Absent, Small, or Homeotic 2-Like–Mediated H3K4 Methylation and Nephrogenesis

Visual Abstract

Abstract

Key Points

• Deficits in nephron numbers are associated with higher risk of adult-onset kidney disease seen in congenital anomalies of the kidney and urinary tract.

• Mouse model experiments suggested that absent, small, or homeotic 2-like was vital for kidney development by activating cell cycle genes through histone methylation.

• Our findings identified absent, small, or homeotic 2-like–regulated genes as a potential target for treating congenital anomalies of the kidney and urinary tract.

Background

Many congenital anomalies of the kidney and urinary tract involve deficits in the number of nephrons, which are associated with a higher risk of hypertension and CKD later in life. Prior work has implicated histone modifications in regulating kidney lineage–specific gene transcription and nephron endowment. Our earlier study suggested that absent, small, or homeotic 2-like (ASH2L), a core subunit of the H3K4 methyltransferase complex, plays a role in ureteric bud morphogenesis during mammalian kidney development. However, the potential involvement of ASH2L in nephron formation remains an open question.

Methods

To investigate the role of ASH2L in nephron development, we inactivated Ash2l specifically in nephron progenitor cells by crossing Six2-e(Kozak-GFPCre-Wpre-polyA)1 mice with Ash2l^fl/fl mice. We used RNA sequencing combined with Cleavage Under Targets and Tagmentation sequencing to screen for gene and epigenomic changes, which were further verified by rescue experiments conducted on ex vivo culture explants.

Results

Inactivating ASH2L in nephron progenitor cells disrupted H3K4 trimethylation establishment at promoters of genes controlling nephron progenitor cell stemness, differentiation, and cell cycle, inhibiting their progression through the cell cycle and differentiation into epithelial cell types needed to form nephrons. Inhibition of the TGF-β/suppressor of mothers against decapentaplegic signaling pathway partially rescued the dysplastic phenotype of the mutants.

Conclusions

ASH2L-mediated H3K4 methylation was identified as a novel epigenetic regulator of kidney development. Downregulation of ASH2L expression or H3K4 trimethylation may be linked to congenital anomalies of the kidney and urinary tract.

Introduction

The precise number of nephrons in the adult human kidney can vary widely among healthy individuals (approximately ten-fold, ranging from 2×10⁵ to 2×10⁶),¹ but too low a number is associated with a higher risk of hypertension and subsequent kidney disease,² and it can compromise the kidney's resilience in the event of injury.^3,4 Nephron deficits occur in many cases of congenital anomalies of the kidney and urinary tract (CAKUT), apparently because of abnormal development of the metanephros. The metanephros in mammals is primarily formed through the coordinated reciprocal induction of two intermediate mesoderm components: the ureteric bud and the metanephric mesenchyme.^5,6 While the ureteric bud derivatives iteratively branch to create the collecting duct system, the SIX2-expressing nephron progenitor cells in the metanephric mesenchyme condense around the branching ureteric bud tips to form the cap mesenchyme. The nephron progenitor cells in the cap mesenchyme then differentiate sequentially into the peritubular aggregate, renal vesicle, comma-shaped body, and S-shaped body, ultimately forming the well-segmented epithelial tubule of the nephron.

Maintaining a delicate balance between the self-renewal and differentiation of the nephron progenitor cell population is crucial for coordinated nephron formation and ensuring a sufficient progenitor niche to support the entire process.⁷ Although previous studies have identified the role of the interplay of numerous transcription and growth factors in regulating nephron progenitor cell self-renewal^8–11 and differentiation,^11,12 the upstream mechanisms coordinating these processes remain incompletely understood.

Epigenetic mechanisms, such as histone modifications, have been found to affect nephron endowment by influencing the transcription of kidney lineage–specific genes. In mice, the nephron progenitor cell–specific deletion of genes involved in histone methylation or acetylation, such as Eed,¹³ Ezh1/2,¹⁴ Dot1L,¹⁵ and Hdac1/2,¹⁶ results in significantly impaired nephron development. The protein “absent, small, or homeotic 2-like” (ASH2L), part of the protein complex responsible for methylating Lys4 on histone 3, has been implicated in multiple developmental processes, including early embryogenesis, amelogenesis, epidermal differentiation, and neural development.^17–20 Our previous research demonstrated that the budding and branching morphogenesis of the ureteric bud was impaired in Ash2l^{Hoxb7Cre−/−} mice, leading to reduced nephron endowment.²¹ We wondered whether ASH2L directly regulates nephron formation in the cap mesenchyme.

Therefore, we explored here the potential role of ASH2L-mediated H3K4 methylation in nephron formation by inactivating it specifically in the nephron progenitor cells of mice.

