Susana S. Lopes

Leukocyte tyrosine kinase functions in pigment cell development.

A fundamental problem in developmental biology concerns how multipotent precursors choose specific fates. Neural crest cells (NCCs) are multipotent, yet the mechanisms driving specific fate choices remain incompletely understood. Sox10 is required for specification of neural cells and melanocytes from NCCs. Like sox10 mutants, zebrafish shady mutants lack iridophores; we have proposed that sox10 and shady are required for iridophore specification from NCCs. We show using diverse approaches that shady encodes zebrafish leukocyte tyrosine kinase (Ltk). Cell transplantation studies show that Ltk acts cell-autonomously within the iridophore lineage. Consistent with this, ltk is expressed in a subset of NCCs, before becoming restricted to the iridophore lineage. Marker analysis reveals a primary defect in iridophore specification in ltk mutants. We saw no evidence for a fate-shift of neural crest cells into other pigment cell fates and some NCCs were subsequently lost by apoptosis. These features are also characteristic of the neural crest cell phenotype in sox10 mutants, leading us to examine iridophores in sox10 mutants. As expected, sox10 mutants largely lacked iridophore markers at late stages. In addition, sox10 mutants unexpectedly showed more ltk-expressing cells than wild-type siblings. These cells remained in a premigratory position and expressed sox10 but not the earliest neural crest markers and may represent multipotent, but partially-restricted, progenitors. In summary, we have discovered a novel signalling pathway in NCC development and demonstrate fate specification of iridophores as the first identified role for Ltk.

Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation

Membrane association with mother centriole (M-centriole) distal appendages is critical for ciliogenesis initiation. How the Rab GTPase Rab11–Rab8 cascade functions in early ciliary membrane assembly is unknown. Here, we show that the membrane shaping proteins EHD1 and EHD3, in association with the Rab11–Rab8 cascade, function in early ciliogenesis. EHD1 and EHD3 localize to preciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is essential for ciliary vesicle formation from smaller distal appendage vesicles (DAVs). Importantly, this step functions in M-centriole to basal body transformation and recruitment of transition zone proteins and IFT20. SNAP29, a SNARE membrane fusion regulator and EHD1-binding protein, is also required for DAV-mediated ciliary vesicle assembly. Interestingly, only after ciliary vesicle assembly is Rab8 activated for ciliary growth. Our studies uncover molecular mechanisms informing a previously uncharacterized ciliogenesis step, whereby EHD1 and EHD3 reorganize the M-centriole and associated DAVs before coordinated ciliary membrane and axoneme growth.

Notch signalling regulates left-right asymmetry through ciliary length control

The importance of cilia in embryonic development and adult physiology is emphasized by human ciliopathies. Despite its relevance, molecular signalling pathways behind cilia formation are poorly understood. We show that Notch signalling is a key pathway for cilia length control. In deltaD zebrafish mutants, cilia length is reduced in Kupffers vesicle and can be rescued by the ciliogenic factor foxj1a. Conversely, cilia length increases when Notch signalling is hyperactivated. Short cilia found in deltaD mutants reduce the fluid flow velocity inside Kupffers vesicle, thus compromising the asymmetric expression of the flow sensor charon. Notch signalling brings together ciliary length control and fluid flow hydrodynamics with transcriptional activation of laterality genes. In addition, our deltaD mutant analysis discloses an uncoupling between gut and heart laterality.

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 cords 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.

Left-Right Organizer Flow Dynamics: How Much Cilia Activity Reliably Yields Laterality?

Internal organs are asymmetrically positioned inside the body. Embryonic motile cilia play an essential role in this process by generating a directional fluid flow inside the vertebrate left-right organizer. Detailed characterization of how fluid flow dynamics modulates laterality is lacking. We used zebrafish genetics to experimentally generate a range of flow dynamics. By following the development of each embryo, we show that fluid flow in the left-right organizer is asymmetric and provides a good predictor of organ laterality. This was tested in mosaic organizers composed of motile and immotile cilia generated by dnah7 knockdowns. In parallel, we used simulations of fluid dynamics to analyze our experimental data. These revealed that fluid flow generated by 30 or more cilia predicts 90% situs solitus, similar to experimental observations. We conclude that cilia number, dorsal anterior motile cilia clustering, and left flow are critical to situs solitus via robust asymmetric charon expression.

