Chapter 8 - Early Development in invertebrates

• The next chapters examine early development in several models, including invertebrates (Ch. 8-9) amphibians (ch 10) and then vertebrates (ch. 11)

1 frog egg becomes 37,000 cells in 43 hours!

Fig. 8.1

1. Cleavage- One cell is subdivided into many cells to form a blastula

2. Gastrulation- Extensive cell rearrangement to form endo-, ecto- and meso-derm

3. Organogenesis- Cells rearranged to produce organs and tissue

4. Gameteogenesis- produce germ cells (sperm/egg) Note: Somatic cells denote all non-germ cells

General Animal Development

Recall from lecture 1

How does egg undergo cleavage without increasing it’s size?

• Answer- it abolishes G1 and G2 phases of cell cycle

• Do you need a cell cycle primer??

Four cell cycle phasesM- mitosisG1- Gap 1S- DNA SynthesisG2- Gap 2

Reminder- mitosis occurs in M phase, DNA replication in interphase

From Mol. Biol of the Cell by Alberts et al, p864

• Cyclin dependent kinases (cdks, cdcs) drive the cell cycle• Cyclins (e.g cycli A, B…) regulate cdk (cdc) activity

Mitosis Promoting Factor (MPF= cyclin B+cdc2)

Example-

MPF

How does egg undergo cleavage without increasing it’s size?

• Answer- it abolishes G1 and G2 phases of cell cycle

Cyclins are synthesized in

eggs

Cyclin is degraded

S phase

M phase

What actually drives the cleavage process?

Answer- Two processes-

1. Karyokinesis (mitotic division of the nucleus)

• The mitotic spindle (microtubules composed of tubulin) does this

2. Cytokinesis (mitotic division of the cell)

• Contractile rings “pinch off” (microfilaments composed of actin)

Fig. 8.3

Cytochalasin B prevents cytokineses

1. Cleavage- One cell is subdivided into many cells to form a blastula

2. Gastrulation- Extensive cell rearrangement to form endo-, ecto- and meso-derm

3. Organogenesis- Cells rearranged to produce organs and tissue

4. Gameteogenesis- produce germ cells (sperm/egg) Note: Somatic cells denote all non-germ cells

General Animal Development

Recall from lecture 1

Gastrulation- cells of blastula are dramatically rearranged

• Three germ layers are producedFive types of movements

1 2 3

GastrulationFive types of movements

4 5

Axis formationThree axes must be determined-

• Anterior-posterior (front-back)• Dorsal-ventral (back-belly)• Right-left (right side-left side)

Fig. 8.7

Now let’s take a look at one beast- the sea urchin

• Cleavages 1 and 2 are through animal/vegetal poles

• Cleavage 3 results in four vegetal and four animal cells

1 2

3

Fig. 8.8

• Cleavage 4 results in four macromeres and and four micromeres only in vegetal cells

4

1. Cleavage

• Post cleavage 5Fig. 8.9

Cell fate map

Micromeres signal other cells via -catenin to influence fate

• Micromere cell fate is autonomous- these become skeletal tissue if placed in a dish

• All other cells have conditional specification

Example- Transplant micromeres to animal pole at 16 cell stage

•Micromeres cause a second invagination•Animal pole cells become vegetal cells

Fig. 8.13

Sea urchin (continued)

EggLate

blastula

Gastrula

Later stages

Fig. 8.16 Sea urchin development

2. Gastrulation

Note-micromemeres produce primary

mesenchyme which will become larval skeleton

How do mesenchyme cells know to migrate inside

blastocoel?

Answer- changing cell attachment proteins

Hyaline layer

Extracellular matrix

Basal lamina

Primary mesenchyme cell-

Blastocoel

98% decrease in hyaline affinity100-fold increase in EM/basal lamina affinity

Fig. 8.19

How does invagination occur?Terms- Invagination region is called archenteronOpening created is called the blastopore

Answer- swelling of inner lamina layer

Inner layerOuter layer

Vegetal cells secrete chondroitin sulfate proteoglycan, causing inner layer to swell and cause buckling

Hyaline

Now let’s take a look at another creature- the nematode C. elegans

• 959 cells at maturity• 1mm long• Produces eggs and sperm (hermaphrodyte)• Transparent• 16 hour from egg to hatch• Entire genome sequences- 19,000 genes

What a great model!

1mm

Mature eggs passes through the sperm on the

way to the vulva

Germ cells undergo mitosis, then begin

meiosis as travel down oviduct

Oviduct

Cleavage

C. elegans

1. Cleavage

1. Cleavage

• Cleavages 1 produces founder cell (AB) and stem cell (P1)

• Cleavage 2 results in three founder cells and one stem cell (P2)

1 2 3

• Remaining cleavages result in a single stem cell and more founder cells

Fig. 8.42

C. elegans

How is the anterior-posterior axis determined? Answer- P-granules- ribonucleoprotein complexes

•P-granules always stay associated with the “P” cell

Fig. 8.43

C. elegans

1

3

5

•PAR proteins- these specify polarity, cell division and cytoplasmic localization

What directs the P granules?

C. elegans

1. Cleavage

• P1 develops autonomously

• AB does not (thus is conditional)

What drives P1 lineage?P granules? No, these do not enter nucleus!

Other possibilities• SKN-1- a bZIP family transcription factor that control

EMS cell fate• PAL-1 - a transcription factor required for P2 lineage• PIE-1- inhibits SKN-1 and PAL-1 in P2

Yes- P2 produces a signal that tells ABp to only neurons and hypodermal cells not neurons or pharynx like ABa

• GLP-1 is the receptor on ABp, and APX-1 is the ligand on P2

GLP-1 is a Notch family proteinAPX-1 is a Delta family protein

Does P2 dictate fate of adjacent cells?