Methods

Mice

All animal experiments were conducted in strict accordance with the guidelines and protocols approved by the Institutional Animal Care and Use Committee at the Center for Excellence in Molecular Cell Science, located at the Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences. The Six2-e(Kozak-GFPCre-Wpre-polyA)1 mouse line was obtained from the Shanghai Model Organisms Center, Inc., while the Ash2l^fl/fl mouse strain was generously provided by Dr. Xiaozhong Peng from the School of Basic Medicine at Peking Union Medical College in Beijing, China.²⁰ Embryo ages were determined on the basis of the detection of vaginal plug, with E0.5 defined as noon on the day when the plug was identified.

Histology and Immunostaining

Kidney samples from mice of various ages were dissected and fixed in 4% paraformaldehyde overnight at 4°C. The tissues were embedded in paraffin, and 5 μm sections were stained with hematoxylin and eosin. For immunostaining, sections were cleared in xylene, rehydrated, and subjected to antigen retrieval in Tris-EDTA buffer (pH 9.0) at 98°C for 30 minutes. After blocking in PBS with 0.1% Triton X-100 and 10% serum, primary antibodies were incubated overnight at 4°C. Sections were then treated with secondary antibodies conjugated to AlexaFluor 405, 488, or 594 (1:600; Jackson ImmunoResearch). Nuclei were counterstained with 4',6-diamidino-2-phenylindole, and images were captured using fluorescence microscopy. A detailed description of the methodology is provided in the Supplemental Methods.

Ex Vivo Organ Culture

For whole-kidney explant cultures, we dissected kidneys from E12.5 mouse embryos under aseptic conditions and placed them on polycarbonate Transwell filters in 12-well plates with DMEM/F-12, 10% FBS, and 1× penicillin-streptomycin-amphotericin B. Cultures were maintained at 37°C in 5% CO₂. For separated metanephric mesenchyme cultures, E11.5 kidneys were incubated in 0.1% trypsin at 37°C for 5 minutes, and then 100 μl FBS was added to halt digestion. The metanephric mesenchyme was separated from the ureteric bud and placed on Transwell filters in DMEM/F-12 with 10% FBS, 10 μM PP2, 50 ng/ml FGF2, 3μM CHIR99021, and 1× penicillin-streptomycin-amphotericin B solution. The metanephric mesenchyme was cultured for 48 hours, followed by a medium change without CHIR99021 for an additional 72 hours. A detailed description of the methodology is provided in the Supplemental Methods.

Cell Proliferation and Apoptosis Assays

Pregnant mice were administered an intraperitoneal injection of PBS containing 2 mg/ml 5-ethynyl-2′-deoxyuridine (EdU; catalog no. ST067, Beyotime) at a dose of 50 mg EdU per kg of body weight. After 1 hour, the kidneys were dissected out and processed for histology and immunostaining as described previously. Proliferating cells were labeled using the BeyoClick EdU Cell Proliferation Kit (catalog no. C0075S, Beyotime) according to the manufacturer's instructions. Alternatively, the dissected tissue was cleared in xylene, rehydrated through a graded series of ethanol solutions, and subjected to antigen retrieval. Apoptotic cells were then labeled using the One Step TUNEL Apoptosis Assay Kit (catalog no. C1090, Beyotime) following the manufacturer's protocol, and the tissue was subsequently immunostained as described above.

Fluorescence-Activated Cell Sorting

Embryonic kidneys from E15.5 mice were dissected, trypsinized, and resuspended in DMEM/F-12 with FBS before sorting green fluorescent protein (GFP)-expressing cells using an MA900 Cell Sorter. A detailed description of the methodology is provided in the Supplemental Methods.

Cell Cycle Analysis

The single-cell suspension was prepared from E15.5 embryonic kidneys as described previously. The cells were then washed and resuspended in 1× PBS. After incubation with 10 μg/ml Hoechst 33342 (catalog no. C1022, Beyotime) for 30 minutes at 37°C, cell analysis was conducted using a BD LSRFortessa flow cytometer, and the data were processed using FlowJO software (version 10.0.7).

RNAscope

RNAscope in situ hybridization was performed on 5-μm thick formalin-fixed, paraffin-embedded sections using the RNAscope 2.5 HD Brown Kit (catalog no. 322300, Advanced Cell Diagnostics, CA) following the manufacturer's instructions. Probes from Advanced Cell Diagnostics were used to target the following genes: Wnt4 (catalog no. 401101) and Ppib (catalog no. 313911) as a positive control and DapB (catalog no. 310043) as a negative control.

RNA Sequencing

GFP-expressing embryonic kidney cells were isolated from control (Six2-GFPCre×Ash2l^fl/+) and Ash2l mutant mice (Six2-GFPCre×Ash2l^fl/fl). RNA sequencing was performed on three biological replicates per genotype, with total RNA extracted using the RNeasy Micro Kit and sequenced/analyzed by OE Biotech. The data are available in the Gene Expression Omnibus (GEO) under accession number GSE269089. A detailed description of the methodology is provided in the Supplemental Methods.

