Retinoic Acid Signaling Mediates Hair Cell Regeneration by Repressing p27kip and sox2 in Supporting Cells.

During development, otic sensory progenitors give rise to hair cells and supporting cells. In mammalian adults, differentiated and quiescent sensory cells are unable to generate new hair cells when these are lost due to various insults, leading to irreversible hearing loss. Retinoic acid (RA) has strong regenerative capacity in several organs, but its role in hair cell regeneration is unknown. Here, we use genetic and pharmacological inhibition to show that the RA pathway is required for hair cell regeneration in zebrafish. When regeneration is induced by laser ablation in the inner ear or by neomycin treatment in the lateral line, we observe rapid activation of several components of the RA pathway, with dynamics that position RA signaling upstream of other signaling pathways. We demonstrate that blockade of the RA pathway impairs cell proliferation of supporting cells in the inner ear and lateral line. Moreover, in neuromast, RA pathway regulates the transcription of p27(kip) and sox2 in supporting cells but not fgf3. Finally, genetic cell-lineage tracing using Kaede photoconversion demonstrates that de novo hair cells derive from FGF-active supporting cells. Our findings reveal that RA has a pivotal role in zebrafish hair cell regeneration by inducing supporting cell proliferation, and shed light on the underlying transcriptional mechanisms involved. This signaling pathway might be a promising approach for hearing recovery. SIGNIFICANCE STATEMENT: Hair cells are the specialized mechanosensory cells of the inner ear that capture auditory and balance sensory input. Hair cells die after acoustic trauma, ototoxic drugs or aging diseases, leading to progressive hearing loss. Mammals, in contrast to zebrafish, lack the ability to regenerate hair cells. Here, we find that retinoic acid (RA) pathway is required for hair cell regeneration in vivo in the zebrafish inner ear and lateral line. RA pathway is activated very early upon hair cell loss, promotes cell proliferation of progenitor cells, and regulates two key genes, p27(kip) and sox2. Our results position RA as an essential signal for hair cell regeneration with relevance in future regenerative strategies in mammals.

Her9 represses neurogenic fate downstream of Tbx1 and retinoic acid signaling in the inner ear.

Proper spatial control of neurogenesis in the inner ear ensures the precise innervation of mechanotransducing cells and the propagation of auditory and equilibrium stimuli to the brain. Members of the Hairy and enhancer of split (Hes) gene family regulate neurogenesis by inhibiting neuronal differentiation and maintaining neural stem cell pools in non-neurogenic zones. Remarkably, their role in the spatial control of neurogenesis in the ear is unknown. In this study, we identify her9, a zebrafish ortholog of Hes1, as a key gene in regulating otic neurogenesis through the definition of the posterolateral non-neurogenic field. First, her9 emerges as a novel otic patterning gene that represses proneural function and regulates the extent of the neurogenic domain. Second, we place Her9 downstream of Tbx1, linking these two families of transcription factors for the first time in the inner ear and suggesting that the reported role of Tbx1 in repressing neurogenesis is in part mediated by the bHLH transcriptional repressor Her9. Third, we have identified retinoic acid (RA) signaling as the upstream patterning signal of otic posterolateral genes such as tbx1 and her9. Finally, we show that at the level of the cranial otic field, opposing RA and Hedgehog signaling position the boundary between the neurogenic and non-neurogenic compartments. These findings permit modeling of the complex genetic cascade that underlies neural patterning of the otic vesicle.

Impaired embryonic haematopoiesis yet normal arterial development in the absence of the Notch ligand Jagged1.

Specific deletion of Notch1 and RBPjkappa in the mouse results in abrogation of definitive haematopoiesis concomitant with the loss of arterial identity at embryonic stage. As prior arterial determination is likely to be required for the generation of embryonic haematopoiesis, it is difficult to establish the specific haematopoietic role of Notch in these mutants. By analysing different Notch-ligand-null embryos, we now show that Jagged1 is not required for the establishment of the arterial fate but it is required for the correct execution of the definitive haematopoietic programme, including expression of GATA2 in the dorsal aorta. Moreover, successful haematopoietic rescue of the Jagged1-null AGM cells was obtained by culturing them with Jagged1-expressing stromal cells or by lentiviral-mediated transduction of the GATA2 gene. Taken together, our results indicate that Jagged1-mediated activation of Notch1 is responsible for regulating GATA2 expression in the AGM, which in turn is essential for definitive haematopoiesis in the mouse.

