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First published online 26 January 2006
doi: 10.1242/dev.02261
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1 Department of Molecular, Cell and Developmental Biology University of
California, Santa Cruz Santa Cruz, CA 95064, USA.
2 Merck Research Laboratories, 126 East Lincoln Avenue, Rahway, New Jersey
07065, USA.
3 Genentech, Incorporated, 1 DNA Way, South San Francisco, CA 94080, USA.
Author for correspondence (e-mail:
hinck{at}biology.ucsc.edu)
Accepted 21 December 2005
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Slit, Robo, Netrin, Neogenin, Mammary, Mouse
|
INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mammary gland undergoes an elaborate and regulated morphogenesis,
establishing a tree-like epithelial structure
(Silberstein, 2001
). This
epithelium is a bi-layered tube comprising an outer layer of MECs that
ultimately contracts under the influence of hormones to squeeze milk into a
central lumen from an inner layer of secretory epithelial cells
(Fig. 1A). Before birth, only a
simple ductal structure forms, which is transformed during puberty into a
mammary tree by the process of ductal elongation, EB bifurcation and secondary
branching. This morphogenesis requires the continuous addition of new cells to
both layers, coordinated with the simultaneous formation of a lumen. The
enlarged termini of ducts, termed EBs, are responsible for both growth and
primary structure of the gland (Fig.
1A). Growth is driven by proliferation of a single layer of
multi-potent progenitor cap cells at the tip of the bud, and by the underlying
LECs. Cap cells differentiate into MECs, generating the outer tubular layer,
and, at the same time, LECs are remodeled, generating a hollowed lumen from a
relatively solid mass of cells present in the EB.
These modifications in shape and form of the gland during ductal
morphogenesis require coordinated cellular interactions. In generating this
double-layered structure, cells interact with each other. Proteins that may
play a role in mediating these interactions are E- and P-cadherins that are
expressed by the LECs and cap/MECs, respectively, and may mediate interactions
between cells within a given layer (Daniel
et al., 1995; Radice et al.,
1997). Interactions between the cell layers, at least in the EB,
are provided by the secreted cue netrin 1 (Ntn1), which is expressed
by LECs, and binds its receptor neogenin (Neo1), present on the
surface of cap cells (Srinivasan et al.,
2003). Loss-of-function mutations in Ntn1 and
Neo1 result in disorganized EBs, characterized by inappropriate
spaces between cap and LEC layers. Significantly, this disorganization does
not extend into the ducts, which appear normal
(Srinivasan et al., 2003).
Consequently, the identity of the adhesion system, if any, that mediates
interactions between the MEC and LEC bi-layers during ductal morphogenesis is
uncertain. Desmosomal constituents are present during postnatal mammary gland
development, but they appear diffuse within cells and are not organized into
mature junctional structures (Dulbecco et
al., 1984; Nanba et al.,
2001). This suggests that desmosomal components of cell adhesion
are held in store and their assembly is delayed during development, perhaps
allowing flexible movement of cells during tissue extension and tube
formation. Later, in the mature gland, adhesion between MECs and LECs is via
desmosomes that provide strong adhesion to maintain tissue architecture
(Runswick et al., 2001).
SLITs, like NTNs, are well known guidance proteins, acting as cues to
direct neurons and their axons to targets during neural development. Although
SLITs and NTNs are structurally dissimilar, they share some of the same
characteristics. They are both proteins that, although secreted, are not
freely diffusible, but instead are immobilized in association with cell
membranes or components of the extracellular matrix
(Kappler et al., 2000;
Zhang et al., 2004). Both act
as bifunctional cues, capable of eliciting attractive and repulsive behaviors
from cells expressing their receptors
(Colamarino and Tessier-Lavigne,
1995; Englund et al.,
2002; Kennedy et al.,
1994; Kidd et al.,
1999; Kramer et al.,
2001). Furthermore, both have receptors that contain extracellular
immunoglobulin domains and fibronectin type III repeats. In many proteins,
these motifs act adhesively. Consequently it is not surprising that, in
addition to their chemotropic guidance functions, SLITs and NTNs act
chemotactically at close range to positively and negatively modulate cell-cell
and cell-ECM interactions (Deiner et al.,
1997; Kang et al.,
2004; Simpson et al.,
2000; Srinivasan et al.,
2003).
