Embigin (Gp70) is a transmembrane glycoprotein within the basigin subgroup of the immunoglobulin superfamily. Embigin facilitates the function of monocarboxylate transporters (MCTs) and, therefore, plays a role in cell metabolism.^1–7 Embigin also acts as an extracellular matrix (ECM) receptor, binding to a specific domain in fibronectin.⁸ Furthermore, embigin has been observed to regulate the function of integrin-mediated cell-substratum adhesion.⁹ According to previous studies, embigin regulates the function of adult mouse stem and progenitor cells in the bone marrow¹⁰ and the sebaceous glands of the skin.⁸ More recently, it has been reported that embigin and CD166 proteins contribute to the hematopoietic function of osteomac-type macrophages.¹¹ In our study Talvi et al.¹², we demonstrated that embigin is widely expressed in the early stage of mouse embryonic development until E10.5 and later in the epithelium of tubular structures of the lungs and kidneys. Embigin-deficient (Emb^−/−) mice were found to exhibit high neonatal mortality, which was explained by delayed lung development. During lung development, the highest levels of embigin expression were observed in the lung primordium, and we suggested that the delay in lung development begins early in primordial stem cells.

The mammalian kidney consists of two major functional components: the collecting system and the nephrons, each with distinct developmental processes. During embryogenesis, these components arise from the intermediate mesoderm, with a complex interaction between the ureteric bud (UB) and the metanephric mesenchyme (MM), leading to kidney formation.¹³ In mice, the metanephric kidney begins to develop on embryonic day 10.5 (E10.5) when the UB extends into the MM. The MM supports the continuous growth and branching of the UB, which eventually forms the collecting duct system of the kidney.¹⁴ The UB tips also maintain nephron progenitors found in the MM and stimulate their differentiation. During the branching morphogenesis of the epithelial UB, each UB tip serves as an inductive centre to initiate nephrogenesis.¹⁵ Embigin protein expression is consistently observed in the tubular structures of mouse kidneys during kidney development and adulthood.¹² Embigin RNA is expressed in the developing rat UB¹⁶ and mouse UB.¹⁷ However, the exact role of embigin in the kidneys or during kidney development remains unclear.

Proper branching of renal epithelial UB is crucial for normal kidney development, as abnormal branching can severely affect kidney formation and function.¹⁸ Branching morphogenesis defines the architecture of several organs and tissues during embryonic development. During this process, epithelial tissues form complex tubular structures in secretory organs and organs that primarily distribute gases or liquids. Branching morphogenesis highly depends on the surrounding mesenchyme and the composition and organization of its ECM.^14,19 Interestingly, embigin is expressed in the epithelial compartments of the prostate and mammary gland in rats, and in the developing prostate, embigin correlates with the appearance of highly organized glandular structures²⁰, raising questions about its potential role in branching morphogenesis.

This study investigates the role of embigin in mouse kidney development. We show that embigin protein localizes to the UB and differentiating nephron precursors during early development of mouse kidneys. The E13.5 Emb^−/− kidneys exhibit a significant reduction in UB branching with concomitant downregulation of several nephron development-related genes based on our RNA sequencing (RNA-seq) results. However, this molecular phenotype appears to be transient, as most of the transcriptional changes were normalised later in development. Furthermore, the reanalysis of single-cell RNA-seq (scRNA-seq) data revealed the presence of embigin in the kidney primordium at E8.75, suggesting a potential role for embigin in renal lineage specification. Finally, our RNA-seq data indicate that embigin regulates Pappa2, Acta2, and Tagln genes during early mouse kidney development.

Embigin protein is expressed in the mouse kidney collecting ducts and distal tubules, as well as in the ureteric bud and differentiating nephron precursors

Previously, we have shown that embigin is expressed at the protein level in mice in the epithelium of tubular structures of kidneys at E13.5-E17.5, at P3, and in adult animals.¹² Here, we display that in postnatal day 3 (P3) WT mouse kidneys, E-cadherin²¹ and embigin colocalize, indicating that embigin protein is expressed in the collecting ducts and distal tubules (Fig. 1a). During early mouse kidney development, embigin protein is highly expressed in the UB and differentiating nephron precursors of mouse embryonic kidneys that have been harvested at E11.5 and cultured for 48 h (Fig. 1b). However, embigin is not detected in the Six2-positive undifferentiated nephron progenitors that cap the branching UB tree during kidney development.²² Accordingly, embigin RNA expression has previously been reported in mouse UB¹⁷ and in the UB of rat embryonic kidneys, too.¹⁶

Embigin is expressed in the vicinity of fibronectin in kidney development

The well-known molecular function of embigin is to interact with many MCT proteins, including MCT1, and assist them to move to the cell membrane.^1–7 To study embigin and MCT1 localization in embryonic mouse kidneys, these proteins were stained with the whole-mount immunofluorescence staining technique in WT kidneys at E13.5 (Fig. 1c). While embigin is expressed in the developing renal UB epithelium and differentiating nephron precursors, MCT1 is mainly expressed in the mesenchyme of the developing kidney, and no obvious colocalization was detected.

