Bioprinting Soft 3D Models of Hematopoiesis using Natural Silk Fibroin-Based Bioink Efficiently Supports Platelet Differentiation.

Hematopoietic stem and progenitor cells (HSPCs) continuously generate platelets throughout one's life. Inherited Platelet Disorders affect ≈ 3 million individuals worldwide and are characterized by defects in platelet formation or function. A critical challenge in the identification of these diseases lies in the absence of models that facilitate the study of hematopoiesis ex vivo. Here, a silk fibroin-based bioink is developed and designed for 3D bioprinting. This bioink replicates a soft and biomimetic environment, enabling the controlled differentiation of HSPCs into platelets. The formulation consisting of silk fibroin, gelatin, and alginate is fine-tuned to obtain a viscoelastic, shear-thinning, thixotropic bioink with the remarkable ability to rapidly recover after bioprinting and provide structural integrity and mechanical stability over long-term culture. Optical transparency allowed for high-resolution imaging of platelet generation, while the incorporation of enzymatic sensors allowed quantitative analysis of glycolytic metabolism during differentiation that is represented through measurable color changes. Bioprinting patient samples revealed a decrease in metabolic activity and platelet production in Inherited Platelet Disorders. These discoveries are instrumental in establishing reference ranges for classification and automating the assessment of treatment responses. This model has far-reaching implications for application in the research of blood-related diseases, prioritizing drug development strategies, and tailoring personalized therapies.

Zebrafish Posterior Lateral Line primordium migration requires interactions between a superficial sheath of motile cells and the skin.

The Zebrafish Posterior Lateral Line primordium migrates in a channel between the skin and somites. Its migration depends on the coordinated movement of its mesenchymal-like leading cells and trailing cells, which form epithelial rosettes, or protoneuromasts. We describe a superficial population of flat primordium cells that wrap around deeper epithelialized cells and extend polarized lamellipodia to migrate apposed to the overlying skin. Polarization of lamellipodia extended by both superficial and deeper protoneuromast-forming cells depends on Fgf signaling. Removal of the overlying skin has similar effects on superficial and deep cells: lamellipodia are lost, blebs appear instead, and collective migration fails. When skinned embryos are embedded in Matrigel, basal and superficial lamellipodia are recovered; however, only the directionality of basal protrusions is recovered, and migration is not rescued. These observations support a key role played by superficial primordium cells and the skin in directed migration of the Posterior Lateral Line primordium.

NetLogo agent-based models as tools for understanding the self-organization of cell fate, morphogenesis and collective migration of the zebrafish posterior Lateral Line primordium.

Interactions between primordium cells and their environment determines the self-organization of the zebrafish posterior Lateral Line primordium as it migrates under the skin from the ear to the tip of the tail forming and depositing neuromasts to spearhead formation of the posterior Lateral Line sensory system. In this review we describe how the NetLogo agent-based programming environment has been used in our lab to visualize and explore how self-generated chemokine gradients determine collective migration, how the dynamics of Wnt signaling can be used to predict patterns of neuromast deposition, and how previously defined interactions between Wnt and Fgf signaling systems have the potential to determine the periodic formation of center-biased Fgf signaling centers in the wake of a shrinking Wnt system. We also describe how NetLogo was used as a database for storing and visualizing the results of in toto lineage analysis of all cells in the migrating primordium. Together, the models illustrate how this programming environment can be used in diverse ways to integrate what has been learnt from biological experiments about the nature of interactions between cells and their environment, and explore how these interactions could potentially determine emergent patterns of cell fate specification, morphogenesis and collective migration of the zebrafish posterior Lateral Line primordium.

Time-lapse imaging beyond the diffraction limit.

