Real-time imaging of the somite segmentation clock: revelation of unstable oscillators in the individual presomitic mesoderm cells

Notch signaling components such as the basic helix-loop-helix gene Hes1 are cyclically expressed by negative feedback in the presomitic mesoderm (PSM) and constitute the somite segmentation clock. Because Hes1 oscillation occurs in many cell types, this clock may regulate the timing in many biological systems. Although the Hes1 oscillator is stable in the PSM, it damps rapidly in other cells, suggesting that the oscillators in the former and the latter could be intrinsically different. Here, we have established the real-time bioluminescence imaging system of Hes1 expression and found that, although Hes1 oscillation is robust and stable in the PSM, it is unstable in the individual dissociated PSM cells, as in fibroblasts. Thus, the Hes1 oscillators in the individual PSM cells and fibroblasts are intrinsically similar, and these results, together with mathematical simulation, suggest that cell-cell communication is essential not only for synchronization but also for stabilization of cellular oscillators.     

Sonic hedgehog in temporal control of somite formation

Vertebrate embryo somite formation is temporally controlled by the cyclic expression of somitogenesis clock genes in the presomitic mesoderm (PSM). The somitogenesis clock is believed to be an intrinsic property of this tissue, operating independently of embryonic midline structures and the signaling molecules produced therein, namely Sonic hedgehog (Shh). This work revisits the notochord signaling contribution to temporal control of PSM segmentation by assessing the rate and number of somites formed and somitogenesis molecular clock gene expression oscillations upon notochord ablation. The absence of the notochord causes a delay in somite formation, accompanied by an increase in the period of molecular clock oscillations. Shh is the notochord-derived signal responsible for this effect, as these alterations are recapitulated by Shh signaling inhibitors and rescued by an external Shh supply. We have characterized chick smoothened expression pattern and have found that the PSM expresses both patched1 and smoothened Shh signal transducers. Upon notochord ablation, patched1, gli1, and fgf8 are down-regulated, whereas gli2 and gli3 are overexpressed. Strikingly, notochord-deprived PSM segmentation rate recovers over time, concomitant with raldh2 overexpression. Accordingly, exogenous RA supplement rescues notochord ablation effects on somite formation. A model is presented in which Shh and RA pathways converge to inhibit PSM Gli activity, ensuring timely somite formation. Altogether, our data provide evidence that a balance between different pathways ensures the robustness of timely somite formation and that notochord-derived Shh is a component of the molecular network regulating the pace of the somitogenesis clock.     

Ultradian oscillations of Stat, Smad, and Hes1 expression in response to serum

Serum response has been used as a model for studying signaling transduction for many biological events such as cell proliferation and survival. Although expression of many genes is up- or down-regulated after serum stimulation, the Notch effector Hes1 displays oscillatory response. However, the precise mechanism and biological significance of this oscillation remain to be determined. Here, we identified serum-induced ultradian oscillators, including molecules in Stat and Smad signaling. Stat and Smad oscillations involve activation of Stat3 and Smad1 and delayed negative feedback by their inhibitors Socs3 and Smad6, respectively. Moreover, Stat oscillations induce oscillatory expression of Hes1 by regulating its half-life, and loss of Hes1 oscillations leads to G(1) phase retardation of the cell cycle. These results indicate that coupled Stat and Hes1 oscillations are important for efficient cell proliferation and provide evidence that expression modes of signaling molecules affect downstream cellular events.     

Heterologous introns can enhance expression of transgenes in mice.

In a previous study we showed that genomic constructs were expressed more efficiently in transgenic mice than constructs that were identical except for the lack of introns. Using the mouse metallothionein promoter-rat growth hormone gene construct as a model, we show that the first intron of the rat growth hormone gene is essential for high-level expression, whereas the other three introns are less effective. Several heterologous introns placed 3' of the coding region of an intronless rat growth hormone gene are also ineffective. However, insertion of some heterologous introns between the metallothionein promoter and the growth hormone gene improves expression. To determine whether addition of heterologous introns would provide a general strategy for improving expression, we have tested them in conjunction with other intronless genes and with different promoters.     

