Gastrulation: making and shaping germ layers.

Gastrulation is a fundamental phase of animal embryogenesis during which germ layers are specified, rearranged, and shaped into a body plan with organ rudiments. Gastrulation involves four evolutionarily conserved morphogenetic movements, each of which results in a specific morphologic transformation. During emboly, mesodermal and endodermal cells become internalized beneath the ectoderm. Epibolic movements spread and thin germ layers. Convergence movements narrow germ layers dorsoventrally, while concurrent extension movements elongate them anteroposteriorly. Each gastrulation movement can be achieved by single or multiple motile cell behaviors, including cell shape changes, directed migration, planar and radial intercalations, and cell divisions. Recent studies delineate cyclical and ratchet-like behaviors of the actomyosin cytoskeleton as a common mechanism underlying various gastrulation cell behaviors. Gastrulation movements are guided by differential cell adhesion, chemotaxis, chemokinesis, and planar polarity. Coordination of gastrulation movements with embryonic polarity involves regulation by anteroposterior and dorsoventral patterning systems of planar polarity signaling, expression of chemokines, and cell adhesion molecules.

Loss of function of FAM177A1, a Golgi complex localized protein, causes a novel neurodevelopmental disorder.

PURPOSE: The function of FAM177A1 and its relationship to human disease is largely unknown. Recent studies have demonstrated FAM177A1 to be a critical immune-associated gene. One previous case study has linked FAM177A1 to a neurodevelopmental disorder in four siblings. METHODS: We identified five individuals from three unrelated families with biallelic variants in FAM177A1. The physiological function of FAM177A1 was studied in a zebrafish model organism and human cell lines with loss-of-function variants similar to the affected cohort. RESULTS: These individuals share a characteristic phenotype defined by macrocephaly, global developmental delay, intellectual disability, seizures, behavioral abnormalities, hypotonia, and gait disturbance. We show that FAM177A1 localizes to the Golgi complex in mammalian and zebrafish cells. Intersection of the RNA-seq and metabolomic datasets from FAM177A1-deficient human fibroblasts and whole zebrafish larvae demonstrated dysregulation of pathways associated with apoptosis, inflammation, and negative regulation of cell proliferation. CONCLUSION: Our data sheds light on the emerging function of FAM177A1 and defines FAM177A1-related neurodevelopmental disorder as a new clinical entity.

Gon4l/Udu regulates cardiomyocyte proliferation and maintenance of ventricular chamber identity during zebrafish development.

Vertebrate heart development requires spatiotemporal regulation of gene expression to specify cardiomyocytes, increase the cardiomyocyte population through proliferation, and to establish and maintain atrial and ventricular cardiac chamber identities. The evolutionarily conserved chromatin factor Gon4-like (Gon4