Department of Genetics, Cell Biology and Development, University of Minnesota, 6-160 Jackson Hall, 321 Church Street, Minneapolis, MN 55455, USA
SUMMARY
The analysis of genetically mosaic worms, in which some cells carry a wild-type gene and others are homozygous mutant, can reveal where in the animal a gene acts to prevent the appearance of a mutant phenotype. In this primer article, we describe how Caenorhabditis elegans genetic mosaics are generated, identified and analyzed, and we discuss examples in which the analysis of mosaic worms has provided important information about the development of this organism.
Introduction
C. elegans derives its power for elucidating animal development
from its suitability for mutant analysis
(Brenner, 1974). A gene reveals
itself as important for development when its loss of function causes a
developmental phenotype. Genetic mosaics allow one to ask what the phenotypic
consequences are when some cells in an animal carry a wild-type gene and the
other cells are homozygous mutant. By analyzing a collection of genetic
mosaics, one can ask which cell or cells in an animal must carry the wild-type
gene to produce a wild-type phenotype. The responsible cell or cells are
referred to as the anatomical focus of the gene's action or function with
respect to the phenotype under study.
As the anatomical focus of a gene's function may well be in only a subset of cells in which the gene is expressed, knowing its complete expression pattern does not necessarily tell one where the gene normally functions with respect to a particular phenotypic effect. Furthermore, the anatomical focus of a gene's action may or may not be in cells that exhibit a mutant phenotype. A gene is said to act cell autonomously when the phenotype of a given cell is affected by the gene's mutation and when that phenotype in mosaic animals depends solely on whether or not the cell has the wild-type gene and is unaffected by the genotypes of other cells. A gene is said to act cell nonautonomously when a cell carrying the wild-type gene in a mosaic animal exhibits a mutant phenotype or when a homozygous mutant cell exhibits a wild-type phenotype. Cell non-autonomy implicates cell-cell interactions, and mosaic analysis can be used to identify the responsible interacting cells. Some genes are used repeatedly during development, and the loss of an early essential role of such a gene in a mutant can lead to developmental arrest that precludes analyzing the gene's role in later developmental events. This problem can be overcome through mosaic analysis, as mosaic animals may be able to complete the early stages of development to reveal a gene's later role.
How to generate C. elegans genetic mosaics
In nearly all mosaic analyses that have been carried out in C.
elegans, mosaic animals have been generated by the spontaneous mitotic
loss of an extrachromosomal genetic element that carries the wild-type allele
of a gene in an otherwise homozygous mutant background. When the
extrachromosomal element and the wild-type gene carried by it
is present in all cells, the worm exhibits a completely wild-type phenotype.
But when, as occurs at low frequency, the extrachromosomal element fails to be
transmitted to one of the daughters of a cell division, all the descendants of
that cell, a clone, will be homozygous mutant. Cells within a mosaic animal
that lack the wild-type gene can be independently identified as mutant if the
extrachromosomal element also carries a marker gene whose absence from a cell
affects the cell's appearance (i. e. acts cell autonomously). Because the
C. elegans cell lineage is invariant
(Sulston et al., 1983), a cell
autonomous marker can allow one to determine precisely where in a worm's
lineage the extrachromosomal element was lost, which helps to verify, by
reference to the known lineage, which cells in the mosaic animal are
homozygous mutant and which are not. The frequency of loss of an
extrachromosomal element per cell division is approximately the same
throughout development (Hedgecock and
Herman, 1995
; Yochem et al.,
1998
). However, extrachromosomal elements are occasionally lost at
two or more consecutive cell divisions to give a pattern of mosaicism that is
somewhat more complicated than that corresponding to a single clone of mutant
cells (Hedgecock and Herman,
1995
; Yochem et al.,
1998
). Possible misinterpretations caused by this effect can be
avoided by scoring for the presence or absence of the extrachromosomal element
in more than a few cells in the lineage of interest.