Chapter 9 - Axis specification in Drosophila

• Drosophila genetics is the groundwork for developmental genetics

• Cheap, easy to breed and maintain

• Drosophila geneticists take pride in being different and in sharing information

• Problems- fairly complex, non-transparent

Fig. 8.1

• Insects tend to undergo superficial cleavage- cleavage occurs at rim of the egg

• In contrast to other creatures, insects form nuclei, then create cells

Fig. 9.1

Drosophila1. Cleavage

1 7

10

• Mitotic divisions #1-#9 - duplicate nuclei (8 min/division• Mitotic division #10 – nuclei migrate to rim

Termed a syncytial blastoderm

• Mitotic division #11-14 – progressively

slower divisions

Fig. 9.3

Drosophila1. Cleavage

14• Mitotic divisions #14 – cells created with each nuclei to create the cellular blastoderm

Note – each nuclei has a territory of cytoskeletal proteins

Nucleistaun

Tubulinstain

Egg plasma membrane folds between nuclei to create individual cells

Cycle 11-14- midblastula transition- nuclear division slows and transcription increases

2. Gastrulation Ventral Dorsal

Ventral furrow(from mesoderm)

It becomes the ventral tube

Segments

3 thorax

8 abdominal

Head

Fig. 9.6

Fig. 9.7

2. GastrulationEstablishment of anterior-posterior

polarity-protein gradients rule the day

Maternal effect- in specific region of egg

Gap- among 1st gene transcribed in embryo

Pair rule – result in 7 bands

Segment polarity – result in 14 segments

Gene family

Fig. 9.8

Examples

bicoidNanoscaudel

fushi tarazuhairy

Kruppelhunchback

engrailedwingless

2. Gastrulation

Active during creation of syncytial blastoderm

Fig. 9.10

Bicoid mRNA injected in anterior

nanos mRNA injected, localize to posterior

Hunchback (diffuse)Caudel (diffuse)

Bicoid prevents caudel mRNA translationNanos prevents hunchback mRNA translation

Maternal effect genes

Oocyte mRNAs

Syncytial Blastoderm proteins

Mechanism

Anterior

Posterior

Maternal effect genes

Fig. 9.11

What if we mess up the bicoid gradient?

Bicoid- mutant

Wild-type

Two tails

Inject bicoid into:

Anterior

Wild-typeBicoid-/- Bicoid-/-

Middle Posterior

Normal Head in middle

Two heads

Thus, bicoid specifies head development

Maternal effect genes

Fig. 9.14

How does nanos specify posterior?Answer- By preventing hunchback translation

Anterior (no nanos)

MechanismIn anterior, Pumilio binds 3’UTR (untranslated region) of hunchback mRNA, and mRNA is polyadenylated and translated

In posterior, nanos prevents polyadenyltation, and thus prevents translation

Posterior (with nanos)

Fig. 9.16

2. Gastrulation Segmentation genesTwo steps in Drosophila development

Bicoid, nanos, hunchback, caudel, etc.

Determination genes

Segmentation genes

Gap genes Pair-rule genes

Segment polarity genes

Specification DeterminationEgg(Cell fate is flexible) (Cell fate is determined)

Maternal effect genes activate gaps genes, which activate pair-rule genes, which activate segment polarity genes

Segmentation genes

establish boundaries Gap Pair-rule Segment polarity

Fig. 9.19

a. Gap Genes• Gap genes respond to maternal effect proteins• Gap proteins interact to define specific regions of embryo• Four major gap proteins- hunchback, giant, Kruppel and

knirp•These are all DNA binding proteins- activate or repress transcription

Fig. 9.21

b. Pair-rule genes

• Gap genes activate and repress pair-rule genes in every other stripe, resulting in seven stripes

• Three major pair rule proteins- hairy, evenskipped, runt•These are all DNA binding proteins- activate or repress transcription•Cells in each parasegment contains a unique set of pair rule genes expression unlike any other parasegment

Gap

Pair-rule

Pair-rule

Why do we observe expression of pair-rule proteins in every other segment?

Answer- pair-rule genes use different enhancer elements

Example- even-skipped (a pair-rule gene) has several enhancers, but only one is active in a given stripe

Fig. 9.22

This enhancer is only active in stripes #1

b. Pair-rule genes

Different concentrations of gap proteins determine pair-rule gene expression

c. Segment polarity genes

Pair-rule Segment polarity

Segment polarity genes act once cells are formed

syncytial blastoderm

Maternal, gap and pair-rule genes operate before cells are formed

14

Fig. 9.1

c. Segment polarity genes

Segment polarity genes encode proteins that make up Hedgehog and Wingless signal transduction pathways

One cell produces wingless

The adjacent cell produces hedgehog

Wingless and hedgehog activate expression of each other

Fig. 9.25

2. Gastrulation Homeotic selector genesResponsible for directing structure formation of each segment

These genes are clustered on chromosome 3 in the homeotic complex (also called Hom-C) in two regions-

• The Antennapedia complex-• The bithorax complex-

Chromosome 3

1. The order of these genes on the chromosome matches order of segmental expression

2. Homeotic genes are regulated by all gene products expressed posterior to it

Two amazing features

What about dorsal ventral polarity??

• This occurs after cells are created (post syncytial blastoderm)•Dorsal (a transcription factor) gradient is established•Dorsal is found throughout syncytial blastoderm, but only in nuclei of ventral cells

How does this occur? By a very complex pathway involving gurkin and torpedo proteins ( and a host of other proteins)

Organs form at the intersection of dorsal-ventral and anterior-posterior regions of gene expression