No abstract

During zebrafish development, the left-right (LR) asymmetric signals are first established around the Kupffer vesicle (KV), a ciliated organ generating directional fluid flow. Then, LR asymmetry is conveyed and stabilized in the lateral plate mesoderm. Although numerous molecules and signaling pathways are involved in controlling LR asymmetry, mechanistic difference and concordance between different organs during LR patterning are poorly understood. Here we show that RA signaling regulates laterality decisions at two stages in zebrafish. Before the 2-somite stage (2So), inhibition of RA signaling leads to randomized visceral laterality through bilateral expression of nodal/spaw in the lateral plate mesoderm, which is mediated by increases in cilia length and defective directional fluid flow in KV. Fgf8 is required for the regulation of cilia length by RA signaling. Blockage of RA signaling before 2So also leads to mild defects of heart laterality, which become much more severe through perturbation of cardiac bmp4 asymmetry when RA signaling is blocked after 2So. At this stage, visceral laterality and the left-sided Nodal remain unaffected. These findings suggest that RA signaling controls visceral laterality through the leftsided Nodal signal before 2So, and regulates heart laterality through cardiac bmp4 mainly after 2So, first identifying sequential control and concordance of visceral and heart laterality.

As one of the model organisms of Parkinson’s disease (PD) research, the zebrafish has its advantages, such as the 87% homology with human genome and transparent embryos which make it possible to observe the development of dopaminergic neurons in real time. However, there is no midbrain dopaminergic system in zebrafish when compared with mammals, and the location and projection of the dopaminergic neurons are seldom reported. In this study, Vmat2:GFP transgenic zebrafish was used to observe the development and distribution of dopaminergic neurons in real time. We found that diencephalons (DC) 2 and DC4 neuronal populations were detected at 24 h post fertilization (hpf). All DC neuronal populations as well as those in locus coeruleus (LC), raphe nuclei (Ra) and telencephalon were detected at 48 hpf. Axons were detected at 72 hpf. At 96 hpf, all the neuronal populations were detected. For the first time we reported axons from the posterior tubercle (PT) of ventral DC projected to subpallium in vivo. However, when compared with results from whole mount tyrosine hydroxylase (TH) immunofluorescence staining in wild type (WT) zebrafish, we found that DC2 and DC4 neuronal populations were mainly dopaminergic, while DC1, DC3, DC5 and DC6 might not. Neurons in pretectum (Pr) and telencephalon were mainly dopaminergic, while neurons in LC and Ra might be noradrenergic. Our study makes some corrections and modifications on the development, localization and distribution of zebrafish dopaminergic neurons, and provides some experimental evidences for the construction of the zebrafish PD model.

The cell cycle needs to strictly coordinate with developmental processes to ensure correct generation of the body plan and different tissues. However, the molecular mechanism underlying the coordination remains largely unknown. In this study, we investigate how the cell cycle coordinates gastrulation cell movements in zebrafish. We present a system to modulate the cell cycle in early zebrafish embryos by manipulating the geminin-Cdt1 balance. Alterations of the cell cycle change the apoptotic level during gastrulation, which correlates with the nuclear level of antiapoptotic nuclear factor κB (NF-κB). NF-κB associates with the Snail1a promoter region on the chromatin and directly activates Snail1a, an important factor controlling cell delamination, which is the initial step of mesendodermal cell movements during gastrulation. In effect, the cell cycle coordinates the delamination of mesendodermal cells through the transcription of Snail1a. Our results suggest a molecular mechanism by which NF-κB and Snail1a coordinate the cell cycle through gastrulation.

In many organisms, transcriptional and post-transcriptional regulation of components of pathways or processes has been reported. However, to date, there are few reports of translational co-regulation of multiple components of a developmental signaling pathway. Here, we show that an RNA element which we previously identified as a dorsal localization element (DLE) in the 3′UTR of zebrafish nodal-related1/squint (ndr1/sqt) ligand mRNA, is shared by the related ligand nodal-related2/cyclops (ndr2/cyc) and the nodal inhibitors, lefty1 (lft1) and lefty2 mRNAs. We investigated the activity of the DLEs through functional assays in live zebrafish embryos. The lft1 DLE localizes fluorescently labeled RNA similarly to the ndr1/sqt DLE. Similar to the ndr1/sqt 3′UTR, the lft1 and lft2 3′UTRs are bound by the RNA-binding protein (RBP) and translational repressor, Y-box binding protein 1 (Ybx1), whereas deletions in the DLE abolish binding to Ybx1. Analysis of zebrafish ybx1 mutants shows that Ybx1 represses lefty1 translation in embryos. CRISPR/Cas9-mediated inactivation of human YBX1 also results in human NODAL translational de-repression, suggesting broader conservation of the DLE RNA element/Ybx1 RBP module in regulation of Nodal signaling. Our findings demonstrate translational co-regulation of components of a signaling pathway by an RNA element conserved in both sequence and structure and an RBP, revealing a ‘translational regulon’.

