DEVELOPMENTAL DYNAMICS 222:341–353 (2001)
Quantitative Axial Profiles of Retinoic Acid in the
Embryonic Mouse Spinal Cord: 9-Cis Retinoic Acid
Only Detected After All-Trans-Retinoic Acid Levels
Are Super-Elevated Experimentally
STINE M. ULVEN,1 THOMAS E. GUNDERSEN,1 AMRIT K. SAKHI,1 JOEL C. GLOVER,2 AND
RUNE BLOMHOFF1*
1
Institute for Nutrition Research, Institute for Basic Medical Sciences, University of Oslo, Oslo, Norway
2
Department of Anatomy, Institute for Basic Medical Sciences, University of Oslo, Blindern, Oslo, Norway
ABSTRACT
Studies using bioassays in normal mice and gene activation in transgenic reporter mice have demonstrated peaks of retinoic
acid receptor (RAR) signaling in the brachial and
lumbar regions of the spinal cord. Recently, Solomin et al. (Solomin et al. [1998] Nature 395:398 – 402)
detected a retinoid X receptor (RXR) signal in the
same region of the developing spinal cord at a
slightly later stage than the RAR signal. This finding raises the question of which retinoid ligands
underlie RAR and RXR signaling in this part of the
embryo. Quantitative measurements of regional
differences in retinoid profiles have not been reported previously due to limitation in the sensitivity and specificity of available retinoid detection
methods. Here, by using a recently developed ultrasensitive HPLC technique (Sakhi et al. [1998]
J. Chromatogr. A 828:451– 460), we address this
question in an attempt to identify definitively the
endogenous retinoids present in different regions
of the spinal cord at the stages when regional differences in RAR and RXR signaling have been reported. We find a bimodal distribution of all-trans
retinoic acid (at-RA), the ligand for RARs, and relate this to the expression of several retinoid-synthesizing enzymes. However, we do not detect
9-cis-retinoic acid (9-cis-RA), the putative RXR ligand, in any region of the spinal cord unless retinoid levels are massively increased experimentally
by gavage feeding pregnant mice with teratogenic
doses of at-RA. This study provides for the first
time quantitative profiles of endogenous retinoids
along the axis of the developing spinal cord,
thereby establishing a foundation for more definitive studies of retinoid function in the future. It
sets definite limits on how much 9-cis-RA potentially is present and demonstrates that at-RA predominates over 9-cis-RA by at least 30- to 180-fold in
different spinal cord regions. © 2001 Wiley-Liss, Inc.
Key words: vitamin A; 9-cis-retinoic acid; alltrans-retinoic acid; spinal cord;
RALDH2
© 2001 WILEY-LISS, INC.
INTRODUCTION
The function of vitamin A during embryonic development is mediated through the action of two classes of
specific retinoid receptors, the retinoic acid receptor
(RAR) and the retinoid X receptor (RXR). Both classes
are ligand-dependent transcription factors belonging to
the nuclear hormone receptor superfamily (Mangelsdorf and Evans, 1995; Mangelsdorf et al., 1995; Chambon, 1996). Little is known about the endogenous retinoid ligands activating these receptors in vivo. A large
number of in vitro experiments have shown, however,
that members of the RAR family are activated by several naturally occurring retinoids, including all-transretinoic acid (at-RA), 9-cis-retinoic acid (9-cis-RA), alltrans-4-oxo-retinoic acid (at-4-oxo-RA), all-trans-4-oxoretinal (at-4-oxo-RAL), all-trans-4-oxo-retinol (at-4oxo-ROH), and all-trans-3,4-didehydro-retinoic acid
(at-dd-RA). Members of the RXR family are more selective, being efficiently activated in vitro by 9-cis-RA,
9-cis-3,4-didehydro-retinoic acid (9-cis-dd-RA) and 9cis-4-oxo-retinoic acid (9-cis-4-oxo-RA) (Mangelsdorf
and Evans, 1995; Mangelsdorf et al., 1995; Chambon,
1996). These ligands are usually synthesized in vivo by
complex metabolic systems, involving numerous enzymes and binding proteins (see Ulven et al., 2000 and
references therein).
The central nervous system (CNS) is a major site of
retinoid action, as both vitamin A deficiency and excess
cause abnormal neural patterning and development
(Durston et al., 1989; Altaba and Jessell, 1991; Maden
et al., 1997; Niederreither et al., 2000). Moreover, null
mutations of specific combinations of nuclear retinoid
receptors produce the same spectrum of abnormalities
Grant sponsor: Norwegian Medical Research Council; Grant sponsor: Throne Holst Fund: Grant sponsor: Nansen Fund.
Drs. Ulven and Glover contributed equally to the execution of this
work.
Drs. Glover and Blomhoff cosupervised the work.
*Correspondence to: Rune Blomhoff, Institute for Nutrition Research, Institute for Basic Medical Sciences, University of Oslo, P. O.
Box 1046 Blindern, 0316 Oslo, Norway.
E-mail: rune.blomhoff@basalmed.uio.no
Received 17 January 2001; Accepted 27 June 2001
Published online 2 October 2001; DOI 10.1002/dvdy.1184
342
ULVEN ET AL.
(Lohnes et al., 1994; Dupe et al., 1999). In addition to
these teratogenic effects, retinoids have been implicated as pivotal regulators of the normal determination
and differentiation of neurons. For example, experimental manipulation of retinoid synthesis modifies
gene expression patterns and the differentiation of specific neuron classes in the developing spinal cord (Forehand et al., 1998; Sockanathan and Jessell, 1998; Pierani et al., 1999). At least some of these effects seem to
derive from the neighboring paraxial mesoderm, either
directly by retinoids synthesized in the mesoderm or
potentially through the action of other factors whose
synthesis in the mesoderm is regulated by retinoids
(Forehand et al., 1998; Gould et al., 1998; Sockanathan
and Jessell, 1998).
Several studies have addressed the axial localization
of retinoid signaling in the developing spinal cord.
Gene activation in transgenic reporter mice (Reynolds
et al., 1991; Colbert et al., 1993; Solomin et al., 1998;
Mata et al., 1999) and bioassay systems (Colbert et al.,
1993; McCaffery and Drager, 1994) have demonstrated
peaks of retinoid signaling in the brachial and lumbar
regions of the spinal cord. In situ hybridization in
mouse embryos and immunohistochemistry in chicken
embryos has demonstrated strong expression of the
retinoic acid synthesizing enzyme RALDH2 in the
somites, the meninges, the roof plate of the neural
tube, and particularly in motoneurons in the brachial
and lumbar regions, the last-mentioned, thus, paralleling the bimodal axial profile of retinoid signaling (Niederreither et al., 1997; Sockanathan and Jessell, 1998;
Berggren et al., 1999).