Since embigin has been shown to function as a fibronectin receptor⁸ and a regulator of integrin function⁹, the localization of embigin and fibronectin was also studied in WT kidneys at E13.5 (Fig. 1d). Fibronectin appears to be highly expressed near embigin in the ECM surrounding the UB. Based on this, embigin may mediate tissue interactions taking place between UB and the surrounding mesenchyme, known to be an essential regulator of both kidney growth via branching and nephron differentiation.²³

The lack of embigin impairs the ureteric bud branching of the mouse kidney

Previously, we have found that the Emb^−/− mice have a high neonatal mortality (72%), which can be explained by delayed lung development. Additionally, the Emb^−/− embryos have been observed to be smaller than the WT embryos.¹² The effect of embigin deficiency was investigated further in mouse kidneys. Based on kidney weights, we observed that the kidneys are significantly smaller (p = 0.026) in Emb^−/− embryos than WT at E17.5 (Fig. 2a). The average kidney weights were 3.4 ± 0.9 mg for Emb^−/− embryos, 4.5 ± 1.2 mg for Emb^+/− embryos, and 4.2 ± 0.9 mg for WT embryos. However, the Emb^−/− and WT kidney weight / embryo weight -ratios had no statistical difference (Fig. 2a). The average body weights were 766 ± 84 mg for Emb^−/− embryos, 1006 ± 127 mg for Emb^+/− embryos, and 931 ± 127 mg for WT embryos. This suggests an overall growth regulatory effect for embigin during development.

Next, we studied the UB branching morphogenesis and analysed the tip formation in the mouse kidneys during early development. The kidneys of WT, Emb^+/−, and Emb^−/− embryos were isolated at E11.5 and then cultured for 48 h. This revealed that the branching of the Emb^−/− UBs was significantly impaired (p = 0.003) during the early stages of kidney development compared to WT (Fig. 2b). Based on these results, embigin might be essential for UB tip formation during early kidney morphogenesis. To investigate whether embigin is a general regulator of tube formation and branching morphogenesis in mice, and as embigin expression has been observed in the mammary glands²⁰, we analysed the mammary glands of 6-week-old mice. In the mammary glands of mice, the epithelial ductal system develops mainly during puberty, starting from 4 weeks of age.²⁴ However, the whole-mount staining and image analysis of branches, junctions, and terminal end-points showed no differences between WT and Emb^−/− mice (Supplementary Fig. 1), suggesting organ-specific effects for embigin during their morphogenesis.

Embigin deficiency significantly reduces the transcription of genes involved in nephron development in E13.5 kidneys

Next, we studied the effect of embigin deficiency on the gene expression of other proteins in embryonic kidneys. The RNA-seq analysis on mouse embryonic kidneys at E13.5 identified 85 downregulated genes and only 16 upregulated genes (Emb^−/− kidneys vs. WT kidneys, log2FC > 0.6 or < -0.6 and adjusted p-value < 0.05; Fig. 3a, 3b, 4a). Metascape enrichment analysis of the RNA-seq data revealed that the downregulated genes were related to the development of nephrons and glomerulus, the regulation of systemic arterial blood pressure, body fluid levels, and hormone levels, the transport of different substances, and tube morphogenesis (Fig. 3c). Importantly, the identified processes are all related to critical functions of mature kidneys and suggest imbalanced differentiation of the renal system. The downregulated genes indeed included several genes related to kidney development (Supplementary Table 1), including Nphs1 (log2FC = -6.2) and R3hdml (log2FC = -5.2). Nphs1 encodes nephrin and R3hdml encodes R3h domain containing-like protein, which are both involved in podocyte development and function.^25,26 Thus, the downregulation of these genes may indicate that at least podocyte development is affected in the Emb^−/− kidneys.

From the MCT-family proteins, Slc16A12 (log2FC = -1.1), encoding MCT12, was also downregulated in the RNA-seq analysis of E13.5 Emb^−/− kidneys. MCT12 is expressed in the nephron proximal tubules and is identified as a transporter for creatine and its precursor, guanidinoacetate.²⁷ MCT12 surface expression is partially dependent on basigin.²⁸ Embigin has not yet been associated with MCT12 function, but as embigin has been shown to regulate the activity and expression of several MCTs^1–7, it may also assist MCT12.

