Gene expression dynamics underlying cell fate emergence in 2D micropatterned human embryonic stem cell gastruloids

Human embryonic stem cells cultured in 2D micropatterns with BMP4 differentiate into a radial arrangement of germ layers and extraembryonic cells. Single-cell transcriptomes demonstrate generation of cell types transcriptionally similar to their in vivo counterparts in Carnegie stage 7 human gastrula. Time-course analyses indicate sequential differentiation, where the epiblast arises by 12 h between the prospective ectoderm in the center and the cells initiating differentiation toward extraembryonic fates at the edge. Extraembryonic and mesendoderm precursors arise from the epiblast by 24 h, while nascent mesoderm, endoderm, and primordial germ cell-like cells form by 44 h. Dynamic changes in transcripts encoding signaling components support a BMP, WNT, and Nodal hierarchy underlying germ-layer specification conserved across mammals, and FGF and HIPPO pathways being active throughout differentiation. This work also provides a resource for mining genes and pathways expressed in a stereotyped 2D gastruloid model, common with other species or unique to human gastrulation

Migration of Zebrafish Primordial Germ Cells: A Role for Myosin Contraction and Cytoplasmic Flow

SummaryThe molecular and cellular mechanisms governing cell motility and directed migration in response to the chemokine SDF-1 are largely unknown. Here, we demonstrate that zebrafish primordial germ cells whose migration is guided by SDF-1 generate bleb-like protrusions that are powered by cytoplasmic flow. Protrusions are formed at sites of higher levels of free calcium where activation of myosin contraction occurs. Separation of the acto-myosin cortex from the plasma membrane at these sites is followed by a flow of cytoplasm into the forming bleb. We propose that polarized activation of the receptor CXCR4 leads to a rise in free calcium that in turn activates myosin contraction in the part of the cell responding to higher levels of the ligand SDF-1. The biased formation of new protrusions in a particular region of the cell in response to SDF-1 defines the leading edge and the direction of cell migration

Gα12/13 regulate epiboly by inhibiting E-cadherin activity and modulating the actin cytoskeleton

Epiboly spreads and thins the blastoderm over the yolk cell during zebrafish gastrulation, and involves coordinated movements of several cell layers. Although recent studies have begun to elucidate the processes that underlie these epibolic movements, the cellular and molecular mechanisms involved remain to be fully defined. Here, we show that gastrulae with altered Gα12/13 signaling display delayed epibolic movement of the deep cells, abnormal movement of dorsal forerunner cells, and dissociation of cells from the blastoderm, phenocopying e-cadherin mutants. Biochemical and genetic studies indicate that Gα12/13 regulate epiboly, in part by associating with the cytoplasmic terminus of E-cadherin, and thereby inhibiting E-cadherin activity and cell adhesion. Furthermore, we demonstrate that Gα12/13 modulate epibolic movements of the enveloping layer by regulating actin cytoskeleton organization through a RhoGEF/Rho-dependent pathway. These results provide the first in vivo evidence that Gα12/13 regulate epiboly through two distinct mechanisms: limiting E-cadherin activity and modulating the organization of the actin cytoskeleton

Gene expression time delays & Turing pattern formation systems

The incorporation of time delays can greatly affect the behaviour of partial differential equations and dynamical systems. In addition, there is evidence that time delays in gene expression due to transcription and translation play an important role in the dynamics of cellular systems. In this paper, we investigate the effects of incorporating gene expression time delays into a one-dimensional putative reaction diffusion pattern formation mechanism on both stationary domains and domains with spatially uniform exponential growth. While oscillatory behaviour is rare, we find that the time taken to initiate and stabilise patterns increases dramatically as the time delay is increased. In addition, we observe that on rapidly growing domains the time delay can induce a failure of the Turing instability which cannot be predicted by a naive linear analysis of the underlying equations about the homogeneous steady state. The dramatic lag in the induction of patterning, or even its complete absence on occasions, highlights the importance of considering explicit gene expression time delays in models for cellular reaction diffusion patterning