Arl13b and the non-muscle myosin heavy chain IIA are required for circular dorsal ruffle formation and cell migration

ABSTRACT The Arf-like protein Arl13b has been implicated in ciliogenesis and Sonic hedgehog signaling. Furthermore, we have previously shown that it regulates endocytic recycling traffic and interacts with actin. Herein, we report that the non-muscle myosin heavy chain IIA, also known as Myh9, is an Arl13b effector. Moreover, we found that both proteins localized to circular dorsal ruffles (CDRs) induced by platelet-derived growth factor stimulation and are required for their formation. CDRs are ring-shaped actin-dependent structures formed on the dorsal cell surface and are involved in diverse processes, such as macropinocytosis, integrin recycling, internalization of receptor tyrosine kinases and cell migration. We found that Arl13b or Myh9 silencing impaired cell migration, suggesting that Arl13b is required for this function through the interaction with Myh9. Moreover, Arl13b silencing impaired neural crest cell migration in zebrafish embryos. Furthermore, we showed that Arl13b is required for the formation of CDRs in migrating cells. Thus, our results indicate a new role for Arl13b in actin cytoskeleton remodeling through the interaction with Myh9, by driving the formation of CDRs necessary for cell migration.

Left-Right Function of dmrt2 Genes Is Not Conserved between Zebrafish and Mouse

Background Members of the Dmrt family, generally associated with sex determination, were shown to be involved in several other functions during embryonic development. Dmrt2 has been studied in the context of zebrafish development where, due to a duplication event, two paralog genes dmrt2a and dmrt2b are present. Both zebrafish dmrt2a/terra and dmrt2b are important to regulate left-right patterning in the lateral plate mesoderm. In addition, dmrt2a/terra is necessary for symmetric somite formation while dmrt2b regulates somite differentiation impacting on slow muscle development. One dmrt2 gene is also expressed in the mouse embryo, where it is necessary for somite differentiation but with an impact on axial skeleton development. However, nothing was known about its role during left-right patterning in the lateral plate mesoderm or in the symmetric synchronization of somite formation. Methodology/Principal Findings Using a dmrt2 mutant mouse line, we show that this gene is not involved in symmetric somite formation and does not regulate the laterality pathway that controls left-right asymmetric organ positioning. We reveal that dmrt2a/terra is present in the zebrafish laterality organ, the Kupffers vesicle, while its homologue is excluded from the mouse equivalent structure, the node. On the basis of evolutionary sub-functionalization and neo-functionalization theories we discuss this absence of functional conservation. Conclusions/Significance Our results show that the role of dmrt2 gene is not conserved during zebrafish and mouse embryonic development.

Organized chaos in Kupffer's vesicle: how a heterogeneous structure achieves consistent left-right patterning.

Successful establishment of left-right asymmetry is crucial to healthy vertebrate development. In many species this process is initiated in a ciliated, enclosed cavity, for example Kupffers vesicle (KV) in zebrafish. The microarchitecture of KV is more complex than that present in the left-right organizer of many other species. While swirling flow in KV is recognized as essential for left-right patterning, its generation, nature and conversion to asymmetric gene expression are only beginning to be fully understood. We recently [Sampaio, P et al. Dev Cell 29:716–728] combined imaging, genetics and fluid dynamics simulation to characterize normal and perturbed ciliary activity, and their correlation to asymmetric charon expression and embryonic organ fate. Randomness in cilia number and length have major implications for robust flow generation; even a modest change in mean cilia length has a major effect on flow speed to due to nonlinear scaling arising from fluid mechanics. Wildtype, and mutant embryos with normal liver laterality, exhibit stronger flow on the left prior to asymmetric inhibition of charon. Our discovery of immotile cilia, taken with data on morphant embryos with very few cilia, further support the role of mechanosensing in initiating and/or enhancing flow conversion into gene expression.

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

ABSTRACT 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 Kupffers 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 Ca2+-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. Summary: Here, we tested the zebrafish Kupffers vesicle (KV) as a model organ to study, through in vivo imaging of KV volume, the stimulation of cystic fibrosis transmembrane conductance regulator (CFTR) in autosomal dominant polycystic kidney disease ADPKD.

Dynamics of cilia length in left–right development

Reduction in the length of motile cilia in the zebrafish left–right organizer (LRO), also known as Kupffers vesicle, has a large impact on left–right development. Here we demonstrate through genetic overexpression in zebrafish embryos and mathematical modelling that the impact of increased motile cilia length in embryonic LRO fluid flow is milder than that of short cilia. Through Arl13b overexpression, which increases cilia length without impacting cilia beat frequency, we show that the increase in cilium length is associated with a decrease in beat amplitude, resulting in similar flow strengths for Arl13b overexpression and wild-type (WT) embryos, which were not predicted by current theory. Longer cilia exhibit pronounced helical beat patterns and, consequently, lower beat amplitudes relative to WT, a result of an elastohydrodynamic shape transition. For long helical cilia, fluid dynamics modelling predicts a mild (approx. 12%) reduction in the torque exerted on the fluid relative to the WT, resulting in a proportional reduction in flow generation. This mild reduction is corroborated by experiments, providing a mechanism for the mild impact on organ situs.