PERSPECTIVE
Retinoid-Enhanced Alveolization
Identifying Relevant Downstream Targets

Richard A. Pierce and J. Michael Shipley

Department of Internal Medicine, Pulmonary Division, Washington University School of Medicine, St. Louis, Missouri

As the basic gas-exchange units of the lung, alveoli must be elastic (to allow expansion and contraction with respiration) and thin (to facilitate gas exchange with the capillary network). Alveolar walls are among the most delicate structures in the vertebrate body, yet they are in constant contact with the external environment (inspired air) and undergo tens of thousands of cycles of inflation each day. Nonetheless, most evidence indicates that alveoli are relatively durable. Alveolization, or the formation of alveoli during lung development, is essentially completed in the neonatal period, and little neo-alveolization occurs in the adult lung. Despite their normal durability, damage to alveolar walls is a root cause of diseases such as emphysema and results in loss of functional gas-exchange capacity, as well as impairing lung mechanics. In premature neonates of very low birth weight, whose lungs are structurally or morphologically immature, alveolization may be impaired, resulting in poor lung function. Therapies capable of promoting alveolization are therefore of great interest.

Until recently, there has been little basis for optimism about therapeutic measures to promote alveolization, either in the premature neonatal lung or in the emphysematous lung. However, it has long been known that vitamin A is essential for normal lung epithelial cell homeostasis. Several studies have shown that retinoids are effective in promoting alveolization in neonatal rats (1), in adult rats with elastase-induced emphysema (2), and in mice with a genetic defect (Tsk) involving impaired alveolar development (3). Vitamin A supplementation has been explored as a therapy for premature infants of very low birth weight as early as 1987 (4), and based on its efficacy in promoting alveolization in animal models, it is currently being tested in clinical trials in humans. There is still much to learn about the role of retinoids in alveolization, or even about how alveolization occurs. In this issue, McGowan and coworkers report that elastin expression by alveolar myofibroblasts is specifically regulated by retinoid treatment in the developing lung, and that specific retinoid receptor subtypes are important for both normal alveolization and normal elastin expression in the neonatal lung (5). These and other emerging reports of retinoid-responsive genes in the developing lung form a new body of data that may lead to more specific treatments to enhance alveolization.

	Steps in Alveolization

Lung development begins as a simple outpouching of epithelium into a sac of surrounding mesenchyme, which then undergoes dichotomous branching through interactions between epithelium and mesenchyme during the pseudoglandular and canalicular stages of development. In a deliberate oversimplification, the process of alveolization may be thought of as a two step process. First, existing walls of distal air spaces become thinner through the flattening of epithelial cells and the reduction in epithelial cell number by apoptosis, generating saccules. At the same time, the capillary network becomes more complex. In neonatal rats and mice, which undergo alveolization postnatally, these saccules function as gas-exchange units.

Next, secondary crests develop and extend to make new alveolar septae, effectively increasing gas-exchange area while simultaneously decreasing mean alveolar size (6). If secondary septae do not form, the lung has the appearance of emphysema, but with the absence of the destructive processes, which are the root causes of adult onset emphysema. Secondary septal formation involves coordinated outgrowth of epithelial cells, a capillary network, and alveolar myofibroblasts at alveolar septal tips. Just as a locomotive might move forward on a new self-made track, these cells must simultaneously synthesize and assemble basement membranes and an elastic interstitial matrix. The signals directing this process are unknown, perhaps because the outgrowth of secondary crests projects into air space, not into a surrounding tissue that could provide a morphogenetic gradient.

	Are There "Stem Sites" for Forming New Alveolar Walls?

Alveoli of the mature lung are similar in size, indicating that secondary septal formation is not a random process, but is directed to specific sites in existing alveolar walls. In contrast to other developmental events, such as neural tube formation, limb budding, or even early branching morphogenesis of lung buds, very little is known about how such sites are "chosen," programmed, or stimulated to begin forming a secondary crest. Fitting the available data into conceptual models may be a reasonable starting point for discussing mechanisms of secondary crest formation. Sites for secondary septae may be determined in at least three possible ways (Figure 1).

View larger version (28K): [in this window] [in a new window]   Figure 1.   Conceptual models of secondary septal formation. (A) During alveolization, secondary septae arise from existing walls of terminal air spaces and extend to increase surface area and increase gas-exchanging capacity of the lung. (B) If sites of secondary septal formation (circles) are programmed, no specific stimulus (shaded area) is required. (C) Sites of secondary septal formation may be determined by a local stimulus (shaded circles). (D) If specific combinations of interacting cells and extracellular environment are required for secondary septal formation, alveolar walls of the adult lung may contain "stem sites" capable of responding specifically to a systemic, nonlocal stimulus (shaded area).

