Developmental Biology Midterm

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Development

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The slow process of PROGRESSIVE change that give rise to a multicellular organism.
Developmental biology studies the initiation and construction of an organism rather than their maintenance.

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Differentiation

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How cells become different from one another and from their precursors. Each cell becomes structurally and functionally different producing distinct cell types, e.g. blood, muscle or skin cells

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Morphogenesis

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The biological process that causes an organism to develop its shape. It is one of three fundamental aspects of developmental biology along with the control of cell growth and cellular differentiation. In morphogenesis cells migrate, alter size and shape, tissues fold and separate into ordered structures. For example during gastrulation, the ball of cells (blastula) is rearrange into a gastrula, cells on the outside of the embryo moves inside, end result is the formation the germ layers, the endoderm, ectoderm and mesoderm.

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Cleavage

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Cell division is tightly regulated process. Each cell divides at the right time and place. The fertilized egg (zygote) rapidly divides into a number of smaller cells forming the blastula - Cleavage.

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Pattern formation

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Overall body plan is laid down (e.g. body axes are specified).

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Phenotypic plasticity

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An organism can express one phenotype under a certain set of condition and another phenotype under other conditions. This is an example of how the environment can influence development. Phenotypic plasticity occurs frequently in plants.

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Fin-to-limb transition

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-Devonian period, 400-600 million years ago
•Proximal skeletal elements (radius, ulna, humerus) of the tetrapod limb are present in the ancestral lobe fin fish

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Some characteristics of a model organism

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1)Size allows for easy manipulation and storage 2) Easily available and inexpensive

3) Short generation time

4) Mutants are easily generated, genetically modified (genetics)

5) Genomics information is available

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Examples of animal model organisms

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• Worm, Caenorhabditis elegans
• Fly, Drosophila melanogaster
• Frog, Xenopus laevis
• Chicken, Gallus gallus
• Fish, Danio rerio
• Mouse, Mus musculus

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Embryology

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The branch of biology that deals with the development of an embryo from the fertilization of the ovum to the fetus stage.

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Factors in choosing a model system

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Embryology-

•Bigger is often better for these experiments
•Some embryos are more robust than others
•Oocytes available in large numbers
•External development or in vitro culturing is important

Cell Biology and Microscopy-

•Need to deal with protective layers (egg shell, vitelline envelope)

•Ease of fixation and staining
(e.g. immunostaining or in situ hybridization)
•Tissue thickness
•Optical clarity
•In vivo imaging (clarity, ability to express transgenes)

Genetics-

•Need to grow for many generations or indefinitely in lab
•Generation time is limiting: the shorter the better

Genomics-

•Low genome complexity (less “junk” DNA and smaller regulatory regions).

•Low amount of gene redundancy makes forward and reverse genetics easier.

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Forward versus reverse genetics

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•Forward genetics (mutational analysis) - need to keep a large number of families in a small space
•Reverse genetics - ability to “knock out” a given gene of interest

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Transgenetics

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The ability to put back new or modified genes into genome.

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Advantages and disadvantages of worms as a model organism (C. elegans)

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• Advantages
- Great genetics - self-fertilizing hermaphrodite, short generation time
- Complete cell lineage known
- Optical clarity
- Sequenced Genome
• Disadvantages
- Small embryos
- Transgenics not as well developed

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Advantages and disadvantages of "Fruit fly: D. melanogaster" as a model organism

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•Advantages
-Great genetics - short generation time, wide array of genetic tool
-Excellent cell biology
-Small genome
•Disadvantages
-Small embryos
-Resistant to transplantation

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Advantages and disadvantages of "Zebrafish: Danio rerio" as a model organism

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• Advantages
- Best option for vertebrate forward genetics
(based on generation time, space and cost)
- Optical clarity and great cell biology
• Disadvantages
- Many genetic tools still in development

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Advantages and disadvantages of "African clawed frog: Xenopus laevis" as a model organism

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• Advantages
- Accessible and cheap
- Large embryo, oocyte available in large numbers, robust, easy
to manipulate
- Excellent embryology and biochemistry
• Disadvantages
- Yolky embryo limits optical clarity
- No genetics

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Advantages and disadvantages of "Chick: Gallus gallus" as a model organism

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• Advantages
-Very convenient and inexpensive to work on
- Eggs are easily maintained
- Excellent for micromanipulation
•Disadvantages
- Genetics not a strength

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Advantages and disadvantages of "Mouse: Mus musculus" as a model organism

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• Advantages
- Good “knockout” and transgenic
technology (homologous recombination)
- Embryos large enough for dissection and
explant assays

- Genetic system that is
evolutionarily closest to
humans
- Phenomenal similarity of
some developmental
genetic mutant phenotypes

•Disadvantages
-In utero development
-Limited quantities of embryos
-Less practical for genetic screens (although these are in progress in a few places)
-Animal care and experimental restrictions can be onerous

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Piebaldism syndrome

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Sterility, deafness, patching skin
coloring caused by mutation in a
gene called kit.

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Aristotle considered two basic developmental questions:

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-Do all parts of a developing organism come into existence together and simply grow larger?
-Is development a stepwise process characterized by progressive organization and an increase in complexity?

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Preformation

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The organism is preformed as a complete miniature structure in the sperm or the egg and simply grows larger as it develops.

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Homunculus

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A miniature, fully formed human. Nicolas Hartsoeker (1694)

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Epigenesis (upon formation)

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New structures arose progressively through a number of different stages. Organs of the embryo form “de novo”. Aristotle was a proponent of this theory.

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Cell Theory

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-Proposed by Theodor Schwann and Matthias Schleiden (1838)

1. All living organisms are composed of one or more cells.
2. The cell is the most basic unit of life.
3. All cells arise from pre-existing, living cells. (this part was added by Rudolph Virchow's (1855)

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Germ-Plasm Theory

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•August Weismann's (1880’s) theory of the germ plasm, which proposed a segregation between germline (germ cells) and somatic (body) cells during development.
• Inheritance in a multicellular organism only takes place by means of the germ cells.

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Anatomical approach to developmental biology

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Describe embryogenesis (comparative/descriptive)

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Experimental approach to developmental biology

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Manipulate the embryo by cutting, grafting, etc.

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Genetic approach to developmental biology

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IsoQlate mutations that change development.

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Quantitative approach to developmental biology

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Mathematical modelling, computational

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Ectoderm

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-(outside) outer layer of embryo

-The ectoderm is the start of a tissue that covers the body surfaces. It emerges first and forms from the outermost of the germ layers.

-The nervous system and the skin derive entirely from the ectoderm. The ectoderm also gives rise to the neural plate.

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Endoderm

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-The endoderm is one of the germ layers formed during animal embryogenesis. Cells migrating inward along the archenteron form the inner layer of the gastrula, which develops into the endoderm

-The endoderm is the innermost layer

-The endoderm ultimately gives rise to the lining of many of the internal organs (viscera)

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Mesoderm

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-From the mesoderm arise the bones of the skeleton and the muscles.

-Middle layer

-The mesoderm germ layer forms in the embryos of animals more complex than cnidarians, making them triploblastic. During gastrulation, some of the cells migrating inward contribute to the mesoderm, an additional layer between the endoderm and the ectoderm.

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Triploblastic

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-Organism that has all three layers in the embryo (ectoderm, endoderm, mesoderm)

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Diploblastic

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-Some phyla such as the cnidarians (sea anemones, hydra and jelly fishes) lack a true mesoderm and are considered diploblastic animals.

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Fate Maps

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-First developed by Walter Vogt (1929)

-A method to label a small number of cells with a dye that would be passed on to all of those cells' direct descendants. Follow the marked cells through development to see what they became.

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How are cells labelled in fate maps?

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-C3 cell injected with a fluorescent die, give rise to mesodermal cells on one side of the embryo.

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Early ablation experiments in experimental embryology

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Destroyed right or left halves of frog embryos. Obtained “half embryos” having a complete right or left side. Wilhelm Roux (1888) concluded that the fate of each cell is determined at cleavage.

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Mosaic Development

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Embryo constructed of individual modules capable of self-differentiation. Cells developed autonomously.

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Theory of nuclear determination

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•Theory put forward by August Weisman (1880’s) provided suppport for mosaic development.
•Unequal distribution of nuclear components called “determinants” drive differentiation.
•Fate of each cell is predetermined at cleavage.

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Was the theory of nuclear determination correct?

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•Weisman’s theory of nuclear determinants is incorrect. However the distribution of cytoplasmic determinants at cleavage do make daughter cells different from each other.

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Early isolation experiments in experimental embryology

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At the two cell stage, Hans Driesch (1892) killed one of the two cells when the embryo was at the two cell stage. The surviving cell developed into a small but otherwise normal larva.

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Regulative Development

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The ability of embryonic cells to change their fate so as to compensate for missing parts

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Is development mosaic or regulative?

