ABSTRACT
Lactation is an essential process for mammals. In sheep, the R96C mutation in suppressor of cytokine signaling 2 (SOCS2) protein is associated with greater milk production and increased mastitis sensitivity. To shed light on the involvement of R96C mutation in mammary gland development and lactation, we developed a mouse model carrying this mutation (SOCS2KI/KI). Mammary glands from virgin adult SOCS2KI/KI mice presented a branching defect and less epithelial tissue, which were not compensated for in later stages of mammary development. Mammary epithelial cell (MEC) subpopulations were modified, with mutated mice having three times as many basal cells, accompanied by a decrease in luminal cells. The SOCS2KI/KI mammary gland remained functional; however, MECs contained more lipid droplets versus fat globules, and milk lipid composition was modified. Moreover, the gene expression dynamic from virgin to pregnancy state resulted in the identification of about 3000 differentially expressed genes specific to SOCS2KI/KI or control mice. Our results show that SOCS2 is important for mammary gland development and milk production. In the long term, this finding raises the possibility of ensuring adequate milk production without compromising animal health and welfare.
INTRODUCTION
The mammary gland has a complex development that is accompanied by significant changes at every reproduction cycle. It is a unique organ in that it only reaches maturity after puberty. The mammary gland goes through several stages of development. At birth, it comprises a rudimentary ductal system that progressively extends into the fat pad until it reaches the periphery during puberty. During pregnancy, mammary epithelial cells (MECs) begin to proliferate and differentiate to form alveolar structures at the ends of side branches; these form future sites of milk production during lactation. After weaning, the mammary gland goes through a process known as involution, which includes massive apoptosis, alveoli collapse and restructuring of mammary epithelium to a simple ductal structure that is similar, but not identical, to that of the virgin state (Watson and Khaled, 2008).
The adult mammary gland comprises several cell types, including epithelial, adipose, fibroblast, immune, lymphatic and vascular cells, that work together to form a fully functional organ (Inman et al., 2015). Two main epithelial cell lineages, luminal and basal epithelial cells, form the mammary epithelial bilayer (Lloyd-Lewis et al., 2017). During the different stages of mammary development, luminal cells restructure the mammary gland by giving rise to the ductal epithelium. During pregnancy in particular, luminal cells differentiate into alveolar cells that then produce and secrete milk at parturition (Inman et al., 2015). Basal cells differentiate into myoepithelial cells that line mammary ducts and alveoli (Inman et al., 2015).
Mammary development is under the control of multiple hormones and cytokines, such as estrogen, progesterone, prolactin (PRL), growth hormone (GH), oxytocin and leptin, among others, which are produced at various stages of development (Inman et al., 2015). These proteins are recognized in a specific manner by receptors on the surface of cells in order to activate different signaling pathways. In the mammary gland, the most important signaling pathway activated by cytokines is the Janus kinase and signal transducer and activator of transcription (JAK-STAT) pathway.
Cytokine signaling is under strict regulation, as it is associated with autoimmune disorders, cancers and hematopoiesis disorders when left unchecked (Letellier and Haan, 2016). This regulation involves several protein families, including suppressor of cytokine signaling (SOCS) proteins. The expression of SOCS proteins is rapidly induced upon activation of the JAK-STAT pathway to negatively regulate cytokine signaling via a feedback loop (Inagaki-Ohara et al., 2013). One of the characteristics of SOCS proteins is their central SH2 domain that allows binding to phosphorylated tyrosine residues, such as those found on cytokine receptors and JAK, thus blocking the signaling cascade. SOCS2 is of particular interest as it regulates growth hormone and prolactin signaling pathways, which are essential when it comes to mammary gland development and function (Metcalf et al., 2000; Pezet et al., 1999). Growth hormone stimulates the proliferation of MECs during development (Kelly et al., 2002). Prolactin plays a role in mammary ductal development, in the differentiation of MECs to form functional acini and in the expression of major milk proteins, such as caseins and whey acidic protein (WAP) (Ormandy et al., 1997; Horseman et al., 1997; Teyssot and Houdebine, 1980; Rosen et al., 1989).
In 2015, Rupp et al. identified a point mutation in SOCS2 that induces the R96C substitution in the SH2 functional domain in sheep (Rupp et al., 2015). This mutation causes a loss of function in SOCS2, as it diminishes its affinity for its ligands. This leads to a significant increase in both the size of the animal and milk production for R96C homozygous sheep; however, it leaves the sheep more susceptible to mastitis (mammary inflammation). This strongly suggests that SOCS2 plays a role in the tradeoff between body growth and milk production, as well as in the inflammatory response of the host to mammary infections, which are regulated by the JAK/STAT signaling pathway (Rupp et al., 2015).
This study focuses on how the SOCS2 R96C mutation affects mammary gland development and milk production. A knock-in (KI) mouse model, SOCS2KI/KI, was used to study the impact of the mutation on mammary gland development and function. Mammary glands from adult virgin KI mice exhibit a ductal branching defect that persists in further stages of development, as well as a lower epithelial to adipose tissue ratio in all stages of development. Furthermore, during lactation, these changes affect milk quality, but not the growth of pups nursed by SOCS2KI/KI mice. In brief, our results show that the loss of function of SOCS2 disrupts mammary gland development and function in a mouse model.
RESULTS
Important morphological changes in the mammary glands of SOCS2KI/KI mice
To characterize the impact of the SOCS2 R96C mutation on mouse mammary gland development, the organ was observed in its entirety. To detect any potential changes in mammary ductal development, whole virgin 8-week-old mammary glands were mounted and the mammary epithelium was stained (Fig. 1A). SOCS2KI/KI mammary glands presented fewer ramifications compared with SOCS2WT/WT mammary glands (Fig. 1B). This shows that the R96C mutation of SOCS2 causes a branching defect during mammary gland development. To further explore this defect and examine ductal structure, Hematoxylin and Eosin-stained mammary gland sections from virgin 8-week-old SOCS2WT/WT (n=11) and SOCS2KI/KI (n=11) mice were compared (Fig. 1C). Once more, fewer mammary ducts were present in SOCS2KI/KI mice; moreover, SOCS2KI/KI mammary glands contain less epithelial tissue than SOCS2WT/WT (Fig. 1D).
