Ian D. Tonks

Transgenic Overexpression of Vascular Endothelial Growth Factor-B Isoforms by Endothelial Cells Potentiates Postnatal Vessel Growth In Vivo and In Vitro

Vascular endothelial growth factors (VEGFs) play significant roles in endothelial growth, survival, and function, and their potential use as therapeutic agents to promote the revascularization of ischemic tissues in being avidly explored. VEGF-A has received most attention, as it is a potent stimulator of vascular growth. Results in clinical trials of VEGF-A as a therapeutic agent have fallen short of high expectations because of serious edematous side effects caused by its activity in promoting vascular permeability. VEGF-B, a related factor, binds some of the VEGF-A receptors but not to VEGF receptor 2, which is implicated in the vascular permeability promoting activity of VEGF-A. Despite little in vitro evidence to date for the ability of Vegf-B to directly promote angiogenesis, recent data indicate that it may promote postnatal vascular growth in mice, suggesting that it may have potential therapeutic application. We have specifically studied the effects of VEGF-B on vascular growth in vivo and on angiogenesis in vitro by analyzing transgenic mice in which individual isoforms (VEGFB167Tg and VEGFB186Tg) of VEGF-B are overexpressed in endothelial cells. VEGFB167Tg and VEGFB186Tg mice displayed enhanced vascular growth in the Matrigel assay in vivo and during cutaneous wound healing. In the aortic explant assay, explants from VEGFB167Tg and VEGFB186Tg mice displayed elevated vascular growth, suggesting a direct effect of VEGF-B isoforms in potentiating angiogenesis. These data support the use of VEGF-B as a therapeutic agent to promote vascular growth, in part, by potentiating angiogenesis. Furthermore, the lack of vascular permeability activity associated with either transgenic overexpression of the VEGF-B gene in endothelial cells or application of VEGF-B protein to the skin of mice in the Miles assay indicates that use of VEGF-B as a therapy should not be associated with edematous side effects.

Smchd1 regulates a subset of autosomal genes subject to monoallelic expression in addition to being critical for X inactivation

BackgroundSmchd1 is an epigenetic modifier essential for X chromosome inactivation: female embryos lacking Smchd1 fail during midgestational development. Male mice are less affected by Smchd1-loss, with some (but not all) surviving to become fertile adults on the FVB/n genetic background. On other genetic backgrounds, all males lacking Smchd1 die perinatally. This suggests that, in addition to being critical for X inactivation, Smchd1 functions to control the expression of essential autosomal genes.ResultsUsing genome-wide microarray expression profiling and RNA-seq, we have identified additional genes that fail X inactivation in female Smchd1 mutants and have identified autosomal genes in male mice where the normal expression pattern depends upon Smchd1. A subset of genes in the Snrpn imprinted gene cluster show an epigenetic signature and biallelic expression consistent with loss of imprinting in the absence of Smchd1. In addition, single nucleotide polymorphism analysis of expressed genes in the placenta shows that the Igf2r imprinted gene cluster is also disrupted, with Slc22a3 showing biallelic expression in the absence of Smchd1. In both cases, the disruption was not due to loss of the differential methylation that marks the imprint control region, but affected genes remote from this primary imprint controlling element. The clustered protocadherins (Pcdhα, Pcdhβ, and Pcdhγ) also show altered expression levels, suggesting that their unique pattern of random combinatorial monoallelic expression might also be disrupted.ConclusionsSmchd1 has a role in the expression of several autosomal gene clusters that are subject to monoallelic expression, rather than being restricted to functioning uniquely in X inactivation. Our findings, combined with the recent report implicating heterozygous mutations of SMCHD1 as a causal factor in the digenically inherited muscular weakness syndrome facioscapulohumeral muscular dystrophy-2, highlight the potential importance of Smchd1 in the etiology of diverse human diseases.

The A-repeat links ASF/SF2-dependent Xist RNA processing with random choice during X inactivation

One X chromosome, selected at random, is silenced in each female mammalian cell. Xist encodes a noncoding RNA that influences the probability that the cis-linked X chromosome will be silenced. We found that the A-repeat, a highly conserved element within Xist, is required for the accumulation of spliced Xist RNA. In addition, the A-repeat is necessary for X-inactivation to occur randomly. In combination, our data suggest that normal Xist RNA processing is important in the regulation of random X-inactivation. We propose that modulation of Xist RNA processing may be part of the stochastic process that determines which X chromosome will be inactivated.

