Department of Cell Biology, PO Box 3709, Duke University Medical Center, Durham, NC 27710, USA
E-mail: b.hogan{at}cellbio.duke.edu
SUMMARY
Santa Fe with its museums and galleries full of art and crafts inspired by natural forms was the perfect setting for a Keystone conference on vertebrate organogenesis in February 2004. Organized by Gail Martin and Cliff Tabin, the conference sessions were loosely subdivided into anatomical systems `skin, hair, teeth', `pancreas, liver, gut', `skeleton', and so on. However, from the outset, common themes emerged that transcended particular organ systems and generated a sense of unity and excitement among the participants.
The first common theme to arise at this conference was clearly articulated by the keynote speaker Mark Krasnow (Stanford University School of Medicine, Stanford, CA, USA). He argued that the only way to identify all the genes controlling the development of a complex organ is to break the process down into simpler events that can be described at the cellular level. In other words, organogenesis must ultimately be reduced to a sequence of changes in parameters such as cell shape, polarity, movement, adhesion and proliferation. This approach enables the investigator to focus on specific events and cell populations in order to achieve a more-complete genetic analysis (e.g. by using gene arrays or proteomics). Other speakers also evoked the concept of deconstructing embryonic development into simple morphometric modules. They did so in the context of understanding how dramatic changes can occur during evolution in the size and shape of organs as diverse as the skeleton and brain.
A second theme to emerge during the meeting was the importance of using organ culture systems to study discrete steps in morphogenesis. The power of in vitro approaches has long been recognized. However, newer technologies for lineage tracing, time-lapse fluorescence microscopy and gene inactivation are making it easier to pinpoint the function of specific factors during morphogenesis. Emerging technologies in mouse genetics, including conditional gene manipulation and small interfering RNA (siRNA) gene knockdown in early embryos, are also having a strong impact on the field of organogenesis. In particular, they are enabling investigators to transcend the early lethality of null mutations and to take on heroic projects to determine the redundant functions of multigene families in specific tissues during organ development.
Finally, several speakers touched on the theme of tissue-specific stem cells in organogenesis, the plasticity of progenitor cells and the regulation of cell fate.
Deconstructing morphogenesis
One of Krasnow's take-home messages to students was the importance of carefully describing the cellular anatomy of organogenesis. He illustrated this concept with recent work from his own laboratory on the formation of the fine terminal branches of the Drosophila larval tracheal system. These branches, which ramify deeply into internal tissues, are generated from single cells containing a lumen 1 µm or less in diameter. This lumen is thought to be formed by the coalescence of multiple apical membrane vesicles that line up in the center of the cell. Using mosaic analysis to circumvent any requirements for tracheal genes early in development, members of his laboratory identified a set of larval mutants with defects in lumen formation. Some mutants completely lack lumens, including those with mutations in genes that encode highly conserved components of the vesicle trafficking machinery. One of the most dramatic tracheal mutants, called `tendrils' because of the supernumerary, convoluted lumena seen in mutant terminal cells (Fig. 1), has mutations in a cytoskeletal protein. Krasnow suggested that this constitutes part of a novel scaffold system, somehow constructed in the center of the terminal cell, on which the vesicles line up before they coalesce. He raised the interesting idea that information on how prelumenal vesicles are aligned could shed light on how endothelial tubes are formed during blood vessel development in vertebrates.
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The concept that simple changes in cell behavior underlie the development
of complex organ systems was also the message of Didier Stainier (University
of California, San Francisco, CA, USA), who works with the zebrafish embryo.
He described how both the development of the heart and the looping of the gut
ultimately depend on mesodermal cell migration and polarization. In the case
of heart formation, for example, mutant analysis shows that fibronectin is
required for the correct epithelial organization of the migrating myocardial
precursors (Trinh and Stainier,
2004). Asymmetric gut looping requires that the right lateral
plate mesoderm (LPM) migrates ventrally underneath the endoderm, pushing it to
the left, while the left LPM simultaneously moves horizontally towards the
midline. The gene heart and soul encodes an atypical protein kinase C
(PKC) required for establishment of cell polarity. Mutations interfere not
only with cell polarization and lumen formation in the gut tube itself, but
also with the epithelialization and migration of the LPM, leading to secondary
effects on the looping of the gut
(Horne-Badovinac et al.,
2003
).
