins and other echinoderms is one of the most intensively studied transcriptional networks in any developing organism. As such, it serves as a preeminent model of GRN architecture and evolution. This review summarizes our current understanding of this developmental network. We describe in detail the most comprehensive model of the skeletogenic GRN, one developed for the euechinoid sea urchin Strongylocentrotus purpuratus, including its initial deployment by maternal inputs, its elaboration and stabilization through regulatory gene interactions, and its control of downstream effector genes that directly drive skeletal morphogenesis. We highlight recent comparative studies that have leveraged the euechinoid GRN model to examine the evolution of skeletogenic programs in diverse echinoderms, studies that have revealed both conserved and divergent features of skeletogenesis within the phylum. Last, we summarize the major insights that have emerged from analysis of the structure and evolution of the echinoderm skeletogenic GRN and identify key, unresolved questions as a guide for future work.
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http://dx.doi.org/10.1002/dvg.23253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294693PMC
October 2018

Global analysis of primary mesenchyme cell cis-regulatory modules by chromatin accessibility profiling.

BMC Genomics 2018 03 20;19(1):206. Epub 2018 Mar 20.

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.

Background: The developmental gene regulatory network (GRN) that underlies skeletogenesis in sea urchins and other echinoderms is a paradigm of GRN structure, function, and evolution. This transcriptional network is deployed selectively in skeleton-forming primary mesenchyme cells (PMCs) of the early embryo. To advance our understanding of this model developmental GRN, we used genome-wide chromatin accessibility profiling to identify and characterize PMC cis-regulatory modules (CRMs).

Results: ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) analysis of purified PMCs provided a global picture of chromatin accessibility in these cells. We used both ATAC-seq and DNase-seq (DNase I hypersensitive site sequencing) to identify > 3000 sites that exhibited increased accessibility in PMCs relative to other embryonic cell lineages, and provide both computational and experimental evidence that a large fraction of these sites represent bona fide skeletogenic CRMs. Putative PMC CRMs were preferentially located near genes differentially expressed by PMCs and consensus binding sites for two key transcription factors in the PMC GRN, Alx1 and Ets1, were enriched in these CRMs. Moreover, a high proportion of candidate CRMs drove reporter gene expression specifically in PMCs in transgenic embryos. Surprisingly, we found that PMC CRMs were partially open in other embryonic lineages and exhibited hyperaccessibility as early as the 128-cell stage.

Conclusions: Our work provides a comprehensive picture of chromatin accessibility in an early embryonic cell lineage. By identifying thousands of candidate PMC CRMs, we significantly enhance the utility of the sea urchin skeletogenic network as a general model of GRN architecture and evolution. Our work also shows that differential chromatin accessibility, which has been used for the high-throughput identification of enhancers in differentiated cell types, is a powerful approach for the identification of CRMs in early embryonic cells. Lastly, we conclude that in the sea urchin embryo, CRMs that control the cell type-specific expression of effector genes are hyperaccessible several hours in advance of gene activation.
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http://dx.doi.org/10.1186/s12864-018-4542-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5859501PMC
March 2018

Functional divergence of paralogous transcription factors supported the evolution of biomineralization in echinoderms.

Elife 2017 11 20;6. Epub 2017 Nov 20.

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, United States.

Alx1 is a pivotal transcription factor in a gene regulatory network that controls skeletogenesis throughout the echinoderm phylum. We performed a structure-function analysis of sea urchin Alx1 using a rescue assay and identified a novel, conserved motif (Domain 2) essential for skeletogenic function. The paralogue of Alx1, Alx4, was not functionally interchangeable with Alx1, but insertion of Domain 2 conferred robust skeletogenic function on Alx4. We used cross-species expression experiments to show that Alx1 proteins from distantly related echinoderms are not interchangeable, although the sequence and function of Domain 2 are highly conserved. We also found that Domain 2 is subject to alternative splicing and provide evidence that this domain was originally gained through exonization. Our findings show that a gene duplication event permitted the functional specialization of a transcription factor through changes in exon-intron organization and thereby supported the evolution of a major morphological novelty.
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http://dx.doi.org/10.7554/eLife.32728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5758115PMC
November 2017

TGF-β sensu stricto signaling regulates skeletal morphogenesis in the sea urchin embryo.

Dev Biol 2017 Jan 10;421(2):149-160. Epub 2016 Dec 10.

Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, United States. Electronic address:

Cell-cell signaling plays a prominent role in the formation of the embryonic skeleton of sea urchins, but the mechanisms are poorly understood. In the present study, we uncover an essential role for TGF-β sensu stricto signaling in this process. We show that TgfbrtII, a type II receptor dedicated to signaling through TGF-β sensu stricto, is expressed selectively in skeletogenic primary mesenchyme cells (PMCs) during skeleton formation. Morpholino (MO) knockdowns and studies with a specific TgfbrtII inhibitor (ITD-1) in both S. purpuratus and Lytechinus variegatus embryos show that this receptor is required for biomineral deposition. We provide pharmacological evidence that Alk4/5/7 is the cognate TGF-β type I receptor that pairs with TgfbrtII and show by inhibitor treatments of isolated micromeres cultured in vitro that both Alk4/5/7 and TgfbrtII function cell-autonomously in PMCs. Gene expression and gene knockdown studies suggest that TGF-β sensu stricto may be the ligand that interacts with TgfbrtII and support the view that this TGF-β superfamily ligand provides an essential, permissive cue for skeletogenesis, although it is unlikely to provide spatial patterning information. Taken together, our findings reveal that this model morphogenetic process involves an even more diverse suite of cell signaling pathways than previously appreciated and show that PMCs integrate a complex set of both generalized and spatially localized cues in assembling the endoskeleton.
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http://dx.doi.org/10.1016/j.ydbio.2016.12.007DOI Listing
January 2017

KirrelL, a member of the Ig-domain superfamily of adhesion proteins, is essential for fusion of primary mesenchyme cells in the sea urchin embryo.

Dev Biol 2017 Jan 17;421(2):258-270. Epub 2016 Nov 17.

Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, United States.

In the sea urchin embryo, primary mesenchyme cells (PMCs) adhere to one another and fuse via filopodia, forming cable-like structures within which skeletal rods are deposited. Although this process was first described more than a century ago, molecules that participate in PMC adhesion and fusion have not been identified. Here we show that KirrelL, a PMC-specific, Ig domain-containing transmembrane protein, is essential for PMC fusion, probably by mediating filopodial adhesions that are a pre-requisite for subsequent membrane fusion. We show that KirrelL is not required for PMC specification, migration, or for direct filopodial contacts between PMCs. In the absence of KirrelL, however, filopodial contacts do not result in fusion. kirrelL is a member of a family of closely related, intronless genes that likely arose through an echinoid-specific gene expansion, possibly via retrotransposition. Our findings are significant in that they establish a direct linkage between the transcriptional network deployed in the PMC lineage and an effector molecule required for a critically important PMC morphogenetic process. In addition, our results point to a conserved role for Ig domain-containing adhesion proteins in facilitating cell fusion in both muscle and non-muscle cell lineages during animal development.
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http://dx.doi.org/10.1016/j.ydbio.2016.11.006DOI Listing
January 2017

Signal-dependent regulation of the sea urchin skeletogenic gene regulatory network.

Gene Expr Patterns 2014 Nov 16;16(2):93-103. Epub 2014 Oct 16.

Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA. Electronic address:

The endoskeleton of the sea urchin embryo is produced by primary mesenchyme cells (PMCs). Maternal inputs activate a complex gene regulatory network (GRN) in the PMC lineage in a cell-autonomous fashion during early development, initially creating a uniform population of prospective skeleton-forming cells. Previous studies showed that at post-blastula stages of development, several effector genes in the network exhibit non-uniform patterns of expression, suggesting that their regulation becomes subject to local, extrinsic cues. Other studies have identified the VEGF and MAPK pathways as regulators of PMC migration, gene expression, and biomineralization. In this study, we used whole mount in situ hybridization (WMISH) to examine the spatial expression patterns of 39 PMC-specific/enriched mRNAs in Strongylocentrotus purpuratus embryos at the late gastrula, early prism and pluteus stages. We found that all 39 mRNAs (including several regulatory genes) showed non-uniform patterns of expression within the PMC syncytium, revealing a global shift in the regulation of the skeletogenic GRN from a cell-autonomous to a signal-dependent mode. In general, localized regions of elevated gene expression corresponded to sites of rapid biomineral deposition. We used a VEGFR inhibitor (axitinib) and a MEK inhibitor (U0126) to show that VEGF signaling and the MAPK pathway are essential for maintaining high levels of gene expression in PMCs at the tips of rods that extend from the ventral region of the embryo. These inhibitors affected gene expression in the PMCs in similar ways, suggesting that VEGF acts via the MAPK pathway. In contrast, axitinib and U0126 did not affect the localized expression of genes in PMCs at the tips of the body rods, which form on the dorsal side of the embryo. Our results therefore indicate that multiple signaling pathways regulate the skeletogenic GRN during late stages of embryogenesis-VEGF/MAPK signaling on the ventral side and a separate, unidentified pathway on the dorsal side. These two signaling pathways appear to be activated sequentially (ventral followed by dorsal) and many effector genes are subject to regulation by both pathways.
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http://dx.doi.org/10.1016/j.gep.2014.10.002DOI Listing
November 2014

Horizontal transfer of the msp130 gene supported the evolution of metazoan biomineralization.

