Richard H. Row

Rho-regulated Myosin phosphatase establishes the level of protrusive activity required for cell movements during zebrafish gastrulation

Rho-dependent amoeboid cell movement is a crucial mechanism in both tumor cell invasion and morphogenetic cell movements during fish gastrulation. Amoeboid movement is characterized by relatively non-polarized cells displaying a high level of bleb-like protrusions. During gastrulation, zebrafish mesodermal cells undergo a series of conversions from amoeboid cell behaviors to more mesenchymal and finally highly polarized and intercalative cell behaviors. We demonstrate that Myosin phosphatase, a complex of Protein phosphatase 1 and the scaffolding protein Mypt1, functions to maintain the precise balance between amoeboid and mesenchymal cell behaviors required for cells to undergo convergence and extension. Importantly, Mypt1 has different cell-autonomous and non-cell-autonomous roles. Loss of Mypt1 throughout the embryo causes severe convergence defects, demonstrating that Mypt1 is required for the cell-cell interactions involved in dorsal convergence. By contrast, mesodermal Mypt1 morphant cells transplanted into wild-type hosts undergo dorsally directed cell migration, but they fail to shut down their protrusive behavior and undergo the normal intercalation required for extension. We further show that Mypt1 activity is regulated in embryos by Rho-mediated inhibitory phosphorylation, which is promoted by non-canonical Wnt signaling. We propose that Myosin phosphatase is a crucial and tightly controlled regulator of cell behaviors during gastrulation and that understanding its role in early development also provides insight into the mechanism of cancer cell invasion.

Suppression of Alk8-mediated Bmp signaling cell-autonomously induces pancreatic β-cells in zebrafish

Bmp signaling has been shown to regulate early aspects of pancreas development, but its role in endocrine, and especially β-cell, differentiation remains unclear. Taking advantage of the ability in zebrafish embryos to cell-autonomously modulate Bmp signaling in single cells, we examined how Bmp signaling regulates the ability of individual endodermal cells to differentiate into β-cells. We find that specific temporal windows of Bmp signaling prevent β-cell differentiation. Thus, future dorsal bud-derived β-cells are sensitive to Bmp signaling specifically during gastrulation and early somitogenesis stages. In contrast, ventral pancreatic cells, which require an early Bmp signal to form, do not produce β-cells when exposed to Bmp signaling at 50 hpf, a stage when the ventral bud-derived extrapancreatic duct is the main source of new endocrine cells. Importantly, inhibiting Bmp signaling within endodermal cells via genetic means increased the number of β-cells, at early and late stages. Moreover, inhibition of Bmp signaling in the late stage embryo using dorsomorphin, a chemical inhibitor of Bmp receptors, significantly increased β-cell neogenesis near the extrapancreatic duct, demonstrating the feasibility of pharmacological approaches to increase β-cell numbers. Our in vivo single-cell analyses show that whereas Bmp signaling is necessary initially for formation of the ventral pancreas, differentiating endodermal cells need to be protected from exposure to Bmps during specific stages to permit β-cell differentiation. These results provide important unique insight into the intercellular signaling environment necessary for in vivo and in vitro generation of β-cells.

Bmp inhibition is necessary for post-gastrulation patterning and morphogenesis of the zebrafish tailbud

Intricate interactions between the Wnt and Bmp signaling pathways pattern the gastrulating vertebrate embryo using a network of secreted protein ligands and inhibitors. While many of these proteins are expressed post-gastrula, their later roles have typically remained unclear, obscured by the effects of early perturbation. We find that Bmp signaling continues during somitogenesis in zebrafish embryos, with high activity in a small region of the mesodermal progenitor zone at the posterior end of the embryo. To test the hypothesis that Bmp inhibitors expressed just anterior to the tailbud are important to restrain Bmp signaling we produced a new zebrafish transgenic line, allowing temporal cell-autonomous activation of Bmp signaling and thereby bypassing the effects of the Bmp inhibitors. Ectopic activation of Bmp signaling during somitogenesis results in severe defects in the tailbud, including altered morphogenesis and gene expression. We show that these defects are due to non-autonomous effects on the tailbud, and present evidence that the tailbud defects are caused by alterations in Wnt signaling. We present a model in which the posteriorly expressed Bmp inhibitors function during somitogenesis to constrain Bmp signaling in the tailbud in order to allow normal expression of Wnt inhibitors in the presomitic mesoderm, which in turn constrain the levels of canonical and non-canonical Wnt signaling in the tailbud.

Completion of the epithelial to mesenchymal transition in zebrafish mesoderm requires Spadetail

The process of gastrulation is highly conserved across vertebrates on both the genetic and morphological levels, despite great variety in embryonic shape and speed of development. This mechanism spatially separates the germ layers and establishes the organizational foundation for future development. Mesodermal identity is specified in a superficial layer of cells, the epiblast, where cells maintain an epithelioid morphology. These cells involute to join the deeper hypoblast layer where they adopt a migratory, mesenchymal morphology. Expression of a cascade of related transcription factors orchestrates the parallel genetic transition from primitive to mature mesoderm. Although the early and late stages of this process are increasingly well understood, the transition between them has remained largely mysterious. We present here the first high resolution in vivo observations of the blebby transitional morphology of involuting mesodermal cells in a vertebrate embryo. We further demonstrate that the zebrafish spadetail mutation creates a reversible block in the maturation program, stalling cells in the transition state. This mutation creates an ideal system for dissecting the specific properties of cells undergoing the morphological transition of maturing mesoderm, as we demonstrate with a direct measurement of cell-cell adhesion.

The zebrafish tailbud contains two independent populations of midline progenitor cells that maintain long-term germ layer plasticity and differentiate in response to local signaling cues.

