Skeletal Staining

Gillian M. Morriss-Kay 1. Introduction

The retinoids comprise a large group of natural and synthetic compounds related to vitamin A (retinol). The family name is derived from the early observation of the necessity of vitamin A for normal vision, and the association of vitamin A deficiency with night blindness (1). With the exception of the visual cycle in the rod photoreceptor cells of the retina, in which protein-bound 11-cis-retinal is reversibly isomerized to free all-trans-retinal, retinoic acid is the active retinoid for biological processes. However, the visual cycle illustrates two characteristics of retinoids that are relevant to the use of this family of molecules as tools in experimental embryology and for the interpretation of results. (1) retinoids are light-sensitive, and will undergo isomerisation or degradation if exposed to light; (2) in nature, their activity and their metabolism are associated with binding to specific proteins.

The most important natural retinoids are retinyl esters, retinol, and retinoic acid (RA). In the all-ira«s form, RA is the ligand for the nuclear RA receptors RAR-a, RAR-P, and RAR-y; in the 9-cis form, it is the ligand for the retinoid "X" receptors (RXRs), which form heterodimers with RARs to form the transcriptionally active complex that binds to RA response elements of target genes, as well as with the RARs (see ref. 2 for details and references). In the past, retinyl esters and retinol have been used for treating embryos (3,4); their effects on developmental pattern probably depend on conversion to RA, although some other natural metabolites, such as 4-oxo-RA, may also be directly active (5). Most recent studies have used RA (mainly in the all-ira«s form) for experimental purposes.

From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ

The disadvantage of using RA, both in vivo and in vitro, is that it is highly toxic. Although it is the principal biologically active retinoid, it is not normally transported long distances from its source of synthesis. Retinoids are stored in the liver as esters and transported in the bloodstream as retinol bound to retinol binding protein (RBP). In early pregnancy in the mouse (i.e., at stages appropriate for studies on hindbrain and limb bud development), the maternal blood is in direct contact with the parietal yolk sac (Reichert's membrane), a thick basement membrane-like structure with dispersed attached cells. Immediately internal to this, the visceral yolk sac endoderm is rich in transcripts of cellular retinol binding protein (CRBP I) (6). The mechanism of transfer involves receptor-mediated uptake of retinol from maternal RBP-retinol by the visceral yolk sac endoderm, where it binds to CRBPI and interacts with the enzymes mediating RA synthesis (7). The assumption that retinol is retained specifically in CRBP I-expressing embryonic tissues has been verified by using 14C-labeled retinyl acetate (delivered intravenously to the pregnant dam) as a source of retinol (8).

RA administered to pregnant rats and mice is rapidly transferred to the embryos, where it remains at high levels for only a few hours. In the mouse, embryonic all-tra«s-RA levels are raised within 30 min of maternal oral administration, reaching a peak at 2 h and then falling rapidly to low levels by 8 h (9). In the rat, the embryonic peak for RA is also at 2 h, falling even more rapidly than in the mouse to a low level at 4 h, and then more gradually to undetectable levels (10). These pharmacokinetic profiles indicate that administration of RA to pregnant rats or mice results in exposure of the embryos to RA for relatively short periods of time; if it is desired to allow development to continue for 24 h or more, we can be confident that they are not exposed to raised RA levels for more than the first few hours after the time of administration, with a peak at 1-2 h. It should also be noted that RA administered in vivo or in vitro undergoes isomerisation to other active retinoids (e.g., 13-ds-RA and 9-ds-RA) both within the maternal tissues and in the embryo (9-11).

The following protocols cover only administration with RA. Retinol can be used in exactly the same way, but higher concentrations are required to induce an equivalent effect (6). It may be more appropriate to use retinol than RA in vitamin A deficiency studies, in which much lower (physiological) concentrations should be used than in studies using RA to alter gene expression. Retinyl esters must be made up as an emulsion; they are only useful in in vivo studies, being inactive in vitro, where the necessary conversion enzymes are not available. In addition to the protocols described below (see Subheading 3.), RA has also been applied on a bead directly to the mouse limb bud using the ex utero technique; the effects on limb development were reduction defects resembling the effects of maternal administration (12).

2. Materials

1. Retinoic acid is available in crystalline form from Sigma (UK). Once open, it must be protected from light and oxygen. Protection from oxygen can be achieved by replacing the air with nitrogen or argon, as available, immediately before sealing the container. All solutions and suspensions must be made up fresh, protected from light, and used within 24 h.

