Transcript Slide 1

XIII. Developmental Genetics
XIII. Developmental Genetics
A. Overview:
- Multicellular organisms have different cell types, but all cells are descended
from a single zygote and have the same DNA.
XIII. Developmental Genetics
A. Overview:
- Multicellular organisms have different cell types, but all cells are descended
from a single zygote and have the same DNA.
- So, cell specialization is really a very special type of gene regulation; the
regulation of genes that influence the developmental fate of a cell.
XIII. Developmental Genetics
A. Overview:
- Multicellular organisms have different cell types, but all cells are descended
from a single zygote and have the same DNA.
- So, cell specialization is really a very special type of gene regulation; the
regulation of genes that influence the developmental fate of a cell.
- This requires two more levels of genetic regulation:
- regulation of cell specialization/differentiation
- co-ordination of this specialization with other cells/tissues
XIII. Developmental Genetics
A. Overview:
- Multicellular organisms have different cell types, but all cells are descended
from a single zygote and have the same DNA.
- So, cell specialization is really a very special type of gene regulation; the
regulation of genes that influence the developmental fate of a cell.
- This requires two more levels of genetic regulation:
- regulation of cell specialization/differentiation
- co-ordination of this specialization with other cells/tissues
- One of the most remarkable revelations in this area of genetics is that all
animals (from flies to worms to mammals, and even radially symmetrical
coral) share the same genes that co-ordinate the basic body plan and its
polarity (anterior-posterior axes and body regions)…. But of course, why
not?
XIII. Developmental Genetics
A. Overview:
B. Genetic Control of Body Plan:
- Drosophila has been the model organism for body plan development, and
resulted in Nobel Prizes in 1995…. (so flies are STILL giving up secrets).
XIII. Developmental Genetics
A. Overview:
B. Genetic Control of Body Plan:
- Drosophila has been the model organism for body plan development, and
resulted in Nobel Prizes in 1995…. (so flies are STILL giving up secrets).
- 1. There are two genomes involved:
the maternal genome produces maternal proteins that form a gradient in
the egg and influence the initial development of the embryo.
XIII. Developmental Genetics
A. Overview:
B. Genetic Control of Body Plan:
- Drosophila has been the model organism for body plan development, and
resulted in Nobel Prizes in 1995…. (so flies are STILL giving up secrets).
- 1. There are two genomes involved:
the maternal genome produces maternal proteins that form a gradient in
the egg and influence the initial development of the embryo.
The zygote’s genome produces proteins that react to these gradient in two
ways:
XIII. Developmental Genetics
A. Overview:
B. Genetic Control of Body Plan:
- Drosophila has been the model organism for body plan development, and
resulted in Nobel Prizes in 1995…. (so flies are STILL giving up secrets).
- 1. There are two genomes involved:
the maternal genome produces maternal proteins that form a gradient in
the egg and influence the initial development of the embryo.
The zygote’s genome produces proteins that react to these gradient in two
ways:
- segmentation genes (gap, pair-rule, segment polarity genes) create
segments in the embryo by defining body regions (gap), then dividing the
embryo into units two segments wide (pair-rule), and then an active
segment polarity gene splits each segment into front and backs.
XIII. Developmental Genetics
A. Overview:
B. Genetic Control of Body Plan:
- Drosophila has been the model organism for body plan development, and
resulted in Nobel Prizes in 1995…. (so flies are STILL giving up secrets).
- 1. There are two genomes involved:
the maternal genome produces maternal proteins that form a gradient in
the egg and influence the initial development of the embryo.
The zygote’s genome produces proteins that react to these gradient in two
ways:
- segmentation genes (gap, pair-rule, segment polarity
genes) create segments in the embryo by defining body regions
(gap), then dividing the embryo into units two segments wide
(pair-rule), and then an active segment polarity gene splits each
segment into front and backs.
- hox genes (shortened from homeobox genes) determine the
developmental fate of cells in a particular segment.
XIII. Developmental Genetics
A. Overview:
B. Genetic Control of Body Plan:
- Drosophila has been the model organism for body plan development, and
resulted in Nobel Prizes in 1995…. (so flies are STILL giving up secrets).
