Noncyclic Notch activity in the presomitic mesoderm demonstrates uncoupling of somite compartmentalization and boundary formation

To test the significance of cyclic Notch activity for somite formation in mice, we analyzed embryos expressing activated Notch (NICD) throughout the presomitic mesoderm (PSM). Embryos expressing NICD formed up to 18 somites. Expression in the PSM of Hes7, Lfng, and Spry2 was no longer cyclic, whereas Axin2 was expressed dynamically. NICD expression led to caudalization of somites, and loss of Notch activity to their rostralization. Thus, segmentation and anterior-posterior somite patterning can be uncoupled, differential Notch signaling is not required to form segment borders, and Notch is unlikely to be the pacemaker of the segmentation clock.     

Oscillator mechanism of Notch pathway in the segmentation clock.

Somites are formed by periodic segmentation of the presomitic mesoderm (PSM). This periodic event is controlled by the segmentation clock, where Notch signaling plays an essential role. The basic helix-loop-helix factor Hes7, a Notch effector, is cyclically expressed by negative feedback and regulates cyclic expression of Lunatic fringe (Lfng), a Notch modulator. Lfng then seems to periodically inhibit Notch, leading to oscillation in Notch activity. It is thought that these coupled negative feedback loops by Hes7 and Lfng are important for sustained and synchronized oscillations in the PSM. Of interest, another Notch effector, Hes1, is cyclically expressed by many cell types such as neuroblasts, suggesting that this clock is widely distributed and regulates many biological events. This review summarizes the recent finding about roles and mechanism of Notch signaling in the segmentation clock and discusses the significance of Hes1 oscillation in non-PSM cells.     

Transcriptional oscillation of lunatic fringe is essential for somitogenesis

A molecular oscillator that controls the expression of cyclic genes such as lunatic fringe (Lfng) in the presomitic mesoderm has been shown to be coupled with somite formation in vertebrate embryos. To address the functional significance of oscillating Lfng expression, we have generated transgenic mice expressing Lfng constitutively in the presomitic mesoderm in addition to the intrinsic cyclic Lfng activity. These transgenic lines displayed defects of somite patterning and vertebral organization that were very similar to those of Lfng null mutants. Furthermore, constitutive expression of exogenous Lfng did not compensate for the complete loss of cyclic endogenous Lfng activity. Noncyclic exogenous Lfng expression did not abolish cyclic expression of endogenous Lfng in the posterior presomitic mesoderm (psm) but affected its expression pattern in the anterior psm. Similarly, dynamic expression of Hes7 was not abolished but abnormal expression patterns were obtained. Our data are consistent with a model in which alternations of Lfng activity between ON and OFF states in the presomitic mesoderm prior to somite segmentation are critical for proper somite patterning, and suggest that Notch signaling might not be the only determinant of cyclic gene expression in the presomitic mesoderm of mouse embryos.     

Control of her1 expression during zebrafish somitogenesis by a delta-dependent oscillator and an independent wave-front activity

Somitogenesis has been linked both to a molecular clock that controls the oscillation of gene expression in the presomitic mesoderm (PSM) and to Notch pathway signaling. The oscillator, or clock, is thought to create a prepattern of stripes of gene expression that regulates the activity of the Notch pathway that subsequently directs somite border formation. Here, we report that the zebrafish gene after eight (aei) that is required for both somitogenesis and neurogenesis encodes the Notch ligand DeltaD. Additional analysis revealed that stripes of her1 expression oscillate within the PSM and that aei/DeltaD signaling is required for this oscillation. aei/DeltaD expression does not oscillate, indicating that the activity of the Notch pathway upstream of her1 may function within the oscillator itself. Moreover, we found that her1 stripes are expressed in the anlage of consecutive somites, indicating that its expression pattern is not pair-rule. Analysis of her1 expression in aei/DeltaD, fused somites (fss), and aei;fss embryos uncovered a wave-front activity that is capable of continually inducing her1 expression de novo in the anterior PSM in the absence of the oscillation of her1. The wave-front activity, in reference to the clock and wave-front model, is defined as such because it interacts with the oscillator-derived pattern in the anterior PSM and is required for somite morphogenesis. This wave-front activity is blocked in embryos mutant for fss but not aei/DeltaD. Thus, our analysis indicates that the smooth sequence of formation, refinement, and fading of her1 stripes in the PSM is governed by two separate activities.     

