Social interactions among epithelial cells during tracheal branching morphogenesis. Nature 441: 746-749. pdf

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Social interactions among epithelial cells during tracheal branching morphogenesis. Nature 441: 746-749. pdf
Vol 441|8 June 2006|doi:10.1038/nature04829
Social interactions among epithelial cells during
tracheal branching morphogenesis
Amin S. Ghabrial1 & Mark A. Krasnow1
Many organs are composed of tubular networks that arise by
branching morphogenesis in which cells bud from an epithelium
and organize into a tube1–3. Fibroblast growth factors (FGFs) and
other signalling molecules have been shown to guide branch
budding and outgrowth4–7, but it is not known how epithelial
cells coordinate their movements and morphogenesis. Here we use
genetic mosaic analysis in Drosophila melanogaster to show that
there are two functionally distinct classes of cells in budding
tracheal branches: cells at the tip that respond directly to Branchless FGF and lead branch outgrowth, and trailing cells that receive
a secondary signal to follow the lead cells and form a tube. These
roles are not pre-specified; rather, there is competition between
cells such that those with the highest FGF receptor activity take the
lead positions, whereas those with less FGF receptor activity assume
subsidiary positions and form the branch stalk. Competition
appears to involve Notch-mediated lateral inhibition that prevents
extra cells from assuming the lead. There may also be cooperation
between budding cells, because in a mosaic epithelium, cells that
cannot respond to the chemoattractant, or respond only poorly,
allow other cells in the epithelium to move ahead of them.
The Drosophila tracheal system develops from epithelial sacs of
about 80 cells from which primary, secondary and terminal branches
sprout without cell division or cell death8,9. Primary branch sprouting is induced by Branchless (Bnl) FGF, a chemoattractant secreted by
clusters of cells surrounding each sac4 (Fig. 1a), which activates
Breathless (Btl) FGF receptor (FGFR), a receptor tyrosine kinase
expressed on tracheal cells10. Primary branches contain 3–20 cells
that organize into a tube as they migrate out from the sac (Fig. 1b).
Bnl also induces the expression of secondary branching genes, such as
the transcription factor pointed (pnt)11, and specifies terminal cells at
the ends of outgrowing branches4,8. Terminal cells ramify in the larva
in response to Bnl to form fine terminal branches12. Other cells at the
ends of primary branches become fusion cells that connect with
neighbouring branches to form a continuous tracheal network.
Terminal and fusion cell fate decisions are also influenced by the
Notch, Dpp and Wingless signalling pathways13–17. Dorsal branches,
the primary branches that we focus on here, typically consist of five or
six cells: two cells near the branch tip, one (DB1) that becomes a
terminal cell and another (DB2) that becomes a fusion cell, and three
or four cells (DB3–DB6) that form the branch stalk (Fig. 1a, b).
In a genetic mosaic screen (A.S.G., B. P. Levi and M.A.K.,
unpublished observations), six mutants (724, 788, 1118, 1187,
1476 and 1684) were identified with a subtle phenotype: mosaic
branches (þ/þ, þ/2, 2/2 cells) were grossly normal, yet homozygous mutant clones (2/2 cells) rarely if ever included terminal
cells (Fig. 1c–e, and Supplementary Tables S1–S4; data not shown for
other primary branches). These were neither general nor terminalcell-specific lethal mutations because homozygous mutant cells were
readily recovered in all other tracheal positions, and there was no
decrease in the overall number of cells in mosaic dorsal branches
(5.3 ^ 1.1 (mean ^ s.d.) versus 4.9 ^ 1.1 in contralateral control
branches; n ¼ 22 pairs of branches) or the number of terminal cells
(98% of both wild-type (n ¼ 127) and 724 or 788 mosaic dorsal
branches (n ¼ 291) had a terminal cell). It was difficult to imagine how
mutations could block clone generation in specific cells. It seemed
more likely that the mutations caused cells otherwise destined to
become terminal cells to switch fates with other tracheal cells.
