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Involvement of DHH and GLI1 inadrenocortical autograft regeneration in
rats
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Nae Takizawa, 1,2
Susumu Tanaka, 1✉
Email tanakass@hirakata.kmu.ac.jp
Souichi Oe, 1
Taro Koike, 1
Takashi Yoshida, 2
Yukie Hirahara, 1
Tadashi Matsuda, 2Hisao Yamada, 1
1 Department of Anatomy and Cell Science, Kansai Medical University,
Hirakata, Osaka, 573-1010 Japan
2 Department of Urology and Andrology, Kansai Medical University,
Hirakata, Osaka, 573-1010 Japan
Received: 5 April 2018 / Accepted: 14 September 2018

Abstract
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Bilateral adrenalectomy forces the patient to undergo glucocorticoid
replacement therapy and bear a lifetime risk of adrenal crisis. Adrenal
autotransplantation is considered useful to avoid adrenal crisis andglucocorticoid replacement therapy. However, the basic process ofregeneration in adrenal autografts is poorly understood. Here, we investigatedthe essential regeneration factors in rat adrenocortical autografts, with a focus
on the factors involved in adrenal development and steroidogenesis, such as
Hh signalling. A remarkable renewal in cell proliferation and increase in
Cyp11b1, which encodes 11-beta-hydroxylase, occurred in adrenocortical
autografts from 2–3 weeks after autotransplantation. Serum corticosterone andadrenocorticotropic hormone levels were almost recovered to sham level at 4
weeks after autotransplantation. The adrenocortical autografts showed
increased Dhh expression at 3 weeks after autotransplantation, but not Shh,which is the only Hh family member to have been reported to be expressed in
the adrenal gland. Increased Gli1 expression was also found in the regenerated
capsule at 3 weeks after autotransplantation. Dhh and Gli1 might function inconcert to regenerate adrenocortical autografts. This is the first report toclearly show Dhh expression and its elevation in the adrenal gland.

Introduction
The adrenal gland comprises the adrenal cortex and the medulla. In humans, theadrenal cortex is composed of three anatomical layers namely the zona
glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis. In the rat adrenal
cortex, aldosterone synthase (Cyp11b2) in ZG and 11-beta-hydroxylase(Cyp11b1) in ZF catalyse the production of aldosterone and corticosterone,
respectively. These CYP genes are regulated by NR5A1 (also known as SF1 or
Ad4BP)[1]. There is also an area with no expression of Cyp11b1 and Cyp11b2
termed the undifferentiated zone (ZU) located between ZG and ZF in rats[2, 3,
4].
AQ1
AQ2
AQ3
AQ4

Familial phaeochromocytoma patients, such as those with multiple endocrine
neoplasia type 2, may require bilateral adrenalectomy, after which the patientsneed to take glucocorticoid replacement therapy throughout their life[5].
Moreover, the risk of adrenal crisis, a life-threatening event, remains as long as
the patients live[6]. Therefore, partial adrenalectomy may be performed as an
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adrenal sparing surgery to avoid adrenal crisis[5]. However, there is the risk thatphaeochromocytoma may recur from the ipsilateral residual adrenal gland evenafter partial adrenalectomy[7]. In this case, autotransplantation of the adrenal
cortex may reduce the risk of phaeochromocytoma recurrence[8] because theadrenal medulla, which is regarded as the origin of phaeochromocytoma, does
not regenerate, whereas adrenocortical cells can be regenerated in adrenal
autografts in animal models[9]. Adrenal transplantation was studied in animals
even in the early 1900s[10]; to our knowledge, the oldest reported adrenaltransplantation was conducted in a patient with Addison’s disease in 1922[11].
Establishment of adrenocortical autotransplantation has the advantage ofreducing recurrence risk and eliminating the need for glucocorticoid replacement
therapy in familial phaeochromocytoma patients[8]. However, the success rate
for adrenal autotransplantation in humans is low, ranging from 20 to 35%[12,13] in contrast to animal models[14, 15]. Consequently, to clarify theregeneration step of adrenocortical autografts after transplantation, and thefactors affecting their remodelling and regeneration, analysis of adrenocortical
autografts in animal models is required.
AQ5

Although adrenocortical autotransplantations have been successful in rodents[14,15], the underlying mechanism has not been clarified yet. Several patterns ofadrenocortical cell zonation, renewal, and remodelling under various conditions
have been reported[2, 16, 17]. Apparently, pools of stem/progenitor cells exist in
the adrenal capsule, the subcapsular, and other regions of the adrenal gland[2,18]. It is well-known that SHH plays a key role in adrenal development and islocalised in the subcapsular region of mice and in the ZU of rats[3, 18, 19].
Shh(+) cells have also been identified as stem/progenitor cells[2, 18, 19, 20]. To
date, only Shh has been reported to be expressed in the adrenal gland among the
Hh family members[2]. SHH regulates Gli1, the effector of Hh signalling and adeterminant of cell fate during embryonic development[21]. Gli1(+) cells havealso been identified as stem/progenitor cells and are localised in the adrenal
capsule[22]. In addition, SHH(+) and GLI1(+) progenitor cells differentiate into
steroidogenic cells. It is conceivable that the Hh signalling pathway is involvedin the regeneration process of adrenocortical autografts as in the development of
the adrenal cortex. In this study, we examined whether Hh signalling functions
as a remodelling and/or regeneration factor for adrenocortical autografts.
Unexpectedly, we found that Shh was suppressed and Dhh was upregulated in
the regeneration step of rat adrenocortical autografts. Our study clearly
demonstrates Dhh expression in the adrenal gland for the first time.

