CMMB 403

DEVELOPMENTAL BIOLOGY

FALL 2001

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)

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Embryologists (W. Roux, 1888; H. Driesch, 1892) conducting experimental manipulations of embryos showed that genetic information is inherited equally by all cells during development.

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Early cytologists (E.B. Wilson, 1896) recognized that embryological changes were caused by cellular changes, which must be directed by genetic information

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Early geneticists (T. Boveri, 1902,; T.H. Morgan, 1927) showed that a full complement of genes was necessary for normal development.

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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.

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The discovery of growth factors (R. Levi-Montalcini and V. Hamburger, 1950’s) 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?

It’s fascinating!!! (That’s why you’re here, isn’t it?)

It has agricultural benefits:

It has medical benefits:

 

A brief aside on model invertebrate organisms

Sea Urchin:

Drosophila:

 

Caenorhabditis elegans:

Figure 13.14 (Gilbert, 1997)

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.

 

Necessary credentials:

Sperm:    mobility

recognition

fertilization

genetic information

 

Ovum:     genetic information

attraction

recognition

prevention of cross-species fertilization

prevention of polyspermy

nuclear fusion

information for development

activation of development

 

Development of Germ Cells

Germ plasm in ovum

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Primordial Germ Cells in embryo

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Established in gonads during development

(may have to migrate to get there)

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Stem cells

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proliferation of gonia (spermatogonia and oogonia)

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gametogenesis (spermatogenesis and oogenesis)

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gametes (sperm and ovum)

 

Important: Review meiosis!

Gametogenesis: differentiation of gametes

Diagrams of Spermiogenesis and Oogenesis

Spermiogenesis Oogenesis
Occurs after meiosis Occurs during meiosis

Resumption of meiosis occurs at oocyte maturation

Diploid spermatogonium

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4 Haploid sperm cells

Diploid oogonium

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1 Hapoid ovum + 3 polar bodies

 

Germ plasm:

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)

 

Germ plasm:

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.

RNA:

mRNA for the "germ cell-less" gene gcl

mRNA for "polar granule component"

mitochondrial  rRNA

Protein:

Nanos protein

Oskar protein

Polar granules determine the formation of "pole cells"

which will become primordial germ cells

 

gcl gene:

Mitochondrial  rRNA:

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)

Nanos protein:

 

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; women’s 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

In mammals:

DNA + histones

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protease digestion of histones

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DNA + basic transition proteins

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transcription stops

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protamines synthesized

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DNA + protamines

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protamines form disulfide bridges

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DNA condensed

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.

Translational regulation:

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)

 

 

Oogenesis

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:

Eg. Drosophila

Meroistic – denotes the involvement of nurse cells

Stem cells

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cystoblasts

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mitosis without cell growth

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16 smaller cells

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1 oocyte + 15 nurse cells

Nurse cells:

Follicle cells:

Surround nurse cells + oocyte

Question:

What factors might determine which cell becomes the oocyte?

fusosome?

 

In mammals, accessory cells are follicle cells/granulosa cells

Primary follicle:

Secondary follicle:

Zona pellucida:

Graafian follicle:

Large, mature follicle with

Ovulation:

Corpus luteum:

Oocyte Structure:

Polarity and Localization are key!

Yolk:

 

Vertebrate liver ® vitellogenin

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yolk platelets in oocyte

Amount and distribution of yolk varies with species:

Oligolecithal/Isolecithal:

small amount of evenly distributed yolk

eg. sea urchin, placental mammals

Telolecithal:

large amount of yolk localized at one end

eg. reptiles, fish, and birds

Mesolecithal:

moderately telolecithal

eg. Xenopus laevis and other amphibians

Centrolecithal:

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

Mitochondria:

- divisions early in oogenesis provide a large store of mitochondria for early development

Germ plasm/germinal granules:

Cortex :

Cortical granules:

 

Pigment granules:

Some eggs, which are eventually exposed to sunlight, contain pigment granules, eg. melanin, which may protect the egg contents against UV irradiation.

Egg Envelopes

Functions:

3 types:

    1. Primary

2. Secondary

3. Tertiary

 

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.

Lampbrush chromosomes:

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?

How do we know RNA polymerase II is involved?

(0.5m g/ml a -amanitin inhibits RNA pol II)

(200m g/ml a -amanitin inhibits RNA pol III)

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.

 

Ribosomes:

Produced in very large numbers (~1000x) compared to somatic cells, to provide translational capacity during early embryogenesis.

Ribosomal components:

5s rRNA

28s rRNA

18s rRNA

ribosomal proteins

5s RNA:

Synthesized earliest, before vitellogenesis, then stored in ribonucleoprotein particles, until ribosome assembly takes place later in oogenesis.

2 sets of 5s RNA genes:

      1. oocyte – transcribed during oogenesis
      2. somatic

Oocyte 5s RNA genes:

TFIIIA:

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

 

 

 

 

 

 

 

 

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:

tRNA:

 

 

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.

    1. Level of polyadenylation

Eg. cyclin and c-mos transcripts

The sequence of the CPE, and its proximity to the AAUAAA, determine the extent and timing of polyadenylation.

    1. Translation inhibitor

Eg. FGFR-1

 

mRNA Localization

A few messages within the oocyte are very specifically localized

Evidence:

Examples:

In Xenopus oocyte

Vg1 mRNA (more later) is localized to the vegetal pole

In Drosophila oocyte

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

1. Cytoskeleton

2. METRO (message transport organizer)

 

 

Oocyte Maturation

In amphibians and most mammals:

Cell cycle events:

GVBD – end of prophase I

Chromosome condensation

Spindle formation

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:

Primary oocyte:

Metaphase I:

Pronucleus (meiosis complete):

 

Regulation of Cell Cycle Events at Maturation

Best studied in Xenopus laevis

Pituitary

 

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Gonadotropins

 

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Follicle cells

 

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Progesterone

 

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Oocyte maturation and ovulation

 

In this system, progesterone acts in a highly atypical way:

    1. It binds to and activates a cell-surface receptor. How do we know? If immature oocytes are bathed in progesterone, they are stimulated to mature. If they are injected with progesterone, they are not.
    2. It affects translation, and not transcription. How do we know? Transcription inhibitors (eg. actinomycin D)have no effect on progesterone-induced maturation, but translation inhibitors (eg. cycloheximide) do.

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:

Homologous complexes were found in diverse organisms, at various cell-cycle regulatory points.

The cdk family:

 

The cyclin family:

 

 

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:

 

 

 

 

This activity was purified from the mature oocyte cytoplasm - CSF

CSF (Cytostatic factor):

 

Activation of MPF in immature oocytes:

What stimulates c-mos activity?

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:

      1. Posterior localization of the oocyte in the egg chamber (mediated by Cadherin based cell adehsion)

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.

MICROTUBULES

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