CUT&Tag Sequencing

The rationale and process of Cleavage Under Targets and Tagmentation sequencing (CUT&Tag-seq) have been previously described elsewhere.²² In brief, libraries were generated using the NovoNGS High-Sensitivity CUT&Tag 3.0 Kit and sequenced on an Illumina platform. The data have been uploaded to GEO under accession number GSE269088. A detailed description of the methodology is provided in the Supplemental Methods.

Statistical Analyses

Statistical analyses were performed using GraphPad Prism software (version 9.0.0). Comparisons between two groups were conducted using an unpaired two-tailed Student's t test. The results are presented as mean±SEM, and a P value of < 0.05 was considered statistically significant.

Results

Inactivation of Ash2l in Nephron Progenitor Cells Led to Their Premature Depletion and Inhibited Nephron Formation in Mice

We crossed Six2-e(Kozak-GFPCre-Wpre-polyA)1 mice with Ash2l^fl/fl mice²⁰ to generate animals (Six2-GFPCre×Ash2l^fl/fl, henceforth referred to as “Ash2l mutant mice”) in which negligible ASH2L was expressed in the nephron progenitor cells during embryonic development, whereas normal levels were expressed in the ureteric bud (Supplemental Figure 1, A and B). Consistently, the nephron progenitor cells from these animals showed significant reductions in H3K4 trimethylation (Supplemental Figure 1C), dimethylation (Supplemental Figure 1D), and monomethylation (Supplemental Figure 1E). Heterozygous mice (Six2-GFPCre×Ash2l^fl/+) were designated as control animals.

The Ash2l mutant mice were born at the expected Mendelian frequency and died shortly after birth. All 14 pups that we examined showed bilateral renal dysplasia in which kidneys lacked a nephrogenic zone, S- or comma-shaped bodies, or mature glomeruli (Figure 1, A and B). This dysplasia was already evident on embryonic days 15.5 and 17.5 (Figure 1C and Supplemental Figure 1F) and was associated with reduced SIX2 signal, indicating premature loss of nephron progenitor cells (Figure 1D). At embryonic day 15.5, expression of Lotus tetragonolobus lectin and uromodulin was negligible, indicating the absence of mature nephron segments, such as proximal tubules and the thick ascending limb of the Henle's loop (Figure 1E). Premature depletion of nephron progenitor cells around the ureteric bud tips was also observed on embryonic day 19.5 (Figure 1F and Supplemental Figure 1G).

Figure 1.

Inactivating Ash2l specifically in nephron progenitor cells in mice led to renal dysplasia similar to that in CAKUT. (A) Representative photomicrographs of the urinary tract in control and mutant mice. Tissues were extracted on postnatal day 0. Scale bar, 1 mm. (B) Representative thin sections of kidneys from mice on postnatal day 0 after staining with hematoxylin-eosin. The black dotted line at the upper left encloses the nephrogenic zone. Red arrows, mature glomeruli; black arrowheads, nascent nephron structures at stages earlier than S- or comma-shaped bodies; red arrowheads, S- or comma-shaped bodies. Scale bar, 40 μm. (C) Representative bright-field micrographs of the urinary tract (left column) and fluorescence micrographs of kidneys (right column) on embryonic day 15.5. Scale bar, 500 μm (left column) or 200 μm (right column). (D) Representative fluorescence micrographs of whole-mount kidney on embryonic day 15.5 after immunostaining against SIX2 to label nephron progenitor cells. The yellow box on the left indicates the region shown at higher magnification on the right. Scale bar, 200 μm (left column) or 50 μm (right column). (E) Representative fluorescence micrographs of thin kidney sections on embryonic day 15.5 after double immunostaining against LTL to label proximal tubules and against uromodulin to label the thick ascending limb of Henle's loop. Scale bar, 40 μm. (F) Representative thin sections of kidneys from mice on embryonic day 19.5 after double immunostaining against SIX2 to label the nephron progenitor cells and against Calbindin to label the ureteric bud derivatives. Scale bar, 40 μm. CAKUT, congenital anomalies of the kidney and urinary tract; DAPI, 4',6-diamidino-2-phenylindole; GFP, green fluorescent protein; LTL, Lotus tetragonolobus lectin.

Consistent with these in vivo observations, we found that the cap mesenchyme of the mutant mouse kidney explants taken on embryonic day 12.5 became increasingly disorganized and its niche volume progressively smaller between 48 and 96 hours in culture (Supplemental Figure 2A) and that these changes were associated with loss of nephron progenitor cells around ureteric bud tips (Supplemental Figure 2, B and C).

Given that defects in the ureteric bud branching can affect the self-renewal and differentiation of nephron progenitors in the cap mesenchyme,²³ we wanted to exclude that the observed loss of progenitors was caused by defects in the ureteric bud development. On embryonic day 11.5, we dissected away the ureteric bud and explanted the metanephric mesenchyme into culture dishes to induce nephron formation chemically²⁴ (Supplemental Figure 2D). Explants from control animals developed well-organized glomeruli as well as proximal and distal tubules, whereas explants from the mutants did not (Supplemental Figure 2E). These results suggested that inactivation of Ash2l in nephron progenitor cells directly inhibits nephron formation, independently of factors derived from the ureteric bud.

