Model Organisms & Experimental Techniques (L1 & L2)

EMBRYOLOGY AND STEM CELL BIOLOGY

What is Developmental Biology?

The study of how an organism develops, from a single fertilised egg to a multi-cellular organism.

What constructs embryos?

Embryonic stem cells.

What can totipotent stem cells form?

The embryonic and extraembryonic tissues.

What can pluripotent stem cells form?

All embryonic tissues including all 3 germ layers, plus gametes.

What are the 2 main types of pluripotent stem cells?

Embryonic stem cells (ESCs) and induced Pluripotent Stem Cells (iPSCs).

What can multipotent stem cells form?

Multiple cell types from one germ layer.

How do Stem Cell Biologists engineer tissues and organs?

By mimicking what happens naturally in the embryo, applying the discoveries of Developmental Biology to solve real-world problems.

What are the 3 major axes of the body?

Ventral/dorsal, anterior/posterior, and left-right.

What does ventral mean?

Belly.

What does dorsal mean?

Back.

What does anterior mean?

Head end. Another word for anterior is rostral.

What does posterior mean?

Tail end. Another word for posterior is caudal.

Which axis is for asymmetrical models?

The left-right axis. Example: the human heart is on the left and the liver is on the right.

Which axis is for symmetrical models?

The lateral-medial axis. Example: the brain is symmetrical.

What does medial mean?

Closer to the middle.

What does lateral mean?

Closer to the edges. Example: fingers are lateral to the arm.

What is a sagittal cross section?

A cut right down the middle.

What is a para-sagittal cros section?

A cut parallel to the sagittal plane.

What is a horizontal/coronal cut section?

A slice splitting back and front.

What can you see in a horizontal/coronal cut section?

Anterior and posterior, but NOT ventral and dorsal.

What is a transverse cross section?

A cut across the body splitting torso from legs.

What is the most common cross section?

Transverse.

What can you see in a transverse cross section?

Left/right and dorsal/ventral, but NOT anterior and posterior.

What does proximal mean?

Close.

What does distal mean?

Far.

What does superior mean?

Above.

What does inferior mean?

Below.

What are the 3 ways to prove a gene is responsible for a type of development?

1. Correlation, 2. Gain of function, 3. Loss of function.

What is necessity in gene function experiments?

Loss of function.

What are the advantages of necessity experiments?

High confidence, and most scientifically compelling.

What is pattern in developmental biology?

Where cells go specifically. Example: fingers go at the end of hands.

What is morphogenesis?

The specific shape formed. Example: the thumb is smaller than other fingers, and fingers are roughly cylindrical in shape.

What comes first: pattern or morphogenesis?

Pattern comes first, then morphogenesis.

Stem cell biologists main interest:

CELL DIFFERENTIATION

What two sides do epithelial tissues have?

An apical side and a basal side.

Where are cortical cell junctions located in epithelial tissues?

Towards the apical side.

Where are extra-cellular matrix (ECM) proteins deposited in epithelial tissues?

At the basal side.

In vitro stem cell lab basal ECM types:

vitronectin, laminin, Matrigel™, etc.

What does the ventricular–marginal axis describe?

The developing brain, which is epithelial and divided into fluid-filled ventricles. Cells closer to the lumen are ventricular; cells further from the lumen are marginal.

In the brain, what does ventricular correspond to?

Ventricular = apical;

In the brain, what does marginal correspond to?

marginal = basal.

What are the stages of embryo development in order?

1. Fertilization → 2. Cleavage → 3. Blastulation → 4. Gastrulation → 5. Neurulation → 6. Organogenesis.

What happens during gastrulation?

Formation of the 3 germ layers: ectoderm covers the embryo, endoderm is inside, and mesoderm is in between.

What happens during neurulation?

Induction of neural tissue from ectoderm.

What happens during organogenesis?

Formation of the organs.

What are the stages of embryonic stem cells as time passes?

1. Totipotent → 2. Pluripotent (naïve) → 3. Pluripotent (primed) → 4. Multipotent → 5. Bipotent.

What is correlation (purely descriptive or observational evidence)?

If A & B occur together, then they might be causally associated,

What is the caution with correlation?

Correlation is not causation! It can suggest one or more hypothetical interactions, but it cannot prove them. A might cause B; B might cause A; A & B might both be caused by something else; or it could just be coincidence and they each have different causes.

