Hypomorphic and dominant-negative impact of truncated SOX9 dysregulates Hedgehog-Wnt signaling, causing campomelia.

Haploinsufficiency for SOX9, the master chondrogenesis transcription factor, can underlie campomelic dysplasia (CD), an autosomal dominant skeletal malformation syndrome, because heterozygous Sox9 null mice recapitulate the bent limb (campomelia) and some other phenotypes associated with CD. However, in vitro cell assays suggest haploinsufficiency may not apply for certain mutations, notably those that truncate the protein, but in these cases in vivo evidence is lacking and underlying mechanisms are unknown. Here, using conditional mouse mutants, we compared the impact of a heterozygous Sox9 null mutation (Sox9+/-) with the Sox9+/Y440X CD mutation that truncates the C-terminal transactivation domain but spares the DNA-binding domain. While some Sox9+/Y440X mice survived, all Sox9+/- mice died perinatally. However, the skeletal defects were more severe and IHH signaling in developing limb cartilage was significantly enhanced in Sox9+/Y440X compared with Sox9+/-. Activating Sox9Y440X specifically in the chondrocyte-osteoblast lineage caused milder campomelia, and revealed cell- and noncell autonomous mechanisms acting on chondrocyte differentiation and osteogenesis in the perichondrium. Transcriptome analyses of developing Sox9+/Y440X limbs revealed dysregulated expression of genes for the extracellular matrix, as well as changes consistent with aberrant WNT and HH signaling. SOX9Y440X failed to interact with ß-catenin and was unable to suppress transactivation of Ihh in cell-based assays. We propose enhanced HH signaling in the adjacent perichondrium induces asymmetrically localized excessive perichondrial osteogenesis resulting in campomelia. Our study implicates combined haploinsufficiency/hypomorphic and dominant-negative actions of SOX9Y440X, cell-autonomous and noncell autonomous mechanisms, and dysregulated WNT and HH signaling, as the cause of human campomelia.

SOX9 and SOX10 control fluid homeostasis in the inner ear for hearing through independent and cooperative mechanisms.

The in vivo mechanisms underlying dominant syndromes caused by mutations in SRY-Box Transcription Factor 9 (SOX9) and SOX10 (SOXE) transcription factors, when they either are expressed alone or are coexpressed, are ill-defined. We created a mouse model for the campomelic dysplasia SOX9Y440X mutation, which truncates the transactivation domain but leaves DNA binding and dimerization intact. Here, we find that SOX9Y440X causes deafness via distinct mechanisms in the endolymphatic sac (ES)/duct and cochlea. By contrast, conditional heterozygous Sox9-null mice are normal. During the ES development of Sox9Y440X/+ heterozygotes, Sox10 and genes important for ionic homeostasis are down-regulated, and there is developmental persistence of progenitors, resulting in fewer mature cells. Sox10 heterozygous null mutants also display persistence of ES/duct progenitors. By contrast, SOX10 retains its expression in the early Sox9Y440X/+ mutant cochlea. Later, in the postnatal stria vascularis, dominant interference by SOX9Y440X is implicated in impairing the normal cooperation of SOX9 and SOX10 in repressing the expression of the water channel Aquaporin 3, thereby contributing to endolymphatic hydrops. Our study shows that for a functioning endolymphatic system in the inner ear, SOX9 regulates Sox10, and depending on the cell type and target gene, it works either independently of or cooperatively with SOX10. SOX9Y440X can interfere with the activity of both SOXE factors, exerting effects that can be classified as haploinsufficient/hypomorphic or dominant negative depending on the cell/gene context. This model of disruption of transcription factor partnerships may be applicable to congenital deafness, which affects ∼0.3% of newborns, and other syndromic disorders.

Tissue distribution and subcellular localization of the family of Kidney Ankyrin Repeat Domain (KANK) proteins.

Kidney Ankyrin Repeat-containing Proteins (KANKs) comprise a family of four evolutionary conserved proteins (KANK1 to 4) that localize to the belt of mature focal adhesions (FAs) where they regulate integrin-mediated adhesion, actomyosin contractility, and link FAs to the cortical microtubule stabilization complex (CMSC). The human KANK proteins were first identified in kidney and have been associated with kidney cancer and nephrotic syndrome. Here, we report the distributions and subcellular localizations of the four Kank mRNAs and proteins in mouse tissues. We found that the KANK family members display distinct and rarely overlapping expression patterns. Whereas KANK1 is expressed at the basal side of epithelial cells of all tissues tested, KANK2 expression is mainly observed at the plasma membrane and/or cytoplasm of mesenchymal cells and KANK3 exclusively in vascular and lymphatic endothelial cells. KANK4 shows the least widespread expression pattern and when present, overlaps with KANK2 in contractile cells, such as smooth muscle cells and pericytes. Our findings show that KANKs are widely expressed in a cell type-specific manner, which suggests that they have cell- and tissue-specific functions.

Differential and overlapping expression pattern of SOX2 and SOX9 in inner ear development.