- 1. There are two genomes involved:
- 2. There is strong homology among animals in these genes:
Runt is a pair-rule gene involved in segmentation. In flies, it later orchestrates
the development of neurons and sex determination. In mammals, it is
needed for bone deposition in segments.
B. Genetic Control of Body Plan:
- 1. There are two genomes involved:
- 2. There is strong homology among animals in these genes:
Runt is a pair-rule gene involved in segmentation. In flies, it later orchestrates
the development of neurons and sex determination. In mammals, it is
needed for bone deposition in segments.
The most dramatic examples of developmental effects are in the hox genes:
On chromosome 3, in order…
Antennapedia cluster
(Ant-C)
Bithorax cluster
(Bx-C)
B. Genetic Control of Body Plan:
- 1. There are two genomes involved:
- 2. There is strong homology among animals in these genes:
Runt is a pair-rule gene involved in segmentation. In flies, it later orchestrates
the development of neurons and sex determination. In mammals, it is
needed for bone deposition in segments.
The most dramatic examples of developmental effects are in the hox genes:
These genes are in all
cells, but each is only
active in it’s respective
segment - switching on
a host of specific
genes that lead to cell,
tissue, and organ
differentiation.
On chromosome 3, in order…
Antennapedia cluster
(Ant-C)
Bithorax cluster
(Bx-C)
B. Genetic Control of Body Plan:
- 1. There are two genomes involved:
- 2. There is strong homology among animals in these genes:
Runt is a pair-rule gene involved in segmentation. In flies, it later orchestrates
the development of neurons and sex determination. In mammals, it is
needed for bone deposition in segments.
The most dramatic examples of developmental effects are in the hox genes:
Each gene codes for a
transcription factor. Each
gene has a 180 bp homeobox
region that encodes a 60 AA
homeodomain in the protein
that takes a Helix-Loop Helix
configuration….
B. Genetic Control of Body Plan:
- 1. There are two genomes involved:
- 2. There is strong homology among animals in these genes:
Runt is a pair-rule gene involved in segmentation. In flies, it later orchestrates
the development of neurons and sex determination. In mammals, it is
needed for bone deposition in segments.
The most dramatic examples of developmental effects are in the hox genes:
And if you turn them on in the
wrong segment, you get an
incorrect pattern of body
segment development!
a.
Normal fly head, with normal
antennae.
b.
Fly with legs for antennae,
cause by activating a hox gene
for leg development that is
normally only on in thoracic
segments. (antennaepedia)
B. Genetic Control of Body Plan:
- 1. There are two genomes involved:
- 2. There is strong homology among animals in these genes:
Runt is a pair-rule gene involved in segmentation. In flies, it later orchestrates
the development of neurons and sex determination. In mammals, it is
needed for bone deposition in segments.
The most dramatic examples of developmental effects are in the hox genes:
Activaton of bithorax in third
thoracic segment …
And the most
dramatic examples
of homology are in
the hox genes, as
well.
And the most dramatic
examples of homology
are in the hox genes,
as well.
And, human diseases
have been identified as
hox mutants by
identifying homology
with fruit fly hox genes,
and they have been
found in the genome
with ss-DNA (probe)
from the fly homeobox
region of that gene.
Cleinocranial dysplasia (CCD),
caused by a dominant mutation in
the runt homeotic gene
And the most dramatic
examples of homology
are in the hox genes,
as well.
And, human diseases
have been identified as
hox mutants by
identifying homology
with fruit fly hox genes,
and they have been
found in the genome
with ss-DNA (probe)
from the fly homeobox
region of that gene.
Synpolydactyly in humans,
caused by a mutation in HOXD13
And the most dramatic
examples of homology
are in the hox genes,
as well.
And, human diseases
have been identified as
hox mutants by
identifying homology
with fruit fly hox genes,
and they have been
found in the genome
with ss-DNA (probe)
from the fly homeobox
region of that gene.
And, because of evolution
and common ancestry, we
can use model organisms
like flies to learn about
how heredity and
development work in all
animals…including
humans.
Ey (eyeless): a selector
gene in Drosophila.
Turn it ON and get eyes
in novel places… fully
functional.