The six mutations compose a single lethal complementation group
that mapped to the left arm of chromosome 3 and failed to
complement breathless LG18. DNA sequencing identified a single
nucleotide change in each mutant resulting in a nonsense or missense
mutation in btl (Fig. 2a). Five mutations (724, 788, 1118, 1476 and
1684) appear to be null btl mutations, whereas the sixth mutation
(1187) causes partial loss of function (Fig. 2b and data not shown).
Thus, the ‘no mutant terminal cells’ gene is btl.
We quantified the distribution of cells homozygous for btl null
mutations (724 and 788), or homozygous for a wild-type btl allele as
a control, in mosaic dorsal branches (Supplementary Tables S1–S4).
Control clones were evenly distributed throughout the branch at the
expected frequencies; for example, the ratio of stalk-cell to terminalcell clones was about 3:1. By contrast, btl 2/2 cells showed a nearly
complete bias against the DB1 position: the ratio of stalk-cell to
terminal-cell clones was 51:1. The three exceptional mutant terminal
cells may be cases in which the clone was induced after btl began to be
expressed, allowing wild-type btl gene products to perdure in mutant
cells. We recovered hundreds of mosaic branches with one or more
btl 2/2 cells present in positions DB2–DB6 without affecting cell or
branch morphology. Indeed, branches composed largely or exclusively of btl 2/2 cells, except for a wild-type terminal cell, were
morphologically indistinguishable from wild-type branches (Fig. 2c,
and Supplementary Tables S3 and S4). Thus, although all tracheal
cells normally express btl, and the receptor is activated by Bnl in most
or all cells of budding branches8,18, the receptor appears to be
required in just a single leading cell (DB1). All other cells can migrate
normally and form tubes in the absence of btl. We conclude that there
are two functionally distinct classes of cells in budding primary
branches: lead cells, which require Btl FGFR and directly respond to
Bnl FGF, and trailing cells, which do not require Btl but follow the
lead cell and form the stalk.
What does it take to become the leader? The lead cell (DB1) is
specified to become a terminal cell by Bnl–Btl signalling4,8. If terminal
cell specification is required, then null mutations in the downstream
gene pnt, which abolish this function8, should have the same effect as
btl mutations. Cell clones homozygous for pnt D88 or two new pnt
alleles isolated in our screen (198 and 1318) failed to develop as
terminal cells, as expected. However, unlike btl mosaic branches, pnt
mosaic branches often lacked a terminal cell (26% of mosaic
branches; n ¼ 97). When a terminal cell was missing, there was
usually (about 80% of the time) a pnt 2/2 cell in the stalk position
nearest the tip (Fig. 2d, and Supplementary Table S5), presumably
Howard Hughes Medical Institute, Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305-5307, USA.
© 2006 Nature Publishing Group
NATURE|Vol 441|8 June 2006
Figure 1 | The ‘no mutant terminal cells’ phenotype. a, Diagram of dorsal
branch (DB) budding from tracheal epithelium (black; DB cells numbered
1–6) at the developmental stages and times indicated. Nearby cells (blue)
secrete Branchless FGF (blue dots), which activates Breathless (Btl) FGFR on
tracheal cells, inducing migration and tube formation. Bnl also induces
secondary branching genes (for example pointed) in cells (green) that form
unicellular secondary branches (stage 15). Subsequently, DB1 (terminal cell)
forms terminal branches in response to Bnl expressed by hypoxic larval
cells. DB2 (fusion cell) forms a branch that fuses (dotted lines) to a
contralateral DB (not shown). DB3–6 cells form DB stalk. b, Micrograph of
budding DB (stage 13). Nuclei are black; cytoplasm is grey. Cells here are
arranged side by side, but subsequently the stalk cells intercalate. Reprinted
with permission (ref. 8). Scale bar, 2.5 mm. c, Fluorescence micrographs of
GFP-marked clones in pairs of larval DBs (dorsal view, anterior up).
