Table Of ContentAdvances in Anatomy, Embryology and Cell Biology
Frederic Shapiro
Disordered
Vertebral and
Rib Morphology
in Pudgy Mice
Structural Relationships to Human
Congenital Scoliosis
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Frederic Shapiro
Disordered Vertebral
and Rib Morphology
in Pudgy Mice
Structural Relationships to
Human Congenital Scoliosis
FredericShapiro
LaboratoryfortheStudyofSkeletalDisorders
HarvardMedicalSchool
Boston,Massachusetts
USA
ISSN0301-5556 ISSN2192-7065 (electronic)
AdvancesinAnatomy,EmbryologyandCellBiology
ISBN978-3-319-43149-9 ISBN978-3-319-43151-2 (eBook)
DOI10.1007/978-3-319-43151-2
LibraryofCongressControlNumber:2016950901
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Abstract
Disorderedvertebralandribmorphologyinthepudgymouse,duetomutationsin
the delta-like 3 (Dll3) gene of the Notch family, has been studied using whole
mount preparations, specimen radiographs, histology, and computerized three-
dimensional reconstructions of serial histologic sections. Studies were done in
68 mice, 37 pudgy (pu/pu) and 31 non-affected (pu/+), from late embryo to
3monthsofage.Vertebralbodiesshowedmalformationsincluding:hemivertebrae;
wedged vertebrae; bifidvertebrae(butterfly, double hemivertebrae at same level);
and fused vertebrae (partial unilateral bony bar or complete bone block).
Intervertebraldiscsadjacenttomalformedvertebraewerealwaysabnormalregard-
ing: size (smaller or larger than normal); position (oblique or vertical); shape
(round, hexagonal, curvilinear, and other patterns); disorganized peripheral fibers
of annulus fibrosus; partial presence across width of vertebral column; and/or
complete absence between adjacent vertebrae. Rib malformations included:
decreased numbers; asymmetric shape and spacing; fused and widened ribs;
branching from fused origins into two, three, or more separate ribs; and
paravertebral continuous longitudinal cartilage accumulations from which fused
ribs originated. The vertebral, intervertebral disc, and rib abnormalities were
differentineachpudgymouse.Thisvariabilityindicatesthatasinglegenemutation
doesnotaccountsolelyforthedeformitiesmakingitnecessarytoexplainbyother
conceptssuchasepigenesis. Mutationsofgenesinvolved intheoscillatoryclock/
segmentation mechanism (delta-ll3, Mesp1, lunatic fringe, and others), expressed
as early as the presomitic mesoderm stage of embryogenesis, have been clearly
implicated in congenital vertebral and rib malformation in mice and humans.
Invariable intervertebral disc abnormalities adjacent to abnormal vertebrae are
observed,leadingtoconsiderationofadditionalpathogeneticmechanisms.Normal
and abnormal structures of vertebral bodies appear directly related to shape and
positionoftheintervertebraldiscs.Thecenterofthecartilagemodelofadevelop-
ing vertebral body and its endochondral bone lie along a line extending (approxi-
mately)perpendiculartothecentralportionofthelongaxisoftheclosestsectionof
theadjacentintervertebraldisc.Hemivertebraeandbifidvertebraeforminrelation
v
vi Abstract
to vertically oriented generally midline discs; the wedged vertebrae, and some
hemivertebrae,forminrelationtoobliquelyorienteddiscs;andbonybars(unilat-
eralorblock)forminrelationtocartilagevertebralbodymodelsnotseparatedby
discs. The more abnormal the intervertebral discs, the more abnormal are the
adjacent vertebral bodies. The final deformity is not fully patterned in the early
embryo;itisduetoacombinationofprimarygenetic/epigeneticfactors,secondary
“obligate”mechanisms,andtertiarybiomechanicalforces.
Findings in pudgy mice involving all vertebrae and ribs and a case of severe
human congenital scoliosis are remarkably similar. Vertebral morphogenesis is
reviewed, including developmental concepts described over several decades and
stressing current molecular concepts of variations in the oscillatory clock which
patterns segmentation. An overview of human congenital scoliosis is outlined
especially the severe deformities (spondylocostal dysplasias). The notochord is a
precursorofthenucleuspulposusoftheintervertebraldisc,andthediscisinvari-
ably abnormal in relation to malformed vertebral bodies. Whether a convoluted
notochordhasaroleinprimarycausationorissolelyapassivedisplacedstructure,
itsabnormalpositioningcouldfurtherinducevertebralbodyvariability.Renewalof
embryonic studies linking notochord/neural tube removal and transplantation
experiments inducing vertebral malformation combined with assessments of
gene/molecular components of the oscillatory segmentation clock and notochord
shouldfurtherclarifyabnormalmorphogenesis.
