Hedgehog signaling is required for commitment but not initial induction of slow muscle precursors

In the embryonic mouse retina, retinoic acid (RA) is unevenly distributed along the dorsoventral axis: RA-rich zones in dorsal and ventral retina are separated by a horizontal RA-poor stripe that contains the RA-inactivating enzyme CYP26A1. To explore the developmental role of this arrangement, we studied formation of the retina and its projections in Cyp26a1 null-mutant mice. Expression of several dorsoventral markers was not affected, indicating that CYP26A1 is not required for establishing the dorsoventral retina axis. Analysis of the mutation on a RA-reporter mouse background confirmed, as expected, that the RA-poor stripe was missing in the retina and its projections at the time when the optic axons first grow over the diencephalon. A day later, however, a gap appeared both in retina and retinofugal projections. As explanation, we found that CYP26C1, another RA-degrading enzyme, had emerged centrally in a narrower domain within the RA-poor stripe. While RA applications increased retinal Cyp26a1 expression, they slightly reduced Cyp26c1. These observations indicate that the two enzymes function independently. The safeguard of the RA-poor stripe by two distinct enzymes during later development points to a role in maturation of a significant functional feature like an area of higher visual acuity that develops at its location.     

Complementary expression patterns of retinoid acid-synthesizing and -metabolizing enzymes in pre-natal mouse inner ear structures

Retinoic acid (RA) plays a pivotal role in patterning and differentiation of the embryonic inner ear. Despite its documented effects during embryonic development, the cellular sites that synthesize or metabolize RA in the inner ear have yet to be determined. Here we describe the distribution of three synthesizing enzymes, retinaldehyde dehydrogenases 1, 2 and 3 (RALDH1, RALDH2 and RALDH3) and two catabolizing enzymes (CYP26A1 and CYP26B1) in the mouse inner ear at embryonic day 18.5 when active cell differentiation is underway. Two detection methods, radioactive and non-radioactive in situ hybridization, were employed to elucidate the tissue distribution and cellular localization of these enzymes, respectively. All of the five enzymes examined, with the exception of CYP26A1, were expressed in both vestibular and cochlear end organs. While expression of the three RALDHs was observed in various cell types, CYP26B1 expression was found only in supporting cells of the vestibular and cochlear end organs. In the cochlea, expression domains of RALDH1-3 and CYP26B1 were complementary to one another. These results reveal specific tissue- and cellular expression patterns of RA synthesizing and catabolizing enzymes in the pre-natal inner ear, and suggest that a precise control of RA concentrations in various cell types of the inner ear is achieved by the balance between RALDHs and CYP26B1 activities.     

Retinoic acid guides eye morphogenetic movements via paracrine signaling but is unnecessary for retinal dorsoventral patterning

Retinoic acid (RA) is required for patterning of the posterior nervous system, but its role in the retina remains unclear. RA is synthesized in discrete regions of the embryonic eye by three retinaldehyde dehydrogenases (RALDHs) displaying distinct expression patterns. Overlapping functions of these enzymes have hampered genetic efforts to elucidate RA function in the eye. Here, we report Raldh1, Raldh2 and Raldh3 single, double and triple null mice exhibiting progressively less or no RA synthesis in the eye. Our genetic studies indicate that RA signaling is not required for the establishment or maintenance of dorsoventral patterning in the retina, as we observe normal expression of Tbx5 and ephrin B2 (Efnb2) dorsally, plus Vax2 and Ephb2 ventrally. Instead, RA is required for the morphogenetic movements needed to shape the developing retina and surrounding mesenchyme. At early stages, Raldh2 expressed in mesenchyme and Raldh3 expressed in the retinal pigmented epithelium generate RA that delivers an essential signal to the neural retina required for morphogenetic movements that lead to ventral invagination of the optic cup. At later stages, Raldh1 expressed in dorsal neural retina and Raldh3 expressed in ventral neural retina (plus weaker expression of each in lens/corneal ectoderm) generates RA that travels to surrounding mesenchyme, where it is needed to limit the anterior invasion of perioptic mesenchyme during the formation of corneal mesenchyme and eyelids. At all stages, RA target tissues are distinct from locations of RA synthesis, indicating that RALDHs function cell-nonautonomously to generate paracrine RA signals that guide morphogenetic movements in neighboring cells.     

Dorsal and ventral retinal territories defined by retinoic acid synthesis, break-down and nuclear receptor expression.

