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First published online September 2, 2003
doi: 10.1242/10.1242/dev.00701

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Investigating C. elegans development through mosaic analysis

Department of Genetics, Cell Biology and Development, University of Minnesota, 6-160 Jackson Hall, 321 Church Street, Minneapolis, MN 55455, USA

View larger version (24K): [in a new window]   Fig. 1. Extrachromosomal arrays for mosaic analysis in C. elegans. (A) Mini-chromosomes, called extrachromosomal arrays, form in vivo from DNA that has been injected into the syncytial part of the gonad of hermaphrodites. Green circles represent plasmids that contain a marker gene that cell-autonomously expresses green fluorescent protein (GFP) in nuclei of transgenic worms; blue circles represent plasmids that contain a wild-type copy of a gene under study. (B) An array can contain multiple copies of each type of injected DNA. Shown in green is the marker gene that expresses GFP in nuclei. In blue are wild-type copies of the gene under study. Endogenous copies of the blue gene have a loss-of-function mutation, as indicated by a red line. The wild-type copies on the array fully complement the mutant copies on the homologous chromosomes.

View larger version (17K): [in a new window]   Fig. 2. An example of mosaic analysis and cell autonomy. (A) In worms that are homozygous for the mutation of the blue gene mentioned in Fig. 1, four hypothetical cells develop an abnormal shape and color. (B) Worms are identified in which the four cells have green fluorescent nuclei, a consequence of inheriting the extrachromosomal array, mentioned in Fig. 1, that expresses GFP cell autonomously. The cells have therefore inherited wild-type copies of the blue gene, because they are also present on the array, and the array is known from preliminary work to complement the mutant phenotype of the blue gene in transgenic worms that are not mosaic. The cells are observed to undergo wild-type development, which involves a change in cell shape and color soon after birth. This pattern of inheritance, however, does not prove that the blue gene must function within the four cells (cell autonomously). Proper development of the four cells may instead depend on the expression of the gene in another cell or cells, which signal to the four cells to change their shape and color. This would be an example of cell non-autonomy. Mosaic worms must therefore be examined carefully for their overall patterns of mosaicism. (C) Loss of the array when the grandmother of the cells divides produces mosaicism within the four cells. The left clone has a wild-type phenotype, and the right clone is mutant. Note that the phenotype correlates with inheritance of the array, as would be expected for the cell autonomous action of the blue gene.

View larger version (98K): [in a new window]   Fig. 3. The Ncl phenotype. (A) Nomarski image of normal nucleoli (arrows) in the nuclei of three neurons that have the genotype ncl-1(e1865)/ncl-1(e1865); sDp3[ncl-1(+)]. Although the endogenous copies of the ncl-1 gene carry e1865, a loss-of-function mutation that is completely recessive to the wild-type gene, each neuron inherited a free duplication (sDp3) that has a wild-type (+) copy of ncl-1, which fully complements the e1865 mutation. (B) Neurons that fail to inherit sDp3 have the genotype ncl-1(e1865)/ncl-1(e1865). They show the Ncl phenotype, a cell-autonomous enlargement of nucleoli (arrowheads). (C) The three nuclei are mosaic for the duplication. The two nuclei on the left have enlarged nucleoli, indicating that they failed to inherit sDp3; the nucleus on the right has a normal nucleolus, indicating its inheritance of the duplication. (D) The opposite pattern of mosaicism results when the two nuclei on the left, but not the nucleus on the right, inherit the duplication. (A'-D') The boundaries of the nuclei (gray) and of the nucleoli (white circles) are indicated for each of the upper panels. Scale bar: 10 µm.

