Stat3/Cdc25a-dependent cell proliferation promotes embryonic axis extension during zebrafish gastrulation

Cell proliferation has generally been considered dispensable for anteroposterior extension of embryonic axis during vertebrate gastrulation. Signal transducer and activator of transcription 3 (Stat3), a conserved controller of cell proliferation, survival and regeneration, is associated with human scoliosis, cancer and Hyper IgE Syndrome. Zebrafish Stat3 was proposed to govern convergence and extension gastrulation movements in part by promoting Wnt/Planar Cell Polarity (PCP) signaling, a conserved regulator of mediolaterally polarized cell behaviors. Here, using zebrafish stat3 null mutants and pharmacological tools, we demonstrate that cell proliferation contributes to anteroposterior embryonic axis extension. Zebrafish embryos lacking maternal and zygotic Stat3 expression exhibit normal convergence movements and planar cell polarity signaling, but transient axis elongation defect due to insufficient number of cells resulting largely from reduced cell proliferation and increased apoptosis. Pharmacologic inhibition of cell proliferation during gastrulation phenocopied axis elongation defects. Stat3 regulates cell proliferation and axis extension in part via upregulation of Cdc25a expression during oogenesis. Accordingly, restoring Cdc25a expression in stat3 mutants partially suppressed cell proliferation and gastrulation defects. During later development, stat3 mutant zebrafish exhibit stunted growth, scoliosis, excessive inflammation, and fail to thrive, affording a genetic tool to study Stat3 function in vertebrate development, regeneration, and disease

Chemokine GPCR signaling inhibits beta-catenin during Zebrafish axis formation

Embryonic axis formation in vertebrates is initiated by the establishment of the dorsal Nieuwkoop blastula organizer, marked by the nuclear accumulation of maternal β-catenin, a transcriptional effector of canonical Wnt signaling. Known regulators of axis specification include the canonical Wnt pathway components that positively or negatively affect β-catenin. An involvement of G-protein coupled receptors (GPCRs) was hypothesized from experiments implicating G proteins and intracellular calcium in axis formation, but such GPCRs have not been identified. Mobilization of intracellular Ca(2+) stores generates Ca(2+) transients in the superficial blastomeres of zebrafish blastulae when the nuclear accumulation of maternal β-catenin marks the formation of the Nieuwkoop organizer. Moreover, intracellular Ca(2+) downstream of non-canonical Wnt ligands was proposed to inhibit β-catenin and axis formation, but mechanisms remain unclear. Here we report a novel function of Ccr7 GPCR and its chemokine ligand Ccl19.1, previously implicated in chemotaxis and other responses of dendritic cells in mammals, as negative regulators of β-catenin and axis formation in zebrafish. We show that interference with the maternally and ubiquitously expressed zebrafish Ccr7 or Ccl19.1 expands the blastula organizer and the dorsoanterior tissues at the expense of the ventroposterior ones. Conversely, Ccr7 or Ccl19.1 overexpression limits axis formation. Epistatic analyses demonstrate that Ccr7 acts downstream of Ccl19.1 ligand and upstream of β-catenin transcriptional targets. Moreover, Ccl19/Ccr7 signaling reduces the level and nuclear accumulation of maternal β-catenin and its axis-inducing activity and can also inhibit the Gsk3β -insensitive form of β-catenin. Mutational and pharmacologic experiments reveal that Ccr7 functions during axis formation as a GPCR to inhibit β-catenin, likely by promoting Ca(2+) transients throughout the blastula. Our study delineates a novel negative, Gsk3β-independent control mechanism of β-catenin and implicates Ccr7 as a long-hypothesized GPCR regulating vertebrate axis formation

MYH3-associated distal arthrogryposis zebrafish model is normalized with para-aminoblebbistatin

Distal arthrogryposis (DA) is group of syndromes characterized by congenital joint contractures. Treatment development is hindered by the lack of vertebrate models. Here, we describe a zebrafish model in which a common MYH3 missense mutation (R672H) was introduced into the orthologous zebrafish gene smyhc1 (slow myosin heavy chain 1) (R673H). We simultaneously created a smyhc1 null allele (smyhc

