The bHLH Protein Nulp1 is Essential for Femur Development Via Acting as a Cofactor in Wnt Signaling in Drosophila.

BACKGROUND: The basic helix-loop-helix (bHLH) protein families are a large class of transcription factors, which are associated with cell proliferation, tissue differentiation, and other important development processes. We reported that the Nuclear localized protein-1 (Nulp1) might act as a novel bHLH transcriptional factor to mediate cellular functions. However, its role in development in vivo remains unknown. METHODS: Nulp1 (dNulp1) mutants are generated by CRISPR/Cas9 targeting the Domain of Unknown Function (DUF654) in its C terminal. Expression of Wg target genes are analyzed by qRT-PCR. We use the Top-Flash luciferase reporter assay to response to Wg signaling. RESULTS: Here we show that Drosophila Nulp1 (dNulp1) mutants, generated by CRISPR/Cas9 targeting the Domain of Unknown Function (DUF654) in its C terminal, are partially homozygous lethal and the rare escapers have bent femurs, which are similar to the major manifestation of congenital bent-bone dysplasia in human Stuve- Weidemann syndrome. The fly phenotype can be rescued by dNulp1 over-expression, indicating that dNulp1 is essential for fly femur development and survival. Moreover, dNulp1 overexpression suppresses the notch wing phenotype caused by the overexpression of sgg/GSK3ß, an inhibitor of the canonical Wnt cascade. Furthermore, qRT-PCR analyses show that seven target genes positively regulated by Wg signaling pathway are down-regulated in response to dNulp1 knockout, while two negatively regulated Wg targets are up-regulated in dNulp1 mutants. Finally, dNulp1 overexpression significantly activates the Top-Flash Wnt signaling reporter. CONCLUSION: We conclude that bHLH protein dNulp1 is essential for femur development and survival in Drosophila by acting as a positive cofactor in Wnt/Wingless signaling.

CXXC5 Associates with Smads to Mediate TNF-α Induced Apoptosis.

Apoptosis is a widespread phenomenon and its dysregulation may result in a variety of human pathologies, such as cancer, autoimmune diseases and neurodegenerative disorders. CXXC-type zinc finger protein 5 (CXXC5) is commonly considered as a tumor suppressor undergoing deregulation or deletion in hematonosis. But it has implied involvement in apoptosis indirectly and the molecular mechanism remains unknown. In this study, we investigated CXXC5-induced apoptosis as well as its underlying mechanism. A fluorescence resonance energy transfer (FRET) assay suggested that CXXC5 induced cell death and caspase-3 activity in primary rat cortical neurons. Further colorimetric TUNEL assay, Hoechst staining and flow cytometric assay indicated a time-dependent apoptosis in which the activities of caspase-8 and caspase-3 were both regulated via CXXC5 according to enzymatic activity assay, Hoechst staining and Western blotting. Transcription reporter assay and Western blotting showed that CXXC5 resulted in activation of tumor necrosis factor-α (TNF-α), initiated the extrinsic apoptosis pathway and cross-linked with the intrinsic mitochondrial pathway. Being a bone morphogenetic protein 4 (BMP-4) downstream regulator, and also a transcription factor, cellular co-localization and co-immunoprecipitation results indicate that CXXC5 co-localized and interacted with Smads. Western blotting and nuclear fraction extraction implied that CXXC5 facilitated Smad3 phosphorylation and Smad4 nuclear translocation, and that co-expression of Smad together with CXXC5 resulted in higher TNF-α reporter activity. In sum, CXXC5 appears to regulate the TNF-α apoptosis pathway by associating with Smads.

Cardiac enhancer activity of the homeobox gene tinman depends on CREB consensus binding sites in Drosophila.

The Drosophila homeobox gene tinman plays a critical role in subdividing the early mesoderm. In particular, tinman is absolutely required for formation of the heart and visceral mesoderm. tinman expression is initiated throughout the mesoderm of the trunk region under the control of the bHLH transcription factor encoded by the twist gene, a determinant of all mesoderm. Later, tinman expression is restricted to the dorsal portion of the mesoderm, a process that is directed by decapentaplegic (dpp) whose product (a TGF-beta-related protein) is secreted by the overlaying ectoderm. Further restriction of tinman expression to the cardiac progenitors, in which it will persist throughout development, involves the secreted segmentation gene product encoded by wingless (wg, a Drosophila Wnt gene). Here, we show that strong early expression depends on the synergistic action of an enhancer element at the 5' end of the gene in conjunction with an element in the first intron. Moreover, two distinct enhancer regions are responsible for tinman expression in the heart: one region confers expression in the heart-tube-associated pericardial cells, the other element drives expression in the contractile, myocardial cells. The latter element contains two CREB consensus binding sites that are essential for cardiac-specific expression. genesis 26:55-66, 2000.

Activation of multifunctional Ca2+/calmodulin-dependent kinase in intact hippocampal slices.

