In our previous studies, we found that knockout of neurodevelopmental genes using Emx1-Cre sometimes exhibited phenotypes that could not be easily explained by compromised brain function. We suspected that Emx1-Cre might mediate recombination in other organs.

To examine the tissue distribution of Emx1-Cre recombination activity, we crossed Emx1-Cre mice with Ai14 mice (hereafter Emx1-Ai14) (Figure 1A). Ai14 is a Cre reporter tool strain that will express robust tdTomato fluorescence after Cre-mediated recombination (Madisen et al., 2010). As expected, tdTomato fluorescence was detected in the neocortex of E18.5 Emx1-Ai14 embryos (n = 3), whereas little fluorescence was detected in E18.5 Ai14 embryos (n = 3) (Figure 1B). After inspecting the whole E18.5 Emx1-Ai14 embryos, we found that tdTomato fluorescence could be also detected in the kidney region (n = 3). The fluorescence intensity was at a comparable level to that in the neocortex (Figure 1C). To examine tdTomato fluorescence in greater detail, we dissected the kidney from E18.5 Emx1-Ai14 embryos. The fluorescence was primarily localized in the renal cortex but not in the medulla (n = 3) (Figure 1D). This preferential localization in the cortex was more prominent in postnatal day (P) 3 (n = 3) (Figure 1E) as well as P7 kidneys (n = 3) (Figure 1F). We concluded that Emx1-Cre mediates robust recombination in the renal cortex during embryogenesis.

By visual inspection, we suspected that tdTomato fluorescence was localized in the proximal and distal tubules. To confirm this observation, five independent experiments were carried out to analyze the colocalization of tdTomato fluorescence with established markers. Kidneys were dissected from P7 Emx1-Ai14 mice. Lotus tetragonolobus lectin (LTL) labels epithelial cells in the proximal convoluted tubules in the renal cortex (Chen et al., 2021). We found that the majority of cells labeled by fluorescein-conjugated LTL also exhibited tdTomato fluorescence (n = 5) (Figures 2A, C). Slc12a3 labels epithelial cells in the distal convoluted tubules (Chen et al., 2021). We found that most Slc12a3-positive cells also exhibited tdTomato fluorescence (n = 5) (Figures 2B, C). These results demonstrated that Emx1-Cre can drive recombination in epithelial cells lining proximal and distal convoluted tubules of the nephron.

These observations prompted us to ask whether other Cre systems used to study brain development also exhibit recombination activity in the kidney. We crossed nestin-Cre mice with Ai14 mice (hereafter Nestin-Ai14) (Figure 3A). Like in Emx1-Ai14 mice, tdTomato fluorescence was detected primarily in the renal cortex of P7 Nestin-Ai14 kidneys (n = 3) (Figure 3B). In addition, we found that the majority of cells labeled by fluorescein-conjugated LTL also exhibited tdTomato fluorescence (n = 5) (Figures 3C, F), and all Slc12a3-positive cells exhibited tdTomato fluorescence (n = 5) (Figures 3D, F). Finally, we carried out a side-by-side comparison of tdTomato fluorescence in P7 Nestin-Ai14 and Emx1-Ai14 kidneys. We found that tdTomato fluorescence could be detected in the glomerulus of the nephron in P7 Nestin-Ai14, but not in Emx1-Ai14 kidneys (n = 3) (Figure 3E). These results demonstrated that nestin-Cre, like Emx1-Cre, can drive recombination in epithelial cells lining proximal and distal convoluted tubules, but also in the glomerulus of the nephron.

In humans, inactivating mutations of LARP7 have been linked to Alazami syndrome, a human NDD with other comorbidities. We recently generated Larp7 knockout mice using either Emx1-Cre or nestin-Cre (hereafter Emx1-Larp7-KO and Nestin-Larp7-KO, respectively) to examine the effect of Larp7 loss on neurodevelopment. In light of findings presented so far, we examined Larp7 protein expression in the kidneys of these mice. The renal cortex and the renal medulla were separately dissected to extract total proteins. In both P90 Emx1-Larp7-KO (n = 3) and Nestin-Larp7-KO kidneys, Larp7 protein levels were greatly reduced in the cortex but not in the medulla (Figure 4A). This result is consistent with the fact that both Emx1-Cre and nestin-Cre can mediate recombination in the renal cortex (Figures 2, 3).

