Summer school „From gametes to organisms“ Part II. Dr. Jiřina Medalová 1 Organogenesis illustrated by histologic slides Musculoskeletal system 2 Musculoskeletal system´s functions •locomotion o 3 support o body shape voice mimic breathing Body axes 4 posterior anterior distal proximal dorsal ventral ventral lateral lateral dorsal Hind (posterior/caudal) Frontal (anterior/rostral/cranial) Sagittal plane - Wikipedia Ascent Descent Afferent Efferent Video time 5 GASTRULATION – three laminar embryo body https://www.youtube.com/watch?v=ADlYn0ImTNg NOTOCHORD FORMATION - first axis https://www.youtube.com/watch?v=73k0k8qXAow NEURULATION – neural tube and crest formation https://www.youtube.com/watch?v=lGLexQR9xGs EMBRYO FOLDING – final shape of embryo https://www.youtube.com/watch?v=4lGq4DkTNko Sources of cells for bones •Bones formed from 3 sources: • oparaxial mesoderm – trunk bones, some head bones olateral plate mesoderm – long bones, sternum oCranial neural crest – head bones 6 Muscles formed from 3 sources: oparaxial mesoderm – trunk and limb muscles, head muscles olateral plate mesoderm – muscle connective tissue oCranial neural crest – head muscles Development of the axial skeleton •Bone development: •mesoderm, development of somites •Trunk bones odevelopment odefects •Head bones odevelopment odefects •Ossification oMembraneous oEndochondral o 7 Axial mesoderm – Notochord formation oNotochord is formed from axial mesoderm oFormation: epiblast cells emigrate from node between epiblast and endoderm and form notochord and prechordal mesoderm oRod-shaped rigid unit stretching along the rostro-caudal embryonic axis onotochord neighbours dorsaly with neural tube and lateraly with paraxial mesoderm o 8 Larsen, Human Embryology Notochord – interspecies comparison ofish and amphibians – formed from cells with big vacuoles, covered by collagen fibers sheath, rigid and flexible structure enabling support and movement o oreptiles, birds, mammals – small and thin notochord, no supportive function o omajority of notochord degrades during development replaced by axial skeleton, notochordal residues form nucleus pulposus of the developing intervertebral discs 9 Introduction to Anatomy and Development, University College London Notochord u ryb a paryb – majority only in larval stage, in some primitive fish (latimerie,chimera) is preserved and function as a backbone, also in hagfish Development of axial skeleton structures •Axial skeleton is formed from 4 sources: • oparaxial mesoderm – trunk bones, some head bones o olateral plate mesoderm – sternum o oaxial mesoderm - notochord o oCranial neural crest – some head bones 10 Neural groove Paraxial mesoderm Paraxial mesoderm Neural tube Lateral plate mesoderm somit Neural crest Edited: Russell, 2018. Chemistry notochord Paraxial mesoderm opara – alongside, axial - axis omesoderm developing on both sides along the longitudinal body axis (neural tube) o oEpiblast cells emigrate from the primitive streak to area between epiblast and endoderm, migrate rostraly (towards the head) and lateraly (to the sides) o oMesodermal cells adjacent to the neural tube condenstate and form paraxial mesoderm – basis for somites 11 Larsen, Human Embryology Axial mesoderm – formation of prechordal mesoderm oFormation: epiblast cells emigrate from node between epiblast and endoderm, migrate rostraly along the central axis and form prechordal mesoderm ocluster of cells rostraly from notochord obasis for thicker prechordal plate - mesenchymal head tissues and rostral cranial mesoderm odifferent names among species – premandibular mesoderm (lamprey, shark), prechordal mesoderm (xenopus, crocodile, chicken), frontal axial mesoderm (zebrafish), ventral cranial mesoderm (mouse) 12 Dr. Staveley, Memorial University Newfoundland Formation of somites - somitogenesis osegmentation of the paraxial mesoderm – formation of paired somites oBasis for bones, cartilage, muscles, tendons, dermis osegmentation begins on cranial end and runs towards the caudal end oCranial paraxial mesoderm – not segmented, basis for facial and neck muscles oSomites form in periodic intervals – used for embryo staging oDifferent number of somites