Mutational analysis of genes with ureteric progenitor cell specific expression in branching morphogenesis of the mouse kidney

Abstract

Background

Ureteric progenitor cells (UPCs) within the branch tips of the arborizing ureteric epithelium of the kidney’s developing collecting system establish the shape and cellular organization of the collecting network, and drive the nephrogenic program through their interactions with nephron progenitor cells. In a previous study, expression screening identified a cohort of genes showing UPC-enriched expression including D17H6S56E-5, Hs3st3a1, Hs3st3b1, and Tmem59l. Each of these is also enriched in branch tips of assembling airways of the developing lungs. Here, we used Crispr-CAS9 directed gene editing to mutate each of these targets to address their potential role(s) in UPC programs.

Results

Single (D17H6S56E-5 and Tmem59l) and double (Hs3st3a1 and Hs3st3b1) mutants were viable, fertile, and displayed varying frequencies of ureter duplications and no overt lung phenotype. Ureter duplications arise spontaneously through multiple outgrowths of the ureteric bud at the onset of kidney development. Tmem59l mutants and Hs3st3a1/Hs3st3b1 compound mutants showed a weakly penetrant, but statistically significant increase in duplicated ureters compared to C57BL6/J and SW wildtype mouse strains.

Conclusions

Tmem59l and Hs3st3a1/Hs3st3b1 activities contribute to the regulatory programs restricting ureteric outgrowth in the developing mouse kidney. However, the low penetrance of the observed phenotype precludes detailed analysis of their specific actions.

INTRODUCTION

Branching morphogenesis is a developmental process that generates a complex arborized epithelial network in a variety of mammalian organ systems, including the kidney and lung.^1,2 In the mouse, kidney development initiates at E10.5 with the outgrowth of the ureteric bud (UB) from the nephric duct at the hindlimb region. The UB uses branching morphogenesis to create the collecting duct system which plays a critical role in water, salt and pH balance, and the transport of urine to the ureter for removal from the kidney. The UB consists of two major cell populations: the highly proliferative branch tips made up of ureteric progenitor cells (UPCs) and non-branching stalk regions where differentiated UPC derivatives form.³ Reciprocal interactions between UPCs and distinct mesenchymal progenitor populations (nephron and interstitial) surrounding the branch tips drives assembly of the mammalian kidney.⁴ Molecular analyzes have highlighted shared features of regulatory interactions in branching morphogenesis of the kidney with other organs with prominent branching epithelial programs, notably the lung.^5–7

In earlier work, we conducted an RNA expression screen comparing the UPC population in ureteric branch tips, with their non-branching cellular derivatives in the ureteric stalk population.⁸ In addition to identifying well established branch tip-enriched gene expression for Wnt11, Ret, Etv4/5, and Sox8/9, and several other genes, the screen identified and validated branch tip-enriched expression for a large number of genes not previously mapped to the UPC population. Comparative analysis showed 48% of the kidney tip-specific genes identified were also expressed in the embryonic lung tip population, suggesting a potential core branching program. For further investigation, we selected four novel genes (D17H6S56E-5, Hs3st3a1, Hs3st3b1, Tmem59l) from this list and generated knockout mouse lines through CRISRP/Cas9 gene editing to determine if these genes are required for normal morphogenesis of kidney and lung.

D17H6S56E-5 (alternatively G7e) was chosen for analysis given the paucity of information on this gene. First identified in 1996 as a gene within the mouse MHC Class III region⁹, D17H6S56E-5 nucleotide sequence has some homology with murine leukemia virus envelope genes, and its predicted protein structure contains viral envelope motifs.⁹ The absence of an obvious ortholog in other mammals suggests a newly acquired mouse gene.

