Let’s fish! Why fish is so useful in biomedical research?

• Introduction to fish models: fugu as a good system for genomic studies, and zebrafish
for genetic analysis.
• Comparative genomics and regulation of gene expression: cis-regulatory regions and
chromatin signatures.
• The fish as a patient: zebrafish as a heart injury model, and as cancer model.
• New avenues using zebrafish as a biomedical research model -development of high
throughput small molecule screens-.
• Imaging fish: modulating fish behavior through light.
• The power of genetics: genetic screenings and the new genome-editing systems -
CRISPR/Cas9
• The fish as human avatar.

Master in Translational Medicine, UB 2022

Cristina Pujades
Department of Medicine and Life Sciences
cristina.pujades@upf.edu
 The Human Genome Project gave an impetus in
vertebrate genomics
• To gain insight into their biology and as models to interpret the human
genome, several other vertebrate genomes have been investigated: mouse
and rat, chick, Xenopus tropicalis and Xenopus laevis, zebrafish, medaka
and fugu.

compaction of the genome??

 Phylogenetic relationships, approximate times of divergence and
genome sizes in the fish and mammalian lineages

The fish lineage (represented by pufferfish species and zebrafish) diverged from the tetrapod lineage (represented by mouse and human)
450 million years ago. After its separation from the tetrapod lineage, but before divergence between zebrafish and pufferfish, the fish
lineage probably underwent an event of whole genome duplication (this is suggested by the presence of numerous ancient gene duplicates
in fish genomes). Genome compaction in the smooth pufferfish lineage possibly took place after separation from the spiny pufferfish lineage
50–70 million years ago.
 Fugu: only a poisonnous delicatessen?
Fugu: a poisonnous delicacy

The fugu or pufferfish is highly toxic, but despite this —or perhaps because of it—
it is considered a delicacy in Japan. The fish contains lethal amounts of a poison in
the internal organs. Therefore, only specially licensed chefs can prepare and sell
fugu to the public. The risk of death does not dissuade the big adepts, ready to
pay more than 300€ for a dish.
 1989
But not only that...

It was S Brenner who realized the need for a simple genome approach
to gene identification and characterization. He reasoned that all
vertebrates would have a similar repertoire of genes, due to the way
in which genomes have evolved. Thus, a vertebrate with a small
genome should be the starting point for investigation. Vertebrates
have a similar early developmental pattern and are physiologically
similar.

He suggested that it was the way in which genes are regulated rather than different genes
that gave rise to vertebrate diversity.

IMPORTANCE OF CIS-REGULATORY REGIONS IN GENE REGULATION

AND THEREFORE IN HUMAN DISEASE
 THE IMPORTANCE OF THE CIS-REGULATORY REGIONS IN
HUMAN DISEASE
Cis-acting regulatory sequences are required for the proper temporal and spatial
control of gene expression. Variation in gene expression is highly heritable and a
significant determinant of human disease susceptibility. The diversity of human
genetic diseases attributed, in whole or in part, to mutations in non-coding
regulatory sequences is on the rise. Improvements in genome-wide methods of
associating genetic variation with human disease and predicting DNA with cis-
regulatory potential are two of the major reasons for these recent advances.
DISCOVERY OF CONSERVED VERTEBRATE CIS-REGULATORY ELEMENTS

Transcription Factor Binding Sites (TFBS) are:

• clusters of 6-10 nucleotides that act at a distance and in any orientation on
transcription;
• complex structure in independent modules.

Inconveniences to detect them:

• They do not have properties comparable to open reading frames, splicing motives…
• Models of transcription regulation do not only involve activation or supression but
also binding activities;
• The structural and functional properties are not always reflected in the nucleotide
sequence;
• The regulatory modules can be widely dispersed in the genome;
• They may be located in introns;