Keywords: Pudgy mouse; Histopathology; Vertebral, intervertebral disc, and
rib abnormalities; Congenital scoliosis; Gene mutations; Segmentation clock;
Notochord
Contents
1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2 MaterialsandMethods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1 Source,Distribution,andAgesofPudgyandNon-affected
Mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2 WholeMountPreparations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.3 RadiographicStudies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.4 HistologicStudies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.4.1 SerialSections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.5 ComputerizedThree-DimensionalReconstructions. . . . . . . . . . . . 5
2.6 PreviousStudiesonChickVertebralDevelopmentandHuman
CongenitalScoliosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3 Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.1 GrossAppearance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.2 WholeMountAppearances. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.2.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.3 RadiographicStudies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.3.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.4 HistologyStudies:Vertebrae,Ribs,IntervertebralDiscs,
andGanglia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.4.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.4.2 SerialSectionAssessments. . . . . . . . . . . . . . . . . . . . . . . 37
3.5 ComputerizedThree-DimensionalReconstructionsofHistologic
Sections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.6 ChickEmbryoVertebralDevelopment(PreviousStudies). . . . . . 58
3.7 RadiologyandHistopathologyofHumanCongenitalScoliosis
oftheSpine(WithNormalComparisons). . . . . . . . . . . . . . . . . . 58
3.7.1 NormalFetalandNeonatalHumanVertebralBodies. . . . . 58
3.7.2 CongenitalScoliosisoftheSpine. . . . . . . . . . . . . . . . . . . 60
vii
viii Contents
4 Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
4.1 NormalMouseVertebralDevelopmentComparedtoPudgy
MouseVertebralDevelopment:GeneralConsiderations. . . . . . . . 67
4.2 TheStudyofVertebral/AxialDevelopmentShowingHistologic
andMolecularProgression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
4.2.1 ConceptsandMechanismsofEmbryonicVertebral
Development.. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . 70
4.2.2 HistogenesisofVertebralPatternDevelopment. . . . . . . . . 73
4.2.3 TiminginEmbryonicAxialDevelopment. . . . . . . . . . . . . 77
4.2.4 CellandTissuePatternsinVertebralDevelopment:
TheNotochordandOtherRegionsInducingAdjacent
EmbryonicTissueDevelopment. . . . . . . . . . . . . . . . . . . . 79
4.2.5 Rib,Ganglia,andIntervertebralDiscDevelopment
inNormalandPudgyMouseSpecimens. . . . . . . . . . . . . . 80
4.2.6 VertebralDevelopmentintheChickEmbryo. . . . . . . . . . 83
4.3 ThePudgyMouse. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
4.3.1 Appearance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
4.3.2 GeneMutations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
4.4 GeneticInfluencesonAxialDevelopment:MutationsIdentified
inMouseModelswithVertebralDeformation. . . . . . . . . . . . . . . 86
4.4.1 HoxGenes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
4.4.2 MouseVertebralMalformations. . . . . . . . . . . . . . . . . . . . 87
4.4.3 MutationsAffectingtheSegmentationClock
andWavefront:TheMolecularOscillator. . . . . . . . . . . . . 88
4.4.4 BovineVertebralMalformations. . . . . . . . . . . . . . . . . . . 88
4.5 CongenitalScoliosis(Human):ItsSimilaritywithPudgyMouse
VertebralAbnormalities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
4.5.1 IncidenceandAssociatedOrganAbnormalities. . . . . . . . . 89
4.5.2 GrossAnatomic,Radiologic,andHistopathologic
AssessmentsinHumanCongenitalScoliosis. . . . . . . . . . . 89
4.5.3 DescriptionandCategorizationofVertebralandRib
AbnormalitiesinCongenitalScoliosis. . . . . . . . . . . . . . . 94
4.5.4 CorrelationofClinical,Radiographic,andHistopathologic
Findings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
4.5.5 ClinicalRecognitionofCongenitalScoliosis. . . . . . . . . . . 95
4.5.6 ClassificationofVertebralDisorders. . . . . . . . . . . . . . . . 96
4.5.7 Types(Patterns)ofVertebralDeformities. . . . . . . . . . . . . 97
4.5.8 AbnormalHumanVertebralDevelopmentandThose
PartsoftheSpectrumMostCloselyAligned
withthePudgyDisorder. . . . . . . . . . . . . . . . . . . . . . . . . 99
4.5.9 GenesRelatedtotheKlippel-FeilSyndrome. . . . . . . . . . . 99
4.6 PathogenesisofPudgyandHumanCongenitalScoliosisBased
onHistopathologicStudies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Contents ix
4.6.1 VertebralMalformation:MechanismsUnderlying
theDevelopmentofStructuralAbnormalities
inCongenitalScoliosisandDescriptiveClassifications
BasedonThem. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
4.6.2 RoleofNeuralTube/SpinalCordandNotochord
inDevelopmentofAxialDeformation:Vertebral
MalformationDuetoErrorsofInduction. . . . . . . . . . . . . 101
4.6.3 RelationshipBetweenShapeandPositioning
oftheIntervertebralDiscs(NucleusPulposus)
[IVD/NP]andAdjacentDevelopingVertebralBodies
inPudgyMice. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 104
4.6.4 StagesofPathogenesisofSpinalDeformity. . . . . . . . . . . 107
4.6.5 SummaryofRibMalformation. . .. . . .. . . .. . . . .. . . .. 108
4.6.6 SummaryofIntervertebralDiscMalformation. . . . . . . . . 109
4.6.7 SummaryofGanglionMalformation. . . . . . . . . . . . . . . . 109
5 Conclusions. . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . 111
5.1 ImplicationsofPudgyVertebralAbnormalitiesforBiologic
Research. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
5.2 ImplicationsofPudgyVertebralAbnormalitiesforClinical
PatientTreatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
