Download Bringing Classical Embryology to C. elegans Gastrulation

Developmental Cell
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infected wild-type flies, these animals are still able to
effectively kill the infecting pathogen. Thus, it is unclear
what is actually causing these NO-inhibited animals to
die. Perhaps some bacteria survive, in a protected niche,
and then go one to cause lethality during pupation. Another possibility is that infection (or the immune response) might cause some irreparable damage, preventing further development. Curiously, if the Toll
pathway is blocked (by mutation) and NO production is
inhibited, naturally infected larvae are no longer able to
kill the infecting bacteria. These results suggest that the
Toll and IMD pathways function redundantly to maximize bactericidal activity in the gut.
The role of NO in Toll signaling was also examined.
NO inhibitors have only a mild inhibitory effect on the
induction of Drosomycin, a target of the Toll pathway.
On the other hand, NO donors can activate expression
of Drosomycin, independent of the IMD pathway. This
argues that NO can activate the Toll pathway, but the
physiological significance of this activation is not yet
clear.
The discovery that immune activation in Drosophila
requires NO raises a number of interesting questions.
How does infection lead to NO production? In the insect
immune response, does NO function by the activation
of sGC, as it does in other mammalian and Drosophila
systems? Or is immune signaling mediated by nitrosylation of other proteins? Lastly, it will be interesting to
Bringing Classical Embryology
to C. elegans Gastrulation
In a recent paper, Lee and Goldstein develop an explant assay that recapitulates key aspects of gastrulation in C. elegans and permits classical embryological
manipulations. The resulting detailed analysis of cell
behavior will ultimately extend to broader issues, such
as, whether morphogenesis can be described as the
sum of single-cell events or if unique phenomena
emerge at the multicellular level.
For an understanding of multicellular rearrangements,
such as gastrulation, neurulation, and organogenesis, it
is important to understand the behaviors of individual
cells. The dissection of complex morphogenetic processes has been facilitated by the development of simplified assays in which cell behavior can be directly
observed and manipulated (e.g., Keller et al., 1991). A
challenge now is to integrate these approaches into
traditionally genetic model systems, such as Drosophila
and C. elegans. In a recent paper published in Development, Lee and Goldstein extend the use of cultured
explant assays in C. elegans to dissect the dynamics of
cell movements during gastrulation (Lee and Goldstein,
2003). This approach will make it possible to assess
how individual mechanisms, such as cell migration, contractility, and adhesion, work together to drive multicellular reorganization.
learn whether, in addition to its role in signaling, NO
also functions as a microbicidal agent in Drosophila
macrophages. Given the extensive genetic analysis of
NO and immune signaling in Drosophila, we can anticipate that we will soon know more about NO.
Neal Silverman
Department of Medicine
Division of Infectious Disease and Immunology
University of Massachusetts Medical School
364 Plantation Road
Worcester, Massachusetts 01605
Selected Reading
Basset, A., Khush, R.S., Braun, A., Gardan, L., Boccard, F., Hoffmann, J.A., and Lemaitre, B. (2000). Proc. Natl. Acad. Sci. USA 97,
3376–3381.
Bogdan, C. (2001). Nat. Immunol. 2, 907–916.
Foley, E., and O’Farrell, P.H. (2003). Genes Dev. 17, 115–125.
Gibbs, S.M., Becker, A., Hardy, R.W., and Truman, J.W. (2001). J.
Neurosci. 21, 7705–7714.
Lemaitre, B., Reichhart, J.M., and Hoffmann, J.A. (1997). Proc. Natl.
Acad. Sci. USA 94, 14614–14619.
Mannick, J.B., and Schonhoff, C.M. (2002). Arch. Biochem. Biophys.
408, 1–6.
Silverman, N., and Maniatis, T. (2001). Genes Dev. 15, 2321–2342.
Wingrove, J.A., and O’Farrell, P.H. (1999). Cell 98, 105–114.
The onset of C. elegans gastrulation is marked by the
ingression of two gut precursor cells into the interior of
the embryo, leaving a space at the surface that is filled
in by neighboring cells (see Figure, panel A). By successfully culturing embryos after removal of the vitelline
membrane, Lee and Goldstein demonstrate that the gut
precursors, Ea and Ep, can still rearrange normally with
respect to two cells, P4 and MSxx, that come to overly
them. Remarkably, this shift in position still occurs in
ablated embryos where more than half of the total cells
are missing. These results demonstrate that gut cell
internalization is not dependent on outside forces from
the vitelline membrane or from several other cell types,
including cells that move together with P4 and MSxx to
close the gap left by Ea and Ep. Interestingly, in cases
where the ablated explants adopt an aberrant linear
orientation, P4 and MSxx can still reposition relative to
Ea and Ep (see Figure, panel B). Therefore, the morphogenetic process of gut cell internalization in C. elegans
can be reduced to the study of interactions among only
four cells.
