1. BIOL 2416 Chapter 19: Development

2. The Big Question
How does a single cell develop into a complex multicellular organism ?
Specifically:
How does a zygote produce many cells of many types?
How do these cells organize themselves into functional 3-D structures?
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3. Embryonic development involves 3 interrelated processes: * cell division * morphogenesis * cell differentiation

4. Cell division multiplies cells (cloning).
Differentiation makes cells “grow up” (specialization of form and function).
Different kinds of cells are organized into tissues and organs.
Morphogenesis, the “creation of form,” gives an organism its shape.
basic body plan laid out very early in embryonic development.
includes establishing the major axes of the embryo: anterior-posterior, dorsal-ventral.
Includes e.g. segmentation

5. Morphogenesis in animals and plants is very different…
In animals, but not in plants, movements of cells and tissues are necessary to transform the embryo.
Ongoing development in adults limited to differentiation (e.g. replenishing blood cells)
E.g. morphogenesis defect = cleft palate
In plants, morphogenesis and growth in overall size are not limited to embryonic and juvenile periods.
Have apical meristems for continual growth of roots, leaves.
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6. Fig. 21.2
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7. Researchers study development in model organisms to identify general principles
Model chosen based on question asked
Frogs traditionally used b/c:
Large eggs easy to see and manipulate
Fertilization and development outside mom’s body.
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8. For developmental genetics:
readily observable embryos,
short generation times,
relatively small genomes, and
preexisting knowledge about the organism and its genes.
Fig. 21.3
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9. What’s so great about the fruit fly Drosophila melanogaster?
small and easily grown in the laboratory.
generation time of only two weeks and produces many offspring.
Embryos develop outside the mother’s body.
vast amounts of information on its genes and other aspects of its biology.
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10. What’s so great about the nematode Caenorhabditis elegans?
easily grown in petri dishes.
Only a millimeter long, it has a simple, transparent body with only a few cell types
grows from zygote to mature adult in only three and a half days.
Its genome has been sequenced.
Because individuals are hermaphrodites, it is easy to detect recessive mutations.
Self-fertilization of heterozygotes will produce some homozygous recessive offspring with mutant phenotypes.
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11. every adult C. elegans has exactly 959 somatic cells.
But the best part is…
every adult C. elegans has exactly 959 somatic cells.
These arise from the zygote in virtually the same way for every individual.
By following all cell divisions with a microscope, biologists have constructed the organism’s complete cell lineage, a type of fate map.
A fate map traces the development of an embryo.
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12. Fig. 21.4
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13. What’s so great about the mouse Mus musculus?
Long history as a mammalian model of development.
Much is known about its biology, including its genes.
Can make transgenic mice and knockout mice in which particular genes are “knocked out” by mutation.
But…
do have a genome as large as ours, and
their embryos develop in the mother’s uterus, hidden from view, and
they are not humans…
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14. What’s so great about the zebrafish Danio rerio?
small fish (2 - 4 cm long) are easy to breed in the laboratory in large numbers.
The transparent embryos develop outside the mother’s body.
Although generation time is two to four months, the early stages of development proceed quickly.
The study of the zebrafish genome is an active area.
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15. What’s so great about the mustard weed Arabidopsis thaliana?
One plant can grow and produce thousands of progeny after eight to ten weeks.
A hermaphrodite means it can self-cross.
Can induce cultured cells to take up foreign DNA (genetic transformation/cloning).
Its relatively small genome, about 100 million nucleotide pairs, has already been sequenced.
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16. Different types of cell in an organism have the same DNA
PARADOX:
Same DNA (genomic equivalence)
BUT different gene expression (DNA > mRNA > protein)
Means different (blocks of) genes activated vs. inactivated during development
Already seen at which levels control is possible
Now the big question: is inactivation irreversible? Or… once a cell has grown up, can we de-program it back into a baby-ish state?
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17. Proving genomic equivalence: differentiated root cells can be
de-programmed to grow into a whole plant (Steward, 1950’s):
Fig. 21.5
WHOA! We’ve cloned a plant! Plant cells remain TOTIPOTENT!