Transcriptional changes in Emb^-/- kidneys are transient

Embigin is also present during the later stages of kidney development. Our earlier RNA-seq results from the E17.5 kidneys¹² were reanalysed to match the analysis workflow used for the RNA-seq of E13.5 kidneys. The results indicated that the genes that were downregulated in the Emb^−/− kidneys at E13.5 (Fig. 4a) did not show statistically significant changes in expression in the Emb^−/− kidneys at E17.5, despite the obvious lack of embigin expression (Fig. 4b). We could not detect apparent morphological abnormalities in Emb^−/− kidneys at E17.5 (Fig. 4c). Thus, the transcriptional changes of Emb^−/− kidneys reach the level of WT during the later developmental stages. Consistently, there were no signs of albuminuria in the newborn (P0) Emb^−/− mice (Fig. 4d). No protein was detected (0 g/L) with Combur 10 UX urine test strips and Urisys 1100 analyzer (Roche) in the urine samples from 5- to 6-month-old adult Emb^−/− mice either. Furthermore, we did not see any obvious alterations in the histology of adult Emb^−/− mouse kidneys.¹² Overall, kidney function, including podocyte-related functions, appears normal in both newborn (P0) and adult Emb^−/− mice.

Embigin regulates genes associated with kidney development and function, including Pappa2, Acta2, and Tagln

Differential gene expression in E13.5 Emb^−/− kidneys may be partially explained by the fact that E13.5 Emb^−/− kidneys exhibit a developmental delay in comparison to E13.5 WT kidneys. We wanted to study whether the lack of embigin has more direct effects on the expression levels of kidney development-related genes. For that purpose, we used specific siRNAs to knockdown embigin expression in a mouse epithelial cell line, namely skin keratinocytes. Two independent siRNAs, Mm_Emb1 (Emb1) and Mm_Emb4 (Emb4) siRNAs (QIAGEN), were selected for experiments. In 48 hours, Emb1 siRNA reduced embigin protein level in cells by 88 ± 4%, while the decrease after Emb4 siRNA treatment was 81 ± 6%, as was demonstrated by western blot analysis (Fig. 5a, 5b).

Since silencing with Emb1 siRNA was more effective than with Emb4 siRNA, the results gained with Emb1 siRNA are presented in the primary RNA-seq analysis; the results are based on the comparison of Emb1 siRNA-silenced cells to negative control siRNA-silenced cells. The knockdown of embigin in mouse epithelial cells led to the upregulation of 439 genes and the downregulation of 85 genes, including embigin (Supplementary Fig. 2). Over-representation analysis of differentially expressed genes in the RNA-seq data indicated that the most significantly enriched biological processes (p-value < 0.01) included developmental processes and morphogenesis of organs (Fig. 5c, 5d). In the upregulated genes, tube morphogenesis, renal system development, regulation of vasculature development, extracellular matrix organization, tissue morphogenesis, and regulation of body fluid levels were represented (Fig. 5c). The downregulated genes were associated with, e.g., nephron and epithelial cell development (Fig. 5d). Indeed, many of the downregulated genes were related to kidney development, including Npnt (log2FC = -0.8) and Hnf1b (log2FC = -0.6) (Supplementary Table 2). These data suggest that in mouse epithelial cells, embigin deficiency may have a significant effect on the expression of the genes that specifically regulate the morphogenesis and function of tissues and organs, including the kidneys.

Next, we studied which downregulated genes in RNA-seq analysis of E13.5 Emb^−/− kidneys were shared with those downregulated in mouse epithelial cells. This identified three genes: Pappa2 [log2FC(E13.5 kidneys) = -1.0, log2FC(mouse epithelial cells) = -1.1], Acta2 [log2FC(E13.5 kidneys) = -1.0, log2FC(mouse epithelial cells) = -1.0], and Tagln [log2FC(E13.5 kidneys) = -0.7, log2FC(mouse epithelial cells) = -1.9] (Fig. 5e). Pappa2 encodes pregnancy-associated plasma protein A2 (PAPP-A2), Acta2 α-smooth muscle actin (α-SMA), and Tagln transgelin, all of which have been associated with kidney development and function^29–32 and could be potential regulatory targets for embigin during early kidney development.

Embigin is present in the earliest stages of mouse kidney morphogenesis

We wanted to analyse the embigin expression during the earliest stages of kidney development when kidney precursors have differentiated from intermediate mesoderm. Therefore, we reanalysed the scRNA-seq data³³ (Supplementary Fig. 3) obtained from the mouse Pax2-expressing renal cells at E8.75 and the caudal urogenital system cells (containing the caudal nephric duct, the UB, the surrounding mesenchyme, and the cloaca) at E11.5 to see whether embigin is expressed in the earlier time points of kidney development. Intriguingly, embigin showed expression at both time points, E8.75 and E11.5, especially in the epithelial cell-type cluster (Supplementary Fig. 3). Thus, it seems that embigin is already present in the kidney pro/mesonephros, which is also known as the kidney primordium, starting at E8.75³³. Hypothetically, basigin may compensate for the lack of embigin in the Emb^−/− kidneys. Therefore, we also observed the presence of basigin in the mouse Pax2-expressing renal cells at E8.75 and the caudal urogenital system cells at E11.5 in addition to embigin (Supplementary Fig. 3). Most of the cells of the epithelial cell-type cluster that express embigin also express basigin.