Two kinds of extrachromosomal element have been used for mosaic analysis:
free chromosome fragments (Herman,
1984) and extrachromosomal arrays
(Lackner et al., 1994
;
Miller et al., 1996
). C.
elegans chromosomes do not have localized centromeres
(Albertson and Thomson, 1982
)
they are said to be holocentric which means that a suitably
large fragment of any part of a chromosome, referred to as a free duplication,
can retain some centromeric function, and can behave as a fairly stable
mini-chromosome and be maintained in genetic stocks.
Extrachromosomal arrays, which are routinely generated to demonstrate
transformation rescue (or complementation) of a mutant phenotype
(Mello and Fire, 1995), are
now often used in preference to free duplications for mosaic analysis. When a
mixture of DNA that contains a wild-type gene and a gene that encodes a cell
autonomous marker is microinjected into the syncytial germline of an adult
hermaphrodite, a mini-chromosome composed of many copies of the DNAs tends to
form spontaneously (Stinchcomb et al.,
1985
) (Fig. 1). One
can then select animals that carry an array that shows good expression of both
the gene to be analyzed, as judged by the rescue of the mutant phenotype, and
the marker gene. Arrays are generally present in one copy per cell. Mitotic
loss leads to mosaic animals in which a clone of cells lacks the array and
therefore lacks both the marker gene and the wild-type gene under study
(Fig. 2). Mitotic losses of
free duplications or extrachromosomal arrays frequently involve
non-disjunction, in which one daughter cell receives no duplication and the
other daughter receives two copies
(Hedgecock and Herman, 1995
;
Yochem et al., 1998
). Some
arrays are lost at a frequency that may be inconveniently high for mosaic
analysis, say >1/50 per cell division, which means every animal will
contain many independent mutant clones. Preliminary work in selecting a
suitable array is therefore a good idea. A crucial assumption when using
extrachromosomal arrays for mosaic analysis is that the expression of the
wild-type gene on the array mimics its normal pattern of expression. [For more
information on this and other issues that are important in C. elegans
mosaic analysis, see also previous reviews
(Herman, 1995
;
Yochem et al., 2000
).]
|
|
To track the mitotic loss of an extrachromosomal element, one wants a cell
autonomous marker that: (1) can be readily scored in virtually all cells
preferably in nuclei of living animals, even when losses occur
very late in the lineage; and (2) has no other phenotypic effect that could
confound mosaic analysis. One excellent marker is a wild-type ncl-1
gene in a mutant ncl-1 background; nearly all homozygous mutant
ncl-1 cells exhibit enlarged nucleoli
(Fig. 3) with no other apparent
effects (Hedgecock and Herman,
1995; Frank and Roth,
1998
). Both free duplications and extrachromosomal arrays carrying
ncl-1(+) have been used for mosaic analysis. Alternatively, the gene
that encodes GFP (green fluorescent protein) when expressed under the control
of a strong promoter, as in sur-5::gfp, can be used to track the
inheritance of extrachromosomal arrays in living animals
(Fig. 4)
(Yochem et al., 1998
).
|
|
|
|
If one wants to delimit the focus of action of a gene that affects the whole animal when mutated, such as by causing uncoordinated movement, lengthened lifespan or inviability, then as many different types of mosaics should be collected as possible to see whether one can correlate the mutant phenotype with the absence of gene expression in a particular cell or set of cells. A good start in such cases is to look first for worms that contain large mutant clones, which have been generated by loss of the extrachromosomal element very early in the embryonic lineage. Loss at P1 (Fig. 7), for example, would give rise to an animal, which we denote as a P1() mosaic, in which 94 of its 95 adult body wall muscle cells would lack the wild-type gene, whereas almost all of its neurons would have it. Thus, P1() mosaics and their complement, AB() mosaics, can quickly distinguish whether a gene acts primarily in the development of muscles or neurons.
|
When a loss-of-function mutation causes a specific cellular abnormality, one can ask whether or not the gene behaves cell autonomously (Fig. 2). In this case, one wants to identify mosaics in which the cell of interest is genotypically different from its neighbors and other potentially interacting cells. If the gene of interest behaves cell non-autonomously, a careful mosaic analysis should be able to identify the interacting cells responsible for the effect.
