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M. Garcia-Castro MIGCrest lab Examining the State of the Science of Mammalian Embryo Model Systems: A Workshop (NAS) Jan 17th 2020 SESSION IV: COMPARATIVE EMBRYONIC DEVELOPMENT ACROSS SPECIES Early Neural Crest Formation: from birds to humans Division of Biomedical SciencesGarcía-Castro Lab UCR, School of Medicine Wilhelm His, 1868 Chick embryo 36 hours dev Head Tail Zwischengstrang, Zwischenrinne Middle cord, Marshall 1879… “NEURAL CREST” Chick embryo Head Neural Crest Migrate extensively through stereotypic patterns Tail Chick embryo Head Neural Crest Derivatives Cartilage, bone, and Connective tissue neurons & glia of the PNS, and Melanocytes Sympathoadrenal cells PATHOLOGIES: 1/3 of all birth defects. Tail Orofacial clefts, rare syndromes, and cancers MIGCrest lab Differentiation Potential of Neural Crest Neurons, Glia, melanocytes, Adipocytes, odontoblast, osteoblasts, chondroblasts, myocytes, Oxigen sensing cells of the Carotid Body, Thymus mesenchyme, etc.… How do NC cells Generate so Many Different Cell types? EPIBLAST ESC Gastrulation Ectoderm Mesoderm Endoderm Skin & CNS Muscle, Bone, Adipo, Blood Gut, Thyroid, Lungs, Pancreas EPIBLAST Melanocytes PNS Gastrulation Neural Crest Cells Ectoderm Mesoderm Endoderm Skin & CNS Muscle, Bone, Adipo, Blood Gut, Thyroid, Lungs, Pancreas Waddington Epigenetics Sequential segregation of potential CRANIOFACIAL Bone , Cartilage, EPIBLAST Muscle, Odontoblast, Revisit the OntogenyAdipocytes, Gastrulation Connective of the Neural Cresttissue, etc. Ectoderm Mesoderm Heart valves Skin & CNS Muscle, Bone, Adipo, Blood Garcia-Castro Lab: Neural Crest Ontogeny Pax3/7 KO and Cre-lines/ROSA26 Rabbit MIGCrest lab ”Ontogeny of Neural Crest " Specification of chick NC before gastrulation. Human NC, Embryonic studies and PSC models suggest an early post epiblast differentiation. Specification of mammalian NC in the Rabbit is evident during gastrulation García-Castro Lab UCR, School of Medicine Neural Crest Formation Classic Induction Wnt BMP Cascade of FGF Transcription Factors and other markers responsible for Neural Crest dev. Non Neural Ecto Neural Ectoderm Mesoderm NCC Induction Neural Crest Formation Our work suggest earlier NC induction (prior to definitive mesoderm and neural tissues) NCC specification evident in early epiblast Specification: initiation of a program, the NC fate program. The specified cell does not display known NC markers yet, but it is able to do so under “undisruptive conditions”. Gain of expression of NC markers used to assess NC fate. NOT COMMITED, could be persuaded to do otherwise Early NCC development MIGCrest lab Early avian NC development Snail2, Sox9, FoxD3 exp Neural plate XIIXII 00 Specified Pax7 expression & requirement Pax7 Snail2 Pax3 Sox9 Ets1 Sox10 FoxD3 Basch et al. 2006 Nature 441:218-222 Early NCC development MIGCrest lab Early avian NC development Snail2, Sox9, FoxD3 exp Neural plate XIIXII 00 Specified Pax7 expression & requirement Earlier specification? Basch et al. Nature 441:218-222 Koller’s sickle Eileen Uribe-Querol (Assistant Professor, UNAM, Mexico) Blastula studies of NCC specification Dissect epiblast tissues, explant into collagen gels Culture in isolation /non inducing conditions CultureCulture 25h45h 1 2 3 4 5 6 7 8 9 10 11 12 HNK1Pax7 Pax7 / Sox9 Snail2 / AP2 HNK1 Pax7 Are NC markers induced during culture? Prospective NCC Dissociate pN pNCC Culture 25h pN Dapi Pax7 Sox9 pNCC pN: 6% Pax7, 5% Sox9. pNCC: 45% Pax7, 23% Sox9. Epiblast Fate Mapping Stephanie Vadasz Pax7 Cell tracing summary Are human Neural Crest also specified before gastrulation? Can we translate the Neural crest Neural ectoderm gains from Chick NC Non-neural ectoderm studies? Mesoderm Medial 0/3 NC Intermediate 7/9 NC Analysis of early human NCC development Neural plate Snail2, Sox9, FoxD3 exp XII * * 0 Specified Pax7 expression & requirement http://embryology.med.unsw.edu.au/wwwhuman/Stages/ Week 2 Week 3 Week 4 Week 5 Carnegie St.6 Carnegie St.13-14 Ronan O’Rahilly Histological analysis Carnegie St.12-18 Dr. Erik Sundström (Karolinska Instituet, Sweden) Analysis of early human NCC development Migratory Primagratory Sox10 HNK1 NCC NCC Week 4 Week 5 Carnegie St.12-18 Pax3, Pax7, Sox9, Sox10, Msx1/2, AP2, HNK1, P75NTR Erin Betters Betters et al, Dev. Biol. 2010 Modeling hNC development with PSC Pomp et al 2005 (R. Goldstein lab Studer, Wysocka, Dalton, Bajpai, Toguchida, Andrews, etc) Fast derivation of hNC from hESC via WNT in defined media accutase 5-day Early migratory NC ±Wnt3A PAX7/SOX10 hESC low density or CHIR99021 PAX7 ~95% of SOX10+ cells co- expressed PAX7 & TFAP2A Validation of NC status Expression of NCC markers Wnt-induced hNCC generate genuine NCC-derivatives Leung et al. 2016, Delopment HISTORY OF NEURAL CREST INDUCTION FROM hPSCS Goldstein Studer Dalton Toguchida/ Garcia-Castro Ikeya CDM CHIR D-2 to D0 CHIR W. Weiss SB Condit Media D-2 to D0 B27 mTeSR1 10-Cell clumps days 5 7 days 11 days (FACS) Huang et al 2016 TFAP2, SOX10, PAX7, PAX3 TFAP2, SOX10 SNAI2, MSX1, MSX2, ZIC1 P75, TWIST, PAX3 Fukuta et al, 2014 Leung et al, 2016 Mica et al 2013, Chambers et al, 2013 Characterizing the signaling parameters Garcia-Castro Transient WNT CHIR first 2 day only 5 Days B27 [CHIR] Low CHIR (WNT) High CHIR (WNT) Anterior NC Posterior NC Gomez et al. 2019a Dev Biology days No Hox-(cartilage) Hox+ (adrenal) Gomez et al. 2019b Development 5 TFAP2, SOX10, PAX7, PAX3 SNAI2, MSX1, MSX2, ZIC1 XENO-Free model high efficiency across multiple ES/iPSC Leung et al, 2016 Hackland et al. 2019 Stem Cell Reports Ontogeny of Wnt induced hNC Gastrulation Ectoderm Mesoderm Endoderm CNS Skin Neural Non- Neural NCC Ontogeny of Wnt induced hNC Classic Induction NNE NE WNT, BMP, FGF, etc GBX2, SP5, Mesoderm pB ZIC3, ZEB2 Ectoderm Mesoderm Endoderm PAX3/7, B MSX, TFAP2 Ontogeny and signaling contributions Leung et al. 2016, Development SOX9/10, FOXD3, ZIC1 NCC Further analyses of hNC Ontogeny EPIGENETIC, TRANSCRIPTOMIC, AND FUNCTIONAL ASSAYS WNT 5 Days ES NC Compare epigenetic regulation and expression of ES and NC markers after WNT induction Repression Activation Transcripts Protein ES H3K27me3 H3K27ac IF Western ES 6hr (OCT4, KLF4, NANOG, SOX2) 12hr pNC ( PAX7/3, Zic3) 18hr CHANGES OCCUR WITHIN THE FIRST DAY, BETWEEN 6 TO 24 HOURS AFTER24hr WNT ADDITION! Hierarchical clustering/ Pheatmap in R-studio program. Upregulation NC proteins in prospective NC soon after WNT activation Leung et al. 2019, BioRxive Prasad et al. 2020, Submitted EPIGENETIC, TRANSCRIPTOMIC, AND FUNCTIONAL ASSAYS WNT 5 Days ES NC INDICATE THAT 6 HOURS AFTER INDUCTION, THE NC PROGRAM IS INITIATED Can we translate the gains from Chick