Genetically altered AMPA-type glutamate receptor kinetics in interneurons disrupt long-range synchrony of gamma oscillation

Gamma oscillations synchronized between distant neuronal populations may be critical for binding together brain regions devoted to common processing tasks. Network modeling predicts that such synchrony depends in part on the fast time course of excitatory postsynaptic potentials (EPSPs) in interneurons, and that even moderate slowing of this time course will disrupt synchrony. We generated mice with slowed interneuron EPSPs by gene targeting, in which the gene encoding the 67-kDa form of glutamic acid decarboxylase (GAD67) was altered to drive expression of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunit GluR-B. GluR-B is a determinant of the relatively slow EPSPs in excitatory neurons and is normally expressed at low levels in gamma-aminobutyric acid (GABA)ergic interneurons, but at high levels in the GAD-GluR-B mice. In both wild-type and GAD-GluR-B mice, tetanic stimuli evoked gamma oscillations that were indistinguishable in local field potential recordings. Remarkably, however, oscillation synchrony between spatially separated sites was severely disrupted in the mutant, in association with changes in interneuron firing patterns. The congruence between mouse and model suggests that the rapid time course of AMPA receptor-mediated EPSPs in interneurons might serve to allow gamma oscillations to synchronize over distance.     

Notch target Hes5 ensures appropriate Notch induced T- versus B-cell choices in the thymus

Notch signaling establishes boundaries in the thymus by inducing T-cell commitment and inhibiting a B-cell choice. Here, we show a significant 1.6-fold increased generation of B-cell precursors in thymuses from mice deficient for Notch target Hes5 compared with wild-type littermates. We further show that culture of bone marrow-derived progenitors with increasing densities of purified immobilized Notch ligand (Delta1(ext-IgG)) induced increased expression of Notch targets Hes1 and Hes5, and that although Hes5-deficient progenitors responded appropriately to high densities of ligand, they misread intermediate and low densities. Together, our results suggest that to ensure an appropriate outcome in the thymus in response to a lower threshold of induced Notch signaling, induction of the additional target Hes5 is required.     

Combinations of closely situated cis-acting elements determine tissue-specific patterns and anterior extent of early Hoxc8 expression.

We have used a transgene mutation approach to study how expression domains of Hoxc8 are established during mouse embryogenesis. A cis-regulatory region located 3 kb upstream from the Hoxc8 translational start site directs the early phase of expression. Four elements, termed A, B, C, and D, were previously shown to direct expression to the neural tube. Here we report that a fifth element, E, located immediately downstream of D directs expression to mesoderm in combination with the other four elements. These elements are interdependent and partially redundant. Different combinations of elements determine expression in different posterior regions of the embryo. Neural tube expression is determined minimally by ABC, ABD, or ACD; somite expression by ACDE; and lateral plate mesoderm expression by DE. Neural tube and lateral plate mesoderm enhancers can be separated, but independent somite expression has not been achieved. Furthermore, mutations within these elements result in posteriorization of the reporter gene expression. Thus, the anterior extent of expression is determined by the combined action of these elements. We propose that the early phase of Hoxc8 expression is directed by two separate mechanisms: one that determines tissue specificity and another that determines anterior extent of expression.     

