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Developmental Biology Tutorial

Determination of Cell Fate: Introduction

by Dr. Caren Helbing
Department of Medical Biochemistry, University of Calgary
and
Leon W. Browder, Department of Biological Sciences, University of Calgary

The following material is a guide to studying Chapter 11 of Browder et al. (1991), Chapter 13 of Gilbert (1997), Chapter 8 of Kalthoff (1996) and Chapter 14 of Shostak (1991). Reference is also made to relevant material in Wolpert et al. (1998).

CELL DETERMINATION is the process by which portions of the genome are selected for expression in different embryonic cells. This involves developmental decisions that gradually restrict cell fate. Cells can progress from TOTIPOTENT to PLURIPOTENT to DETERMINED.

Note the importance of EXTRINSIC versus INTRINSIC factors.

Methods of Studying Cell Determination

1. Fate Maps

A FATE MAP shows us what each part of the embryo becomes at later stages of development and allows us to trace the embryonic origins of specific cells (CELL LINEAGE) .

(See Browder et al., 1991, Figs. 11.1 and 11.2; Gilbert, 1997, Figs. 6.14 and 13.3; Kalthoff, 1996, Figs. 6.2, 6.3; Wolpert et al., 1998, pages 75-81)

Constructing a fate map involves a wide variety of techniques:

1. following pigment granules in some cells
2. vital dye / carbon particle / enzymatic (HRP)/fluorescent dye marking
3. transplantation of genetically or radioactively labelled donors cells to a host
4. using antibody or nucleic acid probes to examine cell- and tissue-specific gene products

The lineage of all cells of some simple organisms has been determined. eg. C. elegans (959 cells in the adult hermaphorodite and 1031 in the adult male)

2. How is Cell Determination Assayed?

1. Kill or remove specific cells and see what is missing later on.
   
2. Dissociate embryonic cells and culture them in isolation (e.g., trochoblast cells in the limpet Patella : Browder et al., 1991, Fig. 11.4; Gilbert, 1997, p. 13.8).
   
   If the isolated cells form only the cells or tissues expected from the normal fate map or cell lineage, then it is DETERMINED. If it forms an entire embryo or more cell types that expected, then the cell is not determined.
   
3. Transplant blastomeres to different parts of the embryo (eg. Spemann's experiments with neural ectoderm induction).

MOSAIC and REGULATIVE Embryos

The difference between mosaic and regulative embryos lies in the timing of when fate restrictions become apparent in the embryo.

In mosaic embryos, the blastomeres become restricted during the first few cleavages. Because cell fates are established early, they cannot compensate for blastomeres that are removed or destroyed. E.g. in tunicates, separated blastomeres from the two cell embryo will develop into half embryos.

In regulative embryos, this restriction occurs later, so regulative embryos can compensate for blastomeres that are removed in early development.

Regulative embryos differ from mosaic embryos in the orientation of the cleavage planes with respect to the distribution of ooplasmic substances involved in cell determination

(See Browder et al., 1991, Fig. 11.5).

Regulative embryo example: Browder et al., 1991, Fig. 11.6; Gilbert, 1997, Fig. 15.5; Kalthoff, 1996, Fig. 8.2; Shostak, 1991, Fig. 14.19, Wolpert, 1998, Fig. 6.20.

Regulation of Cell Determination by Ooplasmic Determinants

History: The Roux-Weisman theory suggested that nuclear determinants were partitioned during cleavage, but Driesch showed that nuclei do not lose their genetic information. Therefore nuclei remain totipotent. (Note the exception to this rule of CHROMATIN DIMINUTION in the process of germ cell determination in some nematodes. (Browder et al., 1991, Figs. 11.8 and 11.9; Kalthoff, 1996, Fig. 7.3).

The cytoplasm is critical in determining what genetic information can be accessed. Herein lie ooplasmic determinants.

Germ Cell Determination (e.g., Drosophila)

This involves the segregation of primordial germ cells with distinct cytoplasmic granules or GERM PLASM.

The pole plasm is incorporated into the pole cells at the posterior pole of the insect embryo. There is considerable evidence that the pole plasm contains ooplasmic determinants for germ cell development. See Germ plasm in insects from Zygote.

Somatic Cell Determination

Ooplasmic determination in Tunicates

See The search for the yellow crescent myogenic factor in Zygote

Spiralian embryos (e.g., Dentalium)

There are several examples in your text that illustrate the ubiquitous nature of cytoplasmic determinants in cell fate determination.

In spirilians, a polar lobe forms before the first cleavage which results in the unequal partitioning of the cytoplasm to the daughter cells.

(See Browder et al., 1991, Figs. 5.30, 11.29; Gilbert, 1997, Fig. 13.9; Kalthoff, 1996, Figs. 8.3 and 8.4; Wolpert et al., 1998, Fig. 6.13)

Polar lobe ablation and isolation experiments suggest that the polar lobe contains substances important in directing the formation of mesodermal structures and other structures that are induced by mesoderm (e.g., velum, heart, intestine, etc.).

(See Browder et al., 1991, Fig. 11.28, Kalthoff, 1996, Fig. 8.6)

If the formation of the polar lobe during the first cleavage is suppressed with low doses of cytochalasin B (disrupts microfilament formation), the cytoplasm is distributed to both AB and CD blastomeres. The end result is the formation of a double embryo which develops into a trochophore larva with two shells.

(See Browder et al., 1991, Fig. 11.30; Gilbert, 1997, Fig. 13.13)

Sequential ablation of the D macromere illustrates the partitioning of cytoplasmic information during cleavage divisions and the concept of recipient cell cytoplasm programming, which yields progeny cells that adopt specific fates and gives the cells the capacity to influence the fate of other cells in the embryo.

(See Browder et al., 1991, Fig. 11.32 and Table 11-1)

Learning Objectives

• Define cell determination
• How are fate maps used to study cell determination? How are fate maps produced?
• Compare and contrast mosaic and regualtive development. Describe examples of both forms of development.
• What is chromatin diminution, and how has it been studied?
• Discuss the experimental evidence that demonstrates that the polar plasm in insects functions to determine germ cells.
• Discuss the experimental evidence that the myoplasm in tunicate eggs contains muscle determinants.
• Describe the polar lobe of Dentalium and discuss the experiments designed to demonstrate its role in cell determination.

Digging Deeper:

Links to Related Material

The organization and cell-lineage of the ascidian egg by E.G. Conklin (from Zygote)

The molecular basis of trochoblast differentiation from Zygote

Modification of spiralian specification from Zygote

Nematode gut determination from Zygote

See related material prepared by Dr. Richard Carthew, University of Pittsburgh: Cell Determination I and Cell Determination II.

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.

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

Wolpert, L., Beddington, R., Brockes, J., Jessell, T., Lawrence, P. and Meyerowitz, E. 1998. Principles of Development. Current Biology. London.

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