MCSF orchestrates branching morphogenesis in developing submandibular gland tissue

During development, the submandibular gland (SMG) tissue undergoes unique morphological changes (termed branching morphogenesis) that share similarities with changes seen in other tissues, including the lungs, kidneys, mammary glands and pancreas. Branching morphogenesis proceeds via spatiotemporal interactions among a variety of cell types including epithelial and mesenchymal cells, neurons and immune cells (Gumbiner, 1992). These cellular interactions involve both biophysical and biochemical dimensions, including direct cell–cell or cell–extracellular matrix (ECM) contacts (Walker et al., 2008), and autocrine or paracrine effects of growth factors, such as epidermal growth factor (EGF), fibroblast growth factor (FGF) and transforming growth factor β (TGF-β), that induce differentiation of cells as well as changes in ECM properties and composition (e.g. in collagen, glycoprotein, fibronectin and laminin) (Kadoya et al., 1995; Nakanishi et al., 1986; Sakai et al., 2003; Yang and Young, 2008). Extracellular signals activate intracellular signaling pathways [e.g. RhoA and Rho-associated protein kinase, mitogen-associated protein kinase, phosphoinositide 3-kinase (PI3K), and wingless (WNT) signaling pathways] (Porazinski et al., 2015) that further induce specific gene expression and protein synthesis, and/or promote cell polarity, movement and tissue self-assembly. Eventually, epithelial bud formation, tissue clefting and branching, and concomitant differentiation and maturation of epithelial cells into acinar and ductal cells occurs (Patel et al., 2011; Steinberg et al., 2005).

Recent studies have highlighted the importance of immune cells, more specifically that of macrophages, in tissue development and growth, as well as in tissue regeneration (Cecchini et al., 1994; Jones et al., 2013). Macrophages have been detected during branching morphogenesis of glandular-like tissues and it has been shown that they can direct tissue growth (Cecchini et al., 1994; Jones et al., 2013). For instance, during normal mammary development, macrophages directly associate with terminal end buds, lining and directing the developing duct. Comparably, at early stages of pancreatic development, macrophages colonize the duct–islet interface, and their numbers increase steadily as the organ development proceeds (Geutskens et al., 2005; Ingman et al., 2006). In the absence of macrophages, terminal buds in the mammary gland are atrophic and poorly branched, and pancreatic islet cells show abnormal morphology (Banaei-Bouchareb et al., 2004; Gouon-Evans et al., 2000; Shibata et al., 2001; Van Nguyen and Pollard, 2002). These studies have indicated that macrophages, mainly the M2 type, play critical roles in tissue development, as well as in the clearance of apoptotic cells and unnecessary matrix (Davies et al., 2013; Shibata et al., 2001).

In this context, macrophage colony stimulating factor (MCSF, also known as CSF1) has been shown to be the key regulator of M2 macrophage differentiation (Fleetwood et al., 2007; Pyonteck et al., 2013). Nevertheless, since most of previous studies have mainly focused on macrophage colonization using mice presenting Csf1 (the MCSF-encoding gene) gene deletion (i.e. Csf1^op/Csf1^op mice), the roles of macrophages on tissue development could have been overemphasized, and the exact effect of MCSF could have been underestimated. Therefore, we hypothesized that MCSF could have a macrophage-independent function in SMG branching and growth. Indeed, by applying both physical and chemical stimuli, we found that MCSF acts as a regulator of SMG development by modulating the expression of the major growth factors involved in SMG growth, FGF7 and FGF10, via the phosphoinositide 3-kinase (PI3K) pathway. Additionally, we found that MCSF promoted neuronal network development by regulating the levels of the neurotrophic factor neurturin (NRTN) during early SMG development.

We first analyzed the MCSF expression pattern during SMG development in embryonic day (E)12.5 to E15.5 mouse SMG tissues by immunohistochemistry and western blotting. Although MCSF was nearly undetectable at E12.5, MCSF expression levels increased markedly in the mesenchyme around the epithelial buds at E13.5 (Fig. 1A; Fig. S1A). Additionally, in order to understand which cells express the receptor for MCSF (CSF1R, also known as MCSFR and CD115), we also analyzed the expression profile of CSF1R, and found it to be mainly present in the mesenchyme in E13 or E13.5, and both in the epithelium and mesenchyme in E14.5 and E15.5 SMGs (Fig. S1B).