Results
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Enlargement of adrenocortical autografts
Macroscopically (Supplementary Fig. 1), we identified the adrenocortical
autograft as a yellowish mass throughout the post-operative period.
Adrenocortical autografts showed thin shapes at post-operative day (POD) 7(Supplementary Fig. 1). At two weeks after surgery, almost all adrenocortical
autografts were flat-shaped and fragile to handle in all rats (Supplementary
Fig. 1). After three weeks (Supplementary Fig. 1), the autografts were stable andincreased in size compared to POD14. There was no difference between POD21
and POD28 with respect to gross appearance.
Haematoxylin and eosin (HE) staining showed (i) partial loss of adrenocorticalcells, (ii) a few remnant adrenocortical cell clusters around the capillaries, and(iii) stromal-like cell proliferation from POD7 to POD14 (Fig. 1). At POD21, we
easily identified the arrangement of the renewing adrenocortical cells in the
cord-like structures similar to the normal ZF (Fig. 1). These cord-like structures
were completely encapsulated in the renewal capsule and in the columns directed
towards the veins (Supplementary Fig. 2). Indeed, a prominent increase in
adrenocortical cells was observed in the region encapsulated within the capsuleafter POD16 (Fig. 1 and Supplementary Fig. 2). Four weeks after surgery, HE
staining indicated fully formed adrenocortical cells (Fig. 1).
Figure 1
Morphological changes in adrenocortical autografts. Microscopic findings ofadrenocortical autografts. Left panels show the low-power fields of HE-stained
sections. Right panels are the expanded views of the square fields in the left panel.
Sections on post-operative day (POD) 7 and POD14 showed only a few remnant
adrenocortical cell clusters over adrenocortical autografts. An obvious renewal
capsule and renewal adrenocortical cell cluster were detected at POD21 and
POD28. Cap: capsule; AC: adrenocortical cells; RAC: renewal adrenocorticalcells; M: muscle. The black broken line shows the boundary between theadrenocortical autograft and muscle tissue.

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Autotransplanted rats recover from Adrenocorticalinsufficiency at POD21
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Serum corticosterone (ng/ml) showed a significant reduction at POD7 (n = 4,
mean ± standard error; 32.6 ± 5.3, p = 0.002, r = 0.74) and POD14 (n = 4, 78.4 ±
8.0, p = 0.008, r = 0.67) compared to the sham group (n = 10, 246.9 ± 33.5)(Fig. 2A). There was a gradual increase in the level of serum corticosterone.
There was no significant difference between the sham group and POD21 (n = 4,
164.9 ± 13.7) or the sham group and POD28 (n = 4, 206.4 ± 28.8). On the other
hand, serum adrenocorticotropic hormone (ACTH) (pg/ml) showed a significant
increase at POD7 (n = 4, mean ± standard error; 215.3 ± 103.7, p = 0.004, r =0.72) and POD14 (n = 4, 197.1 ± 55.7, p = 0.004, r = 0.72) compared to the shamgroup (n = 10, 17.1 ± 6.2) (Fig. 2B). There was no significant difference between
the sham group and POD21 (n = 4, 44.1 ± 23.9) or the sham group and POD28(n = 4, 63.8 ± 17.5).
Figure 2
Change of endocrine function in adrenocortical autografts. (A,B) Serum
corticosterone and ACTH levels in rats (n = 10 in sham group, n = 4 perautografted group). *p < 0.0125 vs. sham group. Each value represents the mean ±standard error. (C–G) The correlation between corticosterone and ACTH at eachtime point. Serum ACTH levels show significantly negative correlation withcorticosterone levels at POD 28 (P = 0.033, R = 0.87). (H,I) Detection of Cyp11b1
and Cyp11b2 in adrenocortical autografts using nCounter technology. The mean
expression at each time point in adrenocortical autografts was compared with thatin the sham-operated adrenal gland. *p < 0.05 vs. sham operation at each time
point. The white bar indicates the copy number of each mRNA in the sham groupand the black bar indicates the same in autografts. Values are presented as themean ±+ standard deviation.

