Normal mammary gland development after MMTV-Cre mediated conditional PAK4 gene depletion

p21-activated kinases (PAKs) are serine/threonine kinases functioning as downstream effectors of the small GTPases Rac1 and Cdc42. Members of the PAK family are overexpressed in human breast cancer, but their role in mammary gland development is not fully explored. Here we examined the functional role of PAK4 in mammary gland development by creating a mouse model of MMTV-Cre driven conditional PAK4 gene depletion in the mammary gland. The PAK4 conditional knock-out mice were born healthy, with no observed developmental deficits. Mammary gland whole-mounts revealed no defects in ductal formation or elongation of the mammary tree through the fat pad. PAK4 gene depletion also did not alter proliferation and invasion of the mammary epithelium in young virgin mice. Moreover, adult mice gave birth to healthy pups with normal body weight upon weaning. This implies that MMTV-Cre induced gene depletion of PAK4 in mice does not impair normal mammary gland development and thereby provides an in vivo model that can be explored for examination of the potential function of PAK4 in breast cancer.

www.nature.com/scientificreports www.nature.com/scientificreports/ mice caused embryonic lethality with severe defects in the heart, brain, and vasculature of the animals 19 ; However, the role of PAK4 in mammary gland development has not been investigated. Due to the early embryonic lethality of conventional PAK4 knock-out mice, conditional PAK4 knock-out mice have been developed to study its role in different tissue's development [20][21][22] . To this end, we created a transgenic mouse model to conditionally deplete PAK4 in the mammary gland by crossing MMTV-Cre mice with PAK4 floxed/floxed mice. We observed that PAK4 conditional knock-out mice were viable, produced standard litter sizes, and pups with normal body weight upon weaning. Moreover, PAK4 knock-out mice exhibited healthy ductal morphology. These results indicate that PAK4 is dispensable for mouse mammary gland development.

Results
MMTV-Cre mediated conditional disruption of PAK4 in the mouse mammary gland. Given that complete depletion of the PAK4 gene in the mouse causes embryonic lethality 19 , we created a mouse model to deplete PAK4 in the mammary epithelium using the Cre/loxP system; by crossing MMTV-Cre mice with PAK4floxed mice 20,23,24 (Fig. 1a) MMTV-Cre mice (line D) have been used in breeding, as this line has minimal effects on mammary gland development compared to other lines 23,25 . The MMTV-Cre mice were used as a control group in this study (Fig. 1a). PAK4 flox was genotyped by PCR analysis of genomic DNA, identifying wild-type mice, as well as homozygous and heterozygous PAK4 knock-out mice (Fig. 1b). When MMTV-Cre; PAK4 fl/+ mice were crossed with PAK4 fl/fl mice, four different genotypes were born approximately at the expected Mendelian ratio, i.e. Wild-type (PAK fl/fl and PAK4 fl/+ ) 49%; Het (MMTV-Cre; PAK4 fl/+ ) 23%; and Homo PAK4 KO (MMTV-Cre; PAK4 fl/fl ) 28% (Table 1). Hereafter, for simplicity, mice with the MMTV-Cre; PAK4 fl/fl genotype will be referred to as PAK4 MEp−/− and MMTV-Cre mice as PAK4 MEp+/+ . Within the same genotypes, female and male displayed an approximately equal distribution; suggesting that loss of PAK4 in the mammary epithelium does not affect survival in any of the sexes.
To test the efficiency of the PAK4 gene depletion in PAK4 MEp−/− mouse mammary glands, MMTV-Cre expression pattern was examined as an indicator of PAK4 depletion. Mosaicism of Cre expression pattern in the MMTV-Cre mice model has been reported 23,25,26 . Based on estimation in both PAK4 MEp+/+ mice and PAK4 MEp−/− mice, more than 90% of the mammary epithelial cells displayed Cre positive staining, and the Cre staining patterns were similar in the two groups (Fig. 1c). Samples from wild-type mice without Cre expression was used here as a negative control. Considering intrinsic limitations of the Cre/LoxP model 23,24,25 , we then performed immunohistochemistry (IHC) using an anti-PAK4 pab and quantified positively labelled cells in the whole mammary duct, displaying a strong reduction of anti-PAK4 labeling in the mammary glands of PAK4 MEp−/− mice ( Fig. 1d-e). Consistently, immunofluorescent labeling using the same anti-PAK4 pab together with labeling of the myoepithelial marker alpha smooth muscle actin (α-SMA) showed significantly lower anti-PAK4 labeling of both luminal and myoepithelial cells in the PAK4 MEp−/− mice compared to PAK4 MEp+/+ mice (Fig. 1f,g). However, there was still a notable fraction of duct cells in the PAK4 MEp−/− mice labeling positive with the anti-PAK4 pab. Thus, the MMTV-Cre mediated recombination was not complete, leading to mosaic expression, consistent with what was previously reported for the MMTV-Cre model 23,25,26 . Nevertheless, these data suggest that our PAK4 conditional knock-out mice have an efficient PAK4 depletion in the vast majority of the mammary epithelial cells.
Considering the possibility that upregulation of other members of the PAK family might compensate for the absence of PAK4, we performed IHC using antibodies against PAK1 27,28 , PAK5, and PAK6. However, we did not detect any increased mammary gland labelling for any of these antibodies in the absence of PAK4 as compared to control glands ( Fig. 1h-m).