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Virtually all embryos undergo both mosaic and regulative development at some point during their growth.

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Gene expression determines cell behavior and development

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1. Cell-cell communication 2. Cell shape changes 3. Cell movement 4. Cell proliferation 5. Cell death (apoptosis)

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Genes expression regulated at several levels

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1. Transcription
2. Selective nuclear RNA processing
3. Selective mRNA export
4. Selective mRNA translation
5. Differential protein modification
6. mRNA or protein degradation

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Cell Identity

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-Differential Utilization of the Genome

-Specific Pattern of Gene Expression and Genes that can be Expressed

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Maternal-effect genes

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-Genes active in the mother whose products are loaded into the egg and are required for the earliest developmental stages.
-The maternal genotype determines the phenotype of the offspring

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Zygotic genes

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Genes active in the embryo, required for its own development. The embryo’s own (zygotic) genes become activated sometime during cleavage.
e.g recessive zygotic gene

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Chimera

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-Organism made up of cells from two different sources.

-If the mutation is present in the germ line the chimera will produce gametes that carry the mutation. Chimera is bred to produce strains of heterozygote transgenic mice.

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Reverse Genetic Approaches

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-Gene Knock-out (mice)

-RNA Interference (RNAi) – introduction of double stranded RNA (dsRNA) triggers the destruction of mRNA of similar sequence.
-Morpholino antisense RNA – short stable RNAs that hybridize to complementary target mRNAs and block translation.
-Dominant negative – the product of a mutated gene adversely affects the function of the wild-type gene.
-Neutralizing antibody – inhibition of protein function by binding of specific antibodies.

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Ectopic Gene Expression

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-Gene is expressed where it is not normally found.

-Aniridia: absence of the colored
part of the eye (the iris). Caused
by mutations in the PAX6 gene.

-Ectopic expression of eyeless

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Neural tube formation

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The neural plate gives rise to the neural tube and neural crest cells.

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Outer ectoderm (epidermis) - ectodermal derivatives

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Neural crest - ectodermal derivatives

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Neural tube - ectodermal derivatives

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Neural Induction

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Interaction between the dorsal lip of the blastopore (the organizer) and the neighboring ectoderm leads to the induction of the nervous system.
Neural induction is due to inhibitory activity of proteins released from the organizer.

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Neural Induction: Default model

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• Ectodermal cells will become neurons if they receive no signals.
• Neural fates is inhibited in prospective epidermal cells by the action of bone morphogenetic proteins (BMPs).
• BMP4 is inhibited in future neural plate when the organizer appears.
• Organizer molecules Noggin
and Chordin inhibit BMP4.

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BMP4

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Regulates the formation of teeth, limbs and bone from mesoderm. It also plays a role in fracture repair, epidermis formation, dorsal-ventral axis formation, and ovarian follical development.

• Neural fates is inhibited in prospective epidermal cells by the action of bone morphogenetic proteins (BMPs).
• BMP4 is inhibited in future neural plate when the organizer appears.
• Organizer molecules Noggin
and Chordin inhibit BMP4.

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Noggin

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-An organizer molecule which inhibits BMP4

Noggin is a signaling molecule that plays an important role in promoting somite patterning in the developing embryo.[3] It is released from the notochord and regulates bone morphogenic protein during development.[4] The absence of BMP4 will cause the patterning of the neural tube and somites from the neural plate in the developing embryo. It also causes formation of the head and other dorsal structures

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Effect of ectoderm, BMP, and inducers on

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Ectoderm alone --------> Neural
Ectoderm + BMP ------> Epidermis
Ectodem + BMP + noggin/chordin ------> with noggin or chordin present to cancel it out, it returns to neural tissue again

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How do noggin and chordin block BMP?

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Noggin and Chordin inhibit BMP by blocking there interaction with the receptor in the ectoderm. They signal from the ventral mesoderm with inducers BMP-4 and Xwnt-8.

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Frizbee

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Also a neural inducer, involves epidermal inducers such as BMP-4, and leads to epidermis.

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How do neural inducers work during gastrulation?

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Inhibitory proteins (noggin/chordin) from the organizer act on adjacent ectoderm at the beginning of gastrulation and cells derived from the organizer (notochord) influence adjacent ectoderm as gastrulation continues.

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How is the neural tube regionalized?

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•The neural tube bulge and constrict to form the chambers of the brain (mid, hind and forebrain) and the spinal cord.
•Cell populations rearrange themselves to form the different functional regions of the brain and the spinal cord.
•Cells differentiate into the numerous types of nerve cells (neurons) and supportive cells.

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Signaling centers of the brain

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ZLI – Zona limitans intrathalamica
MHB – midbrain-hindbrain boundary (Isthmus)

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Hindbrain segmentation

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Hindbrain is segmented along the antero-posterior axis into eight rhombomeres, r1-r8.
Cranial nerves are associated with specific rhombomeres.
Hox genes specify identity of each rhombomere.

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Rhombomeres

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In the vertebrate embryo, a rhombomere is a transiently divided segment of the developing neural tube, within the hindbrain region (a neuromere) in the area that will eventually become the rhombencephalon. Rhombomeres determine the pattern of the following maturation of the rhombencephalon into its final parts. The final parts are defined as the pons, cerebellum and medulla.

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Hox genes are members of the homeobox gene family

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• Regulate developmental processes in diverse organisms (yeast, plants, insects, mammals)
• Homeobox genes code for transcription factors that contain a homeodomain.
•The homeodomain is encoded by a DNA motif called a homeobox.
• Homeobox genes assigns identity to an established segment or region.
• Mutations in homeobox genes causes segments to develop with inappropriate identities – Homeotic transformations.
• Not all genes that contain a homeobox are involved in homeotic transformation!

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Homeosis

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• Coined in 1894 by W. Bateson
“the assumption by one member of a meristic series, of the form or character proper to other members of the series”
In other words: The transformation of one body part into another
• Goethe, 1790 referred to homeotic variation in plants as “abnormal metamorphosis”
• E. B. Lewis 1978, reported a number of mutations that resulted in either the duplication of a distinct body segment or the appearance of an inappropriate structure at an ectopic location in the fly. Received the Nobel Prize in Physiology or Medicine 1995

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Homeotic transformation

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Example, changing antennas into legs (Antennapedia (Antp)) in a fly or adding an extra pair of wings (Bithorax (bx)).

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How Do Homeotic Genes Function?

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•Act as genetic switches that control the choice between different developmental pathways.
• Regulate downstream genes that ultimately control morphology.

Segments where Antp is required for leg formation it disengages antenna genes and a leg is formed.
Expression of Antp in segments designated for antenna, result in legs.

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Homeobox genes exhibit two patterns of localization:

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1.Some are scattered throughout the genome.
2.Other homeobox genes (Hox genes) are clustered within a small region and in a very specific sequence (Hox genes form a Hox complex). These Hox genes are highly conserved in organisms ranging from flies to humans.

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Hox complexes

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Arose by repeated duplication and mutation of an ancestral homeobox gene. This formed an ancestral HOX complex. In some organisms, including most vertebrates, the HOX complex has been duplicated four times.

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Paralogy group

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Hox genes are organized into paralogy and orthology groups.

Are simply the natural clusters of Hox genes on the chromosome. Vertebrates have 4, flies have 1. They are named a, b, c, and d.

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Orthology group

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Hox genes are organized into paralogy and orthology groups.

Sequence comparisons of each gene shows that certain genes are closely related. These are the genes in most vertical columns (for example, Hox a9, b9, c9 and d9). Orthologous Hox genes from different species can replace one another in function (Hox d4 substitutes for Dfd in Drosophila).

Genes of any orthology group have corresponding positions within each complex (if clusters are aligned in rows, the orthology groups form columns. Genes are numbered consecutively from 1-13.

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How do hox gene expression along the antero-posterior axis

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The physical order of Hox genes within the complex is related to their order of expression along the antero-posterior axis of the embryo!! (colinearity). Genes at the 3’ end are expressed in the anterior and genes at the 5’ end are expressed progressively further posteriorly.

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Hoxb cluster

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Genes are expressed in the neural rube and mesoderm. each gene is first expressed at a sharply defined point and expression continues posteriorly and gradually tapers off.

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Homeotic transformations

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-Conversion of one body part to another

-Result from Hox gene loss or over expression.

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Posterior prevalence

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Mutation affects the anterior extent of gene expression. Hox gene loss leads to cells assuming a "more anterior value" i.e. Hoxc8 mutant mice have extra ribs.

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Expression in hox genes for r1 and r2

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No Hox genes are expressed in r1. Expression of Hoxb1 in r2 results in motor axons going to b2 instead of b1 -homeotic transformation.