Here, we demonstrate a functional role for SLIT2 and its ROBO1 receptor
during mammary gland morphogenesis. We show that SLIT2 is distributed
throughout the epithelial compartment, whereas ROBO1 expression is restricted
to cap/MECs. The analysis of glands carrying loss-of-function mutations
reveals similar defects, suggesting this ligand/receptor pair function in the
same pathway. Slit2-/- and Robo1-/-
EBs display inappropriate spaces between the cap and LEC layers, a defect
reminiscent of the phenotypes observed in outgrowths deficient for
Ntn1 and Neo1
(Srinivasan et al., 2003).
Consequently, we generated glands with homozygous deletions in both
Slit2 and Ntn1, and observe, in addition to defects in EB
structure, a synergistic strengthening of the single-mutant phenotypes,
characterized by severe ductal abnormalities that appear to stem from
insufficient adhesion between MECs and LECs. In vitro assays confirm that
Slit2-/-; Ntn1-/- cells are severely
compromised in their ability to form bi-layered organoids, and this deficiency
is rescued by addition of purified SLIT2. Addition of NTN1 does not rescue on
its own, but its addition with SLIT2 dramatically enhances both the number and
size of bi-layered organoids generated, confirming a strong synergism between
NTN1 and SLIT2. These results identify a novel, short-range function for SLIT2
as an adhesive cue acting through its ROBO1 receptor during ductal
morphogenesis. Furthermore our results support a model in which dual `axon'
guidance systems (SLIT2/ROBO1 and NTN1/NEO1) mediate interactions between
cells to preserve the structure of the gland during periods of rapid growth
and morphogenetic modeling.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Leighton et al., 2001;
Plump et al., 2002;
Long et al., 2004). Double
mouse mutants were generated by crossing double heterozygotes.
Transplant techniques
Mammary anlage was rescued from E16-20 embryos and transplanted into
precleared fat pads of athymic nude females
(Robinson et al., 2000).
Tissue fragments from the resulting outgrowths were transplanted into
precleared hosts to generate null and wild-type tissue controls
(Srinivasan et al., 2003).
Tissue analysis
Whole gland preparations were stained for ß-galactosidase activity as
described (Brisken et al.,
1999). Phenotypes were characterized on 6 µm longitudinal
serial sections stained with anti-smooth muscle actin (SMA) and counterstained
with Hematoxylin. Standard error was reported when data from multiple
transplant lines were pooled in penetrance and expressivity studies.
Expression studies
The promoters for Slit1, Slit3, Robo1 and Robo2 drives
the expression of lacZ. Their expression was assessed by whole gland
ß-galactosidase staining (Brisken et
al., 1999). The Slit2 promoter drives the expression of
GFP. Expression was assessed by anti-GFP immunohistochemistry.
Immunohistochemistry
Tissue was fixed in 4% paraformaldehyde. Paraffin embedded tissue was
sectioned at 6 µm and mounted serially. The following antibodies were used
for analysis: anti-SMA, 1:500; anti-laminin-1, 1:50 (Sigma); anti-E-cadherin,
1:500 (BD Transduction Labs); anti-GFP, 1:50 (Molecular Probes); anti-Robo1,
1:250 (DUTT1. gift from Pamela Rabbits); and anti-SLIT2, 1:25 (SCBT). Standard
protocols were followed and Vector ABC kits used for amplification.
RT-PCR of mouse mammary glands
Mammary glands from Robo1+/+ 5-week-old female mice
were used and total RNA was prepared using a Total RNA Purification System
(Invitrogen). cDNA was made from total RNA using iScript cDNA Synthesis Kit
(BioRad) and reverse-transcribed. Robo1 and Dutt1 specific
primers were generated as described (Clark
et al., 2002).