Limitations of mosaic analysis in C. elegans
Two potential problems or complications associated with the analysis of mosaic worms should be noted. The first is called perdurance, which refers to the persistence of a gene product in a cell that lacks the gene. Thus, a wild-type product synthesized in an ancestral cell prior to gene loss might persist and be transmitted to descendant cells even though they did not inherit the gene. Because the effect of perdurance would be to weaken the expected mutant phenotype, mosaics that exhibit a fully expressed mutant phenotype seem to be free of perdurance. Perdurance is also unlikely to be a problem when the loss of a duplication or an array occurs early in the embryonic cell lineage or when the gene product is involved in terminal cellular differentiation and is synthesized only late in development. Very late losses affecting either ncl-1 or sur-5::gfp give clear mosaic phenotypes, indicating that there is very little perdurance of the wild-type gene product for either of these marker genes.
The second complication concerns limitations that occur as a consequence of
the nature of the worm cell lineage. This is generally not a problem when
investigating a cellular phenotype because mosaics can usually be identified
in which the cell under investigation is genotypically different from cells
that are candidates for interaction. However, the worm cell lineage can be
limiting when a gene's focus of action is diffuse and distributed among cells
of disparate lineage. The fully mutant phenotype may then only be apparent in
mosaic animals when the duplication or array is lost by a progenitor of all or
nearly all of the responsible cells, in which case it may be difficult to
pinpoint the responsible cell types. A particularly difficult tissue to
investigate in mosaic worms is the hypodermis, called hyp7, which forms the
skin for the main body of the animal. Hyp7 is a single syncytial cell that is
formed by the fusion of many mononucleate cells that descend from both AB and
P1, the daughters of the very first embryonic cleavage
(Sulston et al., 1983);
therefore, no mosaic animal can contain a single, completely mutant hyp7 clone
(Fig. 7). Mosaic analyses have,
nonetheless, been used to implicate hyp7 as the focus of action of several
genes, as we illustrate below.
Insights into worm development from mosaic analyses
More than 70 genes affecting worm development have been studied in genetic mosaics. Rather than attempt a comprehensive review of those genes here, we will discuss a few examples to illustrate how mosaic analysis has been used to elucidate certain mechanisms of development.
The foci of action of several genes implicated in cell-to-cell signaling
during development have been determined by mosaic analysis. Two classic
examples are glp-1 and lin-12, which encode members of the
Notch family. The proteins encoded by these genes contain copies of a motif
that resembles epidermal growth factor (EGF). Because EGF is an extracellular
factor that affects other cells, and because it is cleaved from a
membrane-bound precursor, it was natural to consider LIN-12 (the protein
encoded by lin-12) and GLP-1 as possible sources of extracellular
signal. However, mosaic analysis indicated that both glp-1 and
lin-12 act cell autonomously in the cells whose fates require their
function (Austin and Kimble,
1987; Seydoux and Greenwald,
1989
), as would be expected if the genes encode receptors and not
precursors for secreted signals.
By contrast, lin-44 encodes a Wnt signal that affects the polarity
of a cell in the worm's tail, called T, and that was shown by mosaic analysis
to act cell non-autonomously not in T but most likely in tail
hypodermal cells (Herman et al.,
1995), the cells believed to secrete the Wnt signal. Another gene
that has been shown by mosaic analysis to act cell nonautonomously is
her-1 (Hunter and Wood,
1992
), a gene that is essential for determining male sexual fate
(Hodgkin 1980
). This result
was consistent with the later molecular characterization of her-1
(Perry et al., 1993
), which
suggested that HER-1 protein is secreted and acts as a signaling molecule in
the worm sex determination pathway.
Loss-of-function mutations in unc-5 lead to defects in the
dorsal-ward guidance of pioneering axons and migrating cells during worm
development. Mosaic analysis has shown that unc-5 acts cell
autonomously (Leung-Hagesteijn et al.,
1992); for example, a migrating cell was found to be defective in
its dorsalward migration if, and only if, it lacked unc-5(+). This
result, together with the molecular characterization of unc-5
(Leung-Hagesteijn et al.,
1992
), led to the proposal that UNC-5 is a transmembrane receptor
that promotes the movement of migrating axons and cells away from a high
ventral concentration of the extracellular matrix protein UNC-6
(Wadsworth et al., 1996
).
