Comparative Lactation BiologyMamExp: an interactive gene expression browser

for collaborative comparative lactation genomics

Christophe Lefèvre , ITRI, BioDeakin, GTP Deakin University

Kevin Nicholas

Julie Sharp

Karensa Menzies

Pragati Sharma

Sanjana Kuruppath

Amit Kumar

Philip Church

James Stillman

Shannu Palamuru

VPAC

Adam Wong

Zoology Uni.Melbourne

Matthew Digby

John Arnould

Sonia Mailer

Cate Pooley

Kylie Cane

Elizabeth Pharo

Coralie Reich

Alison DeLeo

Elie Khalil

Danny Topcic

Joly kwek

Rachael O’Dowd

Amelia Brennan

Karensa Menzies

Maurice Ling

V.B.C, monash

David Powell

Yvan Strahm

Torsten Seeman

Reprogen Uni. Sydney

Keith L. Macmilland

Paul A Sheehy

Peggy Rismiller, University of Adelaide, SA

Katherine Belov, Faculty of Veterinary Science, University of Sydney, NSW platypus genome consortium

Exploring lactation diversity

Comparative genomics & transcriptomics of the lactation system

Molecular interface between mother and child

Evolution/adaptation of the lactation programfrom echidna and wallaby to man...

Comparative lactation biology:

Mammalian diversity…

GENETIC RESOURCES - biodiversity approach

GENETIC RESOURCES - biodiversity approach

Using comparative genomics

Mammal evolution:

fossil dating

Mining 200 Million years of lactation …

Mammal evolution: Mammalian super-tree

The delayed rise of present-day mammals. Olaf R. P. et al. Nature Vol 446 | 29 March 2007

Mammal evolution: Mammalian super-tree

The delayed rise of present-day mammals. Olaf R. P. et al. Nature Vol 446 | 29 March 2007

Mammal evolution: Mammalian super-tree

The delayed rise of present-day mammals. Olaf R. P. et al. Nature Vol 446 | 29 March 2007

Research Platforms

Genomics

Bioinformatics

Proteomics Bioactives

metabolics

Lactation

program

gene discoverygene regulation

gene evolution

sequencing

Genome analysis

sequencing

Expression microarray

Protein prediction

signal prediction

Function prediction

Web query interface generate dynamic annotation (php scripts)

Gene index sequence annotation Bio-activity prediction gene expression regulation

Gene discovery

Raw sequence data

QC sequence

Assemblies Sequence data

Annotation

TRIM

PHRED base calling

CROSS MATCH vector removal

BLAST

Unigene

Nr protein

Genbank

Genomes

PHRAP

Predicted protein data

Signal

prediction

Ipsort,

Spscan,

hmm

Translate

ESTscan

consed

Function

prediction

sequence tags counts Microarray data

annotated

expression tags

annotated

differential expression

Mammary gland

EST sequencingMPSS cDNA Microarray

Tag to EST matching

Normalization

Analysis

- R/Bioconductor

Microarray layout

hmm

Pfam

Peptide analysis

Wallaby

databasemySQL

Discovery engine Machine Learning: marsupial models (hmm)

Pattern matching: gene networks

Data mining: candidate genes

Bioinformatics pipeline

data

Est

sequencesBlast

hits

Pfam

hits

Protein

sequences

Sequence

similarity

search

Blast

HMM

PfamProtein Domain

search

ESTscan2Protein Prediction

Est

sequences

Protein

sequences

contig

sequences

Sequence

assemblyLoad inte

rfa

ce

query

inte

rface

Job management interface

EST-PAC

EST-PAC a web package for EST annotation and protein sequence predictionYvan Strahm , David Powell and Christophe Lefèvre. Source Code for Biology and Medicine 2006, 1:2

Percentage identity

humandog

Distribution of similarity between assembled seal

EST’s

Using estpac…

Lefevre, C. M., Sharp, J. A., & Nicholas, K. R. (2010)Annual review of human genomics

Evolution of lactation

Monotreme

lactation

Monotreme lactation- short gestation period of 21-23 days

- egg incubation for 10-12 days

- lactation 120 to 200 days

milk is delivered to a highly altricial young.

Method

Milk cell cDNA sequencing

EST assembly

EST & contig annotation

Genome mapping

Contig clustering

EST to contig mapping (expression)

Comparative genomics

BLG

CSN2

CSN2b

C6orf58

?