Therefore alignment methods usually perform poorly in cis-regulatory sequences

WHICH ARE THE BASIS OF THE SUITABILITY OF USING FUGU IN COMPARATIVE GENOMICS

1. DNA content indicated a genome size of around 365 Mbp (1/8 size human
genome).
2. The genes are smaller. Although the coding sequence is the same size, intron sizes
are greatly reduced and devoid of repetitive elements (around 20% of all DNA is
probably coding).
3. The genes are densely packed: intergenic distances are much smaller with a gene
every 6-8 Kbp.
4. There is very little repetitive DNA and virtually none of that is dispersed.
5. Similar gene repertoire than there is in the genomes of mammals.
6. High conservation of the order of genes along a chromosome (synteny), but the
precise gene order and orientation has changed.
7. The structure of genes is also conserved, splice sites falling in the identical positions
to those found in man.
8. It is useful in the discovery of conserved regulatory elements.
9. Critically, homology between Fugu and mammalian genes is high enough to
facilitate easy identification both through hybridisation and database comparison.
10. The Fugu sequences will be particularly useful for validating computer-predicted
hypothetical genes in the human genome (HG) and revealing novel genes in the HG
that do not contain any known domains.
S. Brenner and G. Elgar were granted to
generate a landmark map of the Fugu genome
(Fugu Genome Consortium)
COMPLETED
Compaction of the fugu genome
Most of fugu introns are smaller than 200bp (compression of the
fugu genome)
 Comparison of Fugu and Human predicted Proteomes

-The majority of peptides have some degree of match in Fugu

-Aprox 25% predicted human proteins do not appear to have homologs in the Fugu
genome (eg. cytokines underwent either rapid evolution or arose de novo in tetrapods).
 COMPARATIVE GENOMICS
Comparison of gene A in specie 1 and specie 2 (orthologs). Usually flanking genes
are also conserved in the same order in the species being compared. Genome
browsers provide a clear view of a gene’s chromosomal context and are useful to
determine orthology.

Which species should be compared?

Because of the intrinsic limitations of the alignment programs, regulatory regions that
have been rearranged in the genome may be missed. Comparisons of human, rat and
mouse will highlight differences rather than similarities between sequences, but
comparisons of mouse and chicken will identify highly constrained sequences. Better
to use also multiple species for comparison.
 Fugu as a tool for the identification of conserved vertebrate cis-
regulatory elements
A key step in understanding gene regulation is the identification of regulatory elements and
is traditionally addressed by promoter deletion.
However as pufferfish are 400 million years distant from mammals, irrelevant DNA in the
promoter region will be randomized, so the conserved sequence is likely to be functional.

VISTA plot of the pair-wise

comparisons of the human,
mouse and fugu HoxA locus.
Predicted regions of significant
identity are shown as peaks.
Conserved non-coding
sequences.
Alignments
Local or global alignment strategies can be used
AVID, LAGAN, BLASTZ, Dialign-Chaos, PipMaker, VISTA

The rVISTA tool combines TFBS predictions, sequence comparisons and cluster analysis to identify
noncoding DNA regions that are evolutionarily conserved and present in a specific configuration within
genomic sequences
DIRE is a web-server for identifying and visualizing cis-regulatory modules in the promoter regions of a
given set of potentially co-regulated human genes. DIRE relies on a database of putative transcription
factor binding sites that have been carefully annotated across the human genome using evolutionary
conservation with the mouse and rat genomes. An efficient search algorithm is applied to this data set to
identify combinations of transcription factors, whose binding sites tend to co-occur in close proximity
within the promoter regions of the input gene set.
 Small genome is beautiful, and convenient for comparison...

BUT fugu is not a suitable model for functional assay of cis-regulatory elements
Let’s explore the Danio rerio
or zebrafish
 THE IMPORTANCE OF THE CIS-REGULATORY REGIONS
IN HUMAN DISEASE
Cis-acting regulatory sequences are required for the proper temporal and spatial control of
gene expression. Variation in gene expression is highly heritable and a significant
determinant of human disease susceptibility. The diversity of human genetic diseases
attributed, in whole or in part, to mutations in non-coding regulatory sequences is on the
rise. Improvements in genome-wide methods of associating genetic variation with human
disease and predicting DNA with cis-regulatory potential are two of the major reasons for
these recent advances.
 GENOME-WIDE CHROMATIN STRUCTURE

Risca V and Greenleaf W, 2015

Chromatin structure is divided in 3 size scales: i) DNA methylation, sequence features, DNA-bound
factors, nucleosome positions and modifications, and DNA-accessibility; ii) local structures formed by
nucleosome-nucleosome interactions (lack of methodology to address this issue); iii) promoter-enhancer
3D-contacts and mega-base scale chromosome domains.
Whole-Genome Chromatin IP Sequencing (ChIP-Seq)

ChIP-Seq combines chromatin immunoprecipitation

(ChIP) with massively parallel DNA sequencing to
identify binding sites of DNA-associated proteins. It
maps global binding sites for a protein of interest.

