Circadian Genes Are Expressed during Early Development in Xenopus laevis

Circadian oscillators are endogenous time-keeping mechanisms that drive twenty four hour rhythmic changes in gene expression, metabolism, hormone levels, and physical activity. We have examined the developmental expression of genes known to regulate circadian rhythms in order to better understand the ontogeny of the circadian clock in a vertebrate.In this study, genes known to function together in part of the core circadian oscillator mechanism (xPeriod1, xPeriod2, and xBmal1) as well as a rhythmic, clock-controlled gene (xNocturnin) were analyzed using in situ hybridization in embryos from neurula to late tailbud stages. Each transcript was present in the developing nervous system in the brain, eye, olfactory pit, otic vesicle and at lower levels in the spinal cord. These genes were also expressed in the developing somites and heart, but at different developmental times in peripheral tissues (pronephros, cement gland, and posterior mesoderm). No difference was observed in transcript levels or localization when similarly staged embryos maintained in cyclic light were compared at two times of day (dawn and dusk) by in situ hybridization. Quantitation of xBmal1 expression in embryonic eyes was also performed using qRT-PCR. Eyes were isolated at dawn, midday, dusk, and midnight (cylic light). No difference in expression level between time-points was found in stage 31 eyes (p = 0.176) but stage 40 eyes showed significantly increased levels of xBmal1 expression at midnight (RQ = 1.98+/-0.094) when compared to dawn (RQ = 1+/-0.133; p = 0.0004).We hypothesize that when circadian genes are not co-expressed in the same tissue during development that it may indicate pleiotropic functions of these genes that are separate from the timing of circadian rhythm. Our results show that all circadian genes analyzed thus far are present during early brain and eye development, but rhythmic gene expression in the eye is not observed until after stage 31 of development

Characterizing the conformational dynamics of metal-free PsaA using molecular dynamics simulations and electron paramagnetic resonance spectroscopy

Prokaryotic metal-ion receptor proteins, or solute-binding proteins, facilitate the acquisition of metal ions from the extracellular environment. Pneumococcal surface antigen A (PsaA) is the primary Mn2+-recruiting protein of the human pathogen Streptococcus pneumoniae and is essential for its in vivo colonization and virulence. The recently reported high-resolution structures of metal- free and metal-bound PsaA have provided the first insights into the mechanism of PsaA-facilitated metal binding. However, the conformational dynamics of metal-free PsaA in solution remain unknown. Here, we use continuous wave electron paramagnetic resonance (EPR) spectroscopy and molecular dynamics (MD) simulations to study the relative flexibility of the structural domains in metal-free PsaA and its distribution of conformations in solution. The results show that the crystal structure of the metal-free PsaA is a good representation of the dominant conformation in solution, but the protein also samples structurally distinct conformations that are not captured by the crystal structure. Further, these results suggest that the metal binding site is larger and more solvent exposed than indicated by the metal-free crystal structure. Collectively, this study provides atomic-resolution insight into the conformational dynamics of PsaA prior to metal binding and lays the groundwork for future EPR and MD based studies of PsaA in solution

A Dynamic Protein–Protein Coupling between the TonB-Dependent Transporter FhuA and TonB

Bacterial outer membrane TonB-dependent transporters function by executing cycles of binding and unbinding to the inner membrane protein TonB. In the vitamin B₁₂ transporter BtuB and the ferric citrate transporter FecA, substrate binding increases the periplasmic exposure of the Ton box, an energy-coupling segment. This increased exposure appears to enhance the affinity of the transporter for TonB. Here, continuous wave and pulse EPR spectroscopy were used to examine the state of the Ton box in the <i>Escherichia coli</i> ferrichrome transporter FhuA. In its apo state, the Ton box of FhuA samples a broad range of positions and multiple conformational substates. When bound to ferrichrome, the Ton box does not extend further into the periplasm, although the structural states sampled by the FhuA Ton box are altered. When bound to a soluble fragment of TonB, the TonB-FhuA complex remains heterogeneous and dynamic, indicating that TonB does not make strong, specific contacts with either the FhuA barrel or the core region of the transporter. This result differs from that seen in the crystal structure of the TonB–FhuA complex. These data indicate that unlike BtuB and FecA, the periplasmic exposure of the Ton box in FhuA does not change significantly in the presence of substrate and that allosteric control of transporter–TonB interactions functions by a different mechanism than that seen in either BtuB or FecA. Moreover, the data indicate that models involving a rotation of TonB relative to the transporter are unlikely to underlie the mechanism that drives TonB-dependent transport

A temporal summary of the expression patterns of <i>xPer1</i>, <i>xPer2</i>, <i>xBmal1</i>, and <i>Nocturnin</i>.