This simplified explant assay enables the direct manipulation of cells. By removing cells or replacing them
in altered orientations, the authors demonstrate that P4
and MSxx can move independently, indicating that they
do not simply chemotax toward one another. These experiments also reveal an unexpected behavioral difference between P4 and MSxx: the orientation of P4 can
influence its direction of movement with respect to
MSxx, but not vice versa. Analysis of cell interactions in
explants can thus mechanistically distinguish between
Previews
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Early Gastrulation Movements in C. elegans
(A) Illustration of the ingression of gut precursors, Ea and Ep, and the convergence of
neighboring cells, MSxx and P4, in an
embryo.
(B) Simplified schematic to show the movement of just these four cells within an explant
assay, in which more than half of the total
cells have been removed.
movements that appear superficially equivalent in the
intact embryo.
Interestingly, membrane-labeling experiments also
reveal that MSxx is not likely to advance using a fibroblast-type migratory mechanism. Instead, the authors
propose that an actin/myosin-based apical constriction
of Ea and Ep drives their internalization and pulls P4
and MSxx together. In support of this model, membrane
labeling demonstrates an apical constriction in Ea and
Ep. In addition, cell movements are blocked by pharmacological disruption of the actin cytoskeleton and inhibition of the myosin activator, myosin light chain kinase.
Apical constriction is a common early step of cell
ingression during gastrulation in many organisms. For
example, the bottle cells that form the sea urchin vegetal
plate and the Xenopus blastopore lip all apically constrict (Hardin and Keller, 1988; Kimberly and Hardin,
1998). In Drosophila and C. elegans, nonmuscle myosin
accumulates at the apical surface of ingressing cells
(Young et al., 1991; Nance and Priess, 2002), and activation of myosin contractility is required for cell internalization in C. elegans (Lee and Goldstein, 2003). These results raise the possibility that spatial regulation of
myosin-based contractility may be a widely used mechanism for cell internalization. It will be interesting to
determine how this spatial regulation is established and
controlled.
Cell internalization during gastrulation is likely to involve processes in addition to actin/myosin-based apical constriction. Xenopus bottle cells and Drosophila
mesoderm precursors undergo considerable cell elongation along the apical/basal axis, and, in Drosophila,
the basal surface subsequently expands more than
2-fold (Hardin and Keller, 1988; Leptin and Grunewald,
1990; Sweeton et al., 1991). During neurulation in vertebrates, cell shape changes are accompanied by extensive cell rearrangement and cell division (Schoenwolf
and Smith, 1990), suggesting that multiple mechanisms
may contribute to cell internalization. In addition to the
apical constriction of Ea and Ep in C. elegans, membrane-tracking experiments suggest an active rolling
movement of the neighboring MSxx cell, indicating that
MSxx is not just being passively dragged. Furthermore,
MSxx sometimes moves past the Ea/Ep boundary in
explants, suggesting that additional forces can drive cell
translocation. Therefore, even simplified explants can
provide enough complexity for an examination of the
coordinated action of multiple mechanisms during morphogenesis.
During cell rearrangement, where multiple mechanisms are operating and many cells change position,
it is important to establish which cells are the active
participants as well as which mechanism in an active
cell provides the driving force. For example, it is unclear
to what extent the apical constriction of Ea/Ep or the
rolling of MSxx provides the driving force for cell rearrangement in C. elegans. It should now be possible
to address these traditionally difficult questions by generating mosaic explants where individual cells are selectively modified by genetic or pharmacological techniques. It will also be important to determine whether
the active influence is mechanical or signaling in nature,
clues to which may be found by defining the molecular
components involved.
To address how the concerted action of individual
cells can drive complex multicellular rearrangements,
we must first understand the mechanisms that dictate
the behavior of single cells. This understanding may
ultimately reveal that the most useful way to describe
a morphogenetic process may not be at the cellular
level, but at the level of cell sheets or coherent groups
of cells. The findings of Lee and Goldstein also highlight
similarities between cell shape changes of two cells
and the internalization of large cell populations during
Drosophila and vertebrate gastrulation. Further comparison of cell behaviors in simplified assays to those in
intact embryos or to related processes in different species will determine the extent of such similarities and
may reveal mechanistic principles. This work offers hope
for understanding not only the cellular mechanisms of
morphogenesis, but also whether these mechanisms
can account for the dynamics of larger cell populations.
Rachel E. Dawes-Hoang, Jennifer A. Zallen,
and Eric F. Wieschaus
Department of Molecular Biology
Princeton University
Lewis Thomas Lab
Washington Road
Princeton, New Jersey 08544
Developmental Cell
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Selected Reading
Nance, J., and Priess, J.R. (2002). Development 129, 387–397.
Hardin, J., and Keller, R. (1988). Development 103, 211–230.
Schoenwolf, G.C., and Smith, J.L. (1990). Development 109,
243–270.
Keller, R., Clark, W.H.J., and Griffin, F. (1991). Gastrulation: Movements, Patterns, and Molecules (New York: Plenum Press).
Sweeton, D., Parks, S., Costa, M., and Wieschaus, E. (1991). Development 112, 775–789.
Kimberly, E.L., and Hardin, J. (1998). Dev. Biol. 204, 235–250.
