J Mol Hist (2012) 43:95–106 DOI 10.1007/s10735-011-9375-x
ORIGINAL PAPER
Expression profile of NSDHL in human peripheral tissues Marie Morimoto • Christe`le du Souich • Joanne Trinh • Keith W. McLarren • Cornelius F. Boerkoel • Glenda Hendson
Received: 23 September 2011 / Accepted: 9 November 2011 / Published online: 24 November 2011 Ó Springer Science+Business Media B.V. 2011
Abstract NAD(P) steroid dehydrogenase-like (NSDHL) is an X-linked gene that encodes a 3b-hydroxysteroid dehydrogenase in the cholesterol biosynthetic pathway. Loss-of-function mutations in NSDHL cause Congenital Hemidysplasia with Ichthyosiform erythroderma and Limb Defects (CHILD) and CK syndromes. CHILD syndrome is a male lethal X-linked dominant disorder characterized by asymmetric skin and limb anomalies in affected females. CK syndrome is an intellectual disability disorder characterized by disproportionate short stature, brain malformations, and dysmorphic features in affected males. To understand better the relationship of the expression of
Electronic supplementary material The online version of this article (doi:10.1007/s10735-011-9375-x) contains supplementary material, which is available to authorized users. M. Morimoto C. du Souich J. Trinh K. W. McLarren C. F. Boerkoel Department of Medical Genetics, Child and Family Research Institute, University of British Columbia, Vancouver, BC V5Z 4H4, Canada M. Morimoto C. du Souich J. Trinh K. W. McLarren C. F. Boerkoel G. Hendson Rare Disease Foundation, Vancouver, BC, Canada C. F. Boerkoel (&) Provincial Medical Genetics Program, Department of Medical Genetics, Children’s and Women’s Health Centre of BC, 4500 Oak Street, Room C234, Vancouver, BC V6H 3N1, Canada e-mail:
[email protected] G. Hendson Department of Anatomic Pathology, University of British Columbia and Children’s and Women’s Health Centre of British Columbia, Vancouver, BC V6H 3N1, Canada
mRNA and protein encoded by human NSDHL to the peripheral malformations of these disorders, we characterized the peripheral expression of the mRNA and protein by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), immunoblotting and immunohistochemistry. We also profiled the mRNA expression of mouse Nsdhl by in situ hybridization. Expression of the mRNA and protein encoded by human NSDHL parallels that of mouse Nsdhl mRNA for most but not all tissues. Furthermore, human NSDHL protein and mouse Nsdhl mRNA were expressed in tissues synthesizing cholesterol and steroids and in all peripheral tissues affected by CHILD or CK syndromes. Keywords NSDHL Cholesterol biosynthesis CHILD syndrome CK syndrome Immunohistochemistry In situ hybridization
Introduction CHILD and CK syndromes are pleiotropic multisystem disorders (du Souich et al. 2011). CHILD syndrome is an X-linked dominant disorder affecting females and leading to male prenatal lethality. Characteristic features of CHILD syndrome include unilateral distribution of ichthyosiform nevus, limb defects ipsilateral to the skin lesions, visceral and brain malformations, and punctate calcifications of cartilaginous structures (Happle et al. 1980; Happle 2010; du Souich et al. 2011). In contrast, CK syndrome is an X-linked disorder affecting males and is characterized by mild to severe intellectual disability, central nervous system (CNS) malformations, and dysmorphic features including almond shaped eyes, upslanting palpebral fissures, high nasal bridge, high arched palate, crowded dentition, and micrognathia (du Souich et al. 2009; McLarren et al. 2010).
