Genetic studies have shown that retinoic acid (RA) signaling is required for mouse retina development, controlled in part by an RA-generating aldehyde dehydrogenase encoded by Aldh1a2 (Raldh2) expressed transiently in the optic vesicles. We examined the function of a related gene, Aldh1a1 (Raldh1), expressed throughout development in the dorsal retina. Raldh1 ؊/؊ mice are viable and exhibit apparently normal retinal morphology despite a complete absence of Raldh1 protein in the dorsal neural retina. RA signaling in the optic cup, detected by using a RARE-lacZ transgene, is not significantly altered in Raldh1 ؊/؊ embryos at embryonic day 10.5, possibly due to normal expression of Aldh1a3 (Raldh3) in dorsal retinal pigment epithelium and ventral neural retina. However, at E16.5 when Raldh3 is expressed ventrally but not dorsally, Raldh1 embryos lack RARE-lacZ expression in the dorsal retina and its retinocollicular axonal projections, whereas normal RARE-lacZ expression is detected in the ventral retina and its axonal projections. Retrograde labeling of adult Raldh1؊/؊ retinal ganglion cells indicated that dorsal retinal axons project to the superior colliculus, and electroretinography revealed no defect of adult visual function, suggesting that dorsal RA signaling is unnecessary for retinal ganglion cell axonal outgrowth. We observed that RA synthesis in liver of Raldh1 ؊/؊ mice was greatly reduced, thus showing that Raldh1 indeed participates in RA synthesis in vivo. Our findings suggest that RA signaling may be necessary only during early stages of retina development and that if RA synthesis is needed in dorsal retina, it is catalyzed by multiple enzymes, including Raldh1.

Tumor angiogenesis is a key event in cancer progression. Here, we report that tumors can stimulate tumor angiogenesis by secretion of galectin-1. Tumor growth and tumor angiogenesis of different tumor models are hampered in galectin-1-null (gal-1 −/− ) mice. However, tumor angiogenesis is less affected when tumor cells express and secrete high levels of galectin-1. Furthermore, tumor endothelial cells in gal-1 −/− mice take up galectin-1 that is secreted by tumor cells. Uptake of galectin-1 by cultured endothelial cells specifically promotes H-Ras signaling to the Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (Erk) kinase (Mek)/Erk cascade and stimulates endothelial cell proliferation and migration. Moreover, the activation can be blocked by galectin-1 inhibition as evidenced by hampered membrane translocation of H-Ras.GTP and impaired Raf/Mek/Erk phosphorylation after treatment with the galectin-1-targeting angiogenesis inhibitor anginex. Altogether, these data identify galectin-1 as a proangiogenic factor. These findings have direct implications for current efforts on galectin-1-targeted cancer therapies. Cancer Res; 70(15); 6216-24. ©2010 AACR.

Two mouse insulin genes, Ins1 and Ins2, were disrupted and lacZ was inserted at the Ins2 locus by gene targeting. Double nullizygous insulin-deficient pups were growth-retarded. They did not show any glycosuria at birth but soon after suckling developed diabetes mellitus with ketoacidosis and liver steatosis and died within 48 h. Interestingly, insulin deficiency did not preclude pancreas organogenesis and the appearance of the various cell types of the endocrine pancreas. The presence of lacZ expressing ␤ cells and glucagon-positive ␣ cells was demonstrated by cytochemistry and immunocytochemistry. Reverse transcriptioncoupled PCR analysis showed that somatostatin and pancreatic polypeptide mRNAs were present, although at reduced levels, accounting for the presence also of ␦ and pancreatic polypeptide cells, respectively. Morphometric analysis revealed enlarged islets of Langherans in the pancreas from insulin-deficient pups, suggesting that insulin might function as a negative regulator of islet cell growth. Whether insulin controls the growth of specific islet cell types and the molecular basis for this action remain to be elucidated.Insulin is synthesized, stored, and secreted by the pancreatic islet ␤ cells in a highly regulated manner and plays a vital role in glucose homeostasis. Insulin action also results in several other pleiotropic effects that are less well documented. Embryonic insulin synthesis begins early in gestation, but fetal glycemia closely follows maternal blood glucose levels. The question, therefore, arises as to what function embryonic insulin might fulfill during development. For instance, one might ask whether insulin plays an autocrine or paracrine role in pancreatic islet cell growth and differentiation, since insulin is synthesized with other hormones in developing islet cell types (1-3). Recently, this question has been addressed in a few transgenic studies. For instance, the gene encoding PDX-1 (4, 5), a homeodomain transcription factor synthesized in adult ␤ cells and capable of transactivating insulin gene expression, has been inactivated by targeted disruption (6, 7). Agenesis of pancreas resulting from PDX-1 deficiency precluded from addressing the question of the possible role of insulin in islet cell growth and differentiation. Similarly, mice lacking the LIM homeodomain transcription factor ISL1, synthesized in all classes of islet cells in the adult, were arrested in development soon after embryonic day 9.5 (8). The requirement of ISL1 in pancreatic epithelium for the differentiation of all islet cell types was, however, demonstrated by in vitro culture of explants from ISL1-deficient embryonic day 9.5 embryos that gave rise to cells that were negative for glucagon, insulin, and somatostatin. In another study, transgenic mouse embryos expressing the gene encoding the diphteria toxin A chain under control of the rat Ins2 promoter were generated (9). The resulting genetic ablation of the insulin-producing cells did not appear to alter the development of the nontarget...