Recently, Solomin et al. (1998) developed an elegant
transgenic reporter mouse model in which the DNAbinding domain of GAL4 is fused to the ligand-binding
domain of either RAR or RXR. They demonstrated activation of the RAR construct in the brachial and lumbar part of the spinal cord starting at 10.5 days post
coitum (dpc). They also observed activation of the RXR
construct in the same regions slightly later, starting at
11.0 –12.0 dpc.
Transgenic reporter mouse models demonstrate unequivocally whether a specific type of receptor has been
activated, but they provide only qualitative information about the signaling involved. They do not show
which ligands have been bound or in which concentrations. Nor do they provide a reliable indication of temporal dynamics, because the time course of the reporter
gene signal is not only due to ligand-receptor and receptor-response element interactions, but also to reporter gene transcript and protein lifetimes, which
could last hours beyond termination of ligand-receptor
binding. Thus, to advance our understanding of how
retinoid signaling regulates spinal cord development,
direct, quantitative measurements of endogenous retinoid ligands and receptors are essential.
By using high-pressure liquid chromatography
(HPLC), Horton and Maden (1995) detected substantial amounts of at-RA in homogenates from whole spi-
nal cords of 10.5 dpc mouse embryos, but did not detect
other candidate ligands such as 9-cis-RA, 3,4-didehydro-retinoids, or 4-oxo-retinoids. Presumably, the endogenous at-RA is responsible for the various reports of
RAR activation. Because 9-cis-RA is considered the
principal ligand for RXRs, the absence of detectable
9-cis-RA raises the question as to which ligands drive
the RXR activation observed by Solomin et al. (1998)
starting at 11 dpc. One possibility is that 9-cis-RA
appears at later stages than assayed by Horton and
Maden (1995), another is that 9-cis-RA is present but
below the limit of detection, and a third is that RXR
activation is driven by other ligands (Eager et al., 1992;
Harmon et al., 1995; Kitareewan et al., 1996) or by
ligand-independent mechanisms such as phosphorylation (Lefebvre et al., 1995; Rochette-Egly et al., 1995).
As we have recently developed a substantially more
sensitive HPLC method (Sakhi et al., 1998), we now
re-address this question in an attempt to identify definitively the endogenous retinoid ligands present at
the stages when Solomin et al. (1998) report RAR and
RXR receptor activation. Moreover, by microdissection
of over 100 embryonic spinal cords, we extend the findings of Horton and Maden (1995) by measuring different regions of the spinal cord separately to obtain for
the first time quantitative axial profiles. We find a
bimodal distribution of at-RA, which we relate to the
expression of RALDH2 (assayed by immunohistochemistry) and of several other retinoid-synthesizing enzymes (assayed by reverse transcription-polymerase
chain reaction [RT-PCR]). However, we do not detect
9-cis-RA in any region of the spinal cord unless at-RA
levels are massively increased experimentally by gavage feeding pregnant mice with teratogenic doses of
at-RA. Therefore, these results set a definite ceiling on
the maximum amount of 9-cis-RA that could be present
(if indeed 9-cis-RA is normally present at all) in the
different spinal cord regions.
RESULTS
Axial Profiles of Endogenous Retinoids
Studies using bioassays and transgenic reporter
mice have revealed axial profiles of retinoid synthesis
and signaling along the developing spinal cord with
peaks in the brachial and lumbar regions (Reynolds et
al., 1991; Colbert et al., 1993; McCaffery and Drager,
1994; Solomin et al., 1998; Mata et al., 1999). To identify which retinoids are actually present, and to quantitate their axial differences, we used a combination of
microdissection and sensitive HPLC techniques to
measure endogenous retinoids in the brachial, thoracic, and lumbar regions of 11.0 –12.0-dpc spinal cord.
The tissue isolation and preparation, chromatographic
separation, and electrochemical detection methods we
used have been fully validated and show excellent precision and reproducibility (Sakhi et al., 1998). The mole
limits of detection are 70 fmoles for at-ROH, 27 fmoles
for at-RA, and 67 fmoles for 9-cis-RA.
ENDOGENOUS RETINOIDS IN DEVELOPING SPINAL CORD
Fig. 1. Camera lucida drawing of the dorsal aspect of a decapitated
12 days post coitum (dpc) embryo, indicating the regions of spinal cord
(blocked out; B, brachial; T, thoracic; L, lumbar) and the corresponding
regions of body wall (separated by dashed lines) dissected for retinoid
analysis. The shaded regions of the spinal cord represent the regions of
retinoid X receptor (RXR) transactivation reported by Solomin et al.
(1998). The dissected spinal cord regions correspond to segments C1 to
T3 (B), T5 to T12 (T), and LS1-LS11 (L). One segment was removed in
between each region to avoid overlap of the thoracic region with the
regions of RXR transactivation (in dissections for reverse transcriptase
polymerase chain reaction experiments, this was not done). Scale bar ⫽
1 mm.
In initial experiments, we dissected 55 spinal cords
into three regions (Fig. 1), made tissue homogenates,
and injected homogenate volumes equivalent to each
region from 40 spinal cords into the HPLC system
(homogenate volumes equivalent to 4.4 spinal cords
were used for protein measurements and homogenate
volumes equivalent to 4.4 spinal cords were used for
at-ROH quantification, see below). We detected prominent peaks corresponding to at-ROH, at-RA, and an
unidentified compound (Fig. 2A). The identification of
at-ROH and at-RA was confirmed by using HPLC-mass
spectrometry (MS) and full-spectrum ultraviolet (UV)
analysis on a separate homogenate of the lumbar re-
343
gions from 10 spinal cords dissected on a separate
occasion (Fig. 3). By comparison with retention times of
known standards, we could exclude all of the compounds listed in Table 1 as possible candidates for the
unidentified prominent peak, which moreover did not
exhibit the typical UV-spectral characteristics of a retinoid. We also detected minor peaks. One of these corresponded either to at-RAL, or 13-cis-ROH, or both; we
could not discriminate these by full-spectrum UV analysis because the sample was too small. Several other
minor peaks could not be identified, but could be excluded from the list in Table 1 on the basis of retention
time (Fig. 2A). The same results were obtained in a
separate assay of 15 spinal cords dissected on a separate occasion.
