Xianling Zhao

Retinoic acid promotes limb induction through effects on body axis extension but is unnecessary for limb patterning.

Retinoic acid (RA) is thought to be a key signaling molecule involved in limb bud patterning along the proximodistal or anteroposterior axes functioning through induction of Meis2 and Shh, respectively. Here, we utilize Raldh2-/- and Raldh3-/- mouse embryos lacking RA synthesis to demonstrate that RA signaling is not required for limb expression of Shh and Meis2. We demonstrate that RA action is required outside of the limb field in the body axis during forelimb induction but that RA is unnecessary at later stages when hindlimb budding and patterning occur. We provide evidence for a model of trunk mesodermal RA action in which forelimb induction requires RA repression of Fgf8 in the developing trunk similar to how RA controls somitogenesis and heart development. We demonstrate that pectoral fin development in RA-deficient zebrafish embryos can be rescued by an FGF receptor antagonist SU5402. In addition, embryo ChIP assays demonstrate that RA receptors bind the Fgf8 promoter in vivo. Our findings suggest that RA signaling is not required for limb proximodistal or anteroposterior patterning but that RA inhibition of FGF8 signaling during the early stages of body axis extension provides an environment permissive for induction of forelimb buds.

Retinoic acid controls heart anteroposterior patterning by down‐regulating Isl1 through the Fgf8 pathway

Distinct progenitor cell populations exist in cardiac mesoderm important for patterning of the heart. During heart tube formation in mouse, Tbx5 is expressed in progenitors located more laterally, whereas Isl1 and Fgf8 are expressed in progenitors located more medially. Signals that drive mesodermal progenitors into various cardiac lineages include Fgf8, which functions to induce Isl1. Studies in chick and zebrafish have shown that retinoic acid restricts the number of cardiac progenitors, but its role in mammalian cardiac development is unclear. Here, we demonstrate that Raldh2−/− mouse embryos lacking retinoic acid signaling exhibit a posterior expansion of the cardiac Fgf8 expression domain as well as an expansion of Isl1 expression into mesoderm lying posterior to the cardiac field. We provide evidence that retinoic acid acts specifically in the posterior‐medial region of the cardiac field to establish the heart posterior boundary potentially by reducing Fgf8 expression which restricts the Isl1 domain. Developmental Dynamics 237:1627–1635, 2008.

Antagonism between Retinoic Acid and Fibroblast Growth Factor Signaling during Limb Development

The vitamin A metabolite retinoic acid (RA) provides patterning information during vertebrate embryogenesis, but the mechanism through which RA influences limb development is unclear. During patterning of the limb proximodistal axis (upper limb to digits), avian studies suggest that a proximal RA signal generated in the trunk antagonizes a distal fibroblast growth factor (FGF) signal. However, mouse and zebrafish genetic studies suggest that loss of RA suppresses forelimb initiation. Here, using genetic and pharmacological approaches, we demonstrate that limb proximodistal patterning is not RA dependent, thus indicating that RA-FGF antagonism does not occur along the proximodistal axis of the limb. Instead, our studies show that RA-FGF antagonism acts prior to limb budding along the anteroposterior axis of the trunk lateral plate mesoderm to provide a patterning cue that guides formation of the forelimb field. These findings reconcile disparate ideas regarding RA-FGF antagonism and provide insight into how endogenous RA programs the early embryo.

Retinoic acid stimulates myocardial expansion by induction of hepatic erythropoietin which activates epicardial Igf2

Epicardial signaling and Rxra are required for expansion of the ventricular myocardial compact zone. Here, we examine Raldh2–/– and Rxra–/– mouse embryos to investigate the role of retinoic acid (RA) signaling in this developmental process. The heart phenotypes of Raldh2 and Rxra mutants are very similar and are characterized by a prominent defect in ventricular compact zone growth. Although RA activity is completely lost in Raldh2–/– epicardium and the adjacent myocardium, RA activity is not lost in Rxra–/– hearts, suggesting that RA signaling in the epicardium/myocardium is not required for myocardial compact zone formation. We explored the possibility that RA-mediated target gene transcription in non-cardiac tissues is required for this process. We found that hepatic expression of erythropoietin (EPO), a secreted factor implicated in myocardial expansion, is dependent on both Raldh2 and Rxra. Chromatin immunoprecipitation studies support Epo as a direct target of RA signaling in embryonic liver. Treatment of an epicardial cell line with EPO, but not RA, upregulates Igf2. Furthermore, both Raldh2–/– and Rxra–/– hearts exhibit downregulation of Igf2 mRNA in the epicardium. EPO treatment of cultured Raldh2–/– hearts restores epicardial Igf2 expression and rescues ventricular cardiomyocyte proliferation. We propose a new model for the mechanism of RA-mediated myocardial expansion in which RA directly induces hepatic Epo resulting in activation of epicardial Igf2 that stimulates compact zone growth. This RA-EPO-IGF2 signaling axis coordinates liver hematopoiesis with heart development.