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Retinoic Acid (RA)

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• Retinoic acid is a derivative of vitamin A
• Gradient help to pattern the antero- posterior axis.
• Regulates Hox gene expression.

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Retinoic acid in mouse embryo

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Retinoic acid activated gene expression:

Result – gene expression is activated throughout forebrain.

Normal mouse brain:

Abnormal brain of a mouse whose mother ingested this same amount of retinoic acid.

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Retinoic acid acts as both a teratogen and an endogenous signaling molecule

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•Excessive amounts of vitamin A or a vitamin deficiency results in severe brain abnormalities
•Teratogen - agent that can disturb the development of an embryo.
•Accutane in the 1980s: Accutane or isoretinoin a drug used in the treatment of acne. Women who took this drug during pregnancy had an increased number of spontaneous abortions and children born with a range of birth defects due to increased exposure to retinoids.
•The reason for this was not known until retinoic acid was found to activate expression of a number of target genes (e.g. Hox genes).

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Dorso-ventral Organization of the Neural Tube

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•Neural tube that develops into spinal cord is pattern along the
dorso-ventral axis.
•Commissural neurons (C) are located dorsally
•Motor (M) and interneurons (V3) and are located ventrally

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Sonic hedgehog

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A ventral signal in dorsal-ventral patterning.

It occurs in the spine and patterns the ventral half of the tube.

Notochord emits sonic hedgehog that induces floor plate and motor neurons.
Once induced, the floor plate also expresses sonic hedgehog which induce ventral cell types.

BMP is the dorsal signal.
Patterns the dorsal half of the tube.
The epidermis emits BMP which specify the roof plate.
Once induced, the roof plate also expresses BMP and other factors that ensures dorsal differentiation.

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Lateral inhibition

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In neurobiology, lateral inhibition is the capacity of an excited neuron to reduce the activity of its neighbors. Lateral inhibition disables the spreading of action potentials from excited neurons to neighboring neurons in the lateral direction.

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Notch and delta signaling

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• Specification of vertebrate neuronal precursor involves lateral inhibition.
• Involves Delta and Notch signaling
• Once one cell expresses more Delta…
•it inhibits neural differentiation in its neighboring cell.
•it activates genes required neural differentiation.

• Cells can adopt a neural or epidermal fate.
• Each has equal amount of receptor and ligand.
• Delta (ligand) signals to Notch (receptor and transcription factor) which inhibits neuronal differentiation.
• A cell gets an edge of Delta production inhibiting its neighbors from adopting neural fates and at the same time promote its neural fate.
• High Delta, low Notch activity – neuronal differentiation.

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Examples of axial planes

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Ventral-pertaining to the belly. The opposite of dorsal.
Dorsal-refers to the back. The opposite of ventral.
Anterior-refers to the head. The opposite of posterior.
Posterior-refers to the tail. The opposite of anteroir
Lateral-refers to the side of the body.
Medial-refers to a position near the middle or midline.
Distal-farthest from the centre, from the midline or from the point of attachment.
Proximal-nearest to the centre, to the midline, or to the point of attachment.

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Developmental Processes

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Cell Division: producing a multicellular embryo involves an initial cleavage stage followed by regular mitotic divisions.
Morphogenesis: rearranging the embryo (gastrulation and neurulation), change in shape/form. Results in the embryonic organization, the pattern and polarity of cells, organs and tissues.
Cell differentiation: cells acquire distinct identity. Formation of different, specialized cell types.

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Sea Urchin Cleavage

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Cleavage: Egg divides assymetrically producing two cell types; macromeres and micromeres. Blastula consists of cilliated epithelial monolayer surrounding a fluid-filled cavity called blastocoel.

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Sea Urchin Gastrulation

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Gastrulation: compromises a series of cell rearrangements in which the primary germ layers move into their positions and the basic body plan is established, forms a gastrula.

Events in gastrulation
•Primary mesenchyme (mesoderm) cells ingress to form skeletal rods (red).
•Endoderm invaginates forms gut and anus. Gut elongates and fuses with the animal region to form mouth. O-A axis forms.
Secondary mesenchyme (mesoderm) give rise to muscle.
Produces a feeding larva (Pluteus).

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Characteristics of frog egg

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Egg is divided into a dark pigmented hemisphere (the animal pole) and a lightly or unpigmented hemisphere (the vegetal pole).
The pigment granules reside in the cortical cytoplasm of the egg and are more abundant in the animal pole.
The yolk is distributed throughout the cytoplasm of the egg but is much more concentrated toward the vegetal pole.

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Early frog development: cleavage

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Cleavage: Egg is divided into smaller and smaller cells called blastomeres (macromeres and micromeres). Fluid filled blastocoel (Bc) develops in the animal pole (AP) of the blastula.

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Early frog development: blastula

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• Blastula produced after 12 divisions.

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Cells that will give rise to …

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….ectoderm is at the animal pole.
….mesoderm is located around the equator in the marginal zone.
….endodoerm is at the vegetal pole and also overlays the mesoderm in the marginal zone

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Early frog development: gastrulation

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Gastrulation: Extensive rearrangement of the embryo so as to place the germ layers in the correct position. Internalization of the mesoderm and endoderm and spreading of the ectoderm.

Basic phases of frog gastrulation.
1) Formation of a slit-like infold – blastopore with a dorsal lip.
2) Mesoderm and endoderm converge and begin to move inwards at dorsal lip. 3) Ectoderm spreads to cover embryo. 5) A space forms between the yolk cells - archenteron (future gut cavity).
6) Blastocoel is diminished.

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By the end of gastrulation...

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1) 3 germ layers are in position.
2) blastopore is closed with a yolk plug.
3) mesoderm is beneath the ectoderm. 3) ectoderm covers embryo. 4) yolky vegetal cells are internalized (food source).
5) dorsal mesoderm begins to develops into notochord (rod structure along dorsal midline) and somites (segmented blocks of mesoderm along notochord, will give rise to muscle).
6) The body plan is established.

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Early frog development: neurulation

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Neurulation: formation of the neural tube, the precursor for the central nervous system. Neural crest cells are also created. Embryo now called a neurula.

Events in neurulation
1) Formation of a neural plate in the ectoderm located above notochord. 2) The edge of the neural plate forms neural folds which rise towards midline.
3) The neural folds fuse to form neural tube.
4) The neural tube sinks below ectoderm (now epidermis).
5) The anterior neural tube will become the brain. The mid and posterior neural tube will become the spinal cord.

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Formation of a neural tube

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1) Formation of a neural plate in the ectoderm located above notochord. 2) The edge of the neural plate forms neural folds which rise towards midline.
3) The folds fuse to form neural tube.
4) At tube closure, the cells at the junction form neural crest cells.
5) The neural tube sinks below ectoderm (epidermis).

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Early chick development: cleavage

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Cleavage: occurs in small patch of cytoplasm atop the yolk. Produces a blastoderm.
Blastoderm is divided into two areas: 1) the area pellucida (a light area) - area over the subgerminal space and 2) the area opaca (a dark ring).

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Hypoblast, epiblast, and koller's sickle

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Hypoblast – develops over the yolk. Source of extra-embryonic tissues.
Epiblast - forms the embryo.
Koller’s sickle – crescent shaped ridge that forms at the posterior marginal zone, marks the position where gastrulation will begin and posterior end of the embryo.

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Early chick development: gastrulation

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Gastrulation: Allocation of germ layers. Begins with the formation of the primitive streak, a furrow in the epiblast.

1) Hypoblast is replaced by endoblast. Primitive streak begins at the posterior marginal zone specifically at the Koller’s sickle.
2) Streak forms a furrow in the epiblast and extends to the center of the disc.
3) Epiblast cells move towards the streak and internalize at this site. These cell give rise to mesoderm and endoderm.
4) Cells remaining in the epiblast become ectoderm.

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Visualization of gastrulation

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Regression of hensen's node

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Primitive streak regresses, Hensen’s node moves towards the posterior.
Head fold, neural tube and somites form ahead of the regressing node.

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Where does neurulation begin in the chick?

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•Neurulation begins once the notochord has formed.
•Neural tube formation begins at the anterior end of the embryo ahead of the regressing Henson’s node.

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Early mouse development: cleavage

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Cleavage: cell division produces a blastocyst with a fluid filled blastocoel.

Compaction: unique to mammals. Loosely arranged cells become tightly packed. Produces a compacted morula.
For implantation to occur, the zona pellucida is discarded and blastocyst attaches to uterine wall - blastocyst hatching.

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Two cell types in the mouse blastocyst

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1) Inner cell mass

2) Trophectoderm

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Inner cell mass

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Inner cell mass give rise to the primitive ectoderm/epiblast and primitive endoderm/hypoblast.
-Epiblast will form the embryo and give rise to some extra embryonic tissue.
-Primitive endoderm will contribute to the extra-embryonic structure.