Cell behavior during gut development was also the topic of discussion by
Ken Zaret (Fox Chase Cancer Center, Philadelphia, PA, USA), although in
relation to much earlier stages in mouse embryogenesis. By labeling definitive
endoderm cells around the developing anterior foregut (known as the anterior
intestinal portal or AIP) with the lipophilic fluorescent dialkycarbocyanine
dyes DiI or DiO at the two-somite stage and then culturing the embryos in
vitro, members of his laboratory were able to trace the origin of specific
populations of foregut cells that contribute to the liver bud. Unexpectedly,
he found that these liver bud cells come from two locations. Some derive from
the lateral walls of the AIP that come together as the endoderm tube `zippers
up' ventrally. Others arise from a small population of endoderm cells in the
anterior midline that gives rise to a line of cells along the ventral foregut
tube, from the thyroid to the liver. A similar population of cells has
recently been described in the chick embryo at an earlier stage
(Kirby et al., 2003). It
remains to be seen whether these midline foregut cells have organizer
properties or fates different from the more lateral cells.
Morphogenetic modules and vertebrate evolution
The central dogma of evolutionary biology is that changes in the shape of
anatomical structures over time are brought about by natural selection working
on genetic variants. But precisely how such changes are brought about is not
always well understood. Although emphasis is usually placed on changes in
amino acid sequences in proteins, David Kingsley (Stanford University,
Stanford, CA, USA) argued for the importance of variation in DNA regulatory
elements, especially in genes encoding embryonic growth factors and other
morphogenetic signaling proteins. He cited as evidence work from his
laboratory on the mouse bone morphogenetic protein 5 (Bmp5) gene,
which is required for the normal development of many cartilages and bones.
Members of his laboratory have identified discrete DNA regulatory regions that
drive Bmp5 gene expression in specific anatomical sites such as the
perichondrium of the ribs, sternum, ears or thyroid cartilage during mouse
embryogenesis (DiLeone et al.,
2000). In the case of the ribs, two different DNA elements were
identified, lying hundreds of kilobases apart on the chromosome. One element
promotes Bmp5 expression on one lateral surface of the rib, while the
other DNA element drives expression on the geometrically opposite side of the
same rib! The significance of this spatial segregation became obvious when
they investigated how ribs which are cylindrical structures
dynamically change their shape during embryonic and postnatal development: to
increase curvature, bone is deposited on one side of the cylinder and broken
down by osteoclasts on the other. It therefore makes sense for the embryo to
be able to control the activity of genes such as Bmp5 in each domain
independently. Evolutionary changes in the shape of the ribs (or any other
skeletal element) could thus be brought about by selection for changes in the
activity of one regulatory element versus another. Using the three-spined
stickleback as a model genetic system, David Kingsley, Chuck Kimmel
(University of Oregon, Eugene, OR, USA) and others are now testing such ideas
in natural fish populations. They are identifying genes that control the
striking morphological differences in skeletal elements such as spines, jaws
and bony plates that occur in sticklebacks isolated in freshwater and marine
locations around the world (for more information see Stanford Genome Evoution
Center
http://cegs.stanford.edu).
The application of embryology to evolution was also the subject of a
fascinating talk by Cliff Tabin (Harvard Medical School, Boston, MA, USA). His
laboratory is exploring the origin of the differences in beak shape and size
among Darwin's finches in the Galapagos: the warbler finch, for example, has a
thin long beak suited to catching insects, while the large ground finch has a
thick short beak more appropriate for cracking nuts. Studies in chick have
shown that the beak develops from facial buds, each consisting of an
ectodermal jacket filled with neural crest and head mesoderm. The outgrowth of
the buds depends on epithelial-mesenchymal interactions that are driven by
signaling factors such as BMPs, sonic hedgehog (SHH) and fibroblast growth
factors (FGFs) expressed in the different cell populations (e.g.
Schneider and Helms, 2003). It
is not unreasonable to suppose that the beak morphology of different finch
populations depends on variations in the temporal and spatial patterns of
expression of these signaling genes. To test this hypothesis, gene expression
is being analyzed in the developing beaks of finch embryos collected in the
wild.
In vitro organ culture to explore organogenesis
Several investigators described the use of in vitro culture systems to
study specific steps in organogenesis. Organs examined in this way included
kidney and urogenital sinus [Costantini (Columbia University, New York, NY,
USA), Carroll (Harvard University, Boston, MA, USA), Herzlinger (Cornell
University, Ithaca, NY, USA), Mendelsohn (Columbia University, New York, NY,
USA)], submandibular gland [Hoffman (NIH, Bethesda, MD, USA)], and inner ear
[Wu (NIH, Bethesda, MD, USA)]. Joan Brugge (Harvard Medical School, Boston,
MA, USA) described three-dimensional basement membrane cultures in which a
mammary cell line undergoes a morphogenetic program leading to the formation
of a hollow acinar-like structure. Formation of a lumen in these structures
involves selective death of cells within the center of a solid mass
(Fig. 2). Brugge's in vitro
studies suggest that this cell death proceeds by two parallel pathways:
apoptosis and a distinct, but poorly understood, mechanism known as
`autophagy', which is elicted by an extracellular ligand known as TRAIL (tumor
necrosis factor-related apoptosis-inducing ligand)
(Mills et al., 2004). These
findings are important because evasion of programmed cell death by either
pathway appears to be a crucial step in the progression of breast cancer. The
studies also raise the question of whether the processes observed in this in
vitro model recapitulate events associated with clearing of the end buds of
growing mammary glands. Initially, the end bud consists of a dense cluster of
cells that become polarized and organized into an epithelial layer surrounding
a lumen. Anatomical studies of normal mammary gland development by Cheryl
Tickle (University of Dundee, Dundee, UK) had shown that end bud cells that do
not undergo polarization are fated to die
(Hogg et al., 1983
). It will
also be important to see if TRAIL-induced autophagy plays a role in other
examples of lumen formation involving cell death
(Coucouvanis and Martin,
1995
).