<p>The approximate stages of development are represented on the horizontal axis of this figure while the particular tissues and organs are listed on the vertical axis. <i>xPer1</i> is represented by the blue lines, <i>xPer2</i> by the green lines, <i>xBmal1</i> by the red lines, and <i>Nocturnin</i> by the black lines. Dotted lines indicate times during development when a gene may be present, but was not confirmed through sectioning or additional whole mount in situ analysis.</p

<i>xNocturnin</i> is expressed from neural plate to late tailbud stages.

<p>Shown are in situ hybridization results depicting expression of <i>xNocturnin</i> mRNA. All embryos in this figure are shown with the anterior facing left. Side views of the embryos are depicted in panels A,C,E,G,I, and K and dorsal views in panels B,D,F, H, and J. Low levels of <i>xNocturnin</i> were first detected in the neural plate of stage 15/16 embryos A and B. C and D show neural plate staining in a stage 18 embryo. E and F show a neural tube stage embryo (stage 24) with <i>xNocturnin</i> expression in the eyes (black arrow), somites (red arrowhead), and cement gland (blue arrow). G and H show early tailbud stage embryos with staining in the otic vesicle (black arrowhead), pronephric tubules (green arrow), heart (blue arrowhead), olfactory pit (green arrowhead), pineal (orange arrowhead), cement gland (blue arrow) and somites (red arrowhead). Late tailbud stages (I and J; stage 39) show similar results but additional staining in the anus/blastopore (brown arrow) and cement gland staining is absent (blue arrow). Sagittal (L) and transverse sections (M–O) of late tailbud embryos confirm <i>xNocturnin</i> expression in the brain, retina and lens (M), otic vesicle (N, black arrowhead), olfactory pit (L, green arrow), pronephric tubules (N, green arrow), heart (M, blue arrowhead), notochord (O, orange arrow) and in the somites (O, red arrowheads). <i>xNocturnin</i> is absent from the cement gland at late tailbud stages (L, blue arrow). No expression was seen using a sense probe specific to <i>Nocturnin</i> (K).</p

A comparison of somite staining in the posterior of late tailbud embryos (stage 36–38).

<p>Shown are in situ hybridization results depicting RNA expression in paired whole mount and sagittal sections of the posterior of embryos stained with <i>xPer1</i> (A–B), <i>xPer2</i> (C–D), <i>xBmal1</i> (E–F), and <i>Nocturnin</i> (G–H).</p

Isolated eyes show rhythmic expression of <i>xBmal1</i> at stage 40 but not at stage 31.

<p>Eyes were dissected from embryos maintained in a 12L:12D cycle at different stages of development and different circadian times (ZT 0 (dawn), ZT6 (mid-day), ZT12 (dusk),and ZT18 (midnight)). The eyes were analyzed by qRT-PCR. The relative quantitation (RQ) of <i>xBmal1</i> for each sample was calculated with respect to EF1α. No difference in the levels of <i>xBmal1</i> expression was observed in stage 31 embryonic eyes at any time of day tested (ANOVA; df3, F = 1.77, p = 0.176; arrhythmic). A significant difference in <i>xBmal1</i> expression was observed when all ZTs were analyzed in stage 40 embryonic eyes (ANOVA; df3, F12.23, p = 0.00009). The asterisk shows that the level of <i>xBmal1</i> expression at ZT18 was significantly different from ZT0 (ANOVA, df1, F = 27.82, p = 0.0004). Bars in each graph denote standard error.</p

<i>xPer2</i> is expressed from neural plate to late tailbud stages.

<p>Shown are in situ hybridization results depicting expression of <i>xPer2</i> mRNA. Panels A, B, D, and F show a dorsal view of each embryo. Panels C and E show side views. All embryos are oriented with the anterior to the left. G–I show transverse sections and J shows a sagittal section of late tailbud stage embryos. Sections shown in G, H, and J are oriented with the dorsal side at the top right of the panel. Neural plate staining is shown in panel A (stage 16) and B (stage 18). C and D depict early tailbud embryos with continued expression in the CNS as well as in the eye (black arrow), otic vesicle (black arrowhead), cement gland (blue arrow) and somites (red arrowheads). In late tailbud embryos (E and F), <i>xPer2</i> is expressed in the otic vesicle (E,I, black arrowhead), pineal (F,G, orange arrowhead), brain, retina, lens (G), and olfactory pit (I, green arrowhead), although cement gland staining was lost (E, blue arrow). <i>xPer2</i> was also present at low levels in the heart (H, blue arrowhead) and notochord (J, orange arrow). J also shows somite staining (red arrowheads).</p

<i>xClock</i>, <i>xBmal1</i> and <i>xNocturnin</i> are expressed as maternal messages before zygotic expression is observed at stage 24.

<p>Shown are northern blots performed on RNA isolated from whole embryos at the indicated stages in 12L:12D cycle. Three micrograms of total RNA was loaded into each lane. 28S RNA was used as a loading control.</p