Tracheal cells are labelled with DsRED (red), and homozygous clones are
labelled with GFP (green). Arrowheads indicate select clones in DBs and
dorsal trunks (large branches flanking DBs). Top panel: control wild-type
clones. Bottom panel: homozygous 724 mutant clones. DB1 (1, 1 0 ) and DB3
(3, 3 0 ) cells in left and right DBs are indicated. Scale bars, 50 mm.
d, Distribution of control clones (green) in eight representative DBs. Three
contain DB1/terminal cell clones (circled). For simplicity, only DBs with five
cells (DB1–DB5) are shown. e, Distribution of homozygous 724 mutant
clones in eight representative DBs. Clones had no morphological defects but
were excluded from DB1 positions.
the DB1 cell that failed to differentiate into a terminal cell. This suggests
that pnt 2/2 cells are able to assume the lead position but fail to
differentiate as terminal cells, and that the bias against btl mutant
terminal cells is due to the earlier, pnt-independent, function of Btl in
primary branch budding and outgrowth. If cells lacking Btl cannot
migrate in response to Bnl during budding8,10,19, they should not be able
to move to the lead position necessary to be selected as a terminal cell.
Consistent with this, genetic mosaic analysis of stumps (dof/heartbroken), which encodes a Btl adaptor required for cell migration20,21,
showed a dearth of terminal cell clones similar to btl (data not shown).
Two results demonstrate that the ability to sense Bnl and migrate
in response to it is not enough to become the leader: cells compete for
the lead position. The first involves btl BN (E796K mutation in the
kinase domain), a weak btl allele isolated in a separate screen. Unlike
btl 2/2 animals, which die in first larval instar and lack virtually all
branches10 (Fig. 2b), btl BN homozygotes survived until L3 larval stage
or beyond and had a normally patterned tracheal system with a full
complement of terminal cells. The only defects detected were a
reduced number and altered morphology of terminal branches,
presumably due to the dosage-sensitive function of btl in terminal
branch outgrowth12 (Fig. 3a–c). The late and subtle phenotype
demonstrates that BtlBN protein retains sufficient activity for early
migration and terminal cell specification events. However, we found
that in genetic mosaic animals, in which btl BN/BN cells must compete
with btl BN/þ and btl þ/þ tracheal cells, btl BN/BN cells rarely acquired
the lead position (DB1) and developed as terminal cells. Indeed,
homozygosity for btl BN conferred nearly as complete a bias against
becoming a terminal cell as total loss of btl (Fig. 3d–f, and Supplementary Tables S1 and S6). Thus, Btl activity above the threshold
necessary for migration and terminal cell specification is not sufficient to acquire the lead position and become a terminal cell: a cell
must have more Btl activity than other cells in the branch.
Similar conclusions derive from a second experiment in which we
analysed marked wild-type (btl þ/þ) cells in heterozygous (btl þ/2)
animals. Whereas btl þ/þ clones in control (btl þ/þ) animals were
distributed evenly throughout the branch (Fig. 3d), btl þ/þ clones in
btl 788/þ heterozygotes preferentially localized to the tip (Fig. 3g, and
Supplementary Tables S1, S2 and S7). Cells that did not occupy the
lead (DB1) position took positions close to the tip (Fig. 3g, and
Supplementary Table S7). Similar results were obtained for btl þ/þ
clones in animals heterozygous for btl 1187, a partial-loss-of-function
allele (Fig. 3h, and Supplementary Tables S1 and S8). Clones mutant
for sprouty, an FGF feedback inhibitor22,23, also preferentially populated the tip (Supplementary Table S9). Together, the data show that
there is competition for the lead position: cells with highest btl
activity assume positions at or near the tip of the branch, whereas
those with less or no activity segregate towards its base.