Inactivation of Ash2l Inhibited Proliferation of Nephron Progenitor Cells by Halting Their Progression through the Cell Cycle

We wondered whether slower proliferation, greater apoptosis, or both contributed to the observed premature depletion of nephron progenitor cells. We examined the kidneys of control and mutant mice for expression of the proliferation marker Ki67 and for incorporation of the thymidine analog EdU at two stages of kidney development: before obvious loss of progenitor cells (embryonic day 13.5) and after it (embryonic day 15.5). At both time points, kidneys from the mutants contained significantly fewer EdU- or Ki67-positive cells (Figure 2, A and B, and Supplemental Figure 3, A and B), and the progenitors in these kidneys showed a significant shift from G2/M to G1 phase of the cell cycle (Figure 2C), indicating a nonproliferative state. At both time points, apoptotic nephron progenitor cells in the kidneys of both mutant and control mice were nearly undetectable, as assessed by terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling staining and immunostaining for cleaved caspase-3 (Figure 2D and Supplemental Figure 3, C and D).

Figure 2.

Inactivating Ash2l specifically in nephron progenitor cells slowed their proliferation by blocking the cell cycle. (A) Representative fluorescence micrographs of thin sections of the kidney on embryonic day 13.5 after double labeling to detect incorporation of the thymidine analog EdU into DNA and to detect expression of SIX2 to label the nephron progenitor cells. The white arrowheads indicate areas showing colocalization of the EdU and SIX2 signals. Quantification of the full experiment is shown on the right. Scale bar, 40 μm. (B) The same experiment as in (A) except the kidney was analyzed on embryonic day 15.5. Scale bar, 40 μm. (C) Representative distributions of the nephron progenitor cells taken from mice on embryonic day 15.5 at different stages of the cell cycle, based on flow cytometric analysis. Quantification of the full experiment is shown below. (D) Representative fluorescence micrographs of thin sections of the kidney on embryonic day 15.5 after double labeling for expression of SIX2 to label the nephron progenitor cells and for apoptosis with a TUNEL assay. Scale bar, 40 μm. EdU, 5-ethynyl-2′-deoxyuridine; TUNEL, terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling.

Inactivation of Ash2l Inhibited Differentiation of Nephron Progenitor Cells

Our experiments so far suggested that Ash2l inactivation limited the self-renewal of progenitor cells and thereby inhibited the formation of complete nephrons. We wondered whether Ash2l inactivation might also affect differentiation of progenitors into downstream epithelial cell types, labeled by using markers for the different stages of progenitor differentiation²⁵ (Figure 3A).

Figure 3.

Inactivating Ash2l specifically in nephron progenitor cells blocked their differentiation into the various epithelial cell types that give rise to a complete nephron. (A) Gene expression maps in mice nephron progenitor cells and incipient nephron structures. (B) Representative thin sections of kidneys from mice on embryonic day 15.5 after double immunostaining against PAX2 and Calbindin. The white dotted lines indicate nascent nephrons that are PAX2⁺ and Calbindin⁻. Quantification of the full experiment is shown on the right. Scale bar, 40 μm. (C) Representative thin sections of kidneys from mice on embryonic day 15.5 after double immunostaining against LEF1 and Calbindin. The white arrowheads indicate induced nephron progenitor cells that are positive for LEF1. Quantification of the full experiment is shown on the right. Scale bar, 40 μm. (D) Representative thin sections of kidneys from mice on embryonic day 15.5 after double immunostaining against WT1 and JAG1. The yellow arrowheads indicate nascent nephrons at later stages, such as the comma-shaped body or SSB, while the white arrowheads denote RVs. Scale bar, 40 μm. (E) Representative fluorescence micrograph of whole-mount kidneys on embryonic day 15.5 after immunostaining against WT1. Scale bar, 100 μm. RV, renal vesicle; SSB, S-shaped body.

We again examined two stages of kidney development: before obvious loss of progenitor cells (embryonic day 13.5) and after it (embryonic day 15.5). We confirmed the loss of PAX2 expression in the mutants (Figure 3B), indicative of a reduction in the overall nascent nephron structures. At both time points, we detected abundant expression of LEF1, a transcriptional activator of the wingless-related integration site (WNT)/β-catenin signaling pathway, in kidneys of control and mutant mice (Figure 3C and Supplemental Figure 4A). This suggested that Ash2l inactivation did not abolish the induction of the cap mesenchyme that normally induces nephron progenitor cells to begin differentiating. Instead, we found that differentiation was blocked at the renal vesicle stage (Figure 3D and Supplemental Figure 4B), on the basis of the expression patterns of the transcription factor WT1 and Notch receptor JAG1.²⁵ The reduced expression of WT1 was observed in substructures of the nascent nephron and in podocytes (Figure 3E). Consistently, RNAscope analysis revealed significant downregulation of Wnt4 in kidneys from the mutants (Supplemental Figure 4C). These results suggested that Ash2l inactivation may inhibit differentiation of nephron precursor cells by dampening WNT/β-catenin signaling.