Homologous to the Pax6 gene in vertebrates, that affects eye and brain
development.
Query 5 HSGVNQLGGVFVNGRPLPDSTRQKIVELAHSGARPCDISRILQTHADAKVQVLDNQNVSN 64
Sbjct 30 HSG INQLGGVYVNGRPLPDSTRQKIVELAHSGARPCDISRILQ---------------- VSN
75
Query 65 GCVSKILGRYYETGSIRPRAIGGSKPRVATPEVVSKIAQYKRECPSIFAWEIRDRLLSEG 124
Sbjct 76 GCVSKILGRYYETGSIKPRAIGGSKPRVATTPVVQKIADYKRECPSIFAWEIRDRLLSEQ 135
Query 125 VCTNDNIPSVSSINRVLRNLASEKQQMGA---DGMYDKLRMLNGQTGSWGTRPGWYPGTS 181
Sbjct 136 VCNSDNIPSVSSINRVLRNLASQKEQQAQQQNESVYEKLRMFNGQTGGW----AWYPSNT 191
Query 182 VPGQPTqdgcqqqegggeN-----------------------TNSISS--------NGED 210
Sbjct 192 TTAHLTLPPAASVVTSPANLSGQADRDDVQKRELQFSVEVSHTNSHDSTSDGNSEHNSSG
251 Query 211 SDEAQMRLQLKRKLQRNRTSFTQEQIEALEKEFERTHYPDVFARERLAAKIDLPEARIQV 270
Sbjct
252 DEDSQMRLRLKRKLQRNRTSFSNEQIDSLEKEFERTHYPDVFARERLADKIGLPEARIQV 311
Query 271 WFSNRRAKWRREEKLRNQRRQA 292
Sbjct 312 WFSNRRAKWRREEKMRTQRRSA 333
Query = toy (twin of eyeless gene)
Subject = human Pax6
Red = Paired-box domain – DNA binding site
Green = homeodomain DNA binding site
Probability of similarity
occurring by chance:
3.2 x 10-77
In fact, the homology is so good that knock-out flies lacking the eyeless gene
can make eyes when the mammalian Pax6 gene is inserted.
Likewise, a regulatory element upstream from ey can restore Pax6 activity in
vertebrates.
So they gene (Pax6/ey) and the regulation of the gene, have been conserved
through metazoan evolution.
Pax6 / Ey
XIII. Developmental Genetics
A. Overview:
- This requires two more levels of genetic regulation:
- regulation of cell specialization/differentiation
- co-ordination of this specialization with other cells/tissues
B. Genetic control of Body Plan:
C. Co-ordination of cell differentiation with other cells
B. Co-ordination of cell differentiation with other cells
Notch genes code for a transmembrane signal receptor protein that binds
another transmembrane protein called Delta…. So this binding occurs
between cells:
Extracellular domain (NECD)
Transmembrane domain (NTMD)
Intracellular domain (NICD)
Myc
p21
NICD
B. Co-ordination of cell differentiation with other cells
When bound, NICD is cleaved and binds to the Su(H) protein – which passes
into the nucleus and binds and stimualtes transcription factors.
Extracellular domain (NECD)
Transmembrane domain (NTMD)
Intracellular domain (NICD)
Myc
p21
NICD
B. Co-ordination of cell differentiation with other cells
How such signals coordinate gene expression has been determined for the
vinegar worm, Caenorhabditis elegans.
B. Co-ordination of cell differentiation with other cells
How such signals coordinate gene expression has been determined for the
vinegar worm, Caenorhabditis elegans.
This is a great developmental model,
because the animal has a set number
of somatic cells (959) and the fate of
each has been mapped:
Organogenesis of the vulva in C. elegans
There are two cells; one will become the gonadal anchor cell, and the
other will become the first uterine cell. What determines their fate?
Organogenesis of the vulva in C. elegans
There are two cells; one will become the gonadal anchor cell, and the
other will become the first uterine cell. What determines their fate?
Initially, they both produce delta signal protein and notch
receptor protein…
Organogenesis of the vulva in C. elegans
There are two cells; one will become the gonadal anchor cell, and the
other will become the first uterine cell. What determines their fate?
