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Lab PagePostdoctoral Researcher
1996 - Ph.D., Department of Developmental Biology, Stanford University.
1988 - B.A., summa cum laude, Microbiology, University of Minnesota.
Address: Rm. 329 Zoology Research Bldg.; 1117 W. Johnson St., Madison, WI 53706
Phone: (608) 265-2520
Email: jssimske@facstaff.wisc.edu
Introduction
I am interested in the molecules that regulate the behavior of epithelial cells as they migrate, change shape and adhesiveness, and ultimately generate three-dimensional shape during tissue morphogenesis. One paradigm for tissue morphogenesis is ventral enclosure of the embryo by the hypodermis or epidermis. Ventral enclosure involves the wrapping of the six rows of epidermal cells (two rows each of dorsal, seam and ventral epidermis) around the circumference of the embryo and the subsequent formation of adhesive junctions along the ventral midline. Ventral enclosure is a valuable system in which to study epithelial morphogenesis since enclosure is rapid and the changes in cell shape and movement can be monitored in four dimensions either by DIC microscopy or by multiphoton excitation of fluorescent junctional markers (Mohler et al., 1998). Significantly, genes encoding components of the cadherin cell-adhesion complex are required for enclosure, indicating general mechanisms regulating cell adhesion will be uncovered through a genetic and molecular analysis of enclosure (Costa et al., 1998).
Genetic Screen For enclosure Defective Mutants
I have conducted a general screen for enclosure defective mutants
which allows for the recovery of zygotic enclosure defective mutants
(Zens), maternal-effect lethal mutations (Mels), and incompletely
penetrant, variably abnormal homozygous viable mutations (Vabs).
The screen was carried out in a lin-7(e1449)II; jcIs1(jam-1::GFP)IV
strain so that cellular junctions could be visualized in arresting
or arrested embryos. JAM-1 is junction associated molecule 1,
the antigen recognized by the monoclonal antibody MH27. The JAM-1-GFP
fusion protein is localized at the adherens junctions of all
C. elegans epithelia. I screened through approximately 9,000
haploid genomes and recovered 15 alleles: one Vab, 4 apparent
Zens, 9 Mels and one semi-dominant Zen allele. I have mapped one
allele to LGI, two alleles to LGII, five alleles to LGIV, and
four to LGV. Each allele represents a distinct complementation
group; therefore, the screen is far from saturation.
I have identified several characteristic phenotypes while screening
that represent the failure of enclosure at successive stages of
development. The most severe phenotype is full retraction of the
epidermis to a small cap at its original position on the dorsal
posterior region of the embryo. This phenotype represents complete
failure of enclosure. Less severe are partial enclosures, during
which anterior (leading cell) or posterior (pocket cell) regions
remain unenclosed, resulting in the extrusion of embryonic contents
through head and tail ruptures, respectively. Finally, embryos
may enclose completely, but irregularly, resulting in body shape
defects during elongation. Typically, mutations have variable
expressivity and can display more than one of the above arrest
phenotypes.
A major goal of the screen is to identify mutations that specifically
affect enclosure. Mutations that cause broad changes in cell lineage
or cell fate, or mutations that affect the organization of other
tissues may indirectly disrupt enclosure. For example, mutations
in vab-1 and vab-2, which are required for the organization
of neurons underlying the epidermis, disrupt enclosure (George
et al., 1998). To rule out such secondary effects, mutants
are stained with a panel of tissue specific antibodies. For most
mutations we have confirmed the presence of organized muscle and
have observed the normal number and position of epidermal, gut
and pharyngeal cells. Further staining experiments will help determine
which tissues may be aberrant in these mutants and which mutations
specifically disrupt enclosure.
So far I have been able to draw the following conclusions. First,
I have isolated mutations that primarily affect ventral enclosure.
Second, ventral enclosure appears to have a significant maternal
requirement. This was unexpected since existing mutations affecting
enclosure have primarily a zygotic focus. Surprisingly, we recovered
no mutants with a true Zen phenotype. All zygotic mutations identified
in the screen enclosed successfully and thus are classified as
Zels. Many existing Zel mutations primarily affect muscle organization,
position, or function and have only a secondary effect on the
epidermis and are therefore of less immediate interest (Gatewood
and Bucher, 1997). Surprisingly, of the 9 Mel mutations, 3
are temperature sensitive and two are cold-sensitive. Since both
ts and cs mutations were isolated, conditional screens for Mels
of each type could be carried out. Certainly it should be possible
to screen to saturation for Mels that affect enclosure.
Some mutants have developmental defects in processes other than
enclosure. These defects may help to determine the normal function
of these genes. In collaboration with Christina
Thomas, a graduate student in the Hardin lab, we have shown
that in jc7 animals some muscle cells fail to migrate to
their normal position, suggesting that jc7 animals may
have a general cell migration defect.