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CHILD and CK syndromes are caused by deficiency of NSDHL (Konig et al. 2000; Bornholdt et al. 2005; McLarren et al. 2010). NSDHL is a sterol dehydrogenase that is a member of a complex that removes C-4 methyl groups in one of the later steps of the cholesterol biosynthesis pathway (Liu et al. 1999). The protein localizes to the surface of the endoplasmic reticulum and lipid droplets (Caldas and Herman 2003). The divergent clinical phenotypes of CHILD syndrome and CK syndrome can be explained in part by the different alleles of NSDHL; patients with CHILD syndrome have null alleles resulting in complete loss of functional NSDHL (Bornholdt et al. 2005; Kim et al. 2005; Danarti et al. 2010), while patients with CK syndrome have hypomorphic temperature-sensitive alleles (McLarren et al. 2010). However, the mechanism by which NSDHL deficiency causes dysmorphism remains unclear. A first step toward achieving this understanding is defining what tissues human NSDHL is expressed in. Profiling the mRNA and protein encoded by the mouse homologue, Nsdhl, has shown that the mRNA and protein are expressed diffusely in embryonic and extraembryonic tissues (Cunningham et al. 2009; Caldas et al. 2005). It is expressed in steroidogenic tissues such as the liver and Leydig cells as well as in the retina, central and peripheral nervous system, skin, sebaceous glands, hair follicles, epidermis, and rib anlagen (Cunningham et al. 2009). Therefore, mRNA and protein encoded by Nsdhl are expressed in nearly all mouse tissues analogous to those affected in CHILD and CK syndrome patients. Interestingly, however, among the several mouse strains with mutations of Nsdhl (Liu et al. 1999; Lucas et al. 2003), all show significant phenotypic discrepancies from human female patients with CHILD syndrome. Unlike individuals with CHILD syndrome, the mutant mice do not have limb defects or organ hypoplasia. Also, the skin lesions in the mutant mice follow the lines of random X inactivation while the skin lesions in human CHILD patients do not (Liu et al. 1999; Lucas et al. 2003; Happle et al. 1980; Happle 2010; du Souich et al. 2011; Porter and Herman 2011). The question arises therefore as to the relevance of these mice for modeling human disease and whether there are differences in the expression profile between mice and humans. Understanding the relationship between NSDHL expression and disease requires extension of expression studies in mouse (Cunningham et al. 2009; Laubner et al. 2003; Liu et al. 1999; Caldas et al. 2005) to the human. Furthermore, while human NSDHL expression has been profiled previously within the CNS (McLarren et al. 2010), human NSDHL expression in peripheral tissues has not been profiled. Herein therefore we profile human NSDHL protein expression and mouse Nsdhl mRNA expression outside of the CNS.
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Materials and methods Human subjects Anonymized normal human postmortem tissues from a 6-week old female infant and a 39-week gestation male fetus and anonymized normal human postmortem skin from a 16-year old female were supplied in accordance with institutional policies as approved by the Clinical Research Ethics Board (H07-02142) at the University of British Columbia. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) cDNA was synthesized from commercially obtained total RNA (Clontech, Mountain View, CA, USA) extracted from the trachea, lung, stomach, liver, kidney, colon, adipose, skeletal muscle, heart, spleen, thymus, adrenal gland, thyroid, uterus, testis and prostate with qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD, USA). Details of the total RNA from Clontech are provided in Supplementary Table 1. Quantitative PCR was performed using the primers listed in Supplementary Table 2 and SsoFast EvaGreen Supermix (Bio-Rad, Mississauga, ON, Canada). Three technical replicates were performed for all samples using the Applied Biosystems StepOne Plus instrument with the following conditions: 1 cycle of 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 58°C for 10 s; data was collected at the annealing step. The expression of housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was measured as an endogenous control. Immunoblot Commercially obtained human protein medleys (Clontech, Mountain View, CA, USA) were fractionated on a 12% gel by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 19 phosphate buffered saline (PBS) containing 0.2% I-Block (Applied Biosystems, Foster City, CA, USA) and 0.1% Tween 20 overnight at 4°C with gentle agitation. Membranes were incubated with affinity purified rabbit anti-NSDHL serum (1:500, HPA000248, Sigma, St. Louis, MO, USA) for 1 h at room temperature with gentle agitation. Membranes were then incubated with alkaline phosphatase conjugated anti-rabbit IgG (1:10,000, A2556, Sigma, St. Louis, MO, USA) for 1 h at room temperature with gentle agitation. Signal was detected by chemiluminescence using CDP-Star (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s specifications. Membranes were then stripped, blocked and reprobed for GAPDH using mouse anti-GAPDH (1:1,000, 6C5, Advanced ImmunoChemical
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Inc., Long Beach, CA, USA) and alkaline phosphatase conjugated anti-mouse IgG (1:10,000, A3562, Sigma, St. Louis, MO, USA). Details of the human protein medleys from Clontech are provided in Supplementary Table 3.