Next, we analyzed the presence of macrophages by immunohistochemical detection for the macrophage marker F4/80 (Schulz et al., 2012) cells in E13.5 and E15.5 SMGs. As shown in Fig. 1B, there were only a few F4/80⁺ cells at E13.5, but their number increased drastically at E15.5. These cells were located in the mesenchyme close to epithelial buds (Fig. 1B). In an attempt to determine the macrophage subtype, we probed for the expression of inducible nitric oxide synthase II (iNOSII, also known as NOS-2) and arginase 1 (ARG1) as markers for M1 and M2 macrophages, respectively (Hesse et al., 2001; Pyonteck et al., 2013). Although iNOSII-positive cells were rare, we could observe a large number of ARG1-positive cells, indicative of an M2 macrophage phenotype (Fig. 1C,D). Taken together, these data show that MCSF expression precedes the appearance of macrophages in the early stages of SMG development, and suggest that MCSF could be inducing the differentiation or migration of macrophages in the SMG tissue in a subsequent step.

Given that the peak in MCSF expression (E13.5) was earlier than that of macrophage numbers (E15.5), we hypothesized that MCSF could regulate SMG growth via macrophage-independent mechanisms or by modulating the function of other cell types. In an attempt to clarify the effects of MCSF on SMG growth and branching, we performed a series of ex vivo tissue culture studies using track-etched membranes (which induce SMG growth) and a hydrogel culture system. Since the mechanical properties of the SMG tissue change over the course of development due to the accumulation and cross-linking of secreted extracellular molecules (Ingber, 2006), we had previously fabricated hydrogels that differed in terms of mechanical stiffness for ex vivo SMG culture, and demonstrated that stiff gels attenuated, whereas soft gels enhanced, SMG growth (Miyajima et al., 2011).

Therefore, we herein cultured E12.5 SMGs (at a stage earlier than the peak in MCSF levels in vivo) on a stiff hydrogel (Young's modulus=184 kPa) as a negative control, and analyzed the effect of different concentrations of MCSF (5, 10, 20 and 50 ng/ml) on SMG growth for up to 72 h. As shown in Fig. 2A,B, SMG branching was enhanced in a dose-dependent manner up to 20 ng/ml of MCSF. However, an inhibitory effect was observed at 50 ng/ml as compared to 5, 10 or 20 ng/ml concentrations, suggesting the occurrence of a regulatory feedback mechanism. Additionally, as expected, MCSF treatment (20 ng/ml) induced a notable increase in the number of F4/80⁺ macrophages in SMG tissue after 72 h of culture (Fig. S2).

To confirm the effects of MCSF on SMG growth, we cultured SMGs on track-etched membranes as a positive control, to allow tissues to grow, and blocked MCSF function with antibodies against MCSF or CSF1R. As shown in Fig. 2C,D, SMGs showed normal bud and cleft formation, comparable to native tissue growth, when cultured with control medium on a track-etched membrane. However, SMG development was strongly suppressed by the supplementation with MCSF or CSF1R antibodies (Fig. 2C,D). Taken together, these results suggest that MCSF has important roles for not only the attraction or differentiation of macrophages, but also as a chemokine that promotes cell differentiation.

To understand the mechanisms underlying the regulation of SMG growth and branching by MCSF, we analyzed the interaction between MCSF and factors that are essential for SMG formation and growth, namely FGF7 and FGF10, which modulate epithelial growth and ductal elongation, respectively (Steinberg et al., 2005). Interestingly, MCSF enhanced the expression of both growth factors, especially that of FGF10 (Fig. 3A–C). Consistent with previous results, neutralization of FGF7 and FGF10 with their specific antibodies significantly suppressed SMG growth (Taketa et al., 2015), which was, however, partially restored by further MCSF supplementation (Fig. 3D,E). This rescue effect of MCSF supplementation was statistically significant in the case of FGF10, which is in accordance with the fact that MCSF could induce the FGF10 levels more prominently. Interestingly, in an opposite experimental design, FGF7 or FGF10 supplementation could not rescue the inhibition of SMG growth induced by the blockade of MCSF function with anti-MCSF antibody (Fig. 3F,G). Taken together, these results indicate that MCSF works as an upstream factor of FGF7 and FGF10.

Additionally, since MCSF could partially rescue the inhibitory effects of FGF7 and FGF10 neutralization, MCSF could possibly stimulate other factors in the mesenchyme that can replace the function of FGF7 and FGF10, or stimulate the epithelial cells directly. In fact, when we applied MCSF directly on epithelium rudiments, we observed that MCSF markedly enhanced SMG epithelium growth and elongation (Fig. 3H,I). These results strongly support the concept of a macrophage-independent function of MCSF on SMG growth.