2. For oral administration (gavage) of pregnant mice, use a 1-5 in. stainless-steel 18-gage dosing cannula with a luer fitting and a bulbous tip (available from Harvard Apparatus Ltd., Kent, UK), fitted to a standard 1 mL disposable syringe. For intraperitoneal (ip) injection, use a 25-gage x 5/8-in. needle.

3. Methods

The following examples of retinoic acid treatments are from our own studies. The timing is critical. Our timings are included to illustrate what has worked for us in the past. For each new study and for repeats of previous studies, even in the same laboratory, it is essential to invest some time in establishing the time period during which the desired developmental process occurs. Timed matings, in which the male is removed after 2-4 h, minimize but do not abolish the spread of developmental stages between litters; there is also a range of stages within each litter. It is likely that implantation time is more significant than the time of mating for determining the precise developmental stage later on. Time of year, even in an air-conditioned, light- and temperature-controlled animal house, can affect developmental timing in the mouse.

3.1. Production of Hindbrain Abnormalities

Embryos exposed to RA prior to the onset of somitic segmentation fail to develop rhombomeres, and Hoxb-1 is expressed throughout the preotic hind-brain instead of being specific to rhombomere 4; embryos exposed to RA after the onset of somitic segmentation form rhombomeres of variable regularity, with Hoxb-1 expression in rhombomere 2 as well as rhombomere 4, and rhombomeres 1,2 and 5 showing a normal pattern of gene expression with respect to HoxB genes and Krox-20 (13). These abnormalities were induced by oral dosing with RA on d 7.75 and 8.25, respectively (dosing on d 8.0 gave mixed litters), giving 10 or 12 mg RA/kg (a mouse weighs 20-25 g).

Make up crystalline RA as a 1 mg/mL suspension in peanut oil. Mix very thoroughly, using a magnetic stirrer, and use immediately. If kept overnight for using again, the suspension should be gassed, sealed, stored in the dark at 4°C, and mixed thoroughly again immediately before reuse. For 10 mg/kg, give 0.25 mL to a 25 g mouse. For gavage (right-handed person), proceed as follows: place the mouse on the cage laid, holding its tail with your right hand; it will grip the bars of the lid. Pick up the mouse with your left hand so that the skin of its neck and back is held firmly between the thumb and first finger; head movement is restricted, and the snout tilted upward. Crook your little finger around the tail if necessary. The mouse should be comfortable, but immobile; if it struggles the hold is wrong, and you should start again. Introduce the bulb of the cannula between the jaws at the diastema (the gap between the incisors and molars), place it on the tongue, and then use it to tilt the head into a position so that the cannula is in line with the esophagus. Slide the cannula gently down the esophagus so that the tip enters the stomach. This should be easy. If at any stage it is not easy, stop. Deliver the measured amount of RA suspension, and remove the cannula. The mouse should be lively immediately on replacing into the cage, and there should be no sign of oil around the mouth. This is a skilled procedure and should be learned from an experienced user. See Subheading 4.

3.2. Production of Limb Abnormalities

Limb abnormalities can be induced by RA treatment on d 11 or early on d 12. Like all RA-induced effects, the resulting abnormalities of skeletal pattern are stage-related. In our experiments, administration on d 11.0 resulted in partial or complete fusion of digits 4 and 5, administration on d 11.5 to loss of digits 1 and 5, and administration on d 12.0 to partial or complete loss of digit 1; skeletal differentiation was delayed by all of these timed exposures to RA (14).

Make up a 10 mg/mL suspension in peanut oil, and give 100-120 mg/kg (0.25-0.3 mL for a 25-g mouse) by gavage. Limb abnormalities can be analyzed at late fetal stages (d 17 or 18) by double skeletal staining for cartilage and bone (see Subheading 3.5.).

3.3. Intraperitoneal administration

We have used ip injection for low-dose RA supplementation to curly tail mutant mice (15). This treatment results in a decrease in the incidence of neural tube defects and is a good example of the use of RA in studying genotype-phenotype interactions. A thick, oily suspension is unsuitable for delivery through a small-gage needle, so the RA is first dissolved in 5-10% ethanol (this takes 15-20 min), then a small amount of oil, and then mixed with the remainder of the oil. Thorough mixing at each stage is essential, e.g., for administration of 5 mg/kg to a 25 g mouse, dissolve 5 mg RA in 0.8 mL Analar ethanol, add 1.2 mL peanut oil, and mix until dissolved (at least 10 min); add 8 mL peanut oil, and mix again.