What is gain-of-function (functional evidence)?

If we activate A (e.g. over-express A, or otherwise cause A to occur), and B also becomes activated, then activating A is sufficient to cause B.

What does gain-of-function test?

Sufficiency.

What is loss-of-function (functional evidence)?

If we de-activate A (e.g. knock down A, or otherwise prevent A from occurring), and B also becomes de-activated, then activating A is necessary to cause B.

What does loss-of-function test?

Necessity.

What is cell division in the principles of development?

How we go from one cell to many.

What is pattern formation in the principles of development?

How cells are organised in space/time.

What is morphogenesis in the principles of development?

How cells build an organism or organ's shape.

What is differentiation in the principles of development?

How cells become different from each other.

What is growth in the principles of development?

How different organs/body parts grow in the correct proportion

What limit exists on culturing human embryos in vitro?

The 14-day rule (recommended by the Warnock Committee), which prohibits culturing or experimenting on human embryos beyond 14 days of development.

What are the alternative anatomical names for the primary spatial axes?

Rostral–Caudal for Anterior–Posterior, and Medial–Lateral for Left–Right.

What spatial axes are visible in each 2D sectioning plane?

Sagittal plane: Anterior–Posterior and Dorsal–Ventral.
Coronal / Horizontal plane: Anterior–Posterior and Left–Right.
Transverse plane: Dorsal–Ventral and Left–Right (most common cross section).

What are the key advantages of using Xenopus laevis (African clawed frog) as a model?

Produces hundreds of large eggs on demand, develops externally, and allows easy physical micromanipulation/injection.

Why is Xenopus tropicalis used over Xenopus laevis for genetic editing?

Xenopus laevis is allo-tetraploid (4 gene copies), making knockouts difficult, whereas X. tropicalis is diploid (2 gene copies) and ideal for CRISPR/Cas9.

What makes Danio rerio (Zebrafish) an ideal model organism?

Rapid development (1 day to organogenesis), high fecundity, external development, and optical transparency (ideal for live confocal imaging).

How do forward genetics / mutagenesis screens work in Zebrafish?

Chemical mutagens induce recessive mutations in males; breeding through two generations isolates homozygous mutant offspring to discover novel developmental genes.

What are the advantages of Gallus gallus (Chick) as a model organism?

Amniote development closely matching human early 2D flat blastoderm geometry, accessible in-egg or in vitro manipulation, electroporation for gene transfer, and no need to maintain in-house animal breeding colonies.

What is the phylotypic stage?

The developmental stage where chordate embryos most closely resemble each other across species, displaying conserved architecture like the neural tube, notochord, somites, and pharyngeal arches.

What is the developmental hourglass model?

Embryos start with diverse geometries due to varying yolk levels, converge at a narrow "waist" (the phylotypic stage), and then diverge again as adult body plans develop.

What molecular evidence supports the phylotypic stage?

Transcriptomic analyses show that the phylotypic stage exhibits highly conserved gene expression patterns dominated by ancient, slow-evolving genes.

What does "ontogeny recapitulates phylogeny" mean?

Ernst Haeckel's classic theory that the embryonic development of an individual organism mirrors the evolutionary history of its species.

How does RNA Whole-Mount In Situ Hybridisation (ISH) work?

An antisense RNA riboprobe labeled with Digoxigenin (DIG) hybridizes to target mRNA, detected by an anti-DIG antibody bound to Alkaline Phosphatase (AP), which cleaves NBT/BCIP substrate to form a purple cellular precipitate.

What is TOMO-Seq (spatial transcriptomics)?

Slicing an embryo into sequential micro-sections (e.g., ~200 transverse cuts) and performing RNA-Seq on each slice to plot spatial gene expression gradients across axes.

What is fate mapping / lineage tracing?

Labeling embryonic cells with an indelible marker (e.g., fluorescent dye) to track their migration and identify what structures they ultimately form.

How are Loss-of-Function experiments conducted in embryos?

Using antisense Morpholinos (blocking mRNA translation/splicing), antagomiRs (blocking microRNAs), dominant-negative mutant proteins, CRISPR/Cas9 knockouts, or blocking antibodies.

Why is unilateral injection at the 2-cell stage a powerful experimental design?

Injecting morpholinos/plasmids into only 1 of 2 blastomeres alters one half of the developing embryo while leaving the un-injected side as an internal wild-type control.