In vertebrates, limbs develop from structures called limb buds, and the lateral plate mesoderm is where the limb buds are grown from. The limb growth and patterning in zebrafish is under tight regulation of specific signaling pathways which will be discussed in this section. Previously, it was demonstrated that the expression of the T-box transcription factor tbx5 in pectoral fin buds is required and adequate for forelimb formation in zebrafish , , .
In 2010, Grandel and colleague  demonstrated that the signaling pathway that leads to tbx5 expression in zebrafish limb bud is retinoic acid (RA) signaling. Several research groups had previously demonstrated that RA is upstream of tbx5 , and it influences patterning of developing limbs . However, Grandel et al. is the first group to investigate the contribution of RA signaling to zebrafish limb development at different stages of development.
RA is synthesized from its precursor by an enzyme called aldehyde dehydrogenase 1a2 (aldh1a2) (Fig.1). Zebrafish mutants in aldh1a2 are unable to synthesize RA in their early stages of development. It was also reported that aldh1a2 mutants do not develop fins, and they do not express tbx5 [2, 7]. Grandel et al.  investigated the timing of the early RA signaling events that lead to fin precursor specification in zebrafish mutants for aldh1a2 as well as those embryos treated with inhibitors of RA synthesis.
Fig.1- Schematic representation of the intracellular retinoid signaling pathway: Retinol is converted into retinal and retinoic acids, before either it acts through nuclear retinoid receptors or is inactivated by hydroxylation. Abbreviations are: ADH: alcohol dehydrogenase; CRABP: cellular retinoic acid binding protein; CRBP: cellular retinol binding protein; CYP26: cytochrome p450 retinoic acid inducible; RAR: retinoic acid receptor; RALDH: retinaldehyde dehydrogenase; RXR: retinoid X receptor, SDR: short-chain dehydrogenase/reductase.
The requirement for retinoic acid (RA) signaling in fin precursor determination
Zebrafish embryos mutant for aldh1a2 lack pectoral fins at early stages of development (Fig2A,B). aldh1a2 mutants can be recognized as early as 10-somite (s) stage by the lack of tbx5 expression (Fig.2C-H). Since aldh1a2 mutants lack tbx5 expression, this suggests involvement of RA signaling in the process of fin precursor determination. 
Fig.2- Phenotype of Fig.2- Phenotype of nlsu11 mutant and sibling embryos and larvae. A-H: Scanning electron micrograph images (C-H). A: 3 days post fertilization (dpf). Wild type progeny show pectoral fins. A-H: Scanning electron micrograph images (C-H). A: 3 days post fertilization (dpf). Wild type progeny show pectoral fins. B: In nlsu11 mutant, no pectoral fin is observed (arrows in A and B). C: tbx5 expression marks the initially contiguous heart (arrowhead) and pectoral fin (asterisk) in wild-type progeny at the 10-somite stage. E: The fin precursor specific tbx5 expression domain (asterisk) has separated from the heart field domain (arrowhead). D,F: nlsu11 mutant embryos are missing a specific tbx5 expression domain at all stages of development (asterisks). G: Pectoral fin buds continue expressing tbx5 at 32 hr. H: Lack of pectoral fin buds (asterisks) in a nlsu11 mutant embryo.
RA signaling temporally controls fin development
Blocking RA synthesis with a pharmacological inhibitor of RA synthesis, diethylaminobenzaldehyde (DEAB) throughout somitogenesis is sufficient to block tbx5 expression and fin bud formation at 24 hpf or 32 hpf, respectively (Fig.3).
Fig. 3- Application of RA synthesis inhibitor,10−5M diethylaminobenzaldehyde (DEAB), to zebrafish embryos and its effect on fin bud formation. A: Embryos treated with DEAB at shield, 75% epibody and tail bud stage of the development, do not have tbx5 positive fin precursors. Embryos in which the treatment started at the 6-somite stage have detectable tbx5 positive fin precursors. B: Treating embryos with DEAB throughout smitogenesis lead to loss of tbx5 expression and fin bud development, but later treatments lead to reduction of tbx5 expression and the size of fin bud. C: Treating embryos with DEAB during early to midsomitogenesis reduces tbx5 expression and fin bud size. D: Schematics of inhibitor treatments and results.
RA affects the expression of aldh1a2 and its antagonist cyp26a1
RA biosynthesis is initiated with the reversible oxidation of retinol (vitamin A) to retinal by the enzyme called retinol dehydrogenase, and it is followed by the oxidation of retinal to RA by the aldehyde dehydrogenase family of enzymes. Aldehyde dehydrogenase 1a2 is a member of the aldehyde dehydrogenase family which catalyzes the oxidation of retinal to RA. The action of RA is limited by cytochrome P450 26A1 (cyp26a1), a retinoic acid (RA)–metabolizing enzyme.