3.4. Treatment of Embryos and Tissues In Vitro

Exposure of embryos to RA in vitro has the advantage that the precise developmental stage at the time of exposure is known, the concentration in the medium can be precisely calculated, and the duration of the exposure controlled. An exposure time of 2 h is sufficient to induce an effect on morphogenesis and gene expression, and avoids the complication that deleterious effects on the yolk sac placenta may affect development if RA is present throughout the whole culture period. The required concentration and exposure time need to be worked out by using a range of concentrations and times for each new experiment. Hindbrain defects can be induced by a concentration of 0.25 ^g/mL medium. For this, dissolve 1 mg RA in 0.8 mL Analar ethanol and add 1 ^L of this solution to 5 mL culture medium. One microliter ethanol should be added to control cultures. This amount of etha-nol is insufficient to cause abnormalities, and probably evaporates rapidly at 38°C. Some laboratories have used DMSO as a solvent for RA (11). RA should always be added to the medium before the embryos are added. After exposure, embryos should be washed in Tyrode saline (do not use PBS, because they require calcium for normal morphogenesis) and replaced in fresh culture medium.

Both RA and retinol can similarly be introduced to the tissue-culture medium of embryonic cell, tissue, and organ cultures.

3.5. Staining of Fetal and Neonatal Skeletons

The effects of retinoids in vivo include important alterations of skeletal pattern. The following procedures are also used for analysis of skeletal defects of genetic origin. Alcian blue staining alone is appropriate for early stages of skeletogenesis; double staining to show both cartilage and bone is appropriate to late fetal stages, and also works well for early postnatal mice and rats. Alcian blue stains the proteoglycans of cartilage matrix; Alizarin red S stains the mineralized matrix of bone.

3.5.1. Cartilage Staining of Whole Embryos

1. Fix from fresh in Bouin's fluid for 4 h.

2. Drain and wash off fixative with distilled water.

3. Immerse for 1 h in each of four changes of 1% ammonia solution in 70% alcohol (with frequent agitation).

4. Stain in 95 mL 5% acetic acid plus 5 mL of 1% Alcian blue 8GX in 5% acetic acid (use 40-50 mL/embryo) for 1-4 h or overnight (depending on age of embryo).

5. Wash for 1 h in two changes of 5% acetic acid.

6. Transfer to a third change of 5% acetic acid for 16 h.

7. Dehydrate in graded ethanols (70, 95, 100% x 3).

8. Clear and store in three parts methyl salicylate to one part benzyl benzoate.

Result: Cartilage matrix: intense blue/green; other tissues: only very slight staining (of glycosaminoglycans).

3.5.2. Double Staining for Cartilage and Bone

1. Fix in 95% alcohol. Remove skin, including tail skin.

2. Stain in 80 mL 95% alcohol, 20 mL glacial acetic acid, 15 mg Alcian blue 8GX (or 8GN) for 20-48 h.

3. Differentiate in 95% alcohol, three changes over 7 d.

4. Macerate in weak (0.2-2%) KOH until bones are visible (we use 1%).

5. Wash for 12 h in running tap water.

6. Stain in freshly made 0.1% aqueous Alizarin red S + 10-20 drops of 1% KOH 312 h (check color—add acid if necessary to get red color).

7. Wash for 30 min in running tap water.

8. Decolorize 1-2 wk in 20% glycerine in 1% KOH.

9. Dehydrate through mixture of 70% alcohol:glycerine:water sequentially in the following proportions: 1:2:7; 2:2:6; 3:3:4; 4:4:2; 5:5:0, allowing approx 3 d in each mixture. The soft tissues are clear before the end of this procedure, but it is important to complete the process for storage. Skeletal preparations can then be stored for years.

3.5.3 Alternative Double Staining for Cartilage and Bone in Adults

This procedure has been found to work well for adult tissues and can also be used for tissues that have been previously fixed in formalin.

1. Fix in ethanol for 4 d (room temperature).

2. Keep in acetone for 3 d (room temperature).

3. Rinse in distilled autoclaved water.

4. Stain for a minimum of 10 d in a staining solution consisting of:

1 vol of 0.3% alcian blue in 70% ethanol; 1 vol of 0.1% alizarin red in 95% ethanol; 1 vol of 100% acetic acid; 17 vol of 100% distilled autoclaved water.