CSN3

50%

8%

10%

23%

91%

Platypus milk cDNA library

87 ESTs (after removal of 20% rRNA +10% low quality)

1667 ESTs 58 genes

-Milk cell sequencing identifies milk protein sequences

-Milk cell gene expression

-High level of major milk protein

-Overall similar profiles in echidna and platypus

-Consistent with other mammals

-C8orf58:

-new monotreme milk protein ?

- alpha, beta, kappa and beta like caseins in echidna

- alpha-casein in platypus?

- gap in the platypus genome sequence

- CSN1 cDNA sequence confirmed by PCR

Caseins• three classes of phosphorylated micelles forming caseins:

- Related to Ca binding protein involved in bone and dentition

- alpha-caseins (CSN1s1, and CSN1s2 in bovine milk) . precipitated by very low calcium concentrations. have the highest phosphate content. no discernable secondary structure . the least conserved caseins

- Beta-casein: CSN2. precipitated by moderate calcium concentrations . have some secondary structure . moderately conserved

- kappa-caseins: CSN3. insensitive to calcium. essential for micelle formation. most conserved caseins

• rapidly evolving cluster of lactation specific genes • single CSN1 in marsupial• short exons, all in-frame• splice variants common in eutherians• phosphorylation motif straddle exon junction

Ca sensitive

in the platypus genome sequence

casein genes are tightly clustered

on 100kb of ultracontig362

Ensembl, assembly gaps: CSN1, beta 5’UTR & promoter …

short in-frame exons, low similarity with therian caseins

CSN2

CSN1

CSN2b (CSN1s2)

CSN3

Montreme CSN1: alpha-casein

80% identity

1 splice variant long variant platypus pcr is minor in echidna 1/6

>14 exons (gap) in-frame

3 cannonical P sites (exon junction)

Montreme CSN2: beta-casein

64% id.

10 exonscoding in-frame

Long exon VIII

3 echidna variants

2 P sitesP site junctions

Montreme CSN2b: beta-like casein

70% id.

15 exonscoding in-frame

Spice variantsexonIIb echidna

exons IV, VI, VIII, IX

3 P sites2xP sites in CSN1s2

Montreme CSN3: kappa-casein

70% id.

5 exons

1 splice variant

CSN1CSN1S2-like

CSN2

phyllogenetic tree of calcium sensitive caseins

Partial duplication of CSN2 / CSN2b in monotreme lineage

1

5

43

2

6

Sequence similarity in the duplicated region

covering the first 5 exons of CSN2

Presence of P site and in-frame exons in the remaining

of CSN2b suggest an ancestral casein (CSN1s2-like?)

was already present at that position

Comparison of mammalian casein gene clusters

0 0.05 0.1 0.15 0.2 0.25 0.3 Mb

CSN2

CSN2

CSN1S1 CSN3

CSN2

CSN1S2STAT HIS3 HIS1

NP_999876.1

ODAM

FDSCP

CSN1S2b

CSN1 CSN2b CSN3

CSN1 CSN3Odam

CSN1S1

CSN2

CSN3CSN1S2 OdamHIS STAT

csnkcsna Csn1s2a Csn1s2b OdamSTAT HIS

csnb

AK05291

Platypus ultra362

Opossum chr.5

Cattle chr.6

Mouse chr.5

Human chr.4

CSN1 CSN1S2 CSN3CSN2

CSN1 CSN3CSN2

Model 1

Model 2

Ancestral locus

1) CSN2 duplication

2) Homologous recombination CSN2/CSN1S2

1) Transposon invasion

2) CSN1S2 deletion

CSN1S2 duplication

1) CSN1S1 duplication

CSN-s2 duplication

Alternative models for the evolution of caseins(work in progress…)

Monotreme genomics:

- Ancient origin of the casein cluster

- Conserved genomic organisation-Beta-casein promoter un-characterised …

- lineage specific rearrangements

- role in lactation function?

-Comparative genomics and lactation evolution…

-Highly sensitive comparative sequence analysis will be

required together with high resolution genome sequence

EVOLUTION OF WAP GENE STRUCTURE

– proposal of an ancient WAP progenitor

• The apparently dispensable role of WAP in eutherians, in particular humans and domesticated animals such as

the cow, goat and sheep suggests the evolution of WAP may be related to diet of the young which is being

adequately compensated in these species or maybe linked to improved care and hygiene in domesticated life.