Determination of the dorso-ventral dimension of the vertebrate retina is known to involve retinoic acid (RA), in that high RA activates expression of a ventral retinaldehyde dehydrogenase and low RA of a dorsal dehydrogenase. Here we show that in the early eye vesicle of the mouse embryo, expression of the dorsal dehydrogenase is preceded by, and transiently overlaps with, the RA-degrading oxidase CYP26. Subsequently in the embryonic retina, CYP26 forms a narrow horizontal boundary between the dorsal and ventral dehydrogenases, creating a trough between very high ventral and moderately high dorsal RA levels. Most of the RA receptors are expressed uniformly throughout the retina except for the RA-sensitive RARbeta, which is down-regulated in the CYP26 stripe. The orphan receptor COUP-TFII, which modulates RA responses, colocalizes with the dorsal dehydrogenase. The organization of the embryonic vertebrate retina into dorsal and ventral territories divided by a horizontal boundary has parallels to the division of the Drosophila eye disc into dorsal, equatorial and ventral zones, indicating that the similarities in eye morphogenesis extend beyond single molecules to topographical patterns.     

Retinoic acid is required for specification of the ventral eye field and for Rathke's pouch in the avian embryo

We have investigated the role of retinoic acid (RA) in eye development using the vitamin A deficient quail model system, which overcomes problems of retinoic acid synthesising enzyme redundancy in the embryo. In the absence of retinoic acid, the ventral optic stalk and ventral retina are missing, whereas the dorsal optic stalk and dorsal retina develop appropriately. Other ocular abnormalities observed were a thinner retina and the lack of differentiation of the lens. In an attempt to explain this, we studied the expression of various dorsally and ventrally expressed genes such as Pax2, Pax6, Tbx6, Vax2, Raldh1 and Raldh3 and noted that they were unchanged in their expression patterns. In contrast, the RA catabolising enzymes Cyp26A1 and Cyp26B1 which are known to be RA-responsive were not expressed at all in the developing eye. At much earlier stages, the expression domain of Shh in the prechordal plate was reduced, as was Nkx2.1 and we suggest a model whereby the eye field is specified according to the concentration of SHH protein that is present. We also describe another organ, Rathke's pouch which fails to develop in the absence of retinoic acid. We attribute this to the down-regulation of Bmp2, Shh and Fgf8 which are known to be involved in the induction of this structure.     

Cyp26 enzymes generate the retinoic acid response pattern necessary for hindbrain development

Retinoic acid (RA) is essential for normal vertebrate development, including the patterning of the central nervous system. During early embryogenesis, RA is produced in the trunk mesoderm through the metabolism of vitamin A derived from the maternal diet and behaves as a morphogen in the developing hindbrain where it specifies nested domains of Hox gene expression. The loss of endogenous sources of RA can be rescued by treatment with a uniform concentration of exogenous RA, indicating that domains of RA responsiveness can be shaped by mechanisms other than the simple diffusion of RA from a localized posterior source. Here, we show that the cytochrome p450 enzymes of the Cyp26 class, which metabolize RA into polar derivatives, function redundantly to shape RA-dependent gene-expression domains during hindbrain development. In zebrafish embryos depleted of the orthologs of the three mammalian CYP26 genes CYP26A1, CYP26B1 and CYP26C1, the entire hindbrain expresses RA-responsive genes that are normally restricted to nested domains in the posterior hindbrain. Furthermore, we show that Cyp26 enzymes are essential for exogenous RA to rescue hindbrain patterning in RA-depleted embryos. We present a ;gradient-free' model for hindbrain patterning in which differential RA responsiveness along the hindbrain anterior-posterior axis is shaped primarily by the dynamic expression of RA-degrading enzymes.     

Dorsal and ventral rentinoic territories defined by retinoic acid synthesis, break-down and nuclear receptor expression.

Determination of the dorso-ventral dimension of the vertebrate retina is known to involve retinoic acid (RA), in that high RA activates expression of a ventral retinaldehyde dehydrogenase and low RA of a dorsal dehydrogenase. Here we show that in the early eye vesicle of the mouse embryo, expression of the dorsal dehydrogenase is preceded by, and transiently overlaps with, the RA-degrading oxidase creating a trough between very high ventral and moderately high dorsal RA levels. Most of the RA receptors are expressed uniformly throughout the retina except for the RA-sensitive RARbeta, which is down-regulated in the CYP26 stripe. The orphan receptor COUP-TFII, which modulates RA responses, colocalizes with the dorsal dehydrogenase. The organization of the embryonic vertebrate retina into dorsal ventral territories divided by a horizontal boundary has parallels to the division of the Drosophila eye disc into dorsal, equatorial and ventral zones, indicating that the similarities in eye morphogenesis extend beyond single molecules to topographical patterns.     

