WN26 BIOLOGY 205

What are the four organisms that play key roles in our understanding of development?

1. C. elegans (nematode worms)

2. Drosophila (fruit fly)

3. Arabidopsis (mustard plant)

4. Avian species including Chick & Quail

Stages of Animal Development

1. Gametogenesis

2. Fertilization

3. Cleavage

4. Gastrulation

5. Organogenesis

6. Post-embryonic

Gametogenesis

formation of haploid gametes (sperms + eggs) through meiosis

Fertilization

fusion of sperm + egg, creating the zygote

Cleavage

rapid divisions generate many cells from single cell zygote that contribute to gastrulation.

Gastrulation

tissue folding + coordinated cell movement forms different cell layers: ectoderm, mesoderm, endoderm

Organogenesis

different parts of the embryo differentiate into specific combinations of cell types that perform an organ function.

Post-embryonic

transition from newly hatched/born organism to juvenile/adult. Marked by growth and dramatic changes in physical appearance.

Ectoderm

Gives rise to neural tissue (brain, central nervous system, peripheral nervous system)

Mesoderm

Gives rise to mesoderm → muscle, bone, heart, kidney, gonads

Endoderm

Give rise to digestive organs, respiratory organs

How do cells tisssue know how much to divide and where to go

Cell lineage studies (fate mapping)

Cell lineage studies

marking a specific cell and following it & its progeny through development

• early studies used natural markers (e.g. pigment containing cells)

• injection of vital (live) dyes (want to be easliy visualized but doesn’t affect cell health)

• simply looking? can be done in relatively simple, transparent embryos like C. elegans.

Model organisms

an organism with characteristics that make it useful for uncovering fundamental biological principles and processes

What are useful characteristics of a model organism for a developmental studies?

1. short generation time, small and well characterized genome

2. easy to generate lots of embryos

3. easy to maintain in the laboratory

4. relevant to other organisms (esp. humans)

Why choose C. elegans (Nematode worm)?

• small, powerful genetics, microscopically visible, 959 somatic cells

• Embryos are transparent → easy to observe

• Life cycle is quick (embryogenesis 9 hours)

  • Entire cell fate map (1 to 959) was determined by direct observation

Pronuclei

egg and sperm prior to fusing

Use of natural markers to trace lineage for non-transparent cells

natural markers

Chimeric embryos

embryos containing cell types from two sources.

E.g.) fate mapping using chick-chimeras. Quail has pigmentation that chicks don’t have, so you can look at feathers to look at what regions come from what animal without dissection. Le Dourain found that Quail eggs have very obvious nucleus compared to chick, so she can use a regular light microscope to see which cells came from chicks and quails.

Green Fluorescent Protein (GFP)

Fluorescent protein from jellyfish to show specific cells expressed. This is a viable protein as it’s expressed commonly in jellyfish cells. Easier to track cell lineage. Multiple variants and a rainbow of colors are now available.

What are challenges for chick-quail chimeras

1. need exquisite microdissection skill sto make viable chimeras

2. Limited ability to identify individual quail cells in a sea of chicken cells, but many quail cells are visible in a traditional light-microscope

3. Transplantation of quail cells into white chick background demonstrated that regions of the ectoderm and forming neural tissue gave rise to melanocytes and epidermal cells, e.g. feathers (good)

What are challenges for GFP lineage tracing

1. genetic tools needed to inject GFP sequence into the organism’s genome.

2. individual GFP+ cells are readily observed under fluorescence microscopy. (good)

3. different fluorescent proteins can be used to mark different populations of cells in same tissue → track multiple lineages. (good)

4. Not easily visible in adult animals without sacrificing

Apoptosis

programmed cell death

Caspase3 enzyme

cleaving enzyme that cuts up proteins. activates apoptosis

What are some crucial aspects of Drosophila larva?

• it has segements.

Cleavage (drosophila)

after fertilization, there are 13 rounds of rapid nuclear mitosis without cytokinesis, resulting in a syncytium with approximately 6000 nuclei in a single-celled embryo.

1. develop in synctium first. nuclei in same envt.

Synctium

several nuclei sharing cytoplasm

Micropyle

opening through which sperm enters to fertilize egg

At the end of this syncytial stage…

nuclei are enveloped by plasma membrane → cellular blastoderm stage → gastrulation. This is when patterning events take place

Pros and cons of using Drosophila as a model?