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To clarify the recovery of the adrenocortical insufficiency in adrenal autografts,
the correlations between corticosterone and ACTH were analysed at each time
point (Fig. 2C–G). A significant negative correlation was found between
corticosterone and ACTH at POD28 (r = 0.86, p < 0.05) (Fig. 2G). Althoughnormal values of serum corticosterone and improvement of the hyperactive
hypothalamic-pituitary-adrenal axis were found at POD28, latent adrenocorticalinsufficiency was suggested at POD28 based on the negative correlation.
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Increase in Cyp11b1Cyp11b1 (+) cells in adrenocortical
autografts
To confirm the evidence of functional recovery, we examined Cyp11b1
expression in adrenocortical autografts using the RNAscope/Basescope
technique.
RNAscope is a novel RNA in situ hybridisation analysis using a unique doubleZ design probe[23]. The design helps to increase the sensitivity and specificity
for target gene expression. RNAscope signals show as dots, with each dot
corresponding to a single mRNA copy[23].
Cyp11b1 staining in the adrenal gland of a sham-operated rat using theBasescope showed a relatively strong expression in the ZF and a gradually
decreasing expression towards the inner medulla (Fig. 3A). In adrenocortical
autografts at POD7 and POD14, remnant adrenocortical cells around thecapillary circumference showed strong Cyp11b1 expression (Figs 4A and 5A).
There was no signal in the autografts except for remnant adrenocortical cells at
POD7 and POD14 (Figs 4A and 5A). Diffuse Cyp11b1 expression over theadrenocortical autograft was recognised at POD21 (Fig. 6A), and the expression
level per cell was lower than that in the adrenocortical autograft at POD28(Fig. 7A). At POD28, Cyp11b1 was expressed in a polarised manner, which issimilar to its expression pattern in the adrenal gland of sham-operated rats, and astrong Cyp11b1 expression was detected in the regenerated ZF-like region justbeneath the renewing capsular region (Fig. 7A).
Figure 3
IHC and in situ hybridisation in adrenal gland with sham operation. One dot signal
represents one mRNA copy in the RNAscope results. (A) Cyp11b1 expression within situ hybridisation using Basescope. Cyp11b1 was detected in the zonafasciculata of the sham-operated adrenal glands. (B) CYP11B2 expression with
IHC. CYP11B2 signals are indicated as red and autofluorescence as white. (C)DAB2 expression with IHC. (D) PCNA expression with IHC. (E) Gli1 with in situ
hybridisation using RNAscope. (F) Shh with in situ hybridisation using
RNAscope. (G) Dhh with in situ hybridisation using RNAscope. (H) Ihh with insitu hybridisation using RNAscope. In panels (E–H), one dot signal represents one
mRNA copy in the RNAscope results. RNAscope signals are represented as whitecolour and autofluorescences were subtracted from the original figures(Supplementary Fig. 3). The white broken line shows the boundary between theadrenal capsule and adrenocortical cells. Cap: capsule; ZG: zona glomerulosa; ZF:
zona fasciculate; ZU: undifferentiated zone.

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Figure 4
IHC and in situ hybridisation in the adrenocortical autograft at POD7. One dot
signal represents one mRNA copy in the RNAscope results. (A) Cyp11b1
expression with in situ hybridisation using Basescope. Cyp11b1 was detected inremnant adrenocortical cells. (B) CYP11B2 expression with IHC. CYP11B2
signals are indicated as red and autofluorescence as white. No CYP11B2 wasfound. (C) DAB2 expression with IHC. (D) PCNA expression with IHC. (E) Gli1
with in situ hybridisation using RNAscope. (F) Shh with in situ hybridisation
using RNAscope. (G) Dhh with in situ hybridisation using RNAscope. (H) Ihh
with in situ hybridisation using RNAscope. In panels E–H, one dot signal
represents one mRNA copy in the RNAscope results. RNAscope signals are
represented as white colour dots and autofluorescences were subtracted fromoriginal figures (Supplementary Fig. 3). AC: adrenocortical cells.

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Figure 5
IHC and in situ hybridisation in the adrenocortical autograft at POD14. One dot
signal represents one mRNA copy in the RNAscope results. (A) Cyp11b1
expression with in situ hybridisation using Basescope. Cyp11b1 was detected inremnant adrenocortical cells. (B) CYP11B2 expression with IHC. CYP11B2
signals are indicated as red and autofluorescence as white. No CYP11B2 wasfound. (C) DAB2 expression with IHC. (D) PCNA expression with IHC. (E) Gli1
with in situ hybridisation using RNAscope. (F) Shh with in situ hybridisation
using RNAscope. (G) Dhh with in situ hybridisation using RNAscope. (H) Ihh
with in situ hybridisation using RNAscope. In panels E–H, one dot signal
represents one mRNA copy in the RNAscope relults. RNAscope signals are
represented as white colour dots and autofluorescences were subtracted fromoriginal figures (Supplementary Fig. 3). AC: adrenocortical cells.