The zebrafish, with its rapid external development, optical transparency, and the relative ease with which transgenic lines can be created, is rapidly becoming the model of choice for examining developmental processes via time-lapse microscopy. The recent proliferation of techniques for super-resolution imaging now allows for an unprecedented view of embryonic development at high spatial and temporal resolution in live tissues. This review examines both the theoretical basis and practical application of a number of established and emerging super-resolution microscopy techniques, focusing on their application in time-lapse imaging of live zebrafish embryos.

Cxcl12a induces snail1b expression to initiate collective migration and sequential Fgf-dependent neuromast formation in the zebrafish posterior lateral line primordium.

The zebrafish posterior lateral line primordium migrates along a path defined by the chemokine Cxcl12a, periodically depositing neuromasts, to pioneer formation of the zebrafish posterior lateral line system. snail1b, known for its role in promoting cell migration, is expressed in leading cells of the primordium in response to Cxcl12a, whereas its expression in trailing cells is inhibited by Fgf signaling. snail1b knockdown delays initiation of primordium migration. This delay is associated with aberrant expansion of epithelial cell adhesion molecule (epcam) and reduction of cadherin 2 expression in the leading part of the primordium. Co-injection of snail1b morpholino with snail1b mRNA prevents the initial delay in migration and restores normal expression of epcam and cadherin 2 The delay in initiating primordium migration in snail1b morphants is accompanied by a delay in sequential formation of trailing Fgf signaling centers and associated protoneuromasts. This delay is not specifically associated with knockdown of snail1b but also with other manipulations that delay migration of the primordium. These observations reveal an unexpected link between the initiation of collective migration and sequential formation of protoneuromasts in the primordium.

Self-organizing spots get under your skin.

Sixty-five years after Turing first revealed the potential of systems with local activation and long-range inhibition to generate pattern, we have only recently begun to identify the biological elements that operate at many scales to generate periodic patterns in nature. In this Primer, we first review the theoretical framework provided by Turing, Meinhardt, and others that suggests how periodic patterns could self-organize in developing animals. This Primer was developed to provide context for recent studies that reveal how diverse molecular, cellular, and physical mechanisms contribute to the establishment of the periodic pattern of hair or feather buds in the developing skin. From an initial emphasis on trying to disambiguate which specific mechanism plays a primary role in hair or feather bud development, we are beginning to discover that multiple mechanisms may, in at least some contexts, operate together. While the emergence of the diverse mechanisms underlying pattern formation in specific biological contexts probably reflects the contingencies of evolutionary history, an intriguing possibility is that these mechanisms interact and reinforce each other, producing emergent systems that are more robust.

A framework for understanding morphogenesis and migration of the zebrafish posterior Lateral Line primordium.

A description of zebrafish posterior Lateral Line (pLL) primordium development at single cell resolution together with the dynamics of Wnt, FGF, Notch and chemokine signaling in this system has allowed us to develop a framework to understand the self-organization of cell fate, morphogenesis and migration during its early development. The pLL primordium migrates under the skin, from near the ear to the tip of the tail, periodically depositing neuromasts. Nascent neuromasts, or protoneuromasts, form sequentially within the migrating primordium, mature, and are deposited from its trailing end. Initially broad Wnt signaling inhibits protoneuromast formation. However, protoneuromasts form sequentially in response to FGF signaling, starting from the trailing end, in the wake of a progressively shrinking Wnt system. While proliferation adds to the number of cells, the migrating primordium progressively shrinks as its trailing cells stop moving and are deposited. As it shrinks, the length of the migrating primordium correlates with the length of the leading Wnt system. Based on these observations we show how measuring the rate at which the Wnt system shrinks, the proliferation rate, the initial size of the primordium, its speed, and a few additional parameters allows us to predict the pattern of neuromast formation and deposition by the migrating primordium in both wild-type and mutant contexts. While the mechanism that links the length of the leading Wnt system to that of the primordium remains unclear, we discuss how it might be determined by access to factors produced in the leading Wnt active zone that are required for collective migration of trailing cells. We conclude by reviewing how FGFs, produced in response to Wnt signaling in leading cells, help determine collective migration of trailing cells, while a polarized response to a self-generated chemokine gradient serves as an efficient mechanism to steer primordium migration along its relatively long journey.