Morphological changes in the mammary gland caused by SOCS2 p. R96C mutation. (A) Whole mounts of virgin 8-week-old mammary glands from SOCS2WT/WT (n=11) and SOCS2KI/KI (n=11) mice observed under a binocular magnifier. LG, lymphatic ganglion; MD, mammary duct; TEB, terminal end bud. (B) Superplots (Goedhart, 2021) showing the quantification of branching density in whole mounts of virgin 8-week-old mammary glands from SOCS2WT/WT (n=6) and SOCS2KI/KI (n=6) mice. Welch's t-test was used to compare SOCS2WT/WT and SOCS2KI/KI (P=0.024) branching density. Smaller circles represent each technical replicate (i.e. the number of branchings per 200 pixels; three measures per mammary gland). The bigger circles represent the biological replicates (i.e. the mean of the three measures). Data are mean of all biological replicates ±s.d. (C) Hematoxylin and Eosin-stained slides of SOCS2WT/WT and SOCS2KI/KI mammary gland sections. Scale bars: 200 µm for virgin 8-week-old mammary gland sections; 500 µm for pregnancy day 18 (SOCS2WT/WT, n=5, SOCS2KI/KI, n=5) and lactation day 7 (SOCS2WT/WT, n=5, SOCS2KI/KI, n=5). (D) Superplots (Goedhart, 2021) showing the quantification of mammary epithelial tissue density in Hematoxylin and Eosin-stained slides of SOCS2WT/WT and SOCS2KI/KI mammary gland sections. Welch's t-test was used to compare SOCS2WT/WT and SOCS2KI/KI virgin 8-week-old (P=.029), pregnancy day 18 (P=.026) and lactation day 7 (P=.007) mammary gland sections. The circles represent biological replicates. Data are mean of all biological replicates ±s.d. *P<0.05; **P<0.01.
Morphological changes in the mammary gland caused by SOCS2 p. R96C mutation. (A) Whole mounts of virgin 8-week-old mammary glands from SOCS2WT/WT (n=11) and SOCS2KI/KI (n=11) mice observed under a binocular magnifier. LG, lymphatic ganglion; MD, mammary duct; TEB, terminal end bud. (B) Superplots (Goedhart, 2021) showing the quantification of branching density in whole mounts of virgin 8-week-old mammary glands from SOCS2WT/WT (n=6) and SOCS2KI/KI (n=6) mice. Welch's t-test was used to compare SOCS2WT/WT and SOCS2KI/KI (P=0.024) branching density. Smaller circles represent each technical replicate (i.e. the number of branchings per 200 pixels; three measures per mammary gland). The bigger circles represent the biological replicates (i.e. the mean of the three measures). Data are mean of all biological replicates ±s.d. (C) Hematoxylin and Eosin-stained slides of SOCS2WT/WT and SOCS2KI/KI mammary gland sections. Scale bars: 200 µm for virgin 8-week-old mammary gland sections; 500 µm for pregnancy day 18 (SOCS2WT/WT, n=5, SOCS2KI/KI, n=5) and lactation day 7 (SOCS2WT/WT, n=5, SOCS2KI/KI, n=5). (D) Superplots (Goedhart, 2021) showing the quantification of mammary epithelial tissue density in Hematoxylin and Eosin-stained slides of SOCS2WT/WT and SOCS2KI/KI mammary gland sections. Welch's t-test was used to compare SOCS2WT/WT and SOCS2KI/KI virgin 8-week-old (P=.029), pregnancy day 18 (P=.026) and lactation day 7 (P=.007) mammary gland sections. The circles represent biological replicates. Data are mean of all biological replicates ±s.d. *P<0.05; **P<0.01.
Mammary ductal development was also studied using mammary gland transplantation from SOCS2WT/WT (n=6) and SOCS2KI/KI (n=6) donor mice to NMRI nude mice (n=30). Mammary gland tissue from each genotype was grafted in the left and right inguinal fat pad so that each host mouse could serve as its own control. No difference in mammary ductal development was observed 8 weeks post-surgery (Fig. S1). This suggests that the mutation of the mammary epithelial cells alone is insufficient to reproduce the mammary development defect observed in virgin SOCS2KI/KI mice.
The mammary development defect, observed during the virgin stage, was not overcome in subsequent stages of mammary development related to reproduction (pregnancy) and persisted during lactation. Sections from late pregnancy (day 18; SOCS2WT/WT, n=5; SOCS2KI/KI, n=5) and established lactation (day 7; SOCS2WT/WT, n=5; SOCS2KI/KI, n=5) showed less epithelial tissue in SOCS2KI/KI mammary glands compared with SOCS2WT/WT. Pregnancy and lactation are stages of development characterized by a complete invasion of epithelial tissue and replacement of the fat pad. In our mouse model, the mammary gland defect observed in the virgin state, caused by the SOCS2 R96C mutation, cannot be overcome in subsequent stages of development.
Cell composition changes in the mammary glands of SOCS2KI/KI mice
To characterize the effects of the mutation on mammary epithelial cell populations, isolated MECs from 10-week-old SOCS2KI/KI (n=10) and SOCS2WT/WT (n=10) were labeled and sorted. Briefly, CD31+ endothelial cells and CD45+ immune cells were removed. CD31− and CD45− epithelial cells were further differentiated using CD24 as a marker of mammary luminal cells and CD49f as a marker of mammary basal cells. Luminal progenitor cells were positive for both CD24 and CD54 (Fig. 2A). No significant difference in luminal progenitors was observed between SOCS2WT/WT and SOCS2KI/KI mammary glands, suggesting the phenotype observed is not due to a decrease in luminal progenitor cells. However, SOCS2KI/KI mammary glands had more than three times as many basal cells at the expense of luminal cells compared with SOCS2WT/WT glands (Fig. 2B).
MEC subpopulation changes caused by SOCS2 p. R96C mutation. (A) Gating procedure for labeling MEC populations (SSC, side scatter; FSC, forward scatter). (B) Mammary epithelial cells from virgin 10-week-old mammary glands (SOCS2WT/WT: n=10, SOCS2KI/KI: n=10) were sorted into luminal, basal and luminal progenitor cells. The experiment was performed in triplicate and the percentages presented are the averages from all three experiments. *P<0.05 (Mann–Whitney U test; ns, non-significant). Box plot shows mean values (middle bars) and first to third interquartile ranges (boxes); whiskers indicate maximum and minimum values.
MEC subpopulation changes caused by SOCS2 p. R96C mutation. (A) Gating procedure for labeling MEC populations (SSC, side scatter; FSC, forward scatter). (B) Mammary epithelial cells from virgin 10-week-old mammary glands (SOCS2WT/WT: n=10, SOCS2KI/KI: n=10) were sorted into luminal, basal and luminal progenitor cells. The experiment was performed in triplicate and the percentages presented are the averages from all three experiments. *P<0.05 (Mann–Whitney U test; ns, non-significant). Box plot shows mean values (middle bars) and first to third interquartile ranges (boxes); whiskers indicate maximum and minimum values.