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Figure 6
IHC and in situ hybridisation in the adrenocortical autograft at POD21. One dot
signal represents one mRNA copy in the RNAscope results. (A) Cyp11b1
expression with in situ hybridisation using Basescope. Cyp11b1 was detected inrenewal adrenocortical cells. (B) CYP11B2 expression with IHC. CYP11B2
signals are indicated as red and autofluorescence as white. No CYP11B2 wasfound. (C) DAB2 expression with IHC. DAB2 was detected in subcapsular region.
(D) PCNA expression with IHC. (E) Gli1 with in situ hybridisation using
RNAscope. (F) Shh with in situ hybridisation using RNAscope. (G) Dhh with insitu hybridisation using RNAscope. (H) Ihh with in situ hybridisation using
RNAscope. In panels E–H, one dot signal represents one mRNA copy in the
RNAscope results. RNAscope signals are represented as white colour dots andautofluorescences were subtracted from original figures (Supplementary Fig. 3).
Cap: capsule; RAC: renewal adrenocortical cells.

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Figure 7
IHC and in situ hybridisation in the adrenocortical autograft at POD28. One dot
signal represents one mRNA copy in the RNAscope results. (A) Cyp11b1
expression with in situ hybridisation using Basescope. Cyp11b1 was detected inrenewal adrenocortical cells. (B) CYP11B2 expression with IHC. CYP11B2
signals are indicated as red and autofluorescence as white. No CYP11B2 wasfound. (C) DAB2 expression with IHC. DAB2 was detected in subcapsular region.
(D) PCNA expression with IHC. (E) Gli1 with in situ hybridisation using
RNAscope. (F) Shh with in situ hybridisation using RNAscope. (G) Dhh with insitu hybridisation using RNAscope. (H) Ihh with in situ hybridisation using
RNAscope. In panels E–H, one dot signal represents one mRNA copy in the
RNAscope results. RNAscope signals are represented as white colour dots andautofluorescences were subtracted from original figures (Supplementary Fig. 3).
Cap: capsule; RAC: renewal adrenocortical cells.

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Recovery of Cyp11b1Cyp11b1 and absence of
Cyp11b2/CypYP11bB2 in adrenocortical autografts
To confirm the expression of Cyp11b1 and Cyp11b2 in whole adrenocortical
autografts, we conducted digital mRNA counts using nCounter technology(Fig. 2H,I). The Rattus Cyp11b1 (Acc#: NM_012537.3) sequence contains
several deletions compared with the Rattus Cyp11b2 sequence (NM_012538.2);
therefore, we created a unique probe against Rattus Cyp11b2, but we could not
create a unique probe for Cyp11b1, because the homology among Rattus
Cyp11b1, Cyp11b2 (94% identity against Cyp11b1), Cyp11b3 (NM_181824.1,
94% identity), and Cypxib1 (XM_017595365.1) is very high.
No expression of Cyp11b2 was found with the specific Cyp11b2 probe inadrenocortical autografts (mean ± SDstandard deviation, 76.4 ± 76.1 at POD7,
19.7 ± 5.6 at POD14, 117.4 ± 152.9 at POD21, 8.8 ± 5.8 at POD28) (Fig. 2I), as
less than 120 counts with the Cyp11b2 probe are considered negative dependingon the background counts. Cyp11b2 was expressed in the sham-operated adrenal
gland (357.2 ± 310.4 at POD7, 3506.6 ± 754.0 at POD14, 4268.8 ± 1133.9 at
POD21, 3758.7 ± 781.1 at POD28). Saline supplementation might havesuppressed the Cyp11b2 expression at POD7 (see Materials and Methods). In
contrast, the Cyp11b1 probe in nCounter detected a significant reduction incytochrome P450 11B mRNAs in the autografts (17313.8 ± 5046.9) comparedwith that in sham animals (146606.1 ± 19691.6) at POD7 (p = 0.021, r = 0.82)
and demonstrated upregulation of cytochrome P450 11B mRNAs in the
autografts (236321.2 ± 20738.8) compared with that in sham animals (131732.2± 29101.7) at POD14 (p = 0.021, r = 0.82) (Fig. 2H). We had estimated that
Cyp11b1 expression was decreased at POD7 and POD14, which reflected a lossof adrenocortical cells as determined using the Basescope. However, Cyp11b1
expression did not result in a gradual increase in serum corticosterone levels andhad a transient peak at POD14, suggesting that the nCounter Cyp11b1 probe
might have detected other cytochrome P450 11B mRNAs. At POD21, there wasno significant difference in the detection of the nCounter Cyp11b1 probe
between the autografts (128927.5 ± 14906.7) and sham animals (147918.0 ±12235.6). The nCounter Cyp11b1 probe detected a significant increase incytochrome P450 11B mRNAs in the autografts (283069.0 ± 29285.4) comparedwith that in sham animals (132757.8 ± 24891. 9) at POD28 (p = 0.021, r = 0.82).
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To examine the presence of ZG cells in the adrenocortical autograft, we
immunohistochemically stained for rattus ZG specific markers, CYP11B2 andDAB2[24, 25]. CYP11B2 staining in the adrenal gland of a sham-operated rat
showed a strong expression in the ZG (Fig. 3B). The absence of CYP11B2 wasobserved in the adrenocortical autograft as well as the results of nCounter(Figs 4B–7B). However, DAB2 signals were detected not only in the ZG ofsham rat, but also in remnant adrenocortical cells at POD7 and POD14 and in thesubcapsular region at POD21 and POD28 (Figs 3C–7C).

