Transcript Chapter 8 - Early Development in invertebrates

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
General Animal Development
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
Recall from lecture 1
4. Gameteogenesis- produce germ
cells (sperm/egg) Note: Somatic cells
denote all non-germ cells
3. Organogenesis- Cells rearranged to
produce organs and tissue
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 phases
M- mitosis
G1- Gap 1
S- DNA Synthesis
G2- 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)
MPF
activity
ExampleMitosis
Promoting
Factor (MPF=
cyclin B+cdc2)
How does egg undergo cleavage
without increasing it’s size?
• Answer- it abolishes G1 and G2 phases of cell
cycle
M phase
Cyclin is
degraded
Cyclins are
synthesized in
eggs
S phase
What actually drives the cleavage
process?
Answer- Two processes1. 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
General Animal Development
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
Recall from lecture 1
4. Gameteogenesis- produce germ
cells (sperm/egg) Note: Somatic cells
denote all non-germ cells
3. Organogenesis- Cells rearranged to
produce organs and tissue
Gastrulation- cells of blastula are
dramatically rearranged
• Three germ layers are produced
Five types of movements
1
2
3
Gastrulation
Five types of movements
4
5
Axis formation
Three 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
1. Cleavage
• Cleavages 1 and 2 are through animal/vegetal poles
1
3
2
• Cleavage 4
results in four
macromeres and
and four
micromeres only in
vegetal cells
4
• Cleavage 3 results in four
vegetal and four animal cells
Fig. 8.8
Fig. 8.9
• Post cleavage 5
Cell fate map
Micromeres signal other cells via b-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)
2. Gastrulation
Egg
Late
blastula
Gastrula
Later stages
Note-micromemeres
produce primary
mesenchyme which will
become larval skeleton
Fig. 8.16 Sea urchin development
How do mesenchyme cells
know to migrate inside
blastocoel?
Answer- changing cell
attachment proteins
Blastocoel
Fig. 8.19
Extracellular matrix
Basal lamina
Hyaline layer
Primary mesenchyme cell- 98% decrease in hyaline affinity
100-fold increase in EM/basal lamina affinity
How does invagination occur?
Terms- Invagination region is called archenteron
Opening created is called the blastopore
Answer- swelling of inner lamina layer
Hyaline
Inner layer
Outer layer
Vegetal cells secrete
chondroitin sulfate
proteoglycan,
causing inner layer to
swell and cause
buckling
Now let’s take a look at another
creature- the nematode C. elegans
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959 cells at maturity
1mm long
Produces eggs and sperm (hermaphrodyte)
Transparent
16 hour from egg to hatch
Entire genome sequences- 19,000 genes
1mm
C. elegans
1. Cleavage
Oviduct
Germ cells undergo
mitosis, then begin
meiosis as travel down
oviduct
Cleavage
Mature eggs passes
through the sperm on the
way to the vulva
C. elegans
1. Cleavage
• Cleavages 1 produces founder cell (AB) and stem cell (P1)
1
• Cleavage 2 results in three
founder cells and one stem
cell (P2)
2
3
Fig. 8.42
• Remaining
cleavages result in
a single stem cell
and more founder
cells
C. elegans
How is the anteriorposterior axis determined?
Answer- P-granulesribonucleoprotein complexes
•P-granules always stay
associated with the “P” cell
What directs the P granules?
•PAR proteins- these
specify polarity, cell
division and
cytoplasmic localization
1
3
5
Fig. 8.43
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
Does P2 dictate fate of adjacent cells?
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 protein
APX-1 is a Delta family protein
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
1. Cleavage
Drosophila
• Insects tend to undergo superficial cleavagecleavage occurs at rim of the egg
• In contrast to other creatures, insects form nuclei,
then create cells
Termed a syncytial blastoderm
• Mitotic divisions
#1-#9 - duplicate nuclei 1
(8 min/division
• Mitotic division
#10 – nuclei migrate to rim
• Mitotic division
#11-14 – progressively
slower divisions
7
10
Fig. 9.1
1. Cleavage
Drosophila
• Mitotic divisions
#14 – cells created with
each nuclei to create the
cellular blastoderm
14
Note – each nuclei has a territory
of cytoskeletal proteins
Nuclei
staun
Tubulin
stain
Egg plasma membrane folds
between nuclei to create
individual cells
Cycle 11-14- midblastula transition- nuclear division
slows and transcription increases
Fig. 9.3
2. Gastrulation
Ventral
Dorsal
Ventral furrow
(from mesoderm)
Fig. 9.6
Segments
Head
3 thorax
8 abdominal
It becomes the
ventral tube
Fig. 9.7
2. Gastrulation
Establishment of anterior-posterior
polarity-protein gradients rule the day
Gene family
Examples
Maternal effect- in
specific region of egg
bicoid
Nanos
caudel
Gap- among 1st gene
transcribed in embryo
Kruppel
hunchback
Pair rule – result in 7
bands
fushi tarazu
hairy
Segment polarity –
result in 14 segments
engrailed
wingless
Fig. 9.8
2. Gastrulation
Maternal effect genes
Active during creation of syncytial blastoderm
Bicoid mRNA
injected in anterior
Caudel (diffuse)
nanos mRNA
injected, localize
to posterior
Hunchback (diffuse)
Fig. 9.10
Bicoid prevents caudel mRNA translation
Nanos prevents hunchback mRNA translation
Maternal effect genes
Mechanism
Oocyte mRNAs
Anterior
Syncytial Blastoderm proteins
Posterior
Fig. 9.11
Maternal effect genes
What if we mess up the bicoid gradient?
Wildtype
Bicoid
Inject bicoid into:
Bicoid-/-
Bicoid-/-
Wild-type
Anterior
Middle
Posterior
mutant
Head in
Two
middle
heads
Thus, bicoid specifies head
development
Normal
Fig. 9.14
Two tails
How does nanos specify posterior?
Answer- By preventing hunchback translation
Mechanism
In anterior, Pumilio binds 3’UTR (untranslated
region) of hunchback mRNA, and mRNA is
polyadenylated and translated
Anterior (no nanos)
Posterior (with nanos)
In posterior,
nanos prevents
polyadenyltation,
and thus prevents
translation
Fig. 9.16
2. Gastrulation Segmentation genes
Two steps in Drosophila development
Segmentation
genes
Determination
genes
Egg
Specification
(Cell fate is flexible)
Determination
(Cell fate is determined)
Gap genes
Pair-rule genes
Segment polarity genes
Bicoid, nanos,
hunchback, caudel, etc.
Maternal effect genes activate gaps genes, which activate
pair-rule genes, which activate segment polarity genes
Segmentation
genes
establish
boundaries
Gap
Pair-rule
Fig. 9.19
Segment polarity
a. Gap Genes
Gap
• 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
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
Fig. 9.21
Pair-rule
b. Pair-rule genes
Why do we observe expression of pair-rule
proteins in every other segment?
Answer- pair-rule genes use different enhancer elements
Pair-rule
Example- even-skipped (a pair-rule gene) has several
enhancers, but only one is active in a given stripe
This enhancer
is only active
in stripes #1
Different
concentrations
of gap proteins
determine pairrule gene
expression
Fig. 9.22
c. Segment polarity genes
Maternal, gap and pair-rule genes
Pair-rule
operate before cells are formed
syncytial blastoderm
14
Fig. 9.1
Segment polarity
Segment
polarity
genes act
once cells
are formed
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
Fig. 9.25
Wingless and hedgehog activate expression of each other
2. Gastrulation Homeotic selector genes
Responsible 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-
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
Chromosome 3
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