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Axonal guidance molecules in branching morphogenesis
New ideas about ureter bud morphogenesis were also presented by Uta
Grieshammer, from Gail Martin's laboratory (University of California, San
Francisco, CA, USA). She provided evidence for a role in kidney development of
SLIT2 and ROBO2. These evolutionarily conserved proteins are best known as
axonal guidance molecules, although there is evidence that they function in
the branching morphogenesis of the Drosophila tracheal system
(Englund et al., 2002).
Slit2 encodes a secreted protein that either repels or attracts
neurons, whereas Robo2 encodes one of its receptors. Grieshammer
showed that in mouse embryos lacking Slit2 or Robo2,
numerous ectopic ureter buds emerge from the Wolffian duct anterior to the
normal bud (Fig. 3), and some
of them give rise to ectopic kidneys. Slit2 is normally expressed in
the Wolffian duct and in the adjacent intermediate mesoderm, while
Robo2 is expressed only in the mesoderm. In Slit2- and
Robo2-null mutants, Gdnf expression, which initially extends
along the IM, does not become restricted posteriorly to the site where the
single ureter bud normally emerges. However, the mechanism underlying this
defect is not clear. Based on various experiments, including lineage labeling,
Grieshammer argued against the simplest hypothesis, that SLIT2 drives the
posterior repulsion of Gdnf-expressing IM cells so that they
aggregate around the normal site of ureter bud formation, and instead
suggested that SLIT2/ROBO2 controls the extent of the Gdnf expression
domain via effects on transcriptional regulators of Gdnf
(Grieshammer et al, 2004
).
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Genetic redundancy and new technologies in mouse genetics
It is now apparent that the development of most vertebrate organs is regulated by a relatively small set of evolutionarily conserved signaling factors the FGF, BMP, WNT and hedgehog (HH) proteins, for example. However, each class may include more than 20 family members, which are encoded by different genes and/or by multiple splice variants of the same gene. Moreover, several family members may be expressed in the same or overlapping populations of cells at different stages of organogenesis. These redundancies frequently complicate the genetic analysis of vertebrate organogenesis. Sometimes it is possible to overcome the problem by brute force, generating a few embryos with multiple mutant alleles. This approach was used by Ryoichiro Kageyama (Kyoto University, Kyoto, Japan) in his work, to be described later, on the role of basic helix-loop-helix (bHLH) genes in neural stem cell maintenance.
In other cases of genetic redundancy, it is necessary to resort to making
floxed alleles of several gene family members and then inactivating them
simultaneously using a tissue or cell type-specific Cre recombinase. Gail
Martin described a very successful use of this approach. She used a transgene
that drives Cre in the apical ectodermal ridge (AER) of the mouse limb bud to
facilitate inactivation of three out of the four FGF genes co-expressed in
this tissue. Surprisingly, when Fgf4, Fgf9 and Fgf17 are
inactivated, limb patterning is normal, being driven by the remaining family
member, Fgf8. Martin then asked whether there is a special
requirement for Fgf8 or whether its function can be replaced by
Fgf4 if the temporal and spatial expression pattern of this gene is
changed to be more like that of Fgf8. This was achieved using a
conditional gain-of-function transgene to express Fgf4 in the limb
bud in the absence of Fgf8. Under these conditions, Fgf4 is
able to substitute very well for Fgf8. Martin further outlined how
the powerful genetic toolkit that she has generated is being used to test new
ideas and models for the mechanisms patterning the skeletal elements of the
limb (Mariani and Martin,
2003). The talk by Rolf Zeller (University of Basel Medical
School, Basel, Switzerland) also brought home to the audience the fact that,
in spite of all the research on limb development over the past decade, the
story is still undergoing elaborations and revisions. Zeller presented very
detailed and convincing new genetic evidence that the limb deformity phenotype
is not due to inactivation of the formin gene, as previously thought, but to
inactivation of the adjacent gremlin gene (Cktsf1b1 Mouse
Genome Informatics), which is expressed in the developing limb bud and encodes
an antagonist of BMP signaling (Khokha et
al., 2003
; Zuniga et al.,
2004
).