When it comes to mammary ductal structure, no changes in MEC cell polarity were observed. Normal epithelial cell polarity was confirmed with immunostaining for AQP5, a water transporter that marks luminal epithelial cells and, therefore, the apical membrane of mammary ducts. AQP5 was expressed in both SOCS2KI/KI and SOCS2WT/WT mammary glands (Fig. S2).
Mammary epithelial cell structure changes in SOCS2KI/KI mammary glands
To evaluate the effect of the mutation on the shape and intracellular space of MECs, transmission electron microscopy was used (Fig. 3). Sections from SOCS2WT/WT and SOCS2KI/KI mammary glands at three different stages of development were observed: virgin 8-week-old, pregnancy day 8 and lactation day 7. MECs from virgin SOCS2KI/KI mice lacked plasma membrane integrity compared with SOCS2WT/WT. SOCS2WT/WT MECs had easily observable plasma membranes with clear tight junctions in the apical secretory pole delimiting the lumen. Moreover, desmosomes were present along the plasma membrane separating MECs, which was not the case for SOCS2KI/KI MECs (Fig. 3A,B). No changes in membrane integrity were observed in later stages of development. At pregnancy day 18, SOCS2KI/KI MECs showed lipid droplets of abnormally large size that remained in the cell with a few milk fat globules in the lumen (Fig. 3C). SOCS2WT/WT MECs, on the other hand, were producing lipid droplets and secreting them in the lumen to form milk fat globules (Fig. 3D). Similar results were observed at lactation day 7, with SOCS2KI/KI MECs presenting numerous lipid droplets within the cell. Therefore, lumini were filled with casein micelles (Fig. 3E). On the contrary, SOCS2WT/WT MEC lumini contained large fat globules surrounded by casein micelles (Fig. 3F). Quantification of lipid droplets, confirmed electron microscopy (EM) observation: 83% of lipid droplets remained in SOCS2KI/KI MECs, whereas only 41% were found in SOCS2WT/WT MECs. Organelle structure in MECs at lactation day 7 was also evaluated. Lipids in formation can be easily observed in SOCS2KI/KI MECs (Fig. 3G), confirming lipid droplets synthesis, as previously shown. Moreover, the endoplasmic reticulum was enlarged in SOCS2KI/KI MECs compared with SOCS2WT/WT, indicative of greater than usual stress for the cell caused by intense milk component synthesis (Fig. 3G,H).
Mammary epithelial cell structure changes in SCS2KI/KI mice. (A-H) Transmission electron microscopy images of SOCS2WT/WT or SOCS2KI/KI mammary glands at three stages of development: virgin 8-week-old (A,B), pregnancy day 18 (C,D) and lactation day 7 (E-H). Sections from five mice per stage of development and genotype were observed. N, nucleus; m, mitochondria; µv, microvilli; er, endoplasmic reticulum; j, junction (arrows); D, desmosome (small arrows); L, lumen; ld, lipid droplet; fg, fat globule; ldf, lipid droplet in formation; cm, casein micelle; µec, myoepithelial cell.
Mammary epithelial cell structure changes in SCS2KI/KI mice. (A-H) Transmission electron microscopy images of SOCS2WT/WT or SOCS2KI/KI mammary glands at three stages of development: virgin 8-week-old (A,B), pregnancy day 18 (C,D) and lactation day 7 (E-H). Sections from five mice per stage of development and genotype were observed. N, nucleus; m, mitochondria; µv, microvilli; er, endoplasmic reticulum; j, junction (arrows); D, desmosome (small arrows); L, lumen; ld, lipid droplet; fg, fat globule; ldf, lipid droplet in formation; cm, casein micelle; µec, myoepithelial cell.
Gene expression changes in the mammary gland of SOCS2KI/KI mice
In order to look for potential molecular mechanisms behind the observed phenotypes in the mammary gland, the gene expression of virgin 8-week-old (SOCS2WT/WT: n=7 and SOCS2KI/KI: n=7) and pregnancy day 18 (SOCS2WT/WT: n=5 and SOCS2KI/KI: n=6) mammary glands was studied using an Agilent microarray of 36,750 mouse genes. No differentially expressed genes (DEGs) were found between virgin SOCS2KI/KI and SOCS2WT/WT mammary glands. Two DEGs were found between SOCS2KI/KI and SOCS2WT/WT mammary glands at day 18 of pregnancy: Scgb2b2, with a logFC of −1.11, and Scgb2b20, with a logFC of −1.06.
To evaluate potential changes in gene expression, dynamic DEGs induced by mammary development from virgin to pregnancy intra-condition (either SOCS2WT/WT or SOCS2KI/KI) were compared. SOCS2KI/KI mammary glands had 12,204 DEGs from virgin to pregnancy, whereas wild-type glands had 12,666 DEGs. Of these, 9366 DEGs were shared, 3300 were specific to SOCS2WT/WT and 2838 were specific to SOCS2KI/KI. The complete list of DEGs identified in each condition is available in Table S1. Several DEGs from virgin to pregnancy were identified in either SOCS2WT/WT or SOCS2KI/KI mammary tissue, based on current knowledge of genes involved in the regulation of mammary gland processes, such as alveologenesis and branching morphogenesis. In SOCS2WT/WT mammary glands, the genes found have previously been shown to be differentially expressed between the virgin and pregnancy stages of development (Table 1). In SOCS2WT/WT mammary glands, the expression of prolactin (Prlr), progesterone (Pgr), insulin growth factor (Igflr1) receptors and Wnt7b all increased in late pregnancy, whereas the expression of growth hormone releasing hormone (Ghrh), Mmp10 and Wnt3a was reduced. In SOCS2KI/KI mammary glands, those same genes were not significantly differentially expressed; however, other genes were identified whose differential expression could be a result of the SOCS2 R96C mutation. The expression of keratin 14 (Krt14), Wnt7a and Wnt8a was reduced in late pregnancy, whereas the expression of Aldh1a3, Mmp11, and transcription factors Maf1 and Tfam was increased in SOCS2KI/KI mammary glands.