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Rochester, NY, USA) and exposed for 3 weeks at -80°C. Developed slides were counterstained with crystal violet.
Results Immunohistochemistry Human tissues, listed in Supplementary Table 4, were fixed by immersion in 4% paraformaldehyde for 4 days at room temperature. 5 lm sections were cut from paraffin embedded tissue. After deparaffinization in xylene and rehydration through ethanol to water, antigen was retrieved in 10 mM citrate, 0.05% Tween 20, pH 6 at 95°C for 20 min. Endogenous peroxidase activity was blocked with 3% H2O2 before preincubation in blocking solution (10% casein (Vector Laboratories, Burlington, ON, Canada) in Tris buffered saline (TBS, 10 mM Tris–HCl pH 7.0, 150 mM NaCl) and 0.2% Triton X-100) for 1 h at room temperature. The tissue sections were incubated with the primary antibodies diluted with blocking solution overnight at 4°C, washed five times with TBS, and then incubated with biotinylated secondary antibodies (Vector Laboratories, Burlington, ON, Canada). After washing the tissue sections three times with TBS, immune complexes were visualized using peroxidase-conjugated streptavidin (VECTASTAIN Elite ABC standard kit, Vector Laboratories, Burlington, ON, Canada) and 3,30 -diaminobenzidine (DAB, Dako, Mississauga, ON, Canada) according to the manufacturers’ instructions. The specificity of the NSDHL antibody was verified by replacing the NSDHL antibody with the same concentration of normal rabbit serum (R4505, Sigma-Aldrich, St. Louis, MO, USA) (Supplementary Fig. 1).
Quantitative reverse transcriptase PCR and immunoblotting show broad expression of human NSDHL encoded mRNA and protein in peripheral tissues Autologous skin grafts effectively treat the skin lesions of CHILD syndrome, which is consistent with a cell autonomous mechanism of disease (Konig et al. 2010). If the nonCNS features of CHILD and CK syndromes arise cell autonomously, then affected organs must express NSDHL. By qRT-PCR of total RNA extracted from adult human tissues, the adrenal gland had the highest expression of NSDHL mRNA followed by adipose, testis, prostate, lung, liver, uterus, thyroid, stomach, kidney, trachea, colon, spleen, heart, and thymus (Fig. 1a). This expression pattern paralleled the NSDHL protein levels detected by immunoblotting (Fig. 1b) and was generally consistent with NSDHL mRNA
In situ hybridization The bases c.196-1053 of Nsdhl cDNA were subcloned into the pCR4 vector (Invitrogen, Burlington, ON, Canada). This construct was cut at unique NotI and SpeI sites flanking the Nsdhl insert and transcribed by T3 and T7 RNA polymerases to yield antisense and sense in situ hybridization probes, respectively. In situ hybridization was performed by Phylogeny, Inc. (Columbus, OH, USA). C57BL/6 mouse tissue was frozen in isopentane and 10 lm frozen sections were mounted on gelatin-coated slides. Sections were fixed in 4% paraformaldehyde in PBS. cRNA transcripts were synthesized in vitro and labeled with 35S-UTP according to the manufacturer’s instructions (Ambion/Applied Biosystems, Austin, TX, USA). Sections were hybridized overnight at 55°C in 50% deionized formamide, 0.3 M NaCl, 20 mM Tris–HCl pH 7.4, 5 mM EDTA, 10 mM NaH2PO4, 10% dextran sulphate, 19 Denhardt’s, 50 lg/mL total yeast RNA, and 50–80,000 cpm/lL 35S-labeled cRNA probe. Stringent washes were carried out at 65°C. Slides were exposed immersed in photographic emulsion (Kodak,
Fig. 1 Expression profiles of NSDHL mRNA and NSDHL protein in human adult tissues. a Graph showing the relative NSDHL mRNA expression as measured by qRT-PCR. GAPDH mRNA expression was used as an endogenous control; NSDHL mRNA expression in various tissues was normalized to that of the liver. Error bars represent 1 SD. b Photograph of an immunoblot showing NSDHL protein expression in multiple tissues. Membranes probed for NSDHL were subsequently stripped, blocked and probed for GAPDH, which is shown as a loading control