18. Not so with differentiated animal cells…cannot de-program a whole cell…
but you CAN replace the nucleus of an unfertilized egg cell with the nucleus of a differentiated cell (Bridge and King, 1950’s) and grow a new animal (clone).
BUT younger, relatively
undifferentiated cells worked best…so differentiation is harder to un-do in animal cells…
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19. Conclusions: So nuclei do change in some ways as cells differentiate
DNA sequences themselves do not change
But chromatin structure and methylation may change; this affects gene expression (epigenetics)
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20. DOLLY (1997, Ian Wilmut et al)
De-differentiated the nucleus of a 6-year old sheep udder cell by culturing in a nutrient-poor medium, sending the cell into the G0 “resting” phase.
These arrested cells were fused with sheep egg cells whose nuclei had been removed.
The resulting cells divided to form early embryos which were implanted into surrogate mothers.
277 cell fusions
29 embryos
13 surrogate mothers
Only 1 Dolly
Dolly aged more quickly than normal and died at 6 ½ (12 yrs normal life span)
Now know improper DNA methylation in many cloned embryos interferes with normal development (imprinting not 100% right; epigenetics…)
Mitochondrial DNA issues also cause problems

21. SCNT = Somatic Cell Nuclear Transfer Look! No sperm cell!
At best, clone is a delayed
identical twin of the somatic
cell nuclear donor…
If SCNT embryo is implanted into
a womb, it leads to “reproductive cloning”.
If not, it leads to the establishment of an
embryonic stem cell line
(“therapeutic cloning”)
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22. Stem Cells can differentiate into cell types when they grow up

23. Kinds of Stem Cells used for Stem Cell lines
Embryonic Stem Cells (ES)
From leftover IVF embryos
Egg and sperm, but no womb
Cells harvested at 5-day 100-cell blastocyst stage: destroys the embryo
From cloned SCNT embryos
Egg only, no sperm and no womb
Great potential for growing spare tissues or organs (“therapeutic cloning”)
Adult Stem Cells
From adult tissues in the body (not as pluripotent as ES or iPS/piPS cells)
Serve as natural reservoirs of replacement cells in case of injury
Can bank cord blood
Induced Pluripotent Stem Cells (2009!)
Produced by forced induction of genes in an adult somatic cell (iPS cells).
Forcing gene expression/messing with genome carries risks, such as cancer…
Produced by repeated exposure to proteins instead (piPS cells)

25. May also lead to cloned animals for commercial purposes
Beyond the study of differentiation, stem cell research has enormous potential in medicine.
The ultimate aim is to supply cells for the repair of damaged or diseased organs.
No fear of rejection if using SCNT embryos from the patient
Bladders have been grown in culture and successfully implanted in patients (2006)
May also lead to cloned animals for commercial purposes
Enviropigs
Silk-producing goats

26. Different cell types make different proteins, usually as a result of transcriptional regulation
determination is the decision making process that leads up to observable differentiation of a cell.
Cells decide to express tissue specific genes to make tissue-specific proteins.
In most cases, the pattern of gene expression in a differentiated cell is controlled at the level of transcription.
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27. Fig. 21.9
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28. Transcription regulation is directed by maternal molecules in the cytoplasm and signals from other cells
Two sources of information “tell” a cell, like a myoblast or even the zygote, which genes to express at any given time.
First, information is both the RNA and protein molecules, encoded by the mother’s DNA, already in the cytoplasm of the unfertilized egg cell (“cytoplasmic determinants”).
The other important source of developmental information is induction by neighboring cells.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

29. The cytoplasmic determinants regulate the expression of genes that affect the developmental fate of the cell.
Cytoplasmic determinants are maternal proteins or RNA encoded by maternal effect genes, a.k.a egg-polarity genes.
They are deposited into the egg before fertilization, while still in the ovary.
They form gradients, which establish the dorsal-ventral and anterior-posterior axes, and from there trigger formation of correct body part in the right place.

30. For instance, in (segmented) fruit flies:
The bicoid gene, when mutated, produces an embryo with tails at both ends; the normal bicoid gene product must be a morphogen that provides positional info in order to establish the anterior-posterior axis and set up the anterior end.