The vertebrate segmentation clock

Vertebrate somitogenesis has been shown to be associated with a molecular oscillator, the segmentation clock, whose periodicity matches that of the process of somitogenesis. The existence of such a clock in presomitic mesoderm (PSM) cells was originally proposed in theoretical models such as the 'clock and wavefront'. Molecular evidence for the existence of this clock in vertebrates has been obtained on the basis of the periodic expression of several genes, most of which are related to the Notch signalling pathway. These genes are expressed in a dynamic sequence which appears as a wave sweeping caudo-rostrally along the whole PSM once during each somite formation. Notch-pathway mouse and fish mutants lose the dynamic expression of the cycling genes, indicating that Notch signalling is required for their periodic expression, or is required to coordinate the oscillations between PSM cells. Therefore Notch signalling is either part of the mechanism of the oscillator itself or acts as a cofactor required for cycling gene expression. A further potentially important role for the segmentation clock is to periodically activate Notch signalling in the rostral presomitic mesoderm, thereby generating the periodic formation of somite boundaries.     

The vertebrate segmentation clock

Vertebrate somitogenesis has been shown to be associated with a molecular oscillator, the segmentation clock, whose periodicity matches that of the process of somitogenesis. The existence of such a clock in presomitic mesoderm (PSM) cells was originally proposed in theoretical models such as the 'clock and wavefront'. Molecular evidence for the existence of this clock in vertebrates has been obtained on the basis of the periodic expression of several genes, most of which are related to the Notch signalling pathway. These genes are expressed in a dynamic sequence which appears as a wave sweeping caudo-rostrally along the whole PSM once during each somite formation. Notch-pathway mouse and fish mutants lose the dynamic expression of the cycling genes, indicating that Notch signalling is required for their periodic expression, or is required to coordinate the oscillations between PSM cells. Therefore Notch signalling is either part of the mechanism of the oscillator itself or acts as a cofactor required for cycling gene expression. A further potentially important role for the segmentation clock is to periodically activate Notch signalling in the rostral presomitic mesoderm, thereby generating the periodic formation of somite boundaries.     

The winged helix transcription factor Foxc1a is essential for somitogenesis in zebrafish

Previous studies identified zebrafish foxc1a and foxc1b as homologs of the mouse forkhead gene, Foxc1. Both genes are transcribed in the unsegmented presomitic mesoderm (PSM), newly formed somites, adaxial cells, and head mesoderm. Here, we show that inhibiting synthesis of Foxc1a (but not Foxc1b) protein with two different morpholino antisense oligonucleotides blocks formation of morphological somites, segment boundaries, and segmented expression of genes normally transcribed in anterior and posterior somites and expression of paraxis implicated in somite epithelialization. Patterning of the anterior PSM is also affected, as judged by the absence of mesp-b, ephrinB2, and ephA4 expression, and the down-regulation of notch5 and notch6. In contrast, the expression of other genes, including mesp-a and papc, in the anterior of somite primordia, and the oscillating expression of deltaC and deltaD in the PSM appear normal. Nevertheless, this expression is apparently insufficient for the maturation of the presumptive somites to proceed to the stage when boundary formation occurs or for the maintenance of anterior/posterior patterning. Mouse embryos that are compound null mutants for Foxc1 and the closely related Foxc2 have no morphological somites and show abnormal expression of Notch signaling pathway genes in the anterior PSM. Therefore, zebrafish foxc1a plays an essential and conserved role in somite formation, regulating both the expression of paraxis and the A/P patterning of somite primordia.     

Hes1 regulates embryonic stem cell differentiation by suppressing Notch signaling

Embryonic stem (ES) cells display heterogeneous responses upon induction of differentiation. Recent analysis has shown that Hes1 expression oscillates with a period of about 3–5 h in mouse ES cells and that this oscillating expression contributes to the heterogeneous responses: Hes1‐high ES cells are prone to the mesodermal fate, while Hes1‐low ES cells are prone to the neural fate. These outcomes of Hes1‐high and Hes1‐low ES cells are very similar to those of inactivation and activation of Notch signaling, respectively. These results suggest that Hes1 and Notch signaling lead to opposite outcomes in ES cell differentiation, although they work in the same direction in most other cell types. Here, we found that Hes1 acts as an inhibitor but not as an effector of Notch signaling in ES cell differentiation. Our results indicate that sustained Hes1 expression delays the differentiation of ES cells and promotes the preference for the mesodermal rather than the neural fate by suppression of Notch signaling.     