PAK4 gene depletion in the mammary gland does not alter ductal elongation and branching.
To examine the ductal growth within the mammary gland in juvenile virgin and adult virgin mice, mammary glands were isolated from mice at 4 and 10 weeks of age. To determine the potential effect of PAK4 depletion on the morphogenesis of the mammary gland, whole-mount preparations were used for measurement of the area fraction covered by mammary epithelium in the entire mammary fat pad. We found that the mammary fat pads were filled by epithelial tissue in the PAK4 MEp−/− mice to the same extent as in their aged-matched PAK4 MEp+/+ mice (Fig. 2a,b). We further performed a quantitative analysis of mammary tree elongation along the fat pad by measuring mammary tree length from the nipple. Quantitative analysis showed that the relative duct length in whole mounts from PAK4 MEp−/− mice was similar to PAK4 MEp+/+ mice at both 4 and 10 weeks of age (Fig. 2c). To explore potential subtle differences in the ductal structure, we analyzed ductal structures in hematoxylin and eosin (H&E) staining of tissue sections. This staining revealed that the ductal structures in PAK4 MEp−/− mammary glands were evenly distributed along the fat cells and were not distinguishable from those of PAK4 MEp+/+ mice (Fig. 2d). Moreover, the number of mammary ducts was similar between the two groups (Fig. 2e).
Loss of PAK4 does not alter cell proliferation or invasion marker expression in the mammary epithelium. Considering the known role of PAK4 in cell proliferation and invasion 29-31 , we next sought to determine the status of cell proliferation and expression of known markers for invasion within the mammary duct of virgin week 4 mice, a time point where the mammary epithelium is highly proliferative. To quantify cell proliferation within the duct, we labeled tissues for Ki67 and consistent with our previous results, lack of PAK4 did not alter cell proliferation in PAK4 MEp−/− mice (Fig. 3a,b).
Next, we measured matrix metalloproteinases (MMPs) expression level, since MMPs are principal executors of matrix remodeling during mammary gland development 32,33 . Mammary gland tissue lysates from PAK4 MEp+/+ and PAK4 MEp−/− mice showed similar expression levels of MMP2, MMP3, and MMP14, suggesting no expression differences among these key invasion markers (Fig. 3c-e). Consistently, we also did not detect any difference in total MMP activity (Fig. 3f).
www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/ PAK4 knock-out mothers were able to nourish pups sufficiently. We first examined and compared the overall structure of the mammary glands from PAK4 knock-out and wild-type females. In carmine red stained whole-mounts from PAK4 MEp+/+ and PAK4 MEp−/− mothers displayed indistinguishable ductal epithelial development at lactation day 2, and we could not detect any difference in the percentage of epithelial cells coverage of carmine filled fat pads. Moreover, the alveolar units of PAK4 MEp−/− females were as fully developed as in PAK4 MEp+/+ females ( Fig. 4a,b). Both groups produced standard litter sizes (Table 1) and similar pup body weight upon weaning (Fig. 4c). Together, this indicates that MMTV-Cre driven conditional gene depletion of PAK4 caused no defects in mammary gland development or function.