Polarization and migration in the zebrafish posterior lateral line system.

Collective cell migration plays an important role in development. Here, we study the posterior lateral line primordium (PLLP) a group of about 100 cells, destined to form sensory structures, that migrates from head to tail in the zebrafish embryo. We model mutually inhibitory FGF-Wnt signalling network in the PLLP and link tissue subdivision (Wnt receptor and FGF receptor activity domains) to receptor-ligand parameters. We then use a 3D cell-based simulation with realistic cell-cell adhesion, interaction forces, and chemotaxis. Our model is able to reproduce experimentally observed motility with leading cells migrating up a gradient of CXCL12a, and trailing (FGF receptor active) cells moving actively by chemotaxis towards FGF ligand secreted by the leading cells. The 3D simulation framework, combined with experiments, allows an investigation of the role of cell division, chemotaxis, adhesion, and other parameters on the shape and speed of the PLLP. The 3D model demonstrates reasonable behaviour of control as well as mutant phenotypes.

Epb41l5 competes with Delta as a substrate for Mib1 to coordinate specification and differentiation of neurons.

We identified Erythrocyte membrane protein band 4.1-like 5 (Epb41l5) as a substrate for the E3 ubiquitin ligase Mind bomb 1 (Mib1), which is essential for activation of Notch signaling. Although loss of Epb41l5 does not significantly alter the pattern of neural progenitor cells (NPCs) specified as neurons at the neural plate stage, it delays their delamination and differentiation after neurulation when NPCs normally acquire organized apical junctional complexes (AJCs) in the zebrafish hindbrain. Delays in differentiation are reduced by knocking down N-cadherin, a manipulation expected to help destabilize adherens junctions (AJs). This suggested that delays in neuronal differentiation in epb41l5-deficient embryos are related to a previously described role for Epb41l5 in facilitating disassembly of cadherin-dependent AJCs. Mib1 ubiquitylates Epb41l5 to promote its degradation. DeltaD can compete with Epb41l5 to reduce Mib1-dependent Epb41l5 degradation. In this context, increasing the number of NPCs specified to become neurons, i.e. cells expressing high levels of DeltaD, stabilizes Epb41l5 in the embryo. Together, these observations suggest that relatively high levels of Delta stabilize Epb41l5 in NPCs specified as neurons. This, we suggest, helps coordinate NPC specification with Epb41l5-dependent delamination and differentiation as neurons.

Connecting physical cues and tissue patterning.

Several signaling pathways work together, via a protein called Amotl2a, to establish the size and shape of a zebrafish sense organ primordium.

Leading and trailing cells cooperate in collective migration of the zebrafish posterior lateral line primordium.

Collective migration of cells in the zebrafish posterior lateral line primordium (PLLp) along a path defined by Cxcl12a expression depends on Cxcr4b receptors in leading cells and on Cxcr7b in trailing cells. Cxcr7b-mediated degradation of Cxcl12a by trailing cells generates a local gradient of Cxcl12a that guides PLLp migration. Agent-based computer models were built to explore how a polarized response to Cxcl12a, mediated by Cxcr4b in leading cells and prevented by Cxcr7b in trailing cells, determines unidirectional migration of the PLLp. These chemokine signaling-based models effectively recapitulate many behaviors of the PLLp and provide potential explanations for the characteristic behaviors that emerge when the PLLp is severed by laser to generate leading and trailing fragments. As predicted by our models, the bilateral stretching of the leading fragment is lost when chemokine signaling is blocked in the PLLp. However, movement of the trailing fragment toward the leading cells, which was also thought to be chemokine dependent, persists. This suggested that a chemokine-independent mechanism, not accounted for in our models, is responsible for this behavior. Further investigation of trailing cell behavior shows that their movement toward leading cells depends on FGF signaling and it can be re-oriented by exogenous FGF sources. Together, our observations reveal the simple yet elegant manner in which leading and trailing cells coordinate migration; while leading cells steer PLLp migration by following chemokine cues, cells further back play follow-the-leader as they migrate toward FGFs produced by leading cells.