View larger version (68K): [in a new window]   Fig. 4. Expression of sur-5::gfp in nuclei. (A) Fluorescent image of a non-mosaic worm. Arrowheads indicate three intestinal nuclei that have inherited an extrachromosomal array that expresses sur-5::gfp. (B) The corresponding Nomarski image. (C,D) Lack of fluorescence of three intestinal nuclei is indicated with arrowheads in a mosaic worm that lost the array in the embryonic cell E, the progenitor of the gut. (E,F) Patchy mosaicism in the gut. Two positive clones can be seen within an otherwise dark intestine, indicating consecutive losses of the array within the gut cell lineage. Consecutive losses occur more frequently for extrachromosomal arrays than for free duplications. Scale bars: in A, 50 µm for A-D; in F, 150 µm for E,F.

View larger version (28K): [in a new window]   Fig. 5. Correlation of phenotype with mosaic cell lineages. The phenotypes of certain progeny of a worm of genotype unc-36(–); Ex100[unc-36(+) sur-5::gfp] are indicated below diagrams of the early divisions of the invariant cell lineage of C. elegans. Ex100 is an extrachromosomal array that has wild-type (+) copies of the unc-36 gene, which rescue a loss-of-function mutation in the endogenous copies of unc-36. The array also expresses GFP from a cell-autonomous marker gene. Based on which cells are green, the marker allows one to deduce the cell division at which the array was lost in a mosaic animal. (A) Inheritance of the array by all cells, indicated in green in the diagram, produces a non-mosaic worm whose movement is completely coordinated. (B) Failure to inherit the array (indicated in black), owing to meiotic segregation in the mother, results in an uncoordinated (Unc) animal. (C) Loss of the array (indicated in black) in P1 produces a mosaic worm that has normal coordination. The focus of action of the unc-36 gene is therefore not in P1 or its descendants. (D) By contrast, loss of the array in AB, the sister of P1, produces a mosaic worm that is fully uncoordinated. (E) Loss of the array in ABp also produces the uncoordinated phenotype. (F) Loss of the array in ABa and P1 – an example of consecutive losses of the array – gives a coordinated worm. The focus of action of unc-36 is therefore among the descendants of ABp (Kenyon, 1986).

View larger version (32K): [in a new window]   Fig. 6. Using unc-36 to identify specific classes of mosaics. (A) The phenotypes of the progeny of unc-36(–); Ex100[unc-36(+) sur-5::gfp] hermaphrodites, as shown in Fig. 5. The upper two classes of progeny are the most frequent. The non-mutant (fully coordinated), non-mosaic worms have the same genotype as the mother and can be used to propagate the strain. The uncoordinated, non-mosaic progeny derive from zygotes that failed to inherit the array owing to meiotic segregation. (B) The segregation pattern for let-a, a hypothetical gene that is essential for viability (let – lethal when mutant). Homozygosity for a recessive mutation in the gene, designated as let-a(–), results in death soon after hatching. The segregants are from mothers with the genotype unc-36(–); let-a(–); Ex101[unc-36(+) let-a(+) sur-5::gfp]. Ex101 is an extrachromosomal array that has wild-type copies of the unc-36 gene, wild-type copies of the let-a gene and a marker gene that expresses GFP. The segregation of mosaic worms that are fully viable but uncoordinated indicates that the focus of the lethal mutation is not in ABp, because loss of the array in ABp, which affects coordination, has no effect on viability. (C) The segregation pattern for a different lethal gene, let-b. Mutation of this gene also causes death soon after hatching. The segregants derive from mothers with the genotype unc-36(–); let-b(–); Ex102[unc-36(+) let-b(+) sur-5::gfp]. The failure to see older larvae and adults that are uncoordinated indicates that the focus of let-b includes the same part of the cell lineage as the focus of unc-36.

View larger version (32K): [in a new window]   Fig. 7. The embryonic progenitors of several cell types in a hermaphrodite. D, E and P4 each gives rise to only one cell type, and C gives rise only to two neurons, 32 body muscles and nuclei that form part of hyp7, a large syncytium that forms most of the hypodermis, or skin, of the animal. Additional cell types derive from ABa, ABpl, ABpr and MS, but only those cell types discussed in the text are indicated. For a complete picture of the embryonic cell lineage of C. elegans, please go to http://www.wormatlas.org/Sulstonemblin_1983/results.html