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Patterning the vertebrate body axis Regional differentiation of mesoderm

Axial Paraxial Intermediate Chick embryo HD 3 Ed Laufer Somatic

February 24, 2009 1 Splanchnic

Segments organize the body plan during Morphological changes at early post- embryogenesis gastrulation stages

Drosophila Human Mouse Chick Zebrafish

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Axial segments originate from somites Segmentation by sequential addition

Neural tube somites Somites are precursors of: Axial skeleton (ribs, vertebrae) Older, more rostral A caudal growth Trunk/Limb muscles tissue, is allocated zone generates Dermis into segments in a new unpatterned Axial tendons rostrocaudal tissue. progression.

Presomitic mesoderm 5 6 Gilbert (2003) (unsegmented)

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Rhythmic production of somites from the Lateral view of Segmentation presomitic mesoderm (PSM)

Somites form movie here

Rostral Caudal

2-day old chick embryo 7 Olivier Pourquie 8

Somitogenesis is a reiterated process Oscillatory Genes Regulate Somitogenesis

Expression of the hairy gene S+3S+2 in chick Mature somites S+2S+1 S+1S0 Epithelialization S0S-1 Anterior/Posterior patterning Palmerim et al.,1997 S-1 Segment border formation Presomitic (Pourquié Lab) mesoderm (PSM) Segmentation clock= periodicity Wavefront= positional cues

Ish-Horowicz lab animation Tail Extension 9 10

The Notch pathway underlies a The vertebrate segmentation clock “Segmentation Clock” in the PSM

Hes1 promoter Ub Luciferase Delta-like 1,3 PSM cell1 γ-secretase

PSM cell 2

NotchNICD

Hes1,7 Lfng promoter-YFP Lfng Alexander Aulehla and 10 hour movie Olivier Pourquie Two photon time lapse imaging 11 12 Masamiza (2006) PNAS

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Why does the signaling oscillator only lead to The “Wavefront” sets up positional information segmentation at the rostral end of the PSM?

One round of somitogenesis 120 min.

Segmentation Clock Wavefront

FGF8/WNT3a

Phase1 Phase 2 Phase3 Phase 1 ...because there is an inhibitory FGF/Wnt signal 14 produced at the caudal end of the embryo13

The wavefront/determination front controls the spatial response to the segmentation clock Mutations in the Notch pathway/segmentation clock lead to congenital scoliosis

Retinoic acid Mouse Human signaling Differentiation (Myf5, Pax1)

Determination Front Segmentation (Mesp2)

Posterior identity (Brachyury Lfng +/+ Lfng-/- Lfng +/+ Lfng-/- FGF/Wnt Sprouty) SPONDYLOCOSTAL DYSOSTOSIS, AUTOSOMAL RECESSIVE 3; SCDO3; signaling 15 OMIM 609813 16 Bulman Nature Genetics (2000)

Coordinated temporal regulation of signaling modules of Addional Congenital Vertebral Defects the FGF, Notch and Wnt pathways underlies the segmentation clock

JAG1-Alagille syndrome (OMIM Congenital scoliosis & kyphosis 118450) Uncloned, etiology not known (64% display vertebral defects) Erol et al., 2004 17 18 From Nancy Spinner and Ian Krantz

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Somite differentiation Somites differentiate progressively: multiple stages are present in the embryo at any time

19 Day 9.5 mouse embryo 20

Somites differentiate progressively

The sclerotome forms the vertebrae, the myotome forms muscles

By the end of the fifth week, prospective muscles are found divided into two parts:

Epimere - back muscles (from dorsomedial lip of myotome) Hypomere - body wall and Epithelial somite limb muscles (from ventromedial lip of myotome)

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Ribs and Sternum: Two sternal bands condense bilaterally in the lateral plate mesoderm in the ventral body wall, migrate around the Unlike the rest of the axial skeleton (vertebrae and vertebral ribs, developing embryo and fuse at the midline to become the the sternum derives from lateral plate mesoderm manubrium, sternebrae and xiphoid process

somites/ paraxial mesoderm lateral plate intermediate mesoderm mesoderm

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4th Somitic Compartment: The Syndetome Places tendons in correct position in the axial skeleton…

Defined by expression of scleraxis (Scx).

This population arises between myotome (after involution under dermomyotome) and sclerotome.

From sclerotomal compartment.