Knockdown of embigin in mouse epithelial cells also affected critical kidney genes that were not downregulated in E13.5 Emb^−/− kidneys, such as Npnt. The reanalysis of the scRNA-seq data³³ obtained from the mouse caudal urogenital system at E11.5 showed partial co-expression of embigin with Npnt in the epithelial cell-type cluster cells (Supplementary Fig. 4). Npnt gene encodes nephronectin, a key regulator of metanephric kidney induction, regulated by the Pax2/8-Gata3-Lim1-network that establishes the transcriptional program of the kidney primordium.^34,35 Lim1 (gene Lhx1) is a central regulator in primordial kidney morphogenesis, including the nephronectin expression.^34,36 Thus, we analysed the scRNA-seq data³³ obtained from the mouse Pax2-expressing renal cells at E8.75 and the caudal urogenital system cells at E11.5 for embigin and Lhx1 expression. Interestingly, some of these cells in the epithelial cell-type cluster express both embigin and Lhx1 (Supplementary Fig. 4).

Since MCT1 seemed not to clearly colocalize with embigin expression in the kidneys at E13.5 (Fig. 1C), we analysed the scRNA-seq data³³ obtained from the mouse Pax2-expressing renal cells at E8.75 and the caudal urogenital system cells at E11.5 for embigin and MCT1 (gene Slc16a1) expression. Intriguingly, some epithelial cell-type cluster cells showed co-expression of embigin with Slc16a1 (Supplementary Fig. 5), which may indicate some degree of dependence of MCT1 on embigin during early kidney development.

Embigin (Gp70) belongs to the basigin subgroup within the immunoglobulin superfamily, alongside basigin (CD147/EMMPRIN) and neuroplastin (Np65 and Np55). The exact role of these proteins during development and tissue formation has remained unclear. In our recent paper¹², we reported that embigin-deficient C57BL/6N mice have high neonatal mortality, as 72% of the newborn Emb^−/− pups are lost by P3. In that study, the focus was on the developing Emb^−/− lungs, where the structural abnormalities were most obvious. The delayed lung maturation could explain the compromised survival of Emb^−/− mice during the neonatal period. Another recent study reports that the standard C57BL/6N mouse strain harbouring a mutation leading to the lack of embigin shows reduced survival rates, hearing impairment, and defects in the brain and the heart.³⁷ In our knockout model, we did not find evidence that embigin deficiency could cause heart defects. The Emb^−/− mice that survived the neonatal period developed normally and were fertile.¹²

Embigin is widely expressed during mouse development until E10.5 and later, more specifically, in the epithelium of tubular structures of different organs, including the kidneys and lungs.^12,38 Contrary to the lungs, where embigin expression is high only in the lung primordium during mouse development, we have found that embigin protein is present throughout kidney development.¹² In this study, we discovered that in the tubular structures of P3 mouse kidney, embigin is localized in the epithelium of collecting ducts and distal tubules. In addition, during mouse kidney development, embigin protein expression is strong in the epithelial UB structures and differentiating nephron precursors. Interestingly, fibronectin is highly expressed in the ECM surrounding the UB close to the embigin expression. Fibronectin plays a crucial role in kidney branching morphogenesis.³⁹ As we have previously shown that embigin is a fibronectin receptor⁸, embigin may participate in cell-ECM interaction regulation during kidney development.

Increased embigin expression levels have been linked to tubular structure formation in developing rat prostate glands.²⁰ Therefore, we investigated embigin’s role in kidney branching morphogenesis by analysing the UB tip formation of the mouse kidneys in early development. After the kidneys of WT, Emb^+/−, and Emb^−/− embryos were isolated at E11.5 and then cultured for 48 h, we found that the branching of the Emb^−/− UBs was significantly impaired during the early stages of kidney development compared to WT. Thus, embigin may participate in branching morphogenesis during mouse kidney development.