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Trophectoderm

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Trophectoderm will produce extra embryonic structures.
-polar – in contact with inner cell mass.
-mural – not in contact with inner cell mass.

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Early post-implantation development of the mouse embryo

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•Mural trophectoderm gives rise to trophoblast giant cells.
•Polar trophectoderm becomes the extra-embryonic tissue such as placenta.
•The epiblast becomes cup-shaped and the proamniotic cavity forms.
•Primitive endoderm migrates to cover the... …..inner surface of mural trophectoderm to become the parietal endoderm. …….egg cylinder containing epiblast to become the visceral endoderm.

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Basics of mouse gastrulation (similar to gastrulation in chick)

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1) Primitive streak forms in the epiblast at the start of gastrulation.
2) The primitive streak forms at the future posterior end of the embryo.
3) The streak migrates to the bottom of the U-shaped cup, the future anterior end.

4) Epiblast cells move through the primitive streak and spread anteriorly and laterally between the epiblast (ectoderm) and the visceral endoderm to form the mesoderm.
5) At the anterior end of the primitive streak, cells condense forming the node (similar to chick Hensen’s node). 6) Epiblast cells that migrate anteriorly through the node form the notochord.

7) Some epiblast-derived cells that migrate through the primitive streak displace the visceral endoderm to form the definitive endoderm.
8) Epiblast-derived cells that migrate anteriorly will give rise to somites.
9) Definitive endoderm becomes internalized to form gut.

Embryo anterior to the node grows rapidly. Node becomes a stem cell center that produces the post-anal tail.
Neural folds form at the anterior end which give rise to neural tube.
After neurulation the undergoes ‘turning’ which puts the dorsal side on the outside.

Front 113

Early Embryonic Development

Back 113

Cleavage
•Rapid cell division to form the blastula/blastocyst/blastoderm.
Gastrulation
•Rearrangement of the embryo after cleavage to form the gastrula.
•Positioning of the three germ layers
- endoderm, ectoderm and mesoderm
Neurulation
•Rearrangement of the embryo after gastrulation to form the neurula.
•The progenitors of the CNS segregate from the ectoderm to form a neural tube.

Front 114

Major consequences of cleavage

Back 114

•Each cell division amplifies the total number of cells, but the resulting cells become increasingly smaller with each division during the cleavage phase.
•Consequences:
-Production of cells required to form the multicellular animal.
-Acquisition by individual cells or groups of cells of differences that will facilitate their later development into different cell types.

Front 115

Cell Cycle

Back 115

G1 = growth and preparation of the chromosomes for replication
S = synthesis of DNA
G2 = preparation for mitosis
M = mitosis

Front 116

Cleavage Cycle

Back 116

Skips the G1 and G2 growth period between mitotic divisions (biphasic cell cycle).
-rapid
-no growth/increase in size
-synchronous

G1 = growth and preparation of the chromosomes for replication
S = synthesis of DNA
G2 = preparation for mitosis
M = mitosis

Front 117

Mid-blastula transition (MBT)

Back 117

Rapid, synchronous divisions driven by maternal products (RNA and proteins places in the oocyte).

At the mid-blastula transition
-Introduction of gap phases
-Cell cycle lengthens
-Asynchronous divisions

Front 118

Egg activation and the cleavage cycle

Back 118

•Once fertilized, the maternal and paternal genetic material (pronuclei) will fuse, and the now single-celled embryo must divide to produce the cells required to form the multicellular animal.
•Fertilization is the stimulus that triggers the zygote to re-enter the cell cycle (mitosis).

Front 119

Early development depend on maternal products

Back 119

•Maternal RNAs deposited into the egg during oogenesis drive early development.
•Maternal RNAs are degraded during different stages of embryogenesis (blastula and gastrula) and transcription of the zygotic genome begins – Maternal-Zygotic Transition (MZT).
•microRNAs (miRNAs) produced from the zygotic genome promote the elimination of maternal RNA.

Front 120

Maternal-Zygotic Transition (MZT)

Back 120

•The timing of zygotic genome activation differ across species.
•Several mitotic cell cycles occur before the zygotic genome is activated.
•In animals that develop rapidly, more cleavage divisions are completed before the zygotic genome is activated.
•In the mouse, the cell cycles are longer and zygotic contribution begins earlier compared with Xenopus.

Front 121

Cell division - cytokinesis

Back 121

The first visible sign of cytokinesis is when the cell begins to pucker in, a process called furrowing. Furrow is formed by contractile ring.
Furrowing (plane of cleavage) tends to take place at right angles to the axis of the spindle microtubule.
The spindle is the structure that separates the chromosomes into the daughter cells during cell division.

Front 122

Plane of cleavage – why is this important?

Back 122

•Determines the spatial arrangement of daughter cells relative to each other.
•Determines if daughter cells will be of equal or unequal size.
•Partitioning of cytoplasmic determinants to daughter cells, thus determining cell fate.
How are positions of the cleavage plane, i.e. cleavage furrow, determined?

Front 123

Determining Plane of Cleavage

Back 123

Planes of cleavage are determined by the behaviour of the centrosomes in each cell.
Prior to mitosis, the centrosome duplicates and migrate to opposite side of the nucleus and form asters.
The pattern of centrosome duplication and movement determines the plane of cleavage.

Front 124

Patterns of cleavage

Back 124

Early protosome embryo. Its four cells are undergoing cleavages oblique to the original body axis:

e.g. annelids and molluscs

Early deuterostome embryo. Its four cells are undergoing cleavages parallel with and perpendicular to the original body axis.

e.g. echinoderms and chordates

Front 125

Types of cleavage

Back 125

Amount of yolk affects cleavage
1) Very little evenly distributed yolk.
- Holoblastic or complete cleavage.

- Cleavage furrow extends through the entire egg; entire egg is cleaved.
- Example mammals and enchinoderms.
2) Moderate or intermediate amount of unevenly distributed yolk.
- Holoblastic

- The entire egg is cleaved but the yolk free portion divides faster than the yolk filled region.
- Example Xenopus - animal pole cells divide faster than the yolk filled vegetal pole.
3) Large amounts of yolk throughout most of egg.
- Meroblastic or incomplete cleavage

- Cleave furrow does not penetrate the entire egg, only the animal pole divides - yolk impedes/halts division.
- Example chick.

Front 126

Cleavage in sea urchin

Back 126

Sea urchin embryos undergo radial cleavage.
However, the fourth cleavage is unequal producing micromeres at the vegetal pole.

Front 127

Sea urchin blastula

Back 127

•The blastula is a hollow ball of cells organized into an cilliated epithelial monolayer.
•Midway through the blastula stage, the embryo secrete enzymes, which results in proteolytic degradation of the fertilization envelope - hatching.
•The vegetal pole epithelium thickens to form the vegetal plate, which will give rise to primary mesenchyme cells and the archenteron (gut) during gastrulation.

Front 128

Cleavage in mouse

Back 128

•Cleavage also occurs in oviduct. First division at ~24 hours and every 12 hours after that to form the morula (~32 cell stage).
•Cleavage is slower than Xenopus and chick.
•Blastomere compaction happens at 8 cell stage.
•Cleavage continues to produce the blastocyst with a fluid filled blastoceol and inner cell mass.

Front 129

Mouse blastocyst

Back 129

The blastocyst has 2 cell types: Inner Cell Mass and Trophectoderm.
1) Inner cell mass give rise to the primitive ectoderm/epiblast and primitive endoderm/hypoblast.
Epiblast will form the embryo.
Primitive endoderm will contribute to the extra-embryonic structure.
2) Trophectoderm will produce extra embryonic structures.
Embryonic-abembryonic axis

Front 130

Cleavage in Xenopus

Back 130

Egg has distinct polarity, pigmented animal region and a yolky vegetal pole.

•First division occurs about an hour after fertilization along the animal-vegetal axis.
•Second division also occurs in the same animal vegetal axis but a right angles.
•Third cleavage is equatorial and displaced towards the animal pole, this produces four small animal blastomeres (micromeres) and four large vegetal blastomeres (macromeres).
•Division occur every 20 minutes.

Front 131

Xenopus blastula

Back 131

Front 132

Chick blastoderm

Back 132

Blastoderm is divided into two areas: 1) the area pellucida (a light area) - area over the subgerminal space and 2) the marginal area opaca (a dark ring).

Front 133

Cells become polarized in the early embryo

Back 133

Sea urchin: blastula is a hollow sphere with the outer surface covered in cilia. The single layer epithelium is lined by two extracellular matrices: (i) an inner basal lamina and (ii) an exterior hyaline layer.

Cells become polarized in the early embryo.

Mouse: embryo undergo compaction, cell-cell contact/adhesion is increased and microvilli become restricted to the apical (outer) surface.

