Pescando genes: Peces que nos enseñan a entender enfermedades Agustín G Zapata Departamento de Biología Celular Universida...
 
CD4 CD8 CD25 CD44 2.9 42.6 3.1 TIMO POSTNATAL; DIFERENCIACION T 15.6 75.5 5.6 2.5 50.6
EphA4 EphB4 ephrinB1 -/- +/+ wt null ncc null wt Morfogénesis, desarrollo
 
Zebrafish Hematopoiesis <ul><li>Loci hematopoiéticos tempranos </li></ul><ul><li>Aparición y organización de los órganos l...
Hematopoiesis Primitiva Hematopoiesis en Vertebrados Hematopoiesis Definitiva Órganos Intermedios Órganos Linfoides Primar...
 
ICM . 20 hpf N PD
Skin YS Duct of Cuvier (YS blood vessel). 24 hpf
Duct of Cuvier (YS blood vessel). 24 hpf Promonocyte ?
 
 
 
 
 
 
 
72h CV CV CA xa ir ml ml ventral fin nt mitosis? Nikon40xdry caudal-most part of the caudal hematopoietic tissue hematop....
Sudan Black staining of granulocytes 60 hpf 4 dpf Granulopoiesis, as shown by the Sudan Black stained cells, also occurs m...
scl c-myb Scl is expressed in the same space-time pattern as c-myb in the tail then pronephros kidney
48 hpf M  CA CV MC MC M  Histological / ultrastructural analysis of the caudal hematopoietic tissue
End RC RC RC End RC 5 dpf End Rc Rc myeloblasts myelocyte mature neutrophils, among reticular cells (Rc) eosinophil all pr...
End End RC End 2 dpf 5 dpf 5 dpf End 5 dpf
7 dpf End M  5 dpf MB MB
 
 
 
 
 
 
 
B-cell development in teleosts <ul><li>Rag expression </li></ul><ul><li>Species Organ Method Day Reference </li></...
IEL 8 dpf
Spleen. 7 dpf Gut Liver
Spleen. 10 dpf
Spleen. 30 dpf Gut Liver
Spleen. 30 dpf
Spleen. 3 months Liver
3dpf 5dpf 7dpf kidney (dorso-lateral view) tail thymus branchial arches asymetric ? c-myb
A photoactivatable fluorescent tracer for migrating hematopoietic cells Dextran 10.000, -DMNB-caged fluorescein lysine-f...
Photoactivation at 48hpf, fixation 3 days later (5dpf) Immunohistochemical detection of uncaged fluorescein UGO
ear ear Thymus (Uncaged) fluorescein immunodetection (5dpf) ventro-lateral view (?) L-plastin ISH (4dpf) ventral view c-m...
Reminder : 48 hpf Therefore, we now uncage the caged-fluorescein-dextran tracer in these cells runx1, 26 hpf Do precursor...
Laser-mediated photoactivation at 48 hpf Photoactivation of trunk at 24 hpf 5 dpf, thymus 5 dpf, thymus 5 dpf, tail
 
 
 
 
 
Región AGM <ul><li>Contiene precursores linfo-hematopoiéticos, dependientes de Runx1 y Notch, definitivos, que... </li></u...
Región CHT <ul><li>Colonizan, a través de la circulación, la región CHT, </li></ul><ul><li>Donde proliferan, diferencian (...
Rag1+Lck, 5dpf thymus 13 somites left 12 somites left In embryos whose tail + part of the trunk was ablated at 18, 22, 26...
 
 
 
 
 
 
 
Thymus. Cloche. 6 dpf
Thymus. Cloche. 7 dpf
Cloche. 6 dpf Thymic epithelial cells
Cloche. 6 dpf Thymic epithelial cells
Ikaros Mutant 5 dpf
Ikaros Mutant 5 dpf
 
 
 
 
 
Zebrafish mutants with defects in thymus morphogenesis <ul><li>Mutant Defects Gene Coments References </li></ul><u...
MICROCEPHALIC MUTANS <ul><li>Normal expression of SCL/Tal-1, Ikaros, c- myb in ICM (1 dpf) (Stem cell compartment in all...
 
Thymus. Earl grey (egy) 7 dpf
Thymus. Earl grey. 7 dpf
Thymus. Jasmine 6 df
Thymus. Ceylon. 6 dpf
Thymus. Ceylon. 6 dpf
Thymus. Camomile. 6 dpf
 
MICROCEPHALIC MUTANTS Mutant Pharyngeal arches Endoderm Neural crest markers Rhombomeres 3&5 Rhombomeres 5&6 (5) ...
 
