Introduction
All-
trans-retinoic acid (RA)
4The abbreviations used are: RA
all-trans-retinoic acid
SDR
short-chain dehydrogenase/reductase
RDH
retinol dehydrogenase
RALDH
retinaldehyde dehydrogenase
E
embryonic day
P
postnatal day
RAR
retinoic acid receptor
qPCR
quantitative PCR
DKO
double-knockout
VAD
vitamin A–deficient
ESC
embryonic stem cell
CMV
cytomegalovirus.
is the major bioactive form of vitamin A that influences a broad spectrum of physiological processes including embryogenesis and epithelial homeostasis (
1- Al Tanoury Z.
- Piskunov A.
- Rochette-Egly C.
Vitamin A and retinoid signaling: genomic and nongenomic effects.
2- Shannon S.R.
- Moise A.R.
- Trainor P.A.
New insights and changing paradigms in the regulation of vitamin A metabolism in development.
,
3- Metzler M.A.
- Sandell L.L.
Enzymatic metabolism of vitamin A in developing vertebrate embryos.
4Endogenous retinoids in the hair follicle and sebaceous gland.
). In the nucleus, RA regulates gene expression primarily through binding to nuclear transcription factors, retinoic acid receptors (RARs α, β, and γ), which act as heterodimers with retinoid X receptors (
5Gene expression regulation by retinoic acid.
,
6- Benbrook D.M.
- Chambon P.
- Rochette-Egly C.
- Asson-Batres M.A.
History of retinoic acid receptors.
). In the cytoplasm, RA regulates the activity of extracellular signal-regulated kinase (
7Cellular retinoic acid binding proteins: genomic and non-genomic functions and their regulation.
) and exhibits numerous other extranuclear activities (
8- Iskakova M.
- Karbyshev M.
- Piskunov A.
- Rochette-Egly C.
Nuclear and extranuclear effects of vitamin A.
). RA is synthesized from the alcohol form of vitamin A (all-
trans-retinol) via a two-step process. In the first step, retinol dehydrogenases oxidize all-
trans-retinol to all-
trans-retinaldehyde, which is oxidized further by retinaldehyde dehydrogenases (RALDHs) to RA (reviewed in Ref.
9Enzymology of retinoic acid biosynthesis and degradation.
).
The oxidation of retinol to retinaldehyde is the rate-limiting step in RA biosynthesis (
10Retinol metabolism in UC-PKI cells: characterization of retinoic acid synthesis by an established mammalian cell line.
). Several members of the short-chain dehydrogenase/reductase (SDR) superfamily of proteins catalyze this reaction
in vitro (
9Enzymology of retinoic acid biosynthesis and degradation.
), but only one of the retinoid-active SDRs characterized to date, murine retinol dehydrogenase 10 (RDH10), is known to be indispensable for RA biosynthesis, because embryos lacking functional RDH10 do not survive past E12.5 (
11- Rhinn M.
- Schuhbaur B.
- Niederreither K.
- Dollé P.
Involvement of retinol dehydrogenase 10 in embryonic patterning and rescue of its loss of function by maternal retinaldehyde treatment.
). RDH10-null embryos display numerous abnormalities including forelimb, craniofacial, neural, and heart defects (
12- Sandell L.L.
- Sanderson B.W.
- Moiseyev G.
- Johnson T.
- Mushegian A.
- Young K.
- Rey J.-P.
- Ma J.-X.
- Staehling-Hampton K.
- Trainor P.A.
RDH10 is essential for synthesis of embryonic retinoic acid and is required for limb, craniofacial, and organ development.
,
13- Ashique A.M.
- May S.R.
- Kane M.A.
- Folias A.E.
- Phamluong K.
- Choe Y.
- Napoli J.L.
- Peterson A.S.
Morphological defects in a novel Rdh10 mutant that has reduced retinoic acid biosynthesis and signaling.
). The severity of the phenotype indicates that RDH10 functions as the major murine retinol dehydrogenase during mid-embryogenesis. However, whereas the ablation of RDH10 eliminates most of the retinol dehydrogenase activity during the early stages of development, RA synthesis persists in the neural tube of RDH10-null embryos at E9.5 and E10.5 (
14- Cunningham T.J.
- Chatzi C.
- Sandell L.L.
- Trainor P.A.
- Duester G.
Rdh10 mutants deficient in limb field retinoic acid signaling exhibit normal limb patterning but display interdigital webbing.
,
15- Sandell L.L.
- Lynn M.L.
- Inman K.E.
- McDowell W.
- Trainor P.A.
RDH10 oxidation of vitamin A is a critical control step in synthesis of retinoic acid during mouse embryogenesis.
). Importantly,
Rdh10−/− embryos can be rescued by supplementation of maternal diets with retinaldehyde between embryonic stages E7.5 and E9.5. Thus, RDH10 appears to be dispensable during later stages of development and transition to adulthood (
11- Rhinn M.
- Schuhbaur B.
- Niederreither K.
- Dollé P.
Involvement of retinol dehydrogenase 10 in embryonic patterning and rescue of its loss of function by maternal retinaldehyde treatment.
). These data point toward the existence of RDH10-independent sources of RA. However, the identities of additional retinol dehydrogenases accounting for this residual retinaldehyde synthesis remain elusive.
RDH10 belongs to the 16C family of the SDR superfamily of proteins (
16- Wu B.X.
- Chen Y.
- Chen Y.
- Fan J.
- Rohrer B.
- Crouch R.K.
- Ma J.-X.
Cloning and characterization of a novel all-trans-retinol short-chain dehydrogenase/reductase from the RPE.
,
17- Persson B.
- Bray J.E.
- Bruford E.
- Dellaporta S.L.
- Favia A.D.
- Gonzalez Duarte R.G.
- Jörnvall H.
- Kallberg Y.
- Kavanagh K.L.
- Kedishvili N.
- Kisiela M.
- Maser E.
- Mindnich R.
- Orchard S.
- Penning T.M.
- et al.
The SDR (Short-Chain Dehydrogenase/Reductase and Related Enzymes) Nomenclature Initiative.