UNC-6 is a member of the netrin family of proteins
(Ishii et al., 1992
), which
have been shown to affect axon guidance in vertebrate embryos
(Serafini et al., 1994
). The
vab-8 gene has also been shown by mosaic analysis to affect axon
guidance cell autonomously; in this case, Wolf et al. were able to conclude
that vab-8 must be expressed in certain neurons despite the fact that
immunofluorescent staining was only able to detect VAB-8 expression in body
muscle (Wolf et al.,
1998
).
The example of let-23, which encodes an EGF tyrosine kinase
receptor (Aroian et al., 1990),
is also instructive. Activation of LET-23 by an EGF-like ligand occurs
repeatedly during C. elegans development to trigger diverse
developmental events. One of these events is the induction of vulval
development, in which three vulval precursor cells generate 22 cellular
descendants that form the vulva. In the absence of an EGF signal from the
gonadal anchor cell, none of the vulval precursor cells contributes to vulval
development, and no vulva is formed. Because vulval development is not
essential to a worm's survival, it can be analyzed in let-23 mosaics,
even though let-23 is an essential gene. Such an analysis has shown
that normal vulval development can occur when only one vulval precursor cell
contains let-23(+) (Simske and
Kim, 1995
; Koga and Ohshima,
1995
). According to the picture that emerges from these studies,
the vulval precursor cell closest to the signaling anchor cell is activated by
the reception of the EGF signal and then induces its neighboring vulval
precursor cells on each side (through LIN-12-mediated signaling) to embark on
vulval development.
Downstream of LET-23 in the vulval signal-transduction pathway is the small
G protein RAS, which is encoded by let-60
(Beitel et al., 1990;
Han and Sternberg, 1990
). The
lethality of a let-60 loss-of-function mutation has been traced by
mosaic analysis to a single cell, the excretory duct cell
(Yochem et al., 1997
). An
unexpected mosaic phenotype was encountered in this study: when descendants of
ABpl (Fig. 7), which normally
includes the excretory duct cell, were mutant for let-60, a
let-60(+) cell assumed the duct cell fate.
Another example in which mosaicism has given an unexpected phenotype
involves a natural ambiguity in the cell lineage between two cell fates.
Either one of two cells defined by the lineage randomly assumes the anchor
cell (AC) fate, with the other cell becoming VU
(Kimble, 1981). The VU fate is
specified by an AC-to-VU signal and requires lin-12 function: in a
lin-12 loss-of-function mutant, both cells become AC, and in a
lin-12 gain-of-function mutant, both become VU
(Greenwald et al., 1983
;
Seydoux and Greenwald, 1989
).
Mosaic analysis has shown, as expected, that lin-12 function is
required in VU and not in AC (Seydoux and
Greenwald, 1989
). But the collection of mosaics in which one of
the two AC/VU cells was homozygous for a lin-12 loss-of-function
mutation and the other was lin-12(+) gave an unexpected result: in
every animal, the mutant cell became AC and the wild-type cell became VU.
Thus, loss of lin-12 in one cell had a non-autonomous effect that
forced the other cell to become VU. This result is explained by a model in
which the two AC/VU cells signal to each other until the greater activation of
LIN-12 receptor in one (VU) leads to the loss of LIN-12 activity in the other
(AC) (Seydoux and Greenwald,
1989
).
Mosaic analysis also revealed an unexpected anatomical focus for the action
of unc-52, which encodes the worm homolog of mammalian perlecan
(Rogalski et al., 1993). Null
unc-52 mutants are unable to assemble a myofilament lattice and
undergo embryonic arrest typical of that caused by defective body muscle
(Williams and Waterston,
1994
). UNC-52 localizes between muscle and hypodermis, and it had
been concluded that the protein is synthesized by muscle. But mosaic analysis
showed that unc-52 does not function in muscle, because
P1() mosaics (Fig.
7) were viable and fertile
(Spike et al., 2002
). It was
therefore concluded that UNC-52 is synthesized and secreted by hypodermis.