Our RNA-seq analysis of E13.5 Emb^−/− kidneys revealed the downregulation of several nephron development-associated genes, including Nphs1 and R3hdml. Nphs1 encodes nephrin, a protein specifically expressed in podocytes, which are highly specialized epithelial cells in the glomerulus of kidney nephrons that wrap around capillaries.^25,40 Nephrin plays a crucial role in the development and function of the kidney glomerular filtration barrier.²⁵ R3hdml encodes R3h domain containing-like protein, which exhibits a podocyte-specific expression pattern during renal development and has an essential role in glomerular development as well.²⁶ The downregulation of these genes indicates delayed podocyte development in the Emb^−/− kidneys. The reanalysis of RNA-seq results from E17.5 Emb^−/− kidneys did not show statistically significant changes in the expression of the same genes downregulated in the Emb^−/− kidneys at E13.5. Therefore, the development of Emb^−/− kidneys might align with WT kidneys in the later stages of growth, particularly regarding the gene expression patterns. Furthermore, no obvious morphological abnormalities in Emb^−/− kidneys at E17.5 were detected compared to WT kidneys. Embigin protein is present in the tubular structures of the adult mice tissues.¹² However, there were no signs of albuminuria in newborn or adult Emb^−/− mice, and the previous histological analysis of adult Emb^−/− mice kidneys did not reveal morphological anomalies either.¹² Therefore, the kidneys seem to function normally in the surviving Emb^−/− mice. Our results indicate that the impaired branching in embryonic Emb^−/− kidneys likely reflects a transient defect that is overcome by potential compensation from other proteins.

During mouse embryonic development, embigin mRNA and protein expression levels are highest at very early stages.^12,38 We have also described that in mouse lung development, embigin function is crucial in early primordium stem cells¹². Thus, embigin function might be central possibly already in pro/mesonephros, i.e., the kidney primordium, starting from E8.75³³. Therefore, we reanalysed the scRNA-seq data³³ obtained from the mouse Pax2-expressing renal cells at E8.75 and the caudal urogenital system cells at E11.5. Strikingly, embigin was expressed in these renal cells at E8.75 and E11.5, showing that embigin is present in the kidney primordium. In adult mice, embigin has been linked to stem and progenitor cell function, as embigin participates in stem cell niche regulation in the bone marrow¹⁰ and progenitor cell regulation in the sebaceous gland⁸. Besides, embigin is present in the lung primordium.¹² Our results, suggesting that embigin may also have a role in the kidney primordium, are in line with the general idea that embigin supports specific stem and progenitor cells both in adult tissues and during embryonic development.

Basigin may be functionally necessary for Emb^−/− kidneys. The lack of basigin leads to spontaneous polycystic kidney disease in adult mice⁴¹, but its putative role during kidney development is unknown. However, lack of embigin did not elevate basigin mRNA levels based on the RNA-seq analysis of E13.5 or E17.5 Emb^−/− kidneys or embigin-silenced mouse epithelial cells. Surprisingly, based on the reanalysis of the scRNA-seq data³³, most of the embigin-positive renal cells also expressed basigin at E8.75 and E11.5. Thus, it is possible that basigin partially compensates for the lack of embigin, which is why the phenotype is variable and very mild in surviving Emb^−/− mice.

To study the role of embigin in epithelial cells, we performed RNA-seq analysis on embigin-silenced mouse epithelial cells. The data suggested that the lack of embigin significantly affects gene expression related to tissues and organ morphogenesis, particularly in kidney development. However, embigin silencing in mouse epithelial cells resulted in the upregulation of 439 genes, differing greatly from the results seen in the E13.5 kidneys, which can be related to the different origins of the samples. Since embigin is expressed in the renal cells already at E8.75, differential regulation of some critical embigin-related genes might no longer be present at E13.5. Thus, embigin silencing in mouse epithelial cells possibly affected kidney genes that were not downregulated in E13.5 Emb^−/− kidneys. One notable downregulated gene in the mouse epithelial cells was Npnt, which encodes nephronectin, a key regulator of metanephric kidney induction.^34,35 Nephronectin-deficient mice frequently exhibit kidney agenesis due to delayed UB invasion.⁴² Besides, Lhx1 codes for Lim1 protein, an essential regulator of primordial kidney morphogenesis and nephronectin expression.^34,36,35 Reanalysis of scRNA-seq data³³ unveiled that some renal cells express both embigin and nephronectin in the epithelial cell-type cluster at E11.5 and embigin and Lhx1 in the epithelial cell-type cluster at E8.75 and E11.5. Therefore, embigin may have a role in renal lineage specification.

Embigin facilitates the function of MCTs, especially MCT1.^2–4 MCT1 is known to be expressed in the kidneys of adult mice⁴³, but little is known about its expression during embryonic development. In E13.5 WT kidney immunofluorescence staining, MCT1 protein was widely expressed, but the expression did not clearly correlate with embigin. However, the reanalysis of the scRNA-seq data³³ showed that MCT1 is present in early kidney morphogenesis at E8.75 and E11.5, and it is co-expressed with embigin in some of the epithelial-type cells. Thus, MCT1 may have some dependence on embigin during early kidney development.