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J Mol Hist (2012) 43:95–106 b Fig. 2 Photomicrographs of immunohistochemical analysis of human
NSDHL expression in the endochondral cartilage and heart. a–d Within the endochondral cartilage, the maturing chondrocytes of the zone of hypertrophy (ZH) showed strong punctate staining for NSDHL, while the chondrocytes of the zone of proliferation (ZP) and zone of calcification (ZC) were negative for NSDHL staining. e–g No detectable NSDHL staining was observed in the endocardium (En) and myocardium (My) of the cardiac muscle. The boxed regions correspond to the higher magnification images. Scale bars: a, e–g = 100 lm; b–d = 20 lm
expression shown in the NCBI Expressed Sequence Tag (EST) database (http://www.ncbi.nlm.nih.gov/unigene). Immunohistochemistry shows expression of human NSDHL protein in peripheral tissues affected by CHILD and CK syndromes To clarify whether NSDHL is expressed in the tissues affected by NSDHL deficiency, we profiled human NSDHL protein expression by immunohistochemistry in postmortem tissues from a 6-week old female infant and a 39-week gestation male fetus, and postmortem skin from a 16-year old female (Figs. 2, 3, 4, 5, 6, 7, 8 and Supplementary Table 4). Similar to its expression in neural tissue (McLarren et al. 2010), NSDHL, when expressed, had a punctate cytoplasmic subcellular distribution within all cell types analyzed. Bone Individuals with CHILD syndrome have punctate calcifications of cartilaginous structures, bony deficiencies of the affected limbs, scoliosis, and kyphosis (du Souich et al. 2011). Individuals with CK syndrome have micrognathia, a high arched palate, scoliosis, kyphosis, short stature and relatively long digits and limbs (du Souich et al. 2009; McLarren et al. 2010). Proximal tibial growth plate chondrocytes in the zone of hypertrophy strongly expressed NSDHL. Neither chondrocytes in the zones of proliferation or calcification nor cells in the cortical or trabecular bone expressed detectable NSDHL (Fig. 2a–d). Heart Individuals with CHILD and CK syndromes occasionally have cardiac developmental defects (du Souich et al. 2011). NSDHL was undetectable in the heart tissue (Fig. 2e–g). Immunohistochemistry shows expression of human NSDHL protein in peripheral tissues affected by CHILD syndrome but not by CK syndrome Integument Individuals with CHILD syndrome have icthyosiform skin lesions and patchy alopecia (Hummel et al. 2003;
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Avgerinou et al. 2010; Chander et al. 2010). In the epidermis, NSDHL is highly expressed in the stratum granulosum and moderately in the stratum spinosum and stratum germinativum (Fig. 3a, b). Within the dermis, NSDHL was highly expressed in the stratified cuboidal and simple cuboidal epithelium, the myoepithelial cells of the sweat gland duct and in the hair root, papilla, and internal and external root sheath (Fig. 3c–g). Adrenal glands A few individuals with CHILD syndrome have hypoplasia or aplasia of the adrenal gland ipsilateral to their limb defects and skin lesions (Happle et al. 1980; Konig et al. 2002). The fetal and mature adrenal cortex expressed moderate to high levels of NSDHL (Fig. 3h–j), whereas the medulla did not have detectable levels of NSDHL (data not shown). Kidney Most individuals with CHILD syndrome have no renal abnormalities, but among those that do, the renal findings range from unilateral hydronephrosis to renal agenesis (du Souich et al. 2011). Within the kidney, only the cuboidal epithelial cells of the proximal tubules expressed NSDHL (Fig. 4a, b). Lung Several CHILD syndrome patients have had lung hypoplasia without interstitial lung or alveolar disease (Bornholdt et al. 2005; Hummel et al. 2003; Konig et al. 2002; Tang and McCreadie 1974). Within the airways, bronchial