Because small differences in btl dosage or activity affect a cell’s
ability to compete for the lead, we investigated whether lateral
inhibitory mechanisms that amplify small differences in signalling
might be operative. Data suggest that the Notch pathway, a lateral
signalling pathway implicated in cell specification events including
cell fate determination at tracheal branch tips, also affects cell
arrangement15–17. N ts embryos shifted to the restrictive temperature
during budding formed branches in which most DB cells behaved
like lead cells, resulting in large clusters of cells congregated at the
lead position17, whereas expression of constitutively active NACT
throughout the tracheal epithelium had the opposite effect, arresting
outgrowth and stalling cells near the base of the branch (Fig. 4a–c).
We propose that Notch-mediated lateral inhibition among tracheal
cells prevents extra cells from assuming the lead position.
Our results provide evidence for social stratification and dynamic
social interactions between epithelial cells during branching
morphogenesis (Fig. 4d). First, the results show that budding cells
are functionally specialized. A cell at the branch tip requires btl and
leads outgrowth towards the Bnl signalling centre. Trailing cells do
not require btl but nevertheless follow the lead cell towards the Bnl
source. Because tracheal cells do not migrate or form tubes in btl 2/2
animals, trailing cells must receive a secondary signal generated by
the lead cell that induces them to migrate and also activates their
tubulogenesis programme. This could be a secreted molecule or
physical stimulus such as pulling or stretching the trailing cells.
© 2006 Nature Publishing Group
NATURE|Vol 441|8 June 2006
Figure 2 | ‘No mutant terminal cells’ mutations are loss-of-function
mutations in btl. a, Changes in btl coding sequence in ‘no mutant terminal
cells’ mutants. Asterisk, kinase active site. b, Wild-type, homozygous btl LG18
and homozygous btl 724 embryos (10–13 h old) immunostained with
tracheal-specific monoclonal antibody 2A12. Lateral views, anterior left,
dorsal up. Note the absence of branches in both mutants. Scale bar, 50 mm.
c, Fluorescence micrographs of mosaic DB with large btl 788/788 clone (dorsal
view). Tracheal nuclei are labelled with DsRed2nls (red; left and middle
panels), and DB cell nuclei are circled (DB1–DB4, mosaic DB; DB1 0 -DB4 0 ,
contralateral control DB). (DB1 staining is partly obscured by underlying
dorsal trunk staining.) Homozygous btl 788/788 cells are labelled with GFP
(green; middle panel) and comprise the entire mosaic DB except DB1
(see diagram, right panel), yet DB has a normal morphology, like the
contralateral control DB. Scale bar, 50 mm. d, Mosaic DB containing a null
pointed D88 clone (dorsal view). First panel: DsRed2nls-labelled nuclei (red)
are circled (DB1* and DB2, mosaic DB; DB1 0 –DB3 0 , contralateral control
DB). Second panel: pnt D88/D88 clone (green, DB1*) did not develop as a
terminal cell or assume its normal position; instead it formed the distal cell
of the stalk. Third panel: red and green channels superimposed on a
differential interference contrast image, showing air-filled lumens of DBs.
DB1 0 extends terminal branches (arrowhead) into region normally
supplied by missing terminal cell. Fourth panel: diagram of mosaic DB, with
pnt D88/D88 clone shaded green. Scale bar, 50 mm.
Second, these roles are not pre-specified. Rather, there is competition between cells such that those with high Btl FGFR activity
become lead cells whereas those with less or no btl FGFR activity
become trailing cells and form the branch stalk. Competition appears
to involve Notch-mediated lateral inhibitory signalling between
tracheal cells, and it may also be influenced by positive feedback
mechanisms such as increased activation and expression of Btl as cells
approach the Bnl source24. Third, there may be cooperation between
cells, because in a genetically mosaic epithelium, tracheal cells with
less Btl activity allow those with more activity to move ahead of them.