Cyp26b1 within the growth plate regulates bone growth in juvenile mice

Retinoic acid (RA) is an active metabolite of vitamin A and plays important roles in embryonic development. CYP26 enzymes degrade RA and have specific expression patterns that produce a RA gradient, which regulates the patterning of various structures in the embryo. However, it has not been addressed whether a RA gradient also exists and functions in organs after birth. We found localized RA activities in the diaphyseal portion of the growth plate cartilage were associated with the specific expression of Cyp26b1 in the epiphyseal portion in juvenile mice. To disturb the distribution of RA, we generated mice lacking Cyp26b1 specifically in chondrocytes (Cyp26b1^Δchon cKO). These mice showed reduced skeletal growth in the juvenile stage. Additionally, their growth plate cartilage showed decreased proliferation rates of proliferative chondrocytes, which was associated with a reduced height in the zone of proliferative chondrocytes, and closed focally by four weeks of age, while wild-type mouse growth plates never closed. Feeding the Cyp26b1 cKO mice a vitamin A-deficient diet partially reversed these abnormalities of the growth plate cartilage. These results collectively suggest that Cyp26b1 in the growth plate regulates the proliferation rates of chondrocytes and is responsible for the normal function of the growth plate and growing bones in juvenile mice, probably by limiting the RA distribution in the growth plate proliferating zone.     

Expression of the retinoic acid catabolising enzyme CYP26B1 in the chick embryo and its regulation by retinoic acid

We have cloned a fragment of Cyp26B1, a novel retinoic acid (RA) catabolising enzyme, and examined its expression pattern during early stages of chick embryogenesis. It is expressed from stage 7 in the tail bud, an anterior patch of mesenchyme, the heart, the endothelium of the vasculature, the eye, the limb bud, the hindgut and in a complex pattern in the rhombomeres of the hindbrain. As such it has a non-overlapping expression with chick Cyp26A1, the other RA catabolising enzyme, but shows a combination of features of mouse Cyp26A1 and Cyp26B1. We have also examined its expression in the quail embryo and in the RA-free quail embryo. In the absence of RA, Cyp26B1 is only expressed in the hindbrain and fails to be expressed in all the other regions of the embryo, most dramatically in the trunk. Adding back RA rescues Cyp26B1 expression.     

A novel cytochrome P450, zebrafish Cyp26D1, is involved in metabolism of all-trans retinoic acid

Retinoid signaling is essential for development of vertebrate embryos, and its action is mainly through retinoic acid (RA) binding to its RA receptors and retinoid-X receptors, while the critical concentration and localization of RA in embryos are determined by the presence and activity of retinal dehydrogenases (for RA synthesis) and cytochrome P450 RAs (Cyp26s) (for degradation of RA). Previously, we identified a novel cyp26 gene (cyp26d1) in zebrafish that is expressed in hindbrain during early development. Using reverse-phase HPLC analyses, we show here that zebrafish Cyp26D1 expressed in 293T cells could metabolize all-trans RA, 9-cis RA, and 13-cis RA, but could not metabolize retinol or retinal. The metabolites of all-trans RA produced by Cyp26D1 were the same as that produced by Cyp26A1, which are mainly 4-hydroxy-all-trans-RA and 4-oxo-all-trans-RA. Performing mRNA microinjection into zebrafish embryos, we demonstrated that overexpression of Cyp26D1 in embryos not only caused the distance between rhombomere 5 and the first somite of the injected embryos to be shorter than control embryos but also resulted in left-right asymmetry of somitogenesis in the injected embryos. These alterations were similar to those caused by the overexpression of cyp26a1 in zebrafish embryos and to that which resulted from treating embryos with 1 microm 4-diethylamino-benzaldehyde (retinal dehydrogenase inhibitor), implying that cyp26d1 can antagonize RA activity in vivo. Together, our in vitro and in vivo results provided direct evidence that zebrafish Cyp26D1 is involved in RA metabolism.     

Cyp26b1 regulates retinoic acid-dependent signals in T cells and its expression is inhibited by transforming growth factor-β

Background

The vitamin A metabolite, retinoic acid (RA), plays important roles in the regulation of lymphocyte properties. Dendritic cells in gut-related lymphoid organs can produce RA, thereby imprinting gut-homing specificity on T cells and enhancing transforming growth factor (TGF)-β-dependent induction of Foxp3⁺ regulatory T cells upon antigen presentation. In general, RA concentrations in cells and tissues are regulated by its degradation as well. However, it remained unclear if T cells could actively catabolize RA.