Front 134

Forming the blastocyst inner cell mass (ICM)

Back 134

At the 8 cell stage, asymmetric divisions (resulting from a division plane (dashed line) that separates apical and basal domains) produce an inner cell mass cell and a trophectoderm cell, whereas symmetric divisions dividing the apical domain produce two trophectoderm cells. At the 32 cell stage, the position of a cell determines fate, with cells on the inside forming the inner cell mass and cells on the outside taking up a trophectoderm fate.

Front 135

Forming the blastocoel

Back 135

Peripheral sealing of the embryo by the formation of adhesive cell-cell tight junctions on the apical side of the epithelium cell layer. Junctions act as a barrier preventing molecules from diffusing across an epithelial sheet between adjacent cells. Mouse: tight junctions begin to form at the 8 cell stage and the barrier is established by the 32 cell stage of the morula.
Transports of Na+ ions through the epithelium layer into the morula generates an osmotic gradient driving fluid across this epithelium through aquaporin water channels. Fluid accumulates in the intercellular spaces forming a cavity of gradually increasing size.

Front 136

Morphogenesis

Back 136

• Processes that bring about changes/rearrangement of cells in the embryo.
• For example, during gastrulation the primary germ layers move into their positions and the basic body plan is established.
• Achieved through changes in cell adhesion, shape and cell motility.

Front 137

Morphology of embryonic cells: Epithelial morphology

Back 137

•Apical–basal polarity
•Contact with a basal basement membrane
•Extensive cell–cell contacts by specialized junctions
•Stationary

Front 138

Morphology of embryonic cells: Mesenchymal morphology

Back 138

•No distinct polarity
•Reside within a loose extracellular matrix
•No cell–cell contacts by specialized junctions
•Motile

Front 139

Cell Shape

Back 139

•Changes in cell shape are generated by the cytoskeleton.
•The activity of microfilament bundles – e.g. actin filament, microtubules etc. and there associated motor proteins generate force and contraction within the cell which leads to shape change.

Front 140

Apical constriction

Back 140

Apical constriction is caused by contraction of a contractile network (composed of actin and myosin) at the apical end of a cell..
•Apical constriction within an sheet of epithelial cells caused cell movement – invagination.

Front 141

Cell Migration

Back 141

•Migration takes place over a substratum, e.g. extracellular matrix
•Cells move by extending lamellipodium or filopudiium, created by assembly of actin filaments.
•Cells attach/adhere to the substratum through focal contacts.
•Contraction of the actin network in the cell attached to the substratum draw the cell forward.

Front 142

Cadherins and Integrins

Back 142

Cadherins are the main factors adhering embryonic cells. Transmembrane proteins that adhere tightly to the same molecule on other cells in the presence of calcium. Interact with intracellular cytoskeleton via a cytoplasmic tail. Many different types, E-cadherin, N-cadherin, and so on.
Integrins are cell surface proteins which interact with components of the extracelluar matrix such as laminin and fibronectin.

Front 143

Differential adhesion

Back 143

Separation of cell types with different adhesive properties.
Type and number of cadherins molecules on cell surface provide adhesive specificity.

Front 144

The more cohesive ectoderm (epidermis–light blue or grey) remain on the outside...

Back 144

...and the less cohesive cells e.g. mesoderm (red) or neural plate (blue) become internalized.

-Similar cell types sort and aggregate to each other.

Front 145

Differential Adhesion…..

Back 145

…produces a qualitative specificity of adhesion. …provides a mechanism for assembly of different cell types. …stabilize the boundaries between cells.

Front 146

Cadherins aid in ……

Back 146

……the separation of distinct tissue layers or the fusion of tissue masses.
……..the formation of tissue boundaries.

Front 147

Morphogenetic Movements: Invagination

Back 147

Front 148

Morphogenetic Movements: Ingression

Back 148

Front 149

Morphogenetic Movements: Involution

Back 149

Front 150

Morphogenetic Movements: Epiboly

Back 150

Front 151

Morphogenetic Movements: Intercalation

Back 151

Front 152

Morphogenetic Movements: Convergent Extension

Back 152

Front 153

Morphogenetic Movements: Radial Intercalation

Back 153

Front 154

Morphogenetic Movements: Mediolateral Intercalation

Back 154

Front 155

Epithelium to Mesenchymal Transition (EMT)

Back 155

Polarized epithelial cell, which normally interacts with basement membrane, undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype. EMTs is a mechanisms for….
1)dispersing cells in embryos (type 1).
2)forming mesenchymal cells in injured tissues (type 2).
3)initiating the invasive and metastatic behavior of epithelial cancers (type 3).

Front 156

EMT is a reversible process

Back 156

Mesenchymal-to-epithelial transition (MET) involves the conversion of mesenchymal cells to epithelial cells.

Front 157

Movements in Gastrulation: Sea Urchin

Back 157

•Ingression of vegetal cells, primary mesenchymal cells, into the blastocoel.
•Primary messenchymal cells migrate along the blastocoel wall.
•Next the endoderm begin to invaginate as a sheet of cells to form the archenteron (gut).
•Convergent extension of the tip of the gut followed by extension of filopodia from the cylinder to contact the blastocoel wall, fuses forming the mouth region.

Front 158

Formation of primary mesenchymal cells

Back 158

For ingression, primary mesenchyme cells must undergo epithelial to mesenchymal transition to detach from the epithelium of the vegetal plate and ingress into the interior.

Changes in their adhesive properties; lose affinity for neighboring epithelial cells and the hyaline layer and gain affinity for the blastocoel wall.

Front 159

Invagination of the vegetal plate

Back 159

The vegetal plate undergoes invagination forming the blastpore. Started by apical cytoskeleton contraction of the apical ends of some cells in the vegetal plate, forcing the vegetal plate to buckle.

•Primary mesenchyme (mesoderm) cells form skeletal rods (red).
•Endoderm forms gut and anus. Gut elongates and fuses with the animal region to form mouth. O-A axis is established.
•Secondary mesenchyme (mesoderm) give rise to muscle.
•Produces a feeding larva (Pluteus).
Since sea urchins are deuterstomes, the invagination of the vegetal plate (blastopore) forms the anus of the larva later in development.

Front 160

Movements in Gastrulation: Chick

Back 160

•Begins with the formation of the primitive streak.
•Epiblast cells converge on the primitive streak and the ingress (epithelium to mesenchymal transition) into the interior and give rise to the mesoderm and endoderm.

Front 161

Primitive streak

Back 161

•The primitive forms as a slit/line in the epiblast.
•Streak begins to form from the posterior marginal zone (Koller’s sickle) and extend to a point in the central region of the disk.
•Once the streak reaches its greatest length, the anterior end begins to regress back to the posterior end.
•The anterior end of the regressing streak is known as Hensen's Node.

Front 162

Movements in Gastrulation: Mouse

Back 162

Early post-implantation development of the mouse embryo
•Primitive streak forms in the epiblast at the start of gastrulation.
•The primitive streak forms at the future posterior end of the embryo.
•The streak migrates to the bottom of the U-shaped cup, the future anterior end.

Front 163

Basics of mouse gastrulation (similar to gastrulation in chick)

Back 163

1) Epiblast cells move through the primitive streak and spread anteriorly and laterally between the epiblast (ectoderm) and the visceral endoderm to form the mesoderm. 2) Later, the definitive endoderm (derived from epiblast) will replace the visceral endoderm. 3) At the anterior tip of the primitive streak, the node forms (similar to Hensen’s node). 4) Then notochord and somites form anterior to the node.

Front 164

Movements in Gastrulation: Xenopus

Back 164

• Invagination (formation of the dorsal blastopore lip) • Involution of the marginal zone (MZ) • Migration of marginal zone cells on the blastocoel roof • Convergent extension of the MZ • Epiboly (spreading) of the ectoderm

Front 165

Formation of the blastopore

Back 165

Cells at the MZ undergo apical constriction forming bottle cells. This forces the elongated cell bodies inwards, and creates the dorsal lip and a groove called the blastopore.

Front 166

Involution of mesodermal and endodermal cells

Back 166

Presumptive mesoderm and endoderm involute around the blastopore. Involution spreads laterally and ventrally until the blastopore is circular producing the ventral lip. Mesodermal cells migrate anteriorly.

Front 167

Convergent extension elongates the mesoderm

Back 167

The dorsal mesoderm elongates in the anterior-posterior direction by convergent extension.

Front 168

Epiboly spreads the ectoderm

Back 168

The ectoderm spreads by epiboly to cover the entire surface of the embryo. The animal cap spreads towards the vegetal pole. The individual cells also become smaller and more numerous, through cell division.

Front 169

Molecular Basis of Gastrulation: Mesodermal cell migration

Back 169

First mesodermal cells to enter the blastoceol migrate along the blastoceol roof and becomes a part of the anterior/head region. Migratory cells use lamellipodia to migrate along the blastocoel roof as gastrulation proceeds.