and model human NC studies to mammalian embryos? I A B C P A D 7 x a Early NCC development in Mammals P 2 P A I I A B C P D E F P A A D Pax7-cre D 7 x a 2 P Pax7 LacZ / 2 1 P x A Pax7 EGFP s I M P D E F ROSA26 A 3 D x 2 / a Casey DelConte Barbara Murdoch1 x s P M I G D H 3 Mousex E7-E9.5 a EE P P A P A G D I H P D EE NF A P A D 7 V NF NF 7 x V NF x a M M a A, B, H NP P A, B, H NP P 9 Ect C, F, I x 9 Ect D, E o S C, F, I x D, E o S Figure 1. Neural crest marker expression at E7.5. Immunohistochemistry on transverse sections through an E7.5 embryo reveal neural crest marker expression of: AP2 (green) in the neural fold as well as the non-neural ectoderm (B, E, H), Pax7 (red) in the neural fold (E, H, D, G), Pax3 (green) in the neural fold (C, F, I), Msx1/2 (red) in the neural fold, non-neural Figure 1. Neural crest marker expression at E7.5. Immunohistochemistry on transverse ectoderm, and extra-embryonic tissues (C, F, I), and Sox9 (green) in the neural fold (D). A sections through an E7.5 embryo reveal neural crest marker expression of: AP2 (green) in drawing of an E7.5 embryo with planes of section as well as a line drawing of the section is found in G. Single arrowheads indicate cells expressing one marker in the neural folds, the neural fold as well as the non-neural ectoderm (B, E, H), Pax7 (red) in the neural fold (E, double arrowheads indicate cells that coexpress markers. Bars indicated ectodermal ex - pression. A- anterior, D- dorsal, EE- extra-embryonic tissue, Epi- epidermis, M- mesoderm, H, D, G), Pax3 (green) in the neural fold (C, F, I), Msx1/2 (red) in the neural fold, non-neural NF- neural folds, NP- neural plate, P- posterior , V-ventral. Scale bars- 100 µM. ectoderm, and extra-embryonic tissues (C, F, I), and Sox9 (green) in the neural fold (D). A Murdoch et al. J Neuroscience 2010 drawing of an E7.5 embryo with planes of section as well as a line drawing of the section Murdoch et al. PLoS ONE 2012 is found in G. Single arrowheads indicate cells expressing one marker in the neural folds, Pax7KO & Pax3KO DelConte & García-Castro Submitted double arrowheads indicate cells that coexpress markers. Bars indicated ectodermal ex - pression. A- anterior, D- dorsal, EE- extra-embryonic tissue, Epi- epidermis, M- mesoderm, NF- neural folds, NP- neural plate, P- posterior , V-ventral. Scale bars- 100 µM. Rodent specific pre-gastrula development Chick Mouse 150µm 3mm Ecto --- Meso__ Endo--- Rabbit development as a mammalian alternative Chick Rabbit Mouse 700µm 3mm 150µm CHICK MOUSE RABBIT Expression of mesoderm, Neural, and NC markers in the rabbit npb ne AP-2 Pax7/Sox2/3 ne npb St. 6 Rabbit NCC Marker Expression in the Rabbit Expression assessed in embryos from St. 3 to St.11/12 (13ss). Rabbit 5-6ss/St.8 SOX10 Are Rabbit NC specified during gastrulation? midle-inter-lat Sox10 Pax7 Bra Culture, Fix and immunostain Are Rabbit NC specified during gastrulation? 1 45 hr m i l Sox10 Pax7 Bra midle-inter-lat Betters et al., 2018 Culture, Fix and immunostain Developmental Biology MIGCrest lab Conclusions • Chick NC are specified at the blastula stage • Human NC models based on ES/PSC suggest meso- neural independent and fast initiation of the NC fate • Rabbit embryos reveal for the first time the specification of NC during gastrulation in mammals.
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