ciliated mucosa and tracheal mucosal goblet cells and glands expressed NSDHL, whereas hyaline cartilage and muscle did not (Fig. 4c–g and Supplementary Fig. 2). In the alveoli, NSDHL was expressed in cells with the morphology of alveolar type II pneumocytes (Fig. 4h, i). Female gonads and accessory sex organs Occasionally CHILD syndrome patients have unilateral absence or hypoplasia of the ovary and fallopian tubes (Happle et al. 1980; Konig et al. 2002). Within the ovary, NSDHL was detectable in all cells but those of the stroma and tunica albuginea (Fig. 5a–c). Within the oviduct and fallopian tubes, the columnar epithelium expressed moderate levels of NSDHL, whereas the lamina propria and muscularis did not express detectable NSDHL (Fig. 5d, e).
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Fig. 3 Photomicrographs of immunohistochemical analysis of human NSDHL expression in the skin and adrenal gland. a, b Within the epidermis of the skin, the stratum corneum (SC) did not express detectable NSDHL. The stratum germinativum (Sg) and stratum spinosum (SS) stained moderately, and the stratum granulosum (SG) stained strongly for NSDHL. The interpapillary pegs (IP) and the capillary loops (asterisk) of the secondary dermal ridges stained moderately for NSDHL. c–g Within the dermis, the sweat glands and hair follicles stained intensely for NSDHL. The myoepithelial cells (My)
and secretory cells (S) had weak and moderate to strong expression of NSDHL, respectively. The papilla (P), internal root sheath (Int), and external root sheath (Ex) of the hair follicle had moderate to strong expression of NSDHL. h–j In the adrenal gland, the zona reticularis (ZR), zona fasciculata (ZF), and zona glomerulosa (ZG) are the three layers of the cortex (Co); the regressing fetal cortex (fCo) is also present. Strong punctate staining was observed throughout the cortex of the adrenal gland. The boxed regions correspond to the higher magnification images. Scale bars a, c, h–j = 100 lm; b, d, f, g = 20 lm, e = 50 lm
Immunohistochemistry shows expression of human NSDHL in peripheral tissues unaffected by CHILD and CK syndromes
damage is repaired during development, or that the pathology is subtle and has not been noted. Male gonads and accessory sex organs
Besides the tissues affected by CHILD and CK syndromes, several additional peripheral tissues expressed NSDHL. This suggests that NSDHL is not required locally for development or maintenance of those tissues, that the
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Within the testis, NSDHL was highly expressed in Leydig cells but undetectable in Sertoli and germ cells (Fig. 5f, g). Within the male accessory sex organs, it was highly
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Fig. 4 Photomicrographs of immunohistochemical analysis of human NSDHL expression in the kidney and lung. a, b In the kidney, the proximal tubules (PT) showed weak staining for NSDHL, whereas the glomeruli (G) and distal tubules (DT) showed no staining. c–e Within the trachea, the respiratory epithelium (Ep) and the mucous and seromucous glands (Gl) stained weakly for NSDHL. No staining was observed in the perichondrium (P) or C-rings (CR). f, g Within the
intrapulmonary bronchi (IB), the respiratory epithelium had weak staining for NSDHL, and the seromucous glands (Gl) did not stain. h, i Within the terminal bronchioles (B), the simple cuboidal epithelium stained weakly for NSDHL. Within the alveoli (a), type II pneumocytes (P2), but not type I pneumocytes (P1), expressed NSDHL. The boxed regions correspond to the higher magnification images. Scale bars a, f, h = 100 lm; b, d, e, g, i = 20 lm, c = 50 lm
expressed in the epithelium of the efferent ducts and epididymis as well as in some smooth muscle cells surrounding the ducts (Fig. 5h–k). Within the prostate, no cells expressed detectable levels of NSDHL (Fig. 5l, m).