Jagged1 is the pathological link between Wnt and Notch pathways in colorectal cancer.

Notch has been linked to beta-catenin-dependent tumorigenesis; however, the mechanisms leading to Notch activation and the contribution of the Notch pathway to colorectal cancer is not yet understood. By microarray analysis, we have identified a group of genes downstream of Wnt/beta-catenin (down-regulated when blocking Wnt/beta-catenin) that are directly regulated by Notch (repressed by gamma-secretase inhibitors and up-regulated by active Notch1 in the absence of beta-catenin signaling). We demonstrate that Notch is downstream of Wnt in colorectal cancer cells through beta-catenin-mediated transcriptional activation of the Notch-ligand Jagged1. Consistently, expression of activated Notch1 partially reverts the effects of blocking Wnt/beta-catenin pathway in tumors implanted s.c. in nude mice. Crossing APC(Min/+) with Jagged1(+/Delta) mice is sufficient to significantly reduce the size of the polyps arising in the APC mutant background indicating that Notch is an essential modulator of tumorigenesis induced by nuclear beta-catenin. We show that this mechanism is operating in human tumors from Familial Adenomatous Polyposis patients. We conclude that Notch activation, accomplished by beta-catenin-mediated up-regulation of Jagged1, is required for tumorigenesis in the intestine. The Notch-specific genetic signature is sufficient to block differentiation and promote vasculogenesis in tumors whereas proliferation depends on both pathways.

Multiple enhancers located in a 1-Mb region upstream of POU3F4 promote expression during inner ear development and may be required for hearing.

POU3F4 encodes a POU-domain transcription factor required for inner ear development. Defects in POU3F4 function are associated with X-linked deafness type 3 (DFN3). Multiple deletions affecting up to ~900-kb upstream of POU3F4 are found in DFN3 patients, suggesting the presence of essential POU3F4 enhancers in this region. Recently, an inner ear enhancer was reported that is absent in most DFN3 patients with upstream deletions. However, two indications suggest that additional enhancers in the POU3F4 upstream region are required for POU3F4 function during inner ear development. First, there is at least one DFN3 deletion that does not eliminate the reported enhancer. Second, the expression pattern driven by this enhancer does not fully recapitulate Pou3f4 expression in the inner ear. Here, we screened a 1-Mb region upstream of the POU3F4 gene for additional cis-regulatory elements and searched for novel DFN3 mutations in the identified POU3F4 enhancers. We found several novel enhancers for otic vesicle expression. Some of these also drive expression in kidney, pancreas and brain, tissues that are known to express Pou3f4. In addition, we report a new and smallest deletion identified so far in a DFN3 family which eliminates 3.9 kb, comprising almost exclusively the previous reported inner ear enhancer. We suggest that multiple enhancers control the expression of Pou3f4 in the inner ear and these may contribute to the phenotype observed in DFN3 patients. In addition, the novel deletion demonstrates that the previous reported enhancer, although not sufficient, is essential for POU3F4 function during inner ear development.

IkappaBalpha and p65 regulate the cytoplasmic shuttling of nuclear corepressors: cross-talk between Notch and NFkappaB pathways.

Notch and NFkappaB pathways are key regulators of numerous cellular events such as proliferation, differentiation, or apoptosis. In both pathways, association of effector proteins with nuclear corepressors is responsible for their negative regulation. We have previously described that expression of a p65-NFkappaB mutant that lacks the transactivation domain (p65DeltaTA) induces cytoplasmic translocation of N-CoR leading to a positive regulation of different promoters. Now, we show that cytoplasmic sequestration of p65 by IkappaBalpha is sufficient to both translocate nuclear corepressors SMRT/N-CoR to the cytoplasm and upregulate transcription of Notch-dependent genes. Moreover, p65 and IkappaBalpha are able to directly bind SMRT, and this interaction can be inhibited in a dose-dependent manner by the CREB binding protein (CBP) coactivator and after TNF-alpha treatment, suggesting that p65 acetylation is modulating this interaction. In agreement with this, TNF-alpha treatment results in downregulation of the Hes1 gene. Finally, we present evidence on how this mechanism may influence cell differentiation in the 32D myeloid progenitor system.

