Multiple pathways governing Cdx1 expression during murine development.

Cdx1 encodes a mammalian homeobox gene involved in vertebral patterning. Retinoic acid (RA) is likewise implicated in vertebral patterning. We have previously shown that Cdx1 is a direct retinoid target gene, suggesting that Cdx1 may convey some of the effects of retinoid signaling. However, RA appears to be essential for only early stages of Cdx1 expression, and therefore other factors must be involved in maintaining later stages of expression. Based on function and pattern of expression, Wnt family members, in particular Wnt3a, are candidates for regulation of expression of Cdx1. Consistent with this, we confirm prior results which demonstrated that Cdx1 can be directly regulated by Wnt signaling, and identify functional LEF/TCF response motifs essential for this response. We also find that Cdx1 expression is markedly attenuated in a stage- and tissue-specific fashion in the Wnt3a hypomorph vestigial tail, and present data demonstrating that Wnt3a and RA synergize strongly to activate Cdx1. Finally, we show that Cdx1 positively regulates its own expression. These data prompt a model whereby retinoid and Wnt signaling function directly and synergistically to initiate Cdx1 expression in the caudal embryo. Expression is then maintained, at least in part, by an autoregulatory mechanism at later stages.

RARgamma and Cdx1 interactions in vertebral patterning.

Exogenous retinoic acid (RA) can evoke vertebral homeosis when administered during late gastrulation. These vertebral transformations correlate with alterations of the rostral limit of Hox gene expression in the prevertebrae, suggesting that retinoid signaling regulates the combinatorial expression of Hox genes dictating vertebral identity. Conversely, loss of certain RA receptors (RARs) results in anterior homeotic transformations principally affecting the cervical region. Despite these observations, the relationship between retinoid signaling, somitic Hox expression, and vertebral patterning is poorly understood. The members of the murine Cdx family (Cdx1, Cdx2, and Cdx4) are the homologues of Drosophila caudal and encode homeobox-containing transcription factors. Cdx1 homozygous null mutants exhibit anterior homeotic transformations, some of which are reminiscent of those in RARgamma null offspring. In Cdx1 mutants, these transformations occur concomitant with posteriorized prevertebral expression of certain Hox genes. Cdx1 has recently been demonstrated to be a direct RA target, suggesting an indirect means by which retinoid signaling may impact vertebral patterning. To further investigate this relationship, a complete allelic series of Cdx1-RARgamma mutants was generated and the skeletal phenotype assessed either following normal gestation or after administration of RA. Synergistic interactions between these null alleles were observed in compound mutants, and the full effects of exogenous RA on vertebral morphogenesis required Cdx1. These findings are consistent with a role for RA upstream of Cdx1 as regards axial patterning. However, exogenous RA attenuated several defects inherent to Cdx1 null mutants. This finding, together with the increased phenotypic severity of RARgamma-Cdx1 double null mutants relative to single nulls, suggests that these pathways also function in parallel, likely by converging on common targets.

Regulation of gli activity by all-trans retinoic acid in mouse keratinocytes.

Sonic hedgehog (Shh) signaling is essential for many normal developmental processes. The Shh signal is interpreted by the Gli transcription factors. Elevated Gli-1 expression has been associated with several neoplasms, including basal cell carcinoma. All-trans retinoic acid (RA) has strong effects on epidermal growth and differentiation and has been used for the treatment of various epithelial disorders. In this report, we show that RA can inhibit Gli activity in immortalized murine keratinocytes in a RA receptor-specific manner. This inhibition may occur, at least in part, through sequestration of the transcriptional coactivator cyclic AMP-responsive element-binding protein-binding protein and suggests a novel effect of retinoid excess on Shh signaling.

Retinoic acid regulation of Cdx1: an indirect mechanism for retinoids and vertebral specification.