). Notably, two other genes encoding members of the SDR16C family are located adjacent to the gene encoding RDH10 in the human genome on chromosome 8: retinol dehydrogenase epidermal 2 (
RDHE2, SDR16C5) and retinol dehydrogenase epidermal 2-similar (
RDHE2S, SDR16C6) (
18- Belyaeva O.V.
- Lee S.-A.
- Adams M.K.
- Chang C.
- Kedishvili N.Y.
Short chain dehydrogenase/reductase Rdhe2 is a novel retinol dehydrogenase essential for frog embryonic development.
,
19- Belyaeva O.V.
- Chang C.
- Berlett M.C.
- Kedishvili N.Y.
Evolutionary origins of retinoid active short-chain dehydrogenases/reductases of SDR16C family.
). The deduced RDHE2 and RDHE2S proteins share the highest sequence homology (∼43%) with RDH10 (SDR16C4). As we reported previously, the single ortholog of human genes encoding RDHE2 and RDHE2S in
Xenopus laevis functions as a retinol dehydrogenase
in vivo and is essential for embryonic development in frogs (
20- Lee S.-A.
- Belyaeva O.V.
- Kedishvili N.Y.
Biochemical characterization of human epidermal retinol dehydrogenase 2.
). These findings imply that mammalian RDHE2 and RDHE2S complement RDH10 in generating retinaldehyde for RA biosynthesis. However, it remains to be established whether the
in vivo function of amphibian rdhe2 is conserved by its mammalian orthologs. This study was undertaken to assess the catalytic properties of mammalian RDHE2 and RDHE2S as compared with amphibian rdhe2, to determine the expression patterns of RDHE2 and RDHE2S in mice, and to establish whether these enzymes are essential for RA biosynthesis in mammals.
Discussion
Several members of the SDR superfamily of proteins can oxidize retinol to retinaldehyde
in vitro; however, with the exception of RDH10, their physiological relevance for RA biosynthesis remains unclear. This study provides convincing evidence that mammalian RDHE2 and RDHE2S function as physiologically relevant retinol dehydrogenases. The results of this study are important because they highlight the existence of tissue-specific retinol dehydrogenases that are essential for RA biosynthesis in specific cell types while not being critical for survival, as in the case with RDH10 during midgestation (
11- Rhinn M.
- Schuhbaur B.
- Niederreither K.
- Dollé P.
Involvement of retinol dehydrogenase 10 in embryonic patterning and rescue of its loss of function by maternal retinaldehyde treatment.
12- Sandell L.L.
- Sanderson B.W.
- Moiseyev G.
- Johnson T.
- Mushegian A.
- Young K.
- Rey J.-P.
- Ma J.-X.
- Staehling-Hampton K.
- Trainor P.A.
RDH10 is essential for synthesis of embryonic retinoic acid and is required for limb, craniofacial, and organ development.
,
13- Ashique A.M.
- May S.R.
- Kane M.A.
- Folias A.E.
- Phamluong K.
- Choe Y.
- Napoli J.L.
- Peterson A.S.
Morphological defects in a novel Rdh10 mutant that has reduced retinoic acid biosynthesis and signaling.
,
14- Cunningham T.J.
- Chatzi C.
- Sandell L.L.
- Trainor P.A.
- Duester G.
Rdh10 mutants deficient in limb field retinoic acid signaling exhibit normal limb patterning but display interdigital webbing.
15- Sandell L.L.
- Lynn M.L.
- Inman K.E.
- McDowell W.
- Trainor P.A.
RDH10 oxidation of vitamin A is a critical control step in synthesis of retinoic acid during mouse embryogenesis.
). In fact, because RDH10-null embryos can be rescued by supplementation with retinaldehyde (
11- Rhinn M.
- Schuhbaur B.
- Niederreither K.
- Dollé P.
Involvement of retinol dehydrogenase 10 in embryonic patterning and rescue of its loss of function by maternal retinaldehyde treatment.
), the precursor for RA, our findings suggest that other retinol dehydrogenases (
e.g., RDHE2 and RDHE2S) may be as important for postnatal development and maintenance of specific adult tissues as RDH10.
Analysis of substrate and cofactor specificity carried out in this study shows that, in agreement with their functions as oxidative enzymes, epidermal retinol dehydrogenases prefer NAD(H) as cofactors and are more catalytically efficient in the oxidative direction with all-
trans-retinol than in the reductive direction with all-
trans-retinaldehyde as substrate. Interestingly, like human RDH10 (
30- Belyaeva O.V.
- Johnson M.P.
- Kedishvili N.Y.
Kinetic analysis of human enzyme RDH10 defines the characteristics of a physiologically relevant retinol dehydrogenase.
), RDHE2S recognizes 11-
cis-retinol as substrate in addition to all-
trans-retinol. A recent study provided evidence that RDH10 contributes to the oxidation of 11-
cis-retinol in mouse retina, but mice deficient in both RDH10 and RDH5 still convert 11-
cis-retinol to 11-
cis-retinaldehyde in the visual cycle (
31- Sahu B.
- Sun W.
- Perusek L.
- Parmar V.
- Le Y.Z.
- Griswold M.D.
- Palczewski K.
- Maeda A.
Conditional ablation of retinol dehydrogenase 10 in the retinal pigmented epithelium causes delayed dark adaption in mice.
). The identities of the remaining 11-
cis-retinol dehydrogenases are currently unknown. If RDHE2S is expressed in mouse retina, this enzyme could complement RDH10 and RDH5 in oxidizing 11-
cis-retinol.
A surprising finding of this study is that the retinol dehydrogenase activity of murine RDHE2 is undetectable in the isolated microsomal or mitochondrial fractions of RDHE2-expressing Sf9 cells. However, RDHE2 is fully functional as a retinol dehydrogenase when expressed in mammalian living cells. This observation suggests that murine RDHE2 requires additional cellular factors to exhibit its full enzymatic potential. As shown in our previous study, a member of the SDR16C family, human DHRS3 (SDR16C1), requires the presence of human RDH10 (SDR16C4) to display a measurable retinaldehyde reductase activity and, in turn, activates RDH10 (
32- Adams M.K.
- Belyaeva O.V.
- Wu L.
- Kedishvili N.Y.
