maria ina arnone, stazione zoologica anton dohrn napoli, italy EuroMarine 2015 GA meeting, Feb 24th @ SZN Echinoderm models and Gene Regulatory Networks for Development and E volution
maria ina arnone, stazione zoologica anton dohrn napoli, italy EuroMarine 2015 GA meeting, Feb 24th @ SZN
Echinoderm models and Gene Regulatory Networks for Development and Evolution
Outline:
- 1. The sea urchin embryo as laboratory model (historical perspective)
- 2. The sea urchin embryo for gene regulatory biology (key features)
- 3. Genomics, Gene Regulatory Networks, Evolution
- 4. Some examples of GRN studies
- 5. Future directions
Example 1: Evolution of mesoderm and muscle formation
Example 2: Evolution of a GRN controlling gut regionalization
- 1. The sea urchin embryo as laboratory model
http://www.exploratorium.edu/imaging_station/gallery.php?Asset=Sea%20urchin%20-%20pronuclear%20fusion&Group=&Category=Sea%20Urchins&Section=Introduction Sea Urchin Sequencing Consortium. Science, 2006
milestones in sea urchin research
- 1. The sea urchin embryo as laboratory model milestones in sea urchin research
Sea Urchin Sequencing Consortium. Science, 2006
- 1. The sea urchin embryo as laboratory model
amphioxusacorn worm ascidian
The phylogenetic position of the sea urchin relative to other model systems and ourselves
•Defined deuterostome vs chordate-specific vs pan-bilaterian genes •More closely related to chordates than any other non-chordate animals
Sea Urchin Sequencing Consortium. Science, 2006
- 1. The sea urchin embryo as laboratory model
Stongylocentrotus purpuratus
Paracentrotus lividus
- Echinoid species:
T. LepageT.C. Lacalli, University of Saskatchewan Archives
- 1. The sea urchin embryo as laboratory model
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- 2. The sea urchin embryo for gene regulatory biology
>80% of injected zygotes develop normally
DNA is incorporated into a blastomere nucleus at 2nd, 3rd or 4th division
- Biochemistry of TF available
Chromatin IP, ChIP-Seq approaches
Enormous quantities of syncronously developing embryos can be obtained
GFP expression is easily detected in transparent embryos
2-5,000 eggs per experiment can be injected
- Easy gene transfer
x x x2nd
•cis-reg. analysis •reverse genetics • gene knockdown • transgenesis • misexpression
anti-‐SpBra
- 2. The sea urchin embryo for gene regulatory biology
Arnone et al, 1997
cis-regulatory analysis - GFP Intra species cis-reg analysis
CyIIa-GFP P. lividus
• Genomic tools available:
Paracentrotus lividus
• Arrayed cDNA libraries • EST databases (ca 190000) • BAC libraries • BAC sequencing • transcriptomes
Stongylocentrotus purpuratus
• full genome • annotated • Arrayed cDNA libraries • BAC libraries • transcriptomes
<50MY
• Phylogenetic footprint
Baylor College of Medicine
organizers: maria ina arnone mike thorndyke
GENOSCOPE Running genome project
“Sequencing, assembly and annotation of the genome of the sea urchin Paracentrotus lividus”
- 4. Genomics, Gene Regulatory Networks, Evolution
- 3. Genomics, Gene Regulatory Networks, Evolution
http://www.molgen.mpg.de/~ag_seaurchin/
gene expression database
- 3. Genomics, Gene Regulatory Networks, Evolution
The sea urchin endomesoderm GRN
http://www.spbase.org/endomes/#EndomesNetwork/
- 3. Genomics, Gene Regulatory Networks, Evolutionhttp://www.echinobase.org/Echinobase/
- 3. Genomics, Gene Regulatory Networks, Evolution
• Availability of experimentally accessible animals with different/similar mode of development at various evolutionary distance
Arnone, Byrne and Martinez, in press
- 3. Genomics, Gene Regulatory Networks, EvolutionEvolution of regulatory networks: comparison between sea urchin and sea star
Arnone, Byrne and Martinez, in press
- 3. Genomics, Gene Regulatory Networks, Evolution
Evolution of regulatory networks
kernel according to
“GRN and the Evolution of Animal Body Plans”
by Erwin & Davidson, 2006
500 MY
Adapted from Hinman and Davidson, 2007
- 3. Genomics, Gene Regulatory Networks, Evolution
Comparative genomics and transcriptomics in deuterostomes
>540 mya
Ptychodera flava
ECHINODERMATA
Branchiostoma
CHORDATA HEMICHORDATA
ECO-EVO
GRN-EVO
Paracentrotus lividus
EVO-DEVO
CIS-REG
Late development: functional gut and musculature
midgut / stomach
hindgut / intestine esophageal muscles
anus
foregut / esophagus
mouth
72 hpf pluteus larvae
- 4. Some examples of GRN and Evo studies
Example 1: Evolution of mesoderm and muscle formation
Dissecting the GRN controlling muscle formation in sea urchin: 1. defining regulatory state of cells of interest (co-expression with MHC)
phalloidin
MHC AcTub DAPI
48h
72hCarmen Andrikou
Andrikou et al., EvoDevo 2013
Dissecting the GRN controlling muscle formation in sea urchin: 2. perturbation analysis
Example 1: Evolution of mesoderm and muscle formation
FGF signaling through FGFR1 receptor is necessary for muscle formation
A regulatory cascade of Fox family genes is at the core of the GRN controlling muscle formation (quantitative analysis performed with nanostring)
Andrikou et al., submitted
Andrikou et al., submitted
Dissecting the GRN controlling muscle formation in sea urchin: 3. Reconstructing GRN
Developm
ent
Example 1: Evolution of mesoderm and muscle formation
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Example 2: Evolution of a GRN controlling gut regionalization
Rossella Annunziata
Margherita Perillo