Rotary cultures
Primary mammary epithelial cells were prepared from mild collagenase and
dispase digestion, as described (Darcy et
al., 2000). Differential trypsinization was performed to separate
MECs from LECs. These fractions were combined (4 MECs: 1 LEC) and rotated at
60 rpm at 37°C, 5% CO2 in media at 106 cells/ml.
SLIT2 and NTN1 were added prior to rotation. Rotary aggregates were fixed in
4% paraformaldehyde and sectioned for immunostaining (30 µm cryosections)
using a MOM kit (Vector Laboratories). Aggregates were categorized based on
size and whether none or one (or more) MECs were present. At least 10
aggregates were counted for each experiment.
Ductal phenotype quantification
EB arrays were embedded in paraffin and longitudinal 6 µm serial
sections were immunostained for SMA to delineate the MECs layer. Slides with
EBs displaying the null phenotype and with subtending ducts that could be
followed in serial section were analyzed. Phenotype severity was categorized
as sporadic loss of LECs if discontinuous lengths from 20-50 µm were
present or as epithelial separation if lengths from 40-1000 µm of intact
LECs were detached from the MEC layer. Ducts with more than one aberration
were scored with the most severe phenotype.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Park et al., 2003), we did not
examine this family member, nor did we pursue Rig1(Robo3) as
it probably functions as a negative regulator of SLIT responsiveness rather
than as a signaling receptor (Sabatier et
al., 2004). We found using immunohistochemistry that SLIT2 is broadly distributed in wild-type (+/+) tissue throughout the epithelial compartment during the period of ductal outgrowth (5 weeks). It is present in and around both cap and LECs in the EB (Fig. 1B). Little or no background immunostaining was observed in Slit2-/- outgrowths (Fig. 1C). To identify the cells that express Slit2, we took advantage of the expression of GFP under the control of the endogenous promoter in mice targeted for Slit2 and assayed for GFP expression using immunohistochemistry. At 5 weeks of age, we observed GFP expression in Slit2-/- tissue in cap cells of the EB (Fig. 1D, arrowheads) and in LECs (Fig. 1D, arrows). Along the duct, we also observed GFP immunostaining in both MECs (Fig. 1F, arrowheads) and LECs (Fig. 1F, arrow). As this immunostaining was performed on knockout tissue, the morphological structure of the EB and duct was abnormal; these defects are described in detail in the next section. Wild-type (+/+) control tissue displays the normal EB structure, and we observed little or no background GFP staining, indicating that the detection method was specific (Fig. 1E,G). Next, we examined the expression of other Slit family members. We did not detect Slit1 expression at any stage (data not shown). By contrast, taking advantage of the expression of lacZ under the control of the endogenous promoter in mice targeted for Slit3, we observed ß-galactosidase staining in MECs (Fig. 1H, arrowheads) and ductal LECs (Fig. 1H, arrow) of the mature virgin Slit3-/- gland, and during pregnancy in LECs and MECs of alveoli (Fig. 1I). Slit3, however, was not expressed during ductal outgrowth. Moreover, no morphological defects were detected in Slit3-/- mammary glands (P.S., G.S. and L.H., unpublished), probably owing to the presence of residual SLIT2, which is expressed early and may compensate for a lack of SLIT3 later in development.
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), we determined that the Dutt1 isoform is
specifically expressed in the developing mammary gland by performing RT-PCR
analysis on mRNA (Fig. 2E). We
also examined Robo2 expression by staining
Robo2+/- tissue, and, similar to Slit3, found it
was not expressed during ductal outgrowth (data not shown), but was expressed
later in development in a subset of ductal MECs in the mature virgin animal
(Fig. 2F, arrowheads) and
during pregnancy in alveoli (Fig.
2G, arrows).
Loss of either Slit2 or Robo1 results in abnormal EBs that are morphologically similar to Ntn1-/- and Neo1-/- EBs
To investigate the role of SLIT proteins in mammary gland development, we
analyzed glands carrying loss-of-function alleles of Slit2 and
Robo1. The perinatal lethality of the Slit2-/-
mutation prevented the study of mammary glands in mice carrying the homozygous
mutation. Consequently, we followed standard protocols and harvested mammary
anlage from Slit2-/- embryos and transplanted the tissue
into fat pads of immunocompromised mice that had been cleared of their
epithelial tissue (Robinson et al.,
2000; Young,
2000). In all studies, littermate control +/+
outgrowths were generated on the contralateral side for comparison, ensuring
that the +/+ and -/- outgrowths were subjected to the
same systemic environment.
