Dynamic Development

CONTENTS

Main Page Dynamic Development

The Foundations of Developmental Biology

Gametogenesis

From Sperm and Egg to Embryo

Genetic Regulation of Development

Organizing the Multicellular Embryo

Generating Cell Diversity


Dynamic Development at a Glance


Learning Resources

Research Resources

The Developmental Biology Journal Club

Developmental Biology Tutorial

Gene Expression during Amphibian Oogenesis

How does the oocyte nucleus make so much stuff?


One distinction of amphibian oogenesis is the formation of lampbrush chromosomes during the major phase of oocyte growth and differentiation. Meiosis I is suspended at the diplotene stage, and the homologs remain attached by chiasmata. Each homolog is comprised of parallel strands of sister chromatids. At intervals, the chromatin is compacted into chromomeres, and chromatin loops extend laterally from the chromomeres.

(Browder et al., 1991, Fig. 3.27, Gilbert, 1997, Fig. 22.25; Kalthoff, 1996, Fig. 3.12; Shostak, 1991, Fig. 3.13)

A ribonuceoprotein matrix accumulates on the loops, which consists of proteins and nascent RNA (i.e., RNA that was transcribed on the loop chromatin). The matrices on sister loops are identical, but those on different loop pairs are often quite distinctive. The matrix is frequently asymmetrical, being thin at one end and thick at the opposite end .

(Browder et al., 1991, Fig. 3.29).

This asymmetry suggests directionality in transcription, with the thickness of the matrix reflecting the lengths of nascent transcripts. This is confirmed by electron microscopic examination of loops.

(Browder et al., 1991, Fig. 3.30A)

Loops may have more than one transcription unit with opposite polarity. Note how closely packed the polymerases are on the loop chromatin. This suggests that the loops are quite active in transcription. Considering the duration of the lampbrush phase, a tremendous amount of RNA can be synthesized during oogenesis in these organisms.

The image shown below is from Dr. Ulrich Scheer's entry on the University of Wuerzburg Web site. It shows a lampbrush chromosome of the salamander Pleurodeles waltlii, visualized by immunofluorescence microscopy. The fluorescence is due to a fluorescent-labeled antibody that recognizes proteins associated with the nascent transcripts on the chromosome.



Transcription on lampbrush loops has been demonstrated by in situ hybridization. Why does the labeled probe bind only to the nascent RNA and not to the DNA?

(Browder et al., 1991, Fig. 3.31; Kalthoff, 1996, Fig. 15.1)

Alpha-amanitin is a selective inhibitor of RNA polymeras activity: low concentrations (0.5 µg/ml) inhibit polymerase II transcription. Incorporation of label into RNA on lampbrush loops is abolished by this treatment. Incorporation of label on short stretches of loop axes is retained; this is due to polymerase III transcription of 5S RNA genes and is abolished by treatment with 200 µg/ml alpha-amanitin.

(Browder et al., 1991, Fig. 3.32)

Further evidence for polymerase II in transcription on loops is provided by treatment with antibodies to polymerase II, which causes rapid termination of transcription and retraction of lampbrush loops.

(Browder et al., 1991, Fig. 3.33)

Although they have been studied most intensively in amphibians, they are quite widespread in the animal kingdom. This suggests that they play a significant role in synthesis and accumulation of RNA during oogenesis.

Messenger RNA production during oogenesis has been studied extensively by monitoring the accumulation of polyadenylated RNA. This category of RNA is representative of a large fraction of the messenger RNA population. Poly(A) is thought to play two primary roles: protection from 3' exonucleases and promotion of translation. The polyadenylation status of RNA made during oogenesis can change dynamically at later stages, and this has functional significance.

Isolation of poly(A)+ RNA is facilitated by use of oligo (dT), to which the poly(A) tracts bind reversibly.

The majority of poly(A)+ RNA molecules in the full-grown Xenopus oocyte are unusual: they contain interspersed single-copy and repeat sequences that are covalently linked. This suggests that transcription during oogenesis may ignore termination signals and proceed past them into contiguous repetitive sequences.

Perhaps this is an explanation for the large sizes of lampbrush transcripts (which may be up to several kb in length).

Although the interspersed poly(A)+ RNA is not translatable, much of the RNA in full-grown oocytes is potentially translatable. However, the majority of this RNA is not translated in oocytes. Only 20% of the translatable poly(A)+ RNA is in polysomes. Why is 80% of it not being utilized?

The synthesis of poly(A)+ RNA begins early in oogenesis. There is net accumulation of this class of RNA until the beginning of vitellogenesis, when it plateaus. Although transcription persists, there is no net quantitative or qualitative change in poly(A)+ RNA throughout the remainder of oogenesis. The absence of qualitative changes has been demonstrated by use of cloned probes. The constancy of the RNA population indicates that the continued synthesis of poly(A)+ RNA is counterbalanced by degradation. The maintenance of a constant level of RNA is apparently the task of the lampbrush chromosomes with their closely-packed polymerases.

Ribosome Components

The rate of ribosome accumulation during oogenesis is very impressive. It exceeds that of the most active somatic cells by at least 1000-fold. Thus, the ribosomal components (5S, 18S and 28S RNA and ribosomal proteins) are produced in massive amounts.