Hs3st3a1 and Hs3st3b1 are tightly linked genes, encoding closely related type II integral membrane proteins of the heparin sulfate 3-O-sulfotransferase family. These enzymes perform the rare 3-O-sulfation as the final step of heparin sulfate modification within the Golgi apparatus.^10–13 Hs3st3a1 and Hs3st3b1’s sulfotransferase domains are highly similar and act on the same disaccharide, suggesting potential redundant functional activities where expression overlaps.¹⁴ Modifications to heparin sulfate allows for biodiversity within these proteoglycans to assist with their numerous specialized functions. Heparin sulfate proteoglycans are known co-receptors for Fgfr2b, which is a key component in kidney and lung branching morphogenesis within Fgf signaling.¹⁵ Both genes are also expressed in the submandibular gland (SMG) endbuds during salivary grand branching morphogenesis¹⁶ where 3-O-heparin sulfate is suggested to stabilize the Fgf10/Fgfr2b complex to promote MAPK signaling and the expansion of the progenitor population within the endbuds.¹⁶ Hence, Hs3st3a1 and Hs3st3b1 were selected as potential mediators of cell signaling. Additionally, it has been shown that signaling by glial cell line-derived neurotrophic factor (GDNF), an important regulator of kidney branching, requires the presence of heparan sulfate glycosaminoglycans to induce neuronal axonogenesis.^17,18 Thus, heparan sulfate modifying enzymes may impact multiple pathways regulating branching morphogenesis of the mammalian kidney.

Tmem59l (transmembrane protein 59-like) is a single-pass type I membrane glycoprotein expressed within the Golgi apparatus that has not been functionally examined in branching morphogenesis. First identified in the human brain as BSMAP (brain-specific membrane-anchored protein), Tmem59l was suggested to have caspase-dependent apoptotic activity and modulate amyloid precursor protein shedding.^19–21 Furthermore, mouse behavioral studies have implicated the downregulation of Tmem59l in reducing depression and anxiety, while increasing memory.²¹

RESULTS

Identification of Four Ureteric Bud Tip-specific Genes

D17H6S56E-5, Hs3st3a1, Hs3st3b1, and Tmem59l where identified in a bulk RNA sequencing screen comparing the ureteric tip population (Wnt11-RFP⁺/Hoxb7-GFP⁺) to non-branch tip regions (Hoxb7-GFP⁺) in the developing ureteric epithelium of the E16.5 mouse kidney [Fig. 1A–C].^8,22,23 All genes show highly enriched expression in UPCs in ureteric branch tips [Fig. 1D]. Whole-mount in situ hybridization (WISH) showed D17H6S56E-5 and Hs3st3a1 were broadly expressed in the kidney shortly at an early branching stage (E12.5), restricting to the branch tip population by E15.5 [Fig. 1 E, F, I, J]. In contrast, Hs3st3b1 and Tmem59l were tip-restricted from early stages of kidney development [Fig. 1G, H, K, L]. WISH was also performed on mouse embryonic lungs to compare expression patterns between two organs that develop through branching morphogenesis. D17H6S56E-5 and Hs3st3a1 were broadly expressed at E12.5, restricting to airway branch tips by E15.5 [Fig. 1M, N, Q, R], Tmem59l was tip-specific throughout lung development [Fig. 1P, T] and Hs3stsb1 was transiently expressed within the lung epithelium at E12.5, but absent from airway epithelium by E15.5 [Fig. 1O, S].

Fig. 1: Identification of four ureteric bud tip genes through an embryonic kidney bulk RNA sequencing screen.

A-C: Cryosection of a E16.5 kidney from a Hoxb7Venus;Wnt11RFP mouse showcasing ureteric epithelium (GFP) and the tip population (RFP) collected via dissociation and FACs for the bulk RNA sequencing screen comparing tip vs stalk. D: Fold change (FC) and reads per kilobase per million (RPKM) values of four genes of interest from the RNA sequencing screen. E-T: Whole-mount in situ hybridization staining of at E12.5 and E15.5 embryonic kidneys (E-L) and lungs (M-T) of the four genes of interest. Line in A = 20μm; Line in E, I, M, Q = 200μm.

Generation of Knockout Mouse Strains via CRISPR/Cas9 Gene Editing Techniques

To determine the function of D17H6D56E-5, Hs3st3a1, Hs3st3b1, and Tmem59l, knockout mouse strains were generated by CRISPR/Cas9 deletion techniques.