Because retinoid receptors can also be activated by
compounds other than retinoids (Eager et al., 1992;
Harmon et al., 1995; Kitareewan et al., 1996), and we
obtained a prominent peak that is not a retinoid, we
assayed other potential ligands, focusing on relevant
fatty acids. Arachidonic acid (ARA), docosahexaenoic
acid (DHA), and eicosapentaenoic acid (EPA) standards were all detectable with our ECD system, but
none of these corresponded to any of the peaks in the
tissue chromatograms. In general, nonconjugated fatty
acids in physiological plasma concentrations give weak
or negative responses in the described ECD system.
Both at-ROH and at-RA, as well as the unidentified
prominent peak, exhibited bimodal axial profiles with
the highest level in brachial, the lowest level in thoracic, and an intermediate level in the lumbar region
(Table 2). Amounts of at-ROH substantially exceeded
those of at-RA, being approximately 3.5, 6, and 2 times
higher in the brachial, thoracic, and lumbar regions,
respectively, when normalized to protein (Fig. 4A).
To estimate the tissue concentrations of the extant
at-RA, we divided the measured amounts by the approximate volumes of the dissected brachial, thoracic,
and lumbar spinal cord regions. This method provides
a minimum concentration that assumes an even distribution of the at-RA throughout the tissue and probably
underestimates the effective concentrations. The volumetric concentration estimates were 74 nM (brachial),
36 nM (thoracic), and 200 nM (lumbar).
To compare the retinoid profiles in the spinal cord
with those in the body wall, we analyzed the retinoid
contents of the brachial, thoracic, and lumbar regions
of the remainder of the embryos after evisceration and
removal of the spinal cord (Figs. 1, 2B). This analysis
was done three times, each on homogenates from 15
embryos, with samples representing an additional two
or three embryos taken for protein and separate ROH
analyses. The profiles in the body wall (Fig. 4B) differed from those in the spinal cord in two ways. First,
neither at-ROH nor at-RA exhibited bimodal axial profiles in the body wall. Indeed, at-RA levels were higher
in thoracic than in brachial and lumbar regions, despite the inclusion of the extremities in the brachial
and lumbar regions. Second, at-ROH levels exceeded
344
ULVEN ET AL.
Fig. 2. High performance liquid
chromatography analyses of spinal
cord (A, n ⫽ 40) and body wall
(B, n ⫽ 15) dissected from 11.0- to
12.0-day post coitum (dpc) mouse
embryos, by using on-line solid
phase extraction-electrochemical
detection (ECD) system. The peaks
labelled 1, 2, and 3 coeluted with
all-trans-retinal/13-cis-retinol, all-transretinol, and all-trans-retinoic acid,
respectively. The filled arrowheads
indicate the retention time of a prominent unidentified substance, and
the open arrowheads indicate the retention time of the 9-cis-retinoic acid
standard.
at-RA levels by much more in the body wall than in the
spinal cord (being approximately 19, 11, and 10 times
higher in the brachial, thoracic, and lumbar regions,
after normalization to protein).
Nondetection of 9-cis-RA
We did not detect 9-cis-RA in any region of the spinal
cord or body wall (Fig. 2). However, nondetection does
not necessarily mean absence. Given the detection
limit of 9-cis-RA in our electrochemical detection system, if 9-cis-RA were present in any part of the spinal
cord, it would have to be in amounts less then 67
fmoles. This amount would give ratios of at-RA to 9-cisRA greater than 179, 30, and 108 in brachial, thoracic,
and lumbar regions, respectively. As will be described
in a later section, our assay system is capable of detecting 9-cis-RA in embryonic tissue, so the lack of
detection in the normal spinal cord at this stage of
development represents a definite upper limit equivalent to the detection limit.
To test whether the lack of detection of 9-cis-RA was
due to nonenzymatic isomerization during dissection,
we carried a 9-cis-RA standard solution through the
entire procedure. To test whether lack of detection was
due to degradation of 9-cis-RA during postdissection
procedures, we added known amounts of 9-cis-RA to
parallel tissue homogenates (cells in homogenates were
lysed by freeze-thawing to release intracellular contents), both before and after protein extraction. In no
case did we observe degradation or isomerization of the
9-cis-RA.
mRNA Expression of Retinoid
Metabolic Enzymes
By using RT-PCR we analyzed the expression of
genes coding for enzymes known to oxidize retinol into
retinoic acid. In each experiment, we assessed ␤-actin
expression as an internal control, and we used either
mRNA isolated from adult liver, or from whole embryos
(9.5 or 10.5 dpc), tissues known to express the various
enzymes, as positive controls for each primer pair. One
sample in each experiment was run without reverse
transcriptase to ensure that there was no amplification
of genomic DNA.
We analyzed two classes of enzymes. One includes
the alcohol dehydrogenases (ADH) and the short chain
dehydrogenase/reductases (SDR, includes cis-retinol/
androgen dehydrogenases (CRAD) and retinol dehy-
ENDOGENOUS RETINOIDS IN DEVELOPING SPINAL CORD
345
malization to protein (obtained from the same dissection), which raises the relative thoracic amount (Fig.
5). The other transcripts gave more variable patterns
that were not convincingly bimodal.
Axial Profiles of RALDH2 Immunoreactivity
Fig. 3. Confirmation of retinoid identities by high performance liquid
chromatography-diode array detection and high performance liquid chromatography-mass spectrometry. The lumbar region of the spinal cord
was dissected from ten 12.5 days post coitum (dpc) embryos and analyzed as described in the Experimental Procedures section. The on-line
recorded ultraviolet (UV) spectra (A) of peaks 2 and 3 (from the inverted
UV elution profile obtained at 350 nm, shown in upper trace of B) are
identical to the spectra of authentic at-ROH (UV maximum at 325 nm)
and at-RA (UV maximum at 350 nm), respectively. B: Elution profile
resulting from single ion monitoring of m/z ⫽ 269, indicating the presence
of retinol (peak 2, black), and m/z ⫽ 301, indicating the presence of
retinoic acid (peak 3, grey). The black and grey arrowheads indicate the
elution of the at-ROH and at-RA standards, respectively.
drogenases (RDH or RoDH)), which catalyze the conversion of retinols to retinals. Of these, ADH4 and
CRAD2 are able to convert both at-ROH and 9-cisROH, whereas ADH1, RoDH1, and RoDH3 are specific
for all-trans-ROH versus 9-cis-ROH and CRAD1 and
RDH5 have the opposite specificity. The other enzyme
class includes the aldehyde and retinaldehyde dehydrogenases (ALDH, RALDH), which catalyze the conversion of retinals to retinoic acids. Of these, both
ALDH1 and RALDH2 oxidize at-RAL and 9-cis-RAL
into the respective retinoic acids.
