Patterning the vertebrate body axis HD 3 Ed Laufer February 24, 2009 Monday, February 23, 2009 1 Regional differentiation of mesoderm Axial Paraxial Intermediate Chick embryo Somatic Splanchnic Monday, February 23, 2009 2 Morphological changes at early post- gastrulation stages Monday, February 23, 2009 3 Segments organize the body plan during embryogenesis Drosophila Human Mouse Chick Zebrafish Monday, February 23, 2009 4 Axial segments originate from somites Somites are precursors of: Axial skeleton (ribs, vertebrae) Trunk/Limb muscles Dermis Axial tendons Gilbert (2003) Monday, February 23, 2009 5 Segmentation by sequential addition Neural tube somites Presomitic mesoderm (unsegmented) Monday, February 23, 2009 6 Dorsal view of segmenting paraxial mesoderm from chick Segmentation by sequential addition Neural tube somites A caudal growth zone generates new unpatterned tissue. Presomitic mesoderm (unsegmented) Monday, February 23, 2009 6 Dorsal view of segmenting paraxial mesoderm from chick Segmentation by sequential addition Neural tube somites Older, more rostral A caudal growth tissue, is allocated zone generates into segments in a new unpatterned rostrocaudal tissue. progression. Presomitic mesoderm (unsegmented) Monday, February 23, 2009 6 Dorsal view of segmenting paraxial mesoderm from chick Rhythmic production of somites from the presomitic mesoderm (PSM) 2-day old chick embryo Monday, February 23, 2009 7 Rhythmic production of somites from the presomitic mesoderm (PSM) 2-day old chick embryo Monday, February 23, 2009 7 Lateral view of Segmentation Somites form movie here Rostral Caudal Olivier Pourquie Monday, February 23, 2009 8 Lateral view of Segmentation Somites form movie here Rostral Caudal Olivier Pourquie Monday, February 23, 2009 8 Somitogenesis is a reiterated process S+2 Mature somites S+1 S0 S-1 Presomitic mesoderm (PSM) Monday, February 23, 2009 9 Somitogenesis is a reiterated process S+3 Mature somites S+2 S+1 Epithelialization S0 Anterior/Posterior patterning S-1 Segment border formation Presomitic mesoderm (PSM) Tail Extension Monday, February 23, 2009 9 Somitogenesis is a reiterated process S+3 Mature somites S+2 S+1 Epithelialization S0 Anterior/Posterior patterning S-1 Segment border formation Presomitic mesoderm (PSM) Segmentation clock= periodicity Wavefront= positional cues Tail Extension Monday, February 23, 2009 9 Oscillatory Genes Regulate Somitogenesis Expression of the hairy gene in chick Palmerim et al.,1997 (Pourquié Lab) Ish-Horowicz lab animation Monday, February 23, 2009 10 Oscillatory Genes Regulate Somitogenesis Expression of the hairy gene in chick Palmerim et al.,1997 (Pourquié Lab) Ish-Horowicz lab animation Monday, February 23, 2009 10 Oscillatory Genes Regulate Somitogenesis Expression of the hairy gene in chick Palmerim et al.,1997 (Pourquié Lab) Ish-Horowicz lab animation Monday, February 23, 2009 10 The Notch pathway underlies a “Segmentation Clock” in the PSM Hes1 promoter Ub Luciferase Delta-like 1,3 PSM cell1 γ-secretase PSM cell 2 Notch Masamiza (2006) PNAS Monday, February 23, 2009 11 The Notch pathway underlies a “Segmentation Clock” in the PSM Hes1 promoter Ub Luciferase Delta-like 1,3 PSM cell1 γ-secretase PSM cell 2 NICD Hes1,7 Lfng Masamiza (2006) PNAS Monday, February 23, 2009 11 The Notch pathway underlies a “Segmentation Clock” in the PSM Hes1 promoter Ub Luciferase Delta-like 1,3 PSM cell1 γ-secretase PSM cell 2 NICD Hes1,7 Lfng Masamiza (2006) PNAS Monday, February 23, 2009 11 The Notch pathway underlies a “Segmentation Clock” in the PSM Hes1 promoter Ub Luciferase Delta-like 1,3 PSM cell1 γ-secretase PSM cell 2 NICD Hes1,7 Lfng Masamiza (2006) PNAS Monday, February 23, 2009 11 The vertebrate segmentation clock Lfng promoter-YFP Alexander Aulehla 10 hour movie and Olivier Pourquie Two photon time lapse imaging Monday, February 23, 2009 12 The vertebrate segmentation clock Lfng promoter-YFP Alexander Aulehla 10 hour movie and Olivier Pourquie Two photon time lapse imaging Monday, February 23, 2009 12 Why does the signaling oscillator only lead to segmentation at the rostral end of the PSM? Monday, February 23, 2009 13 Why does the signaling oscillator only lead to segmentation at the rostral end of the PSM? ...because there is an inhibitory FGF/Wnt signal produced at the caudal end of the embryo Monday, February 23, 2009 13 The “Wavefront” sets up positional information One round of somitogenesis 120 min. Segmentation Clock Wavefront FGF8/WNT3a Phase1 Phase 2 Phase3 Phase 1 Monday, February 23, 2009 14 Cells in the PSM periodically exhibit high levels of Notch signaling (each round of Notch signaling more caudal cells activate Notch before their cranial neighbors, in a “wave”). In response to Notch signaling the cells epithelialize with their neighbors and form spherical somites. This response is blocked by Fgf produced at the caudal end of the PSM. Thus only the most cranial cells, in the PSM, beyond