D17H6S56E-5 is located on Chromosome 17 and comprises 3 exons spanning 4,023 base pairs encoding a 596 amino acid sequence with a transmembrane feature at amino acid positions 553–579. CRISPR/Cas9 gene editing removed 1,691 base pairs within exon 2 spanning the transmembrane sequence [Fig. 2A]. The predicted protein product results in a 32 amino acid truncated protein with 13 missense amino acids at the C-terminus from the deletion generated frame shift.

Fig. 2: CRISPR/Cas9 gene editing at specific loci resulted in the generation of knockout mice.

A: Wildtype allele of D17H6S56E-5 gene and the CRISPR/Cas9 deletion (highlighted in yellow) that leads to the generation of a truncated missense protein in the D17H6S56E-5−/− mouse model. B: CRISPR/Cas9 deletion (highlighted in yellow) downstream of ATG site leads to the creation of a new STOP codon (labeled with an asterisk) and the generation of a truncated missense protein.

Tmem59l is a 3.5kb gene on Chromosome 8 with 8 exons. Through CRISPR/Cas9 deletion, we removed an 804bp region which includes exon 2 and the N-terminus of exon 3 [Fig. 2B] leading to a new in-frame STOP codon within Exon 3. The resulting allele is predicted to encode a 50 amino acid C-terminal truncated protein product where the final 7 amino acids resulting from the frame shift are missense. The truncated protein would lack the transmembrane sequence encoded by exon 7.

Hs3st3a1 and Hs3st3b1 are both located on Chromosome 11 on opposite strands within 511 kilobases between their promoter regions suggesting that they are likely co-regulated by similar mechanisms in the kidney where co-expression was observed [Fig. 3A]. The two genes are structurally highly related, and both genes are present in the human genome, suggesting a gene duplication event prior to the mammalian radiation. Structurally, each gene comprises two exons with a large intronic region. Due to their highly similar sulfotransferase domain and ability to act on the same disaccharide, we expected functional redundancy and therefore generated a double knockout of the two genes. We acquired the Hs3st3a1−/− mouse from the trans-NIH Knock-Out Mouse Project (KOMP). Through homologous recombination, a deleted sequence (553bp) directly downstream of the ATG site was replaced with an E. coli LacZ gene sequence [Fig. 3B]. This effectively generates LacZ instead of Hs3st3a1 protein during the transcription of this gene. Staining E15.5 Hs3st3a1+/− kidneys for β-galactosidase activity from the lacZ allele demonstrates the ureteric bud tip-specific expression of Hs3st3a1 [Fig. 3C]. By injecting the appropriate gRNA and Cas9 protein into Hs3st3a1−/− fertilized eggs, we generated an 88bp deletion within the first exon of Hs3st3b1 allele [Fig. 3D]. This deletion includes the predicted ATG site, though a second ATG is encoded 37 amino acids downstream from the deletion. Though this could potentially generate an amino-truncated protein, the protein would lack highly-conserved, amino terminal transmembrane domain sequences. Unfortunately, the absence of antibodies recognizing Hs3st3b1 precludes any additional insight though it may be possible in future studies to develop enzymatic assays to test for residual Hs3st3b1 enzyme activity.

Fig 3: Gene editing via CRISPR/Cas9 on the Hs3st3a1−/− mouse resulted in the generation of the Hs3st3a1−/−;Hs3st3b1−/− mouse.

A: The Hs3st3a1 gene is located 511bp downstream from Hs3st3b1 on chromosome 11 B: The Hs3st3a1−/− mouse was generated through the NIH-funded Knock Out Mouse Project, A region directly downstream of the ATG site was deleted and replaced by an E. coli LacZ casette. C: LacZ staining on E15.5 Hs3st3a1+/− kidney demonstrating ureteric bud tip expression D: The Hs3st3b1−/− knockout was produced through a CRISPR/Cas9 deletion that included the ATG site in Exon 1 (highlighted in yellow). A second in-frame ATG site is located downstream in Exon 1, demarcated with the asterisk, which may enable initiation of a truncated protein. Line = 20μm.

Ureter Duplications in Mutant Kidneys

To determine the viability of the knockout mice generated, heterozygote intercrosses were quantified for percentage of each genotype present per sex and compared to Mendelian frequency. Furthermore, to observe any phenotypes, lung and kidneys were collected at E12.5 for each mutant mouse.

D17H6S56E-5−/− mice generated were adult viable and recovered at close to Mendelian frequency (x² = 5.22, p = 0.07): 22% (16/74) of pups born from heterozygous intercrosses [Fig. 4A]. Stage-matched heterozygote and knockout lungs were collected at E12.5 and immunostained to visualize cytokeratin (CK, epithelial marker) and vimentin (Vim, mesenchymal marker). The lungs appeared normal [Fig. 4B–D]. E12.5 stage-matched kidneys also showed no obvious difference in the CK-marked ureteric network or adjacent Six2⁺ nephron progenitor cells though one D17H6S56E-5−/− kidney displayed a double ureter (1/28, 3.6%), a frequency not statistically different from spontaneous double ureter formation in the C57BL6/J mouse strain (1/59, 0.02%) [Fig. 4E–G, Fig.5].

Fig. 4: Knockout mice at E12.5 demonstrate double ureter kidneys at low frequencies with no lung phenotype.

A: D17H6S56E-5 heterozygous intercross results show similar Mendelian frequency to expected (numbers on bar graph are the number of each sex per genotype, x² = 5.22, p = 0.07). B-G: D17H6S56E-5 lungs (B-D) and kidneys (E-G) stained with cytokeratin (CK, epithelial marker), vimentin (Vim, lung mesenchyme marker), and Six2 (kidney nephron progenitor marker). D17H6S56E-5−/− kidneys display a double ureter phenotype in 3.6% (G). H: Percentage of wildtype, heterozygote, and knockout pups born from Tmem59l+/− parents follow expected Mendelian frequency (x² = 1.60, p = 0.45). I-K: Tmem59l lungs stained with CK and Vim. L-N: Tmem59l kidneys stained with CK and Six2. The loss of Tmem59l results in the presence of a double ureter at 8.7% frequency. O-R: Hs3st3a1+/−;Hs3st3b1+/− and Hs3st3a1−/−;Hs3st3b1−/− lungs (O, Q) and kidneys (P, R) stained with CK and either Vim (lung) or Six2 (kidneys). There is no observed phenotype in the lungs, whereas a 10.7% of Hs3st3a1−/−;Hs3st3b1−/− kidneys display a double ureter phenotype. Blue arrow: first branching event within the Six2+ population. White arrow: first branching event outside of Six2+ population in double ureter kidneys. Line = 200μm.

Fig. 5: Frequencies of double ureters across mouse lines.

Swiss Webster (SW) mice have a very low frequency of double ureter formation, none were detected examining 376 E12.5 kidneys. In contrast, C57BL6/J (B6) mice showed a low, spontaneous frequency of unilateral double ureter formation. We have reported^8,24 mutants in Adamts18, a metalloprotease identified in the same UPC-targeted expression screen, display a marked increase in ureter duplications (x² = 369.32, p < 0.0001). Hs3st3a1−/−;Hs3st3b1−/− (x² = 13.67, p = 0.0002) and Tmem59l−/− (x² = 6.77, p = 0.0091) mutant kidneys showed a significantly higher double ureter frequencies than B6 controls. ** = p < 0.01, *** = p < 0.001.

The Tmem59l knockout mice were adult viable but recovered at a frequency (8/47, 17%) slightly lower but not significantly different from Mendelian expectation in heterozygous intercrosses (x² = 1.60, p = 0.45) [Fig. 4H]. At E12.5, no branching phenotype was observed in the developing lungs of homozygote lungs [Fig. 4I–K]. A low percentage of kidneys lacking a normal Tmem59l gene exhibited a double ureter phenotype (2/23, 8.7%) [Fig. 4L–N]. No phenotype was observed in a similar number of heterozygotes (0/19, 0%) and comparison with wild-type C57BL6/J kidneys suggests a statistically significant increase in ureter duplication above background levels [Tmem59l−/− (x² = 6.77, p = 0.0091), Fig.5].

Hs3st3a1/Hs3st3b1 double knockout mice were also adult viable. E12.5 staged-matched lungs did not exhibit any obvious phenotype [Fig. 4O, Q]. On the other hand, embryonic kidneys form double ureters with a low penetrance (3/28, 10.7%), a higher frequency than spontaneous duplications observed in heterozygous compound mutant mice or wildtype C57BL6/J mice [x² = 13.67, p = 0.0002; Fig. 4P, R; Fig. 5]. The double ureter formed is not identical in each case, suggesting a variation in the position of ectopic outgrowth from the mesonephric duct, or from the stalk region of the forming ureteric bud.

DISCUSSION

A Shared Double Ureter Phenotype Amongst the UB Tip Knockout Mice

The UB tip progenitor population is a key cell type for the proper development of the collecting duct system and ultimately, the entire organ. Here, we generated four new knockout alleles complementing a recent study on Adamts18²⁴ which shares with the gene targets in this current study, ureteric and lung branch tip enriched gene expression (excepting Hs3st3b1).⁸ Shared expression in the kidney and lung suggested shared programs of gene regulation, and potentially, function. In this light, the mutational outcomes were surprising: no early lung phenotypes where observed in the mutants analyzed here, in contrast to the pronounced growth phenotype reported for Adamts18 mutants, and in the kidney, a weakly penetrant ureter duplication phenotype (excepting D17H6S56E-5−/− mice), Relative to SW mice, the C57BL6/J background may be “sensitized” for spontaneous ureter duplication, though certitude would require a more extensive interstrain comparison [Fig. 5] suggesting this background may be particular effective at detecting weak interacting genes, the only phenotype observed in this limited study.

In addition to Adamts18, which encodes a secreted, Adams family metalloprotease, ureter duplications are observed in Robo2 and Slit2 mutants. The high penetrance of these phenotypes enabled the identification of a Robo2/Slit2 driven restriction of GDNF expression as the likely cause of ectopic ureteric bud outgrowths.^25,26 Loss of Wnt5a or Bmp4 results in a wide range of kidney phenotypes including the formation of double ureters; phenotypic analysis suggests these signaling pathways normally act to inhibit and confine UB outgrowth.^27–29 Tmem59l has not been linked to any of these proteins or their known pathways of action. In contrast, Hs3st3a1/Hs3st3b1, modulation of heparin sulfate proteoglycans (HSPGs) could link to both FGF and Wnt signaling where HSPGs play important roles.^30,31

Congenital Anomaly of Kidney and Urinary Tract (CAKUT) is an extensive and wide-ranging group of abnormalities that occur during kidney and urinary tract development in humans. Some aberrations include renal agenesis, multicystic dysplasic kidneys, double ureters, congenital megaureter, and vesicouretral reflux. CAKUT patients make up about 20–30% of all developmental defects reported and occur in about 1 in every 500 live births.^32,33 Many of the genes that have been identified to be associated with CAKUT are conserved in mice. Moreover, these genes have also been shown to play a role in kidney development, such as RET, SIX2, and BMP4.^34,35 Currently, none of the genes studied here have been connected to CAKUT in patients.

In summary, while the evidence suggests roles for Tmem59l and Hs3st3a1/Hs3st3b1 in the key initiating step of kidney morphogenesis, ureteric bud outgrowth, the low penetrance of phenotypes seen here presents a significant challenge to obtaining further mechanistic insights into their actions.

EXPERIMENTAL PROCEDURES

Mouse Strains

The Wnt11RFP mouse strain was generated in the McMahon lab (B6;D-Tg(Wnt11-TagRFP/cre/ERT2)28Amc/J; Jackson Laboratory stock 018683).²² The Hoxb7Venus mouse was generated and provided by Dr. Frank Costantini (Tg(Hoxb7-Venus*)17Cos/J; Jackson Laboratory stock 016252).²³ The Hs3st3a1−/− mouse (VG19780) was produced by the trans-NIH Knock-Out Mouse Project (KOMP) and obtained from the KOMP Repository (www.komp.org). Mouse handling, husbandry, and procedures were all performed in compliance with the guidelines established by the Institutional Animal Care and Use Committees (IACUC) at the University of Southern California.

Generation of Knockout Mouse Strains via CRISPR/Cas9 Gene Editing

Standard CRISPR/Cas9 gene editing protocols were used to precisely delete specific regions of the D17H6S56E-5, Hs3st3b1, and Tmem59l genes.³⁶ Exact details of the mutations are reported in the results. A brief generalized description of the approach follows. PCR-based methods were used to synthesize the DNA templates for Cas9 and sgRNA (single guide RNA). Guide RNA (gRNA) sequences for generating sgRNA DNA templates were identified for each target gene on crispr.mit.edu. Purified PCR products for Cas9 and sgRNAs were mixed with dNTPs and T7 enzyme for in vitro transcription. The resulting sgRNA and Cas9 RNA were microinjected into fertilized eggs collected from super ovulating female mice. For D17H6S56E-5 and Tmem59l deletions, injections were performed in C57BL/6J eggs. The Hs3st3b1 injection was performed in Hs3st3a1−/− fertilized eggs. These manipulated zygotes were placed into the oviducts of pseudopregnant foster female mice. Pups were assessed for deleted regions in gene targets by Sanger sequencing of cloned PCR products.

Whole-mount in situ Hybridization

In situ hybridization was completed utilizing our previously published procedure.⁸ Briefly, tissue samples were collected and fixed overnight in 4% paraformaldehyde (PFA), dehydrated in methanol, and stored at −20°C. Tissue was rehydrated, bleached with 6% hydrogen peroxide, incubated in proteinase K (10 μg/ml), fixed in 4% PFA, pre-hybridized in hybridization buffer at 70°C, and then incubated overnight in a RNA probe that recognized the rtTA sequence. Tissue was transferred to a BioLane HTI machine, which performed formamide washes, antibody incubation, and MBST (100 mM maleic acid, 150 mM NaCl, 0.1% Tween-20) washes. Samples were incubated with BM purple for up to 48hr to reveal the in situ hybridization of the RNA probe, fixed in 4% PFA, and stored in 80% glycerol. Tissue was imaged in an AxioZoom.V16 stereozoom microscope (Zeiss).

Antibody Immunofluorescent Staining

Embryonic lungs and kidneys were harvested and subjected to a short fixation in 4% PFA. For whole-mount staining, tissue was incubated in blocking solution (10% sheep serum, 0.1% Triton, PBS) for 1hr and incubated in primary antibodies at 4°C for 24–48hr. Antibodies used were cytokeratin (1:500; Sigma C2562, Novus Biologicals NB120–11213), Six2 (1:500; Proteintech 11562–1-AP), and vimentin (1:500; Abcam ab92547). Samples were washed in PBST (0.1% Triton, PBS) to remove and residual primary antibody for several hours, and then incubated in the corresponding secondary antibody for 24–48hr at 4°C. Tissue was again washed in PBST for several hours to remove any remaining secondary antibody, and then imaged on an AxioZoom.V16 stereozoom microscope (Zeiss).

For cryosections with native fluorescence in the tissue, embryonic kidneys were harvested, fixed in 4% PFA, incubated overnight in 30% sucrose, embedded in OCT, and stored in −80°C. Cryoblocks were sectioned at 12μm thick (Zeiss Microm HM550 cryostat), placed on glass slides (VWR Superfrost Plus Micro Slide), and stored at −80°C. To remove the OCT, slides were washed in PBS, and then incubated in Hoechst 33342 to stain nuclei. Tissue sections were imaged on a confocal SP8 microscope (Leica) to capture native fluorescent signal.