The retinaldehyde reductase activity of DHRS3 is reciprocally activated by retinol dehydrogenase 10 to control retinoid homeostasis.
). However, co-expression of murine RDHE2 or RDHE2S with human DHRS3, which is 97.7% identical to murine DHRS3 in HEK293 cells, had no activating effect on DHRS3 (
19- Belyaeva O.V.
- Chang C.
- Berlett M.C.
- Kedishvili N.Y.
Evolutionary origins of retinoid active short-chain dehydrogenases/reductases of SDR16C family.
), indicating that RDHE2 and RDHE2S do not interact with DHRS3. Like murine RDHE2, human RDHE2 (SDR16C5) also displays a rather low activity (
34- Adams M.K.
- Lee S.A.
- Belyaeva O.V.
- Wu L.
- Kedishvili N.Y.
Characterization of human short chain dehydrogenase/reductase SDR16C family members related to retinol dehydrogenase 10.
). Future affinity purification assays may be warranted to identify possible partners of RDHE2.
It is also interesting that whereas RDHE2S exhibits a higher retinol dehydrogenase activity than RDHE2, a single
Rdhe2s gene knockout does not display the same phenotype as the DKO mice.
Rdhe2s−/− mice have normal eyelids and regular hair growth (data not shown). This observation indicates that first, despite its lower enzymatic activity, RDHE2 is essential for RA biosynthesis in mice and, second, the functions of these two enzymes may be redundant because of the overlapping expression pattern. The reason for such an overlap is unclear at this time. It should be noted that in humans, only
RDHE2 represents a functional gene encoding a stable protein with enzymatic activity, whereas human
RDHE2S (
SDR16C6) appears to have lost its function and is classified as pseudogene (
34- Adams M.K.
- Lee S.A.
- Belyaeva O.V.
- Wu L.
- Kedishvili N.Y.
Characterization of human short chain dehydrogenase/reductase SDR16C family members related to retinol dehydrogenase 10.
). It is possible that in humans,
RDHE2, which exhibits a wider tissue distribution pattern (
https://www.ncbi.nlm.nih.gov/gene/195814#gene-expression) than either murine
Rdhe2 (
https://www.ncbi.nlm.nih.gov/gene/242285) or
Rdhe2s (
https://www.ncbi.nlm.nih.gov/gene/?term=Mus+musculus+Sdr16c6) fulfills the mission of both enzymes.
This study shows that in mice, RDHE2 and RDHE2S together account for up to 80% of the total membrane-associated retinol dehydrogenase activity in skin. Thus, although present in skin, RDH10 is not the major retinol dehydrogenase in this tissue. The absence of RDHE2 and RDHE2S results in significant changes in the hair cycle of mice. Hair cycle occurs in three phases: anagen (growth), catagen (regression), and telogen (resting) (
35Dissecting the bulge in hair regeneration.
36- Chase H.B.
- Rauch R.
- Smith V.W.
Critical stages of hair development and pigmentation in the mouse.
,
37- Plikus M.V.
- Mayer J.A.
- de la Cruz D.
- Baker R.E.
- Maini P.K.
- Maxson R.
- Chuong C.M.
Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration.
,
38- Müller-Röver S.
- Handjiski B.
- van der Veen C.
- Eichmüller S.
- Foitzik K.
- McKay I.A.
- Stenn K.S.
- Paus R.
A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.
,
39- Chen C.C.
- Plikus M.V.
- Tang P.C.
- Widelitz R.B.
- Chuong C.M.
The modulatable stem cell niche: tissue interactions during hair and feather follicle regeneration.
,
40Emerging interactions between skin stem cells and their niches.
,
41FOXC1 maintains the hair follicle stem cell niche and governs stem cell quiescence to preserve long-term tissue-regenerating potential.
,
42- Schneider M.R.
- Schmidt-Ullrich R.
- Paus R.
The hair follicle as a dynamic miniorgan.
,
43- Barker N.
- Tan S.
- Clevers H.
Lgr proteins in epithelial stem cell biology.
,
44Home sweet home: skin stem cell niches.
,
45- Jensen K.B.
- Collins C.A.
- Nascimento E.
- Tan D.W.
- Frye M.
- Itami S.
- Watt F.M.
Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis.
,
46- Genander M.
- Cook P.J.
- Ramsköld D.
- Keyes B.E.
- Mertz A.F.
- Sandberg R.
- Fuchs E.
BMP signaling and its pSMAD1/5 target genes differentially regulate hair follicle stem cell lineages.
47- Ahmed N.S.
- Ghatak S.
- El Masry M.S.
- Gnyawali S.C.
- Roy S.
- Amer M.
- Everts H.
- Sen C.K.
- Khanna S.
Epidermal E-cadherin dependent β-catenin pathway is phytochemical inducible and accelerates anagen hair cycling.
). Typically, development of dorsal hair follicles continues until postnatal day 16, when the first catagen occurs on days 17–20. Telogen spans days 21–25, and anagen lasts from day 28 to day 42. This is followed by the second cycle, during which hair follicles continue to cycle in a nearly synchronized manner. Anagen phase is characterized by thickening of the dermal and epidermal layers of the skin, increased size of hair follicles, extension of follicles deep into the dermal adipose tissue, and initiation of melanin synthesis. All of these features were observed in the dermis and epidermis of DKO skin but not in skin of WT littermates. Consistent with histological assessment, DKO skin showed increased expression of several markers of anagen phase (
26Skin and its regenerative powers: an alliance between stem cells and their niche.
). For example, a strong indication of the anagen phase is the induction of the hedgehog signaling pathway. In DKO skin, both
Gli1 and
Gli2 transcriptional regulators in the hedgehog pathway were up-regulated. Hair cycles are fueled by HF stem cells that reside in the “bulge” niche located at the base of the telogen phase HF (
48- Cotsarelis G.
- Sun T.T.
- Lavker R.M.
Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis.
). In addition to
Gli1, which is expressed in the hair bulge and germ (
49- Levy V.
- Lindon C.
- Harfe B.D.
- Morgan B.A.
Distinct stem cell populations regenerate the follicle and interfollicular epidermis.
,
50- Brownell I.