Characterization of Turing diffusion-driven instability on evolving domains

In this paper we establish a general theoretical framework for Turing diffusion-driven instability for reaction-diffusion systems on time-dependent evolving domains. The main result is that Turing diffusion-driven instability for reaction-diffusion systems on evolving domains is characterised by Lyapunov exponents of the evolution family associated with the linearised system (obtained by linearising the original system along a spatially independent solution). This framework allows for the inclusion of the analysis of the long-time behavior of the solutions of reaction-diffusion systems. Applications to two special types of evolving domains are considered: (i) time-dependent domains which evolve to a final limiting fixed domain and (ii) time-dependent domains which are eventually time periodic. Reaction-diffusion systems have been widely proposed as plausible mechanisms for pattern formation in morphogenesis

Aberrant behaviours of reaction diffusion self-organisation models on growing domains in the presence of gene expression time delays

Turing’s pattern formation mechanism exhibits sensitivity to the details of the initial conditions suggesting that, in isolation, it cannot robustly generate pattern within noisy biological environments. Nonetheless, secondary aspects of developmental self-organisation, such as a growing domain, have been shown to ameliorate this aberrant model behaviour. Furthermore, while in-situ hybridisation reveals the presence of gene expression in developmental processes, the influence of such dynamics on Turing’s model has received limited attention. Here, we novelly focus on the Gierer–Meinhardt reaction diffusion system considering delays due the time taken for gene expression, while incorporating a number of different domain growth profiles to further explore the influence and interplay of domain growth and gene expression on Turing’s mechanism. We find extensive pathological model behaviour, exhibiting one or more of the following: temporal oscillations with no spatial structure, a failure of the Turing instability and an extreme sensitivity to the initial conditions, the growth profile and the duration of gene expression. This deviant behaviour is even more severe than observed in previous studies of Schnakenberg kinetics on exponentially growing domains in the presence of gene expression (Gaffney and Monk in Bull. Math. Biol. 68:99–130, 2006). Our results emphasise that gene expression dynamics induce unrealistic behaviour in Turing’s model for multiple choices of kinetics and thus such aberrant modelling predictions are likely to be generic. They also highlight that domain growth can no longer ameliorate the excessive sensitivity of Turing’s mechanism in the presence of gene expression time delays. The above, extensive, pathologies suggest that, in the presence of gene expression, Turing’s mechanism would generally require a novel and extensive secondary mechanism to control reaction diffusion patterning

Alkylation damage causes MMR-dependent chromosomal instability in vertebrate embryos

SN1-type alkylating agents, like N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU), are potent mutagens. Exposure to alkylating agents gives rise to O6-alkylguanine, a modified base that is recognized by DNA mismatch repair (MMR) proteins but is not repairable, resulting in replication fork stalling and cell death. We used a somatic mutation detection assay to study the in vivo effects of alkylation damage on lethality and mutation frequency in developing zebrafish embryos. Consistent with the damage-sensing role of the MMR system, mutant embryos lacking the MMR enzyme MSH6 displayed lower lethality than wild-type embryos after exposure to ENU and MNU. In line with this, alkylation-induced somatic mutation frequencies were found to be higher in wild-type embryos than in the msh6 loss-of-function mutants. These mutations were found to be chromosomal aberrations that may be caused by chromosomal breaks that arise from stalled replication forks. As these chromosomal breaks arise at replication, they are not expected to be repaired by non-homologous end joining. Indeed, Ku70 loss-of-function mutants were found to be equally sensitive to ENU as wild-type embryos. Taken together, our results suggest that in vivo alkylation damage results in chromosomal instability and cell death due to aberrantly processed MMR-induced stalled replication forks

Kinase-activating and kinase-impaired cardio-facio-cutaneous syndrome alleles have activity during zebrafish development and are sensitive to small molecule inhibitors