The development of the inner ear involves complex processes of morphological changes, patterning and cell fate specification that are under strict molecular control. SOX2 and SOX9 are SOX family transcription factors that are involved in the regulation of one or more of these processes. Previous findings have shown early expression of SOX9 in the otic placode and vesicle at E8.5-E9.5. Here we describe in detail, the expression pattern of SOX9 in the developing mouse inner ear beyond the otocyst stage and compare it with that of SOX2 from E9.5 to E18.5 using double fluorescence immunohistochemistry. We found that SOX9 was widely expressed in the otic epithelium, periotic mesenchyme and cartilaginous otic capsule. SOX2 persistently marked the prosensory and sensory epithelia. During the development of the sensory epithelia, SOX2 was initially expressed in all prosensory regions and later in both the supporting and hair cells up to E15.5, when its expression in hair cells gradually diminished. SOX9 expression overlapped with that of SOX2 in the prosensory and sensory region until E14.5 when its expression was restricted to supporting cells. This initial overlap but subsequent differential expression of SOX2 and SOX9 in the sensory epithelia, suggest that SOX2 and SOX9 may have distinct roles in molecular pathways that direct cells towards different cell fates.

Utility of HoxB2 enhancer-mediated Cre activity for functional studies in the developing inner ear.

The rhombomere 4(r4)-restricted expression of the mouse Hoxb2 gene is regulated by a 1.4-kb enhancer-containing fragment. Here, we showthat transgenic mouse lines expressing cre driven by this fragment (B2-r4-Cre), activated the R26R Cre reporter in rhombomere 4 and the second branchial arch, the epithelium of the first branchial arch, apical ectodermal ridge of the limb buds and the tail region. Of particular interest is Cre activity in the developing inner ear. Cre activity was found in the preotic field and otic placode at E8.5 and otocyst at E9.5-E12.5, in the cochleovestibular and facio-acoustic ganglia at E10.5 and the vestibular and spiral ganglia and all the otic epithelia derived from the otocyst at E15.5 and P0. Our data suggest that the B2-r4-Cre transgenic mice provide an important tool for conditional gene manipulation and lineage tracing in the inner ear. In combination with other transgenic lines expressing cre exclusively in the otic vesicle, the relative contributions of the hindbrain, periotic mesenchyme and otic epithelium in otic development can be dissected.

Aldose reductase regulates hepatic peroxisome proliferator-activated receptor alpha phosphorylation and activity to impact lipid homeostasis.

Aldose reductase (AR) is implicated in the development of a number of diabetic complications, but the underlying mechanisms remain to be fully elucidated. We performed this study to determine whether and how AR might influence hepatic peroxisome proliferator-activated receptor alpha (PPARalpha) activity and lipid metabolism. Our results in mouse hepatocyte AML12 cells show that AR overexpression caused strong suppression of PPARalpha/delta activity (74%, p < 0.001) together with significant down-regulation of mRNA expression for acetyl-CoA oxidase and carnitine palmitoyltransferase-1. These suppressive effects were attenuated by the selective AR inhibitor zopolrestat. Furthermore, AR overexpression greatly increased the levels of phosphorylated PPARalpha and ERK1/2. Moreover, AR-induced suppression of PPARalpha activity was attenuated by treatment with an inhibitor for ERK1/2 but not that for phosphoinositide 3-kinase, p38, or JNK. Importantly, similar effects were observed for cells exposed to 25 mm glucose. In streptozotocin-diabetic mice, AR inhibitor treatment or genetic deficiency of AR resulted in significant dephosphorylation of both PPARalpha and ERK1/2. With the dephosphorylation of PPARalpha, hepatic acetyl-CoA oxidase and apolipoprotein C-III mRNA expression was greatly affected and that was associated with substantial reductions in blood triglyceride and nonesterified fatty acid levels. These data indicate that AR plays an important role in the regulation of hepatic PPARalpha phosphorylation and activity and lipid homeostasis. A significant portion of the AR-induced modulation is achieved through ERK1/2 signaling.

Analysis of imprinted murine Peg3 locus in transgenic mice.

Peg3 is an imprinted gene exclusively expressed from the paternal allele. It encodes a C(2)H(2) type zinc-finger protein and is involved in maternal behavior. It is important for TNF-NFkB signaling and p53-mediated apoptosis. To investigate the imprinting mechanism and gene expression of Peg3 and its neighboring gene(s), we used a 120 kb Peg3-containing BAC clone to generate transgenic mice. The BAC clone contains 20 kb of 5' and 80 kb of 3' flanking DNA, and we obtained three transgenic lines. In one of the lines harboring one copy of the transgene, Peg3 was imprinted properly. In the other two lines, Peg3 was expressed upon both maternal and paternal transmission. Imprinted expression was linked to the differential methylation of a region (DMR) upstream of the Peg3 gene. A second, maternally expressed gene, Zim1, present on the transgene was expressed irrespective of parental inheritance in all lines. These data suggest that, similar to other imprinted genes within domains, Peg3 and Zim1 are regulated by one or more elements lying at a distance from the genes. The imprinting of Peg3 seen in one line may reflect the presence of a responder sequence. Concerning the expression of the Peg3 transgene, we detected appropriate expression in the adult brain. However, this was not sufficient to rescue the maternal behavior phenotype seen in Peg3 deficient animals.