Search

Retinoic acid (RA) has several established functions during cardiac development, including actions in the fetal epicardium required for myocardial growth. An open question is if retinoid effects are limited to growth factor stimulation pathway(s) or if additional actions on uncommitted progenitor/stem populations might drive cardiac differentiation. Here we report the dual effects of RA deficiency on cardiac growth factor signaling and progenitor/stem biology using the mouse retinaldehyde dehydrogenase 2 (Raldh2) knockout model. Although early heart defects in [Raldh2.sup.-/-] embryos result from second-heart-field abnormalities, it is unclear whether this role is transient or whether RA has sustained effects on cardiac progenitors. To address this, we used transient maternal RA supplementation to overcome early [Raldh2.sup.-/-] lethality. By embryonic day 11.5-14.5, [Raldh2.sup.-/-] hearts exhibited reduced venticular compact layer outgrowth and altered coronary vessel development. Although reductions in Fgf2 and target pERK levels occurred, no alterations in Wnt/[beta]-catenin expression were observed. Cell proliferation is increased in compact zone myocardium, whereas cardiomyocyte differentiation is reduced, alterations that suggest progenitor defects. We report that the fetal heart contains a reservoir of stem/progenitor cells, which can be isolated by their ability to efflux a fluorescent dye and that retinoid signaling acts on this fetal cardiac side population (SP). [Raldh2.sup.-/-] hearts display increased SP cell numbers, with selective increases in expression of cardiac progenitor cell markers and reduced differentiation marker levels. Hence, although lack of RA signaling increases cardiac SP numbers, simultaneous reductions in Fgf signaling reduce cardiomyocyte differentiation, possibly accounting for long-term defects in myocardial growth. heart | myocardium | retinoids | stem cells doi: 10.1073/pnas.0910430107

Corepressors play an essential role in nuclear receptor-mediated transcriptional repression. In general, corepressors directly bind to nuclear receptors via CoRNR boxes (L/I-X-X-I/V-I) in the absence of ligand and appear to act as scaffolds to further recruit chromatin remodeling complexes to specific target genes. Here, we describe the identification of the multiple LIM domain protein Ajuba as a unique corepressor for a subset of nuclear hormone receptors. Ajuba contains functional nuclear-receptor interacting motifs and selectively interacts with retinoic acid receptors (RARs) and rexinoid receptor (RXRs) subtypes in a ligand-dependent manner. Simultaneous mutation of these motifs abolishes RAR binding and concomitantly leads to loss of repression on RARE reporter genes. P19 cells depleted of Ajuba are highly sensitized to all-trans retinoic acid (atRA)-induced transcription and differentiation. In the absence of atRA, Ajuba can be readily found at the RARE control elements of RAR endogenous target genes. Stimulation of cells with atRA results in the dissociation of Ajuba from these regions. Moreover, we observed that coexpression of the known Ajuba binding partner Prmt5 (protein arginine methyltransferase-5) inhibited the Ajuba/RAR interaction. The high-affinity Ajuba-RAR/RXR interaction site overlaps the region responsible for Ajuba/Prmt5 binding, and thus binding appears to be mutually exclusive, providing a potential mechanism for these observations. Identification of Ajuba as a unique corepressor for nuclear receptors sheds new light on mechanisms for nuclear receptor-mediated repression and provides a unique target for developing more effective therapeutics to modulate this important pathway. box genes | differentiation | LIM domain | retinoic acid receptors www.pnas.org/cgi/doi/10.1073/pnas.0908656107