5S RNA is synthesized early in Xenopus oogenesis - before the other components are made. It is then stored in 7S and 42S ribonucleoprotein particles and later utilized in ribosome assembly. 5S RNA accounts for ~45% of the total RNA in previtellogenic oocytes.

The genome contains two sets of 5S RNA genes: oocyte-type and somatic-type. There are 24,000 tandemly-repeated oocyte-type coding units per haploid genome. How many templates are there in an oocyte nucleus?

Unlike polymerase II genes with which you are familiar, the 5S genes (which are transcribed by polymerase III) have an internal promoter. Transcription is initiated as the result of an interaction between the promoter site and three oocyte transcription factors. This interaction specifies that transcription will be initiated upstream; i.e., at the transcription start site.

TFIIIA has been especially well studied. TFIIIA is the prototypical zinc finger protein (of which many are now known). This protein has nine of these structures, which are loops formed by periodic folding of the protein due to binding of amino acids within each repeat structure to a zinc ion.

(Browder et al., 1991, Fig. 3.37, Gilbert, 1997, Fig. 10.30)

Binding of TFIIIA to the promoter has been demonstrated by DNase footprinting analysis. DNA that has been labeled at one end is digested with the enzyme deoxyribonuclease I under conditions in which the enzyme makes a single cut in the DNA. Fragments of different lengths are produced, which are separated by electrophoresis and visualized by autoradiography. Bands will be seen that correspond to cleavage at each nucleotide position unless protein protects the site and prevents the enzyme from cutting the DNA. The gap in the sequence is referred to as a "footprint".

(For an example of this technique, see Browder et al., 1991, Fig. 18.16A)

TFIIIA is a bifunctional molecule in that it is also capable of binding to 5S RNA. What consequences would this have for regulation of 5S gene transcription?

The abundance of TFIIIA changes during oogenesis. It is abundant during previtellogenesis and declines in amount thereafter. The abundance of the protein reflects the abundance of the messenger RNA. Its abundance declines 5-10x during postvitellogenesis (Pfaff and Taylor, 1992). How does this differ from most messenger RNA molecules? Trace the consequences of regulation of TFIIA gene transcription for accumulation of 5S RNA.

5S RNA synthesis is no longer detectable after oocyte maturation. Egg extracts will only support 5S gene transcription if exogenous TFIIIA is added, whereas oocyte extracts will support transcription without it addition. How do you interpret these results? Embryos resume 5S RNA synthesis, but only from the somatic genes. This is an excellent example of stage-specific gene expression.

18S and 28S ribosomal RNA are the most abundant components of the oocyte RNA population. Their massive synthesis during oogenesis is facilitated by amplification of the ribosomal RNA genes. Although this phenomenon has been studied most intensively in amphibians, it is also found in some species of insects, mollusks and fish.

(Browder et al., 1991, Fig. 3.39)

Nuclei of somatic cells of Xenopus contain two nucleoli, but hundreds of nucleoli line the inner surface of the nuclear envelope of the germinal vesicle. It has been estimated that it would take 400 years for a frog to produce the amount of ribosomal RNA found in the full-grown oocyte if amplification did not take place.

Transcription of ribosomal RNA is very efficient during vitellogenesis, with polymerase molecules being tightly packed on the chromatin.

(Browder et al., 1991, Fig. 3.40; Gilbert, 1997, Fig. 22.26; Kalthoff, 1996, Fig. 3.13)

By the way, which polymerase molecule is used for ribosomal gene transcription?


Learning Objectives

  • Correlate formation of lampbrush chromosomes with the events of meiosis.
  • How do we know what the matrix of lampbrush chromosomes is composed of?
  • How has transcription been demonstrated on lampbrush chromosomes?
  • What is known about poly(A)+ RNA in oocytes? Why do we care?
  • What is unusual about the ribosome content of oocytes?
  • How many 5S RNA genes are present in the genome?
  • Review the organization of 5S RNA genes.
  • Review the role of TFIIIA in 5S RNA synthesis.
  • How does the oocyte make so much 18S and 28S RNA?


Digging Deeper:

Lampbrush Chromosomes

  • Check out the description of lampbrush chromosomes by Dr. H.C. Macgregor, who studies them.

Ribosome Components

Recent Literature

Brown, D.D. 1997. E.B. Wilson Award Lecture, 1996. Differential gene action. Mol. Biol. Cell 8: 547-553.


References

Browder, L.W., Erickson, C.A. and Jeffery, W.R. 1991. Developmental Biology. Third edition. Saunders College Pub. Philadelphia.

Gilbert, S.F. 1997. Developmental Biology. Fifth Edition. Sinauer. Sunderland, MA.

Kalthoff, K. 1996. Analysis of Biological Development. McGraw-Hill. New York.

Pfaff, S.L. and Taylor, W.L. 1992. Characterization of a Xenopus oocyte factor that binds to a developmentally regulated cis-element in the TFIIIA gene. Develop. Biol. 151: 306-316.

Shostak, S. 1991. Embryology. An Introduction to Developmental Biology. HarperCollins. New York.


Dynamic Development at a Glance
Main Page Dynamic Development

Dynamic Development is a Virtual Embryo learning resource

This material may be reproduced for educational purposes only provided credit is given to the original source.
Leon Browder & Laurie Iten (Ed.) Dynamic Development
Last revised Friday, June 12, 1998