In the mouse, insulin is produced from two similar but nonallelic genes that encode proinsulins I and II. We have investigated expression of these two genes during mouse embryonic development, using a PCR to detect the two gene transcripts and immunocytochemistry to visualize the two corresponding proteins. At appearance of the dorsal pancreatic anlage at day 9.5 of gestation, both mRNAs could be detected in the embryos, and both proteins were present together in the same cells of the developing pancreas. At days 9.5 and 10.5, when the ventral anlage appears, there were fewer proinsulin H mRNAs than proinsulin I mRNAs. At day 12.5 this ratio was reversed. Proinsulin II mRNA, but not proinsulin I mRNA, could be detected at day 8.5 in the prepancreatic embryo. Proinsulin II mRNA, but not proinsulin I mRNA, was also found in the heads of embryos at day 9.5 and at all later stages studied. These results indicate that the two proinsulin genes are regulated independently, at least in part. They also suggest that insulin might play a role as a growth factor in the developing mouse brain.Insulin, a key hormone in metabolic homeostasis, is synthesized, stored, and secreted by beta cells of the pancreatic islets. The protein is synthetized in the form of a precursor, preproinsulin, which is highly conserved among animal species. Unlike most mammals, mice and rats express two nonallelic genes that encode proinsulins I and II. In the mouse these two proteins differ by two amino acids in the B chain and three amino acids in the C peptide. Mouse C peptide I also lacks the Gly-Ala residues present in positions 17 and 18 of C peptide 11(1, 2). The corresponding genes in mouse and rat are highly homologous, and their organization is similar, except that the preproinsulin I gene possesses only the first of two introns present in the preproinsulin II gene (1, 3).On previous studies concerning the regulation of these two nonallelic genes, mRNAs were reported to be present in nearly equal quantities in adult pancreas of mouse (1, 4), as well as of rat (5) Here we have investigated regulation of the expression of the two proinsulin genes during mouse development. In the mouse, the pancreas is derived from the duodenum as two evaginations evolving at days 9.5 (dorsal) and 10.5 (ventral) of the gestation. The evaginations coalesce at day 11, and insulin has been first detected in previous studies at day 11.5 (9, 10). We have used a reverse-transcriptase-PCR (RT-PCR) assay, which allows identification and estimation of the relative amounts of mRNA transcribed from each of the two proinsulin genes. This RT-PCR, using a single pair ofprimers and a single probe for the two transcripts, allowed comparison of the relative amounts of proinsulin I and II mRNAs in the samples (11). Immunocytochemistry with antibodies specific for each of the two proinsulins allowed us to distinguish the products of the two genes in embryo sections. We have, thus, been able to detect insulin mRNAs and proteins much earlier, to distinguish the two forms, ...

Influence of vitamin A (retinol) on growth depends on its sequential oxidation to retinal and then to retinoic acid (RA), producing a ligand for RA receptors essential in development of specific tissues. Genetic studies have revealed that aldehyde dehydrogenases function as tissue-specific catalysts for oxidation of retinal to RA. However, enzymes catalyzing the first step of RA synthesis, oxidation of retinol to retinal, remain unclear because none of the present candidate enzymes have expression patterns that fully overlap with those of aldehyde dehydrogenases during development. Here, we provide genetic evidence that alcohol dehydrogenase (ADH) performs this function by demonstrating a role for Adh3, a ubiquitously expressed form. Adh3 null mutant mice exhibit reduced RA generation in vivo, growth deficiency that can be rescued by retinol supplementation, and completely penetrant postnatal lethality during vitamin A deficiency. ADH3 was also shown to have in vitro retinol oxidation activity. Unlike the second step, the first step of RA synthesis is not tissue-restricted because it is catalyzed by ADH3, a ubiquitous enzyme having an ancient origin.