We sectioned Slit2-/- outgrowths 2-3 weeks
post-transplantation to analyze EBs as whole-mount analysis revealed no
obvious morphological defects (data not shown). The sections were stained with
an SMA antibody to visualize cap and MECs
(Fig. 3A-G). Compared with an
EB from control outgrowths (Fig.
3A), which displayed close apposition of the cap and LEC layers,
Slit2-/- EBs displayed severe abnormalities. In
Slit2-/- EBs, there were significant gaps between the LECs
and cap cells, creating exaggerated subcapsular spaces, ranging from 40-50
µm compared with the 0.1-1 µm space typically observed in +/+
EBs (Fig. 3, compare A and B). Frequently, there were dissociated cells present in this space and
immunostaining with anti-SMA identified these as cap cells
(Fig. 3B). The appearance of
shrunken nuclei in many of these detached cells suggested that they were
apoptotic and probably dying by anoikis, similar to the detached cells present
in Ntn1-/- glands
(Srinivasan et al., 2003). In
some EBs, we observed regions where LECs were completely detached from cap
cells, leaving an intact cap cell layer devoid of underlying LECs
(Fig. 3C, between arrows).
These single layers of cap cells commonly folded inwards, resulting in
double-layered invaginations that disorganized the underlying LECs and
occluded the inner luminal space (Fig.
3D, between arrowheads). The phenotype was 100% penetrant with
60% of the EBs in every outgrowth affected (59.1±26.5%,
n=98 EBs, 12 outgrowths).
A similar analysis was performed on Robo1-/- glands. As
mice carrying the Robo1 mutation were viable, intact glands were
examined, although we confirmed that a similar phenotype was present when
Robo1-/- tissue was transplanted (data not shown). For
these experiments, glands from +/+ littermates served as control
tissue. EBs in Robo1-/- glands were disorganized,
displaying a phenotype that was indistinguishable from the phenotype displayed
in Slit2-/- EBs, characterized by subcapsular spaces,
invaginated cap cell layers and disorganized LECs
(Fig. 3F,G). As is the case for
Slit2-/- outgrowths, the penetrance of the phenotype was
100% with 60% of the EBs in every EB array affected (62±23%,
n=74 EBs, four outgrowths).
A noteworthy aspect of the defects observed in Slit2-/-
and Robo1-/- EBs was their striking similarity to the
defects observed in Ntn-/- and Neo1-/-
EBs (Srinivasan et al., 2003).
One major similarity was that EBs from each homozygous null animal exhibited
loss of adhesion between the LEC and cap layers, with dissociated cap cells
present in the resulting subcapsular space
(Fig. 3B,F). These shared
defects created the impression that Ntn1-/-,
Neo1-/-, Slit2-/- and
Robo1-/- phenotypes were identical, but we detected at
least one unique characteristic in Slit2-/- and
Robo1-/- EBs. The cap cell layer of
Slit2-/- and Robo1-/- EBs folded into
the LEC compartment (Fig.
3D,G), and this was never observed in Ntn1-/-
and Neo1-/- glands
(Srinivasan et al., 2003).
Despite this difference, the overall appearance of EBs from each knock-out
suggested the defects were due to a general loss of cell-cell adhesion between
cap and LEC layers (Srinivasan et al.,
2003). EBs are highly proliferative structures that undergo active
remodeling and are consequently more likely to be sensitive to impaired cell
adhesion. As we discovered disorganization in this sensitive structure when
either NTN/NEO or SLIT2/ROBO1 signaling system was lost, we expect, if these
guidance systems functionally compensate for one another, that loss of both
systems simultaneously will lead to disrupted cell contacts in more stable
regions of the gland that are insensitive to the loss of either system
alone.
