The distribution of the transcripts encoding the RARs have been analysed in the developing mouse limb buds by Dolle and colleagues. At embryonic day 10 (ElO), RAR-a and y are expressed throughout the limb buds, RAR-a transcripts were also seen extending in to the adjacent tissue, whereas RAR-y expression was not seen beyond the
a) Domain A B C D E F Chicken/human 90 96 98 89 97 8 6 Chicken/mouse 79 96 98 91 99 78 Mouse/human 94 1 0 0 1 0 0 98 99 92 b) modulation of transcriptional transactivation
A
B
C
D
E
F
J.
DNA-binding ligand-binding, dimérisation, transcriptional transactivation Figure 1.5.a) Percentage identity between the predicted amino acid sequences of human, mouse and chicken RAR-(î in domains A-F of the protein
(reproduced from Rowe et al, 1992a)
b) Simplified domain structure of the retinoid nuclear receptor proteins (reproduced from Rowe and Brickell, 1993)
proximal region of the limb bud. In contrast RAR-P has a more restricted expression pattern, in the proximal region of the bud (see chapter 4). By E12.5, RAR-y was
predominately restricted to central precartilaginous blastemas and in the distal undifferentiated mesenchyme of the limbs, RA R-a was still distributed throughout the limb bud. By E l 4.5 RAR-y was seen only in the skin and in the cartilage cells of
developing skeletal elements, expression was also located in the developing digits which had not differentiated into muscle, skin or bone. At later stages RAR-y is not expressed in bone. RAR-a was still expressed throughout the limb bud at low levels in developing cartilage (Dolle et al, 1989). In the chick limb, RAR-P transcripts have also been shown
to be restricted to the proximal region of the bud and in the ectoderm (Schofield et al,
1992). The fact that these genes are expressed during limb development and RAR-p and y have restricted expression patterns, suggests their importance in this process.
RARs are also expressed in other areas of embryonic development. Expression of RAR- y in the skin consists of mainly isoform RAR-y2 in early development (E8.5, 9.5) and RAR-yl in later development (Kastner et al, 1990). RAR-y is not expressed in mouse developing neural tissues, but RAR-a and p are. RA R-a was ubiquitously expressed in the spinal cord and brain. At neural tube closure RAR-P transcripts are found only in the closed neural tube and not in the open folds. In the early spinal cord RAR-p is located in
the proliferating neuroepithelium layer. Once motor neurons have begun differentiating RA R-P was seen in the developing motor columns (Ruberte et al, 1993). RAR-p
transcripts are also expressed during development of the facial primordia (Rowe et al,
1992b).
In various human tissues analysed, RA R-a transcripts were present at low levels
(kidney, prostrate, spinal cord, cerebral cortex, adult and foetal liver, spleen, uterus, ovary, testis and breast), in contrast RAR-p transcripts showed high levels of expression
(kidney, prostrate, spinal cord, cerebral cortex), average levels (spleen, liver, uterus, ovary) and low levels (breast and testis) (de The et al, 1989). The examples show the
difference in expression patterns o f the different RARs and also of their isoforms, suggesting each gene has specific functions in development.
The RARs have been tested both in vitro and in vivo to see if they are transcriptionally
upregulated by RA. A hepatoma cell line was treated with RA and the amount of endogenous RAR-a and p in response to this treatment analysed. The levels of RAR-P increased, but the levels of RA R-a remained unaffected, concluding that the increase in RAR-P levels due to RA was due to enhanced transcription. The fact that the levels of R A R -a remained unaltered suggested that these two receptors have distinct roles in
mediating the effects of RA (de The et al, 1989). A similar effect was observed by Zelent
et al (1989) in RA treated F9, embryonal carcinoma, cells, RAR-P was upregulated 30- fold, RA R-a remained the same and RAR-y decreased 2-fold. The responses to RA
differ between RARs.
In vivo RA elevates the levels of RAR-p2 mRNA. Hamish and colleagues (1992) treated
pregnant mice with RA and looked at the subsequent levels of RAR isoforms. The levels of RAR-P2 mRNA increased 7-fold, whilst both RAR-a2 and RAR-yl increased only
2-fold. In contrast to the results seen with RAR-p2, levels of RAR-P 1 and p3 did not
increase in response to RA. The limb buds of day 11 embryos exhibited the greatest increase in RAR-p2 mRNA levels, increasing to 12 times the normal amount. Retinol was also shown to increase the levels of RAR-p2 mRNA in the limb bud. This was
slower acting than RA, presumably because the retinol was being converted to RA. What is interesting from this data, is that the levels of RA administered, result in limb abnormalities, which leaves the question how does RA through the elevated levels of RAR-P2 result in these defects? Soprano and others (1994) showed that mice treated with both teratogenic and non-teratogenic doses of RA caused an elevation of RAR-p2
mRNA levels. They show that just an increase in RAR-p2 mRNA levels is not sufficient
to cause malformations, but that these levels must remain for 6-9 hours post treatment, hence in mice treated with teratogenic doses there is a high probability of
dysmorphogenesis when RAR-p2 and RAR-p protein levels are increased for a
prolonged period of time. This illustrates how RAR-P mediates the effects of RA.
RA also alters the pattern of RAR-p transcripts in the facial primordia. The normal
distribution of RAR-p transcripts in the maxillary primordia is restricted to the anterior part. In response to RA, high levels of RAR-P transcripts are induced in this area. RA causes a truncation of the upper beak and the increase in RAR-p levels is seen before the
morphological changes (Rowe et al, 1991).