The Glycosylphosphatidylinositol-Anchored Serine Protease PRSS21 (Testisin) Imparts Murine Epididymal Sperm Cell Maturation and Fertilizing Ability

An estimated 25%–40% of infertile men have idiopathic infertility associated with deficient sperm numbers and quality. Here, we identify the membrane-anchored serine protease PRSS21, also known as testisin, to be a novel proteolytic factor that directs epididymal sperm cell maturation and sperm-fertilizing ability. PRSS21-deficient spermatozoa show decreased motility, angulated and curled tails, fragile necks, and dramatically increased susceptibility to decapitation. These defects reflect aberrant maturation during passage through the epididymis, because histological and electron microscopic structural analyses showed an increased tendency for curled and detached tails as spermatozoa transit from the corpus to the cauda epididymis. Cauda epididymal spermatozoa deficient in PRSS21 fail to mount a swelling response when exposed to hypotonic conditions, suggesting an impaired ability to respond to osmotic challenges facing maturing spermatozoa in the female reproductive tract. These data suggest that aberrant regulation of PRSS21 may underlie certain secondary male infertility syndromes, such as “easily decapitated” spermatozoa in humans.

Generation and characterization of EphA1 receptor tyrosine kinase reporter knockout mice

Eph receptor tyrosine kinases (RTKs) are a highly conserved family of signaling proteins with functions in cellular migration, adhesion, apoptosis, and proliferation during both adult and embryonic life. Here, we describe a knock‐in mouse in which EphA1 expression is disrupted via the insertion of an internal ribosome entry site (IRES)‐human placental alkaline phosphatase (ALPP) reporter cassette into exon II of the EphA1 gene. This was shown to successfully knockout expression of endogenous EphA1 and enforce expression of the ALPP reporter by the EphA1 promoter. Staining for the ALPP reporter protein demonstrated an epithelially restricted expression pattern in mouse tissues. In EphA1 null mice, two separate phenotypes were identified: abnormal tail development manifesting as a kinky tail was found in ∼80% of homozygous adults. A second, distinct abnormality present in ∼18% of females was characterized by imperforate uterovaginal development with hydrometrocolpos and caused by a resistance of cells to apoptosis during reproductive tract canalization. These results indicate a possible role for EphA1 in tissue patterning and hormone‐induced apoptotic processes. genesis 46:553–561, 2008.

Lack of augmentation of tumor spectrum or severity in dual heterozygous Men1 and Rb1 knockout mice

To identify possible genetic interactions between the mechanisms of tumor suppression of menin and pRb, we intercrossed mice with targeted deletions of Men1 and Rb1, and compared tumor development in cohorts of animals carrying single or dual mutations of these tumor-suppressor genes. In mice lacking one copy of Men1, pancreatic islet and anterior pituitary adenomas are common. In animals lacking one copy of Rb1, intermediate pituitary and thyroid tumors occur at high frequency, with less frequent development of pancreatic islet hyperplasia and parathyroid lesions. In mice heterozygous for both Men1 and Rb1, pancreatic hyperplasia and tumors of the intermediate pituitary and thyroid occurred at high frequency. Serum measurements of calcium and glucose did not vary significantly between genotypic groups. Loss of heterozygosity at the Rb1 locus was common in pituitary and thyroid tumors, whereas loss of menin was observed in pancreatic and parathyroid lesions. The tumor spectrum in the double heterozygotes was a combination of pathologies seen in each of the individual heterozygotes, without decrease in age of onset, indicating independent, non-additive effects of the two mutations. Together with the lack of increased tumor spectrum, this suggests that menin and pRb function in a common pathway of tumor suppression.

Melanocyte homeostasis in vivo tolerates Rb1 loss in a developmentally independent fashion.