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) but
not in Slit2-/- and Robo1-/- EBs. As
cap cells make and secrete the basal lamina, the loss of their integrity in
the Slit2-/-;Ntn1-/- EBs may
destabilize the laminin network surrounding the EB as it does in the
Ntn1-/- EBs
(Srinivasan et al., 2003). To
examine this possibility, we immunostained Slit2-/-,
Robo1-/- and
Slit2-/-;Ntn1-/- outgrowths with
laminin 1 antibody (Fig. 5),
the major laminin in the basal lamina
(Klinowska et al., 1999). As
expected, there were clear breaks in the basal lamina in the
Slit2-/-;Ntn1-/- EBs
(Fig. 5E)
(Srinivasan et al., 2003),
whereas the basal lamina in the Slit2-/- and
Robo1-/- EBs was intact
(Fig. 5A,C). The phenotype
observed in Slit2-/-;Ntn1-/-
outgrowths was 100% penetrant with 80% of the EBs affected
(82.6±15.4% n=103 EBs, eight outgrowths). This represented an
20% increase in expressivity compared with the expressivity exhibited in
Slit2-/- EBs (Fig.
3) or Ntn1-/- EBs
(Srinivasan et al., 2003).
Together with the observation that the
Slit2-/-;Ntn1-/- phenotype was more severe in
EBs (Fig. 4) and ducts (see
below) compared with the phenotype exhibited in outgrowths carrying single
homozygous null alleles, our analysis suggests that NTN/NEO and SLIT/ROBO
pathways function in parallel to mediate contacts between epithelial cell
layers. In addition to abnormal EBs, the ducts of Slit2-/-;Ntn1-/- glands displayed severe adhesion defects. Compared with the ductal structure displayed in +/+ tissue, which illustrates the typically close apposition between LEC and MEC layers (Fig. 6D), SMA immunostaining of double homozygous deficient outgrowths showed significant loss of adhesion between these layers (Fig. 6A,B). In the mildest form, a few LECs were sporadically detached from the MEC layer (Fig. 6A, arrows). A more severe defect was observed when this modest detachment of cells expanded to encompass substantial lengths of the duct, with the LEC layer essentially peeled away from the MEC layer (Fig. 6B, double arrowheads). Interestingly, the MECs (Fig. 6) and basal lamina (Fig. 5B,D,F) along the ducts were intact in Slit2-/-;Ntn1-/- and in Slit2-/- and Robo1-/- ducts, suggesting that loss of adhesion occurs within the duct, affecting the adhesion between MEC and LEC layers. The contralateral control glands never displayed abnormal tissue morphology.
|
).
This loss in adhesion extends into the duct in Slit2-/-,
but not Ntn1-/-, outgrowths
(Fig. 6E,G). The subtle role
for NTN1 in ductal morphogenesis is only revealed by the additional removal of
Ntn1 in a Slit2-/- background which increases the
severity of the ductal defects (Fig.
6A,B). This suggests that, while SLIT2 plays a primary role in
mediating interactions between LEC and MEC layers during ductal morphogenesis,
NTN1 synergistically contributes to these interactions.
SLIT2 and NTN1 mediate contacts between LEC and MEC layers
In the nervous system, studies have shown that SLIT/ROBO signaling
inactivates N-cadherin-mediated adhesion
(Rhee et al., 2002). As we
observe adhesive defects predominantly in the LEC compartment of
Slit2-/-;Ntn1-/- outgrowths where E-cadherin
has been shown to mediate interactions
(Daniel et al., 1995), we
performed immunohistochemistry using anti-E-cadherin. We observed, as
expected, robust membrane staining around LECs on +/+ control
glands. We also observed similar robust immunostaining between LECs of
Slit2-/-;Ntn1-/- EBs and ducts (see Fig. S1A-D
in the supplementary material), indicating that cadherin-mediated contacts are
not altered in double homozygous null tissue.
Next, we focused on proteins that mediate the interaction between LEC and
MEC layers. Although desmosomal components mediate these contacts in mature
ducts (Runswick et al., 2001),
they are not assembled during branching morphogenesis
(Dulbecco et al., 1984;
Nanba et al., 2001).
