Regarding ATRX in the Skeleton
5.2.1
ATRX in Developing Cartilage
In Chapter two, I tested the hypothesis that mice lacking ATRX in developing cartilage will demonstrate dwarfism and other skeletal defects similar to those observed in humans with ATR-X syndrome. I quantified the skeletal phenotypes of mice that lack Atrx
expression in cartilage beginning with the onset of cartilage differentiation from mesenchyme. These mice showed that loss of ATRX in chondrocytes has minimal effects on skeletal development, and that mice lacking ATRX in cartilage were viable and were fertile in adulthood. ATRX loss was confirmed at the level of both mRNA and protein, and shown to have no effect on the expression of important cartilage growth plate genes such as Sox9 and p57. Conditionally ATRX-deficient mice had normal growth plate
morphology, with no defects in the ratios of resting, proliferating, or hypertrophic cells at birth or at weaning. These animals demonstrated only a mild delay in the ossification of
the pelvic girdle, and have none of the associated skeletal phenotypes of ATR-X syndrome. I concluded that skeletal phenotypes of ATR-X syndrome are likely due to secondary effects from tissues like the brain or anterior pituitary.
5.2.2
ATRX in Developing Forelimb
In Chapter three, I investigated the development of the appendicular skeleton from ATRX-deficient mesenchyme. I reported that mice conditionally lacking ATRX in the forelimb mesenchyme demonstrate specific brachydactyly, characterised by a shortening of the cartilage anlage during embryonic development and subsequent shortening of the ossified digit at, and following, birth. While the shortening of distal phalanges persists into adulthood, there are limited disease phenotypes in this model. These animals demonstrate reduced forelimb function, quantified as a significant decrease in forepaw grip strength. Adult animals have a mild alteration in gait which manifests as shorter strides, but do not show any major interlimb coordination defects. The brachydactyly phenotype correlated with increased levels of the DNA-damage marker γ-H2AX, which
appeared as one bright focus within the nucleus of ATRX-deficient embryonic forelimb mesenchyme and chondrocytes. This γ-H2AX focus was often located at the periphery of
the nucleus, and using 3D confocal microscopy, I determined that this focus of damaged DNA was frequently located within one micron of the nuclear lamina. I attribute the digit shortening to an increase in TUNEL-positive apoptotic cell death in proliferating distal tip of the digit. I propose that some of the hand deformities observed in ATR-X syndrome may be due to similar cell death in the developing digit, and predict the cell death is related to the DNA damage associated with loss of ATRX.
5.2.3
ATRX in Developing Bone
In Chapter four, I describe a model of ATRX-deficiency in mineralising osteoblasts, which leads to mild dwarfism but does not cause defects in bone mineral formation during development or in vitro. Although these mice have shortened limbs, this phenotype does not fully recapitulate the variety of skeletal deformities seen in human cases of ATRX depletion. These animals do not show premature aging or the defects in
bone mineral density and trabecular number seen in mice lacking ATRX in the forebrain and anterior pituitary.
5.2.4
Secondary Effects of ATRX on the Skeleton
There are many reasons to expect a skeletal phenotype in a model of ATRX-deficiency. Firstly, mutations to ATRX are associated with a wide variety of severe pathologic
skeletal phenotypes in humans. These include short stature, spinal deformities, clubbed foot, tapered or conical fingers, and delayed bone age (Gibbons et al., 2000). Secondly, genes with similar epigenetic properties to ATRX such as MeCP2, HDAC4 and NIPBL are
also associated with skeletal deformities in humans and defective skeletal development in related mouse models (Alvarez-Saavedra et al., 2010; Kawauchi et al., 2009; Shapiro et al., 2010; Tonkin et al., 2004). Some of these gene products can interact directly with ATRX and affect gene expression (Baker et al., 2013; Kernohan et al., 2010; Nan et al., 2007). Thirdly, there are important roles for proteins with similar functions to ATRX in skeletogenesis, including SWI/SNF chromatin remodeling, histone modifications, and genomic integrity (Williams et al., 2010; Young et al., 2005).
