This document summarizes research on hematopoiesis and lymphopoiesis in zebrafish. It describes how zebrafish are used to study the development of blood and immune cells in early embryonic regions like the intermediate cell mass and caudal hematopoietic tissue, and how these precursors colonize the thymus and kidney. It also discusses mutations affecting thymus morphogenesis and the role of genes like runx1.

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

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

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

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

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 "neo-hematopoiesis" trunk "neo-hematopoiesis" ?

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

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