among species 13 Introduction to Anatomy and Development, University College London Somitogenesis – interspecies comparison Bi6140 Embryology 14 fish (zebrafish): 32 Jian et al. 1998. Curr Biol amphibians (xenopus): 42 Hamilton, 1969 reptiles (anolis): 72-73 Eckalbar et al. 2011. Dev Biol birds (chicken): 55 Cebra-Thomas. Dev Biol mammals (mouse): 65 Indiana Uni reptiles (grass snake): 315 Gomez et al. 2008. Nature mammals (human): 44 The Developing Human 8th edition Clock and wavefront model https://www.youtube.com/watch?v=hRtVae4dwJk&t=348s 15 •https://www.youtube.com/watch?v=9wrBROwoRSk • •FGF is produced by the Hensen´s node cells •Retinoic acid is produced by the somitic cells •FGF and RA are antagonists and their expression determines the wavefront •Clock are the negative feedbackloops - transient expression of EphrinA4 http://www.ncbi.nlm.nih.gov/books/NBK26863/figure/A3943/?report=objectonly Segmentation of somites - part 1 16 Introduction to Anatomy and Development, University College London orostraly – gradual separation and somite formation → rostral somites more differentiated than caudal o oParaxial mesoderm formed of mesenchymal cell mass oSegmentation of somites – sclerotome and dermomyotome oFormation of spherical somites, epithelial sheath, mesenchymal core, somitocoel cavity in early somites Dr. Hill. Uni New South Wales Diferenciace buněk somitů: jakmile se oddělí somit od paraxiálního mezodermu, jakákoliv z buněk v somitu je schopná dát vzniknout jakékoliv struktuře, která se vyvine ze somitu (chrupavky obratlů a žeber, svaly hrudního koše, končetin, svaly břišního lisu, zad a jazyka, šlachy, dermis, vaskulární buňky, které tvoří aortu a meziobratlové cévy, obaly míchy). Tyto buňky jsou multipontentní, do které tkáně budou diferencovat záleží na tom, kde se v somitu nacházejí a tím jak dochází k ovlivnění jejich diferenciaci faktory okolních tkání (neurální trubice, notochord, epidermis, intermediární mezoderm) Segmentation of somites – part 2 17 ventromedial sclerotome vertebral bodies, intervertebral discs, ribs dorsolateral dermomyotome dorsal ventral medial lateral Dr. Hill. Uni New South Wales dorsal dermatom (dermis) ventrolateral myotom (skeletal muscles) dermomyotome → dermatome and myotome Dermomyotome + Sclerotome Development of sclerotome – part 1 oSclerotome cells (S) undergo epithelial -mesenchymal transition (EMT) 18 Scott Gilbert. Developmental Biology 10th edition S NC NT S NC NT oMigrate to notochord (NC) and neural tube (NT) areas S NC NT Development of sclerotome – part 2 19 oSclerotome cells around notochord → vertebral body oSclerotome cells around neural tube → vertebral transversal processes, arch, vertebral spine and ribs Introduction to Anatomy and Development, University College London orostraly formation of the occipital bone at the skull base Vertebral ossification centers Cranial and caudal sclerotome – part 1 oSclerotome compartmentalization along the cranio-caudal body axis 20 ohigher cells density and proliferation rate in caudal sclerotome than in cranial – important for neural crest cells migration and motoric neurons axon growth oPlace of cranial and caudal sclerotome division – von Ebner`s fissure (transversally oriented cells) ocaudal end of first sclerotome fuses with cranial end of the following sclerotome oformation of vertebra from two neighbouring sclerotomes Introduction to Anatomy and Development, University College London Cranial and caudal sclerotome – part 2 21 oformation of vertebra from two neighbouring sclerotomes overtebral mesenchyme encapsulate notochord oformation of cartilaginous vertebral deposits → compression of notochord followed by dissapperance with following ossification onotochord remnants – soft central parts of intervetebral discs (nuclei pulposi) Introduction to Anatomy and Development, University College London Ribs development 22 oribs develop from the transversal processes of the thoracic vertebrae omesenchymal cells permeate between hypomers (myotome part) and differentiate into cartilage olater cartilage ossifies through endochondral ossification, distal cartilage does not ossify – rib cartilage (connection between ribs and sternum) Development of the Vertebral Column. Dr. Károly Altdorfer Development of sternum otwo mesenchymal condensation form on ventral side – cartilage differentiation 23 omedial fusion – begins cranially, formation of cartilaginous basis of sternum oafter fusion – ossification centers formation – endochondral ossification osternum originates from lateral plate mesoderm→ somatic mesoderm (somatopleura) ocells migrate ventrally Somatic mesoderm Marieb et al. Human Anatomy. 