Front 170

Fibronectin

Back 170

•Large glycoprotein, component of extracellular matrix (ECM)
•Serves as an adhesive molecule linking cells to ECM
•Secreted by cells of the blastocoel roof (presumptive ectoderm)
• Required for migration of presumptive mesoderm

Front 171

How do migrating mesoderm cells recognize fibronectin?

Back 171

-with integrin

Integrin

• Cell membrane protein.
• Binds to ECM molecules including fibronectin.
• Expressed on mesodermal cells just prior to and during gastrulation.
• Mesoderm cells use integrin to adhere to fibronectin during movement.

Front 172

Neutralizing antibody (NAb)

Back 172

Inhibition of protein function by binding of specific antibodies.

Front 173

Antibodies that specifically block integrin interactions with the RGD of fibronectin were injected into the blastocoel before the onset of gastrulation....

Back 173

....Normal

Fibronectin (fn) accumulates along the blastocoel (b) roof.
Archenteron (a) forms and the blastocoel (b) is almost eliminated and mesoderm has begun to differentiate

Antibody Injected

Block assembly of FN fibrils in ECM. Gastrula shows misplaced mesoderm that is not adherent to the blastocoel roof (H, arrows). No archenteron is present and a centralized remnant of the blastocoel (b) remains. Dorsal (dm) and ventral (vm) mesoderm still evident.

Front 174

What signals and pathways regulate the effectors?

Back 174

-Platelet derived growth factor A (PDGF)

PDGF signaling is required for directed migration of mesoderm cells across the blastocoel roof.
•PDGF-A (ligand) is expressed by cells in the ectodermal blastocoel roof.
•PDGFRα (receptor) is expressed by cells of the presumptive mesoderm.

Front 175

Dominant negative

Back 175

The product of a mutated gene adversely affects the function of the wild-type gene.

A mutant receptor that, due to dimerization with normal versions of the receptor, has a dominant inhibitory effect upon the normal activity is a dominant negative (dn) mutation.

Front 176

Effects of compromised PDGFA signaling on gastrula.

Back 176

Truncated form of PDGF-A mRNA was injected at the four-cell stage into all four blastomeres. Gastrula of uninjected controls (A) are compared with sibling embryos injected with inhibitory dnPDGF-A mRNA (B).

Mutant embryo: directional migration by mesodermal cells toward the animal pole is replaced by random migration.

Front 177

Inhibition of PDGFR and PDGF-A results in...

Back 177

...diminished head (anterior) structures.

Front 178

Fibronectin, integrin and PDGF signaling are required for...

Back 178

...directional migration of mesoderm cells across the blastocoel roof.

Mesoderm cells extend lamellipodia towards the animal pole. Cells use integrin to make contact with organized fibronectin in the ECM. PDGF signaling provides directionality in the form of a gradient of PDGF-A signal along the blastocoel roof. The dorsal mesoderm elongates in the anterior posterior direction by convergent extension.

Front 179

Convergent Extension and the Keller Sandwich

Back 179

Study of the marginal zone of the amphibian gastrula can be performed using Keller sandwiches. Explants of tissue lying immediately animal to the blastopore lip of an early Xenopus gastrula. Activin treated ectodermal cells behave
like mesodermal cells and undergo
convergent extension.

Front 180

Protocadherins

Back 180

-A factor involved in convergent extension

• Similar to cadherins, regulate cell-cell adhesion.
• Required for convergent extension during mesodermal migration.
Dominant negative protocadherin (DN-PAPC) inhibits activin induced animal cap extension.

Front 181

Wnt/planar cell polarity (PCP) pathway

Back 181

-A factor involved in convergent extension

Signaling leads to changes in actin cytoskeleton and cell polarization. Controls lamella formation and myosin contractility Wnt/PCP signaling is needed during gastrulation for efficient convergent extension movements of mesenchymal cells When Wnt/PCP signaling is compromised by loss or gain of function of Wnt/PCP components, the polarized cell behaviors that drive convergent extension movements are perturbed

Front 182

Interfering with Wnt/PCP function...

Back 182

...blocks the activin-induced elongation of animal cap cells.

The activity of both Protocadherins and PCP signaling are required to coordinate convergent extension movements.

Front 183

Two majors ways to form the neural tube

Back 183

1) Primary neurulation - the neural plate cells proliferate, invaginate and pinch off from the surface to form a hollow tube. 2) Secondary neurulation - the neural tube arises from a solid cord of cells that sinks into the embryo and subsequently hollows out to form a hollow tube

Front 184

Neurulation in various vertebrates

Back 184

• Fish - exclusively secondary • Birds and mammals - anterior portion of the neural tube - primary - posterior portion of the neural tube - secondary

Front 185

Morphogenetic Processes in Neurulation

Back 185

-Columnar thickening of neuroepithelium

-Convergent extension of neural plate and underlying mesoderm

-Apical wedging of neuroepithelium

-Continuing convergent extension

-Medial migration of epidermis

-Continuing convergent extension

-Neural fold fusion

-Shaping of lateral/dorsal neural tissue.

Front 186

Neural Tube Closure

Back 186

Neural tube closure in the mexican salamander (similar to mammalian), the middle of the tube closes first, followed by both ends.

Front 187

Convergent extension drive neural tube formation

Back 187

Wnt/planar cell polarity (PCP) pathway
Activation leads to changes in actin cytoskeleton and cell polarization. This polarization is required for convergent extension.

Dishevelled is required for both neural convergent extension and neural tube closure.

Inhibition of Wnt/planar cell polarity (PCP) pathway results in neural tube defects.

Convergent extension of the neural plate is necessary to reduce the distance between the neural folds, allowing them to meet and fuse.

Mediated by differential expression of cell adhesion molecules.
-Ectoderm expresses E-cadhedrin
-Neural tube expresses N-cadhedrin
N-cadhedrin expressed in epidermal cells -No tube closure

Front 188

Neural tube defects

Back 188

Failure to close the tube results in…
Spina bifida – failure of the posterior neural tube to close completely, severity depends upon how much of the tube remains open.
Anencephaly- lethal condition, failure of the anterior neural tube to close at all, brain tissue is exposed and fore brain does not develop.
Craniorachischisis- lethal condition, the brain and spinal cord are exposed.

Front 189

Totipotent/Pluripotent/Unipotent

Back 189

Cells arising from a common lineage (all arising from the zygote) are able to differentiate into different cell types.

Totipotent – can give rise to any and all cell types.

Pluripotent – can give rise to different cell types.

Unipotent – can give rise to a single cell type.

Front 190

Developmental potential

Back 190

Developmental potential or potency of a cell describes the range of different cell types it CAN become.

Front 191

Progressive Determination

Back 191

As development proceeds…..
…..uncommitted cells become committed to form a specific cell type.
…...the developmental potential of individual cells decrease.
Once determined, the cells pass on this fate to all progeny.

Front 192

Specification map and Fate map

Back 192

Specification map: describes cell behavior in isolation. Differentiation of explants represents the specification of tissue at time of isolation.

Fate map: describes normal development. Does not tell us if cells are specified or determined, i.e. does not indicate developmental commitment.

Front 193

Specification v.s. Fate

Back 193

If you get engaged you become specified to someone, but you can change your mind, and you might find someone else and marry them and become determined.

Fate: describes what it will become in the course of normal development.
Determination: implies a stable change in the internal state of a cell and cannot be influenced by other cells.
Specification: cells are specified when isolated in a neutral environment they develop into their normal fate.

Front 194

Acquisition of Commitment

Back 194

Induction: Cell or group of cells (INDUCER) directs the development of a neighboring cell or group of cells (RESPONDER) -> cell-cell communication.
Lateral Inhibition: Differentiating cell prevents surrounding cells from doing the same -> cell-cell communication.
Cytoplasmic Determinants: Substance located in the egg that guarantees the assumption of a particular state.

Front 195

Cell-cell Communication

Back 195

Three main ways …
1. Cell-to-cell signaling
cell membranes contain specific protein receptors, which bind & transmit extra-cellular signals.
2. Direct contacts cell-cell recognition via receptor proteins which transmit signal.
3. Gap junctions results in cytoplasmic continuity, signaling molecules are passed from one cell to another.

Front 196

Cell-to-cell signaling

Back 196

1.Cell (inducer) synthesize signal molecule.
2.Release of the signaling molecule by the signaling cell (e.g. exocytosis).
3.Transport of the signal to the target cell (responder) .
4.Target cell detects the signal via specific receptor proteins.
5.Transmission of the signal by intracellular molecules – signal transduction.
6.A change in target cell behavior and development - response.

Front 197

PARACRINE (local) SIGNALING

Back 197

• Local regulator/signaling molecules are targeted to specific receptors.
• Often includes: growth factor proteins that promote cell division & growth.