There may be similar social interactions between budding cells in
other branching organs. Studies of other branching processes have
identified genes selectively expressed in tip cells of budding branches,
Figure 3 | Effect of btl dosage on DB cell position. a–c, Fluorescence
micrographs of DB pair (dorsal view) in control btl þ larva (a), larva
homozygous for the weak hypomorphic allele btl BN (b) and larva
transheterozygous for btl BN and null btl 724 allele (c). Tracheal cells were
labelled by expression of a UAS-GFP transgene driven by btl-Gal4
(btl-Gal4 . GFP). The btl BN/BN larva has normal DB primary and
secondary branches, and a normal number of terminal cells, although
terminal branching is reduced. btl BN/724 lacks DBs. Scale bar, 50 mm.
d–h, Relative frequency of GFP-marked clones at different DB positions.
Genotypes of clones and animals in which clones were induced are indicated.
n, number of DBs analysed. Because the average number of cells per clone
varied in different experiments (d, 1.67; e, 1.33; f, 1.36; g, 1.52; h, 1.55), clone
frequencies shown are relative values normalized to one GFP-marked cell
per branch. d, Control clones were evenly distributed between positions
DB1–DB4. e, Clones with no btl activity (btl 724/724) induced in btl 724/þ
animals showed a graded distribution, rarely in DB1 position and increasing
in frequency towards the branch base. f, Clones with mildly reduced btl
activity (btl BN/BN) induced in btl BN/þ animals showed a similar distribution,
even though btl BN/BN cells populate all positions in btl BN/BN animals (b).
g, h, btl þ/þ clones induced in animals heterozygous for null btl 788 allele (g)
or moderate btl 1187 allele (h) showed the opposite distribution, preferentially
in DB1 positions and decreasing in frequency towards the branch base.
and in some cases these cells display morphological specializations
indicating that they might actively lead outgrowth25. However,
because most budding branches contain hundreds or thousands of
cells, it is difficult to track and manipulate individual cells to
investigate social behaviours like those described here. Recent
analyses of chimaeric Ret þ/Ret 2 mouse renal ureteric buds in
culture26 and btl mosaic air sacs27 reveal that cells lacking these
receptor tyrosine kinases are excluded from branch tips, indicating
that RTK-dependent interactions similar to those described here
might be operative in more complex branching events.
Fly stocks. btl LG18, spry D5, pnt D88 and N l1N-ts1(abbreviated N ts) are described in
Flybase (http://flybase.bio.indiana.edu/). New btl and pnt alleles were generated by
ethane methyl sulphonate mutagenesis of a third chromosome containing FLP
recombinase sites FRT2A and FRT82B (Flybase) and isolated in screens described
elsewhere (A.S.G., B. P. Levi and M.A.K., unpublished observations). The Gal4UAS system28 with btl-GAL4 driver (Flybase) was used to express proteins
throughout the tracheal system. UAS responders were as follows: UAS-DsRED
(A.S.G., B. P. Levi and M.A.K., unpublished observations), which expresses a
cytoplasmic red fluorescent protein; UAS-DsRED2nls (M. Galko, G. Fish and
M.A.K, unpublished observations), which expresses a nuclear DsRed2 protein;
UAS-GFP (Flybase); UASi-GFPhp (A.S.G., B. P. Levi and M.A.K., unpublished
observations), which expresses a double-stranded hairpin RNA that inhibits
GFP expression by RNA-mediated interference; and UAS-NACT (Flybase).
Clone generation and labelling. Heterozygous embryos (1–3 h old) carrying the
hsFLP1.22 recombinase transgene (Flybase) were reared at 25 8C and then placed
at 38 8C for 45–60 min to induce mitotic recombination at FRTsites centromereproximal to the mutation, generating clones of homozygous mutant cells29.