A new approach to mouse genetic analysis was well illustrated by Benoit
Bruneau (Hospital for Sick Children, Toronto, Canada) in the context of
exploring the function of one of the components of the SWI/SNF
chromatin-remodelling complex. The particular subunit, SMARCD3
(SWI/SNF-related matrix-associated actin-dependent regulator of chromatin,
subfamily d, member 3), is expressed specifically in the early heart tube. To
knock down the gene encoding this subunit, Bruneau collaborated with Janet
Rossant's laboratory in Mount Sinai Hospital (Toronto, Canada). They used a
recently described method in which DNA that encodes a neomycin-resistance
cassette and the human H1 RNA polymerase III promoter to drive expression of a
short hairpin RNA is electroporated into mouse embryonic stem (ES) cells
(Kunath et al., 2003). Cells
providing good expression of the siRNA are then aggregated with tetraploid
embryos, so that the resulting fetus is derived entirely from the ES cells.
Using another new technique, optical projection tomography
(Sharpe, 2003
), in conjunction
with regular histology, Bruneau found that reduction in SMARCD3 levels leads
to abnormal cardiac chamber morphogenesis, including specific defects in the
outflow tract and ventricular trabeculations of the embryonic heart. Moreover,
by comparing embryos derived from different cell lines, he was able to
generate an `epiallelic series' correlating the extent of gene knockdown with
the severity and localization of the morphological defects. Finally, he used
in vitro assays to show that SMARCD3 functions to potentiate the activation of
Pitx2 and other genes important in heart development.
Stem cells and switches in cell fate
As might be expected, stem cells featured in several of the talks at the
meeting, with the emphasis being on tissue-specific stem cells contributing to
organogenesis and tissue turnover and repair, rather than on pluripotential
embryonic stem cells. Both Bruce Morgan (Massachusetts General Hospital,
Harvard Medical School, Boston, MA, USA) and Fiona Watt (Cancer Research UK,
London, UK) considered stem cells in the epidermis of the adult mouse. One
question is whether the normal turnover of epithelial cells in the skin
relies, in the long term, on only a single source of multipotent stem cells,
located in the bulge of the hair follicle. Morgan described preliminary
studies in which he and his collaborator, Brian Harfe (UFGenetics Institute,
Gainsville, FL, USA) have used a cell-autonomous lineage label to follow the
fate of hair follicle placode cells that once expressed SHH. They found that
all of the cells of the hair follicle, including the bulge, expressed the
label, consistent with them being derived originally from the placode.
However, even after a year, the epidermis between the follicles was not
extensively labeled. This suggests that under normal conditions when there is
no injury, the interfollicular epidermis is renewed from stem cells in this
compartment and does not require migration of progenitor cells from the bulge.
Fiona Watt presented evidence that activation of ß-catenin in
interfollicular epidermal cells could lead to the generation of new hair
follicles and sebaceous gland cells. Whether the ectopic hair follicles have
bulge stem cells is not yet known. However, Watt argued that the level and
duration of ß-catenin signaling in the interfollicular epidermal cells
could lead to a change in the lineage of their descendants from
interfollicular epidermis to hair follicle or sebaceous gland. Brigid Hogan
also presented evidence that transient overexpression of a constitutively
active Lef1/ß-catenin fusion protein in progenitor cells of the embryonic
mouse lung switches their fate to intestinal lineages
(Okubo and Hogan, 2004). Thus,
levels of WNT signaling may affect progenitor cell fate in more than one organ
system.
The regulation of neural stem cell fate was the topic of a talk by Ryoichiro Kageyama. During normal brain development, a pool of stem cells has to be carefully maintained in the ventricular layer. Progenitors leave this pool at different times to migrate to layers where they give rise to different kinds of neurons. Kageyama showed that in animals double mutant for the genes encoding the bHLH transcriptional repressors, HES1 and HES5, progenitor cells in the ventricular layer differentiate into neurons prematurely, thus reducing the pool available to make later-born neurons. In triple mutants also lacking Hes3, the reduction in the pool of stem cells available to generate later neurons was even further reduced, and virtually all neural stem cells become early neurons without generating late neurons and glia. Precisely how the expression of HES genes regulates the individual behavior of stem cells and their topographic organization in the ventricular zone is under investigation.
Conclusion
In the space available here it has not been possible to cover all of the excellent talks, nor to include the data discussed so enthusiastically in the poster sessions. Taken together, the presentations contributed to a most stimulating and inspiring meeting. The feelings of the participants as they left Santa Fe are perhaps best summed up in the words of a Native American people of the Southwest.
May it be beautiful before me.May it be beautiful behind me.
May it be beautiful above me.
May it be beautiful all around me.
In beauty it is finished.
from a Diné Night Chant
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