The cartilage matrisome in adolescent idiopathic scoliosis

The human spinal column is a dynamic, segmented, bony, and cartilaginous structure that protects the neurologic system and simultaneously provides balance and flexibility. Children with developmental disorders that affect the patterning or shape of the spine can be at risk of neurologic and other physiologic dysfunctions. The most common developmental disorder of the spine is scoliosis, a lateral deformity in the shape of the spinal column. Scoliosis may be part of the clinical spectrum that is observed in many developmental disorders, but typically presents as an isolated symptom in otherwise healthy adolescent children. Adolescent idiopathic scoliosis (AIS) has defied understanding in part due to its genetic complexity. Breakthroughs have come from recent genome-wide association studies (GWAS) and next generation sequencing (NGS) of human AIS cohorts, as well as investigations of animal models. These studies have identified genetic associations with determinants of cartilage biogenesis and development of the intervertebral disc (IVD). Current evidence suggests that a fraction of AIS cases may arise from variation in factors involved in the structural integrity and homeostasis of the cartilaginous extracellular matrix (ECM). Here, we review the development of the spine and spinal cartilages, the composition of the cartilage ECM, the so-called "matrisome" and its functions, and the players involved in the genetic architecture of AIS. We also propose a molecular model by which the cartilage matrisome of the IVD contributes to AIS susceptibility

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

Regulator of G Protein Signaling 3 Modulates Wnt5b Calcium Dynamics and Somite Patterning

Vertebrate development requires communication among cells of the embryo in order to define the body axis, and the Wnt-signaling network plays a key role in axis formation as well as in a vast array of other cellular processes. One arm of the Wnt-signaling network, the non-canonical Wnt pathway, mediates intracellular calcium release via activation of heterotrimeric G proteins. Regulator of G protein Signaling (RGS) proteins can accelerate inactivation of G proteins by acting as G protein GTPase-activating proteins (GAPs), however, the possible role of RGS proteins in non-canonical Wnt signaling and development is not known. Here, we identify rgs3 as having an overlapping expression pattern with wnt5b in zebrafish and reveal that individual knockdown of either rgs3 or wnt5b gene function produces similar somite patterning defects. Additionally, we describe endogenous calcium release dynamics in developing zebrafish somites and determine that both rgs3 and wnt5b function are required for appropriate frequency and amplitude of calcium release activity. Using rescue of gene knockdown and in vivo calcium imaging assays, we demonstrate that the activity of Rgs3 requires its ability to interact with Gα subunits and function as a G protein GAP. Thus, Rgs3 function is necessary for appropriate frequency and amplitude of calcium release during somitogenesis and is downstream of Wnt5 activity. These results provide the first evidence for an essential developmental role of RGS proteins in modulating the duration of non-canonical Wnt signaling

Bmp Activity Gradient Regulates Convergent Extension during Zebrafish Gastrulation

AbstractDuring vertebrate gastrulation, a ventral to dorsal gradient of bone morphogenetic protein (Bmp) activity establishes cell fates. Concomitantly, convergent extension movements narrow germ layers mediolaterally while lengthening them anteroposteriorly. Here, by measuring movements of cell populations in vivo, we reveal the presence of three domains of convergent extension movements in zebrafish gastrula. Ventrally, convergence and extension movements are absent. Lateral cell populations converge and extend at increasing speed until they reach the dorsal domain where convergence speed slows but extension remains strong. Using dorsalized and ventralized mutants, we demonstrate that these domains are specified by the Bmp activity gradient. In vivo cell morphology and behavior analyses indicated that low levels of Bmp activity might promote extension with little convergence by allowing mediolateral cell elongation and dorsally biased intercalation. Further, single cell movement analyses revealed that the high ventral levels of Bmp activity promote epibolic migration of cells into the tailbud, increasing tail formation at the expense of head and trunk. We show that high Bmp activity limits convergence and extension by negatively regulating expression of the wnt11 (silberblick) and wnt5a (pipetail) genes, which are required for convergent extension but not cell fate specification. Therefore, during vertebrate gastrulation, a single gradient of Bmp activity, which specifies cell fates, also regulates the morphogenetic process of convergent extension

MZ<i>stat3</i> mutants exhibit transient and mild extension defects in axial mesoderm during gastrulation.