Retinoic acid (RA) receptors (RARs) [alpha], [beta], and [gamma] heterodimerized with rexinoid receptors (RXRs) [alpha], [beta], and [gamma] mediate the RA signal. To analyze the contribution of the transcriptional activity of RXR[alpha], the main RXR during embryogenesis, we have engineered a mouse line harboring a transcriptionally silent RXR[alpha] mutant that lacks the activation functions AF1 and AF2. All homozygous mutants ([Rxra.sup.afo]) display the ocular defects previously observed in compound Rar-null and Rxra/Rar-null mutants, thus demonstrating that a transcriptionally active RXR[alpha] is required during eye development. In contrast, the vast majority of [Rxra.sup.afo] fetuses do not display the Rxra-null mutant hypoplasia of the myocardium, thus demonstrating that RXR[alpha] can act as a transcriptionally silent heterodimerization partner. Similarly, a transcriptionally silent RXR[alpha] mutant can support early embryogenesis, as [Rxra.sup.afo]/Rxrb-null embryos display a normal morphology, contrasting with the severe malformations exhibited by compound Rxra/Rxrb-null embryos. Along the same line, we show that a silent RXR[alpha] mutant is sufficient to allow the initial formation of the placental labyrinth, whereas later steps of trophoblast cell differentiation critically requires the AF2, but not the AF1, function of RXR[alpha]. activation function | gene knockout | nuclear receptor | retinoic acid | transcriptional activity

Retinoic acid (RA) plays important roles in development by modulating gene transcription through nuclear receptor activation. Increasing evidence supports a role for RA and RA receptors (RARs) in synaptic plasticity in the brain. We have recently reported that RA mediates a type of homeostatic synaptic plasticity through activation of dendritic protein synthesis, a process that requires dendritically localized RAR[alpha] and is independent of transcriptional regulation. The molecular basis of this translational regulation by RA/RAR[alpha] signaling, however, is unknown. Here we show that RAR[alpha] is actively exported from the nucleus. Cytoplasmic RAR[alpha] acts as an RNA-binding protein that associates with a subset of mRNAs, including dendritically localized glutamate receptor 1 (GluR1) mRNA. This binding is mediated by the RAR[alpha] carboxyl terminal F-domain and specific sequence motifs in the 5'UTR of the GluR1 mRNA. Moreover, RAR[alpha] association with the GluR1 mRNA directly underlies the translational control of GluR1 by RA: RAR[alpha] represses GluR1 translation, while RA binding to RAR[alpha] reduces its association with the GluR1 mRNA and relieves translational repression. Taken together, our results demonstrate a ligand-gated translational regulation mechanism mediated by a non-genomic function of RA/RAR[alpha] signaling. glutamate receptor 1 | nuclear receptor | synaptoneurosome | RNA binding protein | translational regulation

In eukaryotes, diploid cells give rise to haploid cells via meiosis, a program of two cell divisions preceded by one round of DNA replication. Although key molecular components of the meiotic apparatus are highly conserved among eukaryotes, the mechanisms responsible for initiating the meiotic program have diverged substantially among eukaryotes. This raises a related question in animals with two distinct sexes: Within a given species, are similar or different mechanisms of meiotic initiation used in the male and female germ lines? In mammals, this question is underscored by dramatic differences in the timing of meiotic initiation in males and females. Stra8 is a vertebrate-specific, cytoplasmic factor expressed by germ cells in response to retinoic acid. We previously demonstrated that Stra8 gene function is required for meiotic initiation in mouse embryonic ovaries. Here we report that, on an inbred C57BL/6 genetic background, the same factor is also required for meiotic initiation in germ cells of juvenile mouse testes. In juvenile C57BL/6 males lacking Stra8 gene function, the early mitotic development of germ cells appears to be undisturbed. However, these cells then fail to undergo the morphological changes that define meiotic prophase, and they do not display the molecular hallmarks of meiotic chromosome cohesion, synapsis and recombination. We conclude that, in mice, Stra8 regulates meiotic initiation in both spermatogenesis and oogenesis. Taken together with previous observations, our present findings indicate that, in both the male and female germ lines, meiosis is initiated through retinoic acid induction of Stra8. meiosis | spermatocyte

RIG-I has been implicated in innate immunity by sensing intracellular viral RNAs and inducing type I IFN production. However, we have found a significant RIG-I induction in a biological setting without active viral infection--namely, during RA-induced terminal granulocytic differentiation of acute myeloid leukemias. Here, we present evidence that a significant Rig-I induction also occurs during normal myelopoiesis and that the disruption of the Rig-I gene in mice leads to the development of a progressive myeloproliferative disorder. The initiation of progressive myeloproliferative disorder is mainly due to an intrinsic defect of [Rig-I.sup.-/-] myeloid cells, which are characterized by a reduced expression of IFN consensus sequence binding protein, a major regulator of myeloid differentiation. Thus, our study reveals a critical regulatory role of Rig-I in modulating the generation and differentiation of granulocytes. knockout mice | myelopoiesis | RA-inducible gene |