Discussion
Using MMTV-Cre mediated PAK4 depletion in mammary gland epithelium, we have shown that targeted inactivation of PAK4 in mammary epithelial cells does not impair mammary gland development; this suggests that PAK4 is dispensable for murine mammary gland development and function.
PAK4 is ubiquitously expressed throughout embryonic development and in adult tissues 19,34 . Transgenic mice with constitutive PAK4 gene depletion do not survive past embryonic day 11.5 19 ; therefore conditional gene depletion strategies have been developed in the field to study its role in different organs development [20][21][22]35 . Conditional depletion of PAK4 in heart, vasculature, and brain caused severe defects during embryonic development while PAK4 depletion in the pancreas did not cause any evident defect in the pancreatic tissue development and function [20][21][22]35 . However, the potential function of PAK4 in mammary gland development has remained unclear. To this end, we found that the mammary tree development upon PAK4 depletion was neither affected during pre-puberty nor in young adult virgin female mice; moreover PAK4 knock-out mice gave birth to healthy progeny and were able to nurse them as well as the control mice. However, it is possible that mammary gland development is dependent on other members of the PAK family, since lack of PAK1 in the mammary gland impaired lobuloalveolar development and cell differentiation 36 , while conventional depletion of PAK5 and PAK6 resulted in normal mammary gland development 37,38 . This supports the idea that different members of the PAK family fulfill different functions throughout organ development 34 .
PAK4 is overexpressed in breast cancer cell lines as well as in breast cancer patients, and its overexpression is accompanied by poor patient outcome 11,16,29,39 . However, our understanding of the in vivo function of PAK4 in breast cancer remains limited. Given that our model for conditional PAK4 gene depletion in the mouse mammary gland displays no apparent defect in organ development and function, this can serve as a useful model to study the in vivo role of PAK4 in breast cancer through crossing these mice with mammary tumor models such as MMTV-PyMT and MMTV-Her2 [40][41][42] . In fact, we recently crossed the here presented mouse model of MMTV-Cre driven conditional PAK4 gene depletion with the MMTV-PyMT breast cancer model and observed an increased mammary tumor latency upon PAK4 depletion 39 .
Nevertheless, one should also be aware of the Cre-mosaicism that we observed, consistent with previous reports upon the use of MMTV-Cre and similar models [23][24][25][26] , which could complicate their use in an evolutionary disease like cancer, with the possibility of a selection of cells in which the gene of interest remains expressed 39,43 . Using a reporter gene could be a useful approach to overcome this problem in future studies 44 .
In summary, our data suggest that lack of PAK4 does not alter normal mammary gland development. Therefore, our mouse model of conditional depletion of PAK4 in the mammary epithelium can be useful for testing potential in vivo functions of PAK4 in mammary physiology and diseases such as cancer.