ŅMolecular evolution of Monotreme and marsup ial whey acidic protein genesÓ. Julie Anne

Sharp, Christophe Lefevre, Kevin R Nicholas. Evol Dev. 2007 Jul-Aug;9(4):378-92.

IIBIIII

tammar lactation program phases

Phase 1

Pregnant

Proliferation

Differentiation

Little or no milk

production

Phase 2A

Attached

to nipple

Production of:

Carbohydrates

Protein

Lipid

Bioactives

Phase 2B

In pouch

on and off

nipple

Production of:

Carbohydrates

Protein

Lipid

Bioactives

Phase 3

In and out

of pouch

Large increases in:

Protein productionLipid production

Large decreases in:

Carbohydrate production

WAPELP LLP-A, LLP-B, b-gal

b-1,3 galactosyltransferase

a-casein, b-casein, a-lactalbumin, b-lactoglobulin

-26 0 50 100 150 200 250 300 350

Asynchronous Concurrent Lactation:a model to identify systemic and local regulators of lactation

High Volume

High Lipids

Low Carbohydrates

Higher Protein

Low Volume

Low Lipids

High Carbohydrates

Lower Protein

•Adjacent glands secreting milk of very different composition

•Local control of milk composition

•Local and endocrine control interactions

Does the composition of milk effect

development of the young ?

-26 0 50 100 150 200 250 300 350Phase 1 Phase 3Phase 2A Phase 2B

60 day old pouch young were fostered onto

teats producing milk for 110 day old young

Animals were examined at 172 days of age by which

time they were receiving day 222 milk

Foster Pouch Young

172 days old

Receiving milk from day 222 of lactation

Control Pouch Young

172 days old

Receiving day 172 milk

Milk composition makes a big difference!

Nicholas and al. 1997

Quantitative gene expression from mammary gland EST sequencing

Proportional transcript composition of cDNA libraries

Complexity (singleton)

Good quantification of highly expressed transcripts only

Limitations: sequencing depth and complexity

Gene expression profiling from lactation stage specific

mammary gland transcriptome sequencing

EST library sequencing MPSS

differential expression of the secretome during lactation

Comparative biology

Gene identification

Bioinformatics

e-research platforms

Biotechnology

MammoSapiens

Gene regulation…

NormalizedUn-normalized

Array Fabrication Spotting

•Use PCR to amplify DNA

•Robotic "pen" deposits DNA at defined coordinates on glass slides

•approximately 1-10 ng per spot

Expression series

database

Oligo

microarray

MPSS

cDNA

microarray

Load inte

rfa

ce

query

inte

rface

management interface

eResource for expression data analysis

Preprocessing

Normalisation

Statistical test

EST-PAC

baab

-cas-cas-lac-LG

ELP WAP

100 12515

b -1,3-GAT

b -1,4 GATLLP-B

LLP-A

20017515050 7525 225 2500

b -gal

-26

Phase 2A

Permanent attachment to teat

Phase 2B

On and off teat

Always in pouch

Phase 1

Pregnant

Phase 3

In and out of Pouch

Beta casein

alpha casein

elp

20

40

60

95 96 106 144 144 169 172 174 198 202 231202 242 244 275 276276140

WAP

b-LG623955623955

Lactation (days)

0

WAP mRNA

Phase 2A Phase 2B Phase 3

Condition tree

Distance

1000 (5/70)