5. Rinse in distilled autoclaved water.

6. Add 20% glycerol:2% potassium hydroxide solution and keep at 37°C overnight.

7. Change solution every 2-3 d, keeping at 37°C during the day and at room temperature overnight, for approx 2-3 wk, until the specimens have completely cleared.

8. For storage, transfer specimens to 50, 80, and 100% glycerol in succession.

4. Notes

In the Untied Kingdom, all administration procedures to pregnant mice require Home Office personal and project licenses. Embryo culture beyond midgestation (d 9 in the mouse), and removal of tissues from embryos and fetuses, also require Home Office permission. Licenses will only be granted after attendance at a course on laboratory animal management and welfare (Home Office Training Modules 1-3). Readers from other countries should seek advice from the appropriate authorities.

References

1. Moore, T. (1957) Vitamin A. Elsevier, Amsterdam.

2. Mangelsdorf, D. J., Umesono, K., and Evans, R.M. (1994) The retinoid receptors in The Retinoids: Biology, Chemistry and Medicine, 2nd ed. (Sporn, M. B., Roberts, A. B., and Goodman, D. S., eds.), Raven, New York, pp. 319-349.

3. Morriss, G. M. (1972) Morphogenesis of the malformations induced in rat embryos by maternal hypervitaminosis A. J. Anat. 113, 241-250.

4. Morriss, G. M. and Steele, C. E. (1977) Comparison of the effects of retinol and retinoic acid on postimplantation rat embryos in vitro. Teratology 15, 109-120.

5. Nau, H. (1994) Retinoid teratogenesis: toxicokinetics and structure specificity, in Use of Mechanistic Information in Risk Assessment (Bolt, H. M., Hellman, B., and Denker, L., eds.) (Arch. Toxicol. Suppl. 16), Springer-Verlag, Berlin, pp. 118-127.

6. Ruberte, E., Doll, P., Chambon, P., and Morriss-Kay, G. (1991) Retinoic-acid receptors and cellular binding proteins: II. Their differential pattern of transcription during early morphogenesis in mouse embryos. Development 111, 45-60.

7. Bavik, C., Ward, S. J., and Ong, D. E. (1997) Identification of a mechanism to localize generation of retinoic acid in rat embryos. Mech. Dev. 69, 155-167.

8. Gustafson, A.-L., Dencker, L., and Eriksson, U. (1993) Non-overlapping expression of CRBP I and CRABP I during pattern formation of limbs and craniofacial structures in the early mouse embryo. Development 117, 451-460.

9. Creech-Kraft, J., Lofberg, B., Chahoud, I., Bochert, G., and Nau, H. (1989) Teratogenicity and placental transfer of all-trans-, 13-cis-, 4-oxo-all-trans-, and 4-oxo-13-cis-retinoic acid after administration of a low oral dose during organogenesis in mice. Toxicol. Appl. Pharmacol. 100, 162-176.

10. Ward, S. J. and Morriss-Kay, G. M. (1995) Distribution of all-trans-, 13-cis and 9-cis-retinoic acid to whole rat embryos and maternal serum following oral administration of a teratogenic dose of all-trans-retinoic acid. Pharmacol. Toxicol. 76, 196-201.

11. Klug, S., Creech-Kraft, J., Wildi, E., Merker, H.-J., Persaud, T. V. N., Nau, H., and Neubert, D. (1989) Influence of 13-cis and all-trans retinoic acid on rat embryonic development in vitro: correlation with isomerisation and drug transfer to the embryo. Arch. Toxicol. 63, 185-192.

12. Bryant, S. V. and Gardiner, D. M. (1992) Retinoic acid, local cell-cell interactions, and pattern formation in vertebrate limbs. Dev. Biol. 152, 1-25.

13. Wood, H. B., Pall, G. S., and Morriss-Kay, G. M. (1994) Exposure to retinoic acid before or after the onset of somitogenesis reveals separate effects on rhombomeric segmentation and 3' HoxB gene expression domains. Development 120, 2279-2285.

14. Wood, H. B., Ward, S. J., and Morriss-Kay, G. M. (1996) Effects of retinoic acid on skeletal pattern, 5'HoxD gene expression, and RAR-P2/P4 promoter activity in embryonic mouse limbs. Dev. Genet. 18, 74-84.

15. Chen, W.-H., Morriss-Kay, G. M., and Copp, A. (l995) Genesis and prevention of spinal neural tube defects in the curly tail mutant mouse: involvement of retinoic acid and its nuclear receptors RAR-a and RAR-y. Development 121, 681-691.

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