EpH A3 Morphant 4 dpf
Children´s Hospital. Harvard University Eric C. Liao Barry H. Paw Nikolaus S. Trede Leonard L. Zon Biology Department. MIT...
of 86

Ponencia Agustin Zapata GonzáLez Pescando Genes Santander UIMP 2008

Pescando genes: Peces que nos enseñan a entender enfermedades por Agustín G. Zapata (Departamento de Biología Celular-Universidad Complutense de Madrid) en la Escuela de Verano "Ortega y Gasset" de la Universidad Internacional Menendez Pelayo. UIMP 2008. Palacio de la Magdalena, Santander
Published on: Mar 4, 2016
Published in: Technology      Education      
Source: www.slideshare.net


Transcripts - Ponencia Agustin Zapata GonzáLez Pescando Genes Santander UIMP 2008

  • 1. Pescando genes: Peces que nos enseñan a entender enfermedades Agustín G Zapata Departamento de Biología Celular Universidad Complutense de Madrid
  • 3. CD4 CD8 CD25 CD44 2.9 42.6 3.1 TIMO POSTNATAL; DIFERENCIACION T 15.6 75.5 5.6 2.5 50.6
  • 4. EphA4 EphB4 ephrinB1 -/- +/+ wt null ncc null wt Morfogénesis, desarrollo
  • 6. Zebrafish Hematopoiesis <ul><li>Loci hematopoiéticos tempranos </li></ul><ul><li>Aparición y organización de los órganos linfoides </li></ul><ul><li>Genes implicados </li></ul>
  • 7. Hematopoiesis Primitiva Hematopoiesis en Vertebrados Hematopoiesis Definitiva Órganos Intermedios Órganos Linfoides Primarios ? ? ? (Fetal liver) (AGM/DLP) Intra/Extraembrio-narios (YS, ICM, VBI) (BM, kidney, thymus)
  • 9. ICM . 20 hpf N PD
  • 10. Skin YS Duct of Cuvier (YS blood vessel). 24 hpf
  • 11. Duct of Cuvier (YS blood vessel). 24 hpf Promonocyte ?
  • 19. 72h CV CV CA xa ir ml ml ventral fin nt mitosis? Nikon40xdry caudal-most part of the caudal hematopoietic tissue hematop. precursors xa, xanthocyte; ir, iridocyte; ml, melanocyte (attenuated by PTU treatment); nt, notochord movie4 ventral fin ir
  • 20. Sudan Black staining of granulocytes 60 hpf 4 dpf Granulopoiesis, as shown by the Sudan Black stained cells, also occurs medial to the somite muscles, and not around the part of the CV plexus that lies further ventrally until about 60 hpf. 60 hpf ventral-most channel (=future definitive CV) of the CV plexus ventral lmit of somite muscle and hematopoiesis
  • 21. scl c-myb Scl is expressed in the same space-time pattern as c-myb in the tail then pronephros kidney
  • 22. 48 hpf M  CA CV MC MC M  Histological / ultrastructural analysis of the caudal hematopoietic tissue
  • 23.
  • 24. End RC RC RC End RC 5 dpf End Rc Rc myeloblasts myelocyte mature neutrophils, among reticular cells (Rc) eosinophil all precursors ?
  • 25. End End RC End 2 dpf 5 dpf 5 dpf End 5 dpf
  • 26. 7 dpf End M  5 dpf MB MB
  • 34. B-cell development in teleosts <ul><li>Rag expression </li></ul><ul><li>Species Organ Method Day Reference </li></ul><ul><li>zebrafish Kidney WISH - 1, 2 </li></ul><ul><li> Pronephros ISH on tissue sections First cells 2 wpf 3 </li></ul><ul><li>Clusters 3-4 wpf </li></ul><ul><li>Pronephros ISH on tissue sections 2-3 wpf 4 </li></ul><ul><li>Pronephros Rag-2 GFP 8 dpf 1 </li></ul><ul><li>Whole fish RT-PCR 3 dpf 3 </li></ul><ul><li>Pancreas WISH 4 dpf 5 </li></ul><ul><li>rainbow trout Whole fish RT-PCR 10dpf (before appearance of 6 thymus and kidney) </li></ul><ul><li>Pronephros ISH on tissue sections Hatching 7 </li></ul><ul><li>Ig expression </li></ul><ul><li>Specie Organ Evidence Method Day