Consequently, we entertained the possibility that contact between layers was
mediated, either directly or indirectly, by SLIT2 and NTN1. We investigated by
using an in vitro aggregation assay to examine whether primary cells,
harvested from Slit2-/-;Ntn1-/- outgrowths,
were impaired in their ability to generate bi-layered epithelial structures.
Previous studies have shown that mixtures of wild-type, primary mammary cells
form aggregates of LECs surrounded by single layers of MECs
(Runswick et al., 2001). This
appears to be a timed aggregation with LECs forming clusters that MECs attach
to and surround. In agreement with the previous study, we confirmed that
wild-type mammary cells form bi-layered organoids
(Fig. 7A).
Next, we generated aggregates from
Slit2-/-;Ntn1-/- cells and observed that,
although LEC aggregates formed, few were surrounded by MECs. To quantify this
assay, we considered a structure bi-layered with one or more MECs surrounding
the LEC aggregate. Even with this lenient definition, we found that 70% of
Slit2-/-;Ntn1-/- aggregates lacked a bi-layer
(Fig. 7B). Of the 30%
Slit2-/-;Ntn1-/- aggregates categorized as
having a bi-layer, many contained just one or only a few MECs on the outer
surface, compared with +/+ aggregates, which generally displayed
fully formed MEC layers (Fig.
7A). A second characteristic of
Slit2-/-;Ntn1-/- aggregates was that they
appeared smaller than wild-type aggregates. To quantify aggregate size, we
counted the number of cells comprising each and categorized them as: fewer
than 10 cells, between 10 and 20 cells, and greater than 20 cells
(Fig. 7K). All wild-type
organoids contained greater than 10 cells and the majority contained greater
than 20. By contrast, the majority of aggregates composed of
Slit2-/-;Ntn1-/- cells was small (<10
cells), consistent with a role for the MEC layer, which does not form when
Slit2-/-;Ntn1-/- aggregate, in stabilizing
clustered LECs in rotary culture. We never observed MECs inappropriately mixed
into LEC aggregates, a situation that occurs when desmosomal adhesion in
disrupted (Runswick et al.,
2001). Thus, although
Slit2-/-;Ntn1-/- LECs aggregated properly,
Slit2-/-;Ntn1-/- MECs were severely deficient
in their ability to adhere to the outside of LEC aggregates even though they
express ROBO1 and NEO1 (Fig. 2)
(Srinivasan et al., 2003).
|
|
). At
3 µg/ml and 6 µg/ml of SLIT2, 40% of the aggregates were bi-layered, a
number that increased to 70% and 100%, respectively, with the addition of NTN1
(Fig. 7C-F). The observation
that NTN1 acts in concert with SLIT2 to rescue bi-layered aggregation in vitro
is consistent with the phenotypes observed in vivo. A ductal phenotype is
observed in Slit2-/-, but not Ntn1-/-
outgrowths, and the defects are synergistically strengthened by the additional
loss of Ntn1 in a Slit2-/- background
(Fig. 5). Thus, SLIT2 and NTN1
function in concert during mammary ductal morphogenesis to generate tubular
bi-layers by mediating interactions between distinct epithelial layers. |
DISCUSSION |
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|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Members of functionally-related families act synergistically during mammary ductal morphogenesis
During mammary ductal development, only the Slit2 and
Robo1 members of these gene families are expressed in the gland.
Although secreted SLIT2 protein is widely distributed throughout the
epithelial compartment, ROBO1 is expressed specifically by cap and MECs (Figs
1,
2). Glands harboring
loss-of-function mutations in either gene exhibit similar phenotypes, strongly
supporting a model in which SLIT2 signals through ROBO1 at this stage of
development. Defects in Slit2-/- and
Robo1-/- glands are confined to the EB and, although
relatively modest, are consistent with the loss of stabilizing interactions
(Fig. 3).
Slit2-/- and Robo1-/- EBs exhibit a
general disorganization in cell contacts with inappropriate spaces forming
between the cap and LEC layers. Dissociated cells fill these subcapsular
spaces and layers of cap cells fold inwards, occluding lumenal space. Taken
together, these results suggest that SLIT2-mediated activation of ROBO1 is
required to maintain the proper positioning of cap and LEC layers in the
EB.