the range of Fgfs, respond to Notch activity during each wave. Continued growth at the caudal end results in more PSM cells and also moves the source of Fgf further back, allowing the next somite to form. The “Wavefront” sets up positional information One round of somitogenesis 120 min. Segmentation Clock Wavefront FGF8/WNT3a Phase1 Phase 2 Phase3 Phase 1 Monday, February 23, 2009 14 Cells in the PSM periodically exhibit high levels of Notch signaling (each round of Notch signaling more caudal cells activate Notch before their cranial neighbors, in a “wave”). In response to Notch signaling the cells epithelialize with their neighbors and form spherical somites. This response is blocked by Fgf produced at the caudal end of the PSM. Thus only the most cranial cells, in the PSM, beyond the range of Fgfs, respond to Notch activity during each wave. Continued growth at the caudal end results in more PSM cells and also moves the source of Fgf further back, allowing the next somite to form. The “Wavefront” sets up positional information One round of somitogenesis 120 min. Segmentation Clock Wavefront FGF8/WNT3a Phase1 Phase 2 Phase3 Phase 1 Monday, February 23, 2009 14 Cells in the PSM periodically exhibit high levels of Notch signaling (each round of Notch signaling more caudal cells activate Notch before their cranial neighbors, in a “wave”). In response to Notch signaling the cells epithelialize with their neighbors and form spherical somites. This response is blocked by Fgf produced at the caudal end of the PSM. Thus only the most cranial cells, in the PSM, beyond the range of Fgfs, respond to Notch activity during each wave. Continued growth at the caudal end results in more PSM cells and also moves the source of Fgf further back, allowing the next somite to form. The wavefront/determination front controls the spatial response to the segmentation clock Retinoic acid signaling Determination Front FGF/Wnt signaling Monday, February 23, 2009 15 The wavefront/determination front controls the spatial response to the segmentation clock Retinoic acid signaling Differentiation (Myf5, Pax1) Determination Front Segmentation (Mesp2) Posterior identity (Brachyury FGF/Wnt Sprouty) signaling Monday, February 23, 2009 15 Mutations in the Notch pathway/segmentation clock lead to congenital scoliosis Mouse Human Lfng +/+ Lfng-/- Lfng +/+ Lfng-/- SPONDYLOCOSTAL DYSOSTOSIS, AUTOSOMAL RECESSIVE 3; SCDO3; OMIM 609813 Bulman Nature Genetics (2000) Monday, February 23, 2009 16 Addional Congenital Vertebral Defects JAG1-Alagille syndrome (OMIM Congenital scoliosis & kyphosis 118450) Uncloned, etiology not known (64% display vertebral defects) Erol et al., 2004 From Nancy Spinner and Ian Krantz Monday, February 23, 2009 17 Coordinated temporal regulation of signaling modules of the FGF, Notch and Wnt pathways underlies the segmentation clock Monday, February 23, 2009 18 ESomite 9.5 mouse differentiation embryo Day 9.5 mouse embryo 1mm Monday, February 23, 2009 19 This is a sagital view of a day 9.5 mouse embryo, approximately half way through its gestation period. Here’s the head, trunk and turn around the tail end of the embryo. The entire trunk musculoskeletal system arise from these beads-like segmented structures called somites. Somite is not only a hallmark of all vertebrate embryos, its segmented organization is reflected in the adult body, e.g. the vertebra and ribs. Somites differentiate progressively: multiple stages are present in the embryo at any time Monday, February 23, 2009 20 Somites differentiate progressively Epithelial somite Monday, February 23, 2009 21 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 limb muscles (from ventromedial lip of myotome) Monday, February 23, 2009 22 Ribs and Sternum: Unlike the rest of the axial skeleton (vertebrae and vertebral ribs, the sternum derives from lateral plate mesoderm somites/ paraxial mesoderm lateral plate intermediate mesoderm mesoderm Monday, February 23, 2009 23 Two sternal bands condense bilaterally in the lateral plate mesoderm in the ventral body wall, migrate around the developing embryo and fuse at the midline to become the manubrium, sternebrae and xiphoid process Monday, February 23, 2009 24 4th Somitic Compartment: The Syndetome 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 Monday, February 23, 2009 25 Places tendons in correct position in the axial skeleton… Monday, February 23, 2009 26 Resegmentation: Cells from the caudal half of one somite and cells from the cranial half of the adjacent caudal somite form one vertebral body. This allows the nerves of each segment to project out of phase from the vertebral bodies Monday, February 23, 2009 27 The stereotypic pattern of somite development a presomitic mesoderm (psm) cells b somite formation epithelialization a c b dermomyotome sclerotome c medial myotome d d central myotome lateral myotome Monday, February 23, 2009 28 Here is a diagram of the embryo in slide 19.