Differentially expressed genes in virgin 8-week-old and pregnancy day 18 mammary glands from SOCS2WT/WT or SOCS2KI/KI mice

Next, to see whether specific signaling pathways or functions were enriched in the DEGs detected, functional enrichment analysis was used on DEGs specific to either SOCS2WT/WT or SOCS2KI/KI mammary gland tissue. The 44 most significant gene ontology terms (molecular function, biological process and cellular component) are shown in Table 2. All terms of interest identified as a result of the enrichment analysis are presented in Table S2. When it comes to molecular functions, DEGs specific to SOCS2KI/KI were enriched for multiple functions related to the mitochondria, such as ATP synthase activity and oxidoreduction. DEGs specific to SOCS2WT/WT were enriched for signaling receptor activity and transcription regulator activity. As for biological processes, DEGs identified from both SOCS2KI/KI and SOCS2WT/WT mammary tissue were enriched for regulation of cellular processes; however, only DEGs specific to SOCS2KI/KI were enriched for multiple processes related to the mitochondria, such as oxidative phosphorylation, mitochondrion organization, ATP metabolic process and others. When it comes to cellular components, both SOCS2KI/KI and SOCS2WT/WT had DEGs corresponding to the membrane and cytoplasm. DEGs specific to SOCS2KI/KI were found in different compartments of the mitochondria and endoplasmic reticulum, whereas DEGs specific to SOCS2WT/WT were found in the cytoskeleton, microtubule and mitotic spindle. Finally, in terms of signaling pathways, DEGs specific to SOCS2KI/KI were enriched for multiple pathways related to mitochondrial function and deregulation, such as thermogenesis, oxidative phosphorylation, Parkinson's and Alzheimer's diseases.
Lactation changes in the mammary glands of SOCS2KI/KI mice
After characterizing the multiple changes in mammary gland development observed in SOCS2KI/KI mice, the effect of the mutation on lactation was evaluated. To estimate the quantity of milk produced by SOCS2WT/WT or SOCS2KI/KI mice, SOCS2WT/WT pups were adopted at birth by SOCS2WT/WT or SOCS2KI/KI lactating mothers and the litters were weighed until pups were 15 days old. The growth curves showed no significant changes (P-value=0.5042) in pup growth (Fig. S3), suggesting the milk produced by SOCS2KI/KI mice was sufficient for the growth of pups.
The lipid composition was analyzed to estimate whether the mutation affects milk quality. Therefore, the fatty acid (FA) profiles were determined in milk from SOCS2KI/KI and SOCS2WT/WT mice at 7 days of lactation (Table 3; Table S3). The total FA content is similar between SOCS2KI/KI (198.75±17.78 g/l) and SOCS2WT/WT (171.91±15.97 g/l) milk. Both milks exhibited similar total saturated fatty acid (SFA) contents, regarding both short chain (sc), and medium and long chain (mc and lc) SFAs. In contrast, a sharp rise in the monounsaturated fatty acid (MUFA) content was observed in SOCS2KI/KI milk, with a corresponding increase in oleic acid (44.81±4.64 versus 29.86±3.23% in SOCS2WT/WT milk). Polyunsaturated fatty acid (PUFA) levels were similar in SOCS2KI/KI and SOCS2WT/WT milks. Qualitatively, SFAs were the main FAs present in both SOCS2KI/KI and SOCS2WT/WT milk FA profiles. However, sc SFA levels decreased whereas lc SFA levels remained unchanged in SOCS2KI/KI milk compared with SOCS2WT/WT. MUFAs were present at greater levels in SOCS2KI/KI milk (29.66±2.52 versus 22.02±1.06% in SOCS2WT/WT milk), with more oleic acid (22.89±2.00 versus 17.37±0.77% in SOCS2WT/WT milk). These results show that the SOCS2 R96C mutation affects milk quality by inducing changes in milk lipid composition.
DISCUSSION
SOCS2 is a negative regulator of the JAK-STAT5 pathway, a pathway that controls multiple cellular processes triggered by cytokine signaling. JAK-STAT signaling is a ubiquitous pathway that controls gene expression in multiple tissues and organs, such as the brain, liver, muscle, fat and pancreas (Gurzov et al., 2016). Dysregulation of this pathway and its regulatory proteins is linked to various diseases, including cancer, hematopoiesis disorders and autoimmune diseases (Letellier and Haan, 2016; Yan et al., 2018). In the mammary gland, JAK-STAT signaling plays an important role in multiple stages of its development, as well as in its function (Campo Verde Arboccó et al., 2017; Watson and Burdon, 1996; Jahn et al., 1997). To study SOCS2 regulation, knockout models of SOCS2 (Metcalf et al., 2000; Greenhalgh et al., 2002, 2005) have been generated and its role as a negative regulator of the JAK/STAT pathway has been demonstrated. SOCS2KO/KO mice exhibit gigantism, thereby showing the role of SOCS2 as a repressor of the JAK/STAT-growth hormone signaling pathway. Gigantism is also observed in the R96C SOCS2KI/KI mouse model (see Guzylack-Piriou et al., 2023 preprint). The mammary gland was not studied in the above-mentioned SOCS2 knockout models. In the current study, a SOCS2KI/KI mouse model was used to study effects of this mutation on mammary gland development and function.
A mammary duct branching defect was observed in virgin 8-week-old SOCS2KI/KI mice compared with SOCS2WT/WT. SOCS2KI/KI mammary glands had fewer mammary duct ramifications. At birth, mice mammary glands possess a rudimentary ductal structure. Branching morphogenesis begins at puberty, activated by GH, PRL, estrogen and IGF1 to ultimately create a ductal tree that fills the fat pad (Macias and Hinck, 2012). The implication of GH and PRL in mammogenesis has been well known since the 1930s, when researchers found that pituitary gland extract was able to induce mammogenesis (Trott et al., 2008). Ghr (growth hormone receptor) or Prlr (prolactin receptor) knockout mouse models exhibit delayed mammary gland development and develop only a sparse ductal tree (Gallego et al., 2001). At the same time, overexpression of GH also results in deficient branching in the rodent mammary gland (Bojorge et al., 2021), similar to what we observe in this SOCS2KI/KI mouse model. In earlier years, researchers showed that mammary ductal growth also depended on IGF-1 and estrogen, as branching morphogenesis was disrupted in mice lacking Igf1 and Esr1 (estrogen receptor α) (Trott et al., 2008). All of these hormones rely on the JAK-STAT signaling pathway to exert their effects. Our results show that a defective negative regulation of the JAK-STAT pathway, caused by a SOCS2 mutation, results in a branching defect in virgin adult SOCS2KI/KI mammary glands.