In vitro phosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) converts it to a form that is independent of Ca2+. We demonstrate that significant Ca(2+)-independent CaM kinase activity is present in untreated hippocampal slices. Two manipulations that produce a long-lasting enhancement of neuronal activity in hippocampal slices, elevated extracellular Ca2+ or depolarization with high K+, generate additional Ca(2+)-independent activity. This increase is dependent on extracellular Ca2+ and is correlated with an increased phosphorylation of CaM kinase. In contrast, CaM kinase in posterior pituitary, a brain structure that is not thought to be involved in memory-related processes, is not modulated by depolarization. These results suggest that the Ca(2+)-independent form of CaM kinase may modulate neuronal activity in the hippocampus.

Evidence for separate receptors that mediate parallel effects of serotonin and small cardioactive peptideB (SCPB) on adenylate cyclase in Aplysia californica.

Recent evidence suggests that serotonin (5-HT) and a neuropeptide, small cardioactive peptideB (SCPB), exert parallel modulatory actions on neuronal and muscular function in Aplysia via changes in the levels of cAMP. We have examined the effects of both of these neurotransmitters on the adenylate cyclase activity in membrane homogenates of pleural ganglia. The results indicated that 5-HT and SCPB enhance the production of cAMP via a direct activation of adenylate cyclase. In addition, we found that the increase in cyclase activity produced by 5-HT could be selectively blocked by the S2-serotonergic antagonists, ketanserin and ritanserin. Thus, specific 5-HT receptors are present which appear to be distinct from those that mediate the effects of SCPB on the activity of adenylate cyclase.

Stimuli that produce sensitization lead to elevation of cyclic AMP levels in tail sensory neurons of Aplysia.

Facilitation of synaptic connections between sensory neurons and motor neurons mediating the tail withdrawal reflex in Aplysia is produced by the modulatory effects of sensitizing stimuli. Facilitation can be mimicked by perfusing these neurons with serotonin (5-HT) in a semi-intact preparation. Consequently, 5-HT has been presumed to be acting as an agonist of the modulatory transmitter that mediates sensitizing input in vivo. While the 5-HT effects appear to be mediated by increased cyclic adenosine monophosphate (cAMP) levels in the sensory neurons, a critical issue that has not been examined is whether sensitizing stimuli also increase cAMP levels in these cells. We now report that such sensitizing stimuli delivered to the tail of a semi-intact preparation lead to elevation of cAMP levels in the tail sensory neurons.

Associative conditioning analog selectively increases cAMP levels of tail sensory neurons in Aplysia.

Bilateral clusters of sensory neurons in the pleural ganglia of Aplysia contain cells involved in a defensive tail withdrawal reflex. These cells exhibit heterosynaptic facilitation in response to noxious skin stimulation that can be mimicked by the application of serotonin. Recently it has been shown that this facilitation can be selectively amplified by the application of a classical conditioning procedure to individual sensory neurons. We now report that an analog of this classical conditioning paradigm produces a selective amplification of the cAMP content of isolated sensory neuron clusters. The enhancement is achieved within a single trial and appears to be localized to the sensory neurons. These results indicate that a pairing-specific enhancement of cAMP levels may be a biochemical mechanism for associative neuronal modifications and perhaps learning.

Membrane responses and changes in cAMP levels in Aplysia sensory neurons produced by serotonin, tryptamine, FMRFamide and small cardioactive peptideB (SCPB).

While recent evidence indicates a role for serotonin (5-HT) in modulating the defensive tail-withdrawal reflex in Aplysia, little information exists concerning the specificity of these 5-HT effects. As a first-step in addressing this issue we have examined the dose-response relationship for one aspect of the 5-HT modulation (enhancement of cAMP levels in isolated clusters of sensory neurons) and compared the effects of 5-HT with three potential neurotransmitters: tryptamine, FMRFamide (Phe-Met-Arg-Phe-NH2) and small cardioactive peptideB (SCPB). Cyclic adenosine monophosphate (cAMP) levels were enhanced as a graded function of the concentration of 5-HT with an EC50 of 14 microM. At a concentration of 5 microM, both 5-HT and SCPB produced nearly identical increases in the cAMP content of sensory neurons. In contrast, 5 microM tryptamine or 5 microM FMRFamide had little or no effect on cAMP levels. We also examined the effects of these agents on membrane currents and membrane conductance. Both 5-HT and SCPB produced an inward current associated with a decrease in input conductance. Tryptamine had little or no effect, while FMRFamide produced a response opposite to that of 5-HT and SCPB; an outward current associated with an increase in membrane conductance.

The identification and localization of a catecholamine in the motor neurons of the lobster cardiac ganglion.

The cardiac ganglion from Homarus americanus was investigated for the purpose of providing biochemical and histochemical information as to the identity of the neurotransmitter(s) utilized by this system. Three techniques were employed in this study: (1) the glyoxylic acid histofluorescence staining technique (GA), which showed fluorescence characteristic of catecholamines localized in the five motor neurons; (2) high-voltage electrophoresis (HVE) in one dimension followed by ascending chromatography in the second dimension, which indicated incorporation of label from tritiated tyrosine into norepinephrine (NE) and small amounts of dopamine (DA); (3) high-pressure liquid chromatography with electrochemical detection (HPLC/EC), which indicated the presence of endogenous norepinephrine.