- Guevara E.
- Bai C.B.
- Loomis C.A.
- Joyner A.L.
Nerve-derived sonic hedgehog defines a niche for hair follicle stem cells capable of becoming epidermal stem cells.
), several other markers of the bulge stem cell compartment were up-regulated in DKO skin. Among these, the most notable are SRY (sex-determining region Y)-box 9 (SOX-9), which is indispensable for hair homeostasis (
51- Nowak J.A.
- Polak L.
- Pasolli H.A.
- Fuchs E.
Hair follicle stem cells are specified and function in early skin morphogenesis.
,
52- Vidal V.P.
- Chaboissier M.C.
- Lützkendorf S.
- Cotsarelis G.
- Mill P.
- Hui C.C.
- Ortonne N.
- Ortonne J.P.
- Schedl A.
Sox9 is essential for outer root sheath differentiation and the formation of the hair stem cell compartment.
); T-box 1 (
Tbx1), which is highly enriched in the bulge of developing and cycling HFs (
53- Chen T.
- Heller E.
- Beronja S.
- Oshimori N.
- Stokes N.
- Fuchs E.
An RNA interference screen uncovers a new molecule in stem cell self-renewal and long-term regeneration.
); and leucine-rich repeat-containing G protein–coupled receptor 5 (
Lgr5), a marker of hair-follicle stem cells (
54- Jaks V.
- Barker N.
- Kasper M.
- van Es J.H.
- Snippert H.J.
- Clevers H.
- Toftgård R.
Lgr5 marks cycling, yet long-lived, hair follicle stem cells.
).
The enlargement of sebaceous glands was confirmed by severalfold higher expression of melanocortin-5 receptor (
Mc5r), a marker of sebocyte differentiation (
55- Zhang L.
- Li W.H.
- Anthonavage M.
- Eisinger M.
Melanocortin-5 receptor: a marker of human sebocyte differentiation.
);
Prdm1 gene, which encodes BLIMP1, believed to be a marker of terminally differentiated sebocytes (
56Blimp-1: a marker of terminal differentiation but not of sebocytic progenitor cells.
) and/or a sebocyte progenitor marker (
57- Horsley V.
- O'Carroll D.
- Tooze R.
- Ohinata Y.
- Saitou M.
- Obukhanych T.
- Nussenzweig M.
- Tarakhovsky A.
- Fuchs E.
Blimp1 defines a progenitor population that governs cellular input to the sebaceous gland.
); peroxisome proliferator–activated receptor γ (
Pparγ), which plays an important role in sebaceous gland development; and stearyl CoA-desaturase 1 (
Scd1) and perilipin 2 (
Plin2), which are necessary for sebocyte differentiation and sebum production in sebocytes (reviewed in Ref.
58- Zouboulis C.C.
- Picardo M.
- Ju Q.
- Kurokaw I.
- Törőcsik D.
- Bíró T.
- Schneider M.R.
Beyond acne: current aspects of sebaceous gland biology and function.
).
One of the most up-regulated genes in both males (DKO2 strain) and females (DKO1 strain) was the
Lef1 gene in the Wnt signaling pathway. It is well-known that the regulation of the hair-follicle cycle involves cross-talk of the Wnt/β-catenin, Hedgehog, and Notch signaling pathways (
59- Gat U.
- DasGupta R.
- Degenstein L.
- Fuchs E.
De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated β-catenin in skin.
60Hair cycle regulation of Hedgehog signal reception.
,
61- Silva-Vargas V.
- Lo Celso C.
- Giangreco A.
- Ofstad T.
- Prowse D.M.
- Braun K.M.
- Watt F.M.
β-Catenin and Hedgehog signal strength can specify number and location of hair follicles in adult epidermis without recruitment of bulge stem cells.
,
62- Vauclair S.
- Nicolas M.
- Barrandon Y.
- Radtke F.
Notch1 is essential for postnatal hair follicle development and homeostasis.
,
63- Estrach S.
- Cordes R.
- Hozumi K.
- Gossler A.
- Watt F.M.
Role of the Notch ligand Delta1 in embryonic and adult mouse epidermis.
64- Blanpain C.
- Lowry W.E.
- Pasolli H.A.
- Fuchs E.
Canonical notch signaling functions as a commitment switch in the epidermal lineage.
). However, nonprotein factors, and in particular vitamin A (see Ref.
65Nutrition Classics. The Journal of Experimental Medicine 42: 753–77, 1925. Tissue changes following deprivation of fat-soluble A vitamin. S. Burt Wolbach and Percy R. Howe.
; reviewed in Ref.
66A decade of molecular biology of retinoic acid receptors.
), are also critical for maintenance of skin health. In this respect, there is evidence for mutual antagonism between Wnt/β-catenin signaling and RA signaling (
67Diverse actions of retinoid receptors in cancer prevention and treatment.
). β-Catenin positively regulates CYP26A1, which degrades RA, and conversely, β-catenin undergoes retinoid-dependent interaction with RAR, resulting in competitive inhibition of TCF-binding sites (
67Diverse actions of retinoid receptors in cancer prevention and treatment.
,
68- Easwaran V.
- Pishvaian M.
- Salimuddin
- Byers S.
Cross-regulation of β-catenin-LEF/TCF and retinoid signalling pathways.
). Inhibition of Wnt/β-catenin signaling leads to up-regulation of RA-inducible cellular RA-binding protein type 2 (CRABP2), suggesting increased RA signaling (
69Dynamic regulation of retinoic acid-binding proteins in developing, adult and neoplastic skin reveals roles for β-catenin and Notch signalling.
). It is noteworthy, however, that whereas RA signaling is clearly essential for epidermal differentiation, the mechanisms by which it acts are largely unexplored. This study and the generation of RDHE2 and RDHE2S DKO mouse models should facilitate the investigation of the role RA plays in the regulation of hair-follicle growth and differentiation and in general maintenance of epidermis.
Interestingly, 13-
cis-retinoic acid (isotretinoin) is clinically used for acne treatment and believed to inhibit sebocyte differentiation and lipid synthesis (
70- Clarke S.B.
- Nelson A.M.
- George R.E.
- Thiboutot D.M.