During embryogenesis, the pancreas arises from dorsal and ventral pancreatic protrusions from the primitive gut endoderm upon induction by different stimuli from neighboring mesodermal tissues. Recent studies have shown that Retinoic Acid (RA) signaling is essential for the development of the pancreas in non-mammalian vertebrates. To investigate whether RA regulates mouse pancreas development, we have studied the phenotype of mice with a targeted deletion in the retinaldehyde dehydrogenase 2 (Raldh2) gene, encoding the enzyme required to synthesize RA in the embryo. We show that Raldh2 is expressed in the dorsal pancreatic mesenchyme at the early stage of pancreas specification. RA-responding cells have been detected in pancreatic endodermal and mesenchymal cells. Raldh2-deficient mice do not develop a dorsal pancreatic bud. Mutant embryos lack Pdx1 expression, an essential regulator of early pancreas development, in the dorsal but not the ventral endoderm. In contrast to Pdx1-deficient mice, the early glucagon-expressing cells do not develop in Raldh2 knockout embryos. Shh expression is, as in the wild-type embryo, excluded from the dorsal endodermal region at the site where the dorsal bud is expected to form, indicating that the dorsal bud defect is not related to a mis-expression of Shh. Mesenchymal expression of the LIM homeodolnain protein Is11, required for the formation of the dorsal mesenchyme, is altered in Raldh2-/- embryos. The homeobox gene Hlxb9, which is essential for the initiation of the pancreatic program in the dorsal foregut endoderm, is still expressed in Raldh2-/- dorsal epithelium but the number of HB9-expressing cells is severely reduced. Maternal supplementation of RA rescues early dorsal pancreas development and restores endodermal Pdx1 and mesenchymal Is11 expression as well as endocrine cell differentiation. These findings suggest that RA signaling is important for the proper differentiation of the dorsal mesenchyme and development of the dorsal endoderm. We conclude that RA synthesized in the mesenchyme is specifically required for the normal development of the dorsal pancreatic endoderm at a stage preceding Pdx1 function. Keywords: Pancreas; Raldh2; Retinoids; Foregut; Endoderm; Mesenchymc; Shh; Is11; H1xb9

All-trans retinoic acid (atRA) is an endogenous morphogen that regulates gene transcription. Maternal exposure to atRA results in severe developmental abnormalities by disrupting normal patterns of atRA distribution. Previously, we have shown that the pontine nucleus, which originates from the rhombic lip, is severely atrophied in the mouse on exposure to atRA at gestational days 9 and 10. In this study, we show that this same period of atRA exposure has the contrary effect on the inferior olive and this rhombic lip derivative is expanded in volume and probably contains an increased number of cells. The posterior region of the inferior olive maintains a relatively normal shape but is significantly expanded in size. In contrast, the organization of the anterior inferior olive is severely disrupted. Because endogenous atRA levels are known to be higher in the region of the posterior inferior olive at the time of birth of inferior olivary neurons, these results suggest that endogenous atRA may promote the generation, or select the fate, of posterior neurons of the inferior olive. In support of this concept, a reduction in atRA resulting from vitamin A deficiency results in loss of cells of the posterior inferior olive. Keywords: Hindbrain; Migration; Teratogenesis; Hyperplasia; Vitamin A deficiency; Facial nucleus

