VL 11:Zebrafish: Organogenesis, Pharmacology & Personalized Medicine

Question 1

Zebrafish are used
as a model system for..?

Answer 1

…Oragnogenisis
(Molecular methodology: Clonal Analysis, In vivo imaging, Whole organism lineage tracing by genome editing)

…Pharmacological screening
* Pharmacology: drug screening for clinical trials (example: treatment and prevention of Cerebral Cavernous Malformations)
* Personalized medicine: Zebrafish as tool to directly target and cure human disease (example: The zebrafish avatars as a tools to guide human cancer treatment)

Question 2

Morphogens def.

Answer 2

signaling molecules that act on cells directly to induce distinct cellular responses in a concentration dependent manner.

Question 3

Principels of Development: Organogenesis examples

Answer 3

Example 1: Development of Limbs
* Hox genes establish the polarizing region and also
provide a code for limb patterning
* The polarizing region specifies position along the limb’s antero posterior axis

Example 2: Human heart developmnet and fate map (Pic)

Example 3: Central Nervous System development- Vetrebrate Retina

Question 4

Neurogenesis in the vertebrate retina

Answer 4

• Retinal neurogenesis begins with cell cycle exit and cell-type determination followed by cell migration and differentiation. In vertebrates, retinogenesis progresses in a wave-like manner that spreads across the neuroepithelium, generally in a central-to-peripheral manner (Malicki, 2004).
• During retinal development, neuroepithelial progenitor cells divide in either a symmetric proliferative mode, in which both daughter cells remain mitotic, or in a neurogenic mode, in which at least one daughter cell exits the cell cycle and differentiates as a neuron.
• Neuroepithelial cells move their nuclei in an apical-to-basal manner with reference to the cell cycle. (M phase always occurs at the apical surface, whereas G1, S, and G2 phases occur at more basal locations.)

In this diagram, the white cell produces a postmitotic neuron. After the neurogenic division, one of the daughter cells (gray) exits the cell cycle to become a ganglion cell (red).

Question 5

In vivo cell lineage studies- How to study global dynamics of development?

Answer 5

a Spectrum of Fates approach
* a distinct fluorescent colour is associated with each of the major cell type (by driving the expression of differently tuned FPs from the promoters of a set of transcription factors (TFs), each of which is expressed in one or more cell types)
* useful to study the global dynamics of development and particularly the interactions between cell types
* They utilized different fluorescent proteins (FPs) attached to specific regulatory regions of genes expressed in different retinal cell types.

Question 6

In vivo cell lineage studies - How to study the behaviour of a single or a little group of cells?

Answer 6

Clonal Analysis
* SoFa lines allow precise identification of cell fate within developing clones.
* transplanting a developing SoFa1 clone in an unlabelled wild-type host.

Allows: Lineage Studies, Cell Behaviour, Mode of Cell Division( Symmetric vs Asymmetric), Cell autonomous gene function

Question 7

How to map the complete cell lineage of a complex multicellular organism?

Answer 7

Whole organism lineage tracing by combinatorial and cumulative genome editing
-> Genome Editing of Synthetic Target Arrays for Lineage Tracing (GESTALT)

• DNA barcode consists of multiple CRISPR)/Cas9 target sites.
• Lineage relationships can be readily queried by sequencing the edited barcodes and relating the patterns of edits observed.
  1) injecting fertilized eggs with editing reagents that target a genomic barcode bearing 10 target sites.
  2) accumulation of hundreds to thousands of uniquely edited barcodes per animal
  -> lineage relationships can be inferred on the basis of shared mutations that accumulate over many cell divisions within a compact DNA barcode.

In adult zebrafish, the edited barcodes from ~200,000 cells were evaluated and observed that the majority of cells in each organ are derived from a small number of progenitor cells.
Used aswell to Building a developmental atlas of the zebrafish brain with single-cell transcriptomics.

(Left) A barcode of CRISPR/Cas9 target sites is progressively edited over many cell divisions. (Right) Edited barcode sequences are related to one another on the basis of shared mutations in order to reconstruct lineage trees

Question 8

Cerebral Cavernous Malformations (CCM)

Answer 8

CCM are vascular lesion of the central nervous system vasculature for which a pharmacological cure is not yet available.
* small-molecule suppression screens to test 5,268 unique substances on CCM mutants
* integrated the results with the whole-transcriptome profile of zebrafish CCM2 mutants to identify signaling pathways relevant to the disease and potential targets for small-molecule-based therapies.
* CCM2 mutations lead to defects in vascular structures, resulting in cardiovascular malformations.
* These mutations cause changes in several signaling pathways, including KLF2/4, TLR4, MAPK, b1-integrin, angi
* Screening for heart phenotype rescue in the zebrafishccm2 m201 mutant
-> mutant heart phenotype looks like wild-type herat again
* Indirubin 3 monoxime IR3mo: a novel potential drug for treatment of CCM.
IR3mo reduces the number of caverns in the severe iCCM2 and iCCM3 mouse models

Question 9

The Zebrafish Avatar

Answer 9

Zebrafish implanted with a cancer patient’s tumor could guide cancer treatment, with resolution to reveal intratumor functional cancer heterogeneity.
1) Implantation of labeld Human cancer cells and histological analysis
2) Quantification of Early (EMP) and Late (LMP) Metastatic Potential, through an assay to discriminate the cellular metastatic potential, when placed directly in circulation or not.
3) CRC xenografts show different sensitivities to
standard chemotherapy
-> fast and highly sensitive in vivo model for differential therapy response