Echinoderm models and Gene Regulatory Networks for ... · maria ina arnone, stazione zoologica anton dohrn napoli, italy EuroMarine 2015 GA meeting, Feb 24th @ SZN Echinoderm models

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

http://www.exploratorium.edu/imaging_station/gallery.php?Asset=Sea%20urchin%20-%20pronuclear%20fusion&Group=&Category=Sea%20Urchins&Section=Introduction

- 1. The sea urchin embryo as laboratory model milestones in sea urchin research

Sea Urchin Sequencing Consortium. Science, 2006


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


Stongylocentrotus purpuratus

Paracentrotus lividus

- Echinoid species:

T. LepageT.C. Lacalli, University of Saskatchewan Archives


Arn

one,

Byr

ne a

nd M

artin

ez, i

n pr

ess

- 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:


• 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”



http://www.molgen.mpg.de/~ag_seaurchin/

gene expression database


The sea urchin endomesoderm GRN

http://www.spbase.org/endomes/#EndomesNetwork/

- 3. Genomics, Gene Regulatory Networks, Evolutionhttp://www.echinobase.org/Echinobase/

http://www.echinobase.org/Echinobase/


• 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


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


Comparative genomics and transcriptomics in deuterostomes

>540 mya

Ptychodera flava

ECHINODERMATA

Branchiostoma

CHORDATA HEMICHORDATA

ECO-EVO

GRN-EVO


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


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


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


Annu

nzia

ta e

t al G

enes

is 2

014


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

unzi

ata

& A

rnon

e, D

evel

opm

ent 2

014


Ann

unzi

ata

& A

rnon

e, D

evel

opm

ent 2

014


Annunziata et al Genesis 2014


ParaHox gene cluster evolution

sea urchin

amphioxus

mouse

annelid

sea star

gsx lox cdx

Ann

unzi

ata

et a

l, B

MC

Bio

l 201

3



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


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


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?

?

?


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


A genome wide comparison downstream of Xlox and Cdx in sea urchin and sea star

organizers: maria ina arnone mike thorndyke


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


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

Documents

Echinoderm models and Gene Regulatory Networks for ... · maria ina arnone, stazione zoologica anton dohrn napoli, italy EuroMarine 2015 GA meeting, Feb 24th @ SZN Echinoderm models