1. Zebrafish/Danio rerio

2. Where did zebrafish originate?
Zebrafish were originally found in the Ganges River in India. Currently, the natural habitat of zebrafish is being seriously threatened by altered environmental condition (pollution)

3. Chromosome number and genome size
50 chromosomes 1,5x109 bp genes

6. "Founding Father" of Zebrafish Developmental and Genetic Research
The first who used zebrafish in biological research
The use of zebrafish as a model organism was pioneered at the University of Oregon by George Streisinger.
"Founding Father" of Zebrafish Developmental and Genetic Research
Born:       December 27, 1927, in Budapest, Hungary Died:         August 11, 1984, while scuba-diving at the Oregon Coast near Florence

7. Why Zebrafish?
It’s a vertebrate
Easily injected

8. Zebrafish development
In captivity, zebrafish have a mean lifespan of 42 months.
The maximum age observed in captivity was 66 months

9. Zebrafish development

10. FROM ZYGOTE TO 20 hpf

11. National Institutes of Health (NIH) , Zebrafish Laboratory

13. - CRISPR CAS-9

14. Forward genetics and reverse genetics approaches

15. Forward genetics: Genetics screens
Large-scale genetic screens have been performed using ethyl-nitrosourea (ENU) to generate point mutations.
More than 7000 mutations in 600 genes (not all yet subjected to complementation) have been so generated. The original screens utilized simple dissecting microscopes to find mutations.
Subsequent screens have used probes to highlight particular tissues.
Injection with retroviral vectors has also been used to generate insertional mutations and has the considerable advantage of expediting mutation cloning.
Transparent embryos
e facilmente manipolabili

16. Wild-type vs. golden (gol) mutant.
golden (gol) pigmentation mutant
Wild-type vs. Huli hutu (hht) mutant
In huli hutu (hht) mutants, the retinal cell layers are disorganized and multiple organs have mild to severe nuclear abnormalities that are reminiscent of the atypia of human neoplasia.

17. Reverse genetics: Targeting Induced Local Lesions IN Genomes (TILLING) method

18. Functional analyses by MICROINJECTION
Reverse genetics:
Functional analyses by MICROINJECTION
1) mRNA injection (overexpression) : gain of function phenotypes
2) injection of a specific antisense oligonucleotide (morfololino): loss of function phenotypes (knock down)

19. Microinjection technique: The work station
(functional molecules, functional knock-down of specific genes by morpholino, overexpression assays)

20. Gain of function: mRNA injection (overexpression)
The Drosophila mago nashi gene is required for polarisation of the oocyte and the formation of perpendicular axes.
The zebrafish mago nashi gene encodes a maternal transcript detected in both blastomeres and yolk cell at the 1-2 cell stages, and in the blastoderm during segmentation.

21. Knockdown (KD) by injection of a specific antisense oligonucleotide named morfolino
Morpholinos are antisense synthetic oligonucleotides 
that are the product of a redesign of natural nucleic acid structure. They are highly stable.
In terms of structure, the difference between morpholinos and DNA is that, while morpholinos have standard nucleic acid bases, the bases are bound to morpholine rings instead of deoxyribose rings and linked through phosphorodiamidate groups instead of phosphates.
Usually 25 bases in length, they bind to complementary sequences of RNA by standard nucleic acid base-pairing and can efficiently block translation or splicing acting by "steric blocking”.
2 to 40 ng of an ATG-Blocking morpholino, specifically designed for specifc mRNAs can be injected into fertilized eggs mostly at 1-cell stage (and up to 4-cells stage).
Monitor loss of protein with antibodies and loss of mRNA with RT-PCR
Loss of function mutant phenotypes

22. MO-mediated knockdown
Specific binding to complementary mRNA sequences
MOs act by steric-blocking instead of mRNA degradation
Stable and durable effect during development (not degraded by nucleases)
Low toxicity
This is a summary of the main features of MO mediated knockdown; steric blocking towards ribosome assembly and spliceosome assembly in another case;
Dose dependence: possibility to modulate phenotypes (from mild to severe) by modulating the dose of injected morpholino
Embryos injected with morpholino are called MORPHANTS

23. Microinjection : An Efficient Morpholino
Delivery System
Injection
Site
1.5 hrs
4 hrs
0 hr
Easy to perform:
can inject thousands
of embryos per day
28 hrs
Nasevicius and Ekker (2000, 2001)

24. PAX 6 knock down by morpholino
Degree of escue by mRNA injection

29. Heart: Form, Contractility, and Rhythmicity
The embryonic zebrafish heart at 24 h post fertilization (hpf), as shown is nearly identical to the two-chambered human heart at three weeks of gestation. It is spontaneously contractile, emptying atrium and then ventricle sequentially to generate unidirectional blood flow.
Growth of a muscular septum late in human embryonic development, which divides the primitive atrium and ventricle into left and right parts, differentiates the adult human from the adult zebrafish heart, the latter retaining a single atrium and a single ventricle.
More than 35 mutations in the zebrafish prevent the normal acquisition of contractile function, without disturbing the generation of normal chamber cell fate or formation, and therefore functionally mimic dilated cardiomyopathies.
Pickwick, a mutation in titin, the myofibrillar element around which the actin-myosin arrays assemble and contributor of most of the elastic force to the muscle, has been cloned. It is interesting to note that a human dilated cardiomyopathy family has also recently been documented to have a titin mutation (62, 78).

31. Large number of offspring
Optically clear embryos
Short generation time
Small Size
Reverse Genetics:
Transgenic fish
Tilling with ENU
Morpholino injection
CRISPR-CAS9
Forward Genetics:
ENU mutagenesis
Insertional mutagenesis
Small Molecule Screens:
Predictive of higher vertebrates
Delivery by injection or soaking
Genomics:
Sequenced Genome
cDNA projects
Microarrays
Carcinogenesis

32. The Cfdp1 gene (RLTPR) in Zebrafish
In collboration with Prof. Angel Raya IBEC, Barcelona

33. In Situ Hybridization

34. Developmental expression of Cfdp1 gene

35. The CFDP1 protein is highly abundant in the cephalic structures

36. Knock-down of Cfdp1 gene in Zebrafish

37. Effects of nock-down of Cfdp1 gene in Zebrafish
Delaied development and morphological anomalies of ocular lobes and aberrant somitogenesis
STD-MO
RLTPR-MO

38. Effects of knock-down of Cfdp1 gene in Zebrafish
Malformations of branchial arches
STD-MO
Cfdp1-MO

39. Branchial arches syndromes in humans
Pharyngeal arches are paired structures associated with the pharynx that contribute greatly to the formation of the face, jaw, ear, and neck.
Branchial arches defects are responsible for a large group of genetic syndromes which affect craniofacial development in human such as : Treacher Collins, CHARGE, Goldenhar, Pierre Robin and Nager syndromes.

40. Syndrome de Treacher-Collins
Franceschetti-Zwahlen-Klein ou Dysostose mandibulo-faciale
1/ naissances
Anomalies de l’oreille externe (hypoplasie des pavillons)
Hypoplasie malaire, mandibulaire et zygomatique
Colobome de la paupière inférieure
Fente du palais
Nez large
Microsomie
Transmission autosomique dominante
Pénétrance incomplète
Expressivité variable
Cas de novo 60%

41. Association CHARGE 1/10 000 Anomalies oculaires: colobome ++
Cardiopathie principalement conotroncale
Atrésie des choanes
Retard de croissance et de développement
Hypoplasie génitale
Anomalies de l’oreille/Surdité
Transmission autosomique dominante
Expressivité variable
De novo
CHD7: famille des ADN hélicases à chromodomaine (>75% des cas) (Vissers LE et al. Nat Genet 2004)
Remodelage de la chromatine pour réguler la formation de la crête neurale

42. Dysostose mandibulo-faciale avec microcéphalie
< 1/
Treacher-Collins
Microcéphalie
Fente palatine
Retard de croissance
Retard sévère du langage
Atrésie de l’œsophage
Transmission autosomique dominante ou récessive?

43. Syndrome de Goldenhar 1/6 000 Spectre OAV (Oculo-auriculo-vertébral)
Dermoïde épibulbaire, colobome de la paupière supérieure
Microtie, dysplasie de l’oreille externe et moyenne, condylomes
Surdité de transmission +/- perception
Asymétrie faciale avec microsomie hémifaciale
Anomalies des vertèbres
Fente labiale/palatine
Autres malformations viscérales (cœur, rein)
Retard mental rare
Hypothèse: Transmission autosomique dominante avec variants de novo
Expressivité variable
Pas de gène connu

44. Branchial arches syndromes and chromatin
In the last decade, there is a large body of experimental evidence indicating that mutations in genes encoding chromatin organization and remodeling proteins are important players in cellular differentiation and human developmental disorders, including craniofacial development.
For example, CHD7, a member of the CHD the chromodomain helicase DNA-binding family, encodes a protein mutated in human CHARGE syndrome a multiple anomaly disorder that affects hearing, vision, and cardiac, craniofacial, and nervous system development.
In collaboration with Caroline Rooryck Thambo (Laboratoire de Génétique Moléculaire, Bordeaux France) a screening for Cfdp1 gene mutations will be performed in a cohort of 160 partient with Goldenhar syndrome.

45. Maria Teresa Atterrato Yuri Prozzillo FOREIGNER COLLABORATIONS
FLY LAB
Giovanni Messina
Emanuele Celauro
Maria Teresa Atterrato
Yuri Prozzillo
FOREIGNER COLLABORATIONS
Angel Raya
IBEC, Barcelona
Ana Losada
CNIO, Madrid
COLLABORATIONS
Franco Cotelli
Ruggiero Caizzi
Laura Fanti
University of Sapienz a
Ennio Giordano
Federico II, Napoli