Pros: can be live imaged and many cells tracked with computational methods.

Cons: tracking becomes more difficult once complex folding takes place.

Cellular blastoderm

future cell fate can be predicted at this stage based on their location.

What is the maternal determinant for drosophila

Bicoid mRNA is deposited by the mother’s ovary into the developing oocyte, where it is localized to the anterior. mRNA is translated into protein, protein forms a gradient in the shared yolky cytoplasm. Highest levels of bicoid protein specifies head lower levels specify thorax → patterning!

How do maternal determinants work?

Maternal determinants

loaded by the mother into the embryo to help with patterning

Nurse cells

nurse the development egg by providing ribosomes, lipids, etc that the embryo needs before it can produce those contents themselves

The oocyte is supported by —-

nurse cells

How are nurse cells connected

they are connected to each other and the oocyte through ring canals

During the first 3 hours after fertilization, the embryo is busy dividing. therefore…

no transcription during first 3 hours, the nurse cells provide all the nutrients needed for the embryo.

Ingression

cells release contacts from neighbors and migrate, similar to epithelial to mesenchymal transition

Some cells ingress from ___ and become neuroblasts

ectoderm

Bicoid

(“two tailed”) mutant

Bicoid comes from

the mother

Does the embryo make its own Bicoid for head formation?

No, the decision happens before embryoo develops by the mom

What is development?

A process of progressive change in an organism. The events that enable a single cell (fertilized egg) to give rise to a multicellular organism

What are some important definitions in development?

1. Sperm fertilizes an egg = zygote

2. Zygote grows and divides to produce a multicellular organism

3. This is an embryo

How do we figure out what happens during development?

1. Observation

2. Perturbation

3. Molecular perturbation

Observation

Requirements: to be able to see and record detail

However, it can’t tell you HOW

Perturbation

Disrupting development and ask: what happens?

Requirements: to be precise and to see and record detail.

However, it doesn’t tell you the molecules

Molecular perturbation

Altering individual/combinations of signals with mutations and drugs

However, there are limitations:

1. need mutants or specific drugs

2. Molecules may have multiple functions at different places/times

Preformation theory

organisms develop from miniature, fully-formed versions (homunculi) already existing within a sperm or an egg, which simply unfolded and grew

Epigenesis

Organisms develop from a single cell theory through gradual stages, with cells differentiating and organs forming sequentially, rather than being pre-formed miniatures.

What problem does the amniotic egg solve?

The amniote egg solved the problem of vertebrates being tied to water for reproduction by creating a self-contained, terrestrial aquatic environment, preventing the embryo from drying out, and allowing for gas exchange and waste removal on land.

All embryos have a ____ that controls the microenvironment

protective coating

What makes chicks (chicken embryos) a great model organism?

Easy to work with

• small egg→practical

• cheap to purchase

• embryogenesis outside of mom, can watch embryo in real time.

What technique do you use to modify a chick embryo?

“Windowing” the chick egg. You break the egg, modify the embryo, and then seal the egg back up.

What’s difficult about using the chicken as a model

It’s hard to study the development of the egg in the oviduct.

Blastoderm in the chicken egg

20,000-60,000 cells at layering; single layer of cells that covers the yolk. Incomplete cytokinesis early on

Epiblast

future embryo

Hypoblast

Extra embryonic tissue (e.g. yolk sac), aka endoblast

Primitive streak

Epiblast cell converge on the primitive streak, and the streak moves anteriorly from the posterior marginal zone / Koller’s sickle. Cells in the furrow of the streak move inward and spread out anteriorly and laterally, forming a layer of loosely connected cells called mesenchyme.

Gastrulation occurs here, forming various layers.

EMT

Epithelial to mesenchymal transition. a fundamental process where stationary epithelial cells lose their connections and polarity, transforming into migratory mesenchymal cells.

• Necessary for gastrulation to occur.

Mesenchyme

Migratory cells

What happens in early gastrulation

Formation of Hensen’s node and primitive streak

Hensen’s node

an organizing center that patterns the body → determines fate of cells around. elongates from posterior to anterior node

Primitive streak

Site of cell movement at gastrulation

Gastrulation occurs by cells moving through…

the primitive streak

The ____ gets pushed out of the way by the invaginating cells, which have undergone an ____

hypoblast / endoblast , EMT

What would happen if you blocked EMT?