7th edition Developmental defects of trunk bones oPectus excavatum – sunken chest caused by uneven development of ribs and sternum, 90 % of all congenital chest defects 24 oPectus carinatum – „Bird`s chest“, abnormal growth of rib cartilages cause sternum elevation oJeune syndrome – trunk dystrophy, mutations in wide spectrum of genes, small chest, short risb, short limb bones Tüysüzet al. 2009. AJMG oSternal cleft – insufficient fusion between sternal basis in the midline Pectus excavatum – 1:500, původ není znám, symptomy: bolest na hrudi, srdeční vady, dušnost, řešeno plastickou chirurgií, projevuje se po narození a posléze více v pubertě Pectus carinatum – 1:400, abnormální růst chrupavek, způsobuje utlačování plic a srdce, dýchací problémy Jeunův syndrom – 1:130000, ciliopatie, krátké kosti, polydaktylie, velmi málo pacientů přežije Rozštěp sterna – 1:50000 Development of head bones •Bones and cartilages of head develop from two sources: omesoderm oneural crest 25 oBones of head form two parts: oneurocranium – surrounds brain oviscerocranium – surrounds oral cavity and pharynx oBones of head are formed by two types of ossification: omembraneous – from mesenchyme oendochondral – from cartilage Neural crest neurocranium viscerocranium membraneous endochondral Neurocranium vs. viscerocranium •Neurocranium oSurrounds brain 26 Viscerocranium oSurrounds oral cavity and pharynx membraneous neurocranium cartilaginous neurocranium membraneous viscerocranium cartilaginous viscerocranium Membraneous ossification Membraneous ossification Endochondral ossification Endochondral ossification frontal parietal occipital occipital sphenoid temporal ethmoid zygomatic mandible maxilla premaxilla nasal palatine vomer nasal capsule cartilage ear ossicles Neural crest oNeural crest– forms from ectoderm, from neural fold in developing neural tube oNeural crest cells – transform from epithelial to mesenchymal (epithelial-mesenchymal transition - EMT) → migration oMigration to final destinations – different cell types and tissues oRegions of the neural crest: ocranial + cardiac ovagal otruncal osacral 27 Green et al. 2015. Nature Neurální lišta – neuroektoderm, často nazývaná čtvrtý zárodečný list Cranial neural crest oregions of developing prosencephalon (forebrain), mesencephalon (midbrain) a rhombencephalon (hindbrain) omesencephalic cells and cells from frontal segments of rhombencephalon migrate into prosencephalon → frontal bone, parts of temporal, sphenoid and occipital bones omesencephalic cells and first three rhombencephalic regions cells (R1,2,3) → migrate to frontonasal prominence and first pharyngeal (branchyal) arch → form bones and cartilage of nasal capsule, maxila and mandible, ear ossicles 28 prosencephalon mesencephalon rhombencephalon Neurální lišta – neuroektoderm, často nazývaná čtvrtý zárodečný list Kosti středního ucha 1. faryngeální oblouk – malleus (kladívko) a incus (kovadlinka) se vyvíjejí z Meckelovy chrupavky, endochondrální osifikace Kosti středního ucha 2. faryngeální oblouk – stapes (třmínek) se vyvíjí z Reichertovy chrupavky, enchondrální osifikace Pharyngeal arches 29 oneural crest cells migrate to regions of developing head and neck between surface ectoderm and primitive gut endoderm → 6 pairs of pharyngeal arches opharyngeal clefts osurface depression of ectoderm oexternal auditory canal opharyngeal pouches oprimitive gut endodermal protrusions oEustachian tube olymphatic system (palatal tonsils) oendocrine system (parathyroid glands, thymus) oPharyngeal membrane – broken in fish, communication between oral (pharyngeal) cavity and external environment McGeady et al. Veterinary Embryology. 