To further understand the intracellular pathways involved in MCSF-induced upregulation of FGF7 and FGF10, we investigated the activation of PI3K signaling (p85 subunit, also known as PIK3R1), as this pathway has been reported to be downstream of CSF1R (Yavropoulou and Yovos, 2008) and is also implicated in the regulation of FGF expression (Eswarakumar et al., 2005; Larsen et al., 2003). As shown in Fig. 4A,B, MCSF induced activation of PI3K phosphorylation in whole SMG samples. By contrast, MCSF-induced SMG growth was completely suppressed by treatment with LY294002, a PI3K-specific inhibitor (Vlahos et al., 1994) (Fig. 4C,D). The specificity of the chemical inhibitors is a concern in inhibition studies. Based on a previous report (Larsen et al., 2003), LY294002 and Wortmannin are the major PI3K inhibitors, and significantly inhibit the SMG branching morphogenesis. The LY294002 inhibitory effect, however, was nontoxic and reversible, and thus it was selected in the experiments. As shown in Fig. 4E,F, FGF7 and FGF10 expression was downregulated by LY294002 even in the presence of MCSF. Interestingly, after LY294002-treated SMG samples were washed to remove the inhibitor, and the medium was replaced with MCSF-supplemented medium, the SMG growth was completely restored (Fig. 4G,H).

Taken together, these results demonstrate that MCSF-induced FGF expression and SMG growth is dependent on the activation of PI3K signaling.

Since MCSF could be stimulating factors other than FGFs, we hypothesized that MCSF could also have a stimulatory effect on neurons, based on previous reports showing that MCSF induces proliferation of microglia and Purkinje cells (Murase and Hayashi, 1998) and that neurons play a critical role in SMG organogenesis (Knox et al., 2010). Therefore, we cultured SMGs on stiff hydrogels with or without MCSF supplementation and analyzed neuronal tissue growth. In the absence of MCSF, neuronal growth was strongly inhibited and epithelial bud formation was consequently reduced. In contrast, MCSF treatment significantly enhanced neuronal growth, which prompted normal SMG development (Fig. 5A).

Previous reports showed that NRTN is an important neurotrophic factor (Knox et al., 2013; Liu et al., 2007) and that Nrtn^−/− mice present atrophic innervation of salivary glands (Heuckeroth et al., 1999). Therefore, we investigated whether MCSF regulates neural growth by inducing the expression of NRTN. As shown in Fig. 5B, MCSF strongly enhanced NRTN levels in SMG tissue. To determine whether MCSF and NRTN action are interdependent, we cultured SMGs on stiff hydrogels with NRTN alone or in combination with anti-MCSF antibody, and performed the reverse experiments with MCSF and anti-NRTN antibody. NRTN stimulation enhanced SMG growth and branching, but concomitant blockade of MCSF remarkably suppressed the NRTN effect, thereby inhibiting neuronal outgrowth. In contrast, MCSF promoted branching morphogenesis and the formation of a neuronal network, even in the presence of anti-NRTN antibody (Fig. 5C–E). Furthermore, we mechanically isolated the parasympathetic ganglion (PSG) and cultured the PSG on track-etched membrane, and found that MCSF supplementation can enhance neurite growth (Fig. S3). Collectively, these results indicate that MCSF plays a critical role in promoting neuronal growth, in part by inducing the NRTN expression in developing SMGs. Additionally, MCSF works as an upstream factor of NRTN to promote neuronal growth.

Since MCSF plays pleiotropic roles in different cell types during embryo development, Csf1^op/Csf1^op mice are not suitable models to understand the exact effects of MCSF on initial SMG development, which takes place at ∼E11.5. Therefore, we administered anti-MCSF antibody to pregnant mice by intraperitoneal injection starting at E11 for 4 consecutive days (until E14) to neutralize MCSF at early stages of SMG development. Phenotypic analysis showed that there was no difference in body size between embryos isolated from pregnant mice treated with anti-MCSF antibody or injected with phosphate-buffered saline (PBS). However, SMGs from MCSF-neutralized embryos were notably atrophic (Fig. 6A). Histological analysis revealed a loose mesenchymal tissue along with a disconnected intercellular space and lack of a tight interaction between cells in the epithelium and mesenchyme, a defective neuronal network, and fewer epithelial buds and F4/80⁺ cells (Fig. 6B–D). The expression of FGF7, FGF10 and NRTN was also decreased (Fig. S4), ratifying the results of the organ explant experiments (Figs 3,5). These results confirm that MCSF is essential for SMG development and acts by directly regulating FGF7 and FGF10 levels, as well as neuronal network formation and macrophage differentiation.