Our previous studies have already implicated the NTN/NEO guidance system in
maintaining proper positioning of cap cells at the leading edge of the EB
(Srinivasan et al., 2003). We
proposed that NTN1, expressed by LECs, acts adhesively as a short-range
attractant to maintain the position of NEO1-expressing cap cells. The
similarity in the defects exhibited by Ntn1-/- and
Slit2-/- EBs prompted us to investigate the consequences
of genetically interrupting the expression of genes encoding both guidance
cues. We show that loss of Ntn1 in a Slit2-/-
background results in synergistic strengthening of the single-mutant
phenotypes (Figs 4,
5,
6). Moreover addition of NTN1
synergistically enhances the ability of SLIT2 to rescue bi-layered aggregation
of Slit2-/-;Ntn1-/- mammary cells
(Fig. 7). Our results are
consistent with a model in which two different guidance systems act in
parallel to mediate interactions between distinct epithelial cell types during
organ development, although we have not excluded the possibility that some
crossregulation between these systems occurs
(Stein and Tessier-Lavigne,
2001).
SLIT2 signals through ROBO1 as a short-range adhesive cue
Our experiments support a positive role for SLIT2 in the developing gland.
First, the loss of cell-cell interactions observed in
Slit2-/- and Robo1-/- EBs is
consistent with the loss of a stabilizing interaction
(Fig. 3). Second, we observed
that simple addition of purified SLIT2 rescues the ability of
Slit2-/-;Ntn1-/- cells to form
bi-layered organoids (with NTN1 contributing synergistically). As SLIT2 is not
presented in a way that provides directional information, its role in the
gland appears to be adhesive, which is different from the guidance role that
SLITs play in the nervous system (Fig.
7). This type of short-range adhesive role has already been
proposed for NTN1 and NEO1 (Srinivasan et
al., 2003). At least two models that are not mutually exclusive
can be proposed for the mechanism by which SLIT2/ROBO1 (and NTN1/NEO1) mediate
cell-cell adhesion between epithelial cell layers. One model is that SLIT2
acts directly as an adhesive factor, binding ROBO-expressing cap/MECs and
stabilizing the interaction between these two layers. As SLIT2 is secreted
(Fig. 1F), this model requires
that it associates with LECs. Association probably occurs via heparin sulfate
proteoglycans that have been shown to bind, concentrate and stabilize SLITs
(Ronca et al., 2001;
Steigemann et al., 2004).
Candidate proteoglycans on LECs are glypican and syndecan
(Delehedde et al., 2001), but
there may also be a requirement for proteoglycans on receptor expressing MECs
as genetic studies in Drosophila have shown that syndecan serves as a
necessary co-receptor for ROBO in transducing the SLIT signal
(Steigemann et al., 2004).
The second model is that SLIT2 signals through ROBO1 to affect cell
adhesion indirectly by modulating the expression or function of other cell
adhesion proteins. A candidate cell adhesion protein that could be the target
of SLIT/ROBO signaling is Ep-CAM, which mediates Ca2+ independent,
homotypic cell-cell adhesion (Balzar et
al., 1999a; Balzar et al.,
1999b). One problem with this candidate is that Ep-CAM, like
E-cadherin (see Fig. S1 in the supplementary material), mediates adhesion
between individual LECs and not between LEC and MECs, the contacts of which
appear disrupted in Slit2-/-;Ntn1-/-
glands. Candidates that mediate interactions between LEC and MECs, such as
desmoglein or desmocollin, function in the mature gland
(Runswick et al., 2001), and
although they are present in the developing gland, they have not yet formed
adhesive junctional complexes (Dulbecco et
al., 1984; Nanba et al.,
2001). Consequently, we favor the first model in which SLIT2 and
ROBO1 act directly as cell-adhesion proteins, but currently our data do not
rule out the second model. To distinguish between these two models, we are
currently investigating the signaling events downstream of SLIT2/ROBO1 and
NTN1/NEO1.