The Notch pathway in the developing hematopoietic system.

The main function of the Notch signaling pathway is to generate cell diversity during both embryonic development and adult tissue homeostasis. The extended use of this pathway, together with its conservation during evolution, is indicative of its importance. During embryonic development, the vascular and hematopoietic systems are intimately associated and Notch signals are responsible for the correct specification of both systems. More explicitly, Notch is required for the induction of the arterial program; however, it is simultaneously or consecutively also involved in the generation of hematopoietic stem cells. Although both genetic programs are different, they are both implemented in endothelial cells of the dorsal aorta in the midgestation embryo. This close association during the development of arteries and blood has hindered our understanding of Notch function in the generation of hematopoietic stem cells. Here, we will review the work from recent years showing how Notch participates in the embryonic development of hematopoiesis in the mouse, but also in other organisms such as chick, zebrafish and flies.

Characterization of new otic enhancers of the pou3f4 gene reveal distinct signaling pathway regulation and spatio-temporal patterns.

POU3F4 is a member of the POU-homedomain transcription factor family with a prominent role in inner ear development. Mutations in the human POU3F4 coding unit leads to X-linked deafness type 3 (DFN3), characterized by conductive hearing loss and progressive sensorineural deafness. Microdeletions found 1 Mb 5' upstream of the coding region also displayed the same phenotype, suggesting that cis-regulatory elements might be present in that region. Indeed, we and others have recently identified several enhancers at the 1 Mb 5' upstream interval of the pou3f4 locus. Here we characterize the spatio-temporal patterns of these regulatory elements in zebrafish transgenic lines. We show that the most distal enhancer (HCNR 81675) is activated earlier and drives GFP reporter expression initially to a broad ear domain to progressively restrict to the sensory patches. The proximal enhancer (HCNR 82478) is switched later during development and promotes expression, among in other tissues, in sensory patches from its onset. The third enhancer (HCNR 81728) is also active at later stages in the otic mesenchyme and in the otic epithelium. We also characterize the signaling pathways regulating these enhancers. While HCNR 81675 is regulated by very early signals of retinoic acid, HCNR 82478 is regulated by Fgf activity at a later stage and the HCNR 81728 enhancer is under the control of Hh signaling. Finally, we show that Sox2 and Pax2 transcription factors are bound to HCNR 81675 genomic region during otic development and specific mutations to these transcription factor binding sites abrogates HCNR 81675 enhancer activity. Altogether, our results suggest that pou3f4 expression in inner ear might be under the control of distinct regulatory elements that fine-tune the spatio-temporal activity of this gene and provides novel data on the signaling mechanisms controlling pou3f4 function.

RBPjkappa-dependent Notch function regulates Gata2 and is essential for the formation of intra-embryonic hematopoietic cells.

Definitive hematopoiesis in the mouse embryo originates from the aortic floor in the P-Sp/AGM region in close association with endothelial cells. An important role for Notch1 in the control of hematopoietic ontogeny has been recently established, although its mechanism of action is poorly understood. Here, we show detailed analysis of Notch family gene expression in the aorta endothelium between embryonic day (E) 9.5 and E10.5. Since Notch requires binding to RBPjkappa transcription factor to activate transcription, we analyzed the aorta of the para-aortic splanchnopleura/AGM in RBPjkappa mutant embryos. We found specific patterns of expression of Notch receptors, ligands and Hes genes that were lost in RBPjkappa mutants. Analysis of these mutants revealed the absence of hematopoietic progenitors, accompanied by the lack of expression of the hematopoietic transcription factors Aml1/Runx1, Gata2 and Scl/Tal1. We show that in wild-type embryos, a few cells lining the aorta endothelium at E9.5 simultaneously expressed Notch1 and Gata2, and demonstrate by chromatin immunoprecipitation that Notch1 specifically associated with the Gata2 promoter in E9.5 wild-type embryos and 32D myeloid cells, an interaction lost in RBPjkappamutants. Consistent with a role for Notch1 in regulating Gata2, we observe increased expression of this gene in 32D cells expressing activated Notch1. Taken together, these data strongly suggest that activation of Gata2 expression by Notch1/RBPjkappa is a crucial event for the onset of definitive hematopoiesis in the embryo.