Finally, we used an in vitro SMG tissue synthesis method (Wei et al., 2005) to confirm the effects of MCSF on the early stages of epithelial bud formation of SMG development. In this experiment, epithelial and mesenchymal cells were first dissociated from whole SMG tissue (E13), and subsequently co-cultured in Matrigel. In the control group, self-assembly and epithelial bud formation were observed within 48 h of culture. After 72 h, numerous buds were detected along with a complex network of neurons. However, when the cells were cultured with anti-MCSF antibody, bud formation was suppressed and the distribution of neurons within the tissue was almost completely abolished (Fig. 7).

Organogenesis, including SMG branching morphogenesis, involves a complex and coordinated sequence of events that regulate the growth, proliferation, differentiation, migration and apoptosis of epithelial and mesenchymal cells. These events are mediated by specific and time-dependent activation of genes and intracellular signaling pathways in response to developmental cues. The importance of immune cells in tissue development and regeneration has been revealed by recent studies (Lilla and Werb, 2010; Reed and Schwertfeger, 2010), especially the trophic and scavenging roles of macrophages (Gouon-Evans et al., 2000). In the present study, we showed that M2 macrophages accumulated in developing SMG tissue at E15.5, which was preceded by an upregulation of MCSF and CSF1R levels at E13.5, suggesting that MCSF could be the main trigger for macrophage polarization during SMG tissue development. More importantly, we demonstrated for the first time that MCSF regulated early SMG development by modulating the expression of FGF7 and FGF10, and neural network development.

FGFs are critical for SMG formation and development; in particular, the temporal regulation of FGF7 and FGF10 in branching morphogenesis is a determinant of SMG formation and growth (Ohuchi et al., 2000; Ornitz and Itoh, 2015). The PI3K pathway has been reported to be involved in FGF7 and FGF10 signal transduction (Eswarakumar et al., 2005, 2005; Larsen et al., 2003). Moreover, previous data have also shown that PI3K is downstream of MCSF and CSF1R (Yavropoulou and Yovos, 2008), and regulates cell proliferation. Thus, consistent with these reports, our results indicate that MCSF binding to CSF1R and subsequent activation of PI3K pathway could be the major mechanism associated with MCSF-induced FGF7 and FGF10 expression.

Alternatively, MCSF or CSF1R could act as a co-receptor for the FGF–FGFR interaction and thereby regulate FGF7 and FGF10 function. Since the major receptors for FGF7 and FGF10, FGFR2IIIb (FGFR2b) and FGFR1b respectively, are expressed throughout the epithelium (Steinberg et al., 2005), a co-receptor would probably be necessary to allow FGF to specifically stimulate the proliferation of SMG cells during branching (Steinberg et al., 2005). In this context, a previous study reported that the mitogenic activity of FGF10 is mainly stimulated, whereas that of FGF7 is inhibited by the proteoglycan heparan sulfate (Igarashi et al., 1998), suggesting that the effects of FGF7 and FGF10 could be regulated by heparan sulfate. In fact, heparan sulfate regulates the activity of FGFs by acting as a co-receptor and enhancing FGF–FGFR binding affinity (Patel et al., 2007; Knelson et al., 2014). Since the secreted form of MCSF is also a glycoprotein (Stanley et al., 1994), it may be possible that MCSF also acts as a co-receptor in the FGF–FGFR interaction and thus regulates FGF7 and FGF10 function at the cell membrane level.

MCSF has potent neuroprotective effects following auditory injury and in neuroinflammation (Smith et al., 2013; Yagihashi et al., 2005). We found that MCSF induces the expression of NRTN, which can explain at least in part the MCSF-induced formation of a neuronal network surrounding SMG tissue. Our findings indicate that NRTN-induced neuronal growth or neuroprotection acts downstream of MCSF, since blocking of NRTN did not completely inhibit MCSF-induced SMG growth. On the other hand, blocking MCSF completely suppressed SMG growth even in the presence of NRTN. It is therefore possible that MCSF-induced neuroprotection could involve other neuroprotective or neurotrophic agents. Alternatively, MCSF could also have a NRTN-independent effect on neurons, and promote their growth, in a similar manner to their effects on microglia and Purkinje cells.