Zebrafish GADD45beta genes are involved in somite segmentation

Somites in vertebrates are periodic segmented structures that give rise to the vertebrae and muscles of body. Somites are generated from presomitic mesoderm (PSM), but it is not fully understood how cellular differentiation and segment formation are achieved in the anterior PSM. We report here that zebrafish gadd45beta1 and gadd45beta2 genes are periodically expressed as paired stripes adjacent to the neural tube in the anterior PSM region where presomitic cells mature. In mammals, it is known that GADD45 (growth arrest and DNA damage) family proteins play a role in cell-cycle control. We found that both knockdown and overexpression of gadd45beta genes caused somite defects with different consequences for marker gene expression. Knockdown of gadd45beta genes with antisense morpholino oligonucleotides caused a broad expansion of mesp-a in the PSM, and both cyclic expression of her1 and segmented expression of MyoD were disorganized. On the other hand, injection of gadd45beta1 or gadd45beta2 suppressed expression of mesp-a and her1 in anterior PSM and MyoD in paraxial mesoderm. These results indicate that regulated expression of gadd45beta genes in the anterior PSM is required for somite segmentation.     

Alterations in sympathetic nervous system activity do not regulate adipsin gene expression in mice.

Adipsin gene expression is severely diminished in certain forms of genetic and acquired rodent obesity. Common to many of these models of obesity is decreased sympathetic nervous system (SNS) activity. In addition, treatment of MSG obese mice with the sympathomimetic drug mixture ephedrine and caffeine restores adipsin deficiency to normal, while reversing obesity. Based on these observations, we hypothesized that adipsin gene expression might be regulated through changes in SNS activity with deficient adipsin gene expression in obesity being the result of impaired SNS activity. In the present study we used three models to assess the role of the SNS in regulating adipsin gene expression. First we exposed mice to the cold (4 degrees C), a potent activator of SNS activity. Second, we chemically sympathectomized mice with 60H-dopamine. Third, we treated mice with BRL 26830A, an atypical beta adrenoreceptor agonist. In contrast to our initial hypothesis, these studies demonstrate that alterations of SNS activity do not affect adipsin gene expression in normal mice. Neither increased SNS activity secondary to cold exposure nor decreased SNS activity resulting from sympathectomy alter serum adipsin concentration or adipsin mRNA levels in white (WAT) and brown adipose tissue (BAT). Surprisingly, treatment of lean mice with BRL 26830A decreases both adipsin serum concentrations and adipsin mRNA levels, suggesting a potential role for atypical beta adrenoreceptors in pathways that suppress adipsin expression in vivo. The significance of this observation with respect to adipocyte physiology is unclear at present. Future studies will be aimed at defining the molecular mechanisms by which BRL 26830A suppresses adipsin gene expression and the physiological significance of this effect.     

A splicing defect in the mouse transferrin gene leads to congenital atransferrinemia

We have analyzed the biochemical defect in a mutant line of mice that produces less than 1% of the normal level of serum transferrin. This mouse line (Hp) transcribes the transferrin gene in liver at the same rate observed in normal mice, but the steady state levels of transferrin mRNA sequences are less than 20% of normal. Further hybridization studies reveal that most of the transferrin mRNA sequences present in homozygous Hp mouse liver are in the form of a 5 kb nuclear precursor instead of the mature 2.5 kb transferrin mRNA seen in normal mice. Using several different exon and intron probes from the mouse transferrin gene, we have shown that the 5 kb RNA precursor retains the last two introns of the transferrin gene but that the 5' and middle introns have been removed by processing. The defect in transferrin mRNA processing also extends to nonhepatic tissues and we find the same lack of mature mRNA and increased precursor accumulation in brain RNA. Since Southern blot analysis does not reveal gross changes in the structure of the transferrin gene in Hp mice, we suggest that the Hp defect is due to a small deletion or point mutation that either disrupts splicing signals or uncovers cryptic splice signals that interfere with processing of the last two introns in the transferrin gene. This Hp mouse line provides an opportunity to study the effects of transferrin deficiency on development and iron homeostasis.     