Pharmacologic modulation of sebaceous gland activity: mechanisms and clinical applications.
,
71A topical medication of all-trans-retinoic acid reduces sebum excretion rate in patients with forehead acne.
) by isomerizing to all-
trans-retinoic acid (
58- Zouboulis C.C.
- Picardo M.
- Ju Q.
- Kurokaw I.
- Törőcsik D.
- Bíró T.
- Schneider M.R.
Beyond acne: current aspects of sebaceous gland biology and function.
). The enlarged sebaceous glands observed in DKO mice are consistent with the decreased levels of RA biosynthesis in skin. We have also observed enlarged meibomian glands in DKO eyelids. Meibomian glands are modified sebaceous glands that secrete meibum, a lipid-rich fluid that forms a superficial oily layer on the tear film to prevent the film from evaporating. Of note, severe bilateral lower eyelid retraction and dry eye symptoms were observed in patients with the long-term use of topical retinoids for cosmetic purposes (
72- Winkler K.P.
- Black E.H.
- Servat J.
Lower eyelid retraction associated with topical retinol use.
,
73- Kremer I.
- Gaton D.D.
- David M.
- Gaton E.
- Shapiro A.
Toxic effects of systemic retinoids on meibomian glands.
74- Ding J.
- Kam W.R.
- Dieckow J.
- Sullivan D.A.
The influence of 13-cis-retinoic acid on human meibomian gland epithelial cells.
). Thus, reduced RA biosynthesis in eyelids of DKO mice appears to be consistent with the observed enlargement of their meibomian glands. Previous studies have also noted that systemic use of retinoids for acne treatment or skin rejuvenation was associated with histopathological changes in the eyelids and degenerative changes in the meibomian gland acini (
73- Kremer I.
- Gaton D.D.
- David M.
- Gaton E.
- Shapiro A.
Toxic effects of systemic retinoids on meibomian glands.
,
74- Ding J.
- Kam W.R.
- Dieckow J.
- Sullivan D.A.
The influence of 13-cis-retinoic acid on human meibomian gland epithelial cells.
).
It is worth pointing out that, whereas the in vivo phenotype is clearly consistent with the reduced RA signaling in DKO skin as a result of the 80% reduction in retinol dehydrogenase activity, qPCR analysis of endogenous RA-regulated genes produced a very mixed picture, with some RA-inducible genes being down-regulated, whereas others were unchanged or even up-regulated. Histological analysis of skin sections revealed the cause of this seemingly puzzling outcome. Because the reduced RA signaling led to enlargement of sebaceous glands, which express several RA-sensitive genes (Dhrs9, Lrat, and Stra6), the transcript levels of these genes were likely increased as a result of an expanded number of sebocytes. Thus, measurement of total levels of RA in skin could be misleading, because it would not reflect the localized differences in cell type–specific RA levels in tissue as heterogeneous as skin.
Overall, the results of this study establish RDHE2 and RDHE2S as physiologically relevant retinol dehydrogenases in mammals, which, despite their importance for RA biosynthesis, are not critical for survival during embryogenesis. In view of these findings, the physiological impact of mutations in these genes or changes in their expression can be better appreciated considering their role in RA biosynthesis. As discussed previously (
34- Adams M.K.
- Lee S.A.
- Belyaeva O.V.
- Wu L.
- Kedishvili N.Y.
Characterization of human short chain dehydrogenase/reductase SDR16C family members related to retinol dehydrogenase 10.
), genome-wide association studies have linked the chromosomal region harboring
RDHE2 (
SDR16C5) and seven other genes to stature and growth in cattle, humans, and pigs (
35Dissecting the bulge in hair regeneration.
36- Chase H.B.
- Rauch R.
- Smith V.W.
Critical stages of hair development and pigmentation in the mouse.
,
37- Plikus M.V.
- Mayer J.A.
- de la Cruz D.
- Baker R.E.
- Maini P.K.
- Maxson R.
- Chuong C.M.
Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration.
,
38- Müller-Röver S.
- Handjiski B.
- van der Veen C.
- Eichmüller S.
- Foitzik K.
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,
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,
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,
55- Zhang L.
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,
56Blimp-1: a marker of terminal differentiation but not of sebocytic progenitor cells.
,
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,
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,
59- Gat U.
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,
60Hair cycle regulation of Hedgehog signal reception.
,
61- Silva-Vargas V.
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,
62- Vauclair S.
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Notch1 is essential for postnatal hair follicle development and homeostasis.
,
63- Estrach S.
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Role of the Notch ligand Delta1 in embryonic and adult mouse epidermis.
,
64- Blanpain C.
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Canonical notch signaling functions as a commitment switch in the epidermal lineage.
,
65Nutrition Classics. The Journal of Experimental Medicine 42: 753–77, 1925. Tissue changes following deprivation of fat-soluble A vitamin. S. Burt Wolbach and Percy R. Howe.
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66A decade of molecular biology of retinoic acid receptors.
,
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,
68- Easwaran V.
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Cross-regulation of β-catenin-LEF/TCF and retinoid signalling pathways.
,
69Dynamic regulation of retinoic acid-binding proteins in developing, adult and neoplastic skin reveals roles for β-catenin and Notch signalling.
,
70- Clarke S.B.
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Pharmacologic modulation of sebaceous gland activity: mechanisms and clinical applications.
,
71A topical medication of all-trans-retinoic acid reduces sebum excretion rate in patients with forehead acne.
,
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,
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Toxic effects of systemic retinoids on meibomian glands.
,
74- Ding J.
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The influence of 13-cis-retinoic acid on human meibomian gland epithelial cells.
,
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Polymorphic regions affecting human height also control stature in cattle.
,
76- Nishimura S.
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Genome-wide association study identified three major QTL for carcass weight including the PLAG1-CHCHD7 QTN for stature in Japanese Black cattle.
,
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Many sequence variants affecting diversity of adult human height.
,
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Genome-wide association analysis identifies 20 loci that influence adult height.
,
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Identification of ten loci associated with height highlights new biological pathways in human growth.
,
80- Karim L.
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Variants modulating the expression of a chromosome domain encompassing PLAG1 influence bovine stature.