We did not detect CRAD2, RoDH1, or RoDH3 transcripts in whole 11.5-dpc embryos; therefore, we did not
assay for these in isolated spinal cord regions. Transcripts coding for ADH1, ADH4, RDH5, CRAD1,
ALDH1, and RALDH2 were detected in each region of
the spinal cord (Fig. 5). CRAD1 and RALDH2 were the
only transcripts that in all measurements showed a
consistently bimodal pattern, with a lower amount of
product in the thoracic relative to the brachial and
lumbar regions. This finding was true even after nor-
Antibodies to RALDH2, but not to CRAD1, are available. Previous studies have demonstrated the presence
of RALDH2 immunoreactivity in the spinal meninges,
in the roof plate, and in motoneurons of the lateral
motor column in the brachial and lumbar regions
(Niederreither et al., 1997; Sockanathan and Jessell,
1998; Berggren et al., 1999). We assessed the axial
distribution of RALDH2 immunoreactivity in 11.0- to
12.5-dpc mouse embryos to correlate its spatial distribution with the retinoid and RALDH2 transcript data
described above.
We confirmed the presence of RALDH2 immunoreactivity in brachial and lumbar motoneurons (Fig. 6).
We also found lightly stained cells within the cervical
region of the spinal cord. We saw no RALDH2-positive
cells in the thoracic region of the spinal cord. In contrast to the discontinuous distribution of RALDH2 immunoreactivity within the spinal cord, immunoreactivity in the surrounding mesenchyme, in particular that
which was condensing into the spinal meninges, was
continuous along the entire extent of the spinal cord.
Nevertheless, the meningeal staining was modulated
rostrocaudally, with highest intensity in the cervical
and brachial regions, lowest intensity in the thoracic
region, and an intermediate intensity in the lumbar
region.
Changes in Retinoid Levels After Teratogenic
Doses of at-RA
Solomin et al. (1998) showed that RXR activationmediated lacZ expression, which is normally restricted
to the brachial and lumbar regions, has spread
throughout the spinal cord 12 hr after the administration of a teratogenic dose of at-RA to pregnant mice at
11.0 dpc. To assess how such treatments affect the
levels of the various retinoids in the spinal cord, we
similarly administered teratogenic doses of at-RA following several paradigms. Three and 6 hr after administration of 100 mg/kg at-RA to 11.5-dpc pregnant mice
(Satre and Kochhar, 1989), there was a massive increase (approximately 200 times) of at-RA levels in a
homogenate of five whole spinal cords. Several additional peaks appeared, including at-4-oxo-RA and 13cis-RA. Moreover, a minor peak corresponding to 9-cisRA appeared (Fig. 7). We confirmed the identity of this
peak as 9-cis-RA by using coelution of an added 9-cisRA standard.
The amount of 9-cis-RA per spinal cord was 0.16
pmoles at 3 hr and 0.15 pmoles at 6 hr after administration. Amounts in the rest of the embryo were 0.6
pmoles after 3 hr falling to 0.3 pmoles after 6 hr. In
comparison, the amount of endogenous at-RA per spinal cord in a homogenate of five whole spinal cords
346
ULVEN ET AL.
TABLE 1. List of Retinoids and Fatty Acids
Retinoid isomer
All-trans-retinola
11,13-di-cis-retinol
9-cis-retinol
All-trans-3,4-didehydro-retinol
9-cis-3,4-didehydro-retinol
All-trans-retinal/13-cis-retinolb
13-cis-retinal
9-cis-retinal
All-trans-3,4-didehydro-retinal
9-cis-3,4-didehydro-retinal
All-trans-retinoic acida
13-cis-retinoic acidc
9-cis-retinoic acidc
All-trans-3,4-didehydro-retinoic acid
9-cis-3,4-didehydro-retinoic acid
All-trans-4-oxo-retinoic acidc
Fatty acid
Arachidonic acid
Docosahexaenoic acid
Eicosapentaenoic acid
Abbreviation
Detected
At-ROH
11-cis, 13-cis-ROH
9-cis-ROH
At-dd-ROH
9-cis-dd-ROH
At-RAL/13-cis-ROH
13-cis-RAL
9-cis-RAL
At-dd-RAL
9-cis-dd-RAL
At-RA
13-cis-RA
9-cis-RA
At-dd-RA
9-cis-dd-RA
At-4-oxo-RA
Yes
No
No
No
No
Yes
No
No
No
No
Yes
After gavage feeding
After gavage feeding
No
No
After gavage feeding
ARA
DHA
EPA
No
No
No
For definition of abbreviations, see Introduction section.
a
Based on coelution with authentic standards, full-spectrum ultraviolet analysis, and mass spectrometry analysis.
b
These two retinoids could not be distinguished using electrochemical detection alone. A fullspectrum analysis could not be performed because the amount of tissue was too small.
c
Based on coelution with authentic standards.
TABLE 2. Axial Distributions of Detected Retinoidsa
Retinoid
at-ROHb
at-RA
9-cis-RAc
Brachial
60.4
11.8
⬍0.067
Thoracic
11.2
2.0
⬍0.067
Lumbar
21.0
7.2
⬍0.067
a
Amounts in picomoles per 40 spinal cords (not normalized
to protein). at, all trans; ROH, oxo-retinol; RA, retinoic acid.
b
We were not able to quantify the peak corresponding to
at-ROH in the homogenate of 40 spinal cords because it
saturated the detection system. Instead, we injected a separate homogenate volume equivalent to 4.4 spinal cords for
this purpose. The value from this assay has been multiplied
by 9.1 to represent the equivalent of 40 spinal cords.
c
9-cis-RA could not be detected in any regions, i.e., amounts
must be lower than the limit of detection, which equals 0.067
pmoles.
from untreated control embryos was 0.55 pmoles (this
is nearly the same as for the measurements shown in
Table 2, where addition of brachial, thoracic, and lumbar amounts equals 0.58 pmoles). Thus, despite superelevation of at-RA levels, 9-cis-RA levels do not reach
the normal at-RA level.
Might the 9-cis-RA observed after gavage feeding
represent an artefact? We checked the at-RA we administered to the pregnant mice assiduously for purity
and detected no contamination by 9-cis-RA. To check
whether the 9-cis-RA seen in embryonic tissues after
gavage feeding arose by isomerization of at-RA during
our procedures, we performed control experiments: (1)
at-RA standards in buffer were incubated in Eppendorf
tubes under the same dissection, freezing, and storage
procedures; (2) the same amount of at-RA as we measured in the embryonic tissues after teratogenic doses
was added to homogenates of native embryonic tissue,
either before or after protein extraction. Retinoids were
assayed in these test cases either immediately or after
storage at ⫺70°C. In no case did we observe any
isomerization of exogenous at-RA to 9-cis-RA, indicating that our procedures did not artefactually isomerize
endogenous at-RA to 9-cis-RA.