2009 Bony and cartilaginous derivatives of pharyngeal arches odifferent structures of head and neck develop from mesenchyme of each arch 30 o1. arch obones: maxila (Mx), premaxila (p), mandible (Mn), zygomatic (Z), temporal (t) ocartilage: Meckel`s, malleus, incus Z p Mx Mn Scott Gilbert. Developmental Biology 10th edition o2. arch obones: styloid proces of temporal bone (t) ocartilage: stapes, part of hyoid bone (cartilage) t o3. arch opart of hyoid bone (cartilage) o4. arch oLaryngeal cartilage Processus styloideus – výběžek kosti spánkové Developmental defects of head oCraniosynostosis – premature ossification of cranial sutures (head deformities, brain and eye defects) o oHemifacial microsomy – one part of face incompletely developed (eye, ear, facial bones and muscles) o oCleft lip and/or palate – the most often developmental defects of head, isolated or combined 31 Centers for Disease Control and Prevention Mayo Clinic Family Healt Book, 5th Edition Kraniosynostóza – předčasný srůst lebečních kostí, cca 1:2500, nejčastěji způsobeno mutací v genech fibroblastových růstových faktorů a jejich receptorů, popsáno více než 50 genů způsobující kraniosynostózy, syndromové i nesyndromové Hemifaciální mikrosomie – druhá nejčastější vývojová vada hlavy po rozštěpových vadách, jiný název: Syndrom prvního a druhého faryngeálního oblouku, Goldenhar syndrom Formation of bones oMembraneous ossification oBone forms directly from mesenchyme 32 oEndochondral ossification oCartilage is formed and than replaced by bone cranial vault facial bones clavicles skull base vertebra long bones pelvis ribs sternum Membraneous ossification oBone develops from mesenchyme 33 omesenchymal cells condensation odifferentiation - osteoblasts oossification center formation o oosteoblasts produce minerals oosteoblasts differentiate in ossification center - osteocytes oedges of ossification center – osteogenic progenitors differentiate into osteoblasts oinside – trabecular/spongy bones (osteocytes) osurface – compact bone (periosteum, osteoblasts) Bone formation and development. Oregon State University Endochondral ossification – part 1 oHyaline cartilage transforms into bone 34 omesenchymal cells condensation - chondroblasts ochondroblasts produce matrix - chondrocytes ocartilage is not vascularized – supplied from perichondrium o oosteoblasts migrate through vessels in perichondrium to the edge of cartilage – bone is produced in diaphysis - bone collar obone collar prevents penetration of material to cartilage – chondrocytes are dying and cartilage degrades ospace for vessels – region settled by osteoblasts, formation of primary ossification center Bone formation and development. Oregon State University Endochondral ossification – part 2 oLongitudinal growth of bone - cartilage grows on both ends (future epiphysis), • concurrently is replaced by bone in diaphysis 35 oPrimary ossification terminated – diaphysis ossified, epiphysis cartilaginous oEpiphysal cartilage – ossified later, formation of secondary ossification center oduring development, cartilage remains between epiphysis and diaphysis in form of epiphyseal/growth plate Bone formation and development. Oregon State University Growth plate 36 Bone formation and development. Oregon State University oReserve zone ochondrocytes oMatrix production oProliferative zone ochondrocytes grow ochondrocytes proliferate (mitosis) oHypertrophic zone ochondrocytes grow ochondrocytes maturation oCalcification zone ocalcification of matrix ochondrocytes dying oOssification zone ovascularization oosteoblasts form bone Growth defects oAchondroplasia odwarfism oreduced proliferation of chondrocytes in growth plate ogrowth plate disorganized oshort bones, macrocephaly 37 oThanatophoric dysplasia omore severe form, usually lethal oshort limbs onarrow chest omacrocephaly, brain defects Ornitz and Legeai-Mallet, 2017. Dev Dyn Carrol et al. 2020. Pal Med Rep Development of apendicular skeleton oLimb bones odevelopment odefects 38 Formation and development of limbs oformation of limb buds: o •mesenchyme (mesoderm) covered with epithelium (ectoderm) Bi6140 Embryology 39 Obsah obrázku text, různé Popis byl vytvořen automaticky Obsah obrázku text, různé Popis byl vytvořen automaticky Hamburger and Hamilton, 1951. Embryonic origin of limbs obones and cartilage – lateral plate mesoderm, somatopleura 40 limb skeletal precursors Marieb et al. Human Anatomy. 