Front 198

PARACRINE SIGNALING FACTORS

Back 198

• Small diffusible proteins (called ligands).
• Act by binding to receptors on the target cell.
• Four major families
1) Fibroblast growth factor (FGF)
2) Hedgehog (Hh) family
3) Wingless (Wnt) family
4) Transforming growth factor beta (TGF-beta) superfamily

Front 199

Fibroblast growth factor (FGF) Signaling

Back 199

• 25 FGF genes in vertebrates
•Receptor: FGF receptor (tyrosine kinase)
• Roles at all stages of development, e.g. mesoderm induction and limb bud formation.

Front 200

Transforming growth factor beta (TGF-beta) signaling

Back 200

• Includes, Activin; Vg-1; bone morphogenic protein (BMP); Nodal and nodal-related.
• Receptors: type I and type II (serine threonine kinase).
• Roles at all stages of development, e.g. mesoderm induction and patterning.

Front 201

Wingless (Wnt) family signaling – “canonical” Wnt pathway

Back 201

• 21 Wnt genes in vertebrates
• Receptor: Frizzled family
• Roles at all stages of development,
e.g. dorsal-ventral axis and
limb development

Front 202

Hedgehog (HH) signaling

Back 202

• 3 HH genes in vertebrates (Sonic, Indian and Desert)
• Receptor: Patched (PTCH)
• Roles at many stages of development,
e.g. digit identity, eye development,
tooth development.

All bind to same receptor called patched (PTCH)

The most common one is the sonic hedgehog gene.

Front 203

Responding to Signals: Competence

Back 203

• Competence: the ability of cells to respond to an inductive signal.
• Actively acquired state, e.g. cells may gain competence by repressing the activity of an inhibitor or expressing a particular receptor.
• Often a temporal component.
• Response: change in cell behavior or development
1. Cell shape changes 2. Cell movement 3. Cell proliferation 4. Cell survival (apoptosis)

Front 204

Acquisition of Commitment

Back 204

Induction: Cell or group of cells (INDUCER) directs the development of a neighboring cell or group of cells (RESPONDER)  cell-cell communication.
Lateral Inhibition: Differentiating cell prevents surrounding cells from doing the same  cell-cell communication.
Cytoplasmic Determinants: Substance located in the egg that guarantees the assumption of a particular state.

Front 205

Primary Embryonic Induction

Back 205

The organizer experiment of Mangold and Spemann (1924)
Tissue from the dorsal side of one embryo (dorsal lip of the blastopore) is transplanted to the ventral side of another embryo.
Result - cells on the ventral side of the embryo to form a second neuroaxis.

Front 206

Inductive Event

Back 206

Interaction between an inducing cell/tissue that causes a responding cell/tissue to undergo a change in differentiation.

Front 207

Two types of induction

Back 207

Instructive induction: the responding tissue has a choice of fates, more than one outcome/response to signaling factors.
• morphogenetic gradient: concentration of signal (morphogen) triggers different tissue responses.
Induction
Permissive induction: the responding tissue has only a single outcome in response to a signal.

Front 208

Morphogenic Gradients

Back 208

Cells respond differently to different concentrations of morphogen.
Positional information is translated into the activation and repression of target genes.
Patterning the embryo (pattern formation).

Front 209

Morphogen

Back 209

Substance whose spatial concentration differentially directs cell development.

Front 210

How do morphogens have different transcriptional readouts at different concentrations?

Back 210

Different targets have different affinities for effectors

Front 211

Criteria for a Morphogen

Back 211

1.Secreted molecules - has to form a gradient.
2. It has to be able to direct more than 2 different
states or responses.
3. The cells which it acts upon have to be initially equivalent so that differential responses can be related solely to the differences in concentration of the morphogen.

Front 212

Lateral Inhibition

Back 212

“Telling your neighbors not to be like you”
Differentiating cell secretes an inhibitory molecule.
Molecule acts locally to suppress the signal in surrounding cells.

Front 213

Model for Pattern Formation: Reaction-diffusion Model

Back 213

Activator (P) promotes development of cell type A and stimulates production of inhibitor (S).
Inhibitor (S) diffuses quickly and inhibits the activator (P) in surrounding cells.

Front 214

Cytoplasmic Determinants

Back 214

Substance located in the cell that guarantees the assumption of a particular state of commitment.
mRNA (or protein) that is unevenly distributed in a parent cell.
Unequally distributed to daughter cells, as a result cells are not equal in developmental fates.
The properties of such cells are said to be intrinsic and depend on their lineage
rather than environmental cues.

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The eye has components derived...

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...surface ectoderm, mesoderm and neural ectoderm.
A series of inductive events are required for eye development.

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Pax6

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In situ hybridization showing the expression of the Pax6 gene in the developing mouse eye.

Pax6 (yellow-green) is expressed in both the presumptive lens ectoderm and optic vesicle/cup (presumptive retina).

Pax6 protein is a
competence factor

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Eye field specification

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Prospective eye cells are specified prior to evagination of the optic vesicle.
A single eye field is specified across the anterior neural plate.
Transcription factors such as Pax6 and Otx2 are expressed in the anterior neural plate at late gastrula stage.
SHH (sonic hedgehog) from the prechordal plate suppresses expression of Pax6 and other transcription factors in the center of the embryo, dividing the field in two.

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Reciprocal Induction

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• 1st optic vesicle induces competence and bias to form lens in the ectoderm.
• Lens (actually lens placode) now becomes the inducer and signals back to the optic vesicle (which is now a responder tissue) to become the optic cup.

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Axis Formation: Xenopus

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•Animal-vegetal pole is related to the anterior-posterior axis.
•The dorso-ventral axis is established first.
•The dorso-ventral axis then dictates the orientation of the antero-posterior axis.

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Animal-Vegetal Axis

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• Established by maternal factors.
• mRNA localized to vegetal region.
• e.g Vg-1 (TGF β-family signal), Xwnt (Wnt-Wingless), VegT (transcription factor).

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Cortical Rotation

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Cortical cytoplasm rotates 30 degrees during first cleavage – Cortical Rotation.
Forms gray crescent which specifies the future dorsal side.

Cortical rotation defines the dorso-ventral axis.

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Dorsalizing factors

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Specify the location of the dorsal side.
Specification of signaling centers.

Maternal mRNA and proteins localized to the vegetal region.

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Canonical Wnt signaling pathway

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Without Wnt:
GSK-3 complex phosphorylates beta-catenin and targets it for degradation.
With Wnt bound to Frizzled:
Disheveled inhibits GSK-3 activity. beta-catenin accumulates enters nucleus and activates transcription.
Wnt and disheveled are dorsalizing factors.

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Nieuwkoop Center (NC)

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Beta-catenin can induce a new neural axis (dorsal side).
Beta-catenin specifies a signaling center, the Nieuwkoop Center (NC) or blastula organizer in dorsal vegetal blastomeres.

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Dorso-ventral Axis

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1. Oocyte polarity (Animal-Vegetal) prefigures the germ
layers, but not the body axes.
2. Dorso-ventral axis defined by cortical rotation.
3. Cortical rotation rearranges cytoplasm, resulting in β-catenin stabilization dorsally by local inhibition of GSK3.
4. The dorsalizing ‘Nieuwkoop center’ is established in dorsal vegetal cells of the blastula (blastula organizer).
5. Nieuwkoop center later induces the gastrula stage Spemann organizer in dorsal marginal cells.

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Four divisions of mesoderm

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1. Axial mesoderm (notochord)

2. Paraxial mesoderm (somites) bone, skeletal muscle

3. Intermediate mesoderm, kidneys, gonads

4. Lateral plate mesoderm, blood, connective tissue, cardiovascular system

Animal cells adjacent to vegetal cells become mesoderm not ectoderm.

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Mesoderm is induced from animal cap cells

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Animal cells adjacent to vegetal cells are induced to become mesoderm.
Induction does not require cell-cell contact - diffusible signal.
Inductive signals produced by vegetal cells.

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Mesoderm Induction: Competence

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•Tissues at specific times are competent to receive signals.
•Animal cap cells competent 4-11 hours after fertilization (7 hour window).
•Induction requires at least 2 hours exposure to signal during the blastula stage.
•Expression of mesoderm specific genes occurs at mid-gastrula stage.

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Community Effect

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Cells must be present in sufficient number to be induced.

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Tissues at specific times are competent to receive signals

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•Animal cap cells competent 4-11 hours after fertilization (7 hour window).
•Induction requires at least 2 hours exposure to signal during the blastula stage.
•Expression of mesoderm specific genes occurs at mid-gastrula stage.

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Mesoderm Induction: Four Signal Model

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•Signal 1 : originates from the vegetal cells.
•Signal 2 : originates from the dorsal vegetal cells (Nieuwkoop center).
•Signal 3: originates from the Spemann organizer.
•Signal 4: originates from the ventral region of the mesoderm
•Signals are diffusible, spreads over several cell layers.