First, such sites may be "programmed." In this model, secondary septae could only sprout from specific sites in terminal air-space walls, and such sprouting would always occur unless some untoward event occurs. This model has little support because it is incompatible with the neo-alveolization observed in retinoid-treated lung or in post-pneumonectomy lung growth. If all sites that were "programmed" actually formed secondary septae, there would be no sites available for forming new septae in the adult lung.

A second model is the "local stimulus" model, in which secondary crests could form at any number of sites in existing alveolar walls, but a specific local stimulus "chooses" one site over another. This model is adapted from other morphogenetic events, such as limb bud formation, where a morphogenetic gradient acts locally to induce outgrowth of a new tissue type from relatively undifferentiated surrounding tissue. Cross talk and costimulation between cell types may then control subsequent steps in the process. However, in the alveolizing lung, growth of secondary septae is directed away from existing tissue, not toward a known growth-factor gradient. In this model, if the specific stimulus were directed to multiple sites in alveolar walls by expression of a transgene, an abundance of secondary crests could form, possibly resulting in a dysplastic lung with excessive alveoli of small but variable size.

The third model we propose is the "stem site" model. Much as specific stem cells in tissues may be dormant until needed, is it possible that specific "stem sites" capable of sprouting new alveolar walls exist in the walls of distal air spaces? Stem sites might be determined, for example, by the proximity of a capillary network, alveolar myofibroblasts, and type II alveolar epithelial cells, and activated by a stimulus that elicits unique responses in each different cell type. The different cell types would then respond to signals from the adjacent cell types and coordinately begin to "sprout" a new alveolar septum, complete with the machinery to make an elastic architecture, develop a capillary network, and secrete a basement membrane. This model is consistent with retinoid-stimulated neo-alveolization, as well as some mechanisms such as mechanical stretch, which has been proposed to promote neo-alveolization during postpneumonectomy lung growth. Systemic retinoids, circulating growth factor(s), or mechanical stretch acting on the whole lung could stimulate "stem sites" for alveolization.

	Retinoic Acid Signaling: What Are the Relevant Downstream Targets?

It is clear that normal retinoid metabolism is required for alveolization and for lung homeostasis, and that treatment with retinoic acid can enhance alveolization in several experimental models in rodents. How does treatment with retinoids stimulate alveolization? Vitamin A (retinol) and its derivatives affect vertebrate development, homeostasis, and cell differentiation through the binding of two families of nuclear hormone receptors, the RARs (retinoic acid receptors) , , and , and RXRs (retinoid X receptors) , , and  (7). RARs bind all trans-retinoic acids and 9-cis-retinoic acids, whereas RXR receptors bind only 9-cis-retinoic acid. Typically, heterodimers of RAR and RXR subunits have the highest biologic activity. Their targets are retinoic acid-responsive elements (RAREs) in regulatory regions of downstream genes. The complexity in retinoid signaling results in part from different affinities of various receptor dimers for different ligands, as well as their selectivity in binding to RAREs of different sequences. Retinoic acid or its derivatives activate growth factor signaling pathways (8), regulate transcription factors important in expression of lung-specific genes (9), and enhance expression of Hox genes, which are key patterning genes in development (10). Thus, a number of retinoic acid-responsive genes with roles in lung development and in cellular differentiation are already known. A selection of these retinoid-responsive genes is listed in Table 1.

	Retinoids, Elastin, and Alveolization

Elastin is one candidate gene that may have an essential role in the generation of new alveoli. A series of studies by McGowan and coworkers, including the article in this issue, forge a link between retinoids, alveolization, and elastin expression. These studies show that retinoids upregulate elastin expression in alveolar myofibroblasts (11), and that mice homozygous for a null RAR- allele and heterozygous for a RXR- null allele have diminished lung elastin and impaired alveolization.