Protein ubiquitin-proteasome (ubiquitin-proteasome) system is the major mechanism responsible for protein degradation in eukaryotic cell. During spermatogenesis, the replacement of histone by protamine is vital for normal sperm formation, which is involved in ubiquitination enzymes expressed in testis. Recently, histone ubiquitin ligases have been shown to play critical roles in several aspects of spermatogenesis, such as meiotic sex chromosome inactivation (MSCI), DNA damage response, and spermiogenesis. In this review, we highlight recent progress in the discovery of several histone ubiquitin ligases and elaborate mechanisms of how these enzymes are involved in these processes through knockout mouse model. Using Huwe1, UBR2, and RNF8 as examples, we emphasized the diverse functions for each enzyme and the broad involvement of these enzymes in every stage, from spermatogonia differentiation and meiotic division to spermiogenesis; thus histone ubiquitin ligases represent a class of enzymes, which play important roles in spermatogenesis through targeting histone for ubiquitination and therefore are involved in transcription regulation, epigenetic modification, and other processes essential for normal gametes formation.

Zebrafish dorsal forerunner cells (DFCs) undergo vigorous proliferation during epiboly and then exit the cell cycle to generate Kupffer’s vesicle (KV), a ciliated organ necessary for establishing left–right (L–R) asymmetry. DFC proliferation defects are often accompanied by impaired cilia elongation in KV, but the functional and molecular interaction between cell-cycle progression and cilia formation remains unknown. Here, we show that chemokine receptor Cxcr4a is required for L–R laterality by controlling DFC proliferation and KV ciliogenesis. Functional analysis revealed that Cxcr4a accelerates G1/S transition in DFCs and stabilizes forkhead box j1a (Foxj1a), a master regulator of motile cilia, by stimulating Cyclin D1 expression through extracellular regulated MAP kinase (ERK) 1/2 signaling. Mechanistically, Cyclin D1–cyclin-dependent kinase (CDK) 4/6 drives G1/S transition during DFC proliferation and phosphorylates Foxj1a, thereby disrupting its association with proteasome 26S subunit, non-ATPase 4b (Psmd4b), a 19S regulatory subunit. This prevents the ubiquitin (Ub)-independent proteasomal degradation of Foxj1a. Our study uncovers a role for Cxcr4 signaling in L–R patterning and provides fundamental insights into the molecular linkage between cell-cycle progression and ciliogenesis.

The G protein-coupled receptor APJ/Aplnr has been widely reported to be involved in heart and vascular development and disease, but whether it contributes to organ left-right patterning is largely unknown. Here, we show that in zebrafish, aplnra/b coordinates organ LR patterning in an apela/apln ligand-dependent manner using distinct mechanisms at different stages. During gastrulation and early somitogenesis, aplnra/b loss of function results in heart and liver LR asymmetry defects, accompanied by disturbed KV/cilia morphogenesis and disrupted left-sided Nodal/spaw expression in the LPM. In this process, only aplnra loss of function results in KV/cilia morphogenesis defect. In addition, only apela works as the early endogenous ligand to regulate KV morphogenesis, which then contributes to left-sided Nodal/spaw expression and subsequent organ LR patterning. The aplnra-apela cascade regulates KV morphogenesis by enhancing the expression of foxj1a , but not fgf8 or dnh9 , during KV development. At the late somite stage, both aplnra and aplnrb contribute to the expression of lft1 in the trunk midline but do not regulate KV formation, and this role is possibly mediated by both endogenous ligands, apela and apln . In conclusion, our study is the first to identify a role for aplnra/b and their endogenous ligands apela/apln in LR patterning, and it clarifies the distinct roles of aplnra-apela and aplnra/b-apela/apln in orchestrating organ LR patterning.