7th edition oMuscles and dermis – paraxial mesoderm omyotome – hypaxial part odermatome o hypaxial myotome limb muscle cells precursors Limb bud lateral plate mesoderm endoderm dermatome Epaxial myotome Syndetome ojunction between muscles and bones – tendons formation o ooriginates in paraxial mesoderm – somites o odorsal part of sclerotome - syndetome 41 Nakamichi and Asahara, 2021. Bone Beginning of limb development olimb field – lateral plate mesoderm cells and paraxial mesoderm cells migrate from limb field ocells accumulate under the ectoderm – formation of limb bud 42 Stocum and Fallon, 1982 Science Photo Library oApical ectodermal ridge (AER) formation ◦thickening of surface ectoderm in distal part of limb bud ◦production of growth factors → mesenchymal cells stimulated (proliferation, migration, differentiation) Sadler, 2010. Zone of polarizing activity (ZPA) omesenchyme in posterior part of limb bud oproduction of growth factors omutual influence with apical ectodermal ridge cells odetermines differentiation of limb along the anterio-posterior axis 43 Signaling •Shh is specific for the zone of polarizing activity (ZPA) •En-1 expression blocks development of dorsal part (Wnt7) in ventral part of hand •AER (source of FGF) is formed on the edge of cells producing and not producing r-fng http://www.evol.nw.ru/ AER ZPA Upper Scheme: Mechanisms of D/V Patterning and AER Positioning (A) Gene expression along the limb bud D/V axis. The member of Wnt family Wnt-7a and Radical fringe (r-Fng), which encode secreted factors, are expressed in the dorsal ectoderm. The homeodomain-containing factors encoded by Lmx-1 and Engrailed-1 (En-1) localize to the dorsal mesoderm and ventral ectoderm, respectively. (B) Genetic interactions involved in AER formation and specification of dorsal pattern. En-1 expression in the ventral ectoderm restricts the expression of r-Fng and Wnt-7a to the dorsal ectoderm. Interaction between r-Fng-expressing and r-Fng-nonexpressing cells leads to the specification of the AER. Wnt-7a instructs the dorsal mesoderm to adopt dorsal characteristics, such as Lmx-1 expression, which in turn specifies dorsal pattern. En-1 has a dual function in AER positioning and dorsal specification and hence acts to coordinate the two processes. Lower Scheme: Three Axes and Three Signals: Shh, FGFs, and Wnt-7a Orchestrate Limb Pattern (C) Schematic of a limb bud viewed from the posterior-dorsal aspect showing the localization of Shh to the ZPA, FGFs to the AER, and Wnt-7a to the dorsal ectoderm. (D) Codependence of Shh, FGF, and Wnt-7a signaling and axial patterning. While each secreted factor can be associated with patterning along a single axis, affecting the expression of any single factor will lead to modulation of the other two. For example, reduction of Wnt-7a signaling will lead directly to dorsal patterning defects, but indirectly to posterior defects through a diminution of Shh signaling, and to proliferation defects via a subsequent effect on FGF expression. Primary Signalling Molecules Growth and differentiation of limb bud oProgressive zone model – faith and differentiation of mesenchymal cells determined by time they stay in progressive zone 45 oEarly specification model – faith and differentiation of mesenchymal cells is already determined by formation of three different cell groups in progressive zone McGeady et al. Veterinary Embryology. 