A consequence of the branching defect is that SOCS2KI/KI mammary glands have less epithelial tissue overall, compared with SOCS2WT/WT. Staining of epithelial cells in mammary sections from virgin adult (8-week-old), pregnant (day 18) and lactating (day 7) mice show a higher adipose to epithelial tissue ratio in SOCS2KI/KI mice compared with SOCS2WT/WT. This also shows that the defect in mammary development caused by the SOCS2 mutation is not compensated for in any stage of mammary development, even those during which MEC proliferation is activated. The lack of compensation was not due to a lack of mammary luminal progenitor cells in SOCS2KI/KI glands. Cell sorting showed SOCS2KI/KI and SOCS2WT/WT mammary glands were the same in terms of luminal progenitor cells. However, SOCS2KI/KI glands had more than three times as many basal cells at the expense of luminal cells compared with SOCS2WT/WT. Luminal cells give rise to the ductal epithelium, whereas basal cells differentiate into myoepithelial cells. Cell lineage tracing studies have shown that some basal cells can be derived from luminal cells. This occurs during pregnancy in wild-type mouse mammary glands after estrogen and progesterone stimulation (Song et al., 2019). Thus, a hormonal imbalance in virgin SOCS2KI/KI mice caused by the disruption of SOCS2 function could explain the changes in MEC populations. Moreover, these changes likely play a role in the reduction of epithelial tissue observed in KI mammary glands.
It is interesting to note that, although there are fewer ductal ramifications, the ductal architecture of SOCS2KI/KI mice remains unchanged. In virgin 8-week-old SOCS2KI/KI mammary glands, MECs are able to form the epithelial ductal bilayer, as observed in histological sections. Moreover, AQP5 localization proves that the polarity of MECs in SOCS2KI/KI glands is maintained.
SOCS2KI/KI MECs had changes in cell and organelle structure: a defect in the plasma membrane of MECs from virgin adult 8-week-old SOCS2KI/KI mice was observed. SOCS2WT/WT MECs had clearly visible tight junctions and plasma membranes that clearly delimited every cell, which was not the case for SOCS2KI/KI MECs. The importance of tight junctions is well known when it comes to lactation; their permeability decreases milk secretion, whereas their impermeability increases milk secretion (Nguyen and Neville, 1998). They are also known to be leaky during pregnancy before closing at parturition (Nguyen and Neville, 1998). Despite this observation in virgin SOCS2KI/KI mice, MECs from SOCS2KI/KI mice remained cohesive and no leakage was observed at day 7 of lactation. Nevertheless, MECs from lactating SOCS2KI/KI mice had a dilated endoplasmic reticulum compared with SOCS2WT/WT. The endoplasmic reticulum is responsible for the synthesis and folding of proteins, as well as for signaling and calcium storage. Perturbations in its homeostasis are linked to a stress response and multiple diseases such as heart failure (Ortega et al., 2014; Hamada et al., 2004) and cancer (Hirokawa et al., 2000; Fontana et al., 2020). The presence of dilated endoplasmic reticulum in SOCS2KI/KI MECs shows that they experience more stress during lactation compared with SOCS2WT/WT.
The results presented so far characterize the effect of the mutation on MECs; however, it is important to consider that mammary cell proliferation and differentiation is a result of complex interactions between MECs, immune cells and the stroma (Zwick et al., 2018; Twigger and Khaled, 2021; Wang et al., 2020; Avagliano et al., 2020; Coussens and Pollard, 2011; Gouon-Evans et al., 2000). Mammary gland transplantations of SOCS2KI/KI and SOCS2WT/WT mammary tissue in 3-week-old nude mice resulted in normal mammary branching at 8 weeks (Fig. S1). The rescue of branching when SOCS2KI/KI mammary tissue develops in an environment with normal SOCS2 signaling shows that the disruption of SOCS2 in MECs alone is not sufficient to impede mammary duct ramification.
The SOCS2 mutation disrupted branching morphogenesis and modified MEC structure; however, the mammary gland remained functional. Gene expression in SOCS2KI/KI and SOCS2WT/WT mammary glands at 8 weeks and 18 days of pregnancy showed no DEGs in virgin 8-week-old mammary glands and two DEGs at 18 days of pregnancy: Scgb2b2 and Scgb2b20. These two genes code for proteins in the secretoglobulin family and are not known to play a role in mammary gland function. On the other hand, gene expression dynamic from virgin to pregnant SOCS2KI/KI or SOCS2WT/WT mice shifted. We found that 9366 DEGs were common between SOCS2KI/KI and SOCS2WT/WT; however, 2838 were specific to SOCS2KI/KI and 3300 were specific to SOCS2WT/WT. We identified multiple genes of interest in the DEGs specific to either condition based on current knowledge of genes involved in mammary gland processes, such as alveologenesis and branching morphogenesis.
In the genes specific to SOCS2WT/WT, Prlr, Pgr, Wnt7b and Igflr1 were overexpressed, and Ghrh, Mmp10 and Wnt3a were underexpressed in pregnancy compared with virgin. Prlr, Pgr and Igflr1 are the receptors for PRL, progesterone and IGF1. Their upregulation in the pregnant mammary gland allows MEC differentiation and alveologenesis (Luo et al., 2022; Karayazi Atıcı et al., 2021). Mmp10 is upregulated when cells are stimulated with EGF (epidermal growth factor) (Tajadura-Ortega et al., 2021), which occurs before MEC proliferation (Schroeder and Lee, 1997). The role of Wnt7b is not completely understood; however, its expression in terminal end buds of the ductal epithelium (Weber-Hall et al., 1994) and in luminal cells (Cai et al., 2014) suggests a role in both branching morphogenesis and alveologenesis. Wnt3a, on the other hand, plays a role in branching morphogenesis (Voutilainen et al., 2012); thus, it is downregulated during pregnancy. All of these expression changes are to be expected when the mammary gland is remodeled during pregnancy.
In the genes specific to SOCS2KI/KI, Aldh1a3, Mmp11, Maf1 and Tfam were overexpressed, and Krt14, Wnt7a and Wnt8a were underexpressed in pregnancy compared with virgin. Aldh1a3 is exclusive to the luminal cell compartment (Coradini et al., 2014; Eirew et al., 2012), whereas Krt14 is a basal cell marker (Finot et al., 2019). Their upregulation and downregulation, respectively, suggest an increase of luminal cells at the expense of basal cells. This is likely specific to SOCS2KI/KI mammary glands, as MEC populations are modified in SOCS2KI/KI virgin mice. The downregulation of Krt14 in KI but not SOCS2WT/WT mammary glands is also likely due to the increased number of basal cells found in virgin SOCS2KI/KI glands. Mmp11 controls energy metabolism in vivo and promotes IGF1 signaling (Tan et al., 2020). It also plays a role in mammary gland postnatal development, as its absence reduces ductal growth, alveologenesis and milk production (Tan et al., 2014). Wnt7a activates cell proliferation (Wong et al., 1994) and the expression of Wnt8a is regulated by estrogen (Saitoh et al., 2002). Maf1 is a transcription factor that is known to be overexpressed with PRL and GH treatments in the mammary gland (Malewski et al., 2002). Tfam plays a role in fatty acid oxidation and is normally attenuated by SOCS2 (Zhang et al., 2020). Overall, only two of these genes suggest a dysregulation in gene expression caused by the mutation of SOCS2: Maf1 and Tfam. As for the rest, although they were not found in SOCS2WT/WT mice, no studies link them to any mammary defects. This suggests that the SOCS2 pathway in the mammary gland may be redundant due to its essential role in maintaining the functionality of the mammary gland. Thus, other signaling pathways may partly compensate for any downstream effects of the reduction of efficacy of SOCS2. Nevertheless, the gene enrichment analysis performed on DEGs specific to SOCS2KI/KI mice showed multiple biological processes and signaling pathways related to mitochondrial function, and thus fatty acid oxidation. This suggests a mitochondrial dysregulation and elevated oxidative stress in SOCS2KI/KI mammary glands that is not observed in SOCS2WT/WT.