Initially, they both
produce delta signal
protein (Lag-2) and
notch receptor
protein (lin-12).
By chance, these
cells won’t produce
exactly the same
amount of signal…
the one bombarded
by MORE signal
produces more
receptor... which
causes the genetic
cascade that results
in the cell becoming
the uterine cell.
Organogenesis of the vulva in C. elegans
The anchor cell now interacts with 6 epidermal cells.
The anchor cell
produces another
protein, lin-3. In the
closest cells where
exposure to this protein
are greatest, receptors
trigger a signal
transduction pathway
that turns on 1o vulva
genes.
Organogenesis of the vulva in C. elegans
The anchor cell now interacts with 6 epidermal cells.
The anchor cell
produces another
protein, lin-3. In the
closest cells where
exposure to this protein
are greatest, receptors
trigger a signal
transduction pathway
that turns on 1o vulva
genes.
In the closest cell, a
protein is produced that
activates the lin-12
gene in the neighboring
cells, which arrests 1o
vulvar cell development
– they become 2o vulvar
cells.
Organogenesis in flowers
Arabidopsis thaliana
Thale kress
Mustard family
Organogenesis in flowers
Four whorls
Organogenesis in flowers
‘A’ genes are usually on in first and
second whorls. When active alone
they specify sepal development.
When active with B genes, petals
develop.
Organogenesis in flowers
Organogenesis in flowers
S = sepal, p = petal, st = stamens, c = carpels
Wild = s,p,st,c
APETALA2 = c,st,st,c
PISTILLATA = s,s,c,c AGAMOUS = s,p,s,p
Homeobox Genes:
- have a 180 bp sequence, which codes for 60 aa’s in the
protein called the homeodomain. This sequence creates
the DNA binding site of the protein. As such, it has been
highly conserved over evolutionary history, and all
multicellular organisms (fungi, animals, and plants) have
them… for regulating gene activity in cells that specialize
for different functions.
Parahox
One class of Homeobox
genes are HOX genes.
They were the first class
of homeobox genes
found. They are only in
animals.
NK
MADS-box
58 aa sequence
XIII. Developmental Genetics
A. Overview:
- This requires two more levels of genetic regulation:
- regulation of cell specialization/differentiation
- co-ordination of this specialization with other cells/tissues
B. Genetic control of Body Plan:
C. Co-ordination of cell differentiation with other cells
D. Epigenetic Effects on Development
- Heritable (maternal effects):
snail coiling: dextral vs. sinistral
- Environmental :
Female worker or
female Queen?
KAMAKURA, M. 2011. Royalactin induces queen differentiation in honeybees. Nature.
473:478-483.
Nutritional Control of Reproductive Status in Honeybees via DNA
Methylation, R. Kucharski*, et al. Science 2008.
1. Silencing of DNA methyltransferase
(Dnmt3) using RNAi
(Dnmt3 is an important enzyme in
methylating CpG islands.)
2. Silence methylation in embryos, get a queen phenotype with larger ovaries.
(suggests that royal jelly may disrupt methylation make queen)
Figure 4. Expression profile of an
alternatively spliced and differentially
methylated gene GB18602 in queen and
worker brains.
(A) The CpG methylation pattern
indicating the level of methylation for
individual CpGs (blue squares, workers;
red squares, queens). (B) Gene model of
GB18602 showing the two spliced
variants S (short protein) and L (long
protein) and the positions of PCR primers
used for variant-specific amplifications.
The green and orange arrows indicate
the positions of two alternative Stop
codons. (C) Relative expression of the
two spliced variants in brains of queens
and workers examined by real-time PCR.
The level of transcript S (green) encoding
the truncated protein is significantly upregulated in the queen brain, whereas
the L variant (orange) is expressed at the
same level in both castes. The queen
expression represents a combined set of
data from three independent queen
samples: 4 mo old (1 brain), 12 mo old (2
brains), and swarm queens of unknown
age (2 brains). Workers were 8 d old (6
brains in 3 replicates). The reference
gene was calmodulin [2]. Whisker-box
plot of expression ratio values: dotted
line, median value; box, inter-quartile
range of values; whiskers, outer 50% of
observations. For more details, see Table
S4.