,
81- Littlejohn M.
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Genetic variation in PLAG1 associates with early life body weight and peripubertal weight and growth in Bos Taurus.
82- Jiao S.
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Feed intake, average daily gain, feed efficiency, and real-time ultrasound traits in Duroc pigs: II. Genomewide association.
) and beak deformity in chickens (
83- Bai H.
- Zhu J.
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- Liu R.
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- Wen J.
- Chen J.
Identification of genes related to beak deformity of chickens using digital gene expression profiling.
).
RDHE2 was also identified as the important candidate gene in the pig growth trait by an integrative genomic approach (
84- Xiong X.
- Yang H.
- Yang B.
- Chen C.
- Huang L.
Identification of quantitative trait transcripts for growth traits in the large scales of liver and muscle samples.
). Interestingly, expression of
SDR16C5 was reported to be 4-fold higher than
RDH10 in human lung (pooled RNA from five individuals) (
85- Ashmore J.H.
- Luo S.
- Watson C.J.W.
- Lazarus P.
Carbonyl reduction of NNK by recombinant human lung enzymes: identification of HSD17β12 as the reductase important in (R)-NNAL formation in human lung.
). Altered
SDR16C5 expression is frequently noted in various pathophysiological conditions. For example,
SDR16C5 was decreased in triple-negative breast cancer patient samples (
86- Qi F.
- Qin W.-X.
- Zang Y.-S.
Molecular mechanism of triple-negative breast cancer-associated BRCA1 and the identification of signaling pathways.
). Evidence provided by this study that mammalian RDHE2/E2S function as physiologically relevant retinol dehydrogenases implicates the decrease in RA biosynthesis as at least one of the causes leading to these pathophysiological outcomes.
Experimental procedures
Expression constructs
All primer sequences with corresponding restriction sites used for generation of constructs are listed in
Table S1. Constructs encoding FLAG-tagged murine RDHE2S and
X. laevis rdhe2 in pCMV-Tag 4A vector were described previously (
18- Belyaeva O.V.
- Lee S.-A.
- Adams M.K.
- Chang C.
- Kedishvili N.Y.
Short chain dehydrogenase/reductase Rdhe2 is a novel retinol dehydrogenase essential for frog embryonic development.
,
20- Lee S.-A.
- Belyaeva O.V.
- Kedishvili N.Y.
Biochemical characterization of human epidermal retinol dehydrogenase 2.
). Murine
Rdhe2 cDNA was obtained by RT-PCR of mouse skin and liver mRNA and cloned into SalI and XbaI sites of pBluescript II SK (−) vector.
To generate FLAG-tagged RDHE2 expression construct, the corresponding cDNA was cloned into pCMV-Tag 4A vector in frame with the C-terminal FLAG tag. Subsequently, the FLAG-tagged construct was amplified using primers specified in
Table S1 and cloned into pCS105 and pVL1393 vectors. In addition, a His-tagged RDHE2 construct was generated using a modified pVL1393 vector containing an in-frame C-terminal His
6 tag (
87- Belyaeva O.V.
- Stetsenko A.V.
- Nelson P.
- Kedishvili N.Y.
Properties of short-chain dehydrogenase/reductase RalR1: characterization of purified enzyme, its orientation in the microsomal membrane, and distribution in human tissues and cell lines.
).
Human HA-tagged RALDH1 expression construct in pcDNA3.1-neo (CMV promoter) was a generous gift of Dr. Sylvie Mader (Department of Biochemistry, University of Montreal, Canada). All expression constructs and plasmids were verified by sequencing.
Cell culture models
For activity assays in intact cells, SDR constructs were expressed in human HEK293 cells following the protocols published previously (
30- Belyaeva O.V.
- Johnson M.P.
- Kedishvili N.Y.
Kinetic analysis of human enzyme RDH10 defines the characteristics of a physiologically relevant retinol dehydrogenase.
,
32- Adams M.K.
- Belyaeva O.V.
- Wu L.
- Kedishvili N.Y.
The retinaldehyde reductase activity of DHRS3 is reciprocally activated by retinol dehydrogenase 10 to control retinoid homeostasis.
). The transfected cells were incubated with all-
trans-retinol or all-
trans-retinaldehyde as indicated. Retinoids were extracted and analyzed by normal phase HPLC as described previously (
30- Belyaeva O.V.
- Johnson M.P.
- Kedishvili N.Y.
Kinetic analysis of human enzyme RDH10 defines the characteristics of a physiologically relevant retinol dehydrogenase.
). For kinetic analysis, RDHE2, RDHE2S, and frog rdhe2 were expressed in Sf9 insect cells using pVL1393 transfer vector and the BaculoGold Baculovirus Expression System (BD Biosciences). The subcellular fractions were isolated by differential centrifugation as described (
87- Belyaeva O.V.
- Stetsenko A.V.
- Nelson P.
- Kedishvili N.Y.
Properties of short-chain dehydrogenase/reductase RalR1: characterization of purified enzyme, its orientation in the microsomal membrane, and distribution in human tissues and cell lines.
). The membrane pellets were resuspended in 90 m
m KH
2PO
4, 40 m
m KCl (pH 7.4), and 20% glycerol (w/v).
In vitro activity assays and immunoblotting
Activity assays using subcellular fractions of Sf9 cells were performed as described previously (
30- Belyaeva O.V.
- Johnson M.P.
- Kedishvili N.Y.
Kinetic analysis of human enzyme RDH10 defines the characteristics of a physiologically relevant retinol dehydrogenase.
). The
Km and
Vmax values for all-
trans-retinol, 9-
cis-retinol, 11-
cis-retinol, or all-
trans-retinaldehyde were obtained using seven concentrations of each substrate (0.0625–8 μ
m) in the presence of 1 m
m NAD(H). The
Km and
Vmax values for NAD
+ and NADH were determined at fixed concentration of all-
trans-retinol or all-
trans-retinaldehyde (5 μ
m) and seven concentrations of NAD
+ or NADH (1–500 μ
m).