Surprisingly, when we administered teratogenic doses
of at-RA by using the paradigm of Solomin et al. (1998),
namely analysis 12 hr after administration of 20 mg/kg
at-RA to 11.0-dpc pregnant mice, we found no increase in
at-RA levels in homogenates of brachial, thoracic, or lumbar regions from 20 spinal cords. Nor was 9-cis-RA detected. The absence of an increase is almost certainly
because retinoid elevations generated by this treatment
have already subsided within 12 hr, based on time
courses of retinoid elevation published previously (Creech
Kraft et al., 1987, 1989; Satre and Kochhar, 1989; Kochhar et al., 1995; Ward and Morriss-Kay, 1995).
DISCUSSION
We report here the first quantitated axial profiles of
endogenous retinoids in the developing spinal cord,
obtained with an advanced HPLC system with separation capability and sensitivity sufficient to provide definitive identification of individual retinoids. The main
ENDOGENOUS RETINOIDS IN DEVELOPING SPINAL CORD
Fig. 4. Axial profiles of endogenous retinoids in (A) spinal cord and
(B) the remaining body wall dissected from 11.0- to 12.0-day post coitum
(dpc) embryos. The columns represent the mean values of endogenous
retinoids in the brachial, thoracic, and lumbar regions from two or three
separate analyses. The solid squares indicate the individual measurements. Values are per embryo, normalized to protein. at-RA, all-transretinoic acid; at-ROH, all-trans-retinol; BW, body wall.
findings are (1) Amounts of at-RA normalized to protein are approximately 5 and 4 times higher in brachial
and lumbar regions than in the thoracic region, and
amounts of the precursor at-ROH are approximately 3
and 1.5 times higher in brachial and lumbar regions
than in the thoracic region. These bimodal profiles
support previous reports of retinoid synthetic activity
(McCaffery and Drager, 1994; Berggren et al., 1999)
and retinoid-driven reporter gene assays (Reynolds et
al., 1991; Colbert et al., 1993; Solomin et al., 1998;
Mata et al., 1999). (2) 9-cis-RA is not detected in
the untreated spinal cord. If endogenous 9-cis-RA is
present at all, it cannot exceed 0.6, 3.4, and 0.9% of the
amount of endogenous at-RA in brachial, thoracic, and
347
Fig. 5. Expression of transcripts coding for enzymes potentially involved in retinoic acid production. These are examples taken from one of
several reverse transcriptase polymerase chain reaction runs. Only for
CRAD1 and RALDH2 did we consistently see a bimodal profile of transcript expression. ADH, alcohol dehydrogenase; RDH, retinol dehydrogenase; CRAD, cis-retinol/androgen dehydrogenases; ALDH, aldehyde
dehydrogenase; RALDH, retinaldehyde dehydrogenase.
lumbar regions respectively. (3) 9-cis-RA is detected,
however, after super-elevation of at-RA levels. When
at-RA levels are increased approximately 200-fold by
gavage feeding of pregnant dams, the amount of 9-cisRA in the entire spinal cord reaches approximately 160
fmoles, or approximately 3.5 times lower than the
amount of endogenous at-RA normally present. (4)
Transcripts coding for several retinoid synthesizing enzymes are present in the spinal cord, and transcripts
for two of these, namely CRAD1 and RALDH2, exhibit
bimodal profiles. (5) RALDH2 immunoreactivity exhibits a bimodal profile similar to that seen in the chicken
embryo, as it is expressed in the lateral motor column
(Sockanathan and Jessell, 1998; Berggren et al., 1999).
In addition, there is strong expression in the mesenchyme surrounding the spinal cord, particularly that
348
ULVEN ET AL.
Fig. 6. Retinaldehyde dehydrogenase (RALDH2) immunostaining
in transverse sections of brachial
(B), thoracic (T), and lumbar (L) regions. The upper left panel shows a
dorsal view of a three-dimensional
reconstruction of a decapitated embryonic day (E) 12 mouse embryo
that was sectioned transversely and
immunostained for RALDH2. Rostral
is up. The spinal cord is colored dark
gray, and populations of RALDH2positive spinal neurons are colored
light gray. Individual sections taken
from the brachial (B), thoracic (T),
and lumbar (L) regions are shown at
right, with dorsal up. Motoneurons
are labeled in the brachial and lumbar regions but not the thoracic region. Note that the brachial population of RALDH2⫹ motoneurons may
extend slightly more caudally than
shown in the upper left panel, as a
few sections from the brachial/
thoracic transition were used for immunostaining controls. Roof plate
and condensing meningeal mesenchyme are labeled along the entire
spinal cord. Scale bar ⫽ 1 mm.
which is coalescing into the spinal meninges. Meningeal expression is continuous along the entire length of
the spinal cord but appears to be modulated with a
profile similar to that seen for RALDH2 transcripts
and for endogenous retinoids. Finally, RALDH2 immunoreactivity is present in the roof plate with similar
intensity along the entire spinal cord.
Bimodal Axial Profiles
In previous studies, bioassays and reporter mice
demonstrated the presence of retinoids in the developing neuraxis including the spinal cord, with higher
concentration at brachial and lumbar levels relative to
thoracic levels (Reynolds et al., 1991; Rossant et al.,
ENDOGENOUS RETINOIDS IN DEVELOPING SPINAL CORD
Fig. 7. High performance liquid chromatographic analysis of spinal
cords isolated from embryos carried by pregnant mice, which were gavage-fed with all-trans-retinoid acid. The lower and upper traces are
elution profiles for homogenates equivalent to 4.4 spinal cords isolated 3
and 6 hours, respectively, after feeding. Peak 1 coeluted with all-trans4-oxo-retinoic acid, peak 2 coeluted with 13-cis-retinoic acid, peak 3
coeluted with all-trans-retinol, peak 4 coeluted with 9-cis-retinoic acid,
and peak 5 coeluted with all-trans-retinoic acid.
1991; Colbert et al., 1993; Maden et al., 1998; Solomin
et al., 1998; Mata et al., 1999). This bimodal axial
distribution was correlated with retinoid synthesis
(McCaffery and Drager, 1994) and with RALDH2-positive motoneurons, which are restricted to brachial and
lumbar levels (Niederreither et al., 1997; Sockanathan
and Jessell, 1998; Berggren et al., 1999). However,
none of these studies used techniques that allowed
definitive identification of the retinoids in question. We
have shown here that both at-RA and at-ROH exhibit a
clear bimodal axial distribution along the spinal cord
(see Table 1). An extensive list of other retinoids were
assayed (see Table 1), including all that have been
implicated as ligands for RARs and RXRs. Of these,
only at-RAL and/or 13-cis-ROH were present in detectable amounts. Neither of these functions as ligands.