There has been uncertainty regarding the precise role that the pocket protein Rb1 plays in murine melanocyte homeostasis. It has been reported that the TAT‐Cre mediated loss of exon 19 from a floxed Rb1 allele causes melanocyte apoptosis in vivo and in vitro. This is at variance with other findings showing, either directly or indirectly, that Rb1 loss in melanocytes has no noticeable effect in vivo, but in vitro leads to a semi‐transformed phenotype. In this study, we show that Rb1‐null melanocytes lacking exon 19 do not undergo apoptosis and survive both in vitro and in vivo, irrespective of the developmental stage at which Cre‐mediated ablation of the exon occurs. Further, Rb1 loss has no serious long‐term ramifications on melanocyte homeostasis in vivo, with Rb1‐null melanocytes being detected in the skin after numerous hair cycles, inferring that the melanocyte stem cell population carrying the Cre‐mediated deletion is maintained. Consequently, whilst Rb1 loss in the melanocyte is able to alter cellular behaviour in vitro, it appears inconsequential with respect to melanocyte homeostasis in the mouse skin.

Dual loss of Rb1 and Trp53 in melanocytes perturbs melanocyte homeostasis and genetic stability in vitro but does not cause melanoma or pigmentation defects in vivo.

Dear Sir, We have previously shown that the loss of Rb1 is insufficient to deregulate melanocyte homeostasis in vivo (Tonks et al., 2005), suggesting that further genetic lesions apart from the loss of this important tumour suppressor gene may be required to facilitate spontaneous melanoma. Deregulation of Trp53 appears to be an obvious candidate as the principal melanoma susceptibility gene, CDKN2A, encodes via alternative reading frames p16 and Arf (Quelle et al., 1995), which can regulate the pocket protein (PP) and Trp53 pathways respectively (Sherr and Roberts, 1999; Zhang et al., 1998). Consequently, to test whether the co-ablation of the main tumour suppressor genes of each regulatory pathway was able to yield spontaneous melanoma in vivo, we generated mice with compound ablation of Rb1 and Trp53 in Tyrosinase (Tyr) transcriptional domains using the Cre ⁄ loxP system. In this case, Trp53 ⁄ F2)10 mice (Jonkers et al., 2001) were bred with Rb1 ⁄ :TEC1 mice (Tonks et al., 2005) to yield ultimately Rb1 ⁄ :Trp53 ⁄ :TEC1 pups, where the TEC1 transgene drives Cre recombinase expression and ablation of floxed alleles in a variety of neuroepithelial and neural crest derived tissues, including the melanocytes (Tonks et al., 2003). The Rb1 ⁄ :Trp53 ⁄ :TEC1 pups were born at the expected Mendelian ratios but, in contrast to their control littermates, the Rb1 ⁄ :Trp53 ⁄ :TEC1 mice survived no longer than 6 months. In all cases, mortality was attributable to phaeochromocytomas, but not melanomas (Tonks, I.D., Mould, A., Nurcombe, V., Cool, S.M., Cotterill, A., Keith, P., Walker, G.J., Hayward, N.K. and Kay, G.F., unpublished data). Indeed, at no point did the Rb1 ⁄ :Trp53 ⁄ :TEC1 cohorts manifest any hyperor hypopigmentation defects, even though the floxed alleles were appropriately deleted (Figure S1). Despite the lack of melanoma, the loss of Trp53 alone or in combination with Rb1 does have ramifications on melanocyte homeostasis in vitro. Wild type and Rb1 ⁄ DX2 melanocytes have doubling times of 30 h and 20 h respectively (Tonks et al., 2005). In contrast, the Rb1 ⁄ :Trp53 ⁄ :TEC1 (Rb1 ⁄ :Trp53 ⁄ ) and Rb1 ⁄ :Trp53 ⁄ :TEC1 (Rb1 ⁄ :Trp53 ⁄ ) cultures were found to have an average doubling time of 23.7 h and 17.5 h respectively (Figure S2A). Furthermore, Rb1 ⁄ :Trp53 ⁄ D and Rb1 ⁄ :Trp53 ⁄ D cultures both readily proliferated in substantially reduced levels of mitogens (Figure S2B). The faster growth rates of Rb1 ⁄ :Trp53 ⁄ D and Rb1 ⁄ DX2 cultures compared with Rb1 ⁄ :Trp53 ⁄ D equivalents was unexpected, but was not attributable to increased levels of apoptosis in Rb1 ⁄ :Trp53 ⁄ D cultures. Instead Fluorescent Activated Cell Sorting (FACS) analysis indicated that the differences were underpinned by the marked tendency of Rb1 ⁄ :Trp53 ⁄ D melanocytes to become aneuploid. Indeed, while Rb1 ⁄ :Trp53 ⁄ D melanocytes possessed a relatively constant proportion of hypoploid cells over the passages examined, the Rb1 ⁄ :Trp53 ⁄ D melanocytes showed not only a comparatively and progressively larger population of hypoploid cells for a given passage number but, at higher passage, a significant hyperploid population of cells (Table 1). To assess the potential defects that lead to aneuploidy, melanocytes were probed with anti-a-microtubule and anti-c-tubulin antibodies and counterstained with 4¢,6-diamidino-2-phenylindole (DAPI) to examine their microtubule ⁄ spindle structure, centrosome