Western blot analysis was performed using rabbit polyclonal antibodies against FLAG epitope (Sigma-Aldrich) and β-actin (Abcam, Cambridge, UK) and mouse monoclonal antibodies against His epitope (Clontech, BD Biosciences) and HA epitope (received as a gift from Dr. Hengbin Wang, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham School of Medicine).
Protein samples were separated in 12% polyacrylamide gels in the presence of SDS and transferred to Amersham Biosciences Hybond P polyvinylidene difluoride membranes (GE Healthcare). Following transfer, membranes were blocked with 4% BSA in TBS with Tween 20 (TBST) and incubated with rabbit polyclonal or mouse monoclonal antibodies diluted with 4% BSA in TBST overnight at 4 °C. Dilutions of primary antibodies are indicated in the figure legends. Membranes were rinsed with TBST and incubated for 1 h at room temperature in goat anti-rabbit antibody or goat anti-mouse antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), both of which were diluted 1:10,000 with 4% BSA in TBST. Protein visualization was achieved with Pierce ECL Western blotting substrate (Thermo Fisher Scientific).
Generation of Rdhe2s−/− mouse model
Rdhe2s+/− mice were generated using the
Sdr16c6tm1a(KOMP)Wtsi “knockout-first” allele obtained from the KOMP repository [
https://www.komp.org/pdf.php?projectID=41879,
5Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.
SangerID design 43965]. The knockout-first allele contains an IRES:
lacZ trapping cassette and a floxed promoter-driven
neo cassette inserted into the intron of a gene, disrupting gene function. The targeting vector (KOMP design 43965) was digested with AsiSI and SalI enzymes, which resulted in a 1,431-bp truncation of the 5′ homology arm. Electroporation of the linearized vector in mouse embryonic stem cells (ESCs), selection of ESC-derived clones, and isolation of genomic DNA were performed by the University of Alabama at Birmingham Transgenic and Genetically Engineered Models facility. ESC-derived clones carrying a targeted insertion of the knockout-first cassette were identified by long-range PCR spanning 5′ and 3′ homology arms using the SequalPrep long PCR kit (Invitrogen, Life Technologies, Inc.) with primers listed in
Table S1. Chimeras were generated by the University of Alabama at Birmingham Transgenic and Genetically Engineered Models facility. Male chimeras were crossed to WT C57Bl56/J mice, and pups were genotyped to identify heterozygote founders carrying a knockout-first allele. Crossing of these mice to FLPeR mice carrying a gene encoding FLP recombinase resulted in conversion of the knockout-first allele to a conditional allele, restoring gene activity. Subsequent crossing of FLP-excised mice to EIIa-cre mice, which express Cre recombinase prior to implantations, resulted in a mouse with a deletion of exon 4 and a frameshift mutation (
Fig. S1). Genotyping of mice crossed to FLPeR mice was accomplished using DNA isolated from tails and the primers listed in
Table S1.
Generation of Rdhe2−/−;Rdhe2s−/− mouse models
Murine
Sdr16c6 is located immediately upstream of murine
Sdr16c5 gene. For both genes, CRISPR guide RNAs were designed to target exons 2 and 5, which encode the conserved cofactor and substrate-binding site motifs, respectively (
Table S2,
Fig. 3A, and
Fig S3). Guide RNAs were designed as described previously (
88- Challa A.K.
- Boitet E.R.
- Turner A.N.
- Johnson L.W.
- Kennedy D.
- Downs E.R.
- Hymel K.M.
- Gross A.K.
- Kesterson R.A.
Novel hypomorphic alleles of the mouse tyrosinase gene induced by CRISPR-Cas9 nucleases cause non-albino pigmentation phenotypes.
). Six F0 pups were born following CRISPR-Cas9 microinjections. For the initial screening, the targeted exons 2 and 5 were PCR-amplified from genomic DNA isolated from tail snips and separated by electrophoresis in polyacrylamide gel. In mouse #21855, the electrophoretic mobility shift assay revealed heterogeneous bands in PCR products corresponding to exon 2 of
Sdr16c5 and exon 5 of
Sdr16c6, suggesting the formation of heteroduplexes. In mouse #21853, PCR product for exon 5 of
Sdr16c5 was completely absent, indicating that CRISPR-Cas9 injections generated a larger deletion(s) in this animal and that both copies of the
Sdr16c6-Sdr16c5 locus are mutated.
Subcloning and resequencing of the targeted exons in mouse #21855 identified a 3-bp deletion in exon 2 of Sdr16c5, as well as 1- and 3-bp deletions in exon 5 of Sdr16c6. The loss of a single amino acid resulting from the 3-bp deletion may not lead to a complete loss of enzymatic activity of SDR16C5 (RDHE2), and the frameshift-causing 1-bp deletion in Sdr16c6 was expected to result in a knockout of the Sdr16c6 gene only. As we already had an established knockout strain of Sdr16c6, mouse #21855 was not used as a founder.
To precisely define the alleles generated in mouse #21853, we performed a series of PCR amplifications with primer pairs as indicated in
Fig. 3A and in
Table S1. We assumed that the products would only be generated if the primers’ annealing sites were brought close enough as a result of deletion, because the distance between the annealing sites in the WT type allele is too large for efficient amplification. The primer pair 4 yielded a 585-bp product in mouse #21853, but not in other F0 animals. The product contained a chimeric sequence composed of incomplete exon 5 of
Sdr16c6, incomplete exon 5 of
Sdr16c5 gene, and a 298-bp insertion corresponding to an inverted partial sequence of exon 2 of
Sdr16c5 (
Fig. 3A and
Fig. S3). This result indicated that mouse #21853 contained a large 56,057-bp deletion covering the region between CRISPR targets in exon 5 of
Sdr16c6 and exon 5 of
Sdr16c5. The ORF of the chimeric transcript encodes a truncated polypeptide (
Fig. S3). We have designated this double
Sdr16c6−/−;
Sdr16c5−/− knock-out allele as DKO1.