It was unexpected that mouse models lacking ATRX in cartilage, bone, or forelimb mesenchyme displayed relatively minor phenotypes. I propose that the skeletal phenotypes and dwarfism seen in ATR-X syndrome are more likely due to secondary effects. These effects were later demonstrated succinctly in a forebrain and pituitary- specific Atrx-deletion mouse displaying dwarfism, reduced bone mass, and premature
aging (Watson et al., 2013). In this model of ATR-X syndrome, Atrx is deleted in the
embryonic forebrain starting at E8.5. These mice are born small, and before three weeks of age, display defective and degenerate skeletal phenotypes (Bérubé et al., 2005; Watson et al., 2013). In addition to significant dwarfism, these animals have shortened growth plates, reduced bone density, and reduced trabecular number (Watson et al., 2013). It is crucial to note that these mice lacking ATRX in the forebrain and anterior pituitary have severe endocrine defects, including defects in the TH/IGF1 axis. In this model of ATRX- deficiency, the skeletal defects are attributed to a secondary, systemic defect where skeletal development is affected indirectly.
There are other indirect effects that can influence both skeletal development and growth. Normal regulation of skeletal growth and mineralisation requires sex steroids, including androgens and androgen receptors, as well as estrogens and estrogen receptors [Reviewed in (Callewaert et al., 2010)]. Defects in estrogen sensing in the male mouse leads to decreased radial and longitudinal bone growth, associated with decreased serum levels of IGF-I (Vidal et al., 2000). Skeletal defects are seen in androgen-resistant testicular feminized males, which have low trabecular bone mineral density (Vandenput et al., 2004). These phenotypes are usually seen post-natally, in late pubertal, or adult stages (Vidal et al., 2000). This is similar to the forebrain and pituitary conditional Atrx deletion
mouse, which has normal growth plate morphology and trabecular number early in life, but later develops decreased bone density and other skeletal phenotypes (Watson et al., 2013). In cases of ATR-X syndrome, individuals frequently present with hypogonadisms and in some cases even sex reversal (Gibbons et al., 1995; Ion et al., 1996). These phenotypes are often correlated with delayed or arrested puberty, and manifestation of growth retardation at the time of the pubertal growth spurt (Gibbons, 2006). In these cases, levels of sex steroids and their receptors would be affected, and lead to skeletal defects without a direct effect on gene expression within the cartilage and bone. Thus, it can be concluded that the most ideal model for studying ATRX in skeletal development would be a mouse lacking ATRX in the brain and other indirect systems, rather than removing ATRX directly in the skeleton.
Compared with mouse models of steroid and endocrine defects, mice lacking ATRX in the cartilage, bone, or forelimb demonstrate slight and limited phenotypes. It can be predicted that a model combining both direct and indirect skeletal roles of ATRX may modulate the subtle skeletal phenotypes, such as brachydactyly, towards more severe defects. My data suggest that some of the skeletal defects are treatable via hormonal methods. In cases where circulating levels of hormones are affected, patient skeletal size could be influenced by treatment with growth hormone or drugs to increase endogenous production of growth hormones.
When considering developmental phenotypes caused by other chromatin-modifying proteins, such as MECP2 and HDAC4, it is important to consider that ATRX does not
always affect expression of genes directly. Complexes containing ATRX modulate genome structure and integrity, and have both tissue-specific and chromosome-specific functions. ATRX can affect expression of wide-ranging genetic factors, like imprinting loci and silencing whole chromosomes (Baumann et al., 2009; Kernohan et al., 2010). Additionally, the role of ATRX can be influenced by other factors including variable number tandem repeats (VNTRs), which can vary between individuals (Law et al., 2010). There are very few genotype/phenotype correlations in ATRX syndrome, and even patients with the same mutation may demonstrate different pathological phenotypes [Reviewed in (Gibbons, 2006)]; therefore, it is important to consider the inherently variable nature of ATRX on gene expression when looking at functional phenotypes.