Opl: a zinc finger protein that regulates neural determination and patterning in Xenopus.

In order to study the mechanism of neural patterning in Xenopus, we used subtractive cloning to isolate genes activated early during this process. One gene isolated was opl, (odd-paired-like) that resembles the Drosophila pair-rule gene odd-paired and encodes a zinc finger protein that is a member of the Zic gene family. At the onset of gastrulation, opl is expressed throughout the presumptive neural plate, indicating that neural determination has begun at this stage while, by neurula, opl expression is restricted to the dorsal neural tube and neural crest. opl encodes a transcriptional activator, with a carboxy terminal regulatory domain, which when removed increases opl activity. opl both sensitizes animal cap ectoderm to the neural inducer noggin and alters the spectrum of genes induced by noggin, allowing activation of the midbrain marker engrailed. Consistent with the later dorsal neural expression of opl, the activated form of opl is able to induce neural crest and dorsal neural tube markers both in animal caps and whole embryos. In ventral ectoderm, opl induces formation of loose cell aggregates that may indicate neural crest precursor cells. Aggregates do not express an epidermal marker, indicating that opl suppresses ventral fates. Together, these data suggest that opl may mediate neural competence and may be involved in activation of midbrain, dorsal neural and neural crest fates.

Xenopus hindbrain patterning requires retinoid signaling.

We have asked how posterior neural tissue is patterned in Xenopus by assaying the involvement of endogenous retinoic acid (RA) in this process and by using the labial Hox gene, HoxD1, as a posterior marker. Although RA is able to inhibit anterior gene expression and activate expression of more posterior genes, the normal role of retinoids in anteroposterior (A/P) patterning is unclear. HoxD1 is an early posterior neurectodermal marker, expressed from midgastrula with a later anterior expression limit in the future hindbrain. We previously showed that HoxD1 was induced as an immediate early response to retinoic acid in naive ectoderm (animal caps). Here, we use a truncated RARalpha2.2 receptor (RARDelta) to dominantly interfere with retinoid signaling. In embryos injected with RARDelta expression of HoxD1 is eliminated. Conjugates of ectoderm and dorsolateral mesoderm show that retinoid receptors are required in the ectoderm for HoxD1 induction. Further, expression of Krox-20 in r3 and r5 of the presumptive hindbrain is compressed into a single stripe that suggests elimination of r5. RARalpha2.2 expression almost precisely overlaps that of HoxD1, suggesting that this receptor may normally activate HoxD1. Expression of neither more anterior genes including cement gland, forebrain, and midbrain markers nor a more posterior spinal cord marker is affected by RARDelta. These data suggest that the posterior hindbrain is the region of the nervous system most sensitive to retinoid loss. Finally, we compare the ability of RA and fibroblast growth factor (FGF) to posteriorize isolated anterior neurectoderm and show that both factors can act directly on this substrate. RA acts in a more anterior domain than does FGF; however, neither factor is equivalent to the natural posteriorizing capacity of the posterior mesoderm. We propose that endogenous retinoid and FGF signals pattern largely nonoverlapping regions along the A/P axis and that posterior neural patterning requires multiple inducers.

Antisense RNA to the first N-glycosylation gene, ALG7, inhibits protein N-glycosylation and secretion by Xenopus oocytes.

N-Glycosylation has been shown to affect the rate of glycoprotein transport through the secretory pathway. In order to identify the critical components in the N-glycosylation pathway that directly influence protein secretion, we have studied the effects of downregulation of the first gene in the dolichol pathway, ALG7, on the synthesis, glycosylation and secretion of native and heterologous proteins by Xenopus laevis oocytes. Our strategy involved the use of ALG7 antisense RNA (asRNA) to lower the effective abundance of the ALG7 protein in oocytes. The results showed that there was an inverse dose-response relationship between ALG7 asRNA and the amount of glycosylated and secreted proteins. These effects were also observed for heterologously expressed rat parotid amylase. Since ALG7 asRNA did not inhibit overall protein synthesis, we conclude that downregulation of ALG7 expression directly lowered protein export.

Biosynthesis and secretion of rat salivary proteins by Xenopus laevis oocytes.

Xenopus laevis oocytes injected with poly (A+) RNA isolated from rat parotid and submandibular glands synthesize and secrete salivary proteins. Amylase was identified in the media of cultured oocytes injected with rat parotid mRNA by size and immunoprecipitation with anti-human amylase serum. Secretion of the salivary proteins was detectable in the medium eight h following the parotid mRNA injection and continued in a time-dependent fashion for up to 96 h. In contrast to rat parotid slices in culture, which demonstrate a regulated pathway of secretion highly responsive to the secretagogue isoproterenol, secretion of salivary proteins by oocytes did not respond to the stimulation by isoproterenol. Though parotid mRNA is presumed to contain the templates encoding the regulated pathway of secretion, reconstitution of this pathway of secretion in oocytes was not observed in our experiments. Since Xenopus laevis oocytes secrete constitutively significant amounts of proteins when injected with salivary gland mRNA, they are a useful biological system for the analysis of secretion, processing, and function of salivary proteins.

[Isolation of active gonadotropin fractions from sturgeon hypophysis and procurement of specific immune sera to them]. / Vydelenie aktivnykh fraktsii gonadotropina iz gipofiza osetra i poluchenie k nim spetsificheskikh immunykh syvorotok

The proteins of sturgeon hypophysis form 12 fractions when being separated by disc-electrophoresis. The maturation of the Rana esculenta or Bufo viridis oocytes in vitro served as a criterion of gonadotropic activity of fractions. The gonadotropic activity was shown to reside in two glycoprotein fractions with the mobilities 0.26 and 0.34. The immune serum to the former fraction is monospecific under immunodiffusion and suppresses the oocyte maturation in vitro. The immune serum to the latter fraction is characterized by the antigonadotropic activity as well, but expressed to a lesser extent.