We failed to detect several other compounds that have
been implicated as ligands. We wish to emphasise that
our HPLC system is substantially more sensitive than
any others used for embryonic retinoid measurements
previously. Therefore, it seems that at-RA is the principal, if not the only, retinoid that can generate the
bimodal retinoid signals seen in previous studies. We
cannot exclude that a transient binding of ligand to
RXRs at earlier stages of development may have produced the signal seen at 11.0 –12.0 dpc.
Our RT-PCR assays of enzyme transcripts shed light
on which synthetic pathways may be involved in the
synthesis of RA in the spinal cord. The synthesis of RAs
is a two-step reaction, involving several enzymes that
can oxidize retinols to retinaldehydes, and several enzymes that can further oxidize retinaldehydes to retinoic acids. Transcripts coding for several enzymes participating in each of these steps were present in the
349
spinal cord. CRAD1 and RALDH2 mRNA were the only
transcripts that exhibited a bimodal profile.
Antibodies to RALDH2, but not CRAD1, are available. Therefore, we performed an immunohistochemical analysis of RALDH2 expression to correlate the
spatial distribution of this enzyme with that of retinoids and enzyme gene transcripts. The immunostaining confirms the tissue distribution of RALDH2 demonstrated previously in both mouse and chicken
embryos. RALDH2 is expressed in the roof plate, in
motoneurons of the lateral motor column (restricted to
brachial and lumbar regions), and in the surrounding
meninges and adjacent mesenchyme, as well as other
sites further removed from the spinal cord (Zhao et al.,
1996; Niederreither et al., 1997; Sockanathan and Jessell, 1998; Berggren et al., 1999; Haselbeck et al.,
1999). These earlier studies did not assess axial differences in RALDH2 expression in tissues outside the
neural tube. Our findings supplement these by showing
that the meningeal expression is also modulated along
the length of the cord, in a profile similar to that of
endogenous at-RA. Thus, the bimodal profile of retinoids can be related to intramedullary as well as extramedullary sites of synthesis. However, the detection
of retinoid signaling in reporter mice clearly has a
threshold that is not reached by the lower levels of
retinoids present in the thoracic region.
Several manipulations in chicken embryos have
shown that retinoid signaling plays a pivotal role in
patterning the developing spinal cord. Sockanathan
and Jessell (1998) and Pierani et al. (1999) have demonstrated retinoic acid-dependent regulation of ventral
progenitor proliferation and ventral neuron differentiation. In particular, retinoic acid is required for the
differentiation of motoneurons of the lateral subdivision of the lateral motor column, which are presumably
exposed to retinoic acid as they migrate past RALDH2positive motoneurons in the medial subdivision of the
lateral motor column (Sockanathan and Jessell, 1998).
Thus, the restriction of RALDH2 expression to brachial
and lumbar levels within the ventral part of the spinal
cord can explain why the lateral subdivision of the
lateral motor column only differentiates at these levels.
Pharmacologic experiments using receptor-specific antagonists suggest that this effect of retinoic acid requires the activation of both RARs and of RXRs. Retinoic acid is also required for the differentiation of
specific classes of ventral interneurons (Pierani et al.,
1999), but because these interneuron classes are also
found in the thoracic region, it would seem that extramedullary sources of retinoic acid, such as the meninges, are involved in their differentiation.
In another study, Forehand et al. (1998) applied
at-RA or RA synthesis-blocking drugs to individual
somites at thoracic levels and observed changes in the
differentiation of sympathetic preganglionic neurons.
Of interest here is the combined intrasegmental and
intersegmental pattern of differentiation of these neurons, a feature that led Forehand et al. (1998) to pro-
350
ULVEN ET AL.
pose the combined action of segmentally iterated
signals (presumably from the somites) and a longitudinally graded signal in setting up the pattern. It will be
interesting in this regard to assess the contribution of
meninges-derived and motoneuron-derived retinoic
acid to the longitudinally graded component.
Lack of Detection of 9-cis-RA
Solomin et al. (1998) recently observed a bimodal
activation pattern of a Gal4-RXR fusion protein in the
spinal cord of 11.0- to 12.0-dpc mouse embryos. This
finding demonstrates the activation of RXRs and suggests the presence of an endogenous RXR ligand in the
brachial and lumbar regions of the cord. Because 9-cisRA generally is considered the principal ligand for
RXRs, it is important to determine whether 9-cis-RA is
indeed present in the spinal cord at these stages.
We did not detect 9-cis-RA under normal conditions,
whereas at-RA was readily detectable. The limit of
detection for 9-cis-RA in our HPLC assay system gives
a predominance of at-RA over 9-cis-RA of at least 180fold in the brachial region, 30-fold in the thoracic region, and 110-fold in the lumbar region. These ratios
could in fact be much higher. Although we cannot eliminate the possibility that 9-cis-RA is present, it is
clearly far less abundant than at-RA.
Our estimates of volumetric concentrations of at-RA
ranged from 36 to 200 nM. Such estimates must be
interpreted with caution. First, retinoids may be sequestered into subpopulations of cells within a tissue,
giving potentially higher concentrations. For example,
if at-RA were localized to motoneurons only, our volumetric concentration estimates would be several 10fold too low. On the other hand, our measurements are
of extracted retinoids. Binding of retinoids to proteins
in vivo, for example cellular retinoic acid binding protein, could substantially lower their free concentrations. Therefore, it is entirely speculative to extrapolate from the limit of 9-cis-RA detectability, which
effective concentrations 9-cis-RA might obtain in the
spinal cord. What we do know is that the signal observed by Solomin et al. (1998) at 11.5 dpc was
throughout the dorsoventral extent of the spinal cord,
i.e., it was not limited to a minor subpopulation of
spinal cord cells. Therefore, it seems likely that 9-cisRA, if present, cannot be concentrated to much more
than a few nM, and possibly substantially less.
Whether such concentrations are sufficient to generate
the observed signal remains to be seen.