The anatomical foci of other whole-animal, developmental phenotypes have
been deduced from mosaic analysis. For example, analysis of mosaics of
sma-3, which encodes a component of a TGFß signaling pathway
that regulates body size, lent weight to the conclusion that sma-3
acts in the hypodermis to control body size
(Wang et al., 2002). The
daf-2 gene, which encodes a homolog of insulin-like growth factor I
receptors (Kimura et al.,
1997
), affects two whole-animal phenotypes: lifespan and the
decision of young larvae to enter a state of diapause (the dauer stage) rather
than progressing to adulthood. A partial loss-of-function mutation in
daf-2 leads to extended lifespan
(Kenyon et al., 1993
), whereas
a stronger daf-2 mutation leads to the inappropriate formation of
dauer larvae (Riddle et al.,
1981
). The characterization of many daf-2 mosaics with
different mutant clones has led to the conclusion that the wild-type DAF-2
receptor acts diffusely in multiple cell lineages to regulate the production
or activity of a secondary signal, which then affects lifespan and dauer
formation by affecting the tissues of the whole animal
(Apfeld and Kenyon, 1998
).
Final considerations
Mosaic analysis in C. elegans requires a knowledge of worm anatomy and development, both to pinpoint where in the cell lineage a genetically-marked extrachromosomal element has been lost and to analyze the cellular phenotypes of mosaic animals. This can seem formidable at first, but knowledge of this organism's anatomy can be one of the joys of working with C. elegans. (To begin acquiring such knowledge, we recommend you go to http://www.wormatlas.org/index.htm) One prospect for the future is additional computer-assisted tutorials on worm anatomy. Another future development that would assist the field would be the creation of improved markers to allow for the even more rapid examination of thousands of worms at low magnification for specific mosaics.
Another method for determining the anatomical focus of a gene's action is
to introduce transgenic copies of the wild-type gene under the control of
cell- or tissue-specific promoters into otherwise homozygous mutant animals.
The rescue of a mutant defect by a particular transgene indicates that the
gene's expression in the indicated cell type can provide the required
function. However, one cannot be certain that such promoters are absolutely
specific in their effects, and one must make a new construct when testing each
promoter with a gene of interest. In addition, rescue by expression from the
transgene in this case (generally overexpression because of the multiple gene
copies) does not prove that the wild-type gene normally acts in the same cell
type or tissue. But this approach, especially in combination with mosaic
analysis, can help to build a strong case (e.g.
Zhen and Jin, 1999;
Inoue and Thomas, 2000
;
Zhen et al., 2000
;
Wang et al., 2002
). The
inclusion of mosaic analysis provides greater confidence that one has
identified the cell or tissue that requires the activity of a gene, and we
expect future mosaic analyses to yield more unexpected insights into the
mechanisms of C. elegans development.
ACKNOWLEDGMENTS
We thank Leslie Bell for comments on the manuscript. Our work is supported by a grant from the NIH.
REFERENCES
Albertson, D. G. and Thomson, J. N. (1982). The kinetochores of Caenorhabditis elegans. Chromosoma 86,409 -428.[Medline]
Apfeld, J. and Kenyon, C. (1998). Cell nonautonomy of C. elegans DAF-2 function in the regulation of diapause and life span. Cell 95,199 -210.[Medline]
Aroian, R. V., Koga, M., Mendel, J. E., Ohshima, Y. and Sternberg, P. W. (1990). The let-23 gene necessary for Caenorhabditis elegans vulval induction encodes a tyrosine kinase of the EGF receptor subfamily. Nature 348,693 -699.[CrossRef][Medline]
Austin, J. and Kimble, J. (1987). glp-1 is required in the germ line for regulation of the decision between mitosis and meiosis in Caenorhabditis elegans.Cell 51,589 -599.[Medline]
Beitel, G. J., Clark, S. G. and Horvitz, H. R. (1990). Caenorhabditis elegans ras gene let-60 acts as a switch in the pathway of vulval induction. Nature 348,503 -509.[CrossRef][Medline]
Brenner, S. (1974). The genetics of
Caenorhabditis elegans. Genetics
77, 71-94.
Chen, L., Krause, M., Sepanski, M. and Fire, A.
(1994). The Caenorhabditis elegans MYOD homologue HLH-1
is essential for proper muscle function and complete morphogenesis.