Persistent expression of activated notch in the developing hypothalamus affects survival of pituitary progenitors and alters pituitary structure

Background: As the pituitary gland develops, signals from the hypothalamus are necessary for pituitary induction and expansion. Little is known about the control of cues that regulate early signaling between the two structures. Ligands and receptors of the Notch signaling pathway are found in both the hypothalamus and Rathke's pouch. The downstream Notch effector gene Hes1 is required for proper pituitary formation; however, these effects could be due to the action of Hes1 in the hypothalamus, Rathke's pouch, or both. To determine the contribution of hypothalamic Notch signaling to pituitary organogenesis, we used mice with loss and gain of Notch function within the developing hypothalamus. Results: We demonstrate that loss of Notch signaling by conditional deletion of Rbpj in the hypothalamus does not affect expression of Hes1 within the posterior hypothalamus or expression of Hes5. In contrast, expression of activated Notch within the hypothalamus results in ectopic Hes5 expression and increased Hes1 expression, which is sufficient to disrupt pituitary development and postnatal expansion. Conclusions: Taken together, our results indicate that Rbpj‐dependent Notch signaling within the developing hypothalamus is not necessary for pituitary development, but persistent Notch signaling and ectopic Hes5 expression in hypothalamic progenitors affects pituitary induction and expansion. Developmental Dynamics 244:921–934, 2015. © 2015 Wiley Periodicals, Inc.     

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Background: As the pituitary gland develops, signals from the hypothalamus are necessary for pituitary induction and expansion. Little is known about the control of cues that regulate early signaling between the two structures. Ligands and receptors of the Notch signaling pathway are found in both the hypothalamus and Rathke's pouch. The downstream Notch effector gene Hes1 is required for proper pituitary formation; however, these effects could be due to the action of Hes1 in the hypothalamus, Rathke's pouch, or both. To determine the contribution of hypothalamic Notch signaling to pituitary organogenesis, we used mice with loss and gain of Notch function within the developing hypothalamus. Results: We demonstrate that loss of Notch signaling by conditional deletion of Rbpj in the hypothalamus does not affect expression of Hes1 within the posterior hypothalamus or expression of Hes5. In contrast, expression of activated Notch within the hypothalamus results in ectopic Hes5 expression and increased Hes1 expression, which is sufficient to disrupt pituitary development and postnatal expansion. Conclusions: Taken together, our results indicate that Rbpj‐dependent Notch signaling within the developing hypothalamus is not necessary for pituitary development, but persistent Notch signaling and ectopic Hes5 expression in hypothalamic progenitors affects pituitary induction and expansion. Developmental Dynamics 244:921–934, 2015. © 2015 Wiley Periodicals, Inc.     

Involvement of Notch1/Hes signaling pathway in ankylosing spondylitis

We aimed to investigate the role of Notch1/Hes signaling pathway in the pathogenesis of abnormal ossification of hip ligament in patients with ankylosing spondylitis (AS). 22 AS patients scheduled for artificial hip arthroplasty were randomly chosen as AS group. As controls, we used 4 patients diagnosed with transcervical fracture who underwent hip replacement surgery. Notch1 and Hes mRNA expressions were detected by real-time fluorescent quantitative polymerase chain reaction (RFQ-PCR). Immunohistochemistry (IHC) was used to detect Notch1 and Hes protein expression. Correlation analyses of Notch-l and Hes with AS-related clinical factors were conducted with spearman’s correlation analysis and partial correlation analysis. RFQ-PCR results showed significant differences in Notch1 and Hes mRNA expressions between AS group and the control group (all P < 0.05). IHC analysis further indicated positive nuclear signals of Notch1 and Hes protein, indicating functional activation of the Notch1 and Hes pathways. Semi-quantitative IHC showed a higher Notch1 and Hes expression levels in AS group compared to the control group (all P < 0.05). Correlation analysis suggested that Hes protein expression was positively associated with the clinical course of the disease in AS patients. In conclusion, Notch1 and Hes overexpression was clearly detected in hip joint ligaments of AS patients, Hes protein expression was associated with the clinical course of AS. Taken together, we suggest that signaling pathways mediated by Notch1-Hes may contribute to ligament ossification of hip joints in AS patients.     