Adrenocortical autograft cell proliferation occurs in
subcapsular region
To identify the cell proliferation in adrenal autografts, proliferating cell nuclear
antigen (PCNA) staining was performed. Cell proliferation has been reported to
be dominant in ZG in the adrenal gland,[26] and PCNA signals in adrenal cortex
with sham operation were also found in ZG (Fig. 3D). At POD7 and POD14,
PCNA was broadly and sparsely observed in autografts (Figs 4D and 5D). PCNA
was detected in subcapsular region again at POD21 and POD28 (Figs 6D and
7D).

Upregulation of DhhDhh in adrenocortical autografts
In the adrenal cortex of sham-operated rats, RNAscope findings for Gli1 andShh, which were detected in the adrenal capsule and in the ZU, respectively(Fig. 3E,F), were consistent with a previous report[27]. Gli1 was expressed instromal cells adjacent to the remnant adrenocortical cells at POD7 and POD14(Figs 4E and 5E). Gli1 expression sites were changed to renewal capsular cells
and adrenocortical cells close to the capsule at POD21 (Fig. 6E). Gli1 expression
at POD28 was decreased in the renewal capsular region compared to that at
POD21 (Figs 6E and 7E), and persisted in the adrenocortical cells in thesubcapsular region at POD28 (Fig. 7E). We did not detect any Shh expression
through POD7–21 (Figs 4F–6F). We detected a few Shh signals in theregenerated adrenocortical cells at POD28 (Fig. 7F). In contrast, Dhh was
remarkably and diffusely expressed in the renewal adrenocortical cells at POD21
compared to the sham-operated adrenal gland (Figs 3G and 6G). A few Dhh
expression signals were detected at POD7 and POD14 (Figs 4G and 5G). Dhh
showed scattered expression in both the adrenal cortex with sham operation andadrenocortical autografts at POD28 (Fig. 7G). In contrast to Dhh expression, few
Ihh expression was detected in the adrenal gland upon sham operation and in
adrenocortical autografts. There was also no change throughout the regeneration
period (Figs 3H–7H).

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The dim cytoplasmic fluorescence signals were regarded as autofluorescence ofadrenocortical cells (Supplementary Fig. 3). We used unmixing analysis for
extracting autofluorescence (Supplementary Fig. 4 and method section).

A few Wt1Wt1(+) Gli1Gli1(+) cells in adrenocortical
autografts
WT1 has been reported as a direct regulator of Gli1 without Hh input in theadrenal gland[28]. To clarify the involvement of WT1 in regulating Gli1 in theadrenocortical autograft, double staining with Wt1 (FastGreen) and Gli1
(FastRed) was conducted using RNAscope (Fig. 8). In the adrenal glands ofsham-operated rats, Wt1 expression was found to be weak in the capsule andcortex, and Gli1 was located in the capsule (Fig. 8). Some blurred blue signals in
adrenal cells were regarded as background based on negative control study(Fig. 8). From POD7 to POD21, we found Wt1 expression around the capillaries,
Gli1 expression adjacent to Wt1, and a few Wt1(+) Gli1(+) cells (Fig. 8). Wt1
was suppressed at POD14 and its expression was increased again around theveins at POD21 (Fig. 8). At POD28, Wt1 was re-suppressed around the veins andslightly expressed in renewal adrenocortical cells (Fig. 8).
Figure 8
Wt1 and Gli1 expression in sham-operated adrenal glands and adrenocortical
autografts. Wt1 (FastGreen) and Gli1 (FastRed) were expressed in the adrenal
capsule. Sections on post-operative day (POD)7 to POD21 showed Wt1 expression
around the capillaries, and few Wt1 (+) and Gli1 (+) cells were found. Cap:
capsule; ZG: zona glomerulosa; ZF: zona fasciculata; RAC: renewal
adrenocortical cells; V: vein.