Xlox MO injected vs control embryos
RNA Seq analysis: differential transcriptomics
Xlox MO injected
vs control embryos
late
gas
trula
, 48h
late gastrula, 48h
pluteus, 72h
plut
eus,
72h
Ann
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Example 2: Evolution of a GRN controlling gut regionalization
Ann
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Example 2: Evolution of a GRN controlling gut regionalization
Annunziata et al Genesis 2014
Example 2: Evolution of a GRN controlling gut regionalization
ParaHox gene cluster evolution
sea urchin
amphioxus
mouse
annelid
sea star
gsx lox cdx
Ann
unzi
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Example 2: Evolution of a GRN controlling gut regionalization
Example 2: Evolution of a GRN controlling gut regionalization
anus
mouth
stomach intestine
esophagus
anus
mouth
stomach
intestine
esophagus
intestine
mouth
A comparative study of ParaHox genes within Deuterostomes
Larvae = feeding devices
PmLox - PmCdx
4d
4d 3d
SpLox - SpCdx PfLox2 - PfCdx
CLUSTERNO CLUSTER CLUSTER
LoxCdx
Gsx
Arnone et al Dev. Biol. 2006
ParaHox genes temporal and spatial colinearity: conservation and divergence within Ambulacraria
LoxCdx
GsxLoxCdx
Gsx
Annunziata et al, BMC Biol 2013 Ikuta et al, BMC Evol Biol 2013
Example 2: Evolution of a GRN controlling gut regionalization
Comparison with the sea star embryo and larva
Biphasic expression of cdx in sea star
cdx appears at 24h, before Xlox and before hox11/13b
cdx and hox11/13b overlap in the vegetal plate
lateral view
vegetal view
cdx hox11/13b
cdxhox11/13b
Xlox
Example 2: Evolution of a GRN controlling gut regionalization
Results of preliminary comparison between sea urchin and sea star gut GRN:
High conservation of gene expression topology. Different gene interactions. Divergent GRN? What maintains such an invariant topology? Is the etherochrony of Cdx gene expression cause or consequence of the different GRN architecture?
?
?
Example 2: Evolution of a GRN controlling gut regionalization
Comparison within echinoderms: sea urchin vs sea star gut patterning. High conservation of gene expression topology. Different gene interactions. Divergent GRN? What maintains such an invariant topology? We are using a functional genomics approach to answer these questions
ChIP-Seq PA-RNASeq
PA-RNASeq
Xlox
Xlox
Cdx
Cdx
ChIP-Seq PA-RNASeq
PA-RNASeq
Claudia CuomoElijah Lowe
Example 2: Evolution of a GRN controlling gut regionalization
A genome wide comparison downstream of Xlox and Cdx in sea urchin and sea star
organizers: maria ina arnone mike thorndyke
- 5. Future directions
Paracentrotus lividus• Arrayed cDNA libraries • EST databases (ca 190000) • BAC libraries • BAC sequencing • transcriptomes
Past ---> .... Present
Neptune will train a new generation of young researchers by combining the strengths of modern technologies with a real understanding of traditional approaches.
The Neptune consortium involves seven academic institutions and two industrial partners that will provide Neptune fellows with expertise, specialized equipment and training on a wide range of approaches and methodologies incorporated in evolutionary developmental biology and marine neurobiology.
Neptune is a multidisciplinary training network in evo-devo and neurobiology of marine animal models.Through the use of advanced methods of genetic analysis and imaging technologies, Neptune aims at solving an array of important questions in the evolution, development, neurobiology and ecology of marine invertebrates.
CORBEL (Coordinated Research Infrastructures Building Enduring Life-science Services) proposal presented in response to the Horizon 2020 INFRADEV-4 call (INFRADEV-4-2014/2015: Implementation and operation of cross-cutting services and solutions for clusters of ESFRI and other relevant research infrastructure initiatives)
Future?
Use Case 4 “Marine Metazoan Developmental Models for BioMedical research - from predictive integrated databases to functional testing” EMBRC, ELIXIR, Euro-BioImaging and EU-Openscreen will establish harmonised marine metazoan developmental model databases integrating genomic, transcriptomics, morphological, functional genomic and chemical treatment data for a selected set of marine model species, building on existing community resources the EMBRC partners and their collaborators. One set will cover embryogenesis in 4 marine models developed by European researchers characterised by complementary developmental modes: ascidian (Phallusia), amphioxus (Branchiostoma), sea urchin (Paracentrotus) and jellyfish (Clytia). These species form a complementary and powerful set of models in the fields of embryology, regeneration, stem cell biology, physiology, biomedical toxicology and drug testing
- 5. Future directions
Arnone lab: Claudia Cuomo Elijah Lowe Margherita Perillo Evgeniya Anishchenko Giovanni Annona Alberto Valero-Gracia Salvatore D’Aniello
Former members: Carmen Andrikou Rossella Annunziata Enrique Arboleda Alison Cole Silvia Iannaccone Edmondo Iovene Francesca Rizzo Daniela Lovera Libero Petrone Hector Huerga Berta Canal Teresa Mattiello Josefine Stangberg Aaron Towlson
External collaborators: Detlev Arendt
Sam Dupont Maurice Elphick
Harald Hausen Veronica Hinman
Patrick Lehay Pedro Martinez
Paola Oliveri Florian Raible
Eric Roettinger Kristin Tessmar
Uli Technau Esther Ullrich
Yi-Hsien Su
Thanks to