Dynamic Notch signaling in neural progenitor cells and a revised view of lateral inhibition

In the developing mammalian nervous system, neural progenitor cells first express the Notch effector Hes1 at variable levels and then proneural genes and Notch ligands in salt-and-pepper patterns. Recent real-time imaging analysis indicates that Hes1 expression in these cells oscillates with a period of about 2-3 h. Furthermore, the proneural gene Neurogenin-2 (Ngn2) and the Notch ligand gene Deltalike-1 (Dll1) are expressed cyclically in neural progenitor cells under the control of Hes1 oscillation but are expressed continuously in postmitotic neurons, which lose Hes1 expression. Hes1-driven Ngn2 and Dll1 oscillations seem to be advantageous for maintenance of a group of cells in an undifferentiated state by mutual activation of Notch signaling. This dynamic mode of gene expression would require a revision of the traditional view of how Notch-mediated lateral inhibition operates in the developing mammalian nervous system.     

Notch信号通路相关蛋白在前列腺癌中的表达及临床意义

目的 本研究旨在探讨Notch信号通路相关蛋白在前列腺癌中的表达及临床意义.方法 采用实时定量PCR(Q-PCR)和Western blotting方法检测135例新鲜前列腺癌病理组织标本及临近非肿瘤组织标本中Notch 1、Notch 3及Hes1 mRNA和蛋白表达情况.同时采用免疫组化方法检测135例前列腺癌病理组织标本与临近非肿瘤组织标本中Notch 1、Notch 3及Hes 1蛋白表达情况,并分析各蛋白表达与前列腺癌患者临床病理因素的关系.结果 135例新鲜前列腺癌病理组织标本Q-PCR及Western blotting结果表明,与临近非肿瘤组织相比,前列腺癌组织中Notch 1、Notch 3及Hes 1 mRNA和蛋白表达均上调(P均＜0.05).免疫组化的结果表明,59.26％(80/135)前列腺癌样本为Notch 1阳性,高于临近非肿瘤组织17.78％ (24/135)(P＜0.05);65.19％(88/135)前列腺癌样本为Notch 3阳性,高于临近非肿瘤组织22.22％(30/135) (P＜ 0.05);62.22％ (84/135)前列腺癌样本为Hes 1阳性,高于临近非肿瘤组织17.78％(24/135)(P＜0.05).统计分析证实Notch 1蛋白表达与前列腺癌的肿瘤转移密切相关(x2=7.532,P=0.003);Notch 3蛋白表达与前列腺癌的肿瘤转移密切相关(x2=7.532,P=0.003);Hes 1蛋白表达与前列腺癌的肿瘤大小密切相关(x2=6.781,P=0.012).结论 Notch信号通路相关蛋白Notch 1、Notch 3及Hes 1在前列腺癌患者组织中表达升高,Notch信号通路的激活可能在前列腺癌发生、发展过程中起重要作用.     

Notch inhibition by the ligand DELTA-LIKE 3 defines the mechanism of abnormal vertebral segmentation in spondylocostal dysostosis

Mutations in the DELTA-LIKE 3 (DLL3) gene cause the congenital abnormal vertebral segmentation syndrome, spondylocostal dysostosis (SCD). DLL3 is a divergent member of the DSL family of Notch ligands that does not activate signalling in adjacent cells, but instead inhibits signalling when expressed in the same cell as the Notch receptor. Targeted deletion of Dll3 in the mouse causes a developmental defect in somite segmentation, and consequently vertebral formation is severely disrupted, closely resembling human SCD. In contrast to the canonical Notch signalling pathway, very little is known about the mechanism of cis-inhibition by DSL ligands. Here, we report that Dll3 is not presented on the surface of presomitic mesoderm (PSM) cells in vivo, but instead interacts with Notch1 in the late endocytic compartment. This suggests for the first time a mechanism for Dll3-mediated cis-inhibition of Notch signalling, with Dll3 targeting newly synthesized Notch1 for lysosomal degradation prior to post-translational processing and cell surface presentation of the receptor. An inhibitory role for Dll3 in vivo is further supported by the juxtaposition of Dll3 protein and Notch1 signalling in the PSM. Defining a mechanism for cis-inhibition of Notch signalling by Dll3 not only contributes greatly to our understanding of this ligand's function during the formation of the vertebral column, but also provides a paradigm for understanding how other ligands of Notch cis-inhibit signalling.