Evol Dev 2014 May 15;16(3):139-48. Epub 2014 Apr 15.

Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA, 15213, USA.

It is widely accepted that biomineralized structures appeared independently in many metazoan clades during the Cambrian. How this occurred, and whether it involved the parallel co-option of a common set of biochemical and developmental pathways (i.e., a shared biomineralization "toolkit"), are questions that remain unanswered. Here, I provide evidence that horizontal gene transfer supported the evolution of biomineralization in some metazoans. I show that Msp130 proteins, first described as proteins expressed selectively by the biomineral-forming primary mesenchyme cells of the sea urchin embryo, have a much wider taxonomic distribution than was previously appreciated. Msp130 proteins are present in several invertebrate deuterostomes and in one protostome clade (molluscs). Surprisingly, closely related proteins are also present in many bacteria and several algae, and I propose that msp130 genes were introduced into metazoan lineages via multiple, independent horizontal gene transfer events. Phylogenetic analysis shows that the introduction of an ancestral msp130 gene occurred in the sea urchin lineage more than 250 million years ago and that msp130 genes underwent independent, parallel duplications in each of the metazoan phyla in which these genes are found.
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http://dx.doi.org/10.1111/ede.12074DOI Listing
May 2014

Growth factors and early mesoderm morphogenesis: insights from the sea urchin embryo.

Genesis 2014 Mar 5;52(3):158-72. Epub 2014 Mar 5.

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania.

The early morphogenesis of the mesoderm is critically important in establishing the body plan of the embryo. Recent research has led to a better understanding of the mechanisms that underlie this process, and growth factor signaling pathways have emerged as key regulators of the directional movements of mesoderm cells during gastrulation. In this review, we undertake a comparative analysis of the various essential functions of growth factor signaling pathways in regulating early mesoderm morphogenesis, with an emphasis on recent advances in the sea urchin embryo. We focus on the roles of the vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) pathways in the migration of primary mesenchyme cells and the formation of the embryonic endoskeleton. We compare the functions of VEGF and FGF in sea urchins with the roles that these and other growth factors play in regulating mesoderm migration during gastrulation in Drosophila and vertebrates.
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http://dx.doi.org/10.1002/dvg.22746DOI Listing
March 2014

Genome-wide analysis of the skeletogenic gene regulatory network of sea urchins.

Development 2014 Feb;141(4):950-61

Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.

A central challenge of developmental and evolutionary biology is to understand the transformation of genetic information into morphology. Elucidating the connections between genes and anatomy will require model morphogenetic processes that are amenable to detailed analysis of cell/tissue behaviors and to systems-level approaches to gene regulation. The formation of the calcified endoskeleton of the sea urchin embryo is a valuable experimental system for developing such an integrated view of the genomic regulatory control of morphogenesis. A transcriptional gene regulatory network (GRN) that underlies the specification of skeletogenic cells (primary mesenchyme cells, or PMCs) has recently been elucidated. In this study, we carried out a genome-wide analysis of mRNAs encoded by effector genes in the network and uncovered transcriptional inputs into many of these genes. We used RNA-seq to identify >400 transcripts differentially expressed by PMCs during gastrulation, when these cells undergo a striking sequence of behaviors that drives skeletal morphogenesis. Our analysis expanded by almost an order of magnitude the number of known (and candidate) downstream effectors that directly mediate skeletal morphogenesis. We carried out genome-wide analysis of (1) functional targets of Ets1 and Alx1, two pivotal, early transcription factors in the PMC GRN, and (2) functional targets of MAPK signaling, a pathway that plays an essential role in PMC specification. These studies identified transcriptional inputs into >200 PMC effector genes. Our work establishes a framework for understanding the genomic regulatory control of a major morphogenetic process and has important implications for reconstructing the evolution of biomineralization in metazoans.
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http://dx.doi.org/10.1242/dev.105585DOI Listing
February 2014

Growth factor-mediated mesodermal cell guidance and skeletogenesis during sea urchin gastrulation.