GENE EXPRESSION REGULATION:

ENHANCER CHROMATIN SIGNATURES
GENE EXPRESSION REGULATION:
ENHANCER CHROMATIN SIGNATURES

OPEN CHROMATIN (ACTIVE ENHANCERS) marked by:

• histone H3 lysine 4 mono-methylation
(H3K4me1)
• histone H3 lysine 27 acetylation (H3K27ac)

PUTATIVE PROMOTOR REGIONS marked by:

• H3K4me1/me3

Genomic locations of H3K27ac, H3K4me1, and

H3K4me3.
DIAGRAM OF CHROMATIN ACCESSIBILITY ASSAYS

Tsompana M, Buck M, 2014. Epigenetics and Chromatin.

GENOME WIDE CHROMATIN ACCESSIBILITY ASSAYS

Buenrostro et al, 2013. Nature Methods.

Tsompana M, Buck M, 2014. Epigenetics and Chromatin.
Assay for Transposase-Accesible Chromatin

Buenrostro et al, 2013. Nature Methods.

 COMPARATIVE ANALYSES OF OPEN-CHROMATIN DOMAINS

ATACSeq – nucleosome free DNA (putative binding-sites)

H3K27ac – open chromatin (active enhancers)
H3K4me3 – putative promoters
Letelier et al, PNAS 2018
FROM ZEBRAFISH TO HUMAN: THE FISH AS A PATIENT

• DISEASE MODELLING
• DRUG DISCOVERY
 CAN WE USE ZEBRAFISH FOR CARDIOVASCULAR ANALYSES?

HUMAN MOUSE ZEBRAFISH

While zebrafish bears anatomical differences with mammals it is electro-physiologically

closer to the human heart than the mouse is
 CARDIOVASCULAR DEVELOPMENT

STEPS LEADING TO ASSEMBLY OF THE TRUNK ANGIOGENIC VASCULAR NETWORK

Angiogenic vascular sprouts emerge from the longitudinal trunk axial vessels (dorsal aorta and
posterior cardinal vein) in two spatially and temporally distinct steps. Dorsal aorta derived
sprouts form an initial primary network of vascular segments, followed by emergence of vein-
derived secondary vascular sprouts that interact and interconnect dynamically with the
primary network to initiate vascular flow.
CARDIOVASCULAR DISEASE
CARDIAC REGENERATION
LARVAL MODEL FOR CARDIOMYOCYTE ABLATION
ADULT MODEL FOR CARDIAC INJURY: CRYOINJURY

Freezing of the ventricular apex >

Activation of regenerative programs

non-injured adult heart

injured adult heart

 THE FISH AS A CANCER PATIENT: XENOGRAFTS AND CANCER
TRANSGENIC MODELS

The most common mutation in human melanoma in serine/threonine kinase BRAF

recapitulates the phenotype in zebrafish
 TUMOR TRANSPLANTATION (ALLOGRAFTs)

Optically clear immune-compromised rag2E450fs zebrafish for optimized cell transplantation

and direct visualization of fluorescently labelled cancer cells at single-cell resolution
 TUMOR TRANSPLANTATION (XENOGRAFTs)

• Pediatric brain tumors from mouse models can grow as orthotopic xenografts in the
brains of zebrafish
• Suitable for in vivo high-throughput screening
 SMALL MOLECULE SCREENING IN ZEBRAFISH

THE METHOD OF IDENTIFYING SMALL MOLECULES THAT ALTER THE FUNCTION OF A

BIOLOGICAL PATHWAY, RESULTING IN THE INDUCTION OR RESCUE OF A SPECIFIC PHENOTYPE,
IS CALLED FORWARD CHEMICAL GENETICS.