2009 Bones and cartilage of limb ovariation of the same building plan of vertebral limbs o3 zones on developing limb: ostylopodium (proximal) – humerus, resp. femur ozeugopodium (middle) – radius, ulna, resp. tibia, fibula oautopodium (distal) – metacarpal bones and finger bones o 46 stylopodium zeugopodium autopodium Source of limb cells http://www.utm.utoronto.ca/ http://www.cmrb.eu/media/upload/imatges/ centre-investigacio/linies_treball/6_1_limb.jpg Developmental defects of limb bones oPolydactyly – more than five fingers formed on one limb 48 oSyndactyly – connection between two or more fingers oPhocomelia – missing proximal part of limb J Integr Health Sci Kapoor and Johnson, 2011. N Eng J Med Development of trunk skeletal muscles otrunk muscles have two origins: oParaxial mesoderm (somites) oLateral plate mesoderm o 49 osomites differentiate into sclerotome and dermamyotome odermamyotome differentiates into dermatome and myotome omyotome is divided into: oEpaxial myotome: osome trunk muscles (dorsal) oHypaxial myotome: osome trunk muscles (ventral) olimb muscles o Epaxial and hypaxial trunk muscles oproliferation and migration of myotome cells– muscle cell progenitors formation - myoblasts oEpaxial muscles: oback (dorsal) muscles formation – muscle connective tissue from somites omuscle segments are fusing o oHypaxial muscles: ointercostal muscles – muscle connective tissue from somites, muscle do not fuse oabdominal (ventral) muscles – muscle connective tissue from lateral plate mesoderm, muscles fuse o • • 50 Sefton and Kardon, 2019. Curr Top Dev Biol Development of limb muscles oHypaxial myotome – source of myoblasts for limb muscles omyoblasts emigrate from myotome in the limb field to developing limb bud 51 Hypaxial myotome Deries and Thorsteinsdóttir, 2016. Cell Mol Life Sci Head muscles o3 groups: oextraocular muscles– CM, CNC omuscles originating in pharyngeal arch - CM, CNC oFacial muscles, jaw muscles, neck muscles otongue muscles – PM, CNC 52 oHead muscles develop from 3 sources: ◦nonsegmented cranial mesoderm (CM) ◦Paraxial mesoderm - somites (PM) ◦Cranial neural crest (CNC) Randolph and Pavlath, 2015 Sefton and Kardon, 2019. Curr Top Dev Biol Differentiation of skeletal muscle cells 53 Myotome cells myoblasts mitosis myocytes extension connection Multinuclear myotubules fusion differentiation myotubules maturation Muscle fiber maturation Growth and renewal of muscles – progenitor and muscle stem cells - satellite muscle cells Developmental defects of muscles and muscle dystrophy oDuchene muscle dystrophy – •the most often muscle dystrophy, gradual loose of muscles, mutation in gene for dystrophin – stabilization of muscles oBecker`s muscle dystrophy – less severe form of Duchene dystrophy oPoland syndrome – missing one side of breast muscles, often connected with scapula hypoplasia and other limb bones on the same side 54 Obsah obrázku text Popis byl vytvořen automaticky Obsah obrázku oblečení, osoba, slipy Popis byl vytvořen automaticky Shahi et al. 2020. Cureus Fun facts oHow many bones are in the adult human body? oHow many bones has the newborn? oAnd the skull? oWhat is the smalest bone? o 55 https://www.bioexplorer.net/skeletal-system-fun-facts.html/ oHow many bones are in the adult human body? 206 oHow many bones has the newborn? 270 oAnd the skull? 22 – grow together oWhat is the smalest bone? Staples Information sources Atlas of mouse embryo http://www.emouseatlas.org/eAtlasViewer_ema/application/ema/kaufman/plate_25a.php Chick embryo developmental stages (21 d) https://embryology.med.unsw.edu.au/embryology/index.php/Hamburger_Hamilton_Stages Comparison of human and mouse embryo (21 d) https://embryology.med.unsw.edu.au/embryology/index.php/Category:Mouse_E12 European mole developmental stages (talpa europea) (28 d) https://www.researchgate.net/publication/250068036_Developmental_Stages_and_Growth_Rate_of_the_Mole _Talpa_occidentals_Insectivora_Mammalia https://bio-atlas.psu.edu/ Atlas of danio rerio development (3 d till the egg is hatching) Samples •Homo Sapiens Sapiens •H.S.S. embryos 6th – 22nd week iud (46 weeks) • •Mus Musculus (mouse) •M.M. E12 = 5-6th week iud H.S.S. (21 days) •M.M. E14,5 = 7.-8. week iud H.S.S. • •Gallus Gallus (chicken) •G.G. HH10 (1,5 d) = 3rd week iud H.S.S. (21 days) •G.G. HH20 (3,5 d) = 5th week iud H.S.S. •G.G. HH24 (4,5 d) = 6th week iud H.S.S. •G.G. HH26 (5D) = 6,5th week iud H.S.S. •G.G. Hh28 (5,5-6D) = 7th week iud H.S.S. • •Talpa Europea (Mole) •T.E. 16D = beginning of organogenesis (29 days) •T.E. 27d = just before the birth • •Mesocricetus Auratus (hamster) •M.A. 13,5D= 6. týden iuv H.S.S. (17days) •M.A. 15D= just before the birth • •Danio Rerio (zebrafish) •Zebrafish 5 days – larval stadium (hatching of embryo in 3rd day) • •