•Signal 1: General mesoderm inducer, makes "default" ventral mesoderm.
•Signal 2: Dorsal mesoderm inducer, generates Spemann organizer and notochord.
•Signal 3: Spemann Organizer signal from dorsal region which inhibits the effects of signal 4.
•Signal 4: Ventral mesoderm patterning signal which subdivides the mesoderm.

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Candidate Mesoderm Inducers: Members of TGF-β superfamily

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•Vg1
•Activin
•Nodal (Xnr1, 2, 4-6)
•BMP

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Is Vg1 required?

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•Maternal transcripts (mRNA) are localized to the vegetal pole in Xenopus.
•Embryos with depleted Vg1 lack head and notochord structures (mesoderm derivatives).

• Vg1 pro-protein has no activity.
• Mature Vg1 protein can function as a potent mesoderm inducing agent.
• Endogenous mature Vg1 protein is not detectable in Xenopus embryos.

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Is Activin required?

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Activin is sufficient to induce mesoderm in an dose-dependent manner, BUT the mRNA is not detectable.

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Vg1 vs. Activin

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• Correlation:
Vg-1: mRNA in right place, right time but active mature protein not detected.
Activin: mRNA not detectable.
• Loss of function:
Vg-1, Activin: dominant negative TGF-β receptors block induction.
• Gain of function:
Vg-1: mature Vg1 can induce mesoderm. Activin: potent mesoderm inducer.

Activin activates the Nodal signaling pathway (mimics nodal, another TGF-β signaling protein)

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Xenopus nodal related (Xnr)...

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...proteins turned on in vegetal cells (graded expression) at the appropriate time!

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Activation of Xenopus nodal related (Xnr)

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•Vegetally localized maternal transcription factor VegT activates the zygotic expression of the Xnrs in the vegetal region.
•Xnr then initiate mesoderm formation.
VegT

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Xenopus nodal related (Xnr) dorso-ventral gradient

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•Low level activation of Xnr by VegT.
•High level activation of Xnr by VegT PLUS β-catenin at dorsal side.

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Signals 3 and 4 pattern the mesoderm dorso-ventrally

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• Dorsalizing signals (3) from the Spemann organizer counteract ventralizing signals (4) from ventral mesoderm.
• Candidates for signal 3:
Organizer molecules
- Noggin
- Chordin
- Frizbee
- Follistatin
• Candidates for signal 4:
- BMP-4
- Xwnt-8

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What happens when the tissue from the dorsal side of one embryo (dorsal lip of the blastopore) is transplanted to the ventral side of another embryo?

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Results- This induces cells on the ventral side of the embryo to form a second dorsal axis

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Noggin is an organizer molecule

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• Expressed in the correct place, the organizer
• Secreted protein
• Can rescue can rescue axis in UV-treated eggs (ventralized embryos)
• Does not induce mesoderm from animal cap explants
• Does dorsalize ventral mesoderm (dorsalizing factor)

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Rescue of dorsal structures by Noggin

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UV radiation -> ventralized embryo
Rescue of dorsal structures by Noggin
UV radiated embryo injected with increasing concentration of noggin -> embryo develop dorsal structures.
Too much noggin injected -> embryo produces dorsal anterior tissue at the expense of ventral and posterior tissue.

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Other organizer molecules

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Other organizer molecules, e.g. Chordin and Frizbee, have similar properties as noggin.

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BMP-4 is a ventral mesoderm inducer

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• Members of TGF-β superfamily
• mRNA found throughout blastula.
• Activity becomes restricted to the ventral region of the marginal zone (mesoderm) during gastrulation.
• Blocking BMP-4 activity dorsalizes the embryo.
• Potent ventralizing factor in explants.

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How do signals 3 and 4 interact?

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Purified noggin protein binds
very tightly to purified BMP-4.
Noggin:BMP-4 complex
cannot bind receptor - this prevents receptor activation and results in dorsal mesoderm.
Noggin itself is not an instructive signal.
Inhibition of BMP-4 signaling is sufficient to dorsalize mesoderm.
Chordin and Follistatin also interact with BMP-4 and inhibits activity.

Model:
Noggin, Chordin and Follistatin
creates a dorsal ‘BMP-free zone’.
Result is a gradient of BMP-4
activity from high (ventral) to
low (dorsal).
Frizbee is the analogous inhibitor of
Wnt signals.

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Four Signal Model

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Signal 1: low level of Xnr
Signal 2: high levels Xnr

Signal 3: Spemann organizer molecules ,
noggin, chordin, etc.
Signal 4: BMP-4 and Xwnt-8

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Mesoderm induction and patterning in chick

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Mesoderm induction and patterning occurs at the primitive streak.
Different regions of the streak give rise to different mesodermal tissues.

Factors that initiate primitive streak formation also signal mesoderm induction and patterning.

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Antero-posterior Axis

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Once the dorsal side is established, the movement of the involuting mesoderm (gastrulation) establishes the antero-posterior axis.
Cells at the dorsal lip early in gastrulation –migrate first over the lip and into the embryo – induce and become part of anterior structures.

Once the dorsal side is established, the movement of the involuting mesoderm (gastrulation) establishes the antero-posterior axis.
Cells at the dorsal lip early in gastrulation –migrate first over the lip and into the embryo – induce and become part of anterior structures.

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Regional Specificity of Induction

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As cells involute over the dorsal blastopore lip during gastrulation, they acquire different fates and inducing properties.
Early involuting mesoderm induces head (anterior) structures while later involuting mesoderm induces trunks (posterior).

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Two-inhibitor Model

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Head induction requires dual inhibition of Wnt and BMP signaling.
Trunk induction requires inhibition of BMP signaling.
Organizer produces factors that inhibit both BMP and WNT

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Head Induction

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Head mesoderm induction requires inhibition of BMP and Wnt signaling.
Inhibitors originate from the organizer.

Microinjection of Cerebrus, induces ectopic head.
i.e., BMP and Wnt signals are blocked and head is produced.
No Dickkopf1, reduced anterior structures
i.e., Wnt signals are not inhibited therefore anterior structures are not fully produced.
Secreted Inhibitors: Noggin inhibits BMP; Cerebrus inhibits BMP and Wnt; Frizbee and Dickkopf (Dkk1; thick head) inhibit Wnt

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Noggin, a diffusible protein of the organizer

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Noggin is expressed in head mesoderm and notochord.
Inhibits BMP throughout.

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Trunk Induction

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Trunk induction requires inhibition of BMP signaling BUT require Wnt signaling.

Block BMP and Xwnt, induce head.

Block BMP only, induce trunk.

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Frizbee, a diffusible protein of the organizer

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Frizbee expressed in head endomesoderm but not notochord.
Inhibits Wnt in head region only.
Microinjection of frizbee mRNA into the marginal zone leads to the inhibition of trunk formation.
Wnt is required for trunk formation.
Frizbee inhibits Wnt signalling.

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Endoderm Specification

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•Specified by maternal factors in the vegetal region
•Requires transcription factor VegT
• maternal mRNA is localized to the vegetal region

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Ectoderm Specification

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Ectodermal determinants are located in the embryo’s animal hemisphere and can direct the differentiation of ectoderm derivatives.
• maternal factor(s) localized to the animal region.
• Blocks the TGF-beta signaling pathway (nodal) in presumptive ectoderm cells.

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Ectodermin (Ecto) specifies ectoderm

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Expression of Ectodermin (Ecto) in Xenopus embryo
•Ectodermin RNA is present in the animal pole throughout cleavage stages (D).
•At the blastula stage, Ectodermin mRNA and protein are localized in the animal pole, extending, at lower levels, up to the marginal zone (E)

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Morpholinos (MO)

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Morpholinos (MO) are chemically modified antisense oligonucleotides that have a six-membered morpholine-ring backbone.
Structure makes morpholino oligonucleotides stable in cells and allows them to retain the ability to bind mRNA.

Morpholino oligonucleotides can be designed to bind upstream (5’) of the start codon (AUG) and inhibits progression of the initiation complex, which block translation of the target mRNA.

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Testing the requirement for Ecto in ectoderm specification

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Ectodermin regulates germ-layer identity along the animal-vegetal axis.

Ecto-morpholino (Ecto-MO) (D) and control-MO (C) were injected into one-cell embryos. At the gastrula stage, the mesoderm marker (blue) was upregulated and expanded toward the animal pole in Ecto-MO injected embryos.

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Ectodermin blocks mesoderm inducing signals

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E3 ubiquitin ligase Ectodermin (Ecto)
•Binds to and monoubiquitinates Smad4.
•Ubiquitination prevents Smad4 interaction
with other proteins.
•Blocks Smad4-dependent TGF-β signaling.