Effect of retinoic acid signaling on Wnt/β-catenin and FGF signaling during body axis extension

Cell-cell signaling regulated by retinoic acid (RA), Wnt/beta-catenin, and fibroblast growth factor (FGF) is important during body axis extension, and interactions between these pathways have been suggested. At early somite stages, Wnt/beta-catenin and FGF signaling domains exist both anterior and posterior to the developing trunk, whereas RA signaling occurs in between in the trunk under the control of the RA-synthesizing enzyme retinaldehyde dehydrogenase-2 (Raldh2). Previous studies demonstrated that vitamin A deficient quail embryos and Raldh2(-/-) mouse embryos lacking RA synthesis exhibit ectopic expression of Fgf8 and Wnt8a in the developing trunk. Here, we demonstrate that Raldh2(-/-) mouse embryos display an expansion of FGF signaling into the trunk monitored by Sprouty2 and Pea3 expression, and an expansion of Wnt/beta-catenin signaling detected by expression of Axin2, Tbx6, Cdx2, and Cdx4. Following loss of RA signaling, the caudal expression domains of Fgf8, Wnt8a, and Wnt3a expand anteriorly into the trunk, but no change is observed in caudal expression of Fgf4 or Fgf17 plus caudal expression of Fgf18 and Cdx1 is reduced. These findings suggest that RA repression of Fgf8, Wnt8a, and Wnt3a in the developing trunk functions to down-regulate FGF signaling and Wnt/beta-catenin signaling as the body axis extends.

Retinoic acid controls expression of tissue remodeling genes Hmgn1 and Fgf18 at the digit-interdigit junction.

Previous studies on retinoic acid receptor (RAR) mutants suggested that retinoic acid (RA) is required for loss of interdigital mesenchyme during digit formation. Here, we report that the RA‐generating enzyme retinaldehyde dehydrogenase‐2 (Raldh2) is expressed in the interdigital mesenchyme whereas Cyp26b1, controlling RA degradation, is expressed in digits, limiting autopodal RA action to the interdigital zones. Embryonic day 13.5 Raldh2−/− mouse embryos lose expression of the RARE‐lacZ RA‐reporter transgene and matrix metalloproteinase‐11 (Mmp11) throughout the interdigital mesenchyme, while expression of RARb, Fgf18, and high mobility group N1 (Hmgn1) is lost at the digit–interdigit junction. Raldh2−/− autopods exhibit reduced interdigital apoptosis associated with loss of Bmp7 expression, but Bmp2, Bmp4, Msx2, and Fgf8 were unaffected. Although interdigital expression of Hmgn1 was greatly down‐regulated in Raldh2−/− autopods, complementary expression of Sox9 in digit cartilage was unaffected. Regulation of Hmgn1 and Fgf18 at the digit–interdigit junction suggests RA controls tissue remodeling as well as apoptosis. Developmental Dynamics 239:665–671, 2010.

Uncoupling of Retinoic Acid Signaling From Tailbud Development Before Termination of Body Axis Extension

During the early stages of body axis extension, retinoic acid (RA) synthesized in somites by Raldh2 represses caudal fibroblast growth factor (FGF) signaling to limit the tailbud progenitor zone. Excessive RA down‐regulates Fgf8 and triggers premature termination of body axis extension, suggesting that endogenous RA may function in normal termination of body axis extension. Here, we demonstrate that Raldh2−/− mouse embryos undergo normal down‐regulation of tailbud Fgf8 expression and termination of body axis extension in the absence of RA. Interestingly, Raldh2 expression in wild‐type tail somites and tailbud from E10.5 onwards does not result in RA activity monitored by retinoic acid response element (RARE)‐lacZ. Treatment of wild‐type tailbuds with physiological levels of RA or retinaldehyde induces RARE‐lacZ activity, validating the sensitivity of RARE‐lacZ and demonstrating that deficient RA synthesis in wild‐type tail somites and tailbud is due to a lack of retinaldehyde synthesis. These studies demonstrate an early uncoupling of RA signaling from mouse tailbud development and show that termination of body axis extension occurs in the absence of RA signaling. genesis 49:776–783, 2011.

03-P026 Retinoic acid – planar cell polarity crosstalk during neural tube closure

at the middle stage of regeneration. When we observed the expression of tail marker genes such as DjAbd-Ba, their expression could be detected at the early stage of regeneration, but was not increased after the middle stage of regeneration in DjlimA(RNAi). Based on these findings combined with histological observations, we concluded that DjlimA may be required for maintaining the proper morphological events during the middle stage of regeneration in planarians, but not at the early stage. This is the first report suggesting that initiation and maintenance events during regeneration could be separated by differential activity of transcription factor(s).