In developing chordates, retinoic acid (RA) signaling patterns the rostrocaudal body axis globally and affects gene expression locally in some differentiating cell populations. Here we focus on development of epidermal sensory neurons in an invertebrate chordate (amphioxus) to determine how RA signaling influences their rostrocaudal distribution and gene expression (for AmphiCoe, a neural precursor gene; for amphioxus islet and AmphiERR, two neural differentiation genes; and for AmphiHox1, -3, -4, and -6). Treatments with RA or an RA antagonist (BMS009) shift the distribution of developing epidermal neurons anteriorly or posteriorly, respectively. These treatments also affect gene expression patterns in the epidermal neurons, suggesting that RA levels may influence specification of neuronal subtypes. Although colinear expression of Hox genes is well known for the amphioxus central nervous system, we find an unexpected comparable colinearity for Amphi-Hox1, -3, -4, and -6 in the developing epidermis; moreover, RA levels affect the anteroposterior extent of these Hox expression domains, suggesting that RA signaling controls a colinear Hox code for anteroposterior patterning of the amphioxus epidermis. Thus, in amphioxus, the developing peripheral nervous system appears to be structured by mechanisms parallel to those that structure the central nervous system. One can speculate that, during evolution, an ancestral deuterostome that structured its panepidermal nervous system with an RA-influenced Hox code gave rise to chordates in which this patterning mechanism persisted within the epidermal elements of the peripheral nervous system and was transferred to the neuroectoderm as the central nervous system condensed dorsally.

We used a genetic screening methodology, a human cell line bearing a retinoic-acid-responsive enhanced GFP reporter, and a flow sorter to recover dominant modulators of reporter expression. Four inducers and three suppressors that were fused to the C terminus of a protein scaffold for stability were isolated and their mechanisms of action studied. Mutagenesis experiments indicated that six of these dominant agents exerted their effects at the protein level. The single cDNA coding fragment that was isolated comprised the central 64-amino-acid section of human cyclophilin B, which contained its peptidyl-prolyl isomerase domain; this cyclophilin fragment repressed expression of the retinoic-acid-responsive reporter. The remaining clones encoded peptides shorter than 30 amino acids unrelated to known gene open reading frames. Genetic epistasis studies between the strongest inducer, R3, and a dominant-negative mutant of RAR[alpha] suggest that the two factors function in the same pathway. Transcript microarray analyses suggest that R3 induced a subset of the retinoid-responsive genes in melanoma cells. Finally, yeast two-hybrid assays and co-immunoprecipitation studies of human cell extracts identified PAT1 as a protein that interacts with R3.

In a cell line (NB4) derived from a patient with acute promyelocytic leukemia, all-trans-retinoic acid (ATRA) and interferon (IFN) induce the expression of a novel gene we call RIG-G (for retinoic acid-induced gene G). This gene codes for a 58-kDa protein containing 490 amino acids with several potential sites for post-translational modification. In untreated NB4 cells, the expression of RIG-G is undetectable. ATRA treatment induces the transcriptional expression of RIG-G relatively late (12-24 hr) in a protein synthesis.dependent manner, whereas IFN-a induces its expression early (30 min to 3 hr). Database search has revealed a high-level homology between RIG-G and several IFN-stimulated genes in human (ISG54K, ISG56K, and IFN-inducible and retinoic acid-inducible 58K gene) and some other species, defining a well conserved gene family. The gene is composed of two exons and has been mapped by fluorescence in situ hybridization to chromosome 10q24, where two other human IFN-stimulated gene members are localized. A synergistic induction of RIG-G expression in NB4 cells by combined treatment with ATRA and IFNs suggests that a collaboration exists between their respective signaling pathways.

The effects of retinoic acid on the stability of fatty-acid synthase messenger RNA (mRNA) were analyzed in cultured fetal rat lungs after prolonged incubation in a dexamethasone-containing medium. Northern blot analysis of the extracted RNA from dexamethasone-incubated cells indicated the presence of enhanced FAS mRNA stability compared to cells that were not cultured in dexamethasone. Furthermore, the hormone induced the transcription of FAS gene in dexamethasone-cultured explants of fetal rat lungs.