Clustering of microarrays

Liver

Brain

Seal Preg/Lact

LL

P

EL

3

2b

c

More than 80 secreted proteins during

lactation

2A 2B 31Phase

Sharp et al (2009) Milk proteins: from expression to food p55

Cross-fostering study

Mother at day

120 lactation

Control group

PY day 120 PY day 120 Mother at day

170 lactation

Fostered group

60 days period

Fostered PY had access to

day 170 - 230 milk

Control PY had access to

day 120 - 180 milk

Fostered PY had accelerated growth

Initial 200 days post-partum ~ 230 days post-partum

• Forestomach is

gastric-like

– Has gastric cells

• Forestomach matures

into cardia-like

– Lost gastric cells

Forestomach with

gastric cellsForestomach with

NO�gastric cells

Genes highly expressed /

“unique” to forestomach

with gastric cells

Genes highly expressed /

“unique” to forestomach

with NO gastric cells

ATP4A

GKN2

GHRL

NDRG2

MUC4

KRT20

CSTB

ITLN2

LPLUNC1Kwek et al. (2008) J Exp Zool B Mol Evol Dev

PY 170 PY 230

Gastric phenotype gene expression

* P < 0.001 compared to control PY 180 ＃ P < 0.001 compared to control PY 230

D NDRG2C GHRLB GKN2A ATP4A

Down-regulated in the fostered PY forestomach

Cardia phenotype gene expression

* P < 0.001 compared to control PY 180 ＃ P < 0.001 compared to control PY 230

A MUC4 B KRT20 C CSTB D ITLN2 E LPLUNC1

Remained unchanged in the fostered PY

forestomach

conclusion

Milk switching accelerates growth

And influences forestomach differentiation

• Small body size

• 10-12 month lactation

• Long foraging trips

• Milk production at 20% while foraging

• Controlled at the transcriptional level

• Milk volume increases with each trip

2-3 days

23 days

Otariids (fur seal)

– foraging strategy

Comparative biology

Bioinformatics

e-research platforms

Biotechnology

MammoSapiens

GENE LOSS MAY ACCOUNT FOR REDUCED INVOLUTION

STRATEGY TO REDUCE INVOLUTION / EXTEND LACTATION

Comparative Genomics

cDNA sequencing

canine microarray

Est

sequencesBlast

hits

Sequence

similarity

search

Blast

gene

Sequences

Unigene

ensemblLoad inte

rfa

ce

query

inte

rface

Job management interface

compEXP-PAC

Expression series

database

Oligo

Microarray

cDNA

Microarray

MPSS

EST-PAC

EXP-PAC

MamExp: online retrieval of expression data across experiments and species

Gene mapping

Comparative biology

Bioinformatics

e-research platforms

Biotechnology

Essential pathwaysMilk composition, synthesis and function

MammoSapiens

Comparative Transcriptomics

Folate receptor α

Phase 2B – 3 lactation

1229 1980

711

*9220

seal wallaby

cow

x

*2617 probes

~26%

Preg – lactation

Folate receptor identified:comparative microarray analysis

also up-regulated in

•human & mouse lactating

mammary glands

•IFP cultured cow & mouse

mammary explants

* Limitation: gene transcripts only identified if the gene probe is

present on both the canine and bovine Affymetrix genechips as

well as the wallaby EST array.

Preg – lactation

lactation bioinformatics

-Conclusion

Comparative milk transcriptomics

Developing�on line resources for analysis of genomic data:

-facilitate data storage, visualization and analysis on line

-across: partners, experimental platforms, species..

Learning from the data…

MamExpressionist: portal to gene expression

Challenges:

Integrating expression data analysis, pathway, genomes, other public data & literature…

Comparative genomics … collaborative analysis?

Comparative Lactation BiologyMamExp: an interactive gene expression browser

for collaborative comparative lactation genomics

Christophe Lefèvre , ITRI, BioDeakin, GTP Deakin University

Kevin Nicholas

Julie Sharp

Karensa Menzies

Pragati Sharma

Sanjana Kuruppath

Amit Kumar

Philip Church

James Stillman

Shannu Palamuru

VPAC

Adam Wong

Zoology Uni.Melbourne

Matthew Digby

John Arnould

Sonia Mailer

Cate Pooley

Kylie Cane

Elizabeth Pharo

Coralie Reich

Alison DeLeo

Elie Khalil

Danny Topcic

Joly kwek

Rachael O’Dowd

Amelia Brennan

Karensa Menzies

Maurice Ling

V.B.C, monash

David Powell

Yvan Strahm

Torsten Seeman

Reprogen Uni. Sydney

Keith L. Macmilland

Paul A Sheehy

Peggy Rismiller, University of Adelaide, SA

Katherine Belov, Faculty of Veterinary Science, University of Sydney, NSW platypus genome consortium

?

!

Cluster analysis of in vivo cow mammary and in vitro mammary

explant cultures expression data.

Involution lactation pregnancy in vitro

Separation in vivo / in vitro & sample source.

colostrum ?. sFPI(Karensa) versus IFP(Amelia)