Recapitulation of elements of embryonic development in adult mouse pancreatic regeneration

BACKGROUND & AIMS: The mammalian pancreas has a strong regenerative potential, but the origin of organ restoration is not clear, and it is not known to what degree such a process reflects pancreatic development. To define cell differentiation changes associated with pancreatic regeneration in adult mice, we compared regeneration following caerulein-induced pancreatitis to that of normal pancreatic development. METHODS: By performing comparative histology for adult and embryonic pancreatic markers in caerulein-treated and control pancreas, we addressed cellular proliferation and differentiation (amylase, DBA-agglutinin, insulin, glucagon, beta-catenin, E-cadherin, Pdx1, Nkx6.1, Notch1, Notch2, Jagged1, Jagged2, Hes1), hereby describing the kinetics of tissue restoration. RESULTS: We demonstrate that surviving pancreatic exocrine cells repress the terminal exocrine gene program and induce genes normally associated with undifferentiated pancreatic progenitor cells such as Pdx1, E-cadherin, beta-catenin, and Notch components, including Notch1 , Notch2 , and Jagged2 . Expression of the Notch target gene Hes1 provides evidence that Notch signaling is reactivated in dedifferentiated pancreatic cells. Although previous studies have suggested a process of acino-to-ductal transdifferentiation in pancreatic regeneration, we find no evidence to suggest that dedifferentiated cells acquire a ductal fate during this process. CONCLUSIONS: Pancreatic regeneration following chemically induced pancreatitis in the mouse occurs predominantly through acinar cell dedifferentiation, whereby a genetic program resembling embryonic pancreatic precursors is reinstated.     

PRL gene expression in individual living mammotropes displays distinct functional pulses that oscillate in a noncircadian temporal pattern

PRL gene expression in the anterior pituitary has been the focus of intensive investigation for many years, but very little information is available on the actual dynamics by which this process occurs in individual mammotrope cells. Here, we used single cell bioluminescent imaging microscopy and a recently refined reporter gene strategy to measure PRL promoter-driven gene expression (PRL-GE) in individual living primary mammotropes. Using this approach we report a new phenomenon involving repetitive on/off gene expression bursts that occurred in a distinctly noncircadian oscillatory pattern. Furthermore, we demonstrate a functional basis for these gene expression oscillations, inasmuch as PRL-GE pulses were sensitive to calcium-dependent modulation, which we show arose exclusively as changes in the shape of individual pulse episodes. Our results provide the first clear evidence that PRL-GE, in its homologous cell environment, displays oscillatory bursts of activity. Moreover, they strongly support the idea that these discrete on/off bursts of activity serve as an important determinant of the timing and level of PRL-GE under both basal and stimulated conditions.     

Endothelialization and altered hematopoiesis by persistent Etv2 expression in mice

Etv2 is a master gene for the commitment of hematopoietic/endothelial cells and is a potent inducer of endothelial/hematopoietic cells from embryonic stem cells. Etv2 is highly expressed in endothelial/hematopoietic precursors but is downregulated when they are differentiated, indicating that Etv2 should have transient but not constitutive function. However, relatively little attention has been paid to the importance of transient Etv2 expression. To determine whether transient Etv2 expression is essential to normal development and cell differentiation, we generated mice that constitutively express Etv2 from a Cre-activatable ROSA26 locus in endothelial/hematopoietic, somite, or neuronal lineages. Constitutive Etv2 expression caused profound phenotypes in hematopoietic/endothelial cells, with little effect on somite or neuronal lineages. In hematopoietic/endothelial lineages, constitutive Etv2 expression induced by Tie-2 Cre transgene caused abnormal yolk sac vasculature. Prolonged vascular endothelial cadherin expression and decreased B lymphopoiesis were observed in Etv2 expressing vascular endothelial cadherin(+)/CD45(+) cells, indicating that Etv2 forces endothelial program on hematopoietic cells. Etv2 expression in adult hematopoietic cells by Vav-iCre transgene also conferred an endothelial phenotype on hematopoietic stem cells and suppressed hematopoiesis, with erythropoiesis severely affected. We conclude that constitutive Etv2 expression perturbs vascular development and hematopoiesis. While promoting hematopoiesis/vasculogenesis, Etv2 expression should be tightly regulated to achieve normal vascular development and hematopoiesis.