Solomin et al. (1998) also observed an activation of
the Gal4-RXR fusion protein throughout the longitudinal axis of the embryonic spinal cord after gavage feeding of pregnant dams with at-RA. Their interpretation
was that the exogenous at-RA was isomerized to 9-cisRA and, thereby, activated RXRs throughout the spinal
cord. We assayed the retinoid contents of spinal cords
treated by using a slightly different paradigm (Satre
and Kochhar, 1989) and found a massive increase in
at-RA content and the appearance of a small 9-cis-RA
peak. The amount of 9-cis-RA in this nonphysiological
situation was approximately 30% of the amount of
at-RA present normally. The concurrent super-elevation of at-RA, on the other hand, generated a ratio of
over 600-fold between at-RA and 9-cis-RA. Thus, both
under normal and experimental conditions, 9-cis-RA is
far less abundant than at-RA.
The appearance of 9-cis-RA after gavage feeding with
at-RA suggests a conversion of at-RA to 9-cis-RA. Our
control experiments seem to rule out the possibility of
photoisomerization or chemical isomerization during
handling and preparation of the embryonic tissue.
Moreover, no enzymes are known to catalyze this conversion. Therefore, a likely possibility is that isomerization to 9-cis-RA occurs within the pregnant dam,
with the relatively poor transfer of cis-isomers across
the placenta (Creech et al., 1989; Kochhar et al., 1995),
limiting the amount accumulating in the embryo.
It is important to note that oxidation of 9-cis-ROH to
9-cis-RA in the spinal cord is formally possible given
the presence of CRAD1, ADH4, RDH5, ALDH1, and
RALDH2 transcripts and RALDH2 protein. Nevertheless, the extant enzymatic machinery is clearly generating a more substantial and/or stable pool of at-RA
than 9-cis-RA.
How Are RXRs Activated in the Embryonic
Spinal Cord?
It is clear from several studies that RXRs play important roles in embryonic development. Selective activation of RXRs with RXR-specific ligands affect the
development of embryonic tissues in vivo and in vitro
(Lu et al., 1997; Sockanathan and Jessell, 1998). Gene
knockouts have shown that RXR␣ is necessary for normal development of the mouse, whereas the other
RXRs may be dispensable (Krezel et al., 1996). Selective genetic manipulation of RXR␣, which prevents its
functional interaction with coactivators and, therefore,
its interaction with the general transcriptional machinery, produces specific developmental defects (Mascrez et al., 1998). RXR␣ is expressed in the spinal cord
of both chicken embryos and mouse embryos (Dolle et
al., 1994; Hoover and Glover, 1998). Thus, the demonstration by Solomin et al. (1998) that RXR-mediated
signaling occurs in the spinal cord has important implications for our understanding of retinoid-dependent
differentiation in this part of the central nervous system.
But does 9-cis-RA activate RXRs in the spinal cord?
RXRs are much more efficiently activated by 9-cis-RA
than at-RA in vitro (Heyman et al., 1992; Allenby et al.,
1993). Nevertheless, reporter assays using chimeric
receptor constructs show that all-trans isomers at submicromolar concentrations can bind to RXR homodimers to activate response elements up to 10% or more
of the maximum activation produced by 9-cis-RA (see
Fig. 5 in Allenby et al., 1993). Our estimates of volumetric concentrations for at-RA lie in this range. Thus,
in the absence of 9-cis-RA, or in a situation where
ENDOGENOUS RETINOIDS IN DEVELOPING SPINAL CORD
9-cis-RA is far less concentrated than at-RA, at-RA
could produce at least as strong an activation of RXRs
as 9-cis-RA.
Alternatively, other molecules might function as ligands for RXRs. Phytanic acid and eicosanoids, for
example, have been shown to activate RXRs (Eager et
al., 1992; Kitareewan et al., 1996), and other ligands
may exist as well. It is important to realize that our
chromatograms include only those molecular species
for which the extraction, separation, and detection procedures are designed, and do not rule out the presence
of nonretinoid ligands. Phytanic acid cannot be assayed
by our system. The fatty acids ARA, DHA, and EPA
can, but we did not detect them in the spinal cord.
Additional alternatives are non–ligand-dependent
activation by phosphorylation of specific sites (Lefebvre
et al., 1995; Rochette-Egly et al., 1995) or as yet undiscovered mechanisms involving the RXR ligand-binding
domain (the only domain used in Solomin et al.’s Gal4RXR fusion protein).
EXPERIMENTAL PROCEDURES
Chemicals
All-trans-retinoic acid, 9-cis-retinoic acid, 13-cis-retinoic acid, all-trans-retinol, 13-cis-retinol, all-trans-retinal, 13-cis-retinal, 9-cis-retinal, arachidonic acid, cis5,8,11,14,17-eicosapentaenoic acid, and cis-4,7,10,13,16,19-docosahexaenoic acid were obtained from Sigma
Aldrich (St. Louis, MO). The retinoids 11,13-di-cis-retinol,
9-cis-retinol, all-trans-3,4-didehydro-retinal, 9-cis-3,4-didehydro-retinal, 9-cis-3,4-didehydro-retinoic acid, 9-cis3,4-didehydro-retinol, all-trans-3,4-didehydro-retinol, 4oxo-all-trans-retinoic acid and 4-oxo-13-cis-retinoic acid
were gifts from F. Hoffmann-La Roche (Basel, Switzerland). All-trans-3,4-didehydro-retinoic acid was a generous gift from A. Vahlquist (Uppsala, Sweden).
Animals
Animal protocols were in accordance with the official
governmental guidelines on the care and use of laboratory animals in Norway. Embryos from natural matings of 6- to 8-week-old F1 hybrids (C57BL/6J⫻CBA/J)
were collected at 11.5 days of gestation. The morning of
the day of vaginal plug appearance was defined as 0.5
dpc. For prenatal retinoid treatment, pregnant mice
were gavage-fed with all-trans-RA suspended in 300 ␮l
of corn oil at a concentration of 20 or 100 mg/kg body
weight.
Dissection of Embryos
All of the following procedures, except those involving tissues destined for RT-PCR analysis, were conducted under red lighting, yellow lighting, or both, with
wavelengths above 595 nm to prevent photoisomerization of endogenous retinoids, which occurs within a few
minutes in room light (unpublished results). Pregnant
mice were killed by cervical dislocation after which the
embryos with placentae were removed by means of an
abdominal incision. Before dissection, the embryos in
351
their amnions were maintained in ice-cold saline (0.9%
NaCl). The embryos were staged according to morphology of visceral arches and limb buds (Kaufman, 1992)
and then decapitated. Stages ranged from 11.0 to 12.0
(predominantly 11.5 to 12.0) for HPLC measurements
of endogenous retinoids. Stage 12.5 embryos were used
for control experiments to test for retinoid isomerization, and stage 12.0 to 12.5 embryos were used for
RT-PCR experiments.
