Structure, function, and temporal expression of Fibrillin in Danio rerio.

Christian Francom, Jeffrey Caldwell, Barbara Trask, Ph.D.

Extracellular MatrixFunctions

Structure—tense or elasticCellular AdhesionCellular MotilityMolecular Reservoir

Elastic Extracellular MatricesComponent parts

ElastinMicrofibrils

Fibrillins MAGPs Fibulins Other proteins and proteoglycans

Disorders involving the Elastic ECMElastin SVASMicrofibrils Marfan Syndrome

QuestionHow do the microfibrillar proteins contribute

to the function of the elastic extracellular matrix?When and where is each expressed?What inter-molecular interactions are

maintained?

Model OrganismZebrafish (Danio rerio)

External fertilization and development

Colorless during the first few days of development

Genome sequenced (although not assembled)

**first evolutionary organism to have functional elastic extracellular matrix

Easily maintainedhttp://www.pdn.cam.ac.uk/staff/lewis/zfish3AgesSlide.jpg

Stages of Z-fish Development

Sequences and PrimersFibrillin

atggcagcctgtcctgaattaatcttggaaagagagggaggagaaggagtggatgggggattctttaaggcgaagatgactaaggatttggtggaacgggagattcgcatcacagatgtgcagccggcaaatctgcagcaactgcgtgatgctgtcatctcaaaattggccaaaatctctgaggaatatttccagtaccttgttgaatctttgccacaaagaattgaggcatttgtgaaagcaaaaaggatgtccaacctggagggagccccaggctccaggatattctcagctcaggatgtgaacgagtgcaacgaccccaacacctgcatcagcggtctgtgtctgaacacccctggcagctacatctgcagctgcccccccgactttgagctcaatcccactggagtcggctgcgtcgacactcgctctggaaactgctacctggacgtccagtaccgaggcgatttctctgagagtctgagctgctccaatgagattggagtgggagtgtccaaagcgtcctgctgctgttctaggggtgtggcctggggacagccctgcgaggcctgtccatcactcaactccaagatctgtttcactgcacaacctgaaatctccagtctggtgcagggagagaccagccagtgcttccccaggaggagtatccagtgctgggacacattagcggctcattcccttgggcaagcctggagctcagatggccagctgcacgtttccactcttcatctttgttgtggtgtgaaagaggagaaggagggtttaggaaaacaaaaaagcagtggacagagtgatgttacaaagacaacaggcttcacacagagctctgacagtcgcgtcaaagcgagtgcgctgttattaaaatggtaccgtcccagtgacagctctcacagctttatttttgcagtgtcaggactacggtccaatcatgcaatagtagaggacatccgtctgttcaacagtaagcaatatggagctgagccgctgaaagaactgctcaaactactaccagaagcagatgaggtgaagagattgagagaattcaaaggagacgcaaataagctgacactggttgattccttcatgttccagctcattcaagtaccacgctttgatgtgcgtatcgaggtcatggtacttcaagaagagtttttcccattatgtactgccatgagtcgtgacatcaacattgtgcgccaggcaactgaagctgtgcctgtattagccttctctaagaaaccaatggctcatgaccttttagtcctgacaggtggctatgcaggaaacgctgtaggcttcaaattgtcctctcttctctctctggctgacacaaaggccaacaagcctggcatgaacctgctccattttgtggcattactctcgaggatcttctctgtgttttcacagggtggctatgcaggaaacgctgtaggcttcaaattgtcctctcttctctctctggctgacacaaaggccaacaagcctggcatgaacctgctccattttgtggcattagaagcacagaaaaaggatgaggcacttttaaagttcccagagaagctaacacatattcagagtgcagccagaatttcagtagaaaatatagaaggagagttttcctcactctatgtgaaaacacggtctttggaacagaagattcaagatgacgatgagctgcaaaagcagctagatccatttttgcagagctctgcaaaagccttgcaggaccttaagcaccgcaggctggacttgcgcaaagagggtaatgccctcattgacttcttctgtgaagacaaagacaccttcaaattggatgagtgcttcagaatcttccaagatttctgtctaaagttcaaaaaagctgtgaaggacaactcggacagagaactgaaagaggaagctagacagcgccgcttacgggaactagaggagagacggtgtgcctggggtgcagccaatattgaaacaggtggtaatggaggttttggtcgcagcagcagtgaaaatgatgttgacatcttaaccaaagatggcctcctagacttcctgcttcaatcaagacctcaaagcccccacagtcctttaggacgctcagctagtgctcgccgctaccaccacacatctagtgaccgcagcctccgtggctatatggagctggtctctgggtcttcagcaagtgaatacgccaaattcagcagcctgccacgttcaagcaggaaccatcaaagaaagaccatgccatggcttgtcttgcaggatgataatagagagttgattccacaaagtcaggtttaccaagagaagttagcagtttcacaaagggttgagactgaaccaatcagtcctttggcaagatactcaaatttaggctacaacttgaacagtgatccctatagtaataataataacaattatagttctctttctgaaggtggtcttacacctcgattaaataaaacaaagaatgtctttcaggggacatctggtcaaggcattggacacatgaatgtttctgttgagaaacgcaaactagtgcctgggcttcaaccctttgagataaccagccagatcaataataacagtcatgtttctttggacaatcaacatgaagtgtgtgtgactgatctggaacgagaagaccttcccaaagcttttgttcttgatacaccaccatctagtagaagttctgaggaggggcataagctagaaaaagtatggaatgacaaaatcatctcatcacagagagacgaagcagacacaagtaccttctcttcaacatcttgtgacaccccattgccccttgatccctccagtaaaaaacctgtgttttatgtcatggactgcactgaaacggattgttccattgtgttggactgttctgagattgagagttctcctattataaaagaaggacctggacttatcccaaagttacaggatgtcaactttccaagaatcattcaagacaccaattctctttcatccaattttgagtcttcagactgtcactttgtgtccacatcaagtgatgaaccatttgtaggaagggtcgatcaaacttcagaatcagtcagtccatctgtaacaactgaagaagcagagacagacagctgtgacacagctgaagggcaaagagttgatgagaaaggtgttcagacaggtagccccaaaacgacacttatcaaaaccaagacttcatccaaagggatgactgctcacaccagccgtcctacaagaaccctcacagctactgagactcagaacatgcgaaaagtagtgcccattacaaggcaaaatcgatctagtagcagcatgagaaaggttgataaaccacctgtacatgagagctctgaacccagacgtccatttcgagaccaaagcatgccagccagaagaggcgagagacaaggtagacctacaagacattcaagccttccaccagaggatcctaggacaccaagaggttcgacaccaagcagctctcgttggggaagggactcaattcaacacaggccctccatcaagaaagccaatgccaaacctgttcgtaacattcctaaaccagcaccagaggaaaagatgtgtcgctctacaatgcgtgccttggctcaagctcaggctcaaggaacctcagaaggaggtaccgctgacagccctaacaccaccccaaaaagttcttcacctttgcctagctttgctcgcaacactgtggcatctacttctagatctaaaaaagacatgccatcagcaactgtaaccggaacaccatcaaagagcctatcgagaactagctctcagagattgcctggtggtagaacagagtctgtcagtagcgggcctttaccaaggagtgaggataaactaggaggctcgattaggagggttcaaagtgtaagagcttccagtcgaagcagtgacacaccttcacaatctggtcgggatcactcccttaaaagtagcagtttctcagacagatccacaccaactagagacaccatcatctccagcaggaccagcaaacccacatggaaatag

MAGPatgagactctgttacctgcttctgctgtcactgccgggtctgctggctcaggtgcagaactatgatgaggattatacagacatcttcggtaacagcaacaactacgacaaccccttcttctaccagcagcagatattacagacgcagacgtcagggcctgaacagaacgcgctggatggacagacagagccgactgaacctggaccgctggactgtcgtgaggagcagtatccctgcactcggctctattctgtccatcagccgtgtaaacagtgtctgaacagcatctgcttctacagtctgcgcaggatgtatgtgatcaataaggaggtgtgtgtgcggacggtgtgtgcacatgaggagctcttgagagcggatctgtgtcgtgaccagttcccccgctgtggtgtaatggcgcacattggccagtgtggggcgatggggagcaactgcgccaggagttgtggatcctgctaa