Finally, we could recognize three genes, Pappa2, Acta2, and Tagln, which were downregulated in both E13.5 Emb^−/− kidneys and embigin-silenced mouse epithelial cells. Thus, the expression levels of these genes were considered to be directly associated with embigin expression. Pappa2 encodes pregnancy-associated plasma protein A2 (PAPP-A2), a secreted metalloprotease that increases insulin-like growth factor (IGF) availability.²⁹ IGFs are crucial for normal growth and development, especially for pre- and postnatal kidney development.⁴⁴ Like embigin in mice, PAPPA-A2 is localized in the UB of embryonic rat kidney³⁰. Acta2 codes for α-smooth muscle actin (α-SMA). α-SMA is expressed in smooth muscle cells and fibroblasts. In mice, the deficiency of α-SMA results in normal development, but it causes impaired vascular contractility and leads to hypotension.³¹ Tagln encodes transgelin, which is also abundantly present in smooth muscle cells.⁴⁵ Transgelin has been shown to participate in proteinuria progression and the dynamics of the podocyte foot process.³² Additionally, PAPP-A2, α-SMA, and transgelin have all been connected to cell migration^46–48, a process critically dependent on cell-ECM interaction. Notably, embigin can also participate in the regulation of cell adhesion. Overall, these genes have been linked to kidney development and function, suggesting they may serve as potential regulatory targets for embigin during the early stages of kidney morphogenesis.

In conclusion, our results indicate that the absence of embigin affects cellular behaviour starting from early kidney development. Our data, combined with findings from our previous study on lung development¹², suggest that embigin supports primordial stem cells during early embryonic development. Thus, we hypothesize that the biological function of embigin seems to be facilitating stem cell activities, particularly those related to cell-ECM interactions.

Animals

C57BL/6N mice (Mus musculus, Charles River Laboratories, Wilmington, MA) and embigin knockout (Emb^−/−) mice¹² were maintained in the Central Animal Laboratory at the University of Turku, Finland. All animal experiments were reviewed and approved by the Ethical Committee for Animal Experimentation in Finland, complying with international guidelines for the care and use of laboratory animals. The animal research was conducted in accordance with the ARRIVE guidelines. Mice were anesthetised using CO₂, and cervical dislocation was performed to confirm euthanasia. In timed mating, the day of the vaginal plug appearance was considered embryonic day 0.5 (E0.5). The genotypes of the mice were determined as described previously in detail.¹² The WT and Emb^−/− mouse embryos were examined between embryonic days E11.5-E17.5 and pups at postnatal days P0. The average weight of E17.5 Emb^−/− embryos was 0.77 ± 0.08 g, and that of WT embryos was 0.93 ± 0.13 g. The weight of P0 Emb^−/− pups was 1.14 ± 0.05 g, and that of WT pups was 1.23 ± 0.01 g. Additionally, WT pups were examined at P3. Young female WT and Emb^−/− adult mice were studied at 6 weeks of age. The average weight of 6-week-old Emb^−/− mice was 16.5 ± 0.9 g, while WT mice weighted 19.0 ± 1.0 g. The kidneys of E17.5 embryos from Emb^+/− breedings were weighted and imaged with the AxioZoom.V16 stereo microscope (Zeiss) using AxioCam 105 Colour camera and 1.0x PlanApo Z objective. Image linear brightness and contrast adjustments were performed with ImageJ/Fiji software.⁴⁹

Cell lines

Mouse epithelial keratinocyte cells isolated from adult dorsal skin⁵⁰ were cultured in FAD medium containing three parts DMEM medium (Lonza), one part Ham’s F12 medium (Thermo Fisher Scientific) supplemented with 10% ES screened fetal bovine serum, heat-inactivated (Cytiva), 2 mM L-glutamine (Lonza), 100 units/ml Penicillin-Streptomycin (Lonza), 200 µM adenine (Sigma-Aldrich), 0.5 µg/ml hydrocortisone (Sigma-Aldrich), 5 µg/ml insulin (Sigma-Aldrich), 16,8 ng/ml cholera toxin (Sigma-Aldrich) and 10 ng/ml epidermal growth factor (Sigma-Aldrich). The cells were cultured at 37°C in a humidified atmosphere with 5% CO₂.

Antibodies

Following antibodies were used in our studies: Embigin Monoclonal Antibody, clone G7.43.1, 14-5839-81, eBioscience, Thermo Fisher Scientific (western blotting 1:1000, immunofluorescence 1:200); Monoclonal Anti-β-Tubulin I antibody produced in mouse, clone SAP.4G, T7816, Sigma-Aldrich (western blotting 1:20 000); E-Cadherin Antibody, AF748, R&D Systems (immunofluorescence 1:500); Calbindin D28K Antibody (C-20), sc-7691, Santa Cruz Biotechnology (immunofluorescence 1:400); Six2 Polyclonal antibody, 11562-1-AP, ProteinTech (immunofluorescence 1:500); Anti-Monocarboxylate Transporter 1 Antibody, AB1286-I, Sigma-Aldrich (immunofluorescence 1:1000); Anti-Fibronectin Antibody, AB2033, Sigma-Aldrich (immunofluorescence 1:80); IRDye secondary antibodies, LI-COR Biosciences (western blotting 1:15 000); and Alexa Fluor secondary antibodies, Thermo Fisher Scientific (immunofluorescence 1:400).