Pancreas
Gastrointestinal tract NSDHL was expressed in the epithelium, mucosa and neuronal ganglia throughout the gastrointestinal tract as well as in enteroendocrine cells of the colonic mucosa (Fig. 6a–d, f– i). NSDHL expression was not detected in the intestinal muscle, lymphocytes or connective tissue (Fig. 6a, d, e, g).
The acinar and centroacinar cells of the exocrine pancreas expressed low levels of NSDHL (Fig. 7c, d). However, NSDHL was not detected within the endocrine pancreas (Fig. 7e, f). Adipose In all tissues examined, NSDHL was highly expressed in adjoining adipocytes (Fig. 7g, h). Bladder and urethra
Liver Hepatocytes expressed moderate levels of NSDHL, but Kupffer and bile duct cells did not express detectable levels of NSDHL (Fig. 7a, b).
The epithelium of both the bladder and the urethra expressed low to moderate levels of NSDHL (Fig. 7i–l), while the muscularis of the bladder and the urethra did not express detectable levels (Fig. 7i, k).
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Fig. 5 Photomicrographs of immunohistochemical analysis of human NSDHL expression in the female and male reproductive systems. a–c In the ovary, the stroma (St) showed no detectable staining, the primary oocytes (PO) and granulosa cells (GC) of the ovarian follicles showed weak staining, and the theca cells (Th) showed moderate to strong staining for NSDHL. d, e Within the fallopian tubes, the epithelium (Ep) stained moderately for NSDHL, whereas the lamina propria (LP) did not stain for NSDHL. f, g In the testis, the Leydig cells (LC) stained intensely for NSDHL, whereas the cells of the seminiferous tubules (ST) did not stain for NSDHL. h,
i In the ductuli efferentes (De), the columnar cells stained for NSDHL, whereas the connective tissue (CT) surrounding the tubules did not express detectable NSDHL. j, k The basal cells and principle cells of the ductus epididymis (DE) as well as the smooth muscle (SM) surrounding the tubules stained moderately for NSDHL; the connective tissue (CT) surrounding the tubules did not stain for NSDHL. l, m NSDHL expression was not detected in the prostate epithelium (Ep) or stroma (St). The boxed regions correspond to the higher magnification images. Scale bars a, d, f, h, j, l = 100 lm; b, c, e, g, i, k, m = 20 lm
Hematopoietic tissues
Figs. 3–8). As observed for expression of human NSDHL encoded mRNA, mouse Nsdhl mRNA was expressed in all tissues affected in CHILD and CK syndromes as well as in tissues unaffected by these disorders (Supplementary Fig. 3) (du Souich et al. 2009, 2011; McLarren et al. 2010; Cunningham et al. 2009; Liu et al. 1999). The mouse Nsdhl in situ hybridization showed five patterns of temporal expression (Supplementary Fig. 3). The first pattern was that of expression beginning at or before E9.5 and then increasing throughout development and continuing postnatally; this occurred in the brain, spinal cord, and skin. The second pattern was that of expression throughout development and loss of expression in adulthood; the heart exemplified this. The third pattern of expression was that of expression beginning later in
Within the bone marrow, NSDHL was highly expressed in dendritic cells (Fig. 8a, b) and was undetectable in other bone marrow cells (Fig. 8a, b). It was also undetectable in the Peyer’s patches, lymph nodes, thymus and spleen (Figs. 6d, e, 8c–i). Mouse in situ hybridization shows varying expression of Nsdhl mRNA in tissues throughout development To determine the expression profile during mammalian development, we profiled mouse Nsdhl mRNA expression by in situ hybridization at prenatal stages E9.5, E10.5, E12.5, E15.5 and postnatal stages P1, P10, and P60 (Supplementary