• Zebrafish is an ideal organism for small molecule screenings;

• The ability to use the whole organism allows complex in vivo phenotypes to be assayed;
• Embryos are easily treatable by waterbone exposure; small molecules can penetrate the
chorion of zebrafish embryos and induce embryonic phenotypes within a controlled time
during development.
• The small size and abundance of embryos make them suitable for screening in HTP
manner;
• Small molecules discovered by screening zebrafish disease models may also be useful as
lead compounds for drug development, because it appears to be high level of
conservation of drug activity between mammals and zebrafish.
1. SCREENING FOR CHEMICAL MODIFIERS OF VERTEBRATE DEVELOPMENT (ORGANOGENESIS).

• They provide information about tissue specificity, toxicity, and bioavailability,

• Cells are not transformed and are in their normal physiological environment of cell-cell and
cell-ECM interactions
• They allow the screening of processes not replicated in vitro such as organ development.
 2. SCREENING FOR MODIFIERS OF ONCOGENE-REGULATED HEMATOPIETIC DIFFERENTIATION.

It has been proposed that inhibitors of an oncogene’s effects on multipotent hematopoietic progenitor cell
differentiation may change the properties of the leukemic stem cells and complement the clinical use of
cytotoxic drugs > developping therapeutics targeting oncogene function in leukemia cells

• In vivo hematopoietic differentiation assay that reflects the activity of the oncogene AML1-ETO
• AML1-ETO = AE, leukemic oncogene that robustly converts erythropoiesis to granulopoiesis and blocks the
maturation of the granulocytes in the posterior blood island of the embryonic zebrafish (gata1-positive)
Yeh et al 2009, Nature Chem Biol
3. SCREENING FOR CHEMICAL SUPRESSORS.
Chemical induction of Wnt canonic pathway leads to eyesless phenotype.
 Speeding-up drug discovery pipelines with zebrafish
• Zebrafish screening is typically limited to a few thousand of compunds per week;
• The amount of labor involved in a whole embryo screening is greater thant cell line
screens;
• Zebrafish may often be better used as a secondary screening platform after a HTP
primary screen.
 A MODEL FOR INFLAMATION: Tg[mpx:GFP] let us visualize neutrophil dynamics

1hpw 2hpw 3hpw 6hpw

http://www.jimmunol.org/content/suppl/2013/03/18/jimmunol.1203266.DC1/Movie1.mpg
 OPTOGENETICS: a new enlightenment age for zebrafish neurobiology
(controlling neuronal activity with light)

Aim: Targeting subsets of neurons and monitoring and manipulating their activity.

• New genetically encoded optical tools, fluoresent sensors, and light-gated channels have
been generated – field of optogenetics
• Now, we can monitor and control neuronal activity with minimal perturbation and
unprecedented spatio-temporal resolution.

The application of optogenetics to zebrafish neurobiology has enable for the first time to
functionally test the role of identified neurons in behaviors.

The optogenetic revolution has allowed to silence and to activate neuronal circuits while
observing the neuronal activity or the behavior of live animals.

Optogenetic actuators: Light-gated channels and pumps allow the activation and silencing of
neurons

Optogenetic sensors: Fluorescent proteins engineered to sense calcium or membrane

potential
 OPTOGENETIC ACTUATORS
Photoactivation of ChR2 in zebrafish somatosensory neurons triggers escape behaviors

Isl1:ChR2-YFP
OPTOGENETIC SENSORS
How to monitor neuronal activity: functional imaging during adaptive motor control in
larval zebrafish
 BACK TO GENETICS!!!!

Screens in zebrafish:

• Screen by phenotype: mutagenesis screens, viral-insertion,…

• Screen by genotype: TILLING consortiums
• Other screens going on: zTRAP, FlipTrap

• Development of high throughput small molecule screens;

• The use of forward genetics methods for the study of several disorders, eg.
behavior, aging, etc
THE LONG JOURNEY TO TARGETED MUTAGENESIS
THE ART AND DESIGN OF GENETIC SCREENS: ZEBRAFISH

Outline of large-scale F2 genetic screens.