The mouse glucocorticoid receptor-interacting protein (GRIP1) is a member of the ERAP160 family of nuclear receptor (NR) coactivators (including SRC-1 and TIF2) which function as bridging proteins between ligand-activated NRs bound to cognate hormone-response elements (HREs) and the transcription initiation apparatus (TIA). Although these coactivators bind to several NRs, studies overexpressing these coactivators with these NRs in mammalian cells have not uniformly observed a corresponding enhancement of ligand-dependent transactivation. Here, we show that GRIP1 interacts in vitro in a ligand-dependent manner with thyroid receptor, retinoic acid receptor, and retinoid X receptor. Additionally, in yeast (Saccharomyces cerevisiae) GRIP1 coactivator protein markedly increased the ability of these full-length class II NRs to transactivate [Beta]-galactosidase reporter genes containing cognate HREs. The magnitude of GRIP1 enhancement of liganded NR homodimer was dependent upon NR subtype and HRE configuration. For most HRE configurations, thyroid receptor and retinoic acid receptor homodimers were essentially unresponsive or very weakly active in the absence of GRIP1, but GRIP1 dramatically restored the ligand-dependent function of these NRs. Although GRIP1 exerted no significant effect on NR homodimers in the absence of their cognate ligands, it increased the transactivation of unliganded NR heterodimers. Whether GRIP1 increased ligand-dependent transactivation of a heterodimer to levels greater than that of the cognate homodimer was determined by HRE configuration and copy number. Compared with the limitations of yeast two-hybrid and mammalian coexpression systems, the yeast HRE-assay systems described in this report facilitated both the detection of putative mammalian NR coactivator function and the elucidation of their mechanisms of transactivational enhancement.

By using RAR type ([Alpha], [Beta], or [Gamma])-specific synthetic retinoids and a pan-retinoic X receptor (RXR)-specific ligand, we have investigated the contribution of RARs and RXRs in the activation of RA target genes and the differentiation of embryonal carcinoma cells. We demonstrate cell-type- and promoter context-dependent functional redundancies that differ between the three RAR types for mediating the induction of RAR[Beta]2 and Hoxa-1 in wild-type, [RAR[Gamma].sup.-/-] and [RAR[Alpha].sup.-/-] F9 cells and in P19 cells. The extent of redundancy between RARs is further modulated by the synergistic activation of RXRs with a pan-RXR agonist. We also demonstrate that the expression of RAR[Beta]2 is auto-inducible in [RAR[Gamma].sup.-/-] but not in wild-type F9 cells, indicating that the functional redundancies observed between RARs in gene disruption studies can be artefactually generated. Thus, even though all three RARs can functionally substitute each other for inducing the expression of RA target genes and cell differentiation, one RAR can cell-specifically override the activity of the other RARs. Interestingly, only RAR[Gamma] can mediate the retinoic acid-induced differentiation of wild-type F9 cells, whereas the differentiation of P19 cells can be mediated by either RAR[Alpha] or RAR[Gamma].

A functionally conserved 3' region of mouse Hoxb-1 gene helps reconstruct the early endogenous expression pattern of the gene and regulates its initial ectopic response to the retinoic acid response element (RARE). RARE mediates ectopic response to retinoic acid and provides features of early Hoxb-1 expression pattern. Point mutations in the RARE prevent gene expression in the neuroectoderm.

Antisense oligodeoxynucleotides (ODN) restrict the retinoic-acid-induced stimulation of 3 human HOXB genes present upstream in the four HOX clusters. The stability of messenger RNAs corresponding to the genes remains uninfluenced, and this suggests that the antisense ODN effect on HOX clusters is regulated at the transcriptional revel. Information on HOX inhibition suggests a cascade model of progressive activation of HOX genes with a 3'-to-5' polarity.

Interaction between platelet activating factor (PAF), a lipid mediator which helps the transcriptional activation of primary response genes, and retinoic acid (RA) involves the stimulation of inducible prostaglandin synthase. RA and PAF influence give a unique convergence of nuclear signalling pathways by which specific gene expression is upregulated. PAF-dependent initiation of TIS10/PGS-2 expression is of importance during cell injury, cell differentiation, inflammation and immune responses.