The results of McGowan and colleagues are interesting not only in that they establish a role for a particular subset of RARs in septal formation during normal development of the lung, but they also identify elastin as a potential target of retinoic acid involved in this process. Elastic fiber formation in the lung peaks as septation of the distal air spaces occurs, suggesting that the elastic matrix is critical in this process. Further, additional lines of evidence suggest a link between formation of an elastic fiber network in the developing lung and alveolization. Interfering with elastic fiber synthesis or assembly by blocking the activity of the elastin cross-linking enzyme lysyl oxidase results in failed alveolization (12, 13). Depletion of myofibroblasts from alveolar walls in the platelet-derived growth factor (PDGF)-A-null mouse results in an absence of elastic fibers in alveolar walls and incomplete alveolization (14, 15). Several treatments that suppress alveolization in neonatal rats, such as exposure to hyperoxia (16, 17) or dexamethasone (18) also suppress elastin expression in the alveolar wall. Elastic fibers are primarily composed of insoluble tropoelastin monomers cross-linked into an insoluble polymer, but a scaffold of microfibrils determines their architectureraising the possibility that other components of the elastic fiber, such as microfibrillar glycoproteins, may also be targets of retinoic acid signaling during alveolization.

Fibrillin-1 is a major component of 10-12 nanometer extracellular microfibrils. In many elastic organs including the lung, expression of fibrillin-1, as well as other microfibrillar genes, usually peaks just prior to and during the period of elastin expression. It is therefore intriguing that retinoic acid treatment (3) also reverses the defect in septation associated with a duplication of the central region of fibrillin-1 in the tight skin (Tsk) mouse (19). The mechanism underlying this correction remains to be investigated, but one or more of the genes encoding microfibrillar proteins may also be targets of retinoic acid and be important in alveolization. Regulation of the microfibrillar genes by retinoids has not been studied in the lung, although regulation of fibrillin-1 expression by retinoic acid may impact the establishment of left-right asymmetry during formation of the heart (20, 21). As noted in Table 1, many genes expressed in alveolizing lung are retinoid responsive, leaving investigators with the task of determining if their roles in alveolization are essential or even significant.

	Koch's Postulates Applied to Alveolization

In 1890, Robert Koch proposed that specific criteria be used to test whether an infective agent actually causes a particular disease (22). His principles or postulates have proven valuable when applied to other disciplines as well. Perhaps similar criteria could be applied to test whether a particular candidate factor is required for and actually promotes alveolization:

1. 1. The candidate agent must be present in all models of alveolization.
2. 2. The agent or its receptors must be detected specifically at sites of secondary crest formation.
3. 3. Alveolization must be enhanced when the agent is ectopically administered or expressed.
4. 4. Blocking expression or activity of the agent must block alveolization.

Of course, alveolization is a much more complex process than bacterial infection, in which a single agent can be necessary and sufficient to cause a particular outcome (disease). Although a single master regulator, such as systemically administered retinoids, can promote alveolization in certain models, it is likely that multiple downstream targets must work in concert to direct the formation and extension of secondary septae.

Targets of retinoid signaling such as elastin, as reported by McGowan and associates in this issue, may fulfill a subset of such criteria. For example, elastin is always present and actively expressed in the lung during neo-alveolization, both during development and in models such as postpneumonectomy lung growth, and specifically localizes to sites of secondary septal formation (23). Thus, elastin fulfills Koch's requirements 1 and 2 above. And, as noted above, blocking elastin expression or fiber assembly in a variety of ways results in failed secondary septal formation, fulfilling requirement 4. As a key structural component of the alveolar wall, it is not unreasonable that elastic fiber formation is required for neo-alveolization, but it seems unlikely that ectopic expression of elastin at specific sites in alveolar walls in adult lung (requirement 3) would promote formation of new septae.

	Available and Emerging Technologies: Choices and Challenges

There is an ever-increasing repertoire of technologies to exploit for discerning mechanisms of alveolization and the role of retinoids in this process. Nutritional and pharmaceutical approaches include dietary retinoid depletion and the use of retinoid receptor antagonists. Genetic approaches include generating mice with null alleles (knockouts) for retinoic acid receptors and breeding such strains to generate compound knockouts, the approach used by McGowan and coworkers in this issue. Other genes important in alveolization may also be required for normal development of the lung and other tissues. Thus, the ability to assess the role(s) of such genes in normal postnatal alveolization in mice, or in retinoic acid-mediated neo-alveolization in adult animals may be limited by lethal phenotypes in these knockouts. For example, the elastin-null mouse dies within 24 hours of birth because of circulatory problems and does not live long enough to assess the requirement for elastin in alveolization (24). Similar phenotypes are found in a number of knockout mice of other extracellular matrix, growth factor, and growth factor-receptor genes. Conditional knockout methodology may prove useful in circumventing the problem of lethal developmental phenotypes. Another genetic approach is the use of transgenic mice overexpressing a particular growth factor in a cell type-specific manner in the lung. Both the conditional knockout technology and transgenic approaches require a gene promoter that is highly cell-type specific. Currently, in the lung, promoters which have been well characterized and are being used for this purpose are limited to the CCSP/CC10 gene (Clara cells) and the SP-C gene (type II epithelial cells) (25).