Finally, the effects of the SOCS2 R96C mutation on lactation were evaluated. The redundancy and partial compensation hypothesis is supported by the fact that the mammary gland remains functional even in SOCS2KI/KI mice. The growth curves of SOCS2WT/WT pups ingesting either SOCS2KI/KI or SOCS2WT/WT milk showed no significant difference, showing that the morphological changes caused by the SOCS2 mutation do not interfere with lactation.
However, milk secretion relies, in part, on the formation of lipid droplets in MECs and their secretion in the lumen to form milk fat globules. TEM images showed that lipid droplets were larger in size and were more often retained in SOCS2KI/KI MECs at pregnancy day 18 compared with SOCS2WT/WT. This was also true for SOCS2KI/KI MECs at lactation day 7. The size of lipid droplets and milk fat globules depends on their synthesis and the secretion process (Cohen et al., 2015). Their composition also plays a role, as small milk fat globules are known to contain more SFA compared with large milk fat globules (Mesilati-Stahy and Argov-Argaman, 2014). Our results show a significant increase in MUFA in SOCS2KI/KI milk, confirming this observation. Short chain SFA were also modified in SOCS2KI/KI milk: they were significantly decreased compared with SOCS2WT/WT. No other fatty acid sub-groups were affected (such as PUFA or total SFA). Regulation of milk fat globule size can also be under hormonal control of milk release [such as prolactin and oxytocin (Ollivier-Bousquet, 2002), and progesterone (Argov-Argaman et al., 2020)]. Thus, disrupting SOCS2-mediated regulation of hormonal signaling pathways could in part explain the observed differences in milk fat globule size and composition.
The current scientific consensus is that a high intake of SFA in early life is associated with various diseases (Romieu et al., 2017) and epigenetic aging (Koemel and Skilton, 2022). Conversely, consumption of PUFA may reduce the risk of developing diseases and allergies later in life (Schwarzenberg and Georgieff, 2018; Shek et al., 2012). Thus, although SOCS2WT/WT pups fed by SOCS2KI/KI mothers survived and grew at the same rate as control pups, we cannot predict how they would handle exposure to pathogens or dietary challenges later in life.
In conclusion, our study focuses on determining the effects of the SOCS2 R96C mutation on mammary gland development and function. We report that mice carrying the mutation have impaired mammary gland development in that branching morphogenesis is disrupted, the epithelial tissue is decreased and MEC structure is modified. Despite these changes, the mammary gland remains functional in that KI mice are able to lactate and feed their pups in a way that allows growth comparable with their SOCS2WT/WT counterparts. However, the mitochondrial stress in mammary tissue suggests a detrimental effect on the metabolic health of the mother, potentially diminishing her quality of life and the overall number of lactations she can have. Further characterization of the effects of the SOCS2 R96C mutation on mammary development and function is needed, with a focus on the long-term consequences of the mutation on animal health and welfare.
MATERIALS AND METHODS
Animals and sample collection
This study was performed in compliance with the French regulations on animal experimentation and with the authorization of the French Ministry of Agriculture. All protocols were approved by an Ethics Committee registered within the French Comité National de Réflexion Ethique sur l'Expérimentation Animale. The protocols are referenced here (visa APAFIS#14943-2018051515482700-v5 and visa APAFIS#28001-2020101212102406-v3) by the Comité d'éthique appliqué à l'Expérimentation Animale (COMETHEA Ethics Committee).
C57Bl/6 wild-type (SOCS2WT/WT) and R96C SOCS2KI/KI mice (Guzylack-Piriou et al., 2023 preprint) were housed in a specific pathogen-free (SPF) environment. For mammary gland tissue samples, mice were euthanized via cervical dislocation and the inguinal (gland number 4) mammary glands were removed from adult female mice at 8 weeks of age, 18 days of pregnancy and at 7 days of lactation. The right mammary gland was removed whole and used to prepare whole mounts. The lymphatic ganglion was removed from the left mammary gland and the tissue was prepared accordingly for histology, immunohistochemistry, microscopy and gene expression experiments.
For pup growth curve analysis, SOCS2WT/WT pups were placed at birth under SOCS2KI/KI or SOCS2WT/WT lactating females and litters were weighed daily for 15 days. The litters were adjusted to 10 wild-type pups per nursing mother.
Mammary epithelial cell preparation and flow cytometry
Single cells were prepared according to Di-Cicco et al. (Di-Cicco et al., 2015). Briefly, 10 mammary glands were taken from virgin 10-week-old mice and were minced with scissors and scalpels. Minced tissues were placed in a digestion solution containing 3 mg/ml collagenase A (Roche), 100 units/ml hyaluronidase (Sigma-Aldrich) in CO2-independent medium (Gibco) completed with 5% fetal bovine serum, FBS (Lonza) and 2 mM L-glutamine (Gibco). Samples were incubated for 90 min at 37°C with shaking (150 rpm, rotations per minute). Digested samples were centrifuged at 450 g for 5 min and the supernatant eliminated. Pellets were washed once with CO2-independent medium, treated with a prewarmed 0.25% trypsin solution for 1 min and rinsed with CO2-independent medium containing 5% FBS. Pellets were then resuspended in a solution of 5 mg/ml dispase II (Roche) in CO2-independent medium containing 5% FBS and DNase I (Sigma-Aldrich) was added to a final concentration of 0.1 mg/ml. After a 5 min incubation at 37°C, cells were rinsed once in CO2-independent medium containing 5% FBS and the pellets were treated with an ice-cold ammonium chloride solution (Stem Cell Technologies). Cell suspensions were centrifuged, resuspended in CO2-independent medium and filtered through a nylon mesh cell strainer with 40 µm pores (BD Falcon) before immunolabeling.