Immunofluorescence staining of paraffin sections

To study embigin localization in mouse kidneys, the WT kidney paraffin sections at postnatal day 3 (P3) were stained with embigin and E-cadherin antibodies using fluorescent immunohistochemistry. Formalin-fixed samples were embedded in paraffin, and 4 µm sections were cut using a RM2255 microtome (Leica) and immobilized on adhesion slides (SuperFrost Plus, Thermo Fisher Scientific) overnight at 37°C. The sections were deparaffinized and rehydrated. The antigen retrieval was achieved with 3 min proteinase K treatment (S3020, Agilent). After antigen retrieval, the sections were washed in PBS. The samples were blocked with 1% BSA in PBS for 1 h at RT, and stained with primary antibodies in a blocking buffer overnight at 4°C. A secondary antibody without a primary antibody was applied as the negative control. The samples were washed with PBS and incubated with Alexa Fluor secondary antibodies in a blocking buffer for 1 h at RT. The sections were rinsed in PBS and finally in dH₂O and mounted in Mowiol (Calbiochem) containing 25 mg/ml DABCO anti-fading reagent (Sigma). The samples were imaged with the LSM 880 confocal microscope (Zeiss) using a Plan-Apochromat 20x/0.8 M27 objective. Image stacking, background subtractions, linear brightness, and contrast adjustments were performed with ImageJ/Fiji software.⁴⁹

E13.5 kidney whole-mount immunofluorescence staining

Embigin and MCT1 or fibronectin localization was studied in WT kidneys at E13.5 using the whole-mount immunofluorescence staining technique as described previously in detail for mouse embryos.^51,12 The kidneys were imaged with the LSM 880 confocal microscope (Zeiss) using Plan-Apochromat 20x/0.8 M27 and 40x Zeiss LD LCI Plan-Apochromat objectives. Image stacking, background subtractions, linear brightness, and contrast adjustments were performed with ImageJ/Fiji software.⁴⁹

Kidney explant cultures and whole-mount immunofluorescence staining

Embigin expression in early kidney development was studied in WT kidney explant cultures at E11.5. Additionally, the kidney UB tip formation in WT, Emb^+/−, and Emb^−/− embryos was compared in kidney explant cultures at E11.5. Embryonic kidney cultures were carried out on a Trowell-type system according to published protocols.^52,53 In brief, after isolation of kidneys at E11.5, they were placed on the Transwell insert filter (24 mm Transwell with 0.4 µm Pore Polyester Membrane Insert, 3450, Corning) and cultured in DMEM/F12 medium (Gibco, Thermo Fisher Scientific) supplemented with 10% ES screened fetal bovine serum, heat-inactivated (Cytiva), 2 mM L-glutamine (Lonza), 100 units/ml Penicillin-Streptomycin (Lonza) at 37°C in a humidified atmosphere with 5% CO₂ for 48 h. Whole-mount immunofluorescence staining was performed as previously described.⁵⁴ For staining with embigin and Six2 primary antibodies, the kidneys were fixed for 20 min in 4% paraformaldehyde. For staining with calbindin and Six2 primary antibodies, 10 min ice-cold methanol fixation at 4°C was used. The samples were visualized with Alexa Fluor secondary antibodies and imaged with a Leica SP8 confocal microscope. Image background subtractions, linear brightness, and contrast adjustments were performed with ImageJ/Fiji software.⁴⁹ The number of UB tips was counted and normalisation was done to remove the size variation between different litters from various pregnant dams. Normalisation method for each litter: the number of UB tips / mean number of UB tips of WT samples of the litter *100%.

Mammary gland whole-mount staining and analysis

WT and Emb^−/− abdominal mammary glands of 6-week-old mice at the diestrus stage of the estrous cycle were isolated, spread to adhesion slides (SuperFrost Plus, Thermo Fisher Scientific), and fixed in Carnoy’s solution (60% (v/v) ethanol, 30% (v/v) chloroform, 10% (v/v) glacial acetic acid) for 2 h at RT. The samples were washed in 70% ethanol, hydrated by gradually changing the solution to distilled water, and stained in carmine alum solution (07070, Stemcell Technologies) for 1 day. The samples were dehydrated in graded solutions of ethanol, cleared in xylene for 2 days, and mounted with DPX mountant for histology (Sigma-Aldrich). The mammary glands were imaged with the AxioZoom.V16 stereo microscope (Zeiss) using AxioCam 105 Colour camera and 1.0x PlanApo Z objective. The images were processed with ImageJ/Fiji software’s⁴⁹ skeletonize and analyse skeleton plugins to measure the number of branches, junctions, and terminal end-points of the mammary glands.