Materials and Methods
Animals. All the experimental procedures performed on animals in this study have been performed in accordance with Swedish and European Union guidelines and approved by Stockholm South and Linköping Animal Ethics Committees. To avoid the influence of social isolation, animals were housed in groups with 12:12 light: dark cycle, controlled humidity (55% ± 5%), controlled temperature (21 °C ± 2 °C) and free access to food and water. www.nature.com/scientificreports www.nature.com/scientificreports/  (Table 1).
Genomic DNA was prepared from biopsies using the fast tissue-to-PCR kit (#K1091, Fermentas). Mice were genotyped for heterozygous and homozygous knock-out of PAK4 according to Tian et al 20 . The primer pairs used (synthesized by ThermoFisher) were as follows: Pak4 flox: F, 5′-CGGATATTGTCACCCACACCAG-3′ and R, 5′-CTAACAGGGACAGGAGCT-3′. DNA band was visualized on 2% agar gels stained with GelRed (41003, Biotium). All mammary gland tissues used in this paper are from female mice. tissue collection. Mice were sacrificed by cervical dislocation after anesthesia with isoflurane, and the mammary glands were collected. #1 and #2 thoracic mammary glands were quickly frozen and accordingly used for RNA and protein extraction. The #10 inguinal mammary gland was dissected, flattened on a piece of paper, fixed in 4% Paraformaldehyde overnight, then washed with PBS and kept in 70% ethanol for paraffin embedding and later used for immunohistochemistry.  www.nature.com/scientificreports www.nature.com/scientificreports/ Whole-mount staining of mammary glands. The #4 inguinal mammary gland was collected to determine the area where mammary glands were developed in fat pads. Briefly, the samples were fixed overnight with Carnoy's fixative (100% ethanol/chloroform/glacial acetic acid, 6:3:1). Then samples were hydrated by sequential treatment in 70%; 50%; 30%; and 10% ethanol for 15 min each. After the hydration process, samples were washed in tap water for 5 minutes and placed O/N in staining solution at RT. The staining solution was prepared by dissolving 1 g carmine (C1022, Sigma) and 2.5 g aluminum potassium sulfate (A7167, Sigma) in 500 ml water followed by boiling for 20 min. The samples were then dehydrated by sequential treatment in 70%; 95%; and 100% ethanol for 15 min each, followed by storage in xylene (28975, VWR) until scanning. Scanned images were analyzed using ImageJ/Fiji (Version 1,52i) (National Institutes of Health, NIH). Whole-mount images were cropped to only include #4 inguinal mammary gland then converted to an 8-bit image and sharpened. Area of fat pad occupied by mammary epithelium (carmine staining) was measured by adjustment of threshold automatically and demonstrated as a percentage of the total mammary fat pad occupied by the mammary epithelium.
Quantification of mammary tree branching was done on whole-mount images by measurement of the mammary tree length from the nipple to the last branch in millimeters using ImageJ/Fiji (Version 1,52i) (National Institutes of Health, NIH). H&E staining and immunostaining. Paraffin blocks were cut into 4 μm sections. For H&E staining, sections were stained with hematoxylin and eosin according to a standard protocol 45 . For immunohistochemistry, sections were deparaffinized by incubation in 60 °C incubator for 1 h followed by rehydration steps through washing in xylene and graded ethanol to distilled water. Samples were boiled for 20 min in antigen retrieval solution 0.01 M sodium citrate buffer (100813 M, BDH) (pH 6.0) in water. Endogenous peroxidase activity was blocked via www.nature.com/scientificreports www.nature.com/scientificreports/ treatment with 3% hydrogen peroxide in water (H1009, Sigma). PBS diluted normal serum from the same host species as the secondary antibody was used as a blocking buffer.
Images of α-Ki67 labeling were acquired using a Nikon A1R confocal microscope with a Plan Apo VC 60×/1.4 NA oil objective and NIS-Elements software (Nikon). Proliferation index was calculated as the percentage of Ki67 positive cells relative to the total number of the cells within the ducts.
Images of α-PAK4 labeling were acquired for quantification on a Panaromic MIDI II slide scanner from 3DHISTECH and for display by a Nikon A1R confocal microscope as described above. PAK4 positive labeling was calculated as the percentage of cells positively labelled by the α-PAK4 Pab relative to luminal epithelial and myoepithelial cells, respectively. Myoepithelial cells were defined as the cells labeling positive for α-SMA, while remaining ductal cells were considered to be epithelial cells. Image analysis was performed by counting positively labelled cells in 3 to 7 evenly distributed, similar-sized ducts each of 5 mice for each group. For all image quantifications, images were randomized and visually quantified in a blinded manner.
MMp activity assay. The total MMP activity was measured fluorometrically using an MMP activity assay kit (ab112147, Abcam). Samples collected from inguinal mammary glands of 4 weeks old virgin mice were homogenized in 500 μl Ripa buffer. A black 96-well plate with clear bottom was used to carry out the assay. The enzymatic reaction was performed according to the manufacturer protocol by adding 50 μl of the homogenate to each well, with pre-incubation for 15 minutes. 50 μl of MMP Red Substrate solution was then added to each well. After 1 hour of incubation at 37 °C, fluorescence (relative fluorescence units, RFUs) was measured at 540 nm excitation and 590 nm emissions using a Gemini XPS microplate spectrofluorometer. Each homogenate was analyzed in triplicate. Three substrate control wells were used as negative controls, whose mean value determined the baseline that was subtracted from the sample wells.

Statistics.
A two-tailed unpaired t-test was utilized for statistical analyses. P < 0.05 was considered to represent statistical discernibility of differences.

Data Availability
Underlying data could be obtained from the corresponding author upon reasonable request. www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/