Smchd1 regulates long-range chromatin interactions on the inactive X chromosome and at Hox clusters

The regulation of higher-order chromatin structure is complex and dynamic, and a full understanding of the suite of mechanisms governing this architecture is lacking. Here, we reveal the noncanonical SMC protein Smchd1 to be a novel regulator of long-range chromatin interactions in mice, and we add Smchd1 to the canon of epigenetic proteins required for Hox-gene regulation. The effect of losing Smchd1-dependent chromatin interactions has varying outcomes that depend on chromatin context. At autosomal targets transcriptionally sensitive to Smchd1 deletion, we found increased short-range interactions and ectopic enhancer activation. In contrast, the inactive X chromosome was transcriptionally refractive to Smchd1 ablation, despite chromosome-wide increases in short-range interactions. In the inactive X, we observed spreading of trimethylated histone H3 K27 (H3K27me3) domains into regions not normally decorated by this mark. Together, these data suggest that Smchd1 is able to insulate chromatin, thereby limiting access to other chromatin-modifying proteins.In situ Hi-C and other genome-wide and imaging analyses in different mouse embryonic cell types reveal that the noncanonical SMC protein Smchd1 regulates long-range chromatin interactions and the developmental silencing of Hox genes.

Melanocyte transformation requires complete loss of all pocket protein function via a mechanism that mitigates the need for MAPK pathway activation

Deregulation of p16INK4A is a critical event in melanoma susceptibility and progression. It is generally assumed that the major effect of loss of p16 function is mediated through the CDK−cyclin pathway via its influence on the pocket protein (PP) pRb. However, there are also two other PPs, p107 and p130, which, when phosphorylated by CDK–cyclin complexes, play a role in permitting cell progression. Cohorts of mice carrying melanocyte-specific knockouts (KOs) of various combinations of the three PPs were generated. Mice null for pRb, p107, p130 or any combination of double mutants did not develop melanoma. Surprisingly, melanocyte-specific loss of all three PPs facilitated melanoma development (median age of onset 308 days, penetrance 40% at 1 year). Tumorigenesis was exacerbated by Trp53 co-deletion (median age of onset 275 days, penetrance 82% at 1 year), with cell culture studies indicating that this difference may result from the apoptotic role of Trp53. Melanomas in PP;Trp53-deficient mice lacked either Ras or Braf mutations, and hence developed in the absence of constitutive MAPK pathway activation. The lag period between induction of total PP or PP/Trp53 KO and melanoma development indicates that additional genetic or epigenetic alterations may account for neoplastic progression. However, exome sequencing of PP;Trp53 KO melanomas failed to reveal any additional recurrent driver mutations. Analysis of the putative mutation signature of the PP;Trp53 KO melanomas suggests that melanocytes are primed for transformation via a mutagenic mechanism involving an excess of T>G substitutions, but not involving a preponderance of C>T substitutions at CpG sites, which is the case for most spontaneous cancers not driven by a specific carcinogen. In sum, deregulation of all three PPs appears central to neoplastic progression for melanoma, and the customary reference to the p16INKA/CDK4/pRB pathway may no longer be accurate; all PPs are potentially critical targets of CDK-cyclins in melanoma.