Retinoic acid (RA) is known to act as a signaling molecule during embryonic development, but little is known about the regulation of RA synthesis from retinol. The rate-limiting step in RA synthesis is the oxidation of retinol, a reaction that can be catalyzed by alcohol dehydrogenase (ADH). Ethanol is also a substrate for ADH, and high levels of ethanol inhibit ADH-catalyzed retinol oxidation. This has prompted us to hypothesize that ethanol-induced defects observed in fetal alcohol syndrome involve ethanol inhibition of ADH-catalyzed RA synthesis. Here, we have examined the effect of ethanol on RA levels in cultured mouse embryos by using a bioassay. Treatment with 100 mM ethanol, but no 10 mM, led to a significant decrease in RA detection in 7.5-day-old embryos. Using whole-mount in situ hybridization, we detected mRNA for class IV ADH, but not ethanol-active cytochrome P450 2E1, in 7.5- and 8.5-day-old embryos, indicating that an ADH-linked pathway exists at these stages for metabolizing retinol and ethanol. Thus, the observed ethanol-induced reduction in RA may be caused by ethanol inhibition of retinol oxidation catalyzed by class IV ADH. In our postulated mechanism for fetal alcohol syndrome, this enzyme may well play a crucial role.

Endogenous retinoic acid (RA) has been observed in vertebrate embryos as early as gastrulation, but the mechanism controlling spatiotemporal synthesis of this important regulatory molecule remains unknown. Some members of the alcohol dehydrogenase (ADH) family catalyze retinol oxidation, the rate-limiting step in RA synthesis. Here we have examined mouse embryos for the presence of endogenous RA and expression of ADH genes. RA was not detected in egg cylinder stage embryos but was detected in late primitive streak stage embryos. Detection of class IV ADH mRNA, but not class I or class III, coincided with the onset of RA synthesis, being absent in egg cylinder embryos but present in the posterior mesoderm of late primitive streak embryos. During neurulation, RA and class IV ADH mRNA were colocalized in the craniofacial region, trunk, and forelimb bud. Class IV ADH mRNA was detected in cranial neural crest cells and craniofacial mesenchyme as well as trunk and forelimb bud mesenchyme. The spatiotemporal expression pattern and enzymatic properties of class IV ADH are thus consistent with a crucial function in RA synthesis during embryogenesis. In addition, the finding of endogenous RA and class IV ADH mRNA in the craniofacial region has implications for the mechanism of fetal alcohol syndrome.

The ability of class I alcohol dehydrogenase (ADH1) and class IV alcohol dehydrogenase (ADH4) to metabolize retinol to retinoic acid is supported by genetic studies in mice carrying Adh1 or Adh4 gene disruptions. To differentiate the physiological roles of ADH1 and ADH4 in retinoid metabolism we report here the generation of an Adh1/4 double null mutant mouse and its comparison to single null mutants. We demonstrate that loss of both ADH1 and ADH4 does not have additive effects, either for production of retinoic acid needed for development or for retinol turnover to minimize toxicity. During gestational vitamin A deficiency Adh4 and Adh1/4 mutants exhibit completely penetrant postnatal lethality by day 15 and day 24, respectively, while 60% of Adh1 mutants survive to adulthood similar to wild-type. Following administration of a 50-mg/kg dose of retinol to examine retinol turnover, Adh1 and Adh1/4 mutants exhibit similar 10-fold decreases in retinoic acid production, whereas Adh4 mutants have only a slight decrease. LD 50 studies indicate a large increase in acute retinol toxicity for Adh1 mutants, a small increase for Adh4 mutants, and an intermediate increase for Adh1/4 mutants. Chronic retinol supplementation during gestation resulted in 65% postnatal lethality in Adh1 mutants, whereas only ϳ5% for Adh1/4 and Adh4 mutants. These studies indicate that ADH1 provides considerable protection against vitamin A toxicity, whereas ADH4 promotes survival during vitamin A deficiency, thus demonstrating largely non-overlapping functions for these enzymes in retinoid metabolism.