31. Segmentation involves a CASCADE of gene activations in the fruit fly:
The bicoid protein and other morphogens are transcription factors that regulate the activity of some of the embryo’s own genes.
Gradients of these morphogens bring about regional differences in the expression of segmentation genes, the genes that direct the actual formation of segments after the embryo’s major axes are defined.

32. In the late 1970s, Christiane Nüsslein-Volhard and Eric Weischaus identified 1,200 genes (out of a total of 13,000 genes) essential for embryonic development
About 120 of these were essential for 3-D pattern formation leading to normal segmentation.
1995 Nobel Prize (with Edward B. Lewis).
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33. Sequential activation of three sets of segmentation genes provides the positional information for increasingly fine details of the body plan.
These are gap genes, pair-rule genes, and segment polarity genes.
Fig
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34. Gap genes map out the basic subdivisions along the anterior-posterior axis.
Mutations cause “gaps” in segmentation.
Pair-rule genes define the modular pattern in terms of pairs of segments.
Mutations result in embryos with half the normal segment number.
Segment polarity genes set the anterior-posterior axis of each segment.
Mutations produce embryos with the normal segment number, but with part of each segment replaced by a mirror-image repetition of some other part.
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35. Homeotic genes direct the identity of body parts
In a normal fly, structures such as antennae, legs, and wings develop on the appropriate segments.
The anatomical identity of the segments is controlled by master regulatory genes, the homeotic genes.
Discovered by Edward Lewis, these genes specify the types of appendages and other structures that each segment will form.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

36. Mutations to homeotic genes produce flies with such strange traits as legs growing from the head in place of antennae.
Structures characteristic of a particular part of the animal arise in the wrong place.
Fig
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37. Like other developmental genes, the homeotic genes encode transcription factors that control the expression of genes responsible for specific anatomical structures.
For example, a homeotic protein made in a thoracic segment may activate genes that bring about leg development, while a homeotic protein in a certain head segment activates genes for antennal development.
A mutant version of this protein may label a segment as “thoracic” instead of “head”, causing legs to develop in place of antennae.
So homeotic genes are organ identity genes.
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38. Amazingly, many of the molecules and mechanisms that regulate development in the Drosophila embryo, like the hierarchy below, have close counterparts throughout the animal kingdom.
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39. Homeobox genes have been highly conserved in evolution
All homeotic genes of Drosophila include a 180-nucleotide sequence called the homeobox, which specifies a 60-amino-acid homeodomain.
The homeodomain of the protein is known to bind DNA (= feature of a transcription factor)
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40. Proteins with homeodomains probably regulate development by coordinating the transcription of batteries of developmental genes.
In Drosophila, different combinations of homeobox genes are active in different parts of the embryo and at different times, leading to pattern formation.
Fig
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41. Besides cytoplasmic determinants in the original egg cell, the other important source of developmental information we did not yet discuss is induction by neighboring cells. Signals from neighboring cells cause a change in gene expression leading to observable cellular changes.
Induction brings about differentiation in these cells through transcriptional regulation of specific genes.

42. E.g. induction in the nematode worm:
Already present on the ventral surface of the second-stage larva are six cells from which the vulva will arise.
A single cell in the embryonic gonad, the anchor cell, initiates a cascade of signals that establishes the fate of the vulval precursor cells.
It does so by producing an inducer protein that binds to receptors on the closest cells.
Fig a
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43. The closer to the anchor cell, the more inducer binds; these precursor cells form the inner vulva.
The inducer is a growth factor: the high levels of inducer probably cause division and differentiation of this cell to form this structure.
It also activates a gene for a second inducer.
Receptors on the two adjacent vulval precursor cells bind the second inducer, which stimulates these cells to divide and develop into the outer vulva.
Because the three remaining vulval precursor cells are too far away to receive either signal, they give rise to epidermal cells.

44. Lineage analysis of C. elegans highlights another outcome of cell signaling, programmed cell death or apoptosis.
The timely suicide of cells occurs exactly 131 times in the course of C. elegans’s normal development.
At precisely the same points in development, signals trigger the activation (not production…) of a cascade of “suicide” proteins in the cells destined to die.
Fig a
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