Retinoic acid (RA) is required for diverse developmental programs, including vertebral specification. Both RA receptor disruption and excess RA result in homeotic transformations of the axial skeleton. These effects are believed to occur through altered expression of Hox genes, several of which have been demonstrated to be direct RA targets. Members of the cdx (caudal) homeobox gene family are also implicated in regulating Hox expression. Disruption of cdx1 results in vertebral homeotic transformations and alteration of Hox expression boundaries; similar homeosis is also observed in cdx2 heterozygotes. In Xenopus, gain or loss of Cdx function affects vertebral morphogenesis through a mechanism that also correlates with altered Hox expression. Taken together with the finding of putative Cdx binding motifs in several Hox promoters, these data strongly support a role for Cdx members in direct regulation of expression of at least some Hox genes. Most retinoid-responsive Hox genes have not been demonstrated to be direct RA targets, suggesting that intermediaries are involved. Based on these findings, we hypothesized that one or more cdx members may transduce the effects of RA on Hox transcription. Consistent with this, we present evidence that cdx1 is a direct RA target gene, suggesting an additional pathway for retinoid-dependent vertebral specification.

Cloning and developmental expression of mouse aldehyde reductase (AKR1A4).

The aldo-keto reductase superfamily catalyzes the reduction of a broad range of aldehydes and ketones to their corresponding alcohols. Here we report the cloning of the mouse aldehyde reductase cDNA and its embryonic pattern of expression. From stages E7.5 to E13.5 the gene encoding for this enzyme is expressed at high levels in several tissues, including the neural ectoderm, gut endoderm, somites, branchial arches, otic vesicles, limb buds, and tail bud. In adult mice aldehyde reductase was expressed in all tissues examined.

Characterization of retinoic acid receptor-deficient keratinocytes.

Retinoids are essential for normal epidermal growth and differentiation and show potential for the prevention or treatment of various epithelial neoplasms. The retinoic acid receptors (RARalpha, -beta, and -gamma) are transducers of the retinoid signal. The epidermis expresses RARgamma and RARalpha, both of which are potential mediators of the effects of retinoids in the epidermis. To further investigate the role(s) of these receptors, we derived transformed keratinocyte lines from wild-type, RARalpha, RARgamma, and RARalphagamma null mice and investigated their response to retinoids, including growth inhibition, markers of growth and differentiation, and AP-1 activity. Our results indicate that RARgamma is the principle receptor contributing to all-trans-retinoic acid (RA)-mediated growth arrest in this system. This effect partially correlated with inhibition of AP-1 activity. In the absence of RARs, the synthetic retinoid N-(4-hydroxyphenyl)-retinamide inhibited growth; this was not observed with RA, 9-cis RA, or the synthetic retinoid (E)-4-[2-(5, 5, 8, 8 tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propenyl] benzoic acid. Finally, both RARalpha and RARgamma differently affected the expression of some genes, suggesting both specific and overlapping roles for the RARs in keratinocytes.

Expression and genome organization of resistance gene analogs in soybean.

Sequence analysis of cloned plant disease-resistance genes reveals a number of conserved domains. Researchers have used these domains to amplify analogous sequences, resistance gene analogs (RGAs), from soybean and other crops. Many of these RGAs map in close proximity to known resistance genes. While this technique is useful in identifying potential disease resistance loci, identifying the functional resistance gene from a cluster of homologs requires sequence information from outside of these conserved domains. To study RGA expression and to determine the extent of their similarity to other plant resistance genes, two soybean cDNA libraries (root and epicotyl) were screened by hybridization with RGA class-specific probes. cDNAs hybridizing to RGA probes were detected in each library. Two types of cDNAs were identified. One type was full-length and contained several disease-resistance gene (R-gene) signatures. The other type contained several deletions within these signatures. Sequence analyses of the cDNA clones placed them in the Toll-Interleukin-1 receptor, nucleotide binding domain, and leucine-rich repeat family of disease-resistance genes. Using clone-specific primers from within the 3' end of the LRRs, we were able to map two cDNA clones (LM6 and MG13) to a BAC contig that is known to span a cluster of disease-resistance genes.