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Fig. 6 Photomicrographs of immunohistochemical analysis of human NSDHL expression in the digestive system. a–i Throughout the digestive tract, there was no staining for NSDHL in the lamina propria (LP) and muscularis mucosa (MM) of the mucosa, submucosa (S), and inner circular (IC) and outer longitudinal (OL) muscle layers of the muscularis externa (Me). a–c Moderate NSDHL staining was observed in the epithelium (Ep) of the esophagus and cells adjacent to the basement membrane had the strongest staining. Moderate expression of NSDHL was also observed in the neurons of Auerbach’s myenteric
plexus (Au) of the esophagus. d–f Within the epithelium of the small intestine, weak NSDHL staining was observed in goblet cells, columnar cells, enterocytes, and enteroendocrine cells. Peyer’s patches (PP) did not express detectable NSDHL. g–i Within the epithelium of the large intestine, weak staining for NSDHL was observed in goblet cells and columnar cells, while neurons of Auerbach’s myenteric plexus (Au) stained moderately and the enteroendocrine (EE) cells stained intensely for NSDHL. The boxed regions correspond to the higher magnification images. Scale bars a, d, g = 100 lm; b, c, e, f, h, i = 20 lm
development (E10.5 or later) and continuing into adulthood; this occurred in most tissues including the peripheral ganglia, liver, digestive tract, bladder mucosa, genital tissues, spleen, thymus, brown adipose tissue, and hair follicles. The fourth pattern of expression was that of transient expression during development; this occurred in both the vertebrae and lung. The final pattern of expression was that of no detectable expression during any stage in development; the pancreas exemplified this pattern of expression.
liver and bladder (Supplementary Figs. 3–8). Within bone, mouse Nsdhl mRNA was detected in the mandible and vertebrae (Supplementary Figs. 3, 4b, 5a and 7b); long bones were not studied in the mice and the vertebrae and mandible were not studied in the humans. These expression profiles are generally consistent with prior studies in the mouse (Cunningham et al. 2009; Laubner et al. 2003). Tissues in which mouse Nsdhl mRNA expression was discordant with human NSDHL protein expression included the heart, pancreas, thymus and spleen. In contrast to the complete absence of human NSDHL protein expression within the heart, mouse Nsdhl mRNA was detectable from E9.5 through P10 (Supplementary Figs. 3, 4a, e, 5a, c, 6a, and 7a, b) and undetectable by P60 (Supplementary Fig. 8a). Within the exocrine pancreas, mouse Nsdhl mRNA was not detected at P10 (Supplementary Figs. 3 and 7a) or P60 (Supplementary Figs. 3 and 8a) although human NSDHL protein was weakly expressed in
Mouse Nsdhl mRNA expression parallels human NSDHL protein expression in most but not all tissues Tissues in which mouse Nsdhl mRNA expression was concordant with human NSDHL protein expression included the integument, adrenal cortex, renal cortex, lung, tracheal mucosa, testis, epididymis, esophagus, stomach, intestine,
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Fig. 7 Photomicrographs of immunohistochemical analysis of human NSDHL expression in the liver, pancreas, adipose, bladder, and urethra. a, b Hepatocytes of the liver showed moderate staining for NSDHL. c, d Pancreatic acinar (AC) and centroacinar cells stained weakly for NSDHL. e, f The islets of Langerhans (IL) and connective tissue septa (CT) of the endocrine pancreas did not stain for NSDHL. g,
h Subcutaneous adipose cells had strong punctate staining for NSDHL. i, j In the bladder, the epithelium had weak staining for NSDHL. k, l In the urethra, the epithelium had weak staining for NSDHL. The boxed regions correspond to the higher magnification images. Scale bars a, c, e, g, i, k = 100 lm; b, d, f, h, j, l = 20 lm
this tissue. Unlike NSDHL protein expression observed for the human, mouse Nsdhl mRNA was expressed in the thymus beginning at P1 and in the spleen beginning at P10 (Supplementary Figs. 3, 7a, b, and 8a).