However, as the lipid-laden fibroblast/myofibroblast of the alveolar wall is important in alveolization, the application of conditional knockout and transgenic technology to this problem awaits the identification of alveolar myofibroblast-specific genes and functional characterization of their promoters. Several gene screening technologies such as differential display of mRNAs, gene "chips," and micro-arrays are already being employed to identify retinoid-regulated genes in the lung. These strategies could be applied to whole lungs of animals treated with retinoic acid versus vehicle or to isolated alveolar myofibroblasts in culture to identify retinoid-responsive genes that might be important for neo-alveolization. Another more focused approach would be to use laser-capture microdissection, a technique to isolate particular cells or structures in thin tissue sections (26), to discover which genes are specifically expressed at sites of secondary crest formation. Regardless of the experimental approach, it is likely that a large number of alveolization-associated and retinoid-responsive genes will be identified, and sorting among these positive candidates for key players is a challenge. Perhaps such gene-screening approaches could be applied to multiple models of neo-alveolization in order to derive a consensus of genes that are regulated similarly in each situation. Given the variety of tools, the models now available, and the current interest and focus on mechanisms of alveolization, this decade promises to bring important advances in this field.

	Footnotes

Abbreviations: retinoic acid receptors, RARs; retinoid X receptors, RXRs.

(Received in original form June 6, 2000).

Acknowledgments: This work was supported by grants HL-54049 and HL-60647 from the National Institutes of Health, grants from the Barnes-Jewish Hospital Foundation, and the Alan A. and Edith L. Wolff Charitable Trust.

	References

1. Massaro, G. D., and D. Massaro. 1996. Postnatal treatment with retinoic acid increases the number of pulmonary alveoli in rats [see Comments]. Am. J. Physiol. 270: L305-L310 [Abstract/Free Full Text].

2. Massaro, G. D., and D. Massaro. 1997. Retinoic acid treatment abrogates elastase-induced pulmonary emphysema in rats [see Comments] [published erratum appears in Nat. Med. 1997 Jul;3(7):805]. Nat. Med. 3: 675-677 [Medline].

3. Massaro, G. D., and D. Massaro. 2000. Retinoic acid treatment partially rescues failed septation in rats and in mice. Am. J. Physiol. Lung Cell Mol. Physiol. 278: L955-L960 [Abstract/Free Full Text].

4. Shenai, J. P., K. A. Kennedy, F. Chytil, and M. T. Stahlman. 1987. Clinical trial of vitamin A supplementation in infants susceptible to bronchopulmonary dysplasia. J. Pediatr. 111: 269-277 [Medline].

5. McGowan, S., S. K. Jackson, M. Jenkins-Moore, H.-H. Dai, P. Chambon, and J. M. Snyder. 2000. Mice bearing deletions of retinoic acid receptors demonstrate reduced lung elastin and alveolar number. Am. J. Respir. Cell Mol. Biol. 23: 162-167 [Abstract/Free Full Text].

6. McGowan, S. E.. 1992. Extracellular matrix and the regulation of lung development and repair. FASEB J. 6: 2895-2904 [Abstract].

7. Chambon, P.. 1996. A decade of molecular biology of retinoic acid receptors. FASEB J. 10: 940-954 [Abstract].

8. Han, G. R., D. F. Dohi, H. Y. Lee, R. Rajah, G. L. Walsh, W. K. Hong, P. Cohen, and J. M. Kurie. 1997. All-trans-retinoic acid increases transforming growth factor-beta2 and insulin-like growth factor binding protein-3 expression through a retinoic acid receptor-alpha-dependent signaling pathway. J. Biol. Chem. 272: 13711-13716 [Abstract/Free Full Text].

9. Naltner, A., M. Ghaffari, J. A. Whitsett, and C. Yan. 2000. Retinoic acid stimulation of the human surfactant protein B promoter is thyroid transcription factor 1 site-dependent. J. Biol. Chem. 275: 56-62 [Abstract/Free Full Text].