Previous studies showed that, due to alternative mRNA splicing and post-translational modifications, MCSF can either be secreted as a glycoprotein into the circulation or be expressed on the cell surface as a membrane-spanning glycoprotein (Stanley et al., 1994). Interestingly, transgenic expression of cell surface MCSF rescued most of the defects observed in Csf1^op/Csf1^op mice, including growth retardation and defective tooth eruption (Dai et al., 2004), indicating that this is the major isoform responsible for regulating tissue development. Nevertheless, although this report described a restoration of F4/80⁺ macrophage populations in salivary glands to ∼80% of normal levels upon expression of a full-length MCSF-1 transgene (Dai et al., 2004), the importance of these two isoforms in the process of branching morphogenesis was not examined in detail. Further investigation may clarify the different mechanisms of action of the two MCSF isoforms in SMG branching morphogenesis and in the regulation of FGF and NRTN expression.

Although much has been learned from the study of individual signaling pathways that regulate SMG growth and morphogenesis, the interaction between these pathways and how they fit into a hierarchical signaling system has remained obscure. Here, we demonstrated that MCSF plays crucial roles in SMG development. The proposed model for the MCSF role in SMG growth is that MCSF controls branching morphogenesis in both a direct and indirect manner. MCSF binds to the CSF1R receptor present in the epithelium and controls the epithelium growth directly, indicating that it has a macrophage-independent role. Additionally, when MCSF binds with the receptor present in mesenchymal cells, we suggest that it can promote the expression of FGFs via the PI3K pathway; MCSF also promoted neuronal network growth in developing SMG by inducing the expression of NRTN. Finally, since a similar process of branching morphogenesis occurs in other glandular tissues, MCSF could also play important and direct roles in epithelial branching in the lungs, pancreas and mammary glands.

Pregnant ICR mice were purchased from Charles River Laboratories (Yokohama, Japan). Animal procedures strictly adhered to the Guidelines for Animal Experiments of Okayama University and were carried out with the approval of the Animal Use and Care Committee of Okayama University (OKU-2013033).

The preparation of alginate hydrogel sheets has been previously described (Miyajima et al., 2011). Briefly, sodium alginate solution (4% w/w; Wako Pure Chemical Industries, Osaka, Japan) was poured into a mold made from a porous alumina plate; this was soaked in calcium chloride solution (5% w/w)(Nacalai Tesque, Kyoto, Japan) for 1.5 h. The porous alumina mold effectively prevented any unexpected shrinkage of the formed gel because the mold uniformly supplied calcium chloride solution, a cross-linking agent. The obtained alginate hydrogel sheet was washed with ethanol and Milli-Q ultrapure water (Millipore, Billerica, MA) and stored in Dulbecco's modified Eagle's medium with nutrient mixture F-12 (DMEM/F12; Wako Pure Chemical Industries) supplemented with 1% penicillin-streptomycin (Nacalai Tesque) (DMEM/F12/PS) for at least 24 h. The sheet was then cut into small pieces (10×10×1.5 mm) for organ culture.

SMG tissues extracted from E12.5 ICR mouse embryos were placed directly on the alginate hydrogel sheets with a mechanical stiffness of 184 kPa as negative controls, or on Whatman Nuclepore track-etched membranes (13 mm diameter, 0.1 μm pore size) as positive controls (GE Healthcare, Little Chalfont, UK). The SMG culture was carried out in DMEM/F12/PS at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. The medium was replenished every 24 h. Recombinant MCSF (5, 10, 20 or 50 ng/ml), recombinant NRTN (1 ng/ml), recombinant FGF7 (100 ng/ml), recombinant FGF10 (500 ng/ml) or goat anti-NRTN antibody (25 μg/ml; cat. no AF477) (all from R&D Systems, Minneapolis, MN); goat anti-MCSF (25 µg/ml; cat. no sc-1324), rabbit anti-CSF1R (10 µg/ml; cat. no sc-692), or rabbit anti-FGF7 (1 µg/ml; cat. no sc-7882) antibody (all from Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-FGF10 antibody (1 µg/ml; cat. no ABN44, Millipore); or LY294002 hydrochloride (25 μM) (Sigma-Aldrich, St. Louis, MO) were added to the medium depending on the experiment. SMG growth and morphological changes were detected by light microscopy (TE-2000; Nikon, Tokyo, Japan). The number of buds in developing SMGs was counted at 0, 36 or 72 h, or at 0, 24, 48 or 72 h using Image J software (National Institutes of Health, Bethesda, MD). Terminal end bud numbers were obtained from 5–7 SMGs per group, and each experiment was repeated at least three times.