Tracheal cells were marked with a btl-GAL4 transgene to drive expression of
UAS-DsRED (or UAS-DsRED2nls) and UAS-GFP transgenes. Expression of the
latter was blocked by a UASi-GFPhp transgene present in trans to the mutation
© 2006 Nature Publishing Group
NATURE|Vol 441|8 June 2006
Figure 4 | Effects of Notch (N) activity on cell position. a–c, Fluorescent
micrographs of two DBs (lateral view) in stage 15 wild-type embryo (a), N ts
embryo shifted to non-permissive temperature for 6 h during branch
budding (b) and btlGal4 . UAS-N ACT embryo that expressed activated N
throughout the tracheal system (c). All embryos carried btlGal4 and UASGFP transgenes and were double-stained for GFP (red; tracheal cell marker)
and Vermiform (cyan; luminal marker). a, Cells in wild-type DBs are evenly
distributed (nuclei are numbered and indicated by asterisks). b, N
inactivation caused the migration of extra cells to the DB tip. c, Constitutive
N activity inhibited outgrowth, particularly in posterior metameres in which
some DBs completely failed to bud (arrowhead). Scale bar, 5 mm. d, Social
interactions between tracheal cells during budding. The three panels show
budding tracheal cells expressing the Btl FGFR moving towards a Bnl FGF
signalling centre, as in Fig. 1a. The first panel illustrates cell competition:
cells move towards the lead position and inhibit their neighbours from doing
the same. The second panel illustrates cell cooperation: a cell with less Btl
activity allows one with more to move ahead of it. The third panel illustrates
cell communication: the lead cell sends a secondary (2 8) signal to the trailing
cells, inducing them to follow the lead cell and activating a tubulogenesis
programme. Cells also communicate via Notch-mediated signalling as they
compete for the lead position (inhibition arrow in first panel).
of interest. Homozygous mutant tracheal cells lack the UASi-GFPhp transgene
and so express GFP as well as DsRED. After clone induction, embryos were
returned to 25 8C, and after four or five days the third instar larvae were heatkilled (70 8C for 3–5 s), mounted in 50% glycerol and observed by fluorescence
microscopy to score clone distribution. See Supplementary Methods for
complete descriptions of genotypes and clone analysis.
Tracheal cell counts. Mosaic dorsal branches containing at least one homozygous, GFP-expressing cell were scored for presence of terminal (DB1) cells by
morphological criteria. Cell numbers in mosaic branches and in contralateral
control branches were determined by counting DsRED2nls-labelled nuclei.
Notch experiments. N ts; btl-Gal4, UAS-GFP/þ embryos were collected at 18 8C
for 3 h, aged for 10 h, shifted to the non-permissive temperature (30.5 8C) for 6 h,
then fixed, immunostained and analysed by confocal microscopy. For expression
of NACT throughout the tracheal system, btl-Ga4, UAS-GFP/þ; btl-Ga4, UASCD8GFP/UAS-NACT embryos were raised at 30.5 8C.
Immunostaining and microscopy. Embryo fixation and tracheal staining with
monoclonal antibody 2A12 was performed as described8. For Fig. 4, embryos
were fixed and double-stained with goat anti-GFP (1:200 dilution; Abcam) and
anti-Verm30 (1:600 dilution), and revealed by indirect immunofluorescence with
fluorescein isothiocyanate-conjugated and CY3-conjugated secondary antibodies. Stained embryos and labelled tracheal clones were analysed and photographed with compound or confocal fluorescence microscopes. Where
necessary, a montage of optical sections was assembled (Figs 1c and 4a–c).
Received 30 October 2005; accepted 19 April 2006.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements We thank B. Levi for help with isolation of ‘no mutant
terminal cell’ mutants and for discussion, and S. Artavanis, M. Galko, S. Luschnig
and S. Toering for fly stocks and antisera. This work was supported by an NIH
NRSA fellowship (A.S.G.) and a grant from the N.I.H. M.A.K. is an investigator of
the Howard Hughes Medical Institute.
Author Contributions A.S.G. conceived, designed and performed experiments and
analysed data. M.A.K. advised on the above. A.S.G. and M.A.K. wrote the paper.
Author Information Reprints and permissions information is available at
npg.nature.com/reprintsandpermissions. The authors declare no competing
financial interests. Correspondence and requests for materials should be
addressed to M.A.K. ([email protected])
© 2006 Nature Publishing Group
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