Most studies on SLIT/ROBO signaling have focused on its inhibitory and
chemorepulsive influence on cell migration and axon guidance, although there
are a few examples that demonstrate the outgrowth promoting and
chemoattractive activities of SLIT. For example, human vascular endothelial
cells are attracted to SLIT-expressing tumors
(Wang et al., 2003), and
Drosophila mesodermal cells are attracted to SLIT-expressing muscle
attachment sites (Kramer et al.,
2001). SLITs are also positive regulators of elongation and branch
formation for both rat sensory neurons and Drosophila tracheal cells
(Englund et al., 2002;
Wang et al., 1999). Although
none of these studies demonstrate SLIT acting to increase cell-cell
interactions at short range, the process of guidance at long-range must
involve a series of local interactions as cells or axons move up a gradient
towards the source of cue. Similarly processes such as branch formation must
involve local interactions as a restricted portion of the target membrane
preferentially protrudes and becomes stabilized. In the mammary gland, our
data suggest that SLIT2, which is secreted by target cells, is available on
cell surfaces in the epithelium where it interacts with ROBO1 present on the
surface of cap/MECs. Their interaction maintains tissue architecture and
restricts inappropriate intermingling by mediating contacts between distinct
epithelial cells layers.
These examples establish positive roles for SLITs and ROBOs in cell
migration, branch formation and interepithelial interactions, but studies on
the repellent activity of SLITs have supplied details concerning the
mechanisms by which the SLIT/ROBO signal is transduced. The intracellular
domain of ROBO1 contains four motifs that have been shown to interact with a
number of signaling proteins, including the actin binding protein ENABLED
(murine MENA) (Bashaw et al.,
2000; Yu et al.,
2002), the nonreceptor Abelson tyrosine kinase, c-ABL
(Bashaw et al., 2000), the
adaptor DOCK (Fan et al.,
2003) and the GTPase-activating protein srGAP1
(Wong et al., 2001). All these
signaling proteins are candidates for mediating the attractive and adhesive
activities of ROBO. Indeed, before their roles as negative regulators of ROBO
signaling were revealed, DOCK was identified as a positive regulator in axon
outgrowth and synapse formation (Desai et
al., 1999; Garrity et al.,
1996), and MENA was shown to promote actin dependent motility
(Krause et al., 2002). All
these signaling proteins are also candidates for mediating the interaction of
ROBO1 with the cytoskeleton, leading to changes in the mobility or
adhesiveness of cells.
Concluding remarks
Our discovery that members of functionally related families act in similar
ways during development suggests an explanation for the observation that
single loss-of-function mutations and even multiple loss-of-function mutations
in family members of genes encoding `axon guidance' cues have failed to yield
phenotypes in many vertebrate organ systems. For example, lungs of embryos
carrying loss-of-function alleles in both Ntn1 and Ntn4
display no apparent phenotype, even though treatment of lung explants in vitro
with either NTN1 of NTN4 dramatically reduces lung bud formation
(Liu et al., 2004). Similarly,
early vascular development in embryos carrying loss-of-function alleles in
Ntn1 appears normal, even though treatment of vascular smooth muscle
cells and endothelial cells with NTN1 stimulates proliferation, induces
migration and promotes adhesion of these cells
(Park et al., 2003). In our
studies, the phenotypes exhibited in glands harboring homozygous deletions of
either Slit2 or Netrin1 are relatively mild and largely
confined to the highly specialized EB (Fig.
3) (Srinivasan et al.,
2003). A more dramatic phenotype required deletion of both these
guidance cues as they both appear to mediate adhesive, short-range
associations between neighboring cell types (Figs
4,
5). Taken together, these
results suggest that deciphering the actions of `axon guidance' cues that
function in similar ways (e.g. adhesively during organ development) will
require the analysis of compound homozygous null animals to eliminate the
expression of more than one member of functionally related families. Future
studies in the mammary gland and other organ systems may elucidate other
combinations of Netrin, Slit, Semaphorin or Ephrin proteins that function
synergistically to mediate cell contacts during development.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/5/823/DC1
* These authors contributed equally to the work 
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