The existence of a full-length PCR product for exon 5 of
Sdr16c6, which was obtained during initial F0 screening was inconsistent with the DKO1 allele, which retains only part of this exon. This result suggested that mouse #21853 carries a second mutant allele. PCR amplification of individual exons 4 and 6 of
Sdr16c5 yielded fragments of the expected size, indicating that a shorter-than-DKO1 deletion occurred in the second allele of the same animal. This shorter deletion resulted in a loss of a single exon 5 in
Sdr16c5. Long-range PCR amplification with primers spanning exons 4–6 yielded a 4,912-bp product in WT mouse but a shorter product in mouse #21853. Sequencing of this product confirmed the deletion of 1,837 bp, which covers exon 5 of
Sdr16c5 and a portion of flanking intronic sequences. This allele was designated DKO2 (
Fig. 3A and
Fig. S3).
Thus, mouse #21853 carried two different CRISPR-Cas9–generated alleles, DKO1 and DKO2. This animal was used as the founder, and crossed to WT females to isolate DKO1 and DKO2 strains. Resequencing of individual exons in the DKO2 strain revealed a short 8-bp deletion in exon 2 of
Sdr16c6, in addition to deleted exon 5 in
Sdr16c5. Thus, the ORFs of both
Sdr16c6 and
Sdr16c5 in DKO2 allele are predicted to encode truncated proteins (
Fig. 3A and
Fig. S3). DKO2 represents a second double-knockout strain.
Mice were maintained on either on LabDiet NIH-31 (PMI Nutrition International) containing 22 IU vitamin A/g or on vitamin A–deficient diet Teklad TD.86143 (Envigo) in a facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care. Mice were euthanized by CO2 inhalation followed by cervical dislocation, in accordance with institutional animal care and use committee guidelines at the University of Alabama (Birmingham, AL).
β-Galactosidase staining and in situ hybridizations
Adult mice were arranged in mating pairs, and females were checked for vaginal plugs at noon of the following day. The presence of a plug was considered to represent a developmental stage of E0.5. Genotypes were determined using DNA isolated from yolk sacs. Full-length mouse
Rdhe2 cDNA in pBluescript II SK (−) vector was used for generation of antisense probes for
in situ hybridization. Probes were synthesized using linearized template, T3 RNA polymerase (Promega), and digoxigenin RNA labeling mix (Roche Applied Science). Skin was isolated from the backs of 4-month-old male C57BL/J6 mice, fixed overnight in 4% paraformaldehyde at 4 °C, and embedded in paraffin. The paraffin-embedded skin was cut into 10-μm sections, which were placed on Superfrost Plus microscope slides (Fisher).
In situ hybridization was carried out following standard procedures. β-Galactosidase staining was performed on E14.5
Rdhe2s+/− embryos and skin isolated from 4-month-old
Rdhe2s+/− male mouse tails according to the protocols published previously (
89- Nagy A.
- Gertsenstein M.
- Vintersten K.
- Behringer R.
Manipulating the Mouse Embryo: A Laboratory Manual.
).
qPCR analysis
To determine the expression pattern of Rdhe2 and Rdhe2s genes, two male WT mice fed a VAD diet for 10 weeks were sacrificed with CO2; tissues were collected and stored at −80 °C until RNA extraction. TRIzol reagent (Ambion, catalog no. 15596018) was used for extraction of RNA from all tissues except skin, for which the AurumTM total RNA Fatty and Fibrous Tissue Pack (Bio-Rad, catalog no. 732-6870) was employed. Three μg of RNA per tissue was used for reverse transcription; cDNA was purified with QIAquick Spin columns (Qiagen, catalog no. 1018215). qPCR was performed with 25 ng of cDNA per reaction. Rdhe2 and Rdhe2s expression was normalized to Gapdh and presented as a relative expression of fold-difference from the expression level in the stomach for each gene, which was set to 1.
For analysis of gene expression in skin, ∼75 mg of skin tissue was homogenized, and RNA was extracted with the AurumTM total RNA Fatty and Fibrous Tissue Pack. The concentration of extracted RNA was determined using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific). First-strand cDNA was synthesized from 3.0 μg of total RNA with the Superscript III first-strand synthesis kit (Invitrogen) according to the manufacturer's protocol. For real-time RT-PCRs, the cDNA was diluted 15-fold. Sequences of the primers are available by request. Real-time PCR analysis was conducted on a Roche LightCycler®480 detection system (Roche Applied Science) with SYBR Green as probe (LightCycler®480 CYBR Green I Master, Roche Applied Science). Relative gene expression levels were calculated using the comparative Ct method by normalization to reference genes. Unpaired t test was used to test for statistical significance.
Hair regrowth
The diet for DKO2 heterozygote breeder dams was switched to a VAD diet at mid-gestation (12–14 days post-conception), and the pups were kept on a VAD diet until they were sacrificed at 10 weeks of age. At 8 weeks after birth, WT and DKO2 littermates' dorsal hair was clipped and depilated with Nair® lotion. Littermates were checked daily for hair regrowth until they were sacrificed. Animals that had abnormal hair due to excessive grooming by female breeders were not used for the analysis of hair regrowth.
Statistical analysis
Statistical significance was determined using a two-tailed unpaired t test.
Author contributions
L. W., O. V. B., M. K. A., S.-A. L., R. A. K., K. M. P., and N. Y. K. conceptualization; L. W., O. V. B., M. K. A., A. K., S.-A. L., R. A. K., K. M. P., and N. Y. K. data curation; L. W., O. V. B., M. K. A., A. K., S.-A. L., R. A. K., K. M. P., and N. Y. K. formal analysis; L. W., O. V. B., M. K. A., S.-A. L., K. R. G., R. A. K., and N. Y. K. validation; L. W., O. V. B., M. K. A., A. K., S.-A. L., K. R. G., and N. Y. K. investigation; L. W., O. V. B., M. K. A., S.-A. L., K. R. G., K. M. P., and N. Y. K. visualization; L. W., O. V. B., M. K. A., A. K., S.-A. L., K. R. G., and N. Y. K. methodology; L. W., O. V. B., M. K. A., S.-A. L., K. R. G., R. A. K., and N. Y. K. writing-original draft; L. W., O. V. B., M. K. A., A. K., K. R. G., R. A. K., K. M. P., and N. Y. K. writing-review and editing; R. A. K. and N. Y. K. supervision; R. A. K. and N. Y. K. project administration; N. Y. K. resources; N. Y. K. funding acquisition.