Pa#erningdefectsinDhmiceDh arose as a spontaneousmuta,on inmice (Carter, 1954).Dhanimals exhibit reduced numbers of lumbar vertebrae,asymmetricpreaxialhindlimbdefectssuchasdistalshorteningorabsence of the ,bia, and polydactyly, oligodactyly, andtriphalangismofdigit1(Searle,1964;Green,1967;Rooze,1977).AsymmetryofhindlimbskeletaldefectsisoJenobserved;theleJside is more frequently affected than the right (Owen, 2006).TheseposteriorskeletaldefectsandsoJ,ssuedefects (includingasplenia)suggestaprimarydefectinA-PandL-RpaUerning.

TheroleofDominanthemimeliainmorphogenesisofthegutandthemidguttohindguttransi6on

Pei-ChenEmilyHsiehandMaryH.Owen,Ph.D.DepartmentofBiology,SimmonsCollege,Boston,MA

During gastrula,on, the primi,ve streak defines themajor bodyaxes of the mammalian embryo and cells acquire differentiden,,es,dependingontheirposi,onintheembryo.Theseaxesdetermine correct placement, orienta,on and forma,on oforgans;devia,ons in thenormalpaUernmay result indefects inorgans such as heart and skeleton with clinical implica,ons.Muta,ons in genes that affect paUerning in mice may provideimportantinsightstohumandisorders.Dominanthemimelia(Dh),a spontaneous muta,on in mice, provides such a model. Dhanimals exhibit reduced numbers of lumbar vertebrae,asymmetric preaxial hindlimb defects and asplenia, sugges,ng aprimary defect in anterior-posterior and leJ-right paUerning ofthe mesoderm. Prior reports also suggest that the posteriormidgutanditstransi,ontohindgutmaybeaffectedinDhanimals.Thisstudywasundertakentodescribetherangeofdefectsintheadult midgut and midgut to hindgut transi,on, the associa,onwith skeletal defects, and the developmental expression of keypaUerninggenes(Hoxd10and11) inthegutofDhembryosandtheir wild-type liUermates. Hox genes are known to specifysplanchnic mesoderm, which then signals the underlyingendoderm further refining regional boundaries. Gastro-intes,nal(GI) tractswereremovedfromadultanimalsandthedimensionsof the GI regions recorded. Skeletons of these animals werestained using Alizarin Red - Alcian Blue (McLeod, 1980). Dh/+C3HB6micewerematedwith+/+AKR/JmiceandtheaffectedF1females backcrossed to +/+ AKR/J males. F2 embryos wereharvestedat10to12daysingesta,on(plugdate=0),head,ssuesremoved for genotyping, and carcasses prepared for in situanalysis of Hoxd10 and 11 expression domains. PreliminaryanalysesrevealashorteningofthegutofDhanimalsascomparedtowild-typeliUermates,withsmallerorabsentcaecainthemoreseverely affected Dh animals. Skeletal analyses and in situhybridiza,onstudiesareinprogress.

Results

Methods

C3HB6Animals

AdultDh/+and+/+animals

RemoveDh/+and+/+gut

Measurelengthofsmallintes,neand

dimensionsofcaecum

Stainskeleton

Analyzeskeleton(i.e.,,biallength)

BreedforF1offspringthenF2embryos

Harvestembryos

Obtain,ssueforgenotype

Conductinsituhybridiza,on

Obtain,ssueforgenotyping

Documentgutlengths

Stainskeleton

Euthanizepregnant(F1)

dam

§  Todeterminewhetherthereisshorteningofthesmallintes,neinadultanimalsheterozygousfortheDhallele

§  Toexploretherela,onshipbetweenseverityofgutdefectsandskeletaldefects.

§  Tostudyexpressionofrelevant5’Hoxgenes(Hoxd10andHoxd11)indevelopinggutsofDhandwild-typeliUermates,asdifferencesinHoxexpressionthatmayleadtotheobservedpaUerningdefectsinadults

RoleofHoxgenesinembryonicpa#erning(A-P)andpa#erningofgut§  Hoxgenesarehighlyconservedgenes,ancestrallyrelatedto

homeo,cselectorgenes(Hom-Cgenes)ofDrosophila§  Inmammals,therearefourcopiesoftheHoxcomplexper

haploidcomplement§  Hoxgenesencodetranscrip,onfactors,importanttopaUerning

ofneuraltube,neuralcrestcells,paraxialandsplanchnicmesodermandother,ssues.

§  Hoxgenesarrangedinorderonchromosomeoftheirspa,alexpression,with3’Hoxgenesexpressedmoreanteriorlyand5’Hoxgenemoreposteriorly.