Development 2013 Oct 11;140(20):4214-25. Epub 2013 Sep 11.

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

Growth factor signaling pathways provide essential cues to mesoderm cells during gastrulation in many metazoans. Recent studies have implicated the VEGF and FGF pathways in providing guidance and differentiation cues to primary mesenchyme cells (PMCs) during sea urchin gastrulation, although the relative contributions of these pathways and the cell behaviors they regulate are not fully understood. Here, we show that FGF and VEGF ligands are expressed in distinct domains in the embryonic ectoderm of Lytechinus variegatus. We find that PMC guidance is specifically disrupted in Lv-vegf3 morphants and these embryos fail to form skeletal elements. By contrast, PMC migration is unaffected in Lv-fgfa morphants, and well-patterned but shortened skeletal elements form. We use a VEGFR inhibitor, axitinib, to show that VEGF signaling is essential not only for the initial phase of PMC migration (subequatorial ring formation), but also for the second phase (migration towards the animal pole). VEGF signaling is not required, however, for PMC fusion. Inhibition of VEGF signaling after the completion of PMC migration causes significant defects in skeletogenesis, selectively blocking the elongation of skeletal rods that support the larval arms, but not rods that form in the dorsal region of the embryo. Nanostring nCounter analysis of ∼100 genes in the PMC gene regulatory network shows a decrease in the expression of many genes with proven or predicted roles in biomineralization in vegf3 morphants. Our studies lead to a better understanding of the roles played by growth factors in sea urchin gastrulation and skeletogenesis.
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http://dx.doi.org/10.1242/dev.100479DOI Listing
October 2013

Encoding anatomy: developmental gene regulatory networks and morphogenesis.

Genesis 2013 Jun 28;51(6):383-409. Epub 2013 Mar 28.

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.

A central challenge of developmental and evolutionary biology is to explain how anatomy is encoded in the genome. Anatomy emerges progressively during embryonic development, as a consequence of morphogenetic processes. The specialized properties of embryonic cells and tissues that drive morphogenesis, like other specialized properties of cells, arise as a consequence of differential gene expression. Recently, gene regulatory networks (GRNs) have proven to be powerful conceptual and experimental tools for analyzing the genetic control and evolution of developmental processes. A major current goal is to link these transcriptional networks directly to morphogenetic processes. This review highlights three experimental models (sea urchin skeletogenesis, ascidian notochord morphogenesis, and the formation of somatic muscles in Drosophila) that are currently being used to analyze the genetic control of anatomy by integrating information of several important kinds: (1) morphogenetic mechanisms at the molecular, cellular and tissue levels that are responsible for shaping a specific anatomical feature, (2) the underlying GRN circuitry deployed in the relevant cells, and (3) modifications to gene regulatory circuitry that have accompanied evolutionary changes in the anatomical feature.
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http://dx.doi.org/10.1002/dvg.22380DOI Listing
June 2013

The genomic regulatory control of skeletal morphogenesis in the sea urchin.

Development 2012 Feb 21;139(3):579-90. Epub 2011 Dec 21.

Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.

A central challenge of developmental and evolutionary biology is to understand how anatomy is encoded in the genome. Elucidating the genetic mechanisms that control the development of specific anatomical features will require the analysis of model morphogenetic processes and an integration of biological information at genomic, cellular and tissue levels. The formation of the endoskeleton of the sea urchin embryo is a powerful experimental system for developing such an integrated view of the genomic regulatory control of morphogenesis. The dynamic cellular behaviors that underlie skeletogenesis are well understood and a complex transcriptional gene regulatory network (GRN) that underlies the specification of embryonic skeletogenic cells (primary mesenchyme cells, PMCs) has recently been elucidated. Here, we link the PMC specification GRN to genes that directly control skeletal morphogenesis. We identify new gene products that play a proximate role in skeletal morphogenesis and uncover transcriptional regulatory inputs into many of these genes. Our work extends the importance of the PMC GRN as a model developmental GRN and establishes a unique picture of the genomic regulatory control of a major morphogenetic process. Furthermore, because echinoderms exhibit diverse programs of skeletal development, the newly expanded sea urchin skeletogenic GRN will provide a foundation for comparative studies that explore the relationship between GRN evolution and morphological evolution.
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http://dx.doi.org/10.1242/dev.073049DOI Listing
February 2012

Regulative deployment of the skeletogenic gene regulatory network during sea urchin development.