Histological staining

The kidneys were analysed from WT and Emb^−/− embryos at E17.5. Sections (4 µm) were cut and immobilized on adhesion slides as described earlier. The sections were deparaffinized, rehydrated, and stained with conventional hematoxylin and eosin (H&E), imaged with Pannoramic 250 Flash III slide scanner (3D Histech), and analysed with the CaseViewer program (3D Histech).

Urine analysis of newborn pups and adult mice

Urine samples were collected from P0 pups and 5- to 6-month-old adult mice. The P0 WT, Emb^+/−, and Emb^−/− mice urine samples were run on 12% SDS-PAGE gel, and the gel was stained with InstantBlue Coomassie Protein Stain (ab119211, Abcam) according to the manufacturer’s instructions. Bovine serum albumin (BSA) was used as a reference control. The protein concentrations (g/L) of urine samples from three WT and three Emb^−/− 5- to 6-month-old adult mice were analysed with Combur 10 UX urine test strips (11544373171, Roche) and Urisys 1100 analyzer (Roche).

RNA sequencing of mouse E13.5 kidneys

Total RNA from mouse WT and Emb^−/− kidneys at E13.5 was isolated using the NucleoSpin RNA kit (740955.50, Macherey-Nagel) according to the manufacturer’s instructions. Bead Tubes Type F (740816.50, Macherey-Nagel) were utilized in tissue homogenization. For each WT sample, kidneys from 4 individuals were pooled, and for each Emb^−/− sample, kidneys from 6 individuals were pooled to get enough RNA for five independent biological replicates. The quality and quantity of the RNA were determined using the Nanodrop ND-2000 spectrophotometer (Thermo Scientific).

The samples were prepared for the sequencing from 100 ng of high-quality RNA / sample using Illumina Stranded mRNA Preparation Kit and Illumina Stranded mRNA Library Preparation protocol (Illumina). Sequencing was performed with NovaSeq 6000 SP Sequencing System (Illumina) using paired-end sequencing chemistry and 2 x 50 bp read length. Five independent biological replicates for both WT and Emb^−/− were analysed and run in one lane. The reads obtained from the instrument were base-called using bcl2fastq2 conversion software.

Reanalysis of RNA sequencing results of mouse E17.5 kidneys

The RNA-seq results obtained from WT and Emb^−/− E17.5 kidneys in Talvi et al.¹² were reanalysed as described below to match the analysis workflow used for the RNA-seq of E13.5 kidneys.

RNA interference and RNA sequencing of mouse epithelial cells

Subconfluent mouse epithelial keratinocyte cells were transfected with 75 nM siRNAs: Mm_Emb_1 Flexitube (Emb1; SI00993307, QIAGEN), Mm_Emb_4 Flexitube (Emb4; SI00993328, QIAGEN), or AllStars Negative Control siRNA (1027280; QIAGEN) using siLentFect reagent (1703361, Bio-Rad) according to the manufacturer’s instructions. After 48 h of treatment, cells were detached by scraping them in PBS. Protein samples for western blot analysis were lysed in SDS-PAGE loading buffer (5% (v/v) glycerol, 1.7% (w/v) SDS, 1.6% (w/v) DTT, 0,002% bromophenol blue in 0,05 M Tris-HCl, pH 6,8), denatured by heating at 95°C for 5 min, and loaded on 10% SDS-PAGE gel. Embigin and β-tubulin (diluted in 5% milk and 0.1% Tween-20 in TBS) were stained in the membrane for 2 h at RT. IRDye secondary antibodies and Odyssey CLx imager (LI-COR Biosciences) were used for signal detection. Total RNA was isolated using the NucleoSpin RNA kit (740955.50, Macherey-Nagel) according to the manufacturer’s instructions. The quality and quantity of the RNA were determined using the Nanodrop ND-2000 spectrophotometer (Thermo Scientific).

The samples were prepared for the sequencing from 300 ng of high-quality RNA / sample, and the sequencing was performed as described above. Three samples (negative control, Emb1, and Emb4 siRNA-treated samples) from four independent biological replicates were analysed and run in one lane. The reads obtained from the instrument were base-called using the bcl2fastq2 conversion software. As the siRNA silencing with Emb1 siRNA was more effective than with Emb4 siRNA, the results obtained with Emb1 siRNA are presented in the RNA-seq analysis; the Emb1 siRNA-silenced cells were compared to negative control siRNA-silenced cells.

The RNA-seq datasets generated and/or analysed during the current study are available in the ArrayExpress repository at EMBL-EBI, www.ebi.ac.uk/arrayexpress: