COURSE LECTURE NOTES
The Foundations of Developmental Biology
What is Developmental Biology?
The study of all aspects of development, from the genes and molecular events that control development to the structural changes that an organism undergoes as it develops.
This study is facilitated by a vast array of new technologies, adopted from molecular biology, genetics and cell biology.
Historical roots of developmental biology
Embryology (the descriptive study of development)
Embryologists (W. Roux, 1888; H. Driesch, 1892) conducting experimental manipulations of embryos showed that genetic information is inherited equally by all cells during development.
Early cytologists (E.B. Wilson, 1896) recognized that embryological changes were caused by cellular changes, which must be directed by genetic information
Early geneticists (T. Boveri, 1902,; T.H. Morgan, 1927) showed that a full complement of genes was necessary for normal development.
Embryologists (H. Spemann and H. Mangold, 1924) conducting embryo transplant experiments showed that certain areas of an embryo were responsible for inducing the development of other areas.
The discovery of growth factors (R. Levi-Montalcini and V. Hamburger, 1950s) laid the foundation for the modern study of developmental biology.
Late nineteenth century:
Wilhelm Roux 1888:
Hans Driesch 1892:
What do you think is the explanation for the discrepancy between these two studies?
Theodor Boveri 1902:
Thomas Hunt Morgan, 1927:
Hans Spemann and Hilde Mangold, 1924:
Rita Levi-Montalcini and Viktor Hamburger, 1956
The modern era of developmental biology
The ability to isolate, manipulate, and clone genes has allowed developmental biologists to explore the individual roles of genes in development, and the signals that control expression of those genes.
Some of the most useful tools used by modern developmental biologists include:
Biochemical techniques have allowed the elucidation of "signal transduction pathways" involved in providing cells with stage and site-specific directions during development. Genes which are expressed early in development provide signals to direct the expression of genes later in development
Studies of Drosophila (fruit fly) genetics, combined with molecular biology experiments, have provided a "rosetta stone" of developmental biology. Many genes involved in development are common in insects, animals, and humans.
Eg. the Drosophila compound eye:
Homeotic genes (E.B. Lewis, 1978)
eg. four wings instead of two (bithorax)
eg. legs on the head instead of antennae (antennapedia)
eg. HOX genes in mammals
Segmentation Genes (C. Nüsslein-Volhard and E. Wieschaus, 1980)
What is the significance of developmental biology?
Why study it?
Its fascinating!!! (Thats why youre here, isnt it?)
It has agricultural benefits:
It has medical benefits:
A brief aside on model invertebrate organisms
Figure 13.15 (Gilbert, 1997)
The Ark of Life: The Germ Line
Germ cells (Gametes): Egg (ovum) + Sperm
Provide both the blueprint and the raw materials to form a new organism:
Blueprint: derived from both nuclei
Raw materials: primarily contained in egg cytoplasm
"The germ line is the vehicle of evolution." Explain.
Ovum: genetic information
prevention of cross-species fertilization
prevention of polyspermy
information for development
activation of development
Development of Germ Cells
Germ plasm in ovum
Primordial Germ Cells in embryo
Established in gonads during development
(may have to migrate to get there)
proliferation of gonia (spermatogonia and oogonia)
gametogenesis (spermatogenesis and oogenesis)
gametes (sperm and ovum)
Important: Review meiosis!
Gametogenesis: differentiation of gametes
Diagrams of Spermiogenesis and Oogenesis
|Occurs after meiosis||Occurs during meiosis
Resumption of meiosis occurs at oocyte maturation
4 Haploid sperm cells
1 Hapoid ovum + 3 polar bodies
Germ plasm is responsible for telling cells to be gametes/germ cells.
If a Drosophila (fruit fly) geneticist wanted to isolate a mutation that prevented germ cell development, what phenotype might he/she easily look for?
What might germ plasm be?
Experiment: Illmensee and Mahowold (from Dynamic Development)
Many species seem to have a cytoplasmic determinant that specifies germ cell development in some it is associated with granules with specific staining properties.
In Drosophila, "polar granules" are granules located at the posterior end of the egg, which contain both RNA and protein.
mRNA for the "germ cell-less" gene gcl
mRNA for "polar granule component"
Polar granules determine the formation of "pole cells"
which will become primordial germ cells
discovered in 1992 during screen for female Drosophila that were unable to have grandchildren (Jongens et al)
translated during early cleavage to produce a nuclear protein
protein is essential for pole cell production
Injection of mtlrRNA restores the ability of UV-irradiated embryos to form pole cells (UV-irradiation prevents pole cell formation) (Kobayashi and Okada, 1989)
Polar granule component:
Antisense RNA to pgc prevents pole cells from migrating to gonads (Nakamura et al, 1996)
required for formation of Drosophila abdomen (and thus gonads)
required for migration of pole cells to gonads (Kobayashi et al, 1996)
localizes polar granules to posterior pole
Given these components, develop your own hypothetical story of germ cell development and migration - just has to be plausible - not "right"
Manipulation of Gametes:
Spermatogonia and mature ova can be viably frozen (cryopreserved) in liquid nitrogen. How might this be useful?
Preserve germ lines of endangered species or prize stock
Men about to undergo cancer therapy can bank their spermatogonia for later reimplantation; chemo and radiation therapy are very hard on rapidly dividing cells.
Women could bank their eggs while they are young, for later fertilization when they are ready to have a child; womens eggs deteriorate over time.
Fertilized eggs can also be cryopreserved. What is a potential drawback to this technology?
Spermatogenesis and Spermiogenesis
Spermatogenesis: generation of spermatocytes
Spermiogenesis: generation of sperm cells (spermatozoa)
Mammalian testes (male gonad)
Required to produce very large numbers of highly specialized (mobile, compact) sperm cells
Chromatin condensation during Spermiogenesis
DNA + histones
protease digestion of histones
DNA + basic transition proteins
DNA + protamines
protamines form disulfide bridges
By the spermatid stage, transcription has ceased
(earlier in some species).
All mRNA transcripts needed for spermiogenesis must be transcribed before that time.
After that point, synthesis of proteins is regulated by post-transcriptional mechanisms.
Translation of certain transcripts may be delayed until the appropriate point in spermiogenesis.
For some transcripts, the addition of a long (~160 nucleotide) poly(A) tail inhibits translation, perhaps due to the binding of repressor protein(s). Partial deadenylation allows translation to proceed.
Regulation of protamine synthesis:
3 UTR (untranslated region):
Acrosomal cap forms from Golgi apparatus
Flagellum forms from centriole
Hormone regulation of Spermiogenesis (from Browder Chapter 2)
In mammals, the transformation of oogonia to primary oocytes is complete before or shortly after birth
ie. no more new germ cells (unlike in male)
Oocytes remain arrested in meiotic prophase I until puberty,
when, periodically, a number of oocytes undergo maturation.
Female gonad ovary:
Meroistic Insect Egg Chamber:
Meroistic denotes the involvement of nurse cells
mitosis without cell growth
16 smaller cells
1 oocyte + 15 nurse cells
Surround nurse cells + oocyte
What factors might determine which cell becomes the oocyte?
In mammals, accessory cells are follicle cells/granulosa cells
Large, mature follicle with
Polarity and Localization are key!
Vertebrate liver ® vitellogenin
yolk platelets in oocyte
Amount and distribution of yolk varies with species:
small amount of evenly distributed yolk
eg. sea urchin, placental mammals
large amount of yolk localized at one end
eg. reptiles, fish, and birds
eg. Xenopus laevis and other amphibians
yolk in center of egg
eg. arthropods, including Drosophila
Germinal Vesicle = nucleus of egg
may be displaced to one end in eggs with significant amounts of yolk
- divisions early in oogenesis provide a large store of mitochondria for early development
Germ plasm/germinal granules:
Some eggs, which are eventually exposed to sunlight, contain pigment granules, eg. melanin, which may protect the egg contents against UV irradiation.
Hormonal Regulation of Oogenesis in Mammals
Gene Expression During Amphibian Oogenesis
The major phase of oocyte growth and differentiation, which lasts for several months in Xenopus, takes place during prophase of meiosis I (diplotene phase).
Large amounts of RNA and protein are being synthesized during this period, to be used later during early embryogenesis.
In amphibians and many other animals (though not in mammals or meroistic insects), a very high rate of transcription is made possible by the formation of "lampbrush chromosomes".
How do we know the "bristles" are nascent mRNA transcripts?
- in situ hybridization studies using labeled nucleic acid probes
- 3H-UTP metabolic labeling
How do we know RNA polymerase II is involved?
- 0.5m g/ml a -amanitin abolishes 3H-UTP labeling
(0.5m g/ml a -amanitin inhibits RNA pol II)
(200m g/ml a -amanitin inhibits RNA pol III)
- labeled antibodies to RNA pol II bind to loops
- neutralizing antibodies to RNA pol II abolish 3H-UTP labeling
Unusual poly(A)+ mRNAs occur in fully grown Xenopus oocytes:
If poly(A) RNA is isolated using oligo d(T), the average transcript length is much longer than normal, due to interspersed transcription units and repeated sequences.
This suggests that normal transcription termination signals are being ignored during this phase of oogenesis.
Most of the mRNAs (80%) are not translated in the oocyte, but are stored for later.
Some of the mRNAs are specifically localized within the oocyte.
Produced in very large numbers (~1000x) compared to somatic cells, to provide translational capacity during early embryogenesis.
Synthesized earliest, before vitellogenesis, then stored in ribonucleoprotein particles, until ribosome assembly takes place later in oogenesis.
2 sets of 5s RNA genes:
Oocyte 5s RNA genes:
- many tandem repeats (24,000) per haploid genome
- transcribed by RNA pol III
- internal promoter
- 3 transcription factors
Prototype zinc-finger protein
How do we know TFIIIA binds to the 5s RNA promoter?
DNAase footprinting analysis
TFIIIA is bound by 5s RNA as well
- competes for binding to promoter
- negative regulatory mechanism
Amount of TFIIIA is highest early in oocyte growth phase, then declines. This is consistent with the early transcription of the 5s RNA genes.
18s and 28s RNA:
Regulation of Translation
As in spermiogenesis (but for different reasons), translation of stored transcripts is tightly regulated during oocyte maturation and during embryogenesis.
In Xenopus, there are at least two methods of regulating translation of messages.
Eg. cyclin and c-mos transcripts
The sequence of the CPE, and its proximity to the AAUAAA, determine the extent and timing of polyadenylation.
A few messages within the oocyte are very specifically localized
In Xenopus oocyte
Vg1 mRNA (more later) is localized to the vegetal pole
In Drosophila oocyte
- bicoidmRNA is localized to the anterior pole
- nanosmRNA is localized to the posterior pole
What did you recently learn about nanos function? How does this new information fit in?
Often localization is specified by sequences in the 3'UTR of the mRNA.
How might this be shown?
How are messages localized?
Two pathways in Xenopus for localization to vegetal cortex:
Figure 19.23 in text
2. METRO (message transport organizer)
In amphibians and most mammals:
Cell cycle events:
GVBD end of prophase I
First division to produce first polar body
Realignment of chromosomes in second meiotic metaphase
In other animals, mature eggs rest in various stages of meiosis:
- Many worms
- Dog and Fox
- Some worms
- Many insects
Pronucleus (meiosis complete):
- Sea urchin
Regulation of Cell Cycle Events at Maturation
Best studied in Xenopus laevis
Oocyte maturation and ovulation
In this system, progesterone acts in a highly atypical way:
What acts downstream of progesterone?
Classical experiment by Yoshio Masui:
Purified from the cytoplasm - MPF:
(Maturation promotion factor)
(M-phase promoting factor)
Subsequently, it was shown that MPF consists of:
- cdk1 (cyclin-dependent kinase 1)
- cyclin B
Homologous complexes were found in diverse organisms, at various cell-cycle regulatory points.
The cdk family:
- a family of serine/threonine kinases
- activity is dependent upon association of an active cyclin protein.
The cyclin family:
- a family of very unstable proteins
- amount oscillates with the cell cycle
Phosphorylation of cellular substrates by MPF results in reinitiation of meiosis.
MPF activity ocillates with the cell cycle, due to a corresponding oscillation in the amount of cyclin protein.
Why is meiosis halted at metaphase II in oocyte maturation?
Classic experiment by Yoshio Masui:
- took cytoplasm from a mature oocyte, arrested at metaphase II
- injected it into a frog embryo at the two-cell stage
- the embryo ceased cleaving, arrested at metaphase
This activity was purified from the mature oocyte cytoplasm - CSF
CSF (Cytostatic factor):
- responsible for metaphase arrest
- consists of two subunits
- cdk 2
- c-mos (also a serine/threonine kinase)
- stabilizes cyclin, which would otherwise be degraded, thus preventing exit from metaphase
- present only during oocyte maturation
Activation of MPF in immature oocytes:
- MPF is present, but inactive
- c-mos kinase activates MPF
What stimulates c-mos activity?