Development 2011 Jun;138(12):2581-90

Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.

The well-known regulative properties of the sea urchin embryo, coupled with the recent elucidation of gene regulatory networks (GRNs) that underlie cell specification, make this a valuable experimental model for analyzing developmental plasticity. In the sea urchin, the primary mesenchyme cell (PMC) GRN controls the development of the embryonic skeleton. Remarkably, experimental manipulations reveal that this GRN can be activated in almost any cell of the embryo. Here, we focus on the activation of the PMC GRN during gastrulation by non-skeletogenic mesoderm (NSM) cells and by endoderm cells. We show that most transfating NSM cells are prospective blastocoelar cells, not prospective pigment cells, as was previously believed. Earlier work showed that the regulative deployment of the GRN, unlike its deployment in the micromere-PMC lineage, is independent of the transcriptional repressor Pmar1. In this work, we identify several additional differences in the upstream regulation of the GRN during normal and regulative development. We provide evidence that, despite these changes in the upstream regulation of the network, downstream regulatory genes and key morphoregulatory genes are deployed in transfating NSM cells in a fashion that recapitulates the normal deployment of the GRN, and which can account for the striking changes in migratory behavior that accompany NSM transfating. Finally, we report that mitotic cell division is not required for genomic reprogramming in this system, either within a germ layer (NSM transfating) or across a germ layer boundary (endoderm transfating).
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http://dx.doi.org/10.1242/dev.065193DOI Listing
June 2011

P58-A and P58-B: novel proteins that mediate skeletogenesis in the sea urchin embryo.

Dev Biol 2011 May 26;353(1):81-93. Epub 2011 Feb 26.

Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.

During sea urchin embryogenesis, the skeleton is produced by primary mesenchyme cells (PMCs). PMCs undergo a sequence of morphogenetic behaviors that includes ingression, directed migration, and cell-cell fusion. Ultimately, PMCs deposit the calcite-containing biomineral that forms the endoskeleton of the late embryo and early larva. The endoskeleton has a stereotypical structure and is the major determinant of the distinctive, angular shape of the larva. Although many candidate biomineralization proteins have been identified, functional data concerning these proteins are scant. Here, we identify and characterize two new biomineralization genes, p58-a and p58-b. We show that these two genes are highly conserved in Strongylocentrotus purpuratus and Lytechinus variegatus, two sea urchin species whose ancestors diverged approximately 100 mya. The p58-a and p58-b genes lie in tandem on the chromosome, suggesting that one of the two genes arose via a gene duplication event. The two genes encode closely related, type I transmembrane proteins. We have established by whole mount in situ hybridization that p58-a and p58-b are expressed specifically in the PMCs in both species. Knockdown of either gene by morpholino antisense oligonucleotides leads to profound defects in skeletogenesis, although skeletal elements are not completely eliminated. The P58-A and P58-B proteins do not appear to play a role in the specification, directed migration or differentiation of the PMCs, but most likely are directly involved in biomineralization during sea urchin embryonic development.
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http://dx.doi.org/10.1016/j.ydbio.2011.02.021DOI Listing
May 2011

Cell micromanipulation with an active handheld micromanipulator.

Annu Int Conf IEEE Eng Med Biol Soc 2010 ;2010:4363-6

University of Valladolid, Spain.

The paper describes the use of an active handheld micromanipulator, known as Micron, for micromanipulation of cells. The device enables users to manipulate objects on the order of tens of microns in size, with the natural ease of use of a fully handheld tool. Micron senses its own position using a purpose-built microscale optical tracker, estimates the erroneous or undesired component of hand motion, and actively corrects it by deflecting its own tool tip using piezoelectric actuators. Benchtop experiments in tip positioning show that active compensation can reduce positioning error by up to 51% compared to unaided performance. Preliminary experiments in bisection of sea urchin embryos exhibit an increased success rate when performed with the help of Micron.
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http://dx.doi.org/10.1109/IEMBS.2010.5627111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3252818PMC
March 2011

Activation of the skeletogenic gene regulatory network in the early sea urchin embryo.

Development 2010 Apr 24;137(7):1149-57. Epub 2010 Feb 24.

Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.