More methylation in workers, except in some genes in
brain with respect to one gene.
Ratio = 1, like orange,
means same amount in
queen/worker brains.
More green in queen.
The Honey Bee Epigenomes: Differential Methylation of Brain DNA in Queens and Workers, Lyko, et al. 2010)
Spannhof, et al. 2011. EMBO 12:238-243. Histone deacetylase inhibitor activity in
royal jelly might facilitate caste-switching in bees.
Fas is methylated and
off in K-ras cells.
RJ releases inhibition
So does HDA
But it helps 5-Aza turn on genes
No demethylation by itself
5-Aza is a known methylation inhibitor. When The green fluorescent protein gene
is heavily methylated, 5-Aza reduces methylation BUT 10HDA does NOT. (see bar
charts, and see the number of fluorescing cells in the images). So, 10 HAD is not
really a methylation inhibitor – it must be stimulating transcription another way.
10-HDA is structurally
similar to a known Histone
deacetylase inhibitor
(HDACi)….(don’t let the
common HDA letters fool
you – they stand for
different things!)
AND, as HDA concentration
increases (from right to
left), there is MORE HDAC
inhibition.
Spannhof, et al. 2011. EMBO 12:238-243. Histone deacetylase inhibitor activity in
royal jelly might facilitate caste-switching in bees.
What does RJ do?
- 5% of RJ is 10-hydroxy-2-decenoic acid (HDA)
- acts as a histone deacetylase inhibitor (HDACi)
- histone deacetylases (HDAC’s) remove acetyl groups from lysine amino acids
in the histone proteins – this causes the histones to bind DNA more tightly,
inhibiting polymerases.
- by Inhibiting HDAC’s, histones are relaxed – genes can turn on  Queen.
- So, although 10-HDA is not a methylation inhibitor ITSELF, it relaxes histones
and allows access to genes (and access to demethylating agents, too, like in the
5-Aza experiments).
- Royalactin stimulates growth and reproduction.
XIII. Developmental Genetics
A. Overview:
- This requires two more levels of genetic regulation:
- regulation of cell specialization/differentiation
- co-ordination of this specialization with other cells/tissues
B. Genetic control of Body Plan:
C. Co-ordination of cell differentiation with other cells
D. Epigenetic Effects on Development
- Heritable (maternal effects):
snail coiling: dextral vs. sinistral
- Environmental :
- Heritable Epigenetic Effects: Dutch Hunger Winter
From Nov. 1944 – May 1945:
- Hard winter in northern Europe
- Germany blockaded Netherlands
- People ate ~ 30% of necessary caloric intake – 22,000 died.
Babies conceived in
October, starved during late
development – born small.
Stayed small as adults;
lower incidence of obesity
Babies conceived in February, starved
early – caught up late in development
when food was available to their
mothers – born average size.
Higher incidence of obesity
than population at large
And their children has higher
levels of obesity, too. A
heritable change in epigenetic
modification?
From Carey, N. 2013. The Epigenetic Revolution.
60 years later, children starved early in development had lower methylation of
IGF-2 than their older or younger siblings. Those starved late in development –
no difference.
IGF2 DMR
methylatio
n
Mean methylation fraction (SD)
Exposed (n = 60)
Controls (n = 60)
Relative
change
exposed
Difference
in SDs
P
Average
0.488
(0.047)
0.515
(0.055)
−5.2%
−0.48
5.9 × 10−5
CpG 1
0.436
(0.037)
0.470
(0.041)
−6.9%
−0.78
1.5 × 10−4
CpG 2 and
3
0.451
(0.033)
0.473
(0.055)
−4.7%
−0.41
8.1 × 10−3
CpG 4
0.577
(0.114)
0.591
(0.112)
−2.3%
−0.12
.41
CpG 5
0.491
(0.061)
0.529
(0.068)
−7.2%
−0.56
1.4 × 10−3
Heijmans et al. 2008. Persistent epigenetic differences associated with prenatal
exposure to famine in humans.
So, perhaps changes incurred during
a lifetime can cause a heritable
change to the genome. How long
these epigenetic changes are
imprinted remains to be seen. Also,
they create variation on which
selection can act. Still, it may be
somewhat Lamarckian.