Hoxd4 and Rarg interact synergistically in the specification of the cervical vertebrae.

We show that, relative to single null mutants, mice bearing mutations in both Hoxd4 and Rarg display malformations of the basioccipital bone, and first (C1) and second cervical vertebrae (C2) at increased penetrance and expressivity, demonstrating synergy between Hoxd4 and Rarg in the specification of the cervical skeleton. In contrast to Rarg mutants, retinoic acid (RA) treatment on embryonic day 10.5 of Hoxd4 single or Hoxd4;Rarg double mutants does not rescue normal development of C2. Somitic expression of Hoxd4 is not altered in wild-type or Rarg mutant animals before or after RA treatment on day 10.5, suggesting that Hoxd4 and Rarg act in parallel to regulate the expression of target genes directing skeletogenesis.

Retinoic acid affects left-right patterning.

Our understanding of the means by which the left-right axis is patterned is not fully understood, although a number of key intermediaries have been recently described. We report here that retinoic acid (RA) excess affects heart situs concomitant with alterations in the expression of genes implicated in the establishment of the left-right axis. Specifically, RA exposure during a specific developmental window evoked bilateral expression of lefty-1, lefty-2, nodal, and pitx-2 in the lateral plate mesoderm. Time course experiments, together with analysis of midline markers, suggest that nascent mesoderm constitutes a predominant RA target involved in this process. These events are likely to underlie the perturbations of heart looping provoked by excess RA and suggest a means by which retinoids influence the early steps in establishment of the left-right embryonic axis.

Gene targeting of retinoid receptors.

Gene targeting in embryonic stem (ES) cells has been employed to investigate the role of the retinoid receptors and binding proteins both in the mouse as well as in embryocarcinoma cells. It is a powerful technique for the modification of the mouse genome. With more recent refinements in gene targeting technology, it is now possible to introduce more subtle mutations in the murine genome, as well as to investigate gene function in a tissue and temporally-restricted manner. It should also be possible to modify genes in diverse diploid cell lines, to generate diverse model systems for analysis of retinoid receptor function. In this article, some of the basic principles for gene targeting are described.

A molecular basis for retinoic acid-induced axial truncation.

Dietary deprivation and gene disruption studies clearly demonstrate that biologically active retinoids, such as retinoic acid (RA), are essential for numerous developmental programs. Similar ontogenic processes are also affected by retinoic acid excess, suggesting that the effects of retinoid administration reflect normal retinoid-dependent events. In the mouse, exogenous retinoic acid can induce both anterior (anencephaly, exencephaly) and posterior (spina bifida) neural tube defects depending on the developmental stage of treatment. Retinoic acid receptor gamma (RARgamma) mediates these effects on the caudal neural tube at 8.5 days postcoitum, as RARgamma-/- mice are completely resistant to spina bifida induced by retinoic acid at this stage. We therefore used this null mouse as a model to examine the molecular nature of retinoid-induced caudal neural tube defects by using a panel of informative markers and comparing their expression between retinoic acid-treated wild-type and RARgamma-/- embryos. Our findings indicate that treatment of wild-type embryos led to a rapid and significant decrease in the caudal expression of all mesodermal markers examined (e.g., brachyury, wnt-3a, cdx-4), whereas somite, neuroepithelial, notochord, floorplate, and hindgut markers were unaffected. RARgamma-/- mutants exhibited normal expression patterns for all markers examined, consistent with the notion that mesodermal defects underlie the etiology of retinoid-induced spina bifida. We also found that posterior somitic, but not caudal presomitic, embryonic tissues contained detectable bioactive retinoids, an observation which correlated with the ability of caudal explants to rapidly clear exogenous RA. Interestingly, transcripts encoding mP450RAI, a cytochrome P450, the product of which is believed to catabolize retinoic acid, were abundant in the retinoid-poor region of the caudal embryo. mP450RAI was rapidly induced by retinoic acid treatment in vivo, consistent with previous studies suggesting that it plays a critical role in retinoid signaling. These data suggest that nascent mesoderm is highly sensitive to retinoic acid and that mP450RAI serves to tightly regulate retinoid levels in the caudal embryo. These findings also raise the possibility that RA may play a role in the generation of posterior mesoderm derivatives in part by affecting brachyury expression.

Impaired granulocytic differentiation in vitro in hematopoietic cells lacking retinoic acid receptors alpha1 and gamma.

Transcripts for the retinoic acid receptors (RARs) alpha1, alpha2, gamma1, and gamma2 were found in the granulocytic lineage (Gr-1+ cells) through semiquantitative polymerase chain reaction (PCR) analysis. The screening of single cell cDNA libraries derived from hematopoietic progenitors also showed the presence of RARalpha and, to a lesser extent, RARgamma transcripts in committed granulocyte (colony-forming unit-granulocyte [CFU-G]) or granulocyte-macrophage (CFU-GM) colony-forming cells. The contribution of RARalpha1 and gamma to hematopoietic cell differentiation was therefore investigated in mice bearing targeted disruption of either one or both of these loci. Because RARgamma and RARalpha1gamma compound null mutants die shortly after birth, bone marrow cells were collected from fetuses at 18.5 days postcoitum (dpc) and evaluated for growth and differentiation in culture in the presence of Steel factor (SF), interleukin-3 (IL-3), and erythropoietin (Epo). The frequency of colony-forming cells from bone marrow populations derived from RARalpha1/gamma double null mice was not significantly different from that of RARgamma or RARalpha1 single nulls or from wild-type controls. In addition, the distribution of erythroid, granulocyte, and macrophage colonies was comparable between hematopoietic cells from all groups, suggesting that lineage commitment was not affected by the lack of RARalpha1 and/or RARgamma. Colony cells were then harvested individually and evaluated by morphologic criteria. While terminal granulocyte differentiation was evident in wild-type cells and colonies from either single null mutant, colonies derived from RARalpha1-/-gamma-/- bone marrow populations were blocked at the myelocyte and, to a lesser extent, at the metamyelocyte stages, whereas erythroid and macrophage differentiation was not affected. Together, these results indicate that both RARalpha1 and gamma are required for terminal maturation in the granulocytic lineage in vitro, but appear to be dispensable for the early stages of hematopoietic cell development. Our results raise the possibility that in acute promyelocytic leukemia (APL), the RARalpha fusion proteins cause differentiation arrest at a stage when further maturation requires not only RARalpha, but also RARgamma. Finally, bone marrow cells appear to differentiate normally in vivo, suggesting an effective compensation mechanism in the RARalpha1/gamma double null mice.

Contribution of retinoic acid receptor gamma to retinoid-induced craniofacial and axial defects.

Exogenous retinoic acid (RA) administered during mouse embryogenesis can alter the pattern of the axial skeleton during two developmental periods: an early window (7 to 8.5 days post-coitum; dpc) and a late window (9.5 to 11.5 dpc). Treatment during the early window results in vertebral homeotic transformations (predominantly posteriorizations) concomitant with rostral shifts in Hox gene expression, while treatment at the later window results in similar transformations without detectable alterations in Hox gene expression patterns. Mice null for retinoic acid receptor gamma (RAR gamma) exhibit axial defects, including homeosis of several vertebrae, therefore establishing a role for this receptor in normal axial specification RAR gamma null mutants are also completely resistant to RA-induced spina bifida, which occurs in wildtype embryos treated at 8.5-9.0 dpc, suggesting that this receptor specifically transduces at least a subset of the teratogenic effects of retinoids. To further investigate the role of RAR gamma in RA-induced defects during the early and late windows of retinoid-sensitive vertebral patterning, RAR gamma heterozygotes were intercrossed, pregnant females treated with vehicle or RA at 7.3, 10.5 or 11.5 dpc and full-term fetuses assessed for skeletal defects. Relative to wildtype littermates, RAR gamma null mutants treated at 7.3 dpc were markedly resistant to RA-induced embryolethality, craniofacial malformations, and neural tube defects. Furthermore, while RAR gamma null mutants were modestly resistant to certain vertebral malformations elicited by RA treatment at 7.3, they exhibited more pronounced resistance following treatment at 10.5 and 11.5 dpc. Moreover, several of the vertebral defects inherent to the RAR gamma null phenotype were abolished by RA treatment specifically at 10.5 dpc, suggesting that RAR alpha and/or RAR beta isoforms may substitute for certain RAR gamma functions, and that RAR gamma may elicit its normal effects on vertebral morphogenesis at this developmental stage.

Developmental roles of the retinoic acid receptors.

Retinoic acid, one of the principle active metabolites of vitamin A (retinol), is believed to be essential for numerous developmental and physiological processes. Vitamin A deprivation (VAD) during development leads to numerous congenital defects. Previous studies of retinoic acid receptor (RAR) deficient mice failed to reveal any of these VAD-induced defects. This finding suggested that either the RARs are functionally redundant or that they are not critically required during development. In order to address these possibilities, we derived a number of RAR compound mutants. Unlike RAR single mutants, these compound null mutants died either in utero or shortly following birth. Histological analysis revealed essentially all of the defects characteristic of fetal VAD. A number of additional malformations, not described in previous VAD studies, were also observed. These included defects of the ocular and salivary glands and their ducts, the skeletal elements of the fore- and hindlimbs, and the cervical region of the axial skeleton. In addition, with the exception of derivatives forming within the first pharyngeal arch, most of the elements derived from mesectoderm emanating from cranial and hindbrain levels were affected. A number of these mutants also exhibited supernumerary cranial skeletal elements characteristics of the reptilian skull. A summary of the defects found in these RAR double mutants is presented.

Roles of retinoic acid receptors and of Hox genes in the patterning of the teeth and of the jaw skeleton.

Retinoic acid receptors and transcriptional factors encoded by Hox genes play key roles in vertebrate development and belong to an integrated functional network. To investigate the actual functions of these molecules during ontogenesis and in particular in the patterning of the cranial neural crest cells giving rise to the teeth and to the jaw bones, we have generated null mutant mice lacking functional retinoic acid receptors or Hox genes by gene targeting in embryonic stem cells.

Function of the retinoic acid receptors (RARs) during development (I). Craniofacial and skeletal abnormalities in RAR double mutants.

Numerous congenital malformations have been observed in fetuses of vitamin A-deficient (VAD) dams [Wilson, J. G., Roth, C. B., Warkany, J., (1953), Am. J. Anat. 92, 189-217]. Previous studies of retinoic acid receptor (RAR) mutant mice have not revealed any of these malformations [Li, E., Sucov, H. M., Lee, K.-F., Evans, R. M., Jaenisch, R. (1993) Proc. Natl. Acad. Sci. USA 90, 1590-1594; Lohnes, D., Kastner, P., Dierich, A., Mark, M., LeMeur, M., Chambon, P. (1993) Cell 73, 643-658; Lufkin, T., Lohnes, D., Mark, M., Dierich, A., Gorry, P., Gaub, M. P., Lemeur, M., Chambon, P. (1993) Proc. Natl. Acad. Sci. USA 90, 7225-7229; Mendelsohn, C., Mark, M., Dollé, P., Dierich, A., Gaub, M.P., Krust, A., Lampron, C., Chambon, P. (1994a) Dev. Biol. in press], suggesting either that there is a considerable functional redundancy among members of the RAR family during ontogenesis or that the RARs are not essential transducers of the retinoid signal in vivo. In order to discriminate between these possibilities, we have generated a series of RAR compound null mutants. These RAR double mutants invariably died either in utero or shortly after birth and presented a number of congenital abnormalities, which are reported in this and in the accompanying study. We describe here multiple eye abnormalities which are found in various RAR double mutant fetuses and are similar to those previously seen in VAD fetuses. Interestingly, we found further abnormalities not previously reported in VAD fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)

Function of the retinoic acid receptors (RARs) during development (II). Multiple abnormalities at various stages of organogenesis in RAR double mutants.

Compound null mutations of retinoic acid receptor (RAR) genes lead to lethality in utero or shortly after birth and to numerous developmental abnormalities. In the accompanying paper (Lohnes, D., Mark., M., Mendelsohn, C., Dollé, P., Dierich, A., Gorry, Ph., Gansmuller, A. and Chambon, P. (1994). Development 120, 2723-2748), we describe malformations of the head, vertebrae and limbs which, with the notable exception of the eye defects, were not observed in the offspring of vitamin A-deficient (VAD) dams. We report here abnormalities in the neck, trunk and abdominal regions of RAR double mutant mice, which include: (i) the entire respiratory tract, (ii) the heart, its outlow tract and the great vessels located near the heart, (iii) the thymus, thyroid and parathyroid glands, (iv) the diaphragm, (v) the genito-urinary system, and (vi) the lower digestive tract. A majority of these abnormalities recapitulate those observed in the fetal VAD syndrome described by Joseph Warkany's group more than fourty years ago [Wilson, J. G., Roth, C. B. and Warkany, J. (1953) Am. J. Anat., 92, 189-217; and refs therein]. Our results clearly demonstrate that RARs are essential for vertebrate ontogenesis and therefore that retinoic acid is the active retinoid, which is required at several stages of the development of numerous tissues and organs. We discuss several possibilities that may account for the apparent functional redundancy observed amongst retinoic acid receptors during embryogenesis.

Loss of retinoic acid receptor gamma function in F9 cells by gene disruption results in aberrant Hoxa-1 expression and differentiation upon retinoic acid treatment.

Retinoic acid (RA) signal transduction is believed to be mediated through several high-affinity nuclear receptors [RA receptors (RARs) and retinoid X receptors], which are members of the steroid/thyroid/vitamin D superfamily and function as transcription factors. Why multiple RARs exist and what gene targets are regulated by each of the three receptors remain compelling questions in developmental biology. Through targeted disruption of both RAR gamma alleles, we have identified several differentiation-specific genes that are regulated either directly or indirectly by RAR gamma in F9 embryonal carcinoma cells. These include genes encoding Hoxa-1 (Hox-1.6) and the extracellular matrix proteins laminin B1 and collagen type IV (alpha 1), all of which are RA inducible in wild-type F9 embryonal carcinoma cells but are not significantly induced in the RAR gamma-/- lines. In contrast, transcripts encoding Hoxb-1 (Hox-2.9) and cellular RA binding protein II (CRABPII) are activated by RA for a longer period of time in the RAR gamma-/- lines compared to the wild-type F9 line. Not all RA-responsive genes are aberrantly expressed; Rex-1, RAR beta, and SPARC transcripts are regulated in the RAR gamma-/- lines as they are in F9 wild-type cells. Our results support the idea that each RAR may regulate different subsets of RA-responsive genes, which may explain, in part, the complex regulation of developmental processes by retinoids.

High postnatal lethality and testis degeneration in retinoic acid receptor alpha mutant mice.

Retinoic acid (RA) plays a critical role in normal development, growth, and maintenance of certain tissues. The action of RA is thought to be mediated in part by the three nuclear receptors (RAR alpha, -beta, and -gamma), each of which is expressed as multiple isoforms. To investigate the function of the RAR alpha gene, we have disrupted, in the mouse, the whole gene or the isoform RAR alpha 1. Although RAR alpha 1 is the predominant isoform and is highly conserved among vertebrates, RAR alpha 1-null mice appeared normal. However, targeted disruption of the whole RAR alpha gene resulted in early postnatal lethality and testis degeneration. These results, showing that RAR alpha is indeed involved in the transduction of the RA signal, also suggest an unexpected genetic redundancy.