Inactivation of Ash2l Altered Expression of Genes Involved in the Cell Cycle as Well as in Self-Renewal and Differentiation of Nephron Progenitor Cells

To begin to understand how Ash2l inactivation may inhibit self-renewal and differentiation of nephron progenitor cells, we compared the transcriptomes between fluorescence activated cell sorting–purified nephron progenitor cells from control or mutant mice on embryonic day 15.5. We detected 810 genes that were upregulated by Ash2l inactivation and 756 that were downregulated by it (Figure 4, A–C). The downregulated genes were significantly enriched in gene ontology biological processes related to nuclear division, regulation of the cell cycle, and chromosome segregation (Figure 4D), whereas the upregulated genes were enriched in biological processes related to negative regulation of immune responses, positive regulation of responses to external stimuli, and cascades involving ERK1 and ERK2 (Supplemental Figure 5A).

Figure 4.

Inactivating Ash2l specifically in nephron progenitor cells altered expression of genes related to the cell cycle and to self-renewal and differentiation of progenitors in mice. Gene expression in FACS-purified nephron progenitor cells at embryonic day 15.5 was compared between control and mutant mice. (A) Cluster analysis of differential gene expression. A total of 1566 differentially expressed genes were identified. (B) Cluster analysis results of sample-to-sample distances between pairs of three independent samples from control mice (C1–C3) and pairs of three independent samples from mutant mice (K1–K3). (C) Volcano plot of differentially expressed genes. Genes are colored according to whether their expression was significantly higher (red) or lower (blue) or not significantly different (gray) in mutant kidneys relative to control kidneys. (D) Enrichment of genes downregulated in mutant kidneys in biological processes according to the GO classification. (E) Cluster analysis of differentially expressed genes known to be involved in cell cycle regulation. (F) Comparison of the expression of genes related to self-renewal, survival, and differentiation of the nephron progenitor cells. The red dotted lines mark the minimal fold difference necessary to qualify as differential expression in our analysis. FACS, fluorescence-activated cell sorting; GO, gene ontology.

Further analysis showed that Ash2l inactivation was associated with up- or downregulation of genes encoding cell cycle inhibitors (Cdkn1c and Cdkn1a), cyclins (Ccnd1 and Ccna2), and cyclin-dependent kinases (Figure 4E). It was also associated with up- or downregulation of genes known to be involved in the self-renewal of nephron progenitor cells (Sall1 and Wt1), their survival (Fgf9 and Fgf20), or their differentiation (Fgf8, Wnt4, and Lhx1) (Figure 4F). Consistent with this, gene set enrichment analysis identified metanephros development as one of the processes most affected by Ash2l inactivation (Supplemental Figure 5, B and C). In addition, in line with the results shown in Supplemental Figure 4C, gene set enrichment analysis also identified Wnt-protein binding as another process affected by Ash2l inactivation (Supplemental Figure 5D).

Inactivation of Ash2l Altered H3K4 Methylation Near Promoters of Genes That Regulate the Cell Cycle

Next, we asked whether the ability of Ash2l inactivation to alter gene expression involves the protein's role in H3K4 methylation near gene promoters, which is known to regulate gene expression during development.²⁶ To this end, we profiled genomic sequences at sites of H3K4me3 in fluorescence activated cell sorting–purified nephron progenitor cells from control or mutant mice on embryonic day 15.5. Most sites of trimethylation coincided with transcriptional start sites in promoters, as expected from previous work,²⁷ and significantly fewer sites of trimethylation were detected in progenitors from mutant mice (Figure 5, A–C, and Supplemental Figure 6, A and B). These results were consistent between two independent experiments (Supplemental Figure 6, C and D).

Figure 5.

Landscape of H3K4 trimethylation in the genome of nephron progenitor cells on embryonic day 15.5. (A) Average read density across TSS and chromatin occupancy. (B and C) Distribution of CUT&Tag peaks of H3K4me3 across genomic regions in one of two independent samples from (B) control mice or (C) mutant mice. (D) Overlap between genes whose promoters coincided with H3K4me3 (Cut&Tag) and genes that were downregulated by Ash2l inactivation (DRGs). (E) Enrichment of the overlap genes from (D) in biological processes according to the GO classification. (F) Location of H3K4me3 sites relative to the promoters of certain genes that promote the cell cycle. Results are shown for two independent samples from control mice and two from the mutants. Scale bar, 1 kb. CUT&Tag, Cleavage Under Targets and Tagmentation; DRGs, down-regulated genes; TSS, transcriptional start sites; UTR, untranslated region.