<p>(A) Live images of WT and MZ<i>stat3</i> embryos shown in lateral (a, c) and dorsal view (b). The insets in c show the part of notochord above yolk extension in 30 hpf embryos. Arrowhead, nt, notochord. (B) Morphometric analysis of AP axis extension of 30 hpf embryos shown in A(c). (C) <i>papc</i> in presomitic mesoderm and <i>dlx3b</i> marking neuroectoderm boundary in 1-somite stage WT and MZ<i>stat3</i> embryos (dorsal view). (D) Measurement of ML width of <i>papc</i> expression domain (pink in C). (E) Expression of <i>ntl</i> in notochord and tail in 1-somite stage WT, Z<i>stat3</i>, M<i>stat3</i>, MZ<i>stat3</i>, and MZ<i>stat3</i> embryos overexpressing Stat3-F, as well as WT and MZ<i>stat3</i> embryos injected with 5 ng MO1-<i>stat3</i> (lateral view). Phenol red was used as injection control. (F) Measurement of notochord length in embryos in E (blue lines in E). ****p<0.0001, n.s. = non-significant, error bars = SEM. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006564#pgen.1006564.s004" target="_blank">S3 Fig</a>.</p

Inhibition of cell proliferation using hydroxyurea and aphidicolin leads to axis extension defects in zebrafish gastrulae.

<p>(A) Immunofluorescent anti-pH3 labeling of proliferating cells (red) and total nuclei labeled with DAPI labeling (blue) in DMSO-treated control embryos and hydroxyurea+aphidicolin (H+A)-treated embryos at 6 hpf (animal view) and 10 hpf (dorsal view, anterior to the top). (B-E) Quantification of pH3+ cells (B and D) and DAPI+ cells (C and E) in A. (F and G) Expression of <i>ntl</i> at 1-somite stage (lateral view, anterior to the top). (H) Quantification of notochord length (blue lines in F and G). (I and J) Confocal image of dorsal mesoderm in 3-somite stage embryos expressing mGFP with somite AP dimension illustrated with green arrow, somitic boundaries outlined in green, adaxial cells outlined in orange, adaxial cells and notochord cells between adjacent somitic boundaries numbered in yellow (dorsal view, anterior to the top). (K-M) Quantification of somite AP dimension (K), numbers of adaxial cells (L) and notochord cells (M) in I and J. (N and O) Schema of AP extension of the notochord and presomitic mesoderm. st, somite; nt, notochord. (P-V) Dorsal view showing cells labeled with mGFP in DMSO-treated (P) and drug-treated (Q) embryos at 1-somite stage (anterior to the top). Analyses of notochord cells’ orientation (R), shape (S), long axis (length, T), short axis (width, U) and size (V). ****p<0.0001, n.s. = non-significant, error bars = SEM.</p

Stat3 promotes cell cycle progression during zebrafish embryogenesis.

<p>(A) Immunofluorescent anti-pH3 labeling of proliferating cells (red) and DAPI labeling all nuclei (blue) in WT, M<i>stat3</i>, and MZ<i>stat3</i> embryos at 6 hpf (animal view) and 10 hpf (dorsal view). (B, D) Quantification of mitotic cell number at 6 and 10 hpf. (C, E) Quantification of total cell number at 6 and 10 hpf. (F and G) Average length of each cell cycle (F) and timing of mitosis (G) from Cycle 5 to Cycle 9 in embryos from WT, <i>stat3</i>^{<i>stl27/+</i>}, and <i>stat3</i>^{<i>stl27/+</i>} females. (H-K) Analyses of cell divisions from Cycle 5 to Cycle 9 in 10 pg (H and I) and 25 pg (J and K) <i>stat3-F</i> injected MZ<i>stat3</i> embryos. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s. = non-significant, error bars = SEM. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006564#pgen.1006564.s006" target="_blank">S5 Fig</a>.</p