Billie J. Swalla

bjswalla@u.washington.edu

Transcriptome Sequencing Reveals Heterochronic Shift of

Chordate Gene Networks in Molgulid Ascidians

Tunicates

Ambulacraria Chordata

Cephalochordates

Modified from

Swalla et al. 2001

Xenoturbella

Heterochronyhttp://www.bio.miami.edu/dana/dox/heterochrony.html

Allometric Growth Differential growth in organs and body parts

PaedomorphyRetention of juvenile structures

Heterochronyhetero = otherchronos = time

Haekel – 1875de Beer – 1930 “Embryos and Ancestors”

Gould – 1997 “Ontogeny and Phylogeny”

“Few Evolutionary concepts have generated more confusion and controversy than heterochrony” McKinney, 1999

C. Titus Brown

Evolution and Development of Tailless Ascidians

1. Changes in gene expression in regulatory genes

2. Downstream structural genes become pseudogenes (muscle)

3. Heterochrony - changes in timing of gene activation

4. Gene Loss

Molgula clades

Solitary ascidians have determinant

and invariant cleavage.

Some species have colored cytoplasms.

(Boltenia villosa)

The cell lineage is very similar in Ciona, Phallusia,

Halocythia roretzi &Molgula oculata.

Two Species of Closely Related Ascidians and a Hybrid Larva

Molgula oculata has a head, tail, and otolith - chordate embryo.

M. occulta egg X M. oculata sperm has a head, short tail, and otolith.

Molgula occulta lacks chordate features - head, tail, and otolith.

Swalla and Jeffery, 1996

Notochord formation (convergence & extension)

Jiang and Smith, 2007

Notochord Gene Network

Davidson and Christiaen, Cell (2006)

Molgula oculata notochord(40 cells, converged & extended)

Molgula occulta no notochord(20 cells, not converged & extended)

Hybrid notochord(20 cells, converged & extended)

Notochord Formation in Molgulids

Swalla and Jeffery, 1996

Digital normalization

Digital normalization

Lowe, Swalla, Brown, in preparation

Digi Norm assembly vs Raw read assembly

37 1713623

Digi Norm Raw

2446C. intestinalis

M. occulta

64 1513646

Digi Norm Raw

2398C. intestinalis

M. oculata

Lowe, Swalla, Brown, in preparation

Stage specific express

Lowe, Swalla, Brown, in preparation

Notochord gene expression similar to tailed species

Prickle

Veeman, M., et al., 2007

•Planar cell polarity (PCP) pathway

•Involved in convergence and extension

Prickle expressed in notochord cells

Mita et al Zool. Sci., 2010

M. occulta gastrulation Ciona intestinalis

Satoh Nature Reviews Genetics 4, 2003

Hybrid notochord genes allele specific differential express

Notochord Gene Network

Davidson and Christiaen, Cell 2006

Ascidian Metamorphosis

Heterochronic shift of metamorphosis genes in Molgula

Gene Species Cleavage

Gastrula/Neurula

Tailbud Larva Juvenile

META-2 C. intestinalis

M. oculata

M. occulta

M. tectiformis

Echinonectin C. intestinalis

M. oculata

M. occulta

M. tectiformis

50/168 genes show early differential expression in Molgula compared to C. intestinalis

Larval development

Ascidian Development

Heterochronic Shift inMolgulidae Development

*79 genes examined across of six species

Billie Swalla, Nadine Peyrieras, Alberto Stolfi

M. occulta F+ 12 hatching

M. occulta transgenic NoTrlc Alberto Stolfi & Lionel Christiaen

Summary

Both tail and tailless species contain the majority of known notochord genes

Digital Normalization performs as well or slightly better than raw read assembly

Metamorphosis genes are expressed much earlier than in other ascidian species

Early onset of Metamorphosis may be a pre-adaptation to evolution of a tailless embryo

Thanks to Beacon!!

1. Great colleagues!!2. Revived an exciting research

program that benefited greatly from genomics methods

3. Leadership opportunities and support for female faculty

4. Graduate student support for travel and courses.

2012 Swalla Lab

Acknowledgements

University of Washington Michigan State University

• Dr. Titus Brown

• Dr. Kanchan Pavangadka

• Elijah Lowe

• Dr. Billie J. Swalla

• Dr. Charlotte Konikoff• Kristin Andrykovich • Kelly Hennessey• Max Maliska• Lauren Vandepas New York University

Dr. Lionel Christiaen

Dr. Alberto Stolfi

96/98 Sanger-sequenced Molgulid genes contained by global assembly.

69% of M. oculata transcripts found (BLASTX 1e-12) in Ciona proteome.

55% of M. occulta transcripts => Ciona.

How do we evaluate assembly?

Query: 1MDSSNRSHPNAHLQYHTDYNYPPFRRVMLAAVKEGLYHPRLPSLRRMDMDTATHKLPDEH 60 MDSSNRSHPNAHLQYHTDYNYPPFR+VMLAAVKEGLYHPRLPSLRRMDMDTATHKLPDEHSbjct: 50MDSSNRSHPNAHLQYHTDYNYPPFRKVMLAAVKEGLYHPRLPSLRRMDMDTATHKLPDEH 229

Mapping => quantitation

Reference transcriptome required.

Assembly contig lengths

M. oculata M. occulta

Lowe, Swalla, Brown, in preparation

Tailless allele has high expression in hybrids PCP

pathway