L1: Principles of Vertebrate development

Gastrulation

• when blastula transforms from ball of cells into multi-layered strucuture

• composed of three distinct germ layers

• Requires the control of

  1. differentiation

  2. Morphogenesis

Reductionist vs integrative approach to developmental biology

Reductionist

• break down into smaller manageable parts

Integrative

• info from multiple independent experiments

• performed in different experimental systems

Body plan

• basic organization and arrangement of an animal’s body

• inc:

  • symmetry, segementation and arragement of organs and tissues

Gastrulation

• crucial early stage of embryonic development

• one layered blastula (blastocyst in mammals) is reorganized into 2 or 3 layers gastrula

Blastula

• hollow sphere of cells formed during early embryonic development in aniamls

Germ layers

• fundamental cell layers that form during early embryonic development in most animals

• lead to formation of various tissues and organs

In tripoblastic animals:

1. ectoderm

2. mesoderm

3. endoderm

Differentiation

• process by which cells become specialised

• acquire distinct strucutures and functions

Morphogenesis

• biological process by which a cell, tissue or organism develops its shape and structure

Principle anatomic axes

• Dorsal vs ventral

• Anterior vs posterior

• left vs right

Principal axis in a human (additional axis)

Proximal distal

Overview of Xenopus African clawed frog life cycle

1. Cleavage

   • really fast so no cell growth

2. Gastrulation (large scale morphogenesis (tissue shape changes))

   • Ball/disc—> 3D

3. Neuralation

   • Ectoderm → Neural tube

4. Organogenesis

5. Metamorphosis (in frogs)

Why a good model + cons (idk)

1. Ease of experimental embryology

2. can get lots of proteins from it in biochem analysis

Cons

1. Polyplody

2. metamorphosis?

What happens during cell differentiation

1. cells progressively restricted in array of cell types they can become

   • (their cell fate)

2. determined by signalling and other ascpects of the cell’s environment

What is this modelled by

Conrad Waddington’s Epigenetic landscape

• cells as a ball rolling down a series of valleys

• guided to one fat or another

Key terms of potenecy

1. Totipotent→ generate all embryonic and extraembryonic cells

2. Pluripotent→ generate all embryonic cells

3. Multipotenet→ all cells of multiple lineages

4. Unipotentent→ all cells of a single lineage

Can you go up the landscape

Yes→ transdifferentiation

• experimentally

• e.g induced pluripotency with Yamanaka factors

• back up the landscape

Three types of evidence needed to proove a mechanism in dev biology and how these are exp tested

1. Is it at right place/time (CORRELATION)

   • EXP: e.g gene expression: find correlation between gene and space/time (imaging)

2. Is it necessary for controlling the process? (REQUIREMENT)

   • EXP: knockout/ loss of function

3. Is it enough to drive the process? (SUFFICIENT)

   • EXP: gain of function/ over express the gene

Developmental biology uses many techniques and collaberations

Other sciences

• physistics

• mathematicians (make models)

• computer scientists

increasingthe norm for dev biology

Techniques used by developmental biologists to uncover the mechanisms of development and what questions they try to answer

1. Experimental embryology→ ‘cut and paste’ (classic technique)

   • cut and past tissues

   • Q: what requiremnts of tissue interactions

     • (+ descriptive of just looking which came before experimental embryology)

2. Developmental genetics→ ‘forward and reverse mutagenesis’

   • Q: what is role of gene

3. In vivo cell biology→ looking at cells and their function in their normal context

   • add context to in vitro studies

   • Q: is the gene made in the right time and place?

4. Biochemistry→ ‘extracting and blotting’

   • Q: what does the protein it codes for actually do

5. Neuroscience→ how is the nervous systm formed and wired

   • Q: development of neural connections must explain neurosceince function

   • neural development underpins the logic of how the brain must work

6. Stem cell biology→ recapitualting aspects of developent in vitro

   • What we learn from developmental biology we can apply to stem cell biology

7. Evolutionary developmental biology→ ‘Evo-Devo’ how so evolutionary changes in gene expression or function generate new phenotypes?

   • can only understand how evo works if you understand how dev works and how dev can be altered to form organsims that can change in time

1. Experimental embryology→ ‘cut and paste’: Key technique 1

Making Fate maps

1. mark one cell early in development

2. follow through develop

3. find ultimate fate

1. Experimental embryology→ ‘cut and paste’: Key technique 2

Testing Specification vs determination/competence

1. SPECIFICATION

   1. take cell out

   2. put in neural environment

      1. NOTE: still caveats (pertubation and may still be other signals)

   3. the cell fate observed must be what it is specified to be

   4. THEREFORE: the cell already has received info to make this cell fate before it left

2. DETERMINATION

   1. Take out cell

   2. put in different environment

   3. Receives these signals

   4. IF IT DOES NOT DIFFERENTIATE to cells expected of that environment→ the cell must be determined already

Equipment needed for this manipulations

• binocular microscope

  • microsurgery

• microinjector

  • inject specific mRNA DNA or cells

• Common for Xenopus or Zebrafish

• Use eyelash on a capillary tube to act as a knife

2. Developmental genetics→ ‘forward and reverse mutagenesis’ TECHNIQUES

• Forward

  • find a phenotype

  • find the gene responsible

• Reverse

  • Known gene

  • alter with CRISPR (knockout)
    find what phenotype it is responsible for

• TECHNIQUE: ZEBRAFISH have much mutant data

• many mutagenesis screens

3. In vivo cell biology→ looking at cells and their function in their normal context Technique to find where and right time

mRNA→ in situ hybridisation

1. Probe mRNA expression in cells

2. in situ hybridisation

3. add a probe the attaches to mRNA

4. prob has label

5. secondary antibody can bind

6. has alkaline phosphates

7. akes precipiatte if the mRNA is there

   1. ALTERNATIVE: use fluorescent

proteins (is protein in same place as gene?)→ antibody staining

1. Use first and second antibody

2. attach fluophore directly to the second

3. excite

Advantage of using fluorescent microscopy

• label multiple things

  • organelles, proteins, mRNAs

• All look at the same time→ just use different fluorphores and exite at different wavelength

Problem with using widefeild

• cells in vivo

• will just blur the image

Solution

Confocal microscopy

One section of the embryo at a time

• pin-hole

• only collects light in one focal plane

• can make Z-plane section

• progeress this through

• can form 3D image

4. Biochemistry→ ‘extracting and blotting’ why Xenopus is good for this

• large size

• lots of material can be extracted from single embryo

Why learn about gastrulation

1. Crucial moment → when body plan is established

2. Tech how cells both

   1. Differentiate (cell fate decision)

   2. morphogenesis (Generate shape change)

What happens in gastrulation

Image: with GFP (see how cell division in sync and then out of sync)

1. Ball of cells

2. cavity if formed

   • blastopore→ will become the anus

3. Folds in

4. closes

5. Forms ECTO and ENDO

6. Ecto ingression to form MESO inebtween the two

How does gastrulation become more complex

1. more cells

2. extra-embryonic tissue added (depends on the species)

Cell types from Ectoderm

Neural plate border

1. Nerual crest

   • Pigment

   • craniofacial

   • cranio ganglia

2. Sensory placodes

   • lens, inner ear, olfactory

Skin

CNS

Cell types from Mesoderm

1. Axial

   • Notochord

     • At first→ embryogenic (reigitity)

     • Then→ Spinal column

2. Paraxial

   • somites formed in periodic manner

     • Basis of segmented body plan

3. Intermediate

4. lateral

Cells from the endoderm

1. Gut

2. Respiratory

3. endocrine

Further reading