Differentiation of P19 embryonal carcinoma cells to neural derivatives is stimulated by all-trans-retinoic acid (RA) when subjected to defined culture conditions. Vertebrate anteroposterior axis formation is stimulated by the vitamin A derivative RA. Cellular differentiation and modulation of several gene expression are also performed by the RA. The P19 cells subjected to differentiation clone the neural pathway which, on isolation, yields 14 RA-regulated genes.

Medikin (MK) mRNA was not found in embryonic rat brain cells after the 15th day gestation, though it includes neurite outgrowth on the 17th day. Anti-MK body was used in western blotting to detect MK's presence during mid and later stages of gestation. Immunohistochemical staining confirmed that MK influences neurite growth through a cell surface receptor. Neurite growth was more prominent in dishes coated with MK than with poly-L-lysine. Anti-MK suppressed the growth of Wilms' tumor cells, suggesting that MK was involved in their growth.

The alpha-1 portion of the retinoic acid receptor (RAR)-alpha gene was inactivated by homologous recombination to determine the biological role of RAR-alpha-1. The results showed that mutant mice homozygous for the mutation were viable and exhibited no altered phenotype. These results indicate that the alpha-1 isoform of the RAR gene, which differs by the amino-terminal portion, has no obvious morphogenic or essential function.

The involvement of the retinoid X receptor (RXR) in the binding of peroxisome-proliferator-activated receptor (PPAR) to its PPAR response element (PPRE) was investigated. The results showed that RXR-alpha can facilitate the binding of purified PPAR to PPRE. The results also indicated that additional protein factors are required for this binding activity. Therefore, DR1 type response elements could regulate genes by interactions with protein complexes consisting of elements such as RXR and PPAR.

The development of the hindbrain in fetuses is affected by retinoic acid, due to its influence on the expression of Hox genes. Experiments with transgenic mice show that the anatomical and molecular changes to the hindbrain and branchial region of the head are due to changes in the Hox code, induced by retinoic acid. These changes transform rhombomeres (r)2/3 homeotically to an r4/5 identity. The changes extend to several parts of the first branchial arch.

The roles of theDNA binding site and the transcriptional activation domain in the regulation oftranscription were examined. A strong negative retinoic acid response element (nRARE) was found in the rat oxytocin promoter. The effect of a constitutive transactivating domain was then examined by fusing the herpes simplex viral protein VP16 to the retinoic acid receptor, a transactivating factor. The recombinant protein was able to bind to, but unable to activate transcription from promoters containing the oxytocin nRARE, suggesting that the DNA recognition element determines the activity of bound transactivation domains.

Trophoblast stem cell (TS cell) lines have the ability to differentiate into trophoblast subtypes in vitro and contribute to the formation of placenta in chimeras. In order to investigate the possible role of retinoic acid (RA) in placentation, we analyzed the effects of exogenous RA on TS cells in vitro and the developing ectoplacental cone in vivo. TS cells expressed all subtypes of the retinoid receptor family, with the exception of RAR [Beta], whose expression was stimulated in response to RA. TS cells treated with RA were compromised in their ability to proliferate and exhibited properties of differentiation into trophoblast giant cells. During TS cell differentiation into trophoblast subtypes induced by withdrawal of FGF4, RA treatment further illustrated its role in the specification of cell fate by the promotion of differentiation into giant cells and the suppression of spongiotrophoblast formation. Moreover, administration of RA during pregnancy resulted in the overabundance of giant cells at the expense of spongiotrophoblast cells. RA hereby acts as an extracellular signal whose potential function can be linked to specification events mediating trophoblast cell fate. Taken together with the spatial patterns of giant-cell formation and RA synthesis in vivo, these findings implicate a function for RA in giant-cell formation during placentation. Key Words: trophoblast stem cell (TS cell}; retinoic acid; ectoplacental cone; trophoblast giant cell; spongiotrophoblast cell; cell fate.