Development 120,1631
-1641.
Frank, D. J. and Roth, M. B. (1998).
ncl-1 is required for the regulation of cell size and ribosomal RNA
synthesis in C. elegans. J. Cell Biol.
140,1321
-1329.
Greenwald, I. S., Sternberg, P. W. and Horvitz, H. R. (1983). The lin-12 locus specifies cell fates in Caenorhabditis elegans. Cell 34,435 -444.[Medline]
Han, M. and Sternberg, P. W. (1990). let-60, a gene that specifies cell fates during C. elegans vulval induction, encodes a ras protein. Cell 63,921 -931.[Medline]
Hedgecock, E. M. and Herman, R. K. (1995). The
ncl-1 gene and genetic mosaics of Caenorhabditis elegans.Genetics 141,989
-1006.
Herman, M. A., Vassilieva, L. L., Horvitz, H. R., Shaw, J. E. and Herman, R. K. (1995). The C. elegans gene lin-44, which controls the polarity of certain asymmetric cell divisions, encodes a Wnt protein and acts cell nonautonomously. Cell 83,101 -110.[Medline]
Herman, R. K. (1984). Analysis of genetic
mosaics of the nematode Caenorhabditis elegans.Genetics 106,165
-180.
Herman, R. K. (1995). Mosaic analysis. Methods Cell Biol. 48,123 -146.[Medline]
Hodgkin, J. (1980). More sex-determination
mutants of Caenorhabditis elegans. Genetics
96,649
-664.
Hunter, C. P. and Wood, W. B. (1992). Evidence from mosaic analysis of the masculinizing gene her-1 for cell interactions in C. elegans sex determination. Nature 355,551 -555.[CrossRef][Medline]
Inoue, T. and Thomas, J. H. (2000). Targets of TGF-ß signaling in Caenorhabditis elegans dauer formation. Dev. Biol. 217,192 -204.[CrossRef][Medline]
Ishii, N., Wadsworth, W. G., Stern, B. D., Culotti, J. G. and Hedgecock, E. M. (1992). UNC-6, a laminin-related protein, guides cell and pioneer axon migrations in C. elegans.Neuron 9,873 -881.[Medline]
Kenyon, C. (1986). A gene involved in the development of the posterior body region of C. elegans.Cell 46,477 -487.[Medline]
Kenyon, C., Chang, J., Gensch, E., Rudner, A. and Tabtiang, R. (1993). A C. elegans mutant that lives twice as long as wild type. Nature 366,461 -464.[CrossRef][Medline]
Kimble, J. (1981). Alteration in cell lineage following laser ablation of cells in the somatic gonad of Caenorhabditis elegans. Dev. Biol. 87,286 -300.[Medline]
Kimura, K. D., Tissenbaum, H. A., Liu, Y. and Ruvkun, G.
(1997). daf-2, an insulin receptor-like gene that
regulates longevity and diapause in Caenorhabditis elegans.Science 277,942
-946.
Koga, M. and Ohshima, Y. (1995). Mosaic
analysis of the let-23 gene function in vulval induction of
Caenorhabditis elegans. Development
121,2655
-2666.
Lackner, M. R., Kornfeld, K., Miller, L. M., Horvitz, H. R. and Kim, S. K. (1994). A MAP kinase homologue, mpk-1, is involved in ras mediated induction of vulval cell fates in Caenorhabditis elegans. Genes Dev. 8, 160-173.[Abstract]
Leung-Hagesteijn, C., Spence, A. M., Stern, B. D., Zhou, Y., Su, M.-W., Hedgecock, E. M. and Culotti, J. G. (1992). UNC-5, a transmembrane protein with immunoglobulin and thrombospondin type 1 domains, guides cell and pioneer axon migrations in C. elegans.Cell 71,289 -299.[Medline]
Mello, C. C. and Fire, A. (1995). DNA transformation. Methods Cell Biol. 48,452 -482.
Miller, L. M., Waring, D. A. and Kim, S. K.
(1996). Mosaic analysis using a ncl-1(+)
extrachromosomal array reveals that lin-31 acts in the Pn.p cells
during Caenorhabditis elegans vulval development.