Freshly isolated cells were incubated at 4°C for 20 min with the following conjugated antibodies: anti-CD24-FITC (BD Biosciences, 561777, RRID:AB_10896486), anti-CD49f-PE (BD Biosciences, 561894, RRID:AB_10897164), anti-CD54-PE (BioLegend, 116107, RRID:AB_313698), anti-CD45-APC (BioLegend, 103111, RRID:AB_312976), anti-CD31-APC (BioLegend, 102509, RRID:AB_312916). Labeled cells were sorted on a MoFlo Astrios EQ sorter (Beckman-Coulter) and data were analyzed using Flowlogic software.
Transplantation of mammary epithelium
Mammary epithelium transplant experiments were performed as previously described (Chadi et al., 2016; Kordon and Smith, 1998). Briefly, the proximal part of the inguinal gland of 3-week-old immunodeficient NMRI nude mice (n=30) containing the mammary epithelium was excised (cleared fat pad). Small pieces of mammary tissue collected from gestating female SOCS2WT/WT (n=6) and SOCS2KI/KI (n=6) mice were grafted into the left and right inguinal cleared fat pad of the host mice. In this way, each mouse served as its own control. Mice were sacrificed 8 weeks after the transplantation and the mammary glands were used to make whole mounts as described above. Two independent experiments were performed with 15 nude mice and three donors per genotype for each experiment.
Whole mounts, histology and immunohistochemistry
For whole mounts, one mammary gland was placed in Carnoy's fixative (100% ethanol:chloroform:glacial acetic acid, 6:3:1) for 4 h and acetone overnight at 4°C. It was then rehydrated using 30 min wash cycles of 70%, 50% and 30% ethanol, placed in water for 5 min and stained using a carmine stain overnight at 4°C. Next, it was dehydrated using 30 min wash cycles of 70%, 95% and 100% ethanol before being placed in xylene until the fatty tissue was completely transparent (at least overnight). The slides were mounted with Permount mounting media (Fisher Scientific) and observed under a binocular magnifier (Leica M80 with Leica MC170 HD digital camera, Fisher Scientific). Bright-field images at 0.75× magnification were then taken and processed using a Leica Application Suite LAS EZ software. Ductal branching was measured using ImageJ 1.54 g by counting the number of branches within three measurements from three individual fields of view per whole mount (Wilson et al., 2020). A total of six SOCS2KI/KI and six SOCS2WT/WT mammary glands were processed.
For histology, mammary tissue was fixed in RCl2 (Alphelys) overnight at 4°C before being dehydrated in 70% ethanol and embedded in paraffin wax. This paraffin block was sectioned via a microtome and stained using Hematoxylin and Eosin (H&E). Slides were observed using a Hamamatsu Nanozoomer slide scanner. Mammary gland sections were processed, and the images were analyzed, using both the Hamamatsu Nanozoomer Digital Pathology Virtual Slide Viewer and ImageJ software. Areas occupied by mammary epithelial tissue were measured and quantified using the threshold tool in ImageJ software (Hue-Beauvais et al., 2011). Five mammary glands were processed per developmental stage (virgin 8-week-old, pregnancy day 18 or lactation day 7) and per genotype (SOCS2KI/KI and SOCS2WT/WT) for a total of 30 sections.
For immunohistological staining, mammary tissue was fixed for 10 min in paraformaldehyde (PFA) 4% and then cryopreserved in 40% sucrose for at least 12 h at 4°C. The tissue was then embedded in CryoMATRIX (Thermo Scientific) for frozen sectioning using a cryostat. Five-micrometer frozen cryosections from mammary samples were left at room temperature for several minutes before incubation for 30 min at room temperature with 50 mM NH4Cl in 1×PBS. The sections were then washed in 1×PBS before incubation for 1 h at room temperature with 1×PBS (pH 7), 1% bovine serum albumin (BSA), 0.5% Triton X-100 and 0.05% sodium azide. Primary antibodies AQP5 (Alpha Diagnostic International, AQP51-A, RRID:AB_1609291) to detect the apical membrane of undifferentiated MECs were diluted to 1/100 in the previously described solution and incubated with the sections for 1 h at room temperature. Slides were then washed four times for 5-10 min at room temperature in 1×PBS. The secondary antibody, anti-rabbit IgG coupled to TRITC (Sigma-Aldrich Cat# T6778, RRID:AB_261740), was diluted to 1/300 in a 1×PBS-1% BSA solution and incubated with the sections for 1 h at room temperature. A negative control was present on each slide with the secondary antibody only. Slides were washed as previously described and mounted using Immu-Mount (Fisher Scientific) and DAPI to stain nuclei blue. Slides were observed on an epifluorescence microscope with a DAPI filter to visualize nuclei and a TRITC filter to visualize the apical membrane of MECs.
Transmission electron microscopy
For transmission electron microscopy, tissues were fixed in equal parts 0.2 M sodium cacodylate (pH 7.2) and 4% glutaraldehyde. Contrast was obtained with 0.2% Oolong Tea Extract (OTE) in cacodylate buffer on samples post-fixed with 1% osmium tetroxide containing 1.5% potassium cyanoferrate, gradually dehydrated in ethanol (30-100%) and substituted in a mix of ethanol-epon, then embedded in Epon (Delta microscopy).
After collection on 200 mesh copper grids, thin sections (70 nm) were counterstained with lead citrate and examined with a Hitachi HT7700 electron microscope operated at 80 kV (MILEXIA). Image acquisition was performed using a charge-coupled device camera (AMT). The number of luminal fat globules was measured in relation to the number of lipid droplets within the mammary epithelial cells. A total of 22 MECs and lumina for SOCS2KI/KI and 37 MECs and lumina for SOCS2WT/WT mammary glands were processed.
RNA extraction
Mammary tissue was frozen in liquid nitrogen and stored at −80°C. For nucleotide extraction, 200 mg of frozen mammary tissue were placed in a tube containing 1 ml of RNA NOW and then homogenized using an Ultra-Turrax (IKA) to lyse the cells. This solution was transferred to a different tube containing 200 µl of chloroform, mixed gently and incubated for 5 min on ice. The samples were then centrifuged for 10 min at 15,000 g, and the liquid phases were transferred to tubes containing 1 ml of isopropanol and then mixed gently. Samples were then incubated overnight at −20°C before a second centrifugation for 10 min at 15,000 g and 4°C. The supernatant was eliminated, and 1 ml of 75% ethanol was added to wash the RNA pellet. The tubes were vortexed and centrifuged for 5 min at 5000 g and 4°C. The supernatant was removed, and the pellet was dried for 10-15 min at room temperature. The RNA pellet was dissolved in 100 µl of sterile water. The RNA concentration was measured using a NanoDrop One (Thermo Fisher Scientific).