Some alleles may reveal specific behaviors of different epidermal
cells during enclosure. In jc1 and jc14 mutants,
enclosure of the anterior leading cells is specifically disrupted,
resulting in a phenotype that is reminiscent of the defects in
mutants for the C. elegans cadherin homologue, hmr-1.
Another mutant, jc10, primarily affects enclosure of the
posterior epidermal (pocket) cells. These results suggest there
may be distinct molecular mechanisms underlying the enclosure
of anterior and posterior ventral epidermis.
I am close to cloning two genes. jc1 has been mapped between
egl-20 and egl-38 on LGIVR (a region consisting
of less than 0.16 genetic map units and 300kb of DNA) and, along
with Christina Thomas, jc5
has been mapped between dif-1 and nhr-8 on LGIVR
(a region consisting of less than 0.1 genetic map units and 100kb
of DNA). With the help of undergraduate Ryan
Smith, a third gene, jc11, has been cloned, and its
location has been narrowed to a region of 27kb on LGIIR: there
are three transcription units within the rescuing region.
I have developed an actin::gfp fusion gene under the control
of the jam-1 promoter which allows for the expression and
monitoring of actin dynamics in the epidermis of the enclosing
embryo using multiphoton microscopy (the actin::gfp fusion
gene is a gift from the Seydoux laboratory). Thus it will be possible
to analyze actin dynamics within ventral cell filopodia in mutants
arising from the screen.
Analysis of the Cell Junction Protein VAB-9
A second project involves the characterization of pre-existing
mutations that affect morphogenesis through the alteration of
specific cell behaviors (e.g. cell motility and cell shape changes)
rather than those that alter cell lineage or pattern. One such
mutation is vab-9. vab-9 mutants have defects in
tail, body and vulval morphology but no apparent alterations in
cell lineage. vab-9 has been cloned by cosmid rescue, cDNAs
have been recovered by RT-PCR, and the molecular lesions in e1744
and ju6 (provided by A. Chisholm) have been identified.
A full length vab-9 cDNA expressed under the control of
a heat shock promoter rescues vab-9(e1744) phenotypes.
VAB-9 is a 221aa protein that bears no significant homology to
any known gene in the database but has four strongly predicted
transmembrane spanning domains. A rescuing VAB-9-GFP protein localizes
to cellular junctions with a pattern that bears a striking resemblance
to that of JAM-1, suggesting that VAB-9 is an integral membrane
junctional protein. VAB-9-GFP does not appear to be expressed
in the intestine and expression in the uterus and the male tail
is currently being characterized.
Based on JAM-1 expression, vab-9 larvae with body shape
defects display uneven elongation. In wild-type, seam cells elongate
along the anterior-posterior axis and appear rectangular in shape.
The dramatic change in shape of the two lateral rows of seam cells
is thought to contribute significantly to elongation. The mechanical
force for elongation is generated by the contraction of circumferential
actin filaments and microtubules within the seam cells. In vab-9
animals, the situation is dramatically different: the seam cells
often elongate along the dorsal-ventral axis or display a circular
shape. Where such defects are observed, there is a direct correlation
between seam cell shape and body shape abnormalities. Thus seam
cells in vab-9 animals are able to elongate, but the direction
of seam cell elongation is unregulated. Phalloidin staining of
filamentous actin reveals large gaps in the actin network, presumably
corresponding to regions where seam cells fail to change shape.
We propose that VAB-9 is involved in cell-cell communication during
elongation, and that this communication is required for the smooth,
coordinated elongation of the larva. Since the predicted topology
of VAB-9 is similar to connexins or the tight junction component
occludin, it is possible that VAB-9 may couple hypodermal cells
chemically or adhesively. I hope to address these hypotheses by
expressing VAB-9 in heterologous cultured cells and scoring for
changes in adhesion and conductance in expressing cells.
Interaction Between vab-9 and jam-1
VAB-9 and JAM-1 have overlapping subcellular expression patterns,
suggesting these proteins may directly interact. In addition,
both proteins are expressed in the enclosing embryo, yet inactivation
of these loci by either loss-of function mutations or by RNA mediated
interference (RNA(i)) has no effect on enclosure. jam-1
mutants arrest after elongating to the two-fold or three-fold
stage (two or three times the length of the unelongated embryo);
vab-9 mutants hatch and elongate abnormally. To test whether
these genes may be involved in redundant pathways during enclosure,
I constructed vab-9; jam-1 double mutants or carried out
jam-1 RNA(i) in vab-9 mutants, along with graduate
student Mathias Koeppen. Both approaches
gave the same result: embryos arrest at two-fold and often rupture
at regions of cell-cell contact. Thus the phenotype of the double
mutant is more severe than either single mutant, suggesting (at
least) two models for the normal role of these proteins. In the
first model, VAB-9 may interact with a JAM-1-like molecule (X)
and JAM-1 with a VAB-9-like molecule (Y) in two distinct, functionally
similar protein complexes to mediate cell adhesion during enclosure
and elongation. In the absence of VAB-9 or JAM-1, adhesion mediated
by the remaining complex is sufficient for the embryo to advance
at least to three-fold without rupture. If both adhesion systems
are compromised by inactivation of vab-9 and jam-1,
arrest and rupture occur earlier. Residual adhesive activity between
X and Y may be sufficient for adhesion in the enclosing embryo.