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Discussion
HE staining of adrenocortical autografts showed loss of most adrenocorticalcells by 1 week after operation. The dynamic changes, dependent on the
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regeneration step, occurred between 2 and 3 weeks after surgery as reported
previously[9, 15, 29]. After four weeks, the adrenocortical autografts showedmorphological and functional recovery as shown by the restoration of serum
corticosterone levels with no statistical difference compared to sham animals.
We also found strong expression of Dhh and Gli1 during the remodelling andregeneration process in adrenocortical autografts.
Differentiated ZG cells are known to undergo lineage conversion into ZF
cells[15, 16]. ZG-specific gene deletion of Nr5a1 prevents lineage conversion
into ZF cells, but the production of functional ZF cells is maintained[16]. These
results suggest that ZF cells are produced from ZG cells as well as from othercell populations. There was no obvious ZG in the autograft at POD21 and
POD28 based on our Cyp11b1 and Cyp11b2 /CYP11B2 expression analysis withimmunohistochemistry (IHC), in situ hybridisation, and the nCounter method.
The Cyp11b1 expression pattern in the Basescope analysis and the lack of
Cyp11b2 /CYP11B2 expression in IHC and the nCounter analysis indicated that
almost all the regenerated adrenaladrenocortical cells were ZF cells. Cell
proliferation has been shown dominantly in both mouse ZG[26] and ZF in rat
enucleation models of the adrenal gland[30]. In the present study, PCNA (+)
cells were increased at POD7 and POD14, and strong PCNA (+) cells werelocated in the subcapsular region at POD21 and POD28. These findings wereconsistent with a previous report[31]. These regenerated adrenocortical cells inthe subcapsular region might be directly differentiated into Cyp11b1-expressing
cells in the adrenocortical autografts without the lineage conversion from ZG
cells.
Although no CYP11B2 was found at POD7 and POD14 indicating the absence of
ZG cells, strong DAB2 signals were found in those periods. Absence of ZG inadrenocortical autograft was also previously reported[32]. DAB2 hasmultifaceted roles and is expressed in epithelial cells of several tissues[33].
DAB2 is also induced by physiological stimulations[34]. In addition, it is known
that CYP11B2 expression is restricted to a limited number of ZG cells
expressing DAB2 in humans[35]. Therefore, there might be a dissociation
between CYP11B2 and DAB2 in adrenal cortex remodelling. We observed
DAB2 expression in remnant adrenocortical cells at the early stages. However,subcapsular localisation of DAB2 was found on POD21. It could be said thatimmature ZG cells without CYP11B2 might be regenerated from POD21
onwards.
Not surprisingly, overall Gli1 expression was observed in the adrenocortical
autograft similar to the developmental stage, suggesting the participation of Hhsignalling in the regeneration of adrenocortical autografts. The expression was
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prominent at POD21. Although Shh (+) Gli1 (+) progenitor cells have beenreported to differentiate into steroidogenic cells in the canonical process[18], we
observed only slight expression of Shh in the adrenocortical autografts with nocorrelation between Shh and Gli1 expression in the adrenocortical autografts. We
then performed RNAscope analysis with other Hh signalling ligands, Dhh and
Ihh. Expression of Ihh was hardly found and showed no change in all theexperiments. In contrast, Dhh showed strong and diffused expression in theregenerating adrenocortical cells at 3 weeks after the operation. DHH but not
SHH might be one of the key regulators of GLI1 in adrenocortical autografts as
in gonads[21, 36, 37], and the upregulation of Dhh implies its importance for theregeneration of adrenocortical autografts.
It is well known that WT1 directly controls Gli1 transcription in the adrenal
primordia without Hh signalling[28] and that it is implicated in steroidogenic
cell differentiation in both the adrenal cortex and the gonads[28, 38]. Moreover,WT1 is a tumour suppressor gene and is related to the distant metastasis andprogression of tumour cells by its function as a master regulator of themesenchymal-epithelial transition and epithelial-mesenchymal transition[39].
WT1 is highly expressed in the blood vessels of most human tumours but not in
the blood vessels in normal tissues[40, 41]. In this study, no increase in Wt1
expression was found in the mesenchyme of normal muscle tissues at the site oftransplantation or in the remnant autografted adrenal tissues at POD7. However,WT1 could be involved in graft vascularisation, as a highly specific expressionof Wt1 was observed in possible remnant adrenocortical cells of the perivascularregions in autografts at POD7. Interestingly, an alternative splice variant, WT1KTS, is known to stimulate the expression of the receptor for the vascularendothelial growth factor, Kdr, in murine embryonic gonads[42], and a high Kdr
expression was observed in a previous study on adrenocortical autografts[43].
Accordingly, regulation of vascularisation by WT1 was considered as one of the
key factors for the regeneration step of adrenocortical autografts at the initialstage.
In conclusion, an increase in Dhh and Gli1 expression was observed during theregeneration of adrenocortical tissues. WT1 was suggested to contribute to theregeneration of blood vessels and synergistically potentiate DHH effect. This
report is the first to clearly demonstrate the expression of Dhh and its elevationin the adrenal gland. To clarify whether DHH directly affects adrenocorticalregeneration, further autotransplantation studies using Dhh knockout strains withgenome editing will need to be conducted.

Materials and Methods
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Animals
In total, 37 male Wistar rats (age, 7 weeks; weight, 210–270 g) were purchasedfrom Shimizu Laboratory Supplies (Kyoto, Japan) and housed in a soundattenuated light-controlled room (lights on at 8:00 a.m. and off at 8:00 p.m.; 12 h
light-dark cycle; constant environment at 25 ± 1 °C and 50 ± 10% relative
humidity) for 2 weeks before the operation. Food and water were provided ad
libitum. All animal experiments were approved by the Ethics Committee on
Animal Experiments at Kansai Medical University (approval ID: 17-051) andwere conducted in accordance with the Guide for the Care and Use ofLaboratory Animals of the Institute for Laboratory Animal Research.