Shroom3 is required downstream of FGF signalling to mediate proneuromast assembly in zebrafish.

During development, morphogenetic processes require a precise coordination of cell differentiation, cell shape changes and, often, cell migration. Yet, how pattern information is used to orchestrate these different processes is still unclear. During lateral line (LL) morphogenesis, a group of cells simultaneously migrate and assemble radially organized cell clusters, termed rosettes, that prefigure LL sensory organs. This process is controlled by Fibroblast growth factor (FGF) signalling, which induces cell fate changes, cell migration and cell shape changes. However, the exact molecular mechanisms induced by FGF activation that mediate these changes on a cellular level are not known. Here, we focus on the mechanisms by which FGFs control apical constriction and rosette assembly. We show that apical constriction in the LL primordium requires the activity of non-muscle myosin. We demonstrate further that shroom3, a well-known regulator of non-muscle myosin activity, is expressed in the LL primordium and that its expression requires FGF signalling. Using gain- and loss-of-function experiments, we demonstrate that Shroom3 is the main organizer of cell shape changes during rosette assembly, probably by coordinating Rho kinase recruitment and non-muscle myosin activation. In order to quantify morphogenesis in the LL primordium in an unbiased manner, we developed a unique trainable 'rosette detector'. We thus propose a model in which Shroom3 drives rosette assembly in the LL downstream of FGF in a Rho kinase- and non-muscle myosin-dependent manner. In conclusion, we uncovered the first mechanistic link between patterning and morphogenesis during LL sensory organ formation.

Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy.

We demonstrate three-dimensional (3D) super-resolution in live multicellular organisms using structured illumination microscopy (SIM). Sparse multifocal illumination patterns generated by a digital micromirror device (DMD) allowed us to physically reject out-of-focus light, enabling 3D subdiffractive imaging in samples eightfold thicker than had been previously imaged with SIM. We imaged samples at one 2D image per second, at resolutions as low as 145 nm laterally and 400 nm axially. In addition to dual-labeled, whole fixed cells, we imaged GFP-labeled microtubules in live transgenic zebrafish embryos at depths >45 µm. We captured dynamic changes in the zebrafish lateral line primordium and observed interactions between myosin IIA and F-actin in cells encapsulated in collagen gels, obtaining two-color 4D super-resolution data sets spanning tens of time points and minutes without apparent phototoxicity. Our method uses commercially available parts and open-source software and is simpler than existing SIM implementations, allowing easy integration with wide-field microscopes.

Knockdown of Bardet-Biedl syndrome gene BBS9/PTHB1 leads to cilia defects.

Bardet-Biedl Syndrome (BBS, MIM#209900) is a genetically heterogeneous disorder with pleiotropic phenotypes that include retinopathy, mental retardation, obesity and renal abnormalities. Of the 15 genes identified so far, seven encode core proteins that form a stable complex called BBSome, which is implicated in trafficking of proteins to cilia. Though BBS9 (also known as PTHB1) is reportedly a component of BBSome, its direct function has not yet been elucidated. Using zebrafish as a model, we show that knockdown of bbs9 with specific antisense morpholinos leads to developmental abnormalities in retina and brain including hydrocephaly that are consistent with the core phenotypes observed in syndromic ciliopathies. Knockdown of bbs9 also causes reduced number and length of cilia in Kupffer's vesicle. We also demonstrate that an orthologous human BBS9 mRNA, but not one carrying a missense mutation identified in BBS patients, can rescue the bbs9 morphant phenotype. Consistent with these findings, knockdown of Bbs9 in mouse IMCD3 cells results in the absence of cilia. Our studies suggest a key conserved role of BBS9 in biogenesis and/or function of cilia in zebrafish and mammals.

