Comparative connectomics of dauer reveals developmental plasticity.

A fundamental question in neurodevelopmental biology is how flexibly the nervous system changes during development. To address this, we reconstructed the chemical connectome of dauer, an alternative developmental stage of nematodes with distinct behavioral characteristics, by volumetric reconstruction and automated synapse detection using deep learning. With the basic architecture of the nervous system preserved, structural changes in neurons, large or small, were closely associated with connectivity changes, which in turn evoked dauer-specific behaviors such as nictation. Graph theoretical analyses revealed significant dauer-specific rewiring of sensory neuron connectivity and increased clustering within motor neurons in the dauer connectome. We suggest that the nervous system in the nematode has evolved to respond to harsh environments by developing a quantitatively and qualitatively differentiated connectome.

Circadian expression of clock genes in the rat eye and brain.

The light sensing system in the eye directly affects the circadian oscillator in the mammalian suprachiasmatic nucleus (SCN). To investigate this relationship in the rat, we examined the circadian expression of clock genes in the SCN and eye tissue during a 24 h day/night cycle. In the SCN, rPer1 and rPer2 mRNAs were expressed in a clear circadian rhythm like rCry1 and rCry2 mRNAs, whereas the level of BMAL1 and CLOCK mRNAs decreased during the day and increased during the night with a relatively low amplitude. It seems that the clock genes of the SCN may function in response to a master clock oscillation in the rat. In the eye, the rCry1 and rCry2 were expressed in a circadian rhythm with an increase during subjective day and a decrease during subjective night. However, the expression of Opn4 mRNA did not exhibit a clear circadian pattern, although its expression was higher in daytime than at night. This suggests that cryptochromes located in the eye, rather than melanopsin, are the major photoreceptive system for synchronizing the circadian rhythm of the SCN in the rat.

Cloning and circadian expression of rat Cry1.

In mammals, two types of cryptochrome are involved in the regulation of the circadian rhythm. We previously characterized rat Cry2 and its expression in brain tissue [Eun et al. (2001)]. We report here the cloning of another cryptochrome gene, Cry1, from rat brain by reverse-transcription coupled to polymerase chain reaction (RT-PCR), together with rapid-amplification of cDNA ends (RACE). The cloned Cry1 cDNA consists of 2557 nucleotides and has a single open-reading frame encoding a protein of 588 amino acids with start and stop codons. The deduced amino acid sequence was 70% identical with that of rat Cry2. It also showed 95% identity with mouse and human Cry1 but relatively low identity of 82% with that of zebrafish. Circadian expression of rat Cry1 and Cry2 was examined in the suprachiasma nucleus (SCN) and eye by real-time PCR. Expression of Cry1 and Cry2 mRNA in the SCN displayed a circadian rhythm with a peak at the day/night transition, and there was a slightly different circadian pattern of expression of Cry1 and Cry2 in the eye.

Cloning and expression of cryptochrome2 in the bullfrog (Rana catesbeiana).

Cryptochromes (CRYs) are non-opsin photoactive pigments that have recently been implicated in circadian photo-entrainment in humans, mice and Drosophila. In order to study the mechanism of circadian rhythm in amphibians, we have cloned and characterized a Rana cryptochrome in the bullfrog. We isolated a cDNA of about 2.1 kb from a bullfrog brain cDNA library by screening with a partial cry2 cDNA probe obtained by RT-PCR using degenerate primers. The cloned Rana cry2 cDNA has a complete single open-reading frame consisting of 323 amino acids, and its deduced amino acid sequence has a high degree of homology with human and mouse CRY2. Interestingly, we also isolated two other cry2 cDNAs, which may be splicing variants. Rana cry2 is expressed in all tissues as a 2.2 kb transcript. It is particularly highly expressed in the brain and ovaries, and also showed seasonal variation in expression in ovarian tissue. To examine its involvement in circadian rhythm, we tested whether expression in brain tissue followed a light-dark cycle, and found that expression was higher in the dark than in the light. Rana cry2 should therefore, be useful for studying circadian rhythms in seasonally breeding wild animals.

Preferential ligand selectivity of the monkey type-II gonadotropin-releasing hormone (GnRH) receptor for GnRH-2 and its analogs.

Gonadotropin-releasing hormone (GnRH) regulates the reproductive system through the cognate GnRH receptor (GnRHR) in vertebrates. In this study, we cloned a cDNA encoding the full-length open reading frame sequence for green monkey type-II GnRHR (gmGnRHR-2) from the genomic DNA of CV-1 cells. Transient transfection study showed that gmGnRHR-2 was able to induce both c-fos promoter- and cAMP responsive element-driven transcriptional activities, indicating that gmGnRHR-2 couples to both Gs- and Gq/11-linked signaling pathways. gmGnRHR-2 responded better to GnRH-2 ([His5, Trp7, Tyr8]GnRH) than GnRH-1 ([Tyr5, Leu7, Arg8]GnRH). Substitutions of His5, Trp7, and/or Tyr8 in GnRH-1 increased the potency to activate gmGnRHR-2, suggesting that individual His5, Trp7, and Tyr8 in GnRH-2 contributed to differential ligand sensitivity of gmGnRHR-2. Substitution of D-Ala for Gly6 in GnRH-2 increased the potency to activate the receptor, suggesting that GnRH-2 has a constrained conformation when it binds to the receptor. GnRH-induced gmGnRHR-2 activation was specifically inhibited by GnRH-2 antagonists, Trptorelix-1 and -2, but not by a GnRH-1 antagonist, Cetrorelix. In conclusion, gmGnRHR-2 revealed preferential ligand selectivity for GnRH-2 and its analogs, suggesting that gmGnRHR-2 has a functional activity that is different from mammalian type-I GnRHRs but similar to non-mammalian GnRHRs.

Cloning of ribosomal protein S6 kinase cDNA and its involvement in meiotic maturation in Rana dybowskii oocytes.

Several protein kinases are involved in the meiotic maturation of frog oocytes in order to activate the maturation-promoting factor (MPF). Among these kinases, the 90 kDa ribosomal protein S6 kinase (p90Rsk or Rsk) is directly phosphorylated and activated by the mitogen-activated protein kinase (MAPK). During Xenopus oocyte maturation, the activation of Rsk closely parallels that of MAPK. Both enzymes are dephosphorylated when the cytostatic factor (CSF) disappears after fertilization. Therefore, Rsk seems to play an essential role in the activation of MPF. To evaluate it in other frog oocytes, we cloned and characterized Rsk cDNA in Rana dybowskii oocytes. The cloned Rana Rsk cDNA had 2,961 bp of nucleotides, which contained a complete single open-reading frame with ATG codon and polyadenylation signal. The deduced amino acid sequence of Rana Rsk is 733 amino acids with 83 kDa. Rana Rsk shows a high homology (about 88%) with Xenopus Rsk. It also had two well-conserved kinase domains with specific phosphorylation sites, which are known to be essential for the activation of Rsk. A Northern analysis showed that Rana Rsk mRNA was strongly expressed in ovary tissue, but weakly in other tissue. Rana Rsk protein is expressed with the pTYB1 vector and purified with the IMPACT-CN system. The purified Rana Rsk cross-reacted with Xenopus, a p90Rsk2 antiserum. Therefore, we examined the phosphorylation of Rana Rsk during Rana oocyte maturation. In P4-treated oocytes, Rana Rsk was phosphorylated about 6-9 h, which correlated well with the germinal vesicle breakdown of Rana oocytes. Therefore, it is likely that Rana Rsk plays an important role in the meiotic maturation of seasonal breeding animals.