In F2 screens, a mutagen, such as ethylnitrosourea (ENU), is used to
generate hundreds of point mutations in the male pre-meiotic germ
cells (spermatogonia). ENU-treated males are crossed to wild-type
females to produce the F1 heterozygous progeny. F1 fish are then
crossed to siblings to create F2 families, half of which are
genotypically heterozygous for a specific mutation (m), whereas the
other half are wild type. F2 siblings are crossed, and the resulting F3
progeny are 25% wild type (+/+), 50% heterozygous (+/m) and 25%
homozygous (m/m) for a recessive mutation. Together, the Boston
and Tübingen screens, starting from about 300 ENU founder males,
involved raising more than 5,000 F2 families, analysing more than
6,000 mutagenized genomes and selecting more than 2,000 new
developmental mutants for characterization.
The first large-scale screen published: Tubingen and Boston
Examples of mutants identified in zebrafish large-scale screening efforts

Mutants for one-eyed pinhead (oep), which encodes a member

of the Nodal signalling pathway, lack endoderm, prechordal plate
and ventral neuroectoderm, which results in severe cyclopia
among other defects.

The recessive embryonic-lethal mutation weissherbst (weh)

results in hypochromic blood with decreasing blood cell counts.
Haemoglobin staining shows reduced levels of haemoglobin in
the weh mutant.

A dominant mutation, hagoramo (hag), which results in a

disrupted stripe pattern of adult fish and encodes a protein with
a possible role in proteolysis.
 Other things to screen

The space cadet mutant shows abnormal

swimming behaviours during a stimulus-induced
escape response.

Defects in vascular formation

CRISPR-Cas9 gene edition: overview

How to make CRISPR

… and don’t die trying
CRISPR-Cas9 example: targeting tyrosinase gene as a control

Evaluation of the CRISPR phenotype:

1. Phenotyping the embryos by retina pigmentation deficiency

CONTROL type 1 type 2 type 3

Tol2-BASED TRANSGENESIS

DNA plasmid germline transmission: 5%

DNA plasmid germline transmission of w/
Tol2: 30-70%
THE Gal4/UAS SYSTEM: borrowing the tools from Drosophila

Gal4: yeast transactivator

UAS: Upstream Activator Sequence

ADVANTAGES: versatility, amplification of reporter expression, easy cloning

I. CRISPR/Cas9 genome editing system: Clustered Regularly Interspaced Short Pal-
indromic Repeat associated nuclease 9 (Cas9)
DSB generated by Cas9

In absence of homology: In presence of

Non-Homologous End homology:
Joining (NHEJ) Homologous
Recombination (HR)

Indel mutations Allows the insertion of

sequences flanked by
homology arms

Knock-down
Knock-out
Knock-in

(Wyman and Kanaar, 2006)

GENERATING A KNOCK-IN TRANSGENIC FISH FOR BOUNDARY CELL TRACKING

Hevia et al, Cell Reports 2022

 DESIGNING ZEBRAFISH MODELS FOR HUMAN DISEASE:
ZEBRAFISH AS A HUMAN AVATAR

• Patients: WES, WGS to know where mutations are.

• Find orthologus genes in the zebrafish genome: see whether there is
subfunctionalization
• Reproduce the human mutation in the zebrafish genome using gene-edition
technology CRISPR-Cas9.
• Once transgenic fish line with the mutation is generated, use transgenic line for
doing biochemistry, drug screening, modeling, etc
 IF YOU WANT TO KNOW MORE

• http://www.sanger.ac.uk/Projects/D_rerio/
• http://www.fugu-sg.org/

Patton and Zon, 2001 Nature Reviews Genetics 2, 956-066

Grunwald and Eisen, 2002 Nature Reviews Genetics 3, 717-724
Skromne and Prince, 2009 Developmental Dynamics 237, 861–882
Nüsslein-Volhard, 2012 Development 139, 4099-4103
White et al, 2013 Nature Reviews of Cancer 13, 623-636
Terriente and Pujades, 2013 Developmental Dynamics 242: 97-107