G2 acquisition by transcription-independent mechanism at the zebrafish midblastula transition.

Following fertilization of many animal embryos, rapid synchronous cleavage divisions give way to longer, asynchronous cell cycles at the midblastula transition (MBT). The cell cycle changes at the MBT, including the addition of gap phases and checkpoint controls, are accompanied by activation of the zygotic genome and the onset of cell motility. Whereas the biochemical changes accompanying the MBT in the vertebrate embryo have been extensively documented, the cellular events are not well understood. We show that cell cycle remodeling during the zebrafish MBT includes the transcription-independent acquisition of a G2 phase that is essential for preventing entry into mitosis before S-phase completion in cycles 11-13. We provide evidence from high-resolution imaging that inhibition of Cdc25a and Cdk1 activity, but not Cdk2 activity, is essential for cell cycle lengthening and asynchrony between cycles 9 and 12. We demonstrate that lengthening is not required for initiation of zygotic transcription. Our results are consistent with findings from Drosophila and Xenopus that indicate the central importance of G2 addition in checkpoint establishment, and point to similar mechanisms governing the MBT in diverse species.

Ty3/gypsy-like retrotransposons in Candida albicans and Candida dubliniensis: Tca3 and Tcd3.

Ty3/gypsy retrotransposons are a widespread group of eukaryote mobile genetic elements. They are similar in structure to, and may be ancestors of, the vertebrate retroviruses. Here we describe the first Ty3/gypsy retrotransposons from the pathogenic yeasts Candida albicans and Candida dubliniensis, which we refer to as Tca3 and Tcd3, respectively. Tca3 was first identified in a variety of strains as an element lacking a large part of its coding region. Comparative analyses between C. albicans and C. dubliniensis allowed us to identify the closely related full-length Tcd3 element, and, subsequently, the full-length Tca3 elements. The full-length versions of Tca3 and Tcd3 are broadly similar in structure to other Ty3/gypsy elements, but have several features of special interest, e.g. both elements appear to have a novel mechanism for priming minus-strand DNA synthesis, probably involving conserved secondary structures adjacent to the 5' LTRs. Also, while closely related to each other, the two elements appear to be fairly distantly related to other known Ty3/gypsy-like elements. Finally, the occurrence of the internally deleted forms of Tca3 in many strains raises interesting questions concerning the evolution of these transposable elements in Candida and the evolution of Candida itself. The sequences reported in this paper have been assigned GenBank Accession Nos AF499463, AF499464 and AF510498.

Xena, a full-length basal retroelement from tetraodontid fish.

Mobile genetic elements are ubiquitous throughout the eukaryote superkingdom. We have sequenced a highly unusual full-length retroelement from the Fugu fish, Takifugu rubripes. This element, which we have named Xena, is similar in structure and sequence to the Penelope retroelement from Drosophila virilis and consists of a single long open reading frame containing a reverse transcriptase domain flanked by identical direct long terminal repeat (LTR) sequences. These LTRs show an organization similar to the terminal repeats already described in the Penelope retrotransposon of Drosophila but are structurally and functionally distinct from the LTRs carried by LTR-retrotransposons. In view of their distinctness, we refer to these repeats as PLTRs (Penelope-LTRs). Whereas the element contains a reverse transcriptase, no other domains or motifs commonly associated with retroelements are present. In the full-length Fugu element, the 5' direct PLTR is preceded by an inverted PLTR fragment. Additional elements, many showing various degrees of deletion, are described from the Fugu genome and from that of the freshwater pufferfish Tetraodon nigroviridis. Many of these additional elements are also preceded by inverted PLTR sequences. Xena-like elements are also described from the genomes of several other organisms. The Penelope-Xena lineage is apparently a basal group within the retrotransposons and therefore represents an evolutionarily important class of retroelement.