Disruption of Hox9,10,11 function results in cellular level lineage infidelity in the kidney

Abstract

Hox genes are important regulators of development. The 39 mammalian Hox genes have considerable functional overlap, greatly confounding their study. In this report, we generated mice with multiple combinations of paralogous and flanking Abd-B Hox gene mutations to investigate functional redundancies in kidney development. The resulting mice developed a number of kidney abnormalities, including hypoplasia, agenesis, and severe cysts, with distinct Hox functions observed in early metanephric kidney formation and nephron progenitor maintenance. Most surprising, however, was that extensive removal of Hox shared function in these kidneys resulted in cellular level lineage infidelity. Strikingly, mutant nephron tubules consisted of intermixed cells with proximal tubule, loop of Henle, and collecting duct identities, with some single cells expressing markers associated with more than one nephron segment. These results indicate that Hox genes are required for proper lineage selection/maintenance and full repression of genes involved in cell fate restriction in the developing kidney.

Figure 1

Generation of Hoxc9,10,11 targeting construct. A modified recombineering strategy was utilized to introduce concurrent frameshift mutations in the adjacent genes, Hoxc9, Hoxc10, and Hoxc11. A BAC with the entire HoxC cluster was first altered at the Hoxc9 locus through homologous recombination with a DNA segment including a Kan/Neo selectable marker flanked by the ‘once only’ LoxP sequences LoxP66 and LoxP71. Homologous recombination was driven by two DNA segments with sequence homology to coding regions of the first exon. Inducible Cre recombinase was then used to delete the Kan/Neo selectable marker, leaving behind an inactive LoxP sequence and a small deletion of coding sequence, creating a frameshift mutation. The process was then repeated for Hoxc10 and Hoxc11.

Figure 2

Hox mutant kidneys exhibit hypoplasia, dysplasia, and bilateral agenesis as well as severely cystic kidneys. (a) Gross dissections showed smaller kidneys in Hoxc9,10,11−/− and all heterozygous/homozygous combinations generated (Hoxa9,10,11+/− Hoxc9,10,11−/−, Hoxa9,10,11−/− Hoxc9,10,11+/−, Hoxc9,10,11−/− Hoxd9,10,11+/−, Hoxc9,10,11+/− Hoxd9,10,11−/− as well as triple heterozygotes Hoxa9,10,11+/− Hoxc9,10,11+/− Hoxd9,10,11+/−). Hoxc9,10,11−/− Hoxd9,10,11−/− and Hoxa9,10−/−11+/− Hoxc9,10,11−/− mutants showed very small, rudimentary kidneys and Hoxa9,10,11−/− Hoxc9,10,11−/− showed bilateral renal agenesis. N = 8 for each genotype; arrow – gonads, arrowhead – bladder, asterisk – adrenal gland. (b) Representative H&E images of E18.5 all heterozygous/homozygous mutants generated (genotypes listed above) showed hypoplasia (P’,T’). We observed occasional cystic tubules and dilated Bowmen’s space (T’; asterisk). Hoxc9,10,11−/− Hoxd9,10,11−/− and Hoxa9,10−/−11+/− Hoxc9,10,11−/− mutant kidneys appeared dysplastic, with lobar disorganization (M’,N’) and tubules surrounded by stroma (U’, arrows). E18.5 Hoxa9,10−/−11+/− Hoxc9,10,11−/− mutants lacked a nephrogenic zone (R’; arrowhead), whereas residual areas of nephrogenesis were present in Hoxc9,10,11−/− Hoxd9,10,11−/− mutants (Q’). Hoxa9,10−/−11+/− Hoxc9,10,11−/− mutants also demonstrated dysplastic epithelium in the renal papilla (V’; asterisk). Magnification bar = 50 μm; N = 3 for each genotype. (c) Gross images (W’-Y’; original magnification 6x) and H&Es (Z’-BB’ original magnification 2x; CC’-EE’ original magnification 20x) showed that 10–15% of 2 month old heterozygous/homozygous multi-Hox mutant mice (N = 40 mice total) and all surviving Hoxc9,10,11−/− Hoxd9,10,11−/− mutants (N = 3) developed cystic kidneys.

Figure 3

Ureteric bud (UB) and cap mesenchyme (CM) at E13.5. (a) All heterozygous/homozygous Hox mutant mice generated demonstrated asymmetric branching, either in the dorsal/ventral axis alone (B’) or both the anterior/posterior and dorsal/ventral axes (C’) as quantified by genotype in (Table S3). Hoxc9,10,11−/− Hoxd9,10,11−/− mutants developed elongated branches with limited secondary branching (D’, G’, J’; N = 7/8 mutants examined; with 1 mutant showing absent branching). Hoxa9,10−/−11 +/+ Hoxc9,10,11−/− mutants developed a few branch tips (H’, K’; N = 3). Hoxa9,10,11−/− Hoxc9,10,11−/− mutants lacked secondary branching (E’; N = 3) with a single glomerulus (insert; Wt1 – red). (b) Hoxc9,10,11−/− Hoxd9,10,11−/− kidneys showed increased thickness of CM (J’, arrowhead; Six2 – green) surrounding the reduced number of UB tips (calbindin – purple). CM showed abnormal extension down the length of the UB branch/stalk (J’, arrow). Hoxa9,10−/−11 +/+ Hoxc9,10,11−/− kidneys were severely affected in terms of both UB branching and CM mass (H’,K’). (c) Decreased UB branch tips were observed in all heterozygous/homozygous Hox combinations (N = 3 per genotype, asterisk indicates p < 0.05 from control).

Figure 4

Hox mutant kidney tubules develop ambiguous nephron segmentation. Intermixed DBA (collecting duct) and LTA (proximal tubule) positive cells were present in E18.5 all heterozygous/homozygous mutants generated (Hoxa9,10,11−/− Hoxc9,10,11+/−, Hoxa9,10,11+/− Hoxc9,10,11−/−, Hoxc9,10,11−/− Hoxd9,10,11+/−, Hoxc9,10,11+/− Hoxd9,10,11−/−) (B’,F’; arrows) but were more frequent in Hoxc9,10,11−/− Hoxd9,10,11−/− embryonic kidneys (C’,G’; arrows) and Hoxa9,10−/−11+/− Hoxc9,10,11−/− (data not shown). Adult Hox mutants that develop cysts showed numberous tubules with intermixed DBA and LTA positive cells (H’,L’); (asterisk).

Figure 5

Multi-Hox mutant kidney tubules have intermixed cells with distinct nephron segment identities as well as single cells that express markers of more than one segment. (a) At E18.5, we observed tubules containing DBA (collecting duct) cells interspersed among Villin (proximal tubule) positive cells (B’). Multi-Hox mutants also showed single cells that triple labeled with HNF4a (proximal tubule) as well as DBA and CK (collecting duct) (G’, insert, arrowhead). LTA (proximal tubule) and SLC12A1 (ascending loop of Henle) positive cells were present in a single tubule (L’). (b) Adult cystic kidneys showed cells with LTA and Villin (proximal tubule) adjacent to SLC12A1 (ascending loop of Henle) cells (Q’). Single cells also co-expressed HNF4a (proximal tubule) with CK (collecting duct) (V’), SLC12A1 (ascending loop of Henle) with CK (collecting duct marker) (AA’), and Villin (proximal tubule) with DBA (collecting duct marker) (FF’). Cysts also showed regional expression of proximal tubule markers (Villin and/or LTA) adjacent to CK cells (collecting duct) (KK’). N = 3 per immunoassay.

Figure 6

Scanning electron microscopy confirmed abnormal cell morphologies in adult multi-Hox mutant cystic kidneys. Mutant tubules showed regions of tightly packed microvilli consistent with proximal tubule brush border (i) interspersed among distal tubule/collecting duct cells (ii) with sparse microvilli (C’; higher magnification in C”). Conversely, cystic tubules consisting of primarily proximal tubule cells (i) contained interspersed cells with short, sparse microvilli (ii) consistent with distal tubule/collecting duct cells^, (D’; higher magnification in D”), not observed in control kidney proximal tubules (A’) or collecting ducts (B’). N = 3 controls and 4 mutants.

Figure 7

Dlk1 expression is upregulated in Hox mutant kidneys. Dlk1 expression is very low in the wild type metanephros at E12.5 (N’), E13.5 (B’,D’), E14.5 (E’, image from Eurexpress.org), E15.5 (F’, image from Allen Brain Atlas, brain-map.org), E16.5 (L’), E18.5 (P’) and adult (T’). In contrast, the wild type mesonephros shows DLK1 expression at E12.5 (G’–J’). Hoxa9,10−/−11+/− HoxC−/− mutant kidneys show DLK1 expression in multiple cell types, including proximal tubules, as well as interstitial cells (Q’,R’), and DLK1 is upregulated in Hox cystic kidneys as well (U’,V’). Hoxc9,10,11−/− Hoxd9,10,11−/− show increased DLK1 expression along the ureteric stalks (CC’, arrow) and in developing nephrons (KK’, arrowheads) in E13.5 whole mount IF. N = 3 per immunoassay.

Figure 8

Severe disruption of Hox9,10,11 function results in cellular level lineage infidelity. Hoxc9,10,11−/− Hoxd9,10,11−/− (as well as other multi-Hox mutant combinations of Hoxa9,10,11 Hoxc9,10,11 and Hoxc9,10,11 Hoxd9,10,11) renal tubules consisted of intermixed cells with proximal tubule, loop of Henle, and collecting duct identities, with some single cells expressing markers associated with more than one nephron segment.