The SMG was isolated from E13 mice. To obtain the epithelium rudiment, the SMG was treated with 4 U/ml Dispase (Roche, Basel, Switzerland) for 5 min at room temperature and then separated into epithelial and mesenchymal tissues using fine forceps. Rudiments were placed inside the Matrigel (BD Biosciences, Bedford, MA) and incubated at 37°C for 72 h in DMEM/F12 containing 10% fetal bovine serum (FBS) with or without 20 ng/ml MCSF supplementation. To obtain the epithelial and mesenchymal cell aggregates, whole glands were enzymatically digested for 10 min using collagenase type I (50 U/ml) (Worthington Biochemical Corporation, Lakewood, NJ) followed by digestion with 0.25% trypsin-EDTA (Sigma-Aldrich) for 5 min at 37°C on a rotary shaker. Tissues were dissociated by gentle pipetting, and single cells were obtained by passing the cell suspension through a 70-µm nylon strainer (BD Falcon, Durham, NC). In vitro branching morphogenesis was reconstituted by placing the epithelial–mesenchymal cell aggregates in Matrigel and incubating at 37°C for 72 h in DMEM/F12 containing 10% FBS, 20 ng/ml EGF (R&D Systems) and 100 ng/ml FGF7 (R&D Systems) with or without 25 µg/ml anti-MCSF antibody. The number of buds per mm² in obtained images was counted at 0, 24, 48 and 72 h using ImageJ software.

For immunohistochemical analysis, whole-mount SMGs or epithelial rudiments and mesenchyme alone were fixed with 4% PFA for 20 min at room temperature and washed with PBS (Takara Bio, Otsu, Japan) containing 1% bovine albumin serum (Nacalai Tesque) and 0.1% Triton X-100 (Sigma-Aldrich) (PBSX). Tissue samples were blocked with Blocking One Histo (Nacalai Tesque) and incubated with the following primary antibodies diluted in PBSX: rat anti-F4/80 (1:100; cat. no MCA497, AbD Serotec, Kidlington, UK), rabbit anti-E-cadherin (1:500; cat. no ab76055, Abcam, Cambridge, UK), mouse anti-β-III-tubulin (Tubb3; 1:1000; cat. no MAB1195, R&D Systems), rabbit anti-CSF1R (1:500; cat. no sc-692), rabbit anti-ARG1 (1:500; cat. no sc-101199) and goat anti-MCSF (1:150; cat. no sc-1324) (all from Santa Cruz Biotechnology), and rabbit anti-iNOSII (1:500; cat. no ABN26, Millipore), as well as fluorescein isothiocyanate-conjugated lectin from Arachis hypogaea (1:200) (Sigma-Aldrich). Antibody binding was detected with Alexa Fluor-conjugated secondary antibodies (Life Technologies, Grand Island, NY), and specimens were imaged by confocal microscopy (C1; Nikon).

For western blot analysis, samples were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes using a Mini Trans-Blot system (Bio-Rad, Hercules, CA). Western blotting was performed according to standard procedures using primary antibodies against the following proteins: FGF7 (1:1000; cat. no sc-7882) and MCSF (1:500; cat. no sc-1324) (Santa Cruz Biotechnology); FGF10 (1:2000; cat. no ABN44, Millipore); NRTN (1:1000; cat. no AF477, R&D Systems); PI3K p85 (1:1000; cat. no 4292) and phospho-PI3K p85 (1:1000; cat. no 4228) (Cell Signaling Technology, MA). After incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, protein bands were visualized using Luminata Forte Western HRP substrate (Millipore) and a charge-coupled device-type imager (Image Quant LAS 4000 mini; GE Healthcare) according to the manufacturers' instructions. The blots shown in the figures are representative of three experimental repeats.

The procedure for the and amount of the antibody administration was as described previously (Wei et al., 2005) but with modifications. Anti-MCSF antibody (R&D Systems) (1.5 µg/g of body weight/day) diluted in PBS was administered twice daily for 4 consecutive days to timed pregnant ICR mice (E11–E14) by intraperitoneal injection. PBS (200 µl) was injected as a negative control. At day E14.5, SMG samples were collected, fixed, and processed for analysis. The experiment was repeated three times with similar results.

All SMG data were obtained and mean values with standard deviations were calculated. Statistical significance was taken as P<0.05 as determined with a Student's t-test or one-way ANOVA, with Scheffe's F test, when appropriate.