Young, P.E., Pesacreta, T.C., and Kiehart, D.P. (1991). Development
111, 1–14.
Lee, J.-Y., and Goldstein, B. (2003). Development 130, 307–320.
Leptin, M., and Grunewald, B. (1990). Development 110, 73–84.
In Search of Lipid Translocases
and Their Biological Functions
In plasma membranes, lipids distribute asymmetrically
across the bilayer, a process that requires proteins.
Recent work identified novel lipid translocators in
yeast, and their activity was functionally correlated to
endocytosis, thus boosting investigations on identity,
mechanism, and function of lipid translocases.
The asymmetric distribution of lipids in plasma membranes (PM) of eukaryotic cells is well known. Whereas
neutral phospholipids, like phosphatidylcholine (PC),
sphingomyelin, and (glyco)sphingolipids (GSL), are largely
localized in the outer leaflet, the aminophospholipids
phosphatidylethanolamine (PE) and phosphatidylserine
(PS) reside predominantly in the inner leaflet. Cholesterol
is almost equally distributed over both leaflets and, in
the outer leaflet, is often found in association with GSL,
thereby forming microdomains (“rafts”). Mechanisms
underlying the maintenance and/or regulation of this
distinct lateral and transbilayer lipid distribution are still
poorly understood. Because of thermodynamic constraints, the involvement of integral membrane proteins
in translocating lipids across bilayers appears obvious.
Potential lipid translocators include an ATP-dependent
aminotranslocase (P-type ATPase), a specific inwarddirected pump for aminophospholipids; a lipid nonspecific Ca2⫹-dependent scramblase, which mediates bidirectional translocation; and members of the ATP binding
cassette (ABC) transporter family, which mediate outward lipid migration. As these properties were mostly
revealed through the use of fluorescent or spin-labeled
lipids, direct demonstration of translocation of natural
lipids is as yet poorly supported. Furthermore, incomplete protein purification and poor functional reconstitution has frustrated the functional identification of lipid
translocases.
Blood cells and, more recently, the genetically tractable yeast Saccharomyces cerevisiae are most often employed to study general principles of the mechanism
and regulation of phospholipid translocation. In yeast,
following their initial insertion in the outer PM leaflet,
fluorescent NBD (nitrobenzoxa-diazole) derivatives of
PE and PS, as well as PC, are transported across the
bilayer in a protein- and energy-dependent, but endocytosis-independent, manner (Grant et al., 2001). Interestingly, internalization of PC and PE is inhibited in anterograde sec mutants, suggesting that inward translocation
requires continuous transport and recycling of the translocase(s) and/or accessory elements to the cell surface.
Although it was questioned in early work (Chen et al.,
1999), it now becomes apparent (Gomes et al., 2000;
Pomorski et al., 2003) that Drsp2, a member of a subfamily of P-type ATPases, is one of the key players. Mutated
drs2 generates an aminophospholipid transport-defective phenotype, but a plant homolog from Arabidopsis,
ALA1, can complement for the deficiency in PS translocation (Gomes et al., 2000). Although mainly localized
to the Golgi, Drs2p is rather dynamic and cycles between
PM and late Golgi (Hua et al., 2002; Pomorski et al.,
2003). In an elegant contribution, Pomorski et al. (2003)
now identify two novel drs2-related P-type ATPases,
Dnf1p and Dnf2p, as lipid translocators for PS, PE, and
PC in the yeast PM. Overall, a dynamic picture emerges
of cycling translocases in the endosomal (Dnf) and
Golgi-PM secretory track (Drs2p) (Hua et al., 2002; Pomorski et al., 2003), rationalizing previous observations
that secretory mutants display an inhibition of lipid
translocation (Grant et al., 2001). Most importantly, Pomorski et al. show that, in dnf1/dnf2 deletion mutants,
endogenous PE accumulates at the cell surface and
further increases when drs2 is also deleted, suggesting
that the ATPases may act in concert as a lipid translocation machinery. It is yet not yet clear where Drs2-mediated PE translocation activity is expressed, i.e., at the
Golgi, at the PM, or at both. Neither can it be excluded
that, in the drs2 mutant, vesiculation at the Golgi, which
is facilitated by Drs2p, might have been impaired (Gall
et al., 2002). As a result, PM-directed transport of accessory compounds of the translocation machinery, which
may include “activator proteins” not related to ATPases,
e.g., Rosp3, could have been impeded. Interestingly,
the translocation capacity, but not the apparent affinity
for either lipid class (i.e., PE, PC, and PS), of both translocators differed, with Dnf1p, relative to Dnf2p, displaying
by far the highest activity. Although the noted lipid preference disqualifies these proteins as specific aminophospholipid translocases (but natural PC as substrate
has yet to be determined), sphingolipids, phosphatidic
acid, and phosphatidylglycerol are not substrates (Pomorski et al., 2003). Further work on regulation, including
the potential homo- or heterooligomerization, will be
important to solve issues as to why there should be
multiple translocases, displaying similar affinities for