chordate heart evolution: a basal · pdf filechordate heart evolution: a basal view ......
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Chordate heart evolution: A basal view
Brad Davidson University of California, Berkeley
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Chordate Heart Evolution
A Basal View
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Outline1. Introduction to the Tunicates
2. Why study Ciona?
3. The Ciona Heart
4. Mesp regulation
5. Mesp function
6. Evolutionary implications
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Phylogeny
Echinoderms Hemichordates Tunicates Cephalochordates Vertebrates(ascidians)
Protostomes
Chordates
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Chordate Features in Ascidian Larvae
Notochord Dorsal Nerve Cord
MesenchymeGut Rudiment
Low cell numberSmall, compact genome
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Ascidian Metamorphosis
Settlement (20 minutes)(Loss of Chordate Tail)
Juvenile Differentiation(3-7 days)
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Outline1. Introduction to the Tunicates
2. Why study Ciona?
3. The Ciona Heart
4. Mesp regulation
5. Mesp function
6. Evolutionary implications
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From Satoh ‘04
-Rapid Embryogenesis-Low Cell number-Transparent blastomeres-Can isolate blastomeres-Settle and rear in lab
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- Defined Lineages
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Compact Genome/electroporationRegulatory DNA identification -
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Fertilize500-5000 eggs
Rinse Dechorionate Electroporate10-16 constructs
Grow5-20 hoursStain
10-24 hours
100-500 eggs+
100ug DNA
Electroporation - < 48 Hour reporter assay2000bp Mesp
2000bp Lac-Z
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Stable TransgenicsTime to maturation : 3 months; Life span 1-2years
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Single orthologs/morpholino injections
Echinoderms Hemichordates Tunicates Cephalochordates Vertebrates(ascidians)
Protostomes
Chordates
Duplications
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Tbx6aTbx6b*Tbx6c
Brachyury
Tail muscle genes
Notochord genesTropomyosin*+39 characterized Brachyury target genes
TVC/heart genes3 genes downstreamof Mesp
TLC/blood genes
Endoderm genes
Hand-like
MyoD?
Lhx3
Zic-L
Twist-like1
Mesp
Fgf 9/16/20
Zic-L
10 genes downstream ofTwist-like1
Macho-1
FoxD*
ß-Catenin
10 genes downstream ofTbx6/ZicL
Snail*
Mesenchyme genes10 genes downstream ofTwist-like1 +3 specific to B7.7 lineage
TTF1
Wnt5*Wnt3*Wnte*ADMP*Bmp3*Bmp5/7Lefty*
- Promise of comprehensive gene networks
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Outline1. Introduction to the Tunicates
2. Why study Ciona?
3. Why study Ciona heart development?
4. Mesp regulation
5. Mesp function
6. Evolutionary implications
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2. During gastrulation Bmp (+ ?) secreted by endoderm initiates cardiac specification.
Ant. Lateral (Cardiac) Post. Lateral (blood)Endoderm
A P
3. Induction is prevented in posterior by Wnt signals (blocked by DKK in anterior)
A PWntWntDkk
A P Heart genes:Nkx2.5, Gata, etc.
Chordamesoderm
1. Prior to gastrulation mesodermal cell fates are unspecified.
Current Model for Vertebrate Heart Specification
Posterior Lateral (blood)Ant. Lateral (Cardiac)Paraxial (somite)
Fly
Tinman, Pannier
Dpp Ectoderm maintains
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2. During gastrulation Dpp (+ ?) secreted by dorsal ectoderm maintains Tinman, activates Pannier.
Dorsal Ectoderm
Middle mesoderm - visceral muscle
1. Prior to gastrulation the heart factor Tinman (Nkx2.5) is expressed in all mesoderm.
Current Model for Fly Heart Specification
Dorsal Mesoderm (heart)
Ventral mesoderm - body wall muscle
Middle mesoderm - visceral muscleDorsal Mesoderm (heart)
Ventral mesoderm - body wall muscle
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Wnt-11
Bmp
Fgf
Shh
Notch
Wnt-1
HeartStructural
GenesGata 4
Nkx 2.5
Vertebrate Heart Gene Network
Conundrums...
1. Initial signals?2. Key transcription factors are redundant.3. What are the interconnections? (Black Boxes)
Initial Signal?
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Pericardium / Myocardium
Ciona heart morphology
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Heart movieQuickTime™ and a
DV/DVCPRO - NTSC decompressorare needed to see this picture.
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Ci-Bmp2/4 Ci-GataA Ci-Nkx
Tvc-Hand-like
Tail-Raldh
Ciona heart lineage - conservation of “core” heart network
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?
??
?
??
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Early Middle Late
Constructing a Ciona heart network
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Heart Evolution
Protostomes
Chordates
Dorsal vessel
Conserved “initial” heart specification (Bmp/Nkx/Gata)
Ventral/AnteriorHeart
Multiple chambers
(atria/ventricles)
Modified from Simoes-Costa et al. ‘04
1. Chordate specific heart genes?Why recruited?
2. Chamber evolution.Ancestral 1 or 2?Conserved Genes?
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Outline1. Introduction to the Tunicates
2. Why study Ciona?
3. Why study Ciona heart development?
4. Mesp regulation
5. Mesp function
6. Evolutionary implications
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Mesp (bHLH) and Heart Cell Migration:
Ascidian Mesp Expression: Function: Normal Mesp-KO (Satou et al., 04)
Mouse:(Kitajima, Saga, 1999,2000)
• Mesp1 expression Function: blue cells = Mesp1/2 KO
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Two Questions about Mesp
•What activates Mesp?
•Is Mesp involved in Migration or Specification?
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Outline1. Introduction to the Tunicates
2. Why study Ciona?
3. Why study Ciona heart development?
4. Mesp regulation
5. Mesp function
6. Evolutionary implications
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Manipulation of Mesp function
1. Isolate DNA Binding Domain
Mesp bHLH -WRPW
-Vp16
2. Attach 3’ domain 3. Express in heart lineage
Mesp Enhancer
Mesp Enhancer
Repression
Activation
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MESP Heart SpecificationTBX6c
Repressor X Migration
Mesp KO Mesp Mesp
TVCs (Hndx)migrate and
divide
Anterior Tail Muscle
(Raldh2)
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Outline1. Introduction to the Tunicates2. Why study Ciona?3. Why study Ciona heart development?4. Mesp regulation5. Mesp function
6. Evolutionary implications • a. Early vertebrate heart specification• b. Recruitment of Mesp in chordate heart dev.• c. Mesp and the Germ Plasm• d. Chamber evolution
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In Vertebrates:Mesp initially induces a broad potential cardiac field
Vertebrate Ciona
Tbx6
Mesp on
Cardiac/paraxial (somite) Cardiac/Tail muscleA P A P
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2. During gastrulation Bmp (+ ?) secreted by endoderm initiates cardiac specification.
Ant. Lateral (Cardiac) Post. Lateral (blood)Endoderm
A P
3. Induction is prevented in posterior by Wnt signals (blocked by DKK in anterior)
A PWntWntDkk
A P Heart genes:Nkx2.5, Gata, etc.
Chordamesoderm
1. Prior to gastrulation mesodermal cell fates are unspecified.
Current Model for Vertebrate Heart Specification
Posterior Lateral (blood)Ant. Lateral (Cardiac)Paraxial (somite)
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New Model for Vertebrate Heart Specification
New Vertebrate Model: Endoderm signal for maintenance, not inducer.
1. Tbx -> Mesp creates initial broad potential heart field.
2. Endoderm signal (Bmp, et al.) maintain heart fate, Wnt permits re-specification.
A PWntWntDkk
Tbx6
MespAnt. Paraxial (Cardiac) + Post. Paraxial (blood/somite)
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Heart Evolution
Echinoderms Hemichordates Tunicates Cephalochordates Vertebrates(ascidians)
Protostomes
Dorsal Sac?Dorsal Heart
Vesicle? ChordatesDorsal vessel
Conserved heart maintenance(Bmp/Nkx/Gata)
Ventral/AnteriorHeart
Multiple chambers
(atria/ventricles)
Early Heart SpecificationAnd Migration(Mesp)
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Recruitment of Mesp during chordate heart evolution?
Known: Only Mesp ortholog in flies (Sage) is expressed in salivary glands.
Assumption: Role for Mesp in heart/segmentation clock is specific to chordates.
{Test by expression assays in hemichordates, echinoderms, annelids and arthropods.}
Hypotheses:A. Evolution towards a muscular post-anal tail for chordate locomotion drove…
• Recruitment of Mesp to delay mesoderm differentiation, permitting dense clusters of muscle precursors to form = somites.
B. Lack of posterior heart lineage drove need for delayed heart differentiation.
C. Lack of Mesp in segmentation clock/heart is specific to Drosophila.
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Germ line Silencing(Tomioka et al; 2002)(Nishida)
VegetalSide
Mouse Mesp (Mesoderm Posterior) first isolated in subtractive screen for expression in primordial germ cells (Saga et al. 96)
Mesp and the Germ Plasm
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1. Evolutionary implicationsa. Early vertebrate heart specification
b. Recruitment of Mesp in chordate heart dev.
c. Mesp and the Germ Plasm
d. Chamber evolution
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1. In vertebrates and Ciona, Raldh expression domain is initiallycaudal to heart field.
2. In vertebrates Raldh expression domain later spreads rostrallyto help specify the atrial (inflow) chamber.
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1. Raldh has conserved role in setting posterior/caudal limit of Heart
2. Raldh domain in Ciona (anterior tail muscle) is the precursor tothe inflow (atrial) field in vertebrates.
3. Raldh domain in Ciona is remnant of atrial field.
4. Caudal-Rostral wave of Raldh in Ciona….a. patterned the endoderm and was recruited for heartb. patterned ancestral chambers and was lost in Cionac. patterns chamber precursor domains in Ciona heart
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Thanks to:
Mike Levine
Weiyang Shi
Kasumi Yagi
NIH