Among the H3K4me3 peaks that overlapped between the two independent control samples of nephron progenitor cells, we identified 266 genes that were downregulated by Ash2l inactivation, corresponding to one third of all genes downregulated by the inactivation (Figure 5D). Gene ontology analysis of these genes showed enrichment in processes related to mitotic cell cycle phase transition, mitotic nuclear division, and chromosome segregation (Figure 5E). Kyoto Encyclopedia of Genes and Genomes enrichment analysis of these genes also identified the cell cycle pathway as one of the top enriched pathways (Supplemental Figure 6E). For the genes related to cell cycle progression, H3K4me3 near their promoters was significantly lower in nephron progenitor cells from ASH2L-deficient mice than in those from control animals (Figure 5F).

Inhibition of TGF-β Signaling Partially Rescued Proliferation and Differentiation of Nephron Progenitor Cells in Kidney Explants from the Mutant Mice

We noticed that Ash2l inactivation led to upregulation of the cyclin-dependent kinase inhibitors p21^Cip1 and p57^Kip2 (Supplemental Figure 7, A and B), which may help explain the observed inhibition of cell cycle progression. Given that TGF-β–mediated suppressor of mothers against decapentaplegic signaling activates these inhibitors,^28,29 we wondered whether inhibiting TGF-β would restore proliferation and differentiation of nephron progenitor cells in the mutant mice. We explanted kidneys from the animals on embryonic day 12.5 into culture dishes in the presence or absence of the TGF-β inhibitor SB431542.^30,31 The inhibitor led to significantly more WT1-positive glomerulus-like structures after 3 or 5 days in culture (Figure 6A and Supplemental Figure 8), Lotus tetragonolobus lectin–positive proximal tubule–like structures after only 3 days in culture (Figure 6A), and comma- and S-shaped bodies characteristic of nascent nephrons (Figure 6B). These effects of TGF-β inhibition were associated with significantly increased number of EdU-positive cells (Figure 6, C–C′) as well as with downregulation of p21^Cip1 and p57^Kip2 (Figure 6, D–D′ and E–E′).

Figure 6.

Inhibition of TGF-β signaling in kidney explants partially rescued proliferation and differentiation of nephron progenitor cells lacking ASH2L. Kidneys were excised from mutant mice on embryonic day 12.5 and cultured for 3 days in the presence or absence of an inhibitor of TGF-β signaling (SB431542). (A) Representative fluorescence micrographs of whole-mount explants after double immunostaining against WT1 to label glomeruli and against LTL to label proximal tubules. The white arrowheads indicate proximal tubules that are LTL⁺. Quantification of the full experiment (three replicates) is shown on the right. Scale bar, 40 μm. (B) Representative photomicrographs of whole-mount explants without further labeling or staining. Black arrowheads indicate structures of the developing nephron, such as S- and comma-shaped bodies. Scale bar, 40 μm. (C) Representative fluorescence micrographs of whole-mount explants after double labeling to detect incorporation of the thymidine analog EdU into DNA and to detect the nephron progenitor cell marker SIX2. The white arrowheads indicate areas showing colocalization of the EdU and SIX2 signals, suggesting proliferation of the nephron progenitor cells. Scale bar, 40 μm. (D and E) Representative fluorescence micrographs of whole-mount explants after double immunostaining against GFP to identify the nephron progenitor cells and against either (D) p21^Cip1 or (E) p57^Kip2. Scale bar, 40 μm. (C′–E′) Statistical analyses for the experiments related to (C–E). ASH2L, absent, small, or homeotic 2-like.

Discussion

In this study, we provided evidence that mice lacking ASH2L specifically in nephron progenitor cells died soon after birth and showed renal dysplasia involving severely impaired nephron formation, which could be attributed to inhibition of both the self-renewal of nephron progenitor cells and their differentiation into the various epithelial cell types essential for a functional nephron. These changes were associated with altered H3K4 methylation near the promoters of genes related to cell cycle progression (Supplemental Figure 9). Our work seemed to be the first to implicate Ash2l in nephron development and suggested ASH2L and genes regulated by H3K4 methylation as potential targets in the treatment of CAKUT.

The development of the kidney is a complex process that relies on the coordinated expression of lineage-specific transcription factors, signal transducers, and growth factors. This requires precise regulatory mechanisms to control gene expression both temporally and spatially. Histone modification, an important epigenetic regulation mechanism, is believed to play a vital role in this process.³² One well-established example is the so-called bivalent chromatin structure, which marks core regulatory genes during embryonic development.³³ These genes harbor bivalent domains containing H3K27me3 and H3K4 methylation, which keeps them poised for activation. Because histone modifications are highly reversible,^34,35 this regulatory mechanism enables rapid changes in gene expression during tissue morphogenesis. Previous studies have reported the identification of both H3K27 methylation and H3K4 methylation, as well as the writers that mediate these histone modifications during mouse metanephros development.³⁶ Subsequent studies have shown that nephron progenitor cell–specific inactivation of either Eed or Ezh1/Ezh2, components of the polycomb repressive complex 2 responsible for establishing H3K27 methylation, result in an imbalance of nephron progenitor cell self-renewal and differentiation, ultimately leading to a deficit in nephron formation.^13,14