The spinal cord was dissected in cold saline into
three portions, the first of which (B) encompassed the
brachial region, the second of which (T) was restricted
to the thoracic region, and the third of which (L) encompassed the lumbar region (Fig. 1). All dissections
were performed by the same person (J.C.G.) experienced in microdissection techniques. Dissected spinal
regions were collected, gassed with argon, and frozen in
liquid nitrogen or dry ice, one by one. The time from the
start of dissection to freezing typically ranged from 12
to 20 min and never exceeded 35 min. After all tissues
were collected and frozen they were stored at ⫺70°C
until assays were performed. We wish to emphasize
that these dissections involved a substantial amount of
labor. Tissue for RT-PCR was dissected in cold phosphate buffered saline under RNAse-free conditions,
and then stored at ⫺70°C.
The dissected spinal cord segments were completely
free of surface ectoderm, notochord, and paraxial mesoderm (somites), but there was some contamination
by the developing meninges. All regions were contaminated by slight amounts of ventral meninges, which
was impossible to visualize under the red lighting.
Laterally and dorsally disposed meninges could be
clearly split away from the cord and was consistently
removed from the brachial region, but the lateral portion was difficult to remove from thoracic and lumbar
regions because of higher adhesivity to the spinal cord.
We estimated visually that the thoracic and lumbar
regions were each contaminated by no more than 10%
of the total meningeal covering. By contrast, the meninges were removed completely from all spinal cords
destined for RT-PCR experiments, as these were dissected under normal light.
Protein Assay
Embryonic tissue was homogenized and assayed for
protein levels with the Micro BCA protein assay reagent kit (Pierce). Bovine serum albumin was used as
internal standard.
Solid Phase Extraction - HPLC
To preserve the configuration of the extremely labile
retinoids, on-line solid phase extraction, microcolumns,
and column switching were used in combination with
diode array detection (DAD), coulometric electrochemical detection (ECD), or MS detection.
352
ULVEN ET AL.
Electrochemical Detection
Retinoid analysis was performed according to a recently published method (Sakhi et al., 1998). Tissue
was thawed and homogenized with a motorized pellet
grinder in a clear Eppendorf tube. The homogenate of
330 ␮l was adjusted to 340 ␮l with buffer. Then, 510 ␮l
of cold acetonitrile was added. After thorough mixing
and centrifugation at 5,300 ⫻ g for 10 min (5°C), 750 ␮l
of the clear supernatant was transferred to an amber
glass vial. An aliquot of 250 ␮l of water was added with
subsequent mixing, resulting in a final acetonitrile concentration of 45%. The entire procedure was performed
under red light, and the samples were kept on ice
under argon atmosphere whenever possible. An aliquot
of 1,000 ␮l was then injected into the HPLC system.
The samples were submitted to on-line solid phase
extraction followed by automated transfer of the extract to the analytical column by column switching.
The detection system consisted of three electrochemical
cells. The guard cell was set to ⫹750 mV and was used
to oxidize any trace organic compounds in the separating mobile phase ensuring a very low background. The
screening cell lowered the amount of oxidizeable components in the injected sample and was set to ⫹450
mV. The analytical cell was set to ⫹750 mV relative to
the palladium reference electrode and provided the
signal by oxidizing the double bond in the polyene
chain of the retinoid.
drying gas, 10 L/min; drying gas temperature, 350°C;
capillary voltage, 4,000 V; gain 5.
mRNA Isolation and RT-PCR Analysis
Dissected tissues were frozen in liquid nitrogen immediately and then stored at ⫺70°C until use. mRNA
was isolated by using the MicroFast mRNA isolation
kit (Invitrogen). cDNA synthesis and PCR (40 cycles)
was performed as described by Ulven et al. (1998). The
specific primer pairs are listed in a previous report
(Ulven et al., 2000).
Each RT-PCR analysis was also carried out without
reverse transcriptase as a negative control. ␤-Actin
was used in all experiments as a control. Each experiment was repeated at least three times with similar
results.
Immunohistochemistry
Stage 11.5 to 12.5 embryos were decapitated, fixed in
4% paraformaldehyde in phosphate buffer (pH 7.4) for
several hours at 4°C, rinsed with PBS, and cryoprotected in 20% sucrose in PBS. They were frozen in OTC
compound (ChemiTeknik, Oslo) and sectioned transversely at 14 ␮m. Immunohistochemistry for RALDH2
was performed according to Berggren et al. (1999), by
using fluorescein- or rhodamine-conjugated secondary
antibodies (Amersham, 1:200).
Tissue Volume Estimates
Atmospheric Pressure Electrospray IonizationMass Spectrometry (AP-ESI-MS) and Diode
Array Detection (DAD)
Tissue was homogenized in 30 ␮l of 0.9% NaCl in an
Eppendorf vial by using a motorized pellet grinder
(Kontes) and 60 ␮l of ice-cold acetonitrile was added.
After thorough mixing the vial was snap frozen in
liquid nitrogen before re-homogenization, mixing, and
centrifugation at 10,000 ⫻ g for 1 min at ambient
temperature. An aliquot of 80 ␮l was injected on a
2.1 ⫻ 250-mm suplex pKb-100 column. Separation was
obtained with a gradient starting with 95% solution A
(69.5% AcN-30% water ⫹ 0.5% HCOOH), changing linearly over 40 min to 40% solution B (69.5% AcN-30%
MTBE-0.5% HCOOH). The flow was 0.4 ml/min, and
the temperature was 40°C. Full spectral analysis in the
range 250 – 600 nm was performed with a HP 1100
DAD equipped with a 2-␮l flow cell set to an optical
resolution of 1.2 nm, a bandwidth of 30 nm, and a 2-sec
filter. The liquid chromatograph was interfaced by APESI to the single quadrupole mass spectrometer operated in positive mode (Hewlett Packard). Single ion
monitoring (SIM) was performed for selected retinoids.
For RA, the [M⫹1]⫹ ion of protonated RA with a mass
to charge ratio (m/z) of 301.2 was selected. For ROH,
[M-17]⫹ resulting from dehydration of the alcohol was
selected. Tuned to give maximum sensitivity for these
fragments, the settings of the mass spectrometer was
as follows: fragmentor, 70 V; nebulizer pressure, 30 psi;
The volumes of brachial, thoracic, and lumbar regions dissected for measurement of endogenous retinoids were estimated by measuring cross-sectional areas
in digitized sections obtained for immunohistochemistry and multiplying by the lengths of the respective
regions.
ACKNOWLEDGMENTS
We thank Dr. Peter McCaffery for the gift of
RALDH2 antiserum.
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