Genetics 143,1181
-1191.
Perry, M. D., Li, W., Trent, C., Robertson, A., Fire, A., Hageman, J. M. and Wood, W. B. (1993). Molecular characterization of the her-1 gene suggests a direct role in cell signaling during Caenorhabditis elegans sex determination. Genes Dev. 7,216 -228.[Abstract]
Riddle, D. L., Swanson, M. M. and Albert, P. S. (1981). Interacting genes in nematode dauer larva formation. Nature 290,668 -671.[Medline]
Rogalski, T. M., Williams, B. D., Mullen, G. P. and Moerman, D. G. (1993). Products of the unc-52 gene in Caenorhabditis elegans are homologous to the core protein of the mammalian basement membrane heparan sulfate proteoglycan. Genes Dev. 7,1471 -1484.[Abstract]
Serafini, T., Kennedy, T. E., Galko, M. J., Mirzayan, C., Jessell, T. M. and Tessier-Lavigne, M. (1994). The netrins define a family of axon outgrowthpromoting proteins homologous to C. elegans UNC-6. Cell 78,409 -424.[Medline]
Seydoux, G. and Greenwald, I. (1989). Cell autonomy of lin-12 function in a cell fate decision in C. elegans. Cell 57,1237 -1245.[Medline]
Simske, J. S. and Kim, S. K. (1995). Sequential signalling during Caenorhabditis elegans vulval induction. Nature 375,142 -146.[CrossRef][Medline]
Spike, C. A., Davies, A. G., Shaw, J. E. and Herman, R. K.
(2002). MEC-8 regulates alternative splicing of unc-52
transcripts in C. elegans hypodermal cells.
Development 129,4999
-5008.
Stinchcomb, D. T., Shaw, J. E., Carr, S. H. and Hirsh, D. (1985). Extrachromosomal DNA transformation of Caenorhabditis elegans. Mol. Cell. Biol. 5,3484 -3496.[Medline]
Sulston, J. E., Schierenberg, E., White, J. G. and Thomson, J. N. (1983). The embryonic lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100,64 -119.[Medline]
Wadsworth, W. G., Bhatt, H. and Hedgecock, E. M. (1996). Neuroglia and pioneer neurons express UNC-6 to provide global and local netrin cues for guiding migrations in C. elegans. Neuron 16,35 -46.[Medline]
Wang, J., Tokarz, R. and Savage-Dunn, C.
(2002). The expression of TGFß signal transducers in the
hypodermis regulates body size in C. elegans.Development 129,4989
-4998.
Williams, B. D. and Waterston, R. H. (1994). Genes critical for muscle development and function in Caenorhabditis elegans identified through lethal mutations. J. Cell Biol. 124,475 -490.[Abstract]
Wolf, F. W., Hung, M.-S., Wightman, B., Way, J. and Garriga, G. (1998). vab-8 is a key regulator of posteriorly directed migrations in C. elegans and encodes a novel protein with kinesin motor similarity. Neuron 20,655 -666.[Medline]
Yochem, J., Sundaram, M. and Han, M. (1997). Ras is required for a limited number of cell fates and not for general proliferation in Caenorhabditis elegans. Mol. Cell. Biol. 17,2716 -2722.[Abstract]
Yochem, J., Gu, T. and Han, M. (1998). A new
marker for mosaic analysis in Caenorhabditis elegans indicates a
fusion between hyp6 and hyp7, two major components of the hypodermis.
Genetics 149,1323
-1334.
Yochem, J., Sundaram, M. and Bucher, E. A. (2000). Mosaic analysis in Caenorhabditis elegans.Methods Mol. Biol. 135,447 -462.[Medline]
Zhen, M. and Jin, Y. (1999). The liprin protein SYD-2 regulates the differentiation of presynaptic termini in C. elegans.Nature 401,371 -375.[CrossRef][Medline]
Zhen, M., Huang, X., Bamber, B. and Jin, Y. (2000). Regulation of presynaptic terminal organization by C. elegans RPM-1, a putative guanine nucleotide exchanger with a RING-H2 finger domain. Neuron 26,331 -343.[Medline]
Related articles in Development: