Nephron progenitor number determines nephron endowment; a reduced nephron count is linked to the onset of kidney disease. Several transcriptional regulators including Six2, Wt1, Osr1, Sall1, Eya1, Pax2, and Hox11 paralogues are required for specification and/or maintenance of nephron progenitors. However, little is known about the regulatory intersection of these players. Here, we have mapped nephron progenitor-specific transcriptional networks of Six2, Hoxd11, Osr1, and Wt1. We identified 373 multi-factor associated ‘regulatory hotspots’ around genes closely associated with progenitor programs. To examine their functional significance, we deleted ‘hotspot’ enhancer elements for Six2 and Wnt4. Removal of the distal enhancer for Six2 leads to a ~40% reduction in Six2 expression. When combined with a Six2 null allele, progeny display a premature depletion of nephron progenitors. Loss of the Wnt4 enhancer led to a significant reduction of Wnt4 expression in renal vesicles and a mildly hypoplastic kidney, a phenotype also enhanced in combination with a Wnt4 null mutation. To explore the regulatory landscape that supports proper target gene expression, we performed CTCF ChIP-seq to identify insulator-boundary regions. One such putative boundary lies between the Six2 and Six3 loci. Evidence for the functional significance of this boundary was obtained by deep sequencing of the radiation-induced Brachyrrhine (Br) mutant allele. We identified an inversion of the Six2/Six3 locus around the CTCF-bound boundary, removing Six2 from its distal enhancer regulation, but placed next to Six3 enhancer elements which support ectopic Six2 expression in the lens where Six3 is normally expressed. Six3 is now predicted to fall under control of the Six2 distal enhancer. Consistent with this view, we observed ectopic Six3 in nephron progenitors. 4C-seq supports the model for Six2 distal enhancer interactions in wild-type and Br/+ mouse kidneys. Together, these data expand our view of the regulatory genome and regulatory landscape underpinning mammalian nephrogenesis. Nephrons, the filtering units of the kidney, derive from nephron progenitors. Deficiencies in nephron number increases the risk of kidney disease. An understanding of the regulatory programs governing progenitor actions has important translational potential. Several transcription factors regulate the nephron progenitor population. However, their target interactions are largely unknown. Here, we mapped and intersected the genome-wide binding sites for four such factors in mouse nephron progenitor cells in the developing kidney: Six2, Hoxd11, Osr1, and Wt1. The intersectional data highlight a high-value set of putative enhancer elements linked to genes regulating nephron progenitor properties. We validate the function of two such enhancer elements regulating the levels of Six2, a key transcriptional regulatory factor in nephron progenitor maintenance, and Wnt4, a critical signaling factor controlling the mesenchyme to epithelial transition of induced nephron progenitors. Further characterization of the Six2 regulatory landscape identified higher order regulatory interactions that ensure appropriate enhancer-promoter specificity. CTCF-bound sites between Six2 and the adjacent Six3 locus likely act as boundary elements to define topological interactions domains separating enhancer elements thereby providing distinct tissue specificity to each gene’s expression. An inversion of this region in the Brachyrrhine (Br) mutant mouse reverses Six2 and Six3 expression domains, placing Six3 under control of the Six2 enhancer element above resulting in kidney-specific expression, while Six2 expression shifts to the lens, a normal expression domain for Six3. Together, these data expand our view of the regulatory genome and regulatory landscape underpinning mammalian nephrogenesis.

The Br/+ mutant mouse displays decreased embryological expression of the homeobox transcription factor Six2, resulting in hertitable renal hypoplasia. The purpose of this study was to characterize the renal physiological consequences of embryonic haploinsuffiency of Six2 by analyzing renal morphology and function in the adult Br heterozygous mutant. Adult Br/+ kidneys weighed 50% less than those from wild-type mice and displayed glomerulopathy. Stereological analysis of renal glomeruli showed that Br/+ kidneys had an average of 88% fewer glomeruli than +/+ kidneys, whereas individual glomeruli in Br/+ mice maintained an average volume increase of 180% compared with normal nephrons. Immunostaining revealed increased levels of endothelin-1 (ET-1), endothelin receptors A (ET_A) and B (ET_B), and Na-K-ATPase were present in the dilated renal tubules of mutant mice. Physiological features of chronic renal failure (CRF) including elevated mean arterial pressure, increased plasma creatinine, and dilute urine excretion were measured in Br/+ mutant mice. Electron microscopy of the Br/+ glomeruli revealed pathological alterations such as hypercellularity, extracellular matrix accumulation, and a thick irregular glomerular basement membrane. These results indicate that adult Br/+ mice suffer from CRF associated with reduced nephron number and renal hypoplasia, as well as glomerulopathy. Defects are associated with embryological deficiencies of Six2, suggesting that proper levels of this protein during nephrogenesis are critical for normal glomerular development and adult renal function.

A radiation‐induced mouse mutant,Brachyrrhine(Br), exhibits frontonasal dysplasia and renal hypoplasia, two malformations associated with deficiencies in mesenchymal condensation. The purpose of this study was to resolve theBrlocus, evaluate possible candidate genes, and identify developmental defects in the mutant chondrocranium. Linkage analysis mapped theBrmutation to a critical region distal to D17Mit76, which contains only one gene, the transcription factorSix2. Sequence analysis of theSix2gene, including 1.5 kb of the promoter, failed to reveal theBrmutation. However, homozygousBr/Brembryos showed almost complete absence ofSix2mRNA and protein in craniofacial and renal tissues while heterozygousBr/+ embryos displayed intermediateSix2levels. Mutant embryos displayed malformations of neural crest‐derived structures of the anterior cranium whereSix2is normally expressed. These data suggest a mutation in a novelcis‐acting regulatory region inhibitsSix2expression and is associated with frontonasal dysplasia and renal hypoplasia. Developmental Dynamics 237:1767–1779, 2008.

Computerized animation is becoming an increasingly popular method to provide dynamic presentation of anatomical concepts. However, most animations use artistic renderings as the base illustrations that are subsequently altered to depict movement. In most cases, the artistic rendering is a schematic that lacks realism. Plastinated sections provide a useful alternative to artistic renderings to serve as a base image for animation. The purpose of this study is to describe a method for developing animations by using plastinated sections. This application is used in Project TOUCH as a supplemental learning tool for a problem‐based learning case distributed over the National Computational Science Alliance's Access Grid. The case involves traumatic head injury that results in an epidural hematoma with transtentorial uncal herniation. In addition, a subdural hematoma is animated permitting the student to contrast the two processes for a better understanding of dural hematomas, in general. The method outlined uses P40 plastinated coronal brain sections that are digitized and to which contiguous anatomical structures are rendered. The base illustration is rendered, interpolated, and viewed while audio narration describes the event. This method demonstrates how realistic anatomical animations can be generated quickly and inexpensively for medical education purposes by using plastinated brain sections. Anat Rec (Part B: New Anat) 270B:30–37, 2003.

The adult Brachyrrhine (3H1 Br/+) mouse displays severe midfacial retrognathia, with a “pugnose” external appearance, but information concerning craniofacial morphology of the homozygote (3H1 Br/Br) mutant is lacking. This study characterized craniofacial phenotype and genotypic features of the homozygous condition. Segregation analysis was performed by phenotypic scoring of offspring from 3H1 Br/+ reciprocal matings. Whole‐mount staining was undertaken to determine the presence or absence of cranial base structures in newborn and adult mice, while features of cranial base chondrification were examined using light microscopy and type II collagen immunohistochemistry. Karyotype analysis was performed to determine whether gross chromosomal aberrations were present. Finally, microsatellite mapping analysis was undertaken to provide further resolution of the Br locus. Results showed that Br was inherited as an autosomal semidominant feature. 3H1 Br/Br mice consistently lacked a presphenoid (with its lateral projections, including a preoptic root, postoptic root, and lesser wing). Karyotyping did not reveal major gross aberrations; however, microsatellite analysis localized Br to distal mouse chromosome 17 in the vicinity of D17Mit155. These results indicated that 3H1 Br/Br mice show characteristic features of frontonasal dysplasia, including median facial clefting and bifid cranium, as well sphenoidal malformations. Furthermore, this mutant should serve as a useful model for examining mechanisms of frontonasal dysplasia. Anat Rec Part A 271A:291–302, 2003.

Abdelkarim AZ, et al. Imaging Sci Dent. 2019 Mar;49(1):71-77. https://doi.org/10.5624/isd.2019.49.1.71