- c-mos RNA is stored, untranslated, in oocyte
- c-mos transcript is polyadenylated when the progesterone receptor is activated
Signal transduction cascade
The Origins of Pattern and Embryonic Polarity in Drosophila
(Micklem. 1995. Developmental Biology 172, 377)
How does the Drosophila oocyte become polarized? (how does it change from being symmetric to having a front and back end (ANTERIOR/POSTERIOR) and a top and a bottom (DORSAL/VENTRAL)?
interactions with surrounding somatic tissue that change the oocyte cytoskeleton.
localization of embryonic determinants
Controlled by MATERNAL EFFECT GENES
genes that are transcribed during oogenesis but exert their phenotypic effects in the embryo.
identified in genetic screens as mutations that affect the phenotype of the offspring of mutant mothers rather than affecting the mother's them selves.
how do genes expressed in oogenesis leave a molecular imprint on the embryo?
ANTERIOR-POSTERIOR and DORSAL/VENTRAL asymmetry are established by related mechanisms
A/P asymmetry in the mature oocyte is reflected by the anterior localization bicoid mRNA (anterior determinant) and the posterior localization of nanos mRNA (posterior determinant) and mRNAs essential for the function of the germ plasm, such as oskar
D/V asymmetry is reflected by the dorsal localization of the gurken mRNA
This is accomplished in several key steps:
2. Signaling from oocyte to follicle cells to induce posterior fate 3. Signaling from posterior follicle cells to oocyte to reorganize the oocyte
microtubule cytoskeleton 4. Transport of the gurken mRNA dorsally and anteriorly by the oocyte nucleus 5. Selective, microtubule based localization of the bcd, osk, and
nanos mRNAs to the anterior or posterior poles Posterior localization of the Oocyte in the egg chamber
2. Signaling from oocyte to follicle cells to induce posterior fate
3. Signaling from posterior follicle cells to oocyte to reorganize the oocyte microtubule cytoskeleton
4. Transport of the gurken mRNA dorsally and anteriorly by the oocyte nucleus
5. Selective, microtubule based localization of the bcd, osk, and nanos mRNAs to the anterior or posterior poles
Posterior localization of the Oocyte in the egg chamber
Germarium: somatic sheath surrounding oogonial stem cells
germ line stem cells in the germarium divide to give rise to another stem cell and a cystoblast
Cystoblast gives rise to germ line16 cells. One oocyte and 15 nurse cells ()
Oocyte formation requires the genes Bicaudal-D (Bic-D), egalitarian (egl), and orb. These genes encode proteins that become restricted to the oocyte. Mutants for these genes form 16 nurse cells and no oocyte.
the oocyte forms a Microtubule organizing centre (MTOC) that extends a microtubule network
Oocyte expresses high levels of cadherin, a homophilic cell adhesion molecule
Posterior follicle cells (somatic) also express high levels of cadherin, and the oocyte moves to the posterior follicles cells through Cadherin based cell-cell adhesion
EVIDENCE: the oocyte does not localize properly egg chambers in which either the oocyte or the follicles do not express cadherin.
Each egg chamber has a single interconnecting microtubule complex.
formed from a and b tubulin subunits
inherent polarity because the subunits are organized in a specific orientation in the polymer
"grow" from the plus end
Microtubule associated proteins (MAPs) include motor proteins that can move along the microtubule directed towards either the minus ends (i.e. Dynein) or plus ends (i.e. Kinesin)
(before stage 6) The microtubule organizing centre (MTOC the minus end) localized in the oocyte with the plus end extending into the nurse cells.
(at stage 6) That complex is dismantled, and a new one is assembled with the MTOC at the anterior end of the oocyte and the plus ends of the microtubules extending towards the posterior pole of the oocyte. Both the transport and anchoring of bicoid and oskar transcripts are MT-dependent.
EVIDENCE: oskar and bicoid mRNAs fail to localize when microtubules are disrupted by Colchicine treatment
Re-Polarization of the MT network depends on the posterior location of the oocyte within the egg chamber.
A signal produced by the oocyte (germline) reaches the adjacent follicles. A subset of them, the polar cells, respond to the signal and differentiate with posterior fates.
Posterior follicle cells in turn, signal back to the oocyte nucleus to reorganize the microtubule skeleton and establish the anterior posterior axis.
Signal from the oocyte to the follicle cells depends on gurken
gurken mRNA, produced in the nurse cells, localizes to the oocyte during early oogenesis. gurken encodes a TGF-a like signaling molecule that has an epidermal growth factor (EGF) repeat element. The receptor in the follicle cells is an EGF receptor-like molecule called top/DER.
EVIDENCE: Disruption of this signaling due to mutation in either gurken or top/DER:
prevents determination of the posterior follicle cells.
the oocyte microtubule cytoskeleton fails to become polarized
bcd mRNA becomes distributed to both the anterior and posterior poles
osk mRNA is localized to the middle of the oocyte.
Signal from the posterior follicle cells to the oocyte
Controlled by an unknown ligand
depends on Protein Kinase A (PKA) and mago nashi function in the oocyte
EVIDENCE: when pka or mago nashi is mutant in the oocyte, the oocyte MTOC does not re-organize BUT the posterior follicle cells are still specified properly (this is known because they express posterior specific molecular markers). This means that pka and mago nashi function in the reception of a signal BACK from the posterior follicle to the oocyte as opposed to being part of the initial signal from the oocyte to the follicles
Dorsal-ventral patterning depends on Gurken and the oocyte nucleus
When the MTOC repolarizes, the oocyte nucleus moves to the anterior of the oocyte as a consequence of microtubule function.
At the anterior pole, the nucleus goes at random to one side of the oocyte, and that side is determined as dorsal
The nucleus accompanied by gurken transcripts. As a consequence, Gurken protein is synthesized near the oocyte nucleus.
Dorsalization of the follicle cells occurs in response to a signal produced by gurken.
The grk signal produced by the oocyte, is received by follicle cells via Top/DER
Evidence: grk mRNA is not localized when the nucleus fails to migrate (in top/DER mutant egg chambers). The oocyte microtubule is not properly polarized in egg chambers of these mutants, which lack both A-P and D-V patterning. These observations indicate that the formation of the D-V axis depends upon the prior polarization of the A-P axis, which polarizes the microtubule cytoskeleton the oocyte nucleus and the grk mRNA.
Selective Microtubule Based Localization of the bcd, osk, and nanos mRNAs
How do mRNAs become localized to the anterior versus posterior poles of the oocyte?
oocyte transcripts are synthesized in the nurse cells.
transported into the oocyte, apparently directed by a microtubule minus-end-directed motor proteins.
The reorganization of the microtubule network redirects the minus ends to the anterior end the oocyte and moves the transcripts to the anterior end of the oocyte. Most subsequently become uniformly distributed, with exceptions such as bicoid, staufen, oskar, nanos, gurken and cyclin-B.
when the MT network repolarizes all localized transcripts are at least transiently localized to the anterior pole.
bicoid stays there.
How are transcripts localized to the anterior pole move to the posterior pole?
osk is localized to the posterior cortex, bcd is localized to the anterior.
the 3' untranslated regions of the mRNAs give the messages their different "addresses" in the cell. This is shown by "3' UTR swap experiments"
proteins that recognize and anchor specific transcripts to the microtubules bind distinct elements in the 3' UTRs of the osk and bcd mRNAs, have been identified direct their transport and localization.
Par1 kinase is required to move osk mRNA to the anterior pole
Staufen protein localizes to the anterior pole (possibly by bcd mRNA!). RNA binding protein, binds to oskar mRNA. Required to move osk to the posterior pole. This also requires kinesin.
The localization of oskar mRNA has important implications for formation of the pole plasm.
pole plasm contains elements that are necessary for translation of nanos mRNA, the posterior embryonic determinant and it also contains determinants of the germ line.
Oskar protein induces pole plasm assembly (Ephrussi and Lehmann, 1992).
If mRNA is mislocalized to the anterior pole, it induces polar plasm there.
In mutant ovaries in which oskar mRNA is not localized, ectopic pole plasm does not form (Ephrussi and Lehmann, 1992)
These results suggest that unlocalized osk mRNA fails to be translated and that localization is necessary for its translation.
unlocalized oskar transcripts are inactive because their translation is repressed outside the posterior domain. This repression is dependent upon the binding of a protein called Bruno to elements in the 3' UTR of oskar mRNA.
© Tracy O'Connor 2001