Retinoic acid (RA) is known as teratogen for the brain, but very little information exists about the natural role of endogenously synthesized RA in forebrain development. Overall RA levels in the brain are very low, and RA-synthesizing retinaldehyde dehydrogenases (RALDHs) are restricted to a few sites. The earliest RA sources for the developing telencephalon are RALDH2, located at the anterior neural ridge, and RALDH3, expressed in the placode-forming surface ectoderm of the face. RA synthesis within the telencephalon begins at E11 in the mouse, when RALDH3 appears in its rostrolateral wall. During the first part of neurogenesis, RALDH3 is expressed at high levels in the lateral ganglionic eminence, a structure that protrudes into the ventricular lumen. RA diffusing from here is likely to be dispersed via the central fluid and to act on mitotic precursors throughoue the forebrain. When the telencephalon is enlarging, RALDH2 is very slowly activated in the meninges covering it. Whereas RALDH3 expression in the inner telencephalon disappears in the later embryo, meningeal RALDH2 levels reach a maximum in the first postnatal week, during the period of highest neurite elaboration, and then decline to lower adult levels. The changing RALDH expression patterns are likely to set up radial RA diffusion gradients across the developing cerebral wall, which reverse from early inside-out and to later outside-in patterns. RA diffusing from the RALDH sites across the RA-poor brain tissue provides a mechanism for the coordination of growth processes over an intermediate-distance range. (Supported by R01-EY01938.)

Members of the cellular retinoic acid binding protein (CRABP) family are small proteins involved in the metabolic pathways of retinoic acid (RA). Although there is report describing the cloning of a retinoic-X receptor, no report describes the presence of RA in crustacean. To determine whether RA is also involved in the regulation of development and whether CRABP is also involved in the metabolism of RA in crustacean, we attempt to clone the CRABP cDNAs from the shrimp using a combination of RT-PCR and library screening approach. Several identical cDNA clones encoding the putative MeCRABP were isolated. As confirmed by DNA sequence determination and RNA Northern blot analysis, the size of the CRABP mRNA is 0.9 kb. The cDNA is estimated to encode for a protein of 14 kDa. The size is close to that of the retinoic/fatty acid binding protein reported in the vertebrate and invertebrate. MeCRABP is expressed abundantly in the ovary and moderately in the eyestalk. Other tissues such as the muscles and testis also express these genes. Recombinant protein for the shrimp CRABP was produced in a pRSET bacteria expression system. Purified fusion protein was used to immunize rabbit to raise antibody. The fusion protein binds to both retinoic acid (RA) and retinol (RO) with similar affinity. Anti-rCRABP recognises cells of the eyestalk as well as maturing oocytes of shrimp contain CRABP. Supported by HKU CRCG.

The Polycomb group genes are required for the correct expression of the homeotic complex genes and segment specification during Drosophila embryogenesis and larval development. In mouse, inactivation studies of several Polycomb group genes indicate that they are also involved in Hox gene regulation. We have used our previously generated M33 mutants to study the function of M33, the mouse homologue of the Polycomb gene of Drosophila. In this paper, we show that in the absence of M33, the window of Hoxd4 retinoic acid (RA) responsiveness is opened earlier and that Hoxd11 gene expression is activated earlier in development This indicates that M33 antagonizes the RA pathway and has a function in the establishment of the early temporal sequence of activation of Hox genes. Despite the early activation, A-P boundaries are correct in later stages, indicating a separate control mechanism for early aspects of Hox regulation. This raises a number of interesting issues with respect to the roles of both Pc-G proteins and Hox regulatory mechanisms. We propose that a function of the M33 protein is to control the accessibility of retinoic acid response elements in the vicinity of Hox genes regulatory regions by direct or indirect mechanisms or both. This could provide a means for preventing ectopic transactivation early in development and be part of the molecular basis for temporal colinearity of Hox gene expression. [C] 2000 Academic Press Key Words: Polycomb; M33; Hox genes; temporal colinearity; retinoic acid; chromatin.

Retinoic acid is implicated in the abnormal development of the brain in vertebrates. A short burst of retinoic acid to vertebrate gastrulae respecifies rhombomeres two and three of the hindbrain region, transforming them to four and five. This respecifies anterior brain regions into more posterior hindbrain. This alteration takes place through the Hox genes, suggesting that these genes are important for the form of the head, in addition to the central nervous system.