§  ThesegenesareknowntoplayaroleinpaUerningofposterior,splanchnicmesodermwhichinformsthedevelopmentoffunc,onalregionsofendoderm(posteriormidgutthroughhindgut)

P1:a’Dh/a’+xa+/a+

F1:a’Dh/a+ora’+/a+

P2:a’Dh/a+xa+/a+

F2:a’Dh/a+ora+/a+

Genotyping

Figure3.ThesecrossescreateembryoswithpolymorphismsatspecificDNAmarkerssiteslinkedtogeneofinterest(Dhgene).OriginalstockDh/+C3HB6femalemicewerecrossedwith+/+AKR/Jmales(P1).F1Dhfemaleswerebackcrossedto+/+AKR/JmalestogenerateF2offspringwithpolymorphismsinD1Mit10,265and309microsatellitemarkersthatcanbeusedingenotyping.

D1Mit10 D1Mit265 D1Mit309

C3HB6 139bp 106bp 127bp

AKR/J 133bp 126bp 162bp

InSituHybridiza6on

Figure4.Theprocedureofinsituhybridiza,on.(Gilbert,2006)

1 2 3 4 5 6 7 8 10 11 12 13 14 159

Introduc6on

Abstract

Aims

Figure2.(A)Hoxgenesarehighlyconservedgenesencodingtranscrip,onfactorsthat“specifyA-Ppolarity(Gilbert,2010)“invertebrates(hUp://cnx.org/contents/0Jy9PqKT@8/Features-of-the-Animal-Kingdom#fig-ch27_01_04).(B)Areasoftheendodermwhoseregionaliden,tyisrefinedbyinterac,onswithsurroundingHox-genespecifiedsplanchnicmesoderm.(Gilbert,2004)

•  Dh/+ animals were found to have a range of smallintes,ne lengths. Such a range in effectswaspreviouslyobservedin,biallength(Morinetal.,1999).

•  Lengths of the small intes,nes of Dh/+ animals wereshorterthanthoseof+/+animals: Comparisonofsmallintes,ne length (p=0.0351); Comparison of smallintes,nelength:bodylength(p<0.0001).

•  SomeDh/+animalshadreducedorabsentcaeca.Analysisisongoing.

Discussion

Thank you to Dr. Owen for suppor,ng, direc,ng,troubleshoo,ng, and edi,ng this work. Kelsey Hern,without whose support and help I would not have beenabletocompletethiswork.

Acknowledgements

ThesestudieswereconductedwiththeapprovaloftheSimmonsCollegeIns,tu,onalAnimalCareandUseCommiUee.

IUCACApproval

•  Gilbert,S.F.(2006).DevelopmentalBiology.Sunderland,MassachuseUs:SinauerAssociates,Inc,.358-497.

•  Green,M.C.(1967).AdefectofthemesodermcausedbythemutantgeneDominanthemimelia.DevelopmentalBiology,15,62-89.

•  Owen,M.H.,Coull,B.A.,&Holmes,L.B.(2006).Asymmetryofskeletal effects of Dominant hemimelia. Birth DefectsResearch. (Part A). Clinical and Molecular Teratology, 76(6),474-482.

•  Rooze, M. A. (1977) . The effects of the Dh gene on limbmorphogenesis in the mouse. Birth defects: Original Ar,cleSeries,13(1),69-95.

References

Figure6.(A)HindlimbofDh/+animaland(C)itsgut(stomachtocaecum).(B)Hindlimbofwild-typeanimaland(D)itsgut(stomachtocaecum),1=stomachand2-caecum.Tissueswerefixedpriortophotographing.

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A

A B

Figure1.(A)Dh/+adult.ThisimagewascontributedforMouseGenomeDatabaseuse.Note:Dh/DhanimalsdieaJerbirth.(hUp://www.informa,cs.jax.org/image/phenoSummary/marker/MGI:94889).(B)Hindlimbskeletalelements(Alizarinred/Alcianbluestain)of18dayDh/+embryo.Absenceof,biaandoligodactylyofdigitoneisfoundontheleJ;,bialshorteningandsyndactylyofdigits1and2ontheright(Owenetal.,2006).

A.

Figure 5. Ra,os of small intes,ne length to body length forDh/+ and +/+animals (p<0.0001). Length of small intes,ne was determined prior tofixa,on.

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Figure7.Genotypingresults.AmplifiedD1Mit10fromDNApurifiedfromhead,ssueofF2embryos(1:20dilu,onofDNA).Dh/+animalstwobands;+/+singleband.

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