The Ras/MAPK pathway is critical for human development and plays a central role in the formation and progression of most cancers. Children born with germ-line mutations in BRAF, MEK1 or MEK2 develop cardio-facio-cutaneous (CFC) syndrome, an autosomal dominant syndrome characterized by a distinctive facial appearance, heart defects, skin and hair abnormalities and mental retardation. CFC syndrome mutations in BRAF promote both kinase-activating and kinase-impaired variants. CFC syndrome has a progressive phenotype, and the availability of clinically active inhibitors of the MAPK pathway prompts the important question as to whether such inhibitors might be therapeutically effective in the treatment of CFC syndrome. To study the developmental effects of CFC mutant alleles in vivo, we have expressed a panel of 28 BRAF and MEK alleles in zebrafish embryos to assess the function of human disease alleles and available chemical inhibitors of this pathway. We find that both kinase-activating and kinase-impaired CFC mutant alleles promote the equivalent developmental outcome when expressed during early development and that treatment of CFC-zebrafish embryos with inhibitors of the FGF-MAPK pathway can restore normal early development. Importantly, we find a developmental window in which treatment with a MEK inhibitor can restore the normal early development of the embryo, without the additional, unwanted developmental effects of the drug

Excess PLAC8 promotes an unconventional ERK2-dependent EMT in colon cancer

The epithelial-to-mesenchymal transition (EMT) transcriptional program is characterized by repression of E-cadherin (CDH1) and induction of N-cadherin (CDH2), and mesenchymal genes like vimentin (VIM). Placenta-specific 8 (PLAC8) has been implicated in colon cancer; however, how PLAC8 contributes to disease is unknown, and endogenous PLAC8 protein has not been studied. We analyzed zebrafish and human tissues and found that endogenous PLAC8 localizes to the apical domain of differentiated intestinal epithelium. Colon cancer cells with elevated PLAC8 levels exhibited EMT features, including increased expression of VIM and zinc finger E-box binding homeobox 1 (ZEB1), aberrant cell motility, and increased invasiveness. In contrast to classical EMT, PLAC8 overexpression reduced cell surface CDH1 and upregulated P-cadherin (CDH3) without affecting CDH2 expression. PLAC8-induced EMT was linked to increased phosphorylated ERK2 (p-ERK2), and ERK2 knockdown restored cell surface CDH1 and suppressed CDH3, VIM, and ZEB1 upregulation. In vitro, PLAC8 directly bound and inactivated the ERK2 phosphatase DUSP6, thereby increasing p-ERK2. In a murine xenograft model, knockdown of endogenous PLAC8 in colon cancer cells resulted in smaller tumors, reduced local invasion, and decreased p-ERK2. Using MultiOmyx, a multiplex immunofluorescence-based methodology, we observed coexpression of cytosolic PLAC8, CDH3, and VIM at the leading edge of a human colorectal tumor, supporting a role for PLAC8 in cancer invasion in vivo

Zebrafish Mutants calamity and catastrophe Define Critical Pathways of Gene–Nutrient Interactions in Developmental Copper Metabolism

Nutrient availability is an important environmental variable during development that has significant effects on the metabolism, health, and viability of an organism. To understand these interactions for the nutrient copper, we used a chemical genetic screen for zebrafish mutants sensitive to developmental copper deficiency. In this screen, we isolated two mutants that define subtleties of copper metabolism. The first contains a viable hypomorphic allele of atp7a and results in a loss of pigmentation when exposed to mild nutritional copper deficiency. This mutant displays incompletely penetrant skeletal defects affected by developmental copper availability. The second carries an inactivating mutation in the vacuolar ATPase that causes punctate melanocytes and embryonic lethality. This mutant, catastrophe, is sensitive to copper deprivation revealing overlap between ion metabolic pathways. Together, the two mutants illustrate the utility of chemical genetic screens in zebrafish to elucidate the interaction of nutrient availability and genetic polymorphisms in cellular metabolism