GENE223 - Mod 2, Animal Development

What are some of the most popular model multicellular organisms?

Drosophila (flies), C. Elegans (roundworms), Danio rerio (Zebrafish), S. cerevisiae (Yeast), A. thaliana (mustard plant)

Describe the embryonic strategy employed by Zenopus (frogs)

Xenopus females lay large eggs with provisions for 12 cell divisions

• The cells divide holoblastically: the whole embryo is sliced into cells - this is cleavage.

Describe cleavage during development

The whole embryo is sliced into cells. This is called cleavage.Cleavage cycles are shortened cell cycles which are rapid and synchronous.

What is the most difficult stage of triblastic organisms’ development?

Gastrulation, this is the switching of many cells from blastrulation into three different segments, exoderm, mesoderm and ectoderm. These segments can develop further into different body parts.

Describe the different ways an embryo develops into multicelularity

Cohesive multicellularity:

Starts with a single cell, spore or fertilized egg, that divides by mitosis to become an embryo. Cells remain together and feed in the multicellular stage.

Aggregative multicellularity:

Single cells feed and proliferate, only coming together when starved. Aggregates form fruiting bodies that can become dormant cysts or spores.

Describe the process of D. discoideum switching to aggregate multicellularity

The trigger is High pre-starvation factor and high cell density. This is facilitated by a chemotaxis, as amobae move towards high [cAMP].

cAMP triggers the GtaC, a GATA transcription factor which upregulates three proteins to be produced for cell adhesion.

~10^5 cells gather, cAMP pulses from the top shape the slime slug development.

Describe what germ cells are and why they are set aside early in development

Germ cells are pre-sex cells used to form gametes, they have different development patterns as they perform meiosis and their different developmental pattern causes them to be differentiated early.

What does a fate map show?

Inside a drawing of an embryo different portions of the system will be coloured or otherwise indicated to show what system they will develop into.

What is determination versus specification

Determination is the process where a cell develops into a certain kind of cell regardless of it environment, its cell type is already determined.

Specification is development to a kind of cell type in response to its environment. Usually the developing cell receives chemical messengers telling it to express certain genes and develop to be part of a tissue.

What is the main process through which cells segregate to different fates?

a) Localized determinants: proteins/RNA elements that push expression of a cell towards one tissue type. It is not cloned during mitosis and so only ends up in one of the daughter cells.

b) Inductive signalling: the localised determinant in the cell makes the cell produce chemical messengers that specify its surrounding cells.

c) Different Environments: After cell polarization and embryo compaction, the inner cells are cut off from the rest of the organism. This environment can cause specification of all those cells into a tissue.

What are some different chemical signalling mechanisms used by cells to specifiy others during development?

1. Paracrine Signalling. This is generla release of chemical messages by a cell and it is released into extracellular space, binding to any cells with correct receptors.

2. Juxtacrine Signalling. This only works through on adjacent cells and induces them through gap or tight junctions.

What is the cell called containing a determinant that specifies surrounding cells to form a specific organ/tissue?

The Anchor cell

What are some genetics experiments used to understand how to vulva develops in C. elegans?

• By lasering and destroying the anchor cell that signals vulva development, a phenotype formed is a vulva-less worm that isn’t able to excrete embryos.

• Mutant-testing. Finding mutants via their phenotype and making deductions from there, using reduced function mutations and loss of function mutations to see how development is affected by certain proteins. (Lin3 is mentioned in slides)

• Let23 encodes the Lin3 receptor, so non-functional Let23 causes no vulva development.

Label the early axes of bilatarians (6) like xenopus

Anterior - front

posterior - back

Dorsal - top like dorsal fin

Ventral - bottom, containing more organs while dorsal is spine.

Explain the experiment that lead us to understand how axes develop in amphibian embryos

Spemann-Mangold Organiser: By splitting embryos very gently at different angles to separate different parts, a grey section of the embryo was found to be required to be partially in each split part of the embryo to ensure proper development of the organism. This was the discovery of the Organiser.

How does the Organiser turn mesoderm cells from blood to dorsal muscle tissue?

Regular mesoderm cells produce BMPs (Bone morphogenetic proteins) that instruct cells to become and stay ventral mesoderm (blood cells). The organiser produces diffusing BMP inhibitor, which creates a [BMP] gradient which is interpreted by transcription factor promoters.

What is a morphogen

An information (chemical) gradient across cells, a long range signal that is secreted from cells at one loaction and diffuse away, forming a signal gradient. Cells along different points of the gradient directly respond to the signal, developing along different fates.

How is chemical concentration across the signal gradient from a morphogen interpreted by cells.

Cells will have multiple receptors with different binding affinities to the chemical signal, so they will have different responses based on their location in the signal gradient.

Explain how symmetry is broke in early amphibian development by the gray crescent containing a dorsal determinant

The grey crescent containing the dorsal determinant forms opposite the point of sperm entry due to cortical rotation, the dorsal determinant will determine the fates of surrounding cells and forming the dorsal region tissues.

How do transcription factors set off from morphogens switch genes on/off?

By allowing the transcription a gene; if this gene encodes a transcription factor as its protein product, it could potentially upregulate its own expression, thus starting a positive feedback loop that continues, that same transcription factor could block other genes from ever being expressed.

Describe and interpret experiments in zebrafish that demonstrate morphogen gradients.

By using expression markers to show what transcription factors are being expressed, experimenters injected different concentrations of an mRNA called Nodal. There were three distinct TFs and phenotypes at different signal concentrations, expressed in different cells depending on the concentration of Nodal added to the start cell.

Outline ways we can show where a gene is expressed in developing embryos.

Immunohistochemistry (for proteins), in situ hybridization (for mRNA)

Using reporter genes, getting a promoter for a gene expressed in nerve cells, then have that promoter express GFP and you can observe where those genes are usually expressed.

What is one method for finding the candidate genes within a molecular organiser?

Gene “fishing” to find genes expressed within the molecular organiser. On a dorsal blastopore area, there is a ‘lip’ where the organiser sits. You can dissect out organiser regions to extract mRNA being produced there, RT it into cDNA to make an expression library.

Explain and interpret experiments that led to the identification of the organiser candidate gene that encoded the TF in zebrafish.

Gene “fishing” in the organiser: discovery of Goosecoid gene
1. Isolate homeobox sequences from expression library: identify Goosecoid

2. Gsc is expressed only in the organiser.

3. Inject synthetics Gsc mRNA into ventral blastomeres, this causes two axes to form, indicating that Goosecaod is a candidate gene in vertebrates. Gsc is a TF tho, so it can’t be the actual morphogen.

Describe the experiment used to determine the morphogen element in the organiser of xenopus (amphibian) embryos.

using UV ventrilation, which destroys the dorsal determinant, causing only a belly of a zebrafish to form. They then injected pools of mRNA from their expression library, those that restored regular development were split in half and injected again, thus narrowing down the candidate genes until we could identify the determinant one.

How were candidate genes identified in zebrafish via experiments

Via mutagenic screening, we identified mutants with defects in the ventral regions organiser gene.

What drives assymetric cell division?

The positioning of the mitotic spindle during cytokinesis can change if a determinant is split between or goes to one daughter cell.