10. Packer, A. I., K. G. Mailutha, L. A. Ambrozewicz, and D. J. Wolgemuth. 2000. Regulation of the Hoxa4 and Hoxa5 genes in the embryonic mouse lung by retinoic acid and TGFbeta1: implications for lung development and patterning. Dev. Dyn. 217: 62-74 [Medline].

11. McGowan, S. E., M. M. Doro, and S. K. Jackson. 1997. Endogenous retinoids increase perinatal elastin gene expression in rat lung fibroblasts and fetal explants. Am. J. Physiol. 273: L410-L416 [Abstract/Free Full Text].

12. Das, R. M.. 1980. The effect of -aminopropionitrile on lung development in the rat. Am. J. Pathol. 101: 711-720 [Abstract].

13. Kida, K., and W. M. Thurlbeck. 1980. Lack of recovery of lung structure and function after administration of BAPN in the postnatal period. Am. Rev. Respir. Dis. 122: 467-473 [Medline].

14. Bostrom, H., K. Willets, M. Pekny, P. Leveen, P. Lindahl, H. Hedstrand, M. Pekna, M. Hellstrom, S. Gebremedhin, M. Schalling, M. Nilsson, S. Kurland, J. Tornell, J. K. Heath, and C. Betscholtz. 1996. PDGF-A signalling is a critical event in lung alveolar myofibroblast development and alveogenesis. Cell 85: 863-873 [Medline].

15. Lindahl, P., L. Karlsson, M. Hellstrom, S. Gebre-Medhin, K. Willets, J. K. Heath, and C. Betsholtz. 1997. Alveogenesis failure in PDGF-A-deficient mice is coupled to a lack of distal spreading of alveolar smooth muscle progenitors during lung development. Development 124: 3943-3953 [Abstract].

16. Bruce, M. C., E. N. Bruce, K. Janiga, and A. Chetty. 1993. Hyperoxic exposure of developing rat lung decreases tropoelastin mRNA levels that rebound postexposure. Am. J. Physiol. (Lung Cell. Mol. Physiol.) 9: L293-L300 .

17. Bruce, M. C., R. Pawlowski, and J. F. Tomashefski Jr.. 1989. Changes in elastic fiber structure and concentration associated with hyperoxic exposure in the developing rat lung. Am. Rev. Respir. Dis. 140: 1067-1074 [Medline].

18. Blanco, L. N., and L. Frank. 1993. The formation of alveoli in rat lung during the third and fourth postnatal weeks: effect of hyperoxia, dexamethasone, and deferoxamine. Pediatr. Res. 34: 334-340 [Medline].

19. Siracusa, L. D., R. McGrath, Q. Ma, J. J. Moskow, J. Manne, P. J. Christner, A. M. Buchberg, and S. A. Jimenez. 1996. A tandem duplication within the fibrillin 1 gene is associated with the mouse tight skin mutation. Genome Research 6: 300-313 [Abstract/Free Full Text].

20. Eisenberg, L. M., and R. R. Markwald. 1995. Molecular regulation of atrioventricular valvuloseptal morphogenesis. Circ. Res. 77: 1-6 [Free Full Text].

21. Smith, S. M., E. D. Dickman, R. P. Thompson, A. R. Sinning, A. M. Wunsch, and R. R. Markwald. 1997. Retinoic acid directs cardiac laterality and the expression of early markers of precardiac asymmetry. Dev. Biol. 182: 162-171 [Medline].

22. Brock, T. 2000. Robert Koch: a life in medicine and bacteriology. ASM, Washington, DC. 364.

23. Koh, D., J. Roby, B. Starcher, R. Senior, and R. Pierce. 1996. Postpneumonectomy lung growth: a model of reinitiation of tropoelastin and type I collagen in a normal pattern in adult rat lung. Am. J. Respir. Cell Mol. Biol. 15: 611-623 [Abstract].

24. Li, D. Y., G. Faury, D. G. Taylor, E. C. Davis, W. A. Boyle, R. P. Mecham, P. Stenzel, B. Boak, and M. T. Keating. 1998. Novel arterial pathology in mice and humans hemizygous for elastin. J. Clin. Invest. 102: 1783-1787 [Medline].

25. Whitsett, J. A., and L. Zhou. 1996. Use of transgenic mice to study autocrine-paracrine signaling in lung morphogenesis and differentiation. Clin. Perinatol. 23: 753-769 [Medline].

26. Simone, N. L., R. F. Bonner, J. W. Gillespie, M. R. Emmert-Buck, and L. A. Liotta. 1998. Laser-capture microdissection: opening the microscopic frontier to molecular analysis. Trends Genet. 14: 272-276 [Medline].