was expressed in all non-CNS tissues with malformations in CHILD and CK syndromes and also in multiple other peripheral tissues. Consistent with the known function of NSDHL, each of the tissues expressing NSDHL has been shown in humans or model organisms to synthesize cholesterol (Bloch et al. 1946; Srere et al. 1950; Dietschy and Siperstein 1965; Dirksen 1969). Similar to prior observations of Nsdhl protein expression in the mouse (Cunningham et al. 2009; Laubner et al. 2003;
Discussion We profile for the first time NSDHL protein expression in peripheral human tissue (Supplementary Table 4). NSDHL
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Fig. 8 Photomicrographs of immunohistochemical detection of NSDHL expression in the hematopoietic system. a, b Within the bone marrow, only dendritic cells (DC) stained positive for NSDHL. c, d Lymphocytes in the lymph node did not express detectable NSDHL. e, f In the thymus, there was no staining for NSDHL in the medulla (M), cortex (C) or Hassall’s corpuscles (H). g–i In the spleen,
no expression was observed in the capsule (Ca), septum (Se), white pulp (WP), or red pulp (RP) for NSDHL. Yellow cells without nuclei are circulating red blood cells within the spleen. The boxed regions correspond to the higher magnification images. Scale bars a, c, g = 100 lm; b, d, f, h, i = 20 lm; e = 200 lm
Liu et al. 1999; Caldas et al. 2005), peripheral human NSDHL protein expression is spatially restricted to specific cells. This raises the possibility that NSDHL and other human cholesterogenesis genes form a developmental synexpression group and regulate human development as the homologues do in mice (Laubner et al. 2003; Gawantka et al. 1998; Sakakura et al. 2001). As proposed by Laubner et al., this would provide a conceptual basis for the conservation of some malformations among disorders of cholesterogenesis, although full evaluation of this hypothesis in humans will require analysis of cholesterogenic gene expression in human tissue from earlier developmental stages. Overall NSDHL expression in the human infant and the late gestation fetus paralleled that in the mouse. Exceptions included expression in the mouse but not in the human heart, spleen, or thymus and expression in the human but not in the mouse pancreas. These differences could arise because of species differences, the lack of tissues from earlier stages of human development, differences between mRNA and protein expression, or differences in sensitivity
between immunohistochemistry and in situ hybridization. Partially consistent with the last possibility, comparison of the human qRT-PCR and immunoblotting in Fig. 1 suggests that either the NSDHL antibody we used is not as sensitive at detecting NSDHL protein as qRT-PCR is at detecting NSDHL mRNA or that there is post-transcriptional regulation of NSDHL translation. Alternatively or in addition, because the distribution of skin lesions is different between Nsdhl deficient mice and CHILD syndrome patients and because Nsdhl deficient mice do not have skeletal lesions but CHILD syndrome patients do (Liu et al. 1999; Lucas et al. 2003), some of these expression differences could be attributable to species differences. In summary, the expression pattern of human NSDHL protein is consistent with both cell autonomous and nonautonomous mechanisms underlying the malformations associated with NSDHL deficiency. Both mechanisms can arise from impairment of normal biological processes by cholesterol deficiency, direct toxicity of accumulated cholesterol pathway intermediates, and toxicity of
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intermediate-derived metabolites (Porter and Herman 2011). Therefore, further studies are needed to determine the relative contribution of each mechanism to disease. Acknowledgments The authors thank Drs. Gail Herman and David Cunningham for critical review of this manuscript. This work was supported in part by a British Columbia Children’s Hospital Foundation Telethon Award (C.D.S.), a Scottish Rite Foundation Award (C.D.S.) and a Child & Family Research Institute Establishment Award (C.F.B.). C.F.B. is a scholar of the Michael Smith Foundation for Health Research.
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