Vertebrate body axis formation depends on a population of bipotential neuromesodermal cells along the posterior wall of the tailbud that make a germ layer decision after gastrulation to form spinal cord and mesoderm. Despite exhibiting germ layer plasticity, these cells never give rise to midline tissues of the notochord, floor plate and dorsal endoderm, raising the question of whether midline tissues also arise from basal posterior progenitors after gastrulation. We show in zebrafish that local posterior signals specify germ layer fate in two basal tailbud midline progenitor populations. Wnt signaling induces notochord within a population of notochord/floor plate bipotential cells through negative transcriptional regulation of sox2. Notch signaling, required for hypochord induction during gastrulation, continues to act in the tailbud to specify hypochord from a notochord/hypochord bipotential cell population. Our results lend strong support to a continuous allocation model of midline tissue formation in zebrafish, and provide an embryological basis for zebrafish and mouse bifurcated notochord phenotypes as well as the rare human congenital split notochord syndrome. We demonstrate developmental equivalency between the tailbud progenitor cell populations. Midline progenitors can be transfated from notochord to somite fate after gastrulation by ectopic expression of msgn1, a master regulator of paraxial mesoderm fate, or if transplanted into the bipotential progenitors that normally give rise to somites. Our results indicate that the entire non-epidermal posterior body is derived from discrete, basal tailbud cell populations. These cells remain receptive to extracellular cues after gastrulation and continue to make basic germ layer decisions. Summary: Progenitor cells that generate the midline tissues of the zebrafish floor plate, notochord, and hypochord make germ layer decisions after gastrulation based on local canonical Wnt and Notch signaling.

FGF and canonical Wnt signaling cooperate to induce paraxial mesoderm from tailbud neuromesodermal progenitors through regulation of a two-step epithelial to mesenchymal transition

Mesoderm induction begins during gastrulation. Recent evidence from several vertebrate species indicates that mesoderm induction continues after gastrulation in neuromesodermal progenitors (NMPs) within the posteriormost embryonic structure, the tailbud. It is unclear to what extent the molecular mechanisms of mesoderm induction are conserved between gastrula and post-gastrula stages of development. Fibroblast growth factor (FGF) signaling is required for mesoderm induction during gastrulation through positive transcriptional regulation of the T-box transcription factor brachyury. We find in zebrafish that FGF is continuously required for paraxial mesoderm (PM) induction in post-gastrula NMPs. FGF signaling represses the NMP markers brachyury (ntla) and sox2 through regulation of tbx16 and msgn1, thereby committing cells to a PM fate. FGF-mediated PM induction in NMPs functions in tight coordination with canonical Wnt signaling during the epithelial to mesenchymal transition (EMT) from NMP to mesodermal progenitor. Wnt signaling initiates EMT, whereas FGF signaling terminates this event. Our results indicate that germ layer induction in the zebrafish tailbud is not a simple continuation of gastrulation events. Summary: FGF signaling is required for post-gastrula paraxial mesoderm induction through a unique mechanism that differs from its role during gastrula stage mesoderm induction.

BMP and FGF signaling interact to pattern mesoderm by controlling basic helix-loop-helix transcription factor activity

The mesodermal germ layer is patterned into mediolateral subtypes by signaling factors including BMP and FGF. How these pathways are integrated to induce specific mediolateral cell fates is not well understood. We used mesoderm derived from post-gastrulation neuromesodermal progenitors (NMPs), which undergo a binary mediolateral patterning decision, as a simplified model to understand how FGF acts together with BMP to impart mediolateral fate. Using zebrafish and mouse NMPs, we identify an evolutionarily conserved mechanism of BMP and FGF-mediated mediolateral mesodermal patterning that occurs through modulation of basic helix-loop-helix (bHLH) transcription factor activity. BMP imparts lateral fate through induction of Id helix loop helix (HLH) proteins, which antagonize bHLH transcription factors, induced by FGF signaling, that specify medial fate. We extend our analysis of zebrafish development to show that bHLH activity is responsible for the mediolateral patterning of the entire mesodermal germ layer.

PS5.85Canonical Wnt signaling and Sox2 establish a lineage primed state in neuromesodermal progenitors

Sox2 is known to play a major role in maintaining neural stem cells in a multipotent state, functioning both as transcriptional activator and repressor. We are interested in understanding how Sox2 can switch between these two functions and, in particular, we want to determine whether this activity change is linked to SUMOylation. We have compared the transcriptional activation ability of wildtype (wt) Sox2 to the activity of a mutant Sox2 construct which should not be SUMOylated (K247R) and to Sox2-SUMO1/2/3 fusion constructs. The activity of wt Sox2 resulted comparable to the one of the K247R, while all the fusion constructs showed reduced activation ability. These results suggest that SUMOylation of Sox2 could cause a loss of its transcriptional activator activity or, potentially, that it causes a switch from activator to repressor function. However, does Sox2 SUMOylation affect Sox2 transcriptional activity in hNSC? In order to answer this question, we performed RNA sequencing on hNSC transfected with wt Sox2, K247R or K247R-SUMO2 fusion construct. We are now analysing the results of this experiment and comparing them with publicly available data from published literature. Only a very small amount of the total Sox2 present within a single cell is expected to be SUMOylated. For this reason, showing that Sox2 is indeed SUMOylated in vivo is a very challenging task. In order achieve this, we are testing different experimental approaches, including subcellular fractionation, protein pull-down followed by western-blot and mass spectrometry. It is known that Sox2 regulates the multipotency of NSC and that cells expressing high levels of Sox2 progress slowly through cell cycle. In order to determine whether SUMOylation plays a role in this regulation, we are performing cell cycle analysis and proliferation assays on hNSC transiently transfected with either Sox2, K247R or K247R-SUMO2.