Adrenocortical autotransplantation
Adrenocortical autotransplantation was performed in 9-week-old rats as
described previously[44]. Briefly, bilateral adrenal glands were resected undergeneral anaesthesia by inhalation of 2% isoflurane (Pfizer Japan Inc., Tokyo,
Japan) and 3 L/minute oxygen. The adrenal gland was divided into four pieces
and the medulla was discarded. The adrenocortical autografts retaining thecapsule, ZG, and ZU[4] were autotransplanted into the right biceps femoris.
Sham operations without adrenalectomy were performed simultaneously. The
animals could survive on saline without any glucocorticoid replacement
following the adrenalectomy[45]. We then performed blood collection from therat heart and tissue sampling at the following designated periods. Sera wereisolated by centrifugation at 1,500 × g for 30 min at 4 °C and stored at −80 °C
until use.

HE staining
Adrenal tissues were immersion-fixed overnight in 4% formaldehyde inphosphate-buffered saline (PBS), and frozen tissues, embedded in OCT
compound, were cut into 10 µm-thick sections. Each 10- µm-thick frozen section
was hydrated and stained with HE according to the general method.

Immunohistochemistry
The procedure of fixing and sectioning were the same as the procedure in thesection above. Heat-induced epitope retrieval was conducted with 10 mM TrisHCl (pH 6.0) for 5 min for PCNA and DAB2 staining. Inactivation ofendogenous peroxidases with 0.1% phenylhydrazine in PBS was performed for
30 min. Sections were then incubated with mouse anti-PCNA antibody (610664,
BD biosciences, CA, USA; dilution at 1:1000) with PBS containing 0.3% triton
X-100 or mouse anti-Disabled-2/p962 antibody (610464; BD biosciences, CA,
USA; dilution at 1:1000) with PBS containing 0.3% triton X-100 overnight at
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room temperature. After washing with PBS containing 0.3% triton X-100,
sections were incubated with ImmPRESS REAGENT anti-mouse IgG (MP7422; VECTOR LABORATORIES, INC., CA, USA) for 30 min, followed bystaining with diaminobenzidine. Sections were counterstained withhaematoxylin.
For CYP11B2, heat-induced epitope retrieval step was omitted. After
inactivation of endogenous peroxidases, sections were blocked with 10% goat
serum and 0.5% SDS in 0.1 M Tris pH 7.4 for 1 h. Then, sections were incubatedwith rabbit anti-CYP11B2 antibody[24] (dilution 1:6000 with 0.1 M Tris, 10%goat serum and 0.2% Tween-20) supplied by C. Gomez-Sanchez (University ofMississippi Medical Center) overnight at room temperature. After washing with
PBS containing 0.2% Tween-20, sections were incubated with goat anti-rabbit
IgG-Alexa flour 488 for 1 h (A27034; Thermo Fisher Scientific, MA, USA;
dilution 1:1000 with 0.1 M Tris, 10% goat serum and 0.2% Tween-20).
Control experiments with omission of the antibodies were conducted to extract
the reference spectral profile.

Enzyme immunoassay for serum corticosterone and ACTH
levelsWe measured the rat serum corticosterone and ACTH levels in duplicate using a
Corticosterone EIA Kit (YK240; Yanaihara Institute Inc., Shizuoka, Japan) andan ACTH ELISA kit (M046006; MD bioproducts, Zurich, Switzerland)following the manufacturer’s protocol, respectively. The range of detection forthe corticosterone concentration was from 0.21 to 50 ng/mL and for ACTH
concentration was from 1.0 to 640 pg/ml, respectively. The intra-assay
coefficient of variation and inter-assay coefficient of variation were 2.5–4.7%
and 7.7–9.8% in Corticosterone EIA Kit and 2.24–6.71% and 6.9–7.1% in
ACTH ELISA kit, respectively.

RNAscope and Basescope (in situ hybridisation)
Adrenal tissues were treated according to the Sample Preparation Technical Note
for Fixed Frozen Tissue (ACD, Hayward CA, USA). In brief, fixed frozen
tissues embedded in OCT compound were cut into 10- µm-thick sections andmounted on Superfrost Plus slides (Fisher Scientific, Waltham MA, USA) for
RNA in situ hybridisation. These sections were air-dried at −20 °C and stored at−80 °C until use.
We performed RNAscope analysis with rat probes (Supplementary Table 1) toreveal the mRNA expression and localisation in adrenocortical autografts using
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the BaseScope Reagent Kit, RNAscope 2.5HD Reagent Kit (Single-plex, RED),
or RNAscope 2-plex Reagent Kit (ACD) with the HybEZ Hybridisation system
according to the kit manual. Each mRNA was detected with FastRed or
FastGreen. Counterstaining was conducted with 33% Mayer’s haematoxylin or
50% Gill’s haematoxylin.