27. Federspiel, S. J., S. J. DiMari, M. L. Guerry-Force, and M. A. Haralson. 1990. Extracellular matrix biosynthesis by cultured fetal rat lung epithelial cells: II. Effects of acute exposure to epidermal growth factor and retinoic acid on collagen biosynthesis. Lab. Invest. 63: 455-466 [Medline].

28. Redlich, C. A., H. M. Delisser, and J. A. Elias. 1995. Retinoic acid inhibition of transforming growth factor-beta-induced collagen production by human lung fibroblasts. Am. J. Respir. Cell Mol. Biol. 12: 287-295 [Abstract].

29. Liu, B., C. S. Harvey, and S. E. McGowan. 1993. Retinoic acid increases elastin in neonatal rat lung fibroblast cultures. Am. J. Physiol. 265: L430-L437 [Abstract/Free Full Text].

30. Schoenermark, M. P., T. I. Mitchell, J. L. Rutter, P. R. Reczek, and C. E. Brinckerhoff. 1999. Retinoid-mediated suppression of tumor invasion and matrix metalloproteinase synthesis. Ann. NY Acad. Sci. 878: 466-486 [Abstract/Free Full Text].

31. Zhang, L. X., K. J. Mills, M. I. Dawson, S. J. Collins, and A. M. Jetten. 1995. Evidence for the involvement of retinoic acid receptor (RAR) alpha-dependent signaling pathway in the induction of tissue transglutaminase and apoptosis by retinoids. J. Biol. Chem. 270: 6022-6029 [Abstract/Free Full Text].

32. Oberg, K. C., and G. Carpenter. 1991. Dexamethasone and retinoic acid regulate the expression of epidermal growth factor receptor mRNA by distinct mechanisms. J. Cell. Physiol. 149: 244-251 [Medline].

33. Ravi, R. K., F. M. Scott, F. Cuttitta, E. Weber, G. P. Kalemkerian, B. D. Nelkin, and M. Mabry. 1998. Induction of gastrin releasing peptide by all-trans retinoic acid in small cell lung cancer cells. Oncol. Rep. 5: 497-501 [Medline].

34. Glick, A. B., B. K. McCune, N. Abdulkarem, K. C. Flanders, J. A. Lumadue, J. M. Smith, and M. B. Sporn. 1991. Complex regulation of TGF beta expression by retinoic acid in the vitamin A-deficient rat. Development 111: 1081-1086 [Abstract/Free Full Text].

35. Jonk, L. J., M. E. de Jonge, C. E. Pals, S. Wissink, J. M. Vervaart, J. Schoorlemmer, and W. Kruijer. 1994. Cloning and expression during development of three murine members of the COUP family of nuclear orphan receptors. Mech. Dev. 47: 81-97 [Medline].

36. Lee, H. Y., D. F. Dohi, Y. H. Kim, G. L. Walsh, U. Consoli, M. Andreeff, M. I. Dawson, W. K. Hong, and J. M. Kurie. 1998. All-trans retinoic acid converts E2F into a transcriptional suppressor and inhibits the growth of normal human bronchial epithelial cells through a retinoic acid receptor- dependent signaling pathway. J. Clin. Invest. 101: 1012-1019 [Medline].

37. Bogue, C. W., I. Gross, H. Vasavada, D. W. Dynia, C. M. Wilson, and H. C. Jacobs. 1994. Identification of Hox genes in newborn lung and effects of gestational age and retinoic acid on their expression. Am. J. Physiol. 266: L448-L454 [Abstract/Free Full Text].

38. Nevrivy, D. J., V. J. Peterson, D. Avram, J. E. Ishmael, S. G. Hansen, P. Dowell, D. E. Hruby, M. I. Dawson, and M. Leid. 2000. Interaction of GRASP, a protein encoded by a novel retinoic acid-induced gene, with members of the cytohesin family of guanine nucleotide exchange factors. J. Biol. Chem. 275: 16827-16836 [Abstract/Free Full Text].

39. Walpole, S. M., K. T. Hiriyana, A. Nicolaou, E. L. Bingham, J. Durham, M. Vaudin, M. T. Ross, J. R. Yates, P. A. Sieving, and D. Trump. 1999. Identification and characterization of the human homologue (RAI2) of a mouse retinoic acid-induced gene in Xp22. Genomics 55: 275-283 [Medline].