Gives rise to the tendons. Scx 25 26

Resegmentation: The stereotypic pattern of somite development

Cells from the caudal half of a presomitic surface ectoderm one somite and cells from neural tube mesoderm (psm) cells the cranial half of the notochord adjacent caudal somite form b one vertebral body. somite formation epithelialization

This allows the nerves of each a c segment to project out of phase b dermomyotome from the vertebral bodies c sclerotome

medial myotome d d central myotome

27 lateral myotome28

Inductive interactions subdivide the somite Patterning the axial skeleton - specification of the - Shh and noggin (BMP antagonist), secreted by the notochord and somite along the anteroposterior (AP) axis floor plate cause the ventral part of the somite to form sclerotome Although the basic cellular differentiation pattern of somites at different (Pax1 txn factor). axial positions is very similar, unique vertebral structures form along the - Wnt, noggin (dorsal neural tube) and low Shh (notochord) induce craniocaudal axis, indicating that somites acquire specific identities dermamyotome (Pax3). according to their axial position. - Wnts also direct the dorsomedial portion of the somite to form epaxial (back) muscles. - NT-3 directs dermatome Axial identity is differentiation. regulated by Hox gene - Hypaxial (limb and body expression wall) muscles are formed from dorsolateral portion of the somite in response to Wnt and BMP signaling. 29 30

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(2) The fly leg and antenna are homologous What are Hox genes? structures A little bit of background...

Wild type fruit fly T2 has a wing T3 has a haltere (rudimentary wing)

(1) A fruit fly carrying a “homeotic mutation”: the third thoracic segment develops like the second thoracic segment 31 32

Homeotic mutant with legs in place of antennae The homeotic genes are arranged in a cluster that reflects the order of the body parts they regulate

Drosophila

Chromosome

lab pb Dfd Scr Antp Ubx AbdA AbdB 3 ANT-C BX-C

They encode transcription factors that contain a conserved DNA binding motif, the homeobox.

“Antennapedia” 33 34

Vertebrates have homologous genes, arrayed along Vertebrate Hox axial expression patterns also four chromosomes: The paralogous Hox gene reflect their chromosomal order clusters Expression is progressively induced Drosophila during gastrulation, and the expression patterns are nested in a Chromosome chromosomal and morphological lab pb Dfd Scr Antp Ubx AbdA AbdB progression. This is called ANT-C BX-C 3 “colinearity”. Mouse/ Human The combination of Hox genes A1 A2 A3 A4 A5 A6 A7 A9 A10 A11 A13 Evx1 expressed in a tissue forms a Hox HoxA 6 code.

B1 B2 B3 B4 B5 B6 B7 B8 B9 B13 HoxB 11 Note that Hox10 paralogs have an anterior (rostral) expression C4 C5 C6 C8 C9 C10 C11 C12 C13 boundary at the thoracic/lumbar HoxC 15 border, and Hox11 at lumbar/sacral

D1 D3 D4 D8 D9 D10 D11 D12 D13 Evx2 2 HoxD 35 36 paralogs

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‘Segment transformation’ Loss of paralogous function Lumbar --> Thoracic Hox expression is conserved and boundaries correlate with T12 L3 S2 C5 results in complete loss of changes in vertebral morphology regional identity in axial column T13 } T13 * * * *

Sacral 10aaccdd

* } * * * {

Lumbar{ T13 } Control * * * * T12 L3 S2 C5 { 11aaccdd 11aaccdd T12 L3 S2 C5

‘Segment transformation’37 38 Wellik and Capecchi, Science, 2003 Sacral --> Lumbar

Hox genes do not specify cartilage/bone directly Key Ideas (I): Rather they modulate the way skeletal progenitors respond to other signals. The axial body plan is organized into repeating mesodermal structures Similar to inductive signals, they are used repeatedly to called somites modulate pattern in many developing tissues. Somites form progressively from presegmental mesoderm at the caudal end of the growing embryo, through a “clock and wavefront” mechanism that involves Notch, FGF and Wnt signaling

Malfunctioning of this signaling system cause vertebral segmentation defects

Somites are subdivided by inductive signals emanating from the surrounding embryonic structures

Somites differentiate into axial skeleton, muscles, dermis and tendons

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Key Ideas (II): Cells at different cranial-caudal and different dorsal-ventral locations in the embryo express different combinations of transcription factors “telling” cells where they are and hence modulating the structures they produce

Along the cranial-caudal (“anterior-posterior”) axis, Hox genes provide a combinatorial code for cell fate

The homeobox is a highly conserved DNA-binding domain of the Hox proteins

There are 4 clusters of Hox genes (A->D) with a total of 13 gene families (low numbered Hox genes are expressed more cranially)

Hox genes are also used in adult cells as transcription factors that regulate growth and differentiation, but not pattern 41

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