Direct count of mRNA copies
Adrenal glands of sham-operated rats and adrenocortical autografts were quickly
removed. The removed adrenal tissues were isolated from the surrounding fat
and muscle tissues as much as possible, and total RNA was extracted using
Sepasol-RNA I Super G reagent (Nacalai Tesque, Inc., Kyoto, Japan). Cyp11b1
and Cyp11b2 mRNAs in each sample were hybridised with the target probes(Supplementary Table 2) and reporter tags (Elements XT TagSet system,
NanoString Technologies, Inc. N Seattle, WA). After trapping, the RNA copy
number was counted in a digital analyser with the nCounter system (NanoString
Technologies, Inc. N Seattle, WA) according to the provided manual. We
analysed the data using nSolver analysis software (NanoString Technologies,
Inc. N Seattle, WA). Each mRNA count was normalised to Hprt1 as the internal
control gene. Background subtraction was performed using the raw counts of
RNase-free water.

Microscope imaging
Optical images were collected using ECLIPSE E1000M (Nikon, Tokyo, Japan),
ECLIPSE Ci (Nikon, Tokyo, Japan), or LSM700 (ZEISS, Oberkochen,
Germany) microscopes. Autofluorescence frequency interferes with theidentification of specific signals in fluorescence imaging and varies in eachorgan[46]. Some autofluorescence are derived from endogenous molecules
related to metabolism, such as nicotinamide adenine dinucleotide phosphate, andlipofuscin[46, 47, 48]. Therefore, autofluorescence has been reported to be
useful for monitoring metabolic changes[48]. The adrenal gland is one of theorgans known to produce autofluorescence[47]. We found autofluorescence inthe adrenal gland and adrenocortical autograft tissues (Supplementary Fig. 3).
Recently, “unmixing” technique by spectral imaging was reported to separate
desired signals from autofluorescence[49]. The autofluorescence spectral profilein adrenocortical cells could be clearly discriminated from the fluorescence with
FastRed chromogen by RNAscope and from the fluorescence with Alexa 488
green fluorophore profile using lambda mode and spectral unmixing algorithms
equipped with Zeiss LSM700 microscope. The autofluorescence spectral profile,
as a reference, was extracted from the control experiments described above(Supplementary Fig. 4). Positive signal spectral profile of the fluorescence with
FastRed chromogen in RNAscope was extracted from ovary tissues
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Statistical analysis
Normality was analysed by the Shapiro-Wilk normality test for all groups.
The distributions of the Cyp11b1 and Cyp11b2 expression levels via nCounter
technology were compared between the sham group and PODautograft groups ateach time point using the Mann-Whitney U-test because the data from one groupwere not normally distributed (P < 0.05). P < 0.05 was considered to indicate astatistically significant difference in each comparison of gene expression. The
distributions of corticosterone and ACTH concentrations between the sham andautograft groups waswere compared using ANOVA and the Mann-Whitney U
test using SPSS for Windows. Significance levels were set at p < 0.0125 forcorticosterone and ACTH concentrations after Bonferroni’s correction for
multiple comparisons. The correlations between corticosterone and ACTH
concentrations at each time were examined using the Spearman correlation
coefficient test. A p value less than 0.05 was considered as statisticallysignificant.
Electronic supplementary material
Supplementary information accompanies this paper athttps://doi.org/10.1038/s41598-018-32870-9.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.

Acknowledgements
The present study was supported by the Japan Society for the Promotion of
Science KAKENHI fund (Grant Number 15K08224 and 16K08533 to ST, Grant
Number 16K10483 to TY), a research grant from Yamaguchi Endocrine
Research Foundation and Kansai Medical University to NT, and a MEXTSupported Program for the Strategic Research Foundation at Private Universities(Grant Number S1101034 and S1201038) to HY. The authors would like to thankthe Department of Pathology at Kansai Medical University and Prof. Kiyoshi
Kurokawa (Osaka International University) for their helpful comments, and theCentral Research Center and Kansai Medical University research consortium fortheir support. We thank Ms. Ayako Nagata for providing technical assistance.
Author Contributions
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Study concept and design: N.T., S.T. and H.Y.; acquisition of data: N.T., S.T.,
S.O. and T.K.; processing of data: N.T., and T.K. and S.T.; analysis andinterpretation of data: N.T. and S.T.; drafting of the manuscript: N.T., S.T. and
H.Y.; critical revision of the manuscript for important intellectual content: N.T.,
S.T., S.O., T.K., T.Y., Y.H., T.M. and H.Y.; statistical analysis: N.T. and S.T.;
materials: N.T., S.T., T.Y., T.M. and H.Y.; study supervision: S.T. and H.Y.; All
authors have approved the final draft of this manuscript that was submitted.
Competing Interests The authors declare no competing interests.

Electronic supplementary material
supplementary information

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