RA controls cyp26a1 and aldh1a2 transcription and it operates in the hypoblast (during gastrulation stage). Blocking RA synthesis with diethylaminobenzaldehyde (DEAB) purturbs cyp26a1 and aldh1a2 expression domains in the gastrula hypoblast (Fig.4). Upon inhibitor treatment, cyp26a1 domain is decreased by 75%, and the expression of cyp26a1 is lost in the dorsal hypoblast. Overall, there was a notable reduction of the marginal hypoblast expression domain for cyp26a1 (Fig4. A-F).  The aldh1a2 pattern of expression behaves in a complementary manner to cyp26a1,and the staining for aldh1a2 increases in the ventral side of the gastrula (Fig.4J-K).
Fig.4- A–K: Changes in cyp26a1 (A–F) and aldh1a2 (G–K) expression domains in the gastrula hypoblast with 10−5M diethylaminobenzaldehyde (DEAB) treatments from 30% epiboly until the stage of fixation. A-F: Treating embryos with DEAB lead to an absence of the paraxial cyp26a1 expression domain at 75% and a considerable reduction of the marginal hypoblast expression domain (compare expression domains in A,C,D to B,E,F arrowheads). G,H,J,K: Treating embryos from 30% epiboly on with DEAB, show a stronger aldh1a2 expression domain at 90% epiboly and tail bud stage (compare G,J to H,K). I: Relative enhancement of marginal aldh1a2 expression at 90% epiboly. experimental embryos (red, n=10) are compared to control embryos (blue, n=10). Asterisks indicate significant differences in conrol vs. experimental embryos.
Fin precursor determination is triggered by RA signaling during gastrulation
Application of RA synthesis inhibitor, DEAB, during gastrulation or somitogenesis results in down-regulation of marginal cyp26a1 and paraxial hoxb5a expression after 1 hour of incubation (Fig.5A,B). Removing DEAB at mid-gastrulation stage (after 2 hours of treatment), restores the wild type levels of hoxb5a at the 10-somite stage (Fig.5C).
Fig.5- Kinetics of RA synthesis inhibitor (DEAB) effects on the transcription of RA target gene after incubation embryos with 10-5M DEAB with different time point treatments: gastrulation (A) somitogenesis (B), and after inhibitor removal. Duration (in hours) is given on the lower right side of each frame.
RA signaling is able to trigger a delayed fin precursor determination during somitogenesis
Embryos treated with the inhibitor for the RA signaling (DEAB) lack development of observable fin buds by 32 hours post fertilization (Fig. 6A). The same embryos, at the gene expression level show a few fin precursors with reduced tbx5 expression (Fig. 6B).  The results of Grandel et al.  work (refer to Fig. 6 and Fig.7) suggests that in the wild-type embryo the RA signal acts before the end of gastrulation to initiate the process of fin bud precursor determination and tbx5 expression at the 10-somite stage. On the other hand, it is possible that the activation of RA signaling after gastrulation might be enough to stimulate fin precursor development. 
Fig.6- Effects of treating zebra fish embryos with DEAB before the 10-somite stage. myoD, tbx5 double staining at the 10-somite (10s) stage. A: DEAB was added in the later stages of development. If embryos are treated with DEAB at the gastrulation stage then, fin precursor are absent. If embryos are treated with DEAB after the gastrulation stage then, fin precursor are present. B: 2-hr inhibitor treatments: If inhibitors are added during the gastrulation stage then, fin precursors are absent. If treatments are performed during early early somitogenesis then, fin precursors are present. C: Schematics of inhibitor treatments.
Fig.7- Response to delayed RA signal. A: Treating embryos from 30% epiboly to bud tail stage with DEAB. They lack fin buds. B: In situ hybridization. tbx5 is expressed in the mesoderm of the presuming region of fin development.
Finally, Grandel et al.  proposed a model for zebra fish limb development by RA signaling. In this model, RA (graded shading) triggers fin precursor (FP) development in the late gastrulation stage (hypoblast). These precursors start expressing tbx5 (red) lateral to somites 1-3 during somitogenesis (Fig. 8A). In the absence of RA signaling during all stages of development, there are no fin precursor determination as well as bud morphogenesis (Fig. 8B). Activation of RA signaling during gastrulation, initiates the process of fin precursor determination. Since such embryos are receiving early RA only, they fail to maintain the fin precursor (Fig.8C). Late activation of RA signaling from somites and intermediate mesoderm results in a weakly tbx5-positive domain in the lateral plate but fails ti undergo regular fin bud morphogenesis.
There are still areas that have not been addressed by Grandel’s paper. There needs to be more in depth research done to characterize other signaling pathways that will get activated as a result of cross-talk between RA signaling and those unknown signaling pathways.
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