In support of this model, overexpression of VAB-9 under control
of a heat shock promoter leads to arrest during enclosure. This
results suggests that excess VAB-9 may sequester JAM-1 and X (and
perhaps other JAM-1-like molecules as well) away from functional
adhesion sites, resulting in defects in epidermal cell adhesion
or communication during elongation. In addition, I found that
overexpression of JAM-1 enhances the elongation defect of vab-9
mutants. Normally vab-9 animals survive to the adult
stage. In contrast, 40 percent of vab-9 animals overexpressing
JAM-1 arrest as grossly misshapen L1 larvae. This result suggests
that relative levels of VAB-9 and JAM-1 are critical for normal
development.
A second, less elaborate model predicts that VAB-9 and JAM-1 are
not components of similar protein complexes. Instead, the synergistic
phenotype of vab-9; jam-1 double mutants may result simply
from an additive weakening of general cell adhesion through defects
in two distinct adhesion systems. I plan to test for direct protein-protein
interactions between VAB-9 and JAM-1.
Additional Materials
Images and additional information about experiments described
here may be found at:
http://worms.zoology.wisc.edu/labwebpage/Simske.html
References
Costa, M., Raich, W., Agbunag,
C., Leung, B., Hardin, J., and Priess, J. R. (1998). A putative
catenin-cadherin system mediates morphogenesis of the Caenorhabditis
elegans embryo. J Cell Biol 141, 297-308. [PDF]
Gatewood, B. K., and Bucher, E. A. (1997). The mup-4 locus in Caenorhabditis elegans is essential for hypodermal integrity, organismal morphogenesis and embryonic body wall muscle position. Genetics 146, 165-83.
George, S. E., Simokat, K., Hardin, J., and Chisholm, A. D. (1998). The VAB-1 Eph receptor tyrosine kinase functions in neural and epithelial morphogenesis in C. elegans. Cell 92, 633-43. [PDF]
Mohler, W. A., Simske, J. S., Williams-Masson, E. M., Hardin, J. D., and White, J. G. (1998). Dynamics and ultrastructure of developmental cell fusions in the Caenorhabditis elegans hypodermis. Curr Biol 8, 1087-90.
Hardin, J., Raich, W.B. and Simske, J.S. (1999) Morphogenesis at single-cell resolution: studying changes in the shape of the embryo in the tradition of Hörstadius. In press.
Simske, J. S. and Kim, S.K. (1998) Pattern Formation by Sequential Signaling During C. elegans Vulval Induction. In "Hormones and Growth Factors in Development and Neoplasia" R.B. Dickson and D.S. Salomon, Editors, 3-17.
Mohler, W.A., Simske, J.S., Williams-Masson, E.M., Hardin, J.D., White, J.G. (1998) Dynamics and ultrastructure of developmental cell fusions in the Caenorhabditis elegans hypodermis. Curr Biol. 8, 1087-90
Eisenmann, D. M., Maloof, J. N., Simske, J. S., Kenyon, C. and Kim, S. K. (1998). The beta-catenin homolog BAR-1 and LET-60 Ras coordinately regulate the Hox gene lin-39 during Caenorhabditis elegans vulval development. Development 125, 3667-3680
Simske, J. S., Kaech, S. M., Harp, S. A., and Kim, S. K. (1996). LET-23 receptor localization by the cell junction protein LIN-7 during C. elegans vulval development. Cell,. 85, 195-204.
Simske, J. S. and Kim, S. K. (1995). Sequential signalling during Caenorhabditis elegans vulval induction. Nature 375, 142-146.
Charles, C.H., Yoon, J.K., Simske, J.S., & Lau, L.F. (1992). Genomic structure, cDNA sequence, and expression of g1y96, a growth factor inducible immediate-early gene encoding a short-lived glycosylated protein. Oncogene, 8, 797 - 801.
Charles, C.H., Simske, J.S., O'Brien, T.P. & Lau, L.F. (1990). Pip92: A Short-Lived, Growth Factor-Inducible Protein in BALB/c 3T3 and PCl2 Cells. Mol. Cell. Biol., 10, 6769 - 6774
Simske, J.S. & Scherer, S. (1989). Pulsed-field gel electrophoresis of circular DNA. Nucleic Acids Res., 17, 4359 - 4365.
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