Expression DataFibrillin MAGP

Fibrillin

Actin Control

MAGP

0

50

100

150

200

8 Som 21 Som 24 hpf 35 hpf 52 hpf 75 hpf 96 hpf 120 hpf

Developmental Timepoint

Rela

tive

Inte

nsity

Fibrillin

0

50

100

150

200

8 Som 21Som

24 hpf 35 hpf 52 hpf 75 hpf 96 hpf 120 hpf

Developmental Timepoint

Aver

age

Rela

tive

Inte

nsity

MAGPGAPDH Control

Future PossibilitiesMore replicates should be done

Analysis with Real-Time PCRAlternative primers should be madeIn situ hybridization should localize

expression

QuestionHow do the microfibrillar proteins contribute

to the function of the elastic extracellular matrix?When and where is each expressed?What inter-molecular interactions are

maintained?

Intermolecular interactionsFibrillin interacts with: MAGP interacts with:

MAGP CSPG LTGF-β Others?

Fibrillins CSPG Elastin Others?

Mammalian Structure of Fibrillin-1

Fibrillin-1

3 5 6

Isolated Bands of LTGF-β Domains

3 5 6

300bp200bp100bp

LTGF-β Binding Domains….MAACPELILEREGGEGVDGGFFKAKMTKDLVEREIRITDVQPANLQQLRDAVISKLAKISEEYFQYLVESLPQRIEAFVKAKRMSNLEGAPGSRIFSAQDVNECNDPNTCISGLCLNTPGSYICSCPPDFELNPTGVGCVDTRSGNCYLDVQYRGDFSESLSCSNEIGVGVSKASCCCSRGVAWGQPCEACPSLNSKICFTAQPEISSLVQGETSQCFPRRSIQCWDTLAAHSLGQAWSSDGQLHVSTLHLCCGVKEEKEGLGKQKSSGQSDVTKTTGFTQSSDSRVKASALLLKWYRPSDSSHSFIFAVSGLRSNHAIVEDIRLFNSKQYGAEPLKELLKLLPEADEVKRLREFKGDANKLTLVDSFMFQLIQVPRFDVRIEVMVLQEEFFPLCTAMSRDINIVRQATEAVPVLAFSKKPMAHDLLVLTGGYAGNAVGFKLSSLLSLADTKANKPGMNLLHFVALLSRIFSVFSQGGYAGNAVGFKLSSLLSLADTKANKPGMNLLHFVALEAQKKDEALLKFPEKLTHIQSAARISVENIEGEFSSLYVKTRSLEQKIQDDDELQKQLDPFLQSSAKALQDLKHRRLDLRKEGNALIDFFCEDKDTFKLDECFRIFQDFCLKFKKAVKDNSDRELKEEARQRRLRELEERRCAWGAANIETGGNGGFGRSSSENDVDILTKDGLLDFLLQSRPQSPHSPLGRSASARRYHHTSSDRSLRGYMELVSGSSASEYAKFSSLPRSSRNHQRKTMPWLVLQDDNRELIPQSQVYQEKLAVSQRVETEPISPLARYSNLGYNLNSDPYSNNNNNYSSLSEGGLTPRLNKTKNVFQGTSGQGIGHMNVSVEKRKLVPGLQPFEITSQINNNSHVSLDNQHEVCVTDLEREDLPKAFVLDTPPSSRSSEEGHKLEKVWNDKIISSQRDEADTSTFSSTSCDTPLPLDPSSKKPVFYVMDCTETDCSIVLDCSEIESSPIIKEGPGLIPKLQDVNFPRIIQDTNSLSSNFESSDCHFVSTSSDEPFVGRVDQTSESVSPSVTTEEAETDSCDTAEGQRVDEKGVQTGSPKTTLIKTKTSSKGMTAHTSRPTRTLTATETQNMRKVVPITRQNRSSSSMRKVDKPPVHESSEPRRPFRDQSMPARRGERQGRPTRHSSLPPEDPRTPRGSTPSSSRWGRDSIQHRPSIKKANAKPVRNIPKPAPEEKMCRSTMRALAQAQAQGTSEGGTADSPNTTPKSSSPLPSFARNTVASTSRSKKDMPSATVTGTPSKSLSRTSSQRLPGGRTESVSSGPLPRSEDKLGGSIRRVQSVRASSRSSDTPSQSGRDHSLKSSSFSDRSTPTRDTIISSRTSKPTWK

LTGF-β Binding Domains….MAACPELILEREGGEGVDGGFFKAKMTKDLVEREIRITDVQPANLQQLRDAVISKLAKISEEYFQYLVESLPQRIEAFVKAKRMSNLEGAPGSRIFSAQDVNECNDPNTCISGLCLNTPGSYICSCPPDFELNPTGVGCVDTRSGNCYLDVQYRGDFSESLSCSNEIGVGVSKASCCCSRGVAWGQPCEACPSLNSKICFTAQPEISSLVQGETSQCFPRRSIQCWDTLAAHSLGQAWSSDGQLHVSTLHLCCGVKEEKEGLGKQKSSGQSDVTKTTGFTQSSDSRVKASALLLKWYRPSDSSHSFIFAVSGLRSNHAIVEDIRLFNSKQYGAEPLKELLKLLPEADEVKRLREFKGDANKLTLVDSFMFQLIQVPRFDVRIEVMVLQEEFFPLCTAMSRDINIVRQATEAVPVLAFSKKPMAHDLLVLTGGYAGNAVGFKLSSLLSLADTKANKPGMNLLHFVALLSRIFSVFSQGGYAGNAVGFKLSSLLSLADTKANKPGMNLLHFVALEAQKKDEALLKFPEKLTHIQSAARISVENIEGEFSSLYVKTRSLEQKIQDDDELQKQLDPFLQSSAKALQDLKHRRLDLRKEGNALIDFFCEDKDTFKLDECFRIFQDFCLKFKKAVKDNSDRELKEEARQRRLRELEERRCAWGAANIETGGNGGFGRSSSENDVDILTKDGLLDFLLQSRPQSPHSPLGRSASARRYHHTSSDRSLRGYMELVSGSSASEYAKFSSLPRSSRNHQRKTMPWLVLQDDNRELIPQSQVYQEKLAVSQRVETEPISPLARYSNLGYNLNSDPYSNNNNNYSSLSEGGLTPRLNKTKNVFQGTSGQGIGHMNVSVEKRKLVPGLQPFEITSQINNNSHVSLDNQHEVCVTDLEREDLPKAFVLDTPPSSRSSEEGHKLEKVWNDKIISSQRDEADTSTFSSTSCDTPLPLDPSSKKPVFYVMDCTETDCSIVLDCSEIESSPIIKEGPGLIPKLQDVNFPRIIQDTNSLSSNFESSDCHFVSTSSDEPFVGRVDQTSESVSPSVTTEEAETDSCDTAEGQRVDEKGVQTGSPKTTLIKTKTSSKGMTAHTSRPTRTLTATETQNMRKVVPITRQNRSSSSMRKVDKPPVHESSEPRRPFRDQSMPARRGERQGRPTRHSSLPPEDPRTPRGSTPSSSRWGRDSIQHRPSIKKANAKPVRNIPKPAPEEKMCRSTMRALAQAQAQGTSEGGTADSPNTTPKSSSPLPSFARNTVASTSRSKKDMPSATVTGTPSKSLSRTSSQRLPGGRTESVSSGPLPRSEDKLGGSIRRVQSVRASSRSSDTPSQSGRDHSLKSSSFSDRSTPTRDTIISSRTSKPTWK

Future DirectionsClone for expressionProtein binding studies

AcknowledgementsJosh McBrideAric ElmerOUR