Extracted RNA was treated with DNase using the rDNase set kit (Macherey-Nagel) according to the manufacturer's instructions. Treated RNA was then purified using the NucleoSpin RNA Clean-up kit (Macherey-Nagel) according to the manufacturer's instructions. RINs (RNA integrity number) were assessed for each sample using the RNA 6000 Nano kit (Agilent) and the 2100 Bioanalyzer (Agilent), according to the manufacturer's instructions. Samples with RIN higher than 8 were used for further experiments (Fleige and Pfaffl, 2006).
Microarray hybridization and gene expression analysis
The SurePrint G3 Mouse Gene Expression v2 8×60 K Microarray kit (Agilent) was used for microarray hybridization. Cyanine-3 (Cy3)-labeled cRNA was prepared from 100 ng total RNA using the One-Color Low Input Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions. Labeled cRNA was purified using an RNeasy Mini kit (Qiagen) and Cy3-dye incorporation, and the yield of cRNA was quantified with a Nanodrop ND-2000 spectrophotometer (Thermo Fisher Scientific). The quality of synthesized cRNA was assessed using the RNA 6000 Nano kit (Agilent) and the 2100 Bioanalyzer (Agilent). cRNA (600 ng with a specific activity>6.0 pmol Cy3/µg of cRNA) was fragmented at 60°C for 30 min and hybridized to the SurePrint G3 Mouse Gene Expression v2 8×60 K Microarray kit (Agilent) at 65°C for 17 h with rotation at 10 rpm, followed by washing according to the manufacturer's instructions. Microarray slides were scanned immediately with the G2505CA microarray scanner (Agilent) using a scan protocol with a resolution of 3 µm and a dynamic range of 20 bit. The resulting .tiff images were analyzed with Feature Extraction Software 12.0.3.2 (Agilent) using default parameters.
Milk fatty acid composition analysis
Milk was collected from female mice at 10 days of lactation. Mice were separated from their pups for 4 h before receiving intraperitoneal injections of an analgesic (Fynadine, 10 µg/g body weight) and oxytocin 10 IU/ml (150 µl per female) to stimulate milk secretion. Gaseous anesthetic (isoflurane, rate of flow 0.8 l/min O2 supplemented with 2.5% isoflurane) was then administered for the length of the milking procedure. Milk was collected as previously described and stored at −80°C (Boumahrou et al., 2009).
Milk samples were analyzed for fatty acid (FA) content. Lipid extraction was performed on 25 µl of whole milk using a method adapted from Folch et al. (Folch et al., 1957), by decantation in chloroform:methanol (2:1) and saline overnight. The fatty acids of the total lipids extracted were then trans-methylated using 7% methanol in boron trifluoride (Sigma-Aldrich) for 1 h at 100°C, as previously described (Morrison and Smith, 1964). The resulting fatty acid methyl esters were analyzed by gas chromatography (Auto Sampling AI/AS1310 Gas Chromatograph Trace 1310; Thermo Scientific) coupled to a flame ionization detector on an Econo-Cap EC capillary column – WAX (30 m, 0.32 mm internal diameter, 0.25 µm film, reference 19654; ALLTECH Associates). The identification of the fatty acids was made with reference to the known FA profiles obtained from the injection of a standard FAME mixture (FA methyl esters, Supelco 37 components FAME mix, ref 47885-U, Sigma). The FA profile was established for each sample and expressed as a percentage of total FA. Before lipid extraction, 1000 µg of heptadecanoic acid (C17:0) was added to whole-fat samples and then used as an internal standard to measure total milk FA concentration in the process of analysis of chromatograms.
Statistical analysis
A Mann–Whitney U test was performed on mammary epithelial cell subtype (FACS) data as well as milk fatty acid composition data to determine whether results were significant at a risk of 2.5%. A Shapiro-Wilk test of normality was used to verify the normal distribution of data in whole-mount branching density measures. Welch's t-test was used to compare epithelial tissue in virgin 8-week-old, pregnancy day 18 and lactation day 7 mammary gland Hematoxylin and Eosin sections. Statistical analysis to study gene expression from microarray data was performed using the limma package (Ritchie et al., 2015) in R (R Core Team, 2020). Enrichment analysis was performed using gProfiler's Functional profiling (Raudvere et al., 2019). Pup growth curves were analyzed using a linear mixed effects model using the REML algorithm in R (R Core Team, 2020).
Acknowledgements
Microarray and histology experiments were performed at the @BRIDGE facility (INRAE, Jouy-en-Josas, France, http://abridge.inrae.fr). This work has benefitted from the facilities and expertise of MIMA2 (MIMA2, 2018) (GABI, INRAE, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France) and the Imagerie-Gif core facility [Cytometry Facility, Imagerie-Gif, Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France] supported by l'Agence Nationale de la Recherche (ANR-11-EQPX-0029/Morphoscope, ANR-10-INBS-04/FranceBioImaging; ANR–11–IDEX–0003–02/ Saclay Plant Sciences). The mice used in this study were under the care of the staff of UE0907 IERP (Infectiologie Expérimentale des Rongeurs et Poissons, INRAE, Jouy-en-Josas, France).
Footnotes
Author contributions
Conceptualization: E.I., C.H.-B., G.F., M.C., F.L.P.; Methodology: E.I., C.H.-B., J.C., J. Laubier, S.L.G., E.A., J. Lecardonnel, L.L., D.R.-R., C.P., M.L., M.C.; Validation: E.I., C.H.-B., F.J., F.L.P.; Formal analysis: E.I., S.L.G., E.A., J. Lecardonnel, F.J., D.R.-R., C.P., M.L., M.C., F.L.P.; Investigation: F.L.P.; Resources: E.I.; Data curation: E.I.; Writing - original draft: E.I.; Writing - review & editing: M.C., F.L.P.; Visualization: E.I.; Supervision: M.C., F.L.P.; Project administration: G.F., M.C., F.L.P.; Funding acquisition: G.F., F.L.P.
Funding
This work was funded by the Agence Nationale de la Recherche [REIDSOCS project, the MeMoFlaMa project (APIS-GENE) and the department of Animal Genetics (INRAE)].
Data availability
Microarray data files have been deposited in the GEO under the accession number GSE225441.
Peer review history
The peer review history is available online at https://journals.biologists.com/dev/lookup/doi/10.1242/dev.202332.reviewer-comments.pdf
References
Competing interests
The authors declare no competing or financial interests.