However, the role of H3K4 methylation during nephron development is still not well studied. A recent study using a zebrafish model revealed that the PENK-A–H₂O₂ pathway regulates the expression of tcf21, a core gene mediating the formation of renal progenitor cell aggregates, through promoter H3K4me3 remodeling.³⁷ In humans, a rare genetic disorder called Kabuki syndrome has been reported to be associated with de novo pathogenic variants in KMT2D, the coding gene of one complex proteins associated with set1 (COMPASS) family H3K4 methyltransferase, and CAKUT phenotypes have been observed in a considerable proportion of patients with Kabuki syndrome.^38–40 On the basis of a recently reported long-term expandable mouse and human-induced nephron progenitor cells platform, researchers have established the role of KMT2A, another COMPASS family protein, in nephron progenitor cells' self-renewal.⁴¹ In mammals, the methyltransferase activity of the six KMT2 family lysine methyltransferases relies heavily on a cofactor known as the WDR5, RbBP5, ASH2L, and DPY-30 complex. This complex comprises four subunits: WDR5, RbBP5, ASH2L, and DPY30, with ASH2L being an essential component.⁴² Therefore, it is reasonable to selectively remove this core subunit to investigate the role of H3K4 methylation during the development of the mammalian metanephros. In this study, we provided direct evidence that in the developing kidney, ASH2L-mediated H3K4me3 fine-tunes the self-renewal and differentiation of the nephron progenitor cells primarily through the regulation of cell cycle–related genes. Inactivation of Ash2l resulted in a striking reduction of H3K4me3 at the promoter regions of key cell cycle activator genes, such as Ccna2, Ccnd1, Cdc25a, Cdc6, Wee1, and Plk1 (Figure 5F). Concurrently, the expression of two important cell cycle inhibitors, p21^Cip1 and p57^Kip2, was significantly upregulated upon Ash2l inactivation. In a previous study by Liu et al., a dramatic 220-fold upregulation of the cell cycle inhibitor CDKN2a/p16 was observed in Ezh1^−/− NPC^Ezh2−/− nephron progenitor cells,¹⁴ indicating that both H3K4me3 and H3K27me3 are critical for the expression control of cell cycle–related genes, particularly cell cycle inhibitors. However, the situation seems to be somewhat different in Ash2l-deficient nephron progenitor cells. We observed an abnormal upregulation of p21^Cip1 and p57^Kip2 against a backdrop of downregulated expression of numerous cell cycle activators, which cannot be fully explained by the well-characterized role of H3K4me3 in transcription initiation.²⁶ We speculated that the aberrant upregulation of these two cell cycle inhibitors may arise from the moonlighting, methyltransferase-independent activities of ASH2L,^43,44 although this hypothesis remains purely speculative.

Previous studies have established a connection between TGF-β signaling and kidney development in mice. For instance, Tgfb1^−/− or Tgfb3^−/− mice display no significant kidney malformations,^45,46 whereas Tgfb2^+/− and Tgfbr3^+/− mice exhibit accelerated ureteric bud branching and increased nephron endowment.^47,48 In addition, Six2Cre, Tgfbr2^loxP/− mice show moderately impaired nephron endowment.⁴⁹ These findings suggest a significant yet complex role for TGF-β signaling in nephrogenesis. Our RNA-seq data indicate that Ash2l inactivation does not directly alter the transcription of TGF-β signaling–related genes, including Tgfb1, Tgfb2, Tgfb3, Tgfbr1, Tgfbr2, Tgfbr3, and Smad1–9. However, inhibition of the TGF-β/suppressor of mothers against decapentaplegic signaling pathway significantly reduced the expression of p21^Cip1 and p57^Kip2, restored cell proliferation, and partially rescued the dysplastic phenotype in kidney explants from ASH2L-deficient mice. If our findings can be reproduced in vivo, they may have therapeutic implications of targeting specific signaling pathways to mitigate CAKUT and, by extension, other developmental disorders involving epigenetic dysregulation.

In addition, inactivation of Ash2l led to expression changes in core genes critical for nephron progenitor stemness and differentiation, notably the downregulation of Sall1, Wt1, Lef1, Fgf8, Wnt4, Lhx1, and Hey1, along with the upregulation of Meox1, Fgf9, and Fgf20 (Figure 4F). These observations suggest that the effects on core gene expression are more likely secondary consequences of cell cycle arrest after Ash2l inactivation. Previous studies have indicated that WT1-mediated Wnt4 expression serves as a critical molecular signal that initiates the differentiation of nephron progenitor cells.⁵⁰ In the absence of Eed or with dual inactivation of Ezh1/2, premature Wnt4 expression was observed in the nephron progenitor cells, leading to an unscheduled mesenchyme-to-epithelium transition and subsequent depletion of the nephron progenitor cell pool.^13,14 By contrast, our present investigation revealed a significant reduction in Wnt4 expression within nephron progenitor cells or early nephron progenitors of the mutants (Figure 4F and Supplemental Figure 4C). Concomitant with the downregulation of Wnt4, we also observed a marked decrease in Wt1 expression in the mutant nephron progenitor cells (Figures 3D and 4F). We speculated that in the absence of WT1, the repressive function of SIX2 on Wnt4 predominates over WT1's activating role, ultimately resulting in a blockage of nephron differentiation in the mutants.