The gene regulatory network (GRN) that underlies the development of the embryonic skeleton in sea urchins is an important model for understanding the architecture and evolution of developmental GRNs. The initial deployment of the network is thought to be regulated by a derepression mechanism, which is mediated by the products of the pmar1 and hesC genes. Here, we show that the activation of the skeletogenic network occurs by a mechanism that is distinct from the transcriptional repression of hesC. By means of quantitative, fluorescent whole-mount in situ hybridization, we find that two pivotal early genes in the network, alx1 and delta, are activated in prospective skeletogenic cells prior to the downregulation of hesC expression. An analysis of the upstream regulation of alx1 shows that this gene is regulated by MAPK signaling and by the transcription factor Ets1; however, these inputs influence only the maintenance of alx1 expression and not its activation, which occurs by a distinct mechanism. By altering normal cleavage patterns, we show that the zygotic activation of alx1 and delta, but not that of pmar1, is dependent upon the unequal division of vegetal blastomeres. Based on these findings, we conclude that the widely accepted double-repression model is insufficient to account for the localized activation of the skeletogenic GRN. We postulate the existence of additional, unidentified repressors that are controlled by pmar1, and propose that the ability of pmar1 to derepress alx1 and delta is regulated by the unequal division of vegetal blastomeres.
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http://dx.doi.org/10.1242/dev.048652DOI Listing
April 2010

The expression and distribution of Wnt and Wnt receptor mRNAs during early sea urchin development.

Gene Expr Patterns 2010 Jan 22;10(1):60-4. Epub 2009 Oct 22.

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

The protein beta-catenin plays a critically important role in establishing axial polarity during early animal development. In many organisms, beta-catenin is degraded preferentially on one side of the cleavage stage embryo. On the opposite side of the embryo, beta-catenin is stabilized and accumulates in the nucleus, where it functions in concert with members of the LEF/TCF family to activate the transcription of diverse target genes. Genes that are activated by beta-catenin play an essential role in the specification of endomesoderm and in the establishment of key signaling centers in the early embryo. In several organisms, the asymmetric distribution of maternal components of the canonical Wnt pathway has been shown to be responsible for the polarized stabilization of beta-catenin. In this study, we identified all Wnt and Wnt receptor mRNAs that are present in unfertilized sea urchin eggs and early embryos and analyzed their distributions along the primary (AV) axis. Our findings indicate that the asymmetric distribution of a maternal Wnt or Wnt receptor mRNA is unlikely to be a primary determinant of the polarized stabilization of beta-catenin along the AV axis. This contrasts sharply with findings in other organisms and points to remarkable evolutionary flexibility in the molecular mechanisms that underlie this otherwise very highly conserved patterning process.
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http://dx.doi.org/10.1016/j.gep.2009.10.004DOI Listing
January 2010

Lessons from a gene regulatory network: echinoderm skeletogenesis provides insights into evolution, plasticity and morphogenesis.

Development 2009 Jan;136(1):11-21

Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.

Significant new insights have emerged from the analysis of a gene regulatory network (GRN) that underlies the development of the endoskeleton of the sea urchin embryo. Comparative studies have revealed ways in which this GRN has been modified (and conserved) during echinoderm evolution, and point to mechanisms associated with the evolution of a new cell lineage. The skeletogenic GRN has also recently been used to study the long-standing problem of developmental plasticity. Other recent findings have linked this transcriptional GRN to morphoregulatory proteins that control skeletal anatomy. These new studies highlight powerful new ways in which GRNs can be used to dissect development and the evolution of morphogenesis.
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http://dx.doi.org/10.1242/dev.023564DOI Listing
January 2009

Mesenchymal cell fusion in the sea urchin embryo.

Methods Mol Biol 2008 ;475:315-34

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA.

Mesenchymal cells of the sea urchin embryo provide a valuable experimental model for the analysis of cell-cell fusion in vivo. The unsurpassed optical transparency of the sea urchin embryo facilitates analysis of cell fusion in vivo using fluorescent markers and time-lapse three-dimensional imaging. Two populations of mesodermal cells engage in homotypic cell-cell fusion during gastrulation: primary mesenchyme cells and blastocoelar cells. In this chapter, we describe methods for studying the dynamics of cell fusion in living embryos. These methods have been used to analyze the fusion of primary mesenchyme cells and are also applicable to blastocoelar cell fusion. Although the molecular basis of cell fusion in the sea urchin has not been investigated, tools have recently become available that highlight the potential of this experimental model for integrating dynamic morphogenetic behaviors with underlying molecular mechanisms.
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http://dx.doi.org/10.1007/978-1-59745-250-2_18DOI Listing
November 2008
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