Reference </li></ul><ul><li>zebrafish Whole fish VDJ (  ) rearrangements PCR amplification of 4 dpf 5 </li></ul><ul><li>genomic DNA </li></ul><ul><li>Whole fish IgM expression RT-PCR 7 dpf (membrane) 5 </li></ul><ul><li>13 dpf (secreted) </li></ul><ul><li>Whole fish IgM expression ISH on tissue sections 10 dpf (pancreas) 5 </li></ul><ul><li>19 dpf (pronephros) </li></ul><ul><li>Whole fish IgL isotypes RT-PCR 3 dpf 3 </li></ul><ul><li>Kidney IgL isotypes ISH on tissue sections 3 wpf 3 </li></ul><ul><li>rainbow trout Whole fish cIgH- cells Immunohistochemistry 12-14 dpf 8 </li></ul><ul><li>Whole fish IgL ELISA 8 d pre-hatching 9 </li></ul><ul><li>Pronephros sIgM+cells Immunohistochemistry 4 d post-hatching 10 </li></ul><ul><li>1.-Willett et al´97 Dev Biol 182, 331. 2.-Trede et al´04 Immunity 20, 367. 3.-Lam et et al DCI 28, 9. 4.-Willett et al´94 Dev Dynam 214,323. </li></ul><ul><li>5.-Danilova and Steiner´02 PNAS 99, 13711. 6.-Hansen´97 Immunogenetics 46, 365. 7.-Hansen and Zapata´98 Immunol Rev 166, 199. </li></ul><ul><li>8.-Castilo et al´93 DCI 17,419. 9.-Sánchez et al´95 DCI 19,79. Razquin et al´90 J Fish Biol 36, 159. </li></ul>
  • 35. IEL 8 dpf
  • 36. Spleen. 7 dpf Gut Liver
  • 37. Spleen. 10 dpf
  • 38. Spleen. 30 dpf Gut Liver
  • 39. Spleen. 30 dpf
  • 40. Spleen. 3 months Liver
  • 41. 3dpf 5dpf 7dpf kidney (dorso-lateral view) tail thymus branchial arches asymetric ? c-myb
  • 42. A photoactivatable fluorescent tracer for migrating hematopoietic cells Dextran 10.000, -DMNB-caged fluorescein lysine-fixable UV 1cell UV irradiation ‘uncages’ the fluorescein, restoring its fluorescence K. Kissa-Marin DMNB Covalent bond cleaved by UV Fluorescein Detection of uncaged fluorescein by immunohistochemistry PA Fluorescein NBT/BCIP
  • 43. Photoactivation at 48hpf, fixation 3 days later (5dpf) Immunohistochemical detection of uncaged fluorescein UGO
  • 44. ear ear Thymus (Uncaged) fluorescein immunodetection (5dpf) ventro-lateral view (?) L-plastin ISH (4dpf) ventral view c-myb 5dpf, dorso-lateral? ikaros, 5 dpf, dorso-lateral? L-plastin, 5 dpf, ventral
  • 45. Reminder : 48 hpf Therefore, we now uncage the caged-fluorescein-dextran tracer in these cells runx1, 26 hpf Do precursors from the trunk seed the hematopoietic tail ?
  • 46. Laser-mediated photoactivation at 48 hpf Photoactivation of trunk at 24 hpf 5 dpf, thymus 5 dpf, thymus 5 dpf, tail
  • 52. Región AGM <ul><li>Contiene precursores linfo-hematopoiéticos, dependientes de Runx1 y Notch, definitivos, que... </li></ul><ul><li>Se generan en el mesénquima entre DA y PCV no en clusters intra-aórticos </li></ul><ul><li>Dividen asincrónicamente y una de las dos células sale a la circulación por PCV (endotelio fenestrado) no por DA, </li></ul><ul><li>y....... </li></ul>
  • 53. Región CHT <ul><li>Colonizan, a través de la circulación, la región CHT, </li></ul><ul><li>Donde proliferan, diferencian (trombocitos, granulocitos, precursores linfoides) y migran a los OLP (timo, riñón) a través del mesénquima </li></ul><ul><li>HSCs pueden migrar directamente desde AGM al riñón </li></ul><ul><li>CHT funciona durante 2 semanas, aunque el riñón sea hematopoiética funcional desde el día 4 </li></ul>
  • 54. Rag1+Lck, 5dpf thymus 13 somites left 12 somites left In embryos whose tail + part of the trunk was ablated at 18, 22, 26, or 48 hpf, hematopoietic colonization of the thymus and kidney is normal (even possibly faster…) L-plastin, 5dpf Gata1, 5dpf kidney Control embryo thymus kidney trunk &quot;neo-hematopoiesis&quot; trunk &quot;neo-hematopoiesis&quot; ?
  • 62. Thymus. Cloche. 6 dpf
  • 63. Thymus. Cloche. 7 dpf
  • 64. Cloche. 6 dpf Thymic epithelial cells
  • 65. Cloche. 6 dpf Thymic epithelial cells
  • 66. Ikaros Mutant 5 dpf
  • 67. Ikaros Mutant 5 dpf
  • 73. Zebrafish mutants with defects in thymus morphogenesis <ul><li>Mutant Defects Gene Coments References </li></ul><ul><li>Van gogh Morphogenesis of endoderm Tbx-1 One of the earliest gene involved in Piotrowski et al´03 </li></ul><ul><li>pharyngeal arch development. It mimics Development 130,5043 </li></ul><ul><li>the Di George syndrome </li></ul><ul><li>Faust Gut morphogenesis. Development GATA-5 Regulate the numbers of endodermal Reiter et al´01 </li></ul><ul><li>of liver, pancreas, thymus, thyroid cells . Nodal signalling pathway Development 128, 125 </li></ul><ul><li>One-eyed Absence of endoderm Nodal signalling pathway Schier et al´97 </li></ul><ul><li>pinhead (oep) Development 124, 327 </li></ul><ul><li>Casanova Morphogenesis of endoderm Nodal signalling pathway Alexander et al´99 </li></ul><ul><li>(cas) Dev Biol 215, 343 </li></ul><ul><li>Lazarus Segmental patterning in the Pbx Mediate Hox gene function Popperl et al 2000 </li></ul><ul><li>hindbrain and pharyngeal arcs Mol Cell 6, 255 </li></ul>
  • 74. MICROCEPHALIC MUTANS <ul><li>Normal expression of SCL/Tal-1, Ikaros, c- myb in ICM (1 dpf) (Stem cell compartment in all mutants is intact). </li></ul><ul><li>Erythropoiesis in all mutants is unaffected. </li></ul><ul><li>Alymphoid thymus in all mutants (Ikaros, Rag-1, Lck negative). </li></ul>
  • 76. Thymus. Earl grey (egy) 7 dpf
  • 77. Thymus. Earl grey. 7 dpf
  • 78. Thymus. Jasmine 6 df
  • 79. Thymus. Ceylon. 6 dpf
  • 80. Thymus. Ceylon. 6 dpf
  • 81. Thymus. Camomile. 6 dpf
  • 83. MICROCEPHALIC MUTANTS Mutant Pharyngeal arches Endoderm Neural crest markers Rhombomeres 3&5 Rhombomeres 5&6 (5) Earl grey Rostral 3 arches N Pax 9a Crestin/Forkhead (+) Krox 20 (+) Hoxa 3 (+) (CZ3 , CZ5 , Caudal 4 arches CZ11) incompletely developed (13) Jasmine 7 arches N Pax 9a(+) Crestin/Forkhead (+) Krox 20 (+) Hoxa 3 (+) (CZ18) (3) Ceylon Rostral 3 arches deformed Pax 9a Crestin (-) in caudal Krox 20 (+) Hoxa 3 (+) (CZ26) Caudal 4 , 5 arches shortened arches Caudal 6, 7 arches absent Forkhead (+) (2) Camomile Rostral 3 arches N Pax 9a Crestin/forkhead (+) Krox 20 (+) Hoxa 3 (+) (CZ32) Caudal 4 arches incompletely developed -Hindbrain architecture normal -Primitive TECs form 1 or 2 compact layers (Early grey, Jasmine) Empty spaces among the TEC network (Ceylon, Camomile) -Normal arches (Jasmine) (defect in early T lymphopoiesis/ thymus organogenesis ?) Abnormal arches (Early grey, Ceylon, Camomile) (underdevelopement of 3th pharyngeal pouch ?)
  • 85. EpH A3 Morphant 4 dpf
  • 86. Children´s Hospital. Harvard University Eric C. Liao Barry H. Paw Nikolaus S. Trede Leonard L. Zon Biology Department. MIT Catherine E. Willett Nadia Danilova Lisa A. Steiner Max-Plank-Institute for Immunobiology Thomas Boehm Queensland Institute of Medical Research Andrew W. Boyd Pasteur Institute. Paris Philippe Herbomel Emi Murayama Karima Kissa Dpt. Cell Biology. Complutense University Alfonso Cortés Agustín Zapata