To assess the renal function, blood tests were carried out to measure the levels of blood urea nitrogen (BUN), serum creatinine (Scr), and electrolytes (potassium (K⁺), sodium (Na⁺) and chloride (Cl⁻)). Emx1-Larp7-KO and wild-type mice exhibited comparable levels of these five parameters (Figure 4B). In contrast, Nestin-Larp7-KO mice exhibited increased BUN levels, compared to wild-type littermates. The Scr and electrolyte levels were normal in Nestin-Larp7-KO mice (Figure 4C). Considering that nestin-Cre (but not Emx1-Cre) can mediate recombination in the renal glomeruli (Figure 3D), these observations indicated that under homeostatic conditions, the filtering functions of the nephron may be compromised by Larp7 genetic ablation.

Emx1-Cre (B6.129S2-Emx1^tm1(cre)Krj/J; Jackson Lab, #005628) and nestin-Cre (B6. Cg-Tg (Nes-cre) 1Kln/J; Jackson Lab, #003771) mice were previously described, and obtained from Cyagen Biosciences (Suzhou, China). To identify tissue expression patterns of Emx1-Cre and nestin-Cre, they were crossed with Ai14(B6.Cg-Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/J; Jackson Lab, #007914) reporter mice. In Ai14 reporter mice, a 3X STOP codons flanked by two loxP sites are localized upstream of the tdTomato coding sequence, thus preventing tdTomato expression without Cre recombinase activity. To generate conditional knockout of Larp7, Larp7 ^flox/flox mice were crossed with Emx1-Cre or nestin-Cre mice, respectively. Noon on the day of the vaginal plug was defined as E0.5. Embryos and offsprings were genotyped by PCR of genomic DNA. The following primers are used: Genome-Ai14-F: GGC​ATT​AAA​GCG​CTA​TCC; Genome-Ai14-R: CTG​TTC​CTG​TAC​GGC​ATG​G; Genome-Cre-F: ACC​CTG​TTA​CGT​ATA​GCC​GA; Genome-Cre-R: CTC​CGG​TAT​TGA​AAC​TCC​AG; Genome-Larp7-floxed-F: ATG​TAT​CAG​CTC​TGG​GGA​AAG​TAG; Genome-Larp7-floxed-R: GTA​TCT​TCA​AAC​AGC​TAG​GGC​TCC.

Samples were fixed with 4% paraformaldehyde overnight at 4°C, and cryopreserved in 25% sucrose for 1 day before embedding in Tissue-Tek O.C.T. Compound (Sakura Finetek, USA). Samples were sectioned on a cryostat at 8 μm. The glass slides were washed three times (5 min each time) with phosphate buffer saline (PBS), and incubated with blocking solution containing 0.1% Triton X-100% and 10% fetal bovine serum (FBS) for 1 h at room temperature. Then, slides were incubated at 4°C overnight with fluorescein-labeled lotus tetragonolobus lectin (LTL) (Vector Laboratory, FL-1321) to detect proximal convoluted tubules, and with anti-Slc12a3 (Abcam, AB95302) to detect distal convoluted tubules. The next day, slides were washed in PBS for three times (5 min each time), incubated with donkey-anti-rabbit secondary antibodies (Abcam, ab150073) at room temperature for 2 h, and then counterstained by 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. Finally, slides were washed three times with PBS before mounting with Fluoromount (Southern Biotech, 0100-01). Images were documented by inverted fluorescence microscopy (DMi8, Leica, Germany), and analyzed by Leica Application Suite X. The percentage of tdTomato⁺;LTL⁺ among LTL⁺ cells, and tdTomato⁺;Slc12a3⁺ among Slc12a3⁺ cells were quantified by ImageJ. For protein blotting, P90 WT and Emx1-Larp7 KO mice were used. The renal cortex was separated from medulla by micro-dissection, grinded by cryogenic grinder (Ningbo Xinyi ultrasonic equipment Co., Ltd, Xlnyl-24N) in RIPA buffer (Sigma, R0278), supplemented with 0.5 mM DTT, 0.1% PMSF, 5 mM NaF, 2 mM Na₃VO₄, cleared by centrifugation at 4°C (13,000 g, 10 min), and the supernatant was removed for analysis. The specificity of anti-Larp7 polyclonal antibodies were previously verified (Dai et al., 2014).

The values of each parameter within a group were expressed as the mean ± SD. For comparisons between groups with normally distributed data, statistical significance was assessed using the two-tailed, unpaired t-test. p < 0.05 were regarded as statistically significant.