In contrast to Ash2l inactivation, the overexpression of ASH2L is unlikely to significantly affect the overall methyltransferase activity of the KMT2 complex and, in our view, would not substantially influence nephrogenesis as in mammalian cells—the expression levels of ASH2L already significantly exceed those of other components of the KMT2 complex.⁴² Furthermore, the embryonically lethal effects associated with global inactivation of Ash2l explain the absence of evidence regarding ASH2L mutations in patients with CAKUT,¹⁷ as noted in our previous study.²¹

Future work should clarify how ASH2L in the ureteric bud controls branching morphogenesis,²¹ whereas the same protein in the metanephric mesenchyme regulates self-renewal and differentiation of the nephron progenitor cells. That the same protein should perform different roles in different components of the intermediate mesoderm may be determined by one or more signals during kidney development, such as different concentrations of FGF9 or retinoic acid or different durations of exposure to Wnt signals.⁵¹ Alternatively, it is possible that not all H3K4 methylation is mediated by ASH2L-containing complexes; other methyltransferases besides the COMPASS complex may also contribute to H3K4 methylation of core or lineage-specific genes. To elucidate the tissue-specific roles of ASH2L, emerging technologies such as Spatial-CUT&Tag sequencing and spatial transcriptomics may be required.^52,53

Supplementary Material

jasn-36-798-s001.pdf ^{(2.2MB, pdf)}

jasn-36-798-s002.pdf ^{(1.3MB, pdf)}

Disclosures

Disclosure forms, as provided by each author, are available with the online version of the article at http://links.lww.com/JSN/F17.

Funding

F. Lin: Shanghai Scientific and Technological Committee (22ZR1441000), Shanghai Jiao Tong University School of Medicine Research Physician Project (2024), and National Natural Science Foundation of China (82470706). Z. Zhao: Shanghai Scientific and Technological Committee (24YF2727300).

Author Contributions

Conceptualization: Gengru Jiang, Fujun Lin, Ziyi Zhao.

Data curation: Xuantong Dai, Ziyi Zhao.

Formal analysis: Xuantong Dai, Ziyi Zhao.

Funding acquisition: Fujun Lin, Ziyi Zhao.

Investigation: Xuantong Dai, Ziyi Zhao.

Methodology: Xuantong Dai, Ziyi Zhao.

Project administration: Fujun Lin.

Resources: Fujun Lin.

Software: Xuantong Dai, Ziyi Zhao.

Supervision: Gengru Jiang, Fujun Lin.

Validation: Xuantong Dai, Ziyi Zhao.

Visualization: Xuantong Dai, Ziyi Zhao.

Writing – original draft: Ziyi Zhao.

Writing – review & editing: Gengru Jiang, Fujun Lin.

Data Sharing Statement

The data supporting the findings of this study are available from the corresponding author upon reasonable request. The CUT&Tag-seq datasets have been deposited in the GEO database and are accessible at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc5GSE269088. The RNA-seq datasets have been shared and can be accessed via the National Center for Biotechnology Information GEO repository at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc5GSE269089.

Supplemental Material

This article contains the following supplemental material online at http://links.lww.com/JSN/F16.

Supplemental Methods

Supplemental Figure 1. Inactivation of Ash2l specifically in the nephron progenitor cells led to deficient H3K4 methylation and loss of the nephron progenitor cell markers.

Supplemental Figure 2. Inactivating Ash2l specifically in nephron progenitor cells led to their premature depletion in explants.

Supplemental Figure 3. Loss of the nephron progenitor cells due to Ash2l inactivation primarily arose from decreased proliferation, rather than increased apoptosis.

Supplemental Figure 4. Ash2l inactivation in the nephron progenitor cells blocked nephron differentiation at the renal vesicle stage.

Supplemental Figure 5. Gene expression changes in the nephron progenitor cells after Ash2l inactivation.

Supplemental Figure 6. Alterations in H3K4me3 modifications after Ash2l inactivation in the nephron progenitor cells.

Supplemental Figures 7 and 8. Abnormal expression of p21^Cip1 and p57^Kip2 played a central role in the cell cycle blockage of the nephron progenitor cells in the mutants.

Supplemental Figure 9. Model of how Ash2l inactivation in nephron progenitor cells leads to deficiency of complete nephrons.