All cells become ectoderm

Can cells reverse EMT?

Yes, cells can under MET transition to become epithelial cells.

Cells moving through the primitive streak and Hensen’s node receive signals that….

determine their cell fate.

What allows us to trace cell movement

Lineage tracing, GFP

How does the embryo elongate?

Convergent extension and intercalation

Convergent Extension

Process which a tissue narrows along one axis and lengthens in a perpendicular (anti to post) axis, cells moving toward each other and intercalating; transforms the embryo from round to elongated

Intercalation

mechanism by which cells move together, establishing new contacts. Cells get new neighbors

What happens at the anterior of the primitive streak?

Cells condense, forming Henson’s node which secretes signals that influence cell fate. The streak regresses and Henson’s node moves posteriorly. The notochord is laid down in its wake, and mesoderm on each side of the node forms somites

Notochord

signature structures of chordates. Helps to pattern the neural tube (gives rise to the CNS0

Somites

blocks of mesodermal tissue that give rise to muscles and bone. During gastrulation, epiblast cells migrate through the primitive streak and displace the hypoblast (endoblast) from the center of the embryo.

What do the hypoblast/endoblast cells give rise to?

Extraembryonic tissue (e.g. yolk), which is packed with nutrients

In chicken egg, is fate pre-specified?

No, but cells that enter the streak early are more likely to become endoderm than cells that move through later.

Which events happen at the same time?

Gastrulation, formation of primitive streak, cells moving through the streak.

How does mammalian fertilization occur?

Mammalian egg = oocyte. Oocyte is released from ovary with follicle cells and zona pellucida. Fertilization in oviduct, then preimplantation development. ‘Hatching’ followed by implantation and formation of placenta.

In humans, initial cleavages are ___ ___ and zygotic transcription begins at the ___

very slow , two-cell stage

What similarities are there for the chicken and human embryogenesis

Both embryo derived from a flat epithelia cell.

14 day rule

“in vitro vulture of the human embryo is not allowed to proceed beyond the equivalent of day 14 of embryonic development, or the approximate time at which the primitive streak appears”

What makes mammalian development hard to study?

Everything happens in the utero

Trophectoderm

gives rise to extraembryonic tissues, eg. placenta. Doesn’t happen in birds

Inner cell mass

gives rise to the epiblast and primitive endoderm

Epiblast

gives rise to the three germ layers of the embryo

At the 16-cell stage…

The fate of cells haven’t been restricted yet

At the 64-cell stage…

the fate of cells have been set.

If the mother did not provide clues, then how does the anterior-posterior organization emerge?

Expression of Vg1 and Wnt8C (both signaling molecules) precedes streak and predicts where it forms

Self-organization

emergence of ordered structures and body axes emerge from dynamic local cell-cell interactions and movements, without a pre-specified blueprint or centralized instruction

Human embryonic stem cell (hESCs)

an undifferentiated cell from an early-stage embryo that can develop into different specialized cell types, including blood, neurons, or heart cells. They are regenerative, offering potential for disease treatment and research

Induced human embryonic stem cell (iPSCs)

a type of cell derived from adult cells that have been genetically reprogrammed to an embryonic stem cell-like state, allowing them to develop into any cell type.

What is a key difference between the way mesoderm forms in the fly vs. amniotes?

Mesoderm as a continuation of the epiblast.

Metazoan

Animalia kingdom - eukaryotes, multicellular organism.

How many layers of tissue are there possible?

A few animals only have one layer (e.g. sponges)

Some animals (e.g. Hydra) have diploblast with Oral-Aboral Polarity (endo and ecto) adhered by extracellular matrix.

Most Metazoans have a triploblast body plan - mesoderm, ectoderm, endoderm. There’s an empty space called the coelum

Cortical Reaction of Ferilization

the release of enzymes by the egg after the fusion of the sperm and oocyte membranes, which prevents further binding and entry of other sperm cells. This forms a protective envelope to prevent polyspermy.

Cortical Granule Exocytosis - Sea urchin fertilization

Wave of Calcium release triggers this. Sperm entry effects Calcium signal wave in egg. The calcium wave also carries positional information for some animals.

Briefly, what happens after fertilization for the sea urchin?

• Cortical granule exocytosis → calcium signal → increase in respiration

• Increase in protein synthesis, polyadenylation

• Nuclear fusion

• Mitosis