Fluid extraction from the left-right organizer uncovers mechanical properties needed for symmetry breaking.

Humans and other vertebrates define body axis left-right asymmetry in the early stages of embryo development. The mechanism behind left-right establishment is not fully understood. Symmetry breaking occurs in a dedicated organ called the left-right organizer (LRO) and involves motile cilia generating fluid-flow therein. However, it has been a matter of debate whether the process of symmetry breaking relies on a chemosensory or a mechanosensory mechanism (Shinohara et al., 2012). Novel tailored manipulations for LRO fluid extraction in living zebrafish embryos allowed us to pinpoint a physiological developmental period for breaking left-right symmetry during development. The shortest critical time-window was narrowed to one hour and characterized by a mild counterclockwise flow. The experimental challenge consisted in emptying the LRO of its fluid, abrogating simultaneously flow force and chemical determinants. Our findings revealed an unprecedented recovery capacity of the embryo to re-fil and re-circulate new LRO fluid. The embryos that later developed laterality problems were found to be those that had lower anterior angular velocity and thus less anterior-posterior heterogeneity. Next, aiming to test the presence of any secreted determinant, we replaced the extracted LRO fluid by a physiological buffer. Despite some transitory flow homogenization, laterality defects were absent unless viscosity was altered, demonstrating that symmetry breaking does not depend on the nature of the fluid content but is rather sensitive to fluid mechanics. Altogether, we conclude that the zebrafish LRO is more sensitive to fluid dynamics for symmetry breaking.

Zebrafish Model as a Screen to Prevent Cyst Inflation in Autosomal Dominant Polycystic Kidney Disease.

In autosomal dominant polycystic kidney disease (ADPKD), kidney cyst growth requires the recruitment of CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel that is defective in cystic fibrosis. We have been studying cyst inflation using the zebrafish Kupffer's vesicle (KV) as model system because we previously demonstrated that knocking down polycystin 2 (PC2) induced a CFTR-mediated enlargement of the organ. We have now quantified the PC2 knockdown by showing that it causes a 73% reduction in the number of KV cilia expressing PC2. According to the literature, this is an essential event in kidney cystogenesis in ADPKD mice. Additionally, we demonstrated that the PC2 knockdown leads to a significant accumulation of CFTR-GFP at the apical region of the KV cells. Furthermore, we determined that KV enlargement is rescued by the injection of Xenopus pkd2 mRNA and by 100 µM tolvaptan treatment, the unique and approved pharmacologic approach for ADPKD management. We expected vasopressin V2 receptor antagonist to lower the cAMP levels of KV-lining cells and, thus, to inactivate CFTR. These findings further support the use of the KV as an in vivo model for screening compounds that may prevent cyst enlargement in this ciliopathy, through CFTR inhibition.

Notch/Her12 signalling modulates, motile/immotile cilia ratio downstream of Foxj1a in zebrafish left-right organizer.

Foxj1a is necessary and sufficient to specify motile cilia. Using transcriptional studies and slow-scan two-photon live imaging capable of identifying the number of motile and immotile cilia, we now established that the final number of motile cilia depends on Notch signalling (NS). We found that despite all left-right organizer (LRO) cells express foxj1a and the ciliary axonemes of these cells have dynein arms, some cilia remain immotile. We identified that this decision is taken early in development in the Kupffer's Vesicle (KV) precursors the readout being her12 transcription. We demonstrate that overexpression of either her12 or Notch intracellular domain (NICD) increases the number of immotile cilia at the expense of motile cilia, and leads to an accumulation of immotile cilia at the anterior half of the KV. This disrupts the normal fluid flow intensity and pattern, with consequent impact on dand5 expression pattern and left-right (L-R) axis establishment.

The zebrafish Kupffer's vesicle as a model system for the molecular mechanisms by which the lack of Polycystin-2 leads to stimulation of CFTR.

In autosomal dominant polycystic kidney disease (ADPKD), cyst inflation and continuous enlargement are associated with marked transepithelial ion and fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR). Indeed, the inhibition or degradation of CFTR prevents the fluid accumulation within cysts. The in vivo mechanisms by which the lack of Polycystin-2 leads to CFTR stimulation are an outstanding challenge in ADPKD research and may bring important biomarkers for the disease. However, hampering their study, the available ADPKD in vitro cellular models lack the three-dimensional architecture of renal cysts and the ADPKD mouse models offer limited access for live-imaging experiments in embryonic kidneys. Here, we tested the zebrafish Kupffer's vesicle (KV) as an alternative model-organ. KV is a fluid-filled vesicular organ, lined by epithelial cells that express both CFTR and Polycystin-2 endogenously, being each of them easily knocked-down. Our data on the intracellular distribution of Polycystin-2 support its involvement in the KV fluid-flow induced Ca(2+)-signalling. Mirroring kidney cysts, the KV lumen inflation is dependent on CFTR activity and, as we clearly show, the knockdown of Polycystin-2 results in larger KV lumens through overstimulation of CFTR. In conclusion, we propose the zebrafish KV as a model organ to study the renal cyst inflation. Favouring its use, KV volume can be easily determined by in vivo imaging offering a live readout for screening compounds and genes that may prevent cyst enlargement through CFTR inhibition.

Dll1 and Dll4 function sequentially in the retina and pV2 domain of the spinal cord to regulate neurogenesis and create cell diversity.

Signalling mediated by Notch receptors is known to have multiple functions during vertebrate neural development, regulating processes like progenitor differentiation and cell type diversification. Various Notch ligands are expressed in the developing nervous system and their activities might contribute to this multiplicity of functions. Here, we show that two Delta-like genes, Dll1 and Dll4, are sequentially expressed in differentiating neurons of the embryonic mouse retina and spinal cord's pV2 domain, with Dll1 starting to be expressed before Dll4. Analysis of Dll1 mutants reveals this gene is necessary and sufficient to maintain a pool of progenitors in the embryonic neuroepithelium. Accordingly, in the spinal cord domains where Dll1 is the only expressed Notch ligand, its inactivation leads to an increased rate of neurogenesis and premature differentiation of neural progenitors. In contrast, in the pV2 domain and retina where Dll1 is co-expressed with Dll4, progenitors are not exhausted and cell diversity is maintained. Together, our results support a model where Dll1 and Dll4 are part of a unique genetic circuitry that regulates subsequent steps of neurogenesis in the retina and pV2 domain: while Dll1 serves to prevent the untimely differentiation of neural progenitors, Dll4 might function to generate diversity within the population of differentiating neurons.