Methodology/Principal Findings

We assessed the expression of known RA-catabolizing enzymes in T cells from mouse lymphoid tissues. Antigen-experienced CD44⁺ T cells in gut-related lymphoid organs selectively expressed Cyp26b1, a member of the cytochrome P450 family 26. However, T cells in the spleen or skin-draining lymph nodes did not significantly express Cyp26b1. Accordingly, physiological levels of RA (1–10 nM) could induce Cyp26b1 expression in naïve T cells upon activation in vitro, but could not do so in the presence of TGF-β. Overexpression of Cyp26b1 significantly suppressed the RA effect to induce expression of the gut-homing receptor CCR9 on T cells. On the other hand, knocking down Cyp26b1 gene expression with small interfering RNA or inhibiting CYP26 enzymatic activity led to enhancement of the RA-induced CCR9 expression.

Conclusions/Significance

Our data demonstrate a role for CYP26B1 in regulating RA-dependent signals in activated T cells but not during TGF-β-dependent differentiation to Foxp3⁺ regulatory T cells. Aberrant expression of CYP26B1 may disturb T cell trafficking and differentiation in the gut and its related lymphoid organs.     

Retinoic acid regulation by CYP26 in vertebrate lens regeneration

Xenopus laevis is among the few species that are capable of fully regenerating a lost lens de novo. This occurs upon removal of the lens, when secreted factors from the retina are permitted to reach the cornea epithelium and trigger it to form a new lens. Although many studies have investigated the retinal factors that initiate lens regeneration, relatively little is known about what factors support this process and make the cornea competent to form a lens. We presently investigate the role of Retinoic acid (RA) signaling in lens regeneration in Xenopus. RA is a highly important morphogen during vertebrate development, including the development of various eye tissues, and has been previously implicated in several regenerative processes as well. For instance, Wolffian lens regeneration in the newt requires active RA signaling. In contrast, we provide evidence here that lens regeneration in Xenopus actually depends on the attenuation of RA signaling, which is regulated by the RA-degrading enzyme CYP26. Using RT-PCR we examined the expression of RA synthesis and metabolism related genes within ocular tissues. We found expression of aldh1a1, aldh1a2, and aldh1a3, as well as cyp26a1 and cyp26b1 in both normal and regenerating corneal tissue. On the other hand, cyp26c1 does not appear to be expressed in either control or regenerating corneas, but it is expressed in the lens. Additionally in the lens, we found expression of aldh1a1 and aldh1a2, but not aldh1a3. Using an inhibitor of CYP26, and separately using exogenous retinoids, as well as RA signaling inhibitors, we demonstrate that CYP26 activity is necessary for lens regeneration to occur. We also find using phosphorylated Histone H3 labeling that CYP26 antagonism reduces cell proliferation in the cornea, and using qPCR we find that exogenous retinoids alter the expression of putative corneal stem cell markers. Furthermore, the Xenopus cornea is composed of an outer layer and inner basal epithelium, as well as a deeper fibrillar layer sparsely populated with cells. We employed antibody staining to visualize the localization of CYP26A, CYP26B, and RALDH1 within these corneal layers. Immunohistochemical staining of these enzymes revealed that all 3 proteins are expressed in both the outer and basal layers. CYP26A appears to be unique in also being present in the deeper fibrillar layer, which may contain cornea stem cells. This study reveals a clear molecular difference between newt and Xenopus lens regeneration, and it implicates CYP26 in the latter regenerative process.     

Regulation of retinoic acid distribution is required for proximodistal patterning and outgrowth of the developing mouse limb

Exogenous retinoic acid (RA) induces marked effects on limb patterning, but the precise role of endogenous RA in this process has remained unknown. We have studied the role of RA in mouse limb development by focusing on CYP26B1, a cytochrome P450 enzyme that inactivates RA. Cyp26b1 was shown to be expressed in the distal region of the developing limb bud, and mice that lack CYP26B1 exhibited severe limb malformation (meromelia). The lack of CYP26B1 resulted in spreading of the RA signal toward the distal end of the developing limb and induced proximodistal patterning defects characterized by expansion of proximal identity and restriction of distal identity. CYP26B1 deficiency also induced pronounced apoptosis in the developing limb and delayed chondrocyte maturation. Wild-type embryos exposed to excess RA phenocopied the limb defects of Cyp26b1(-/-) mice. These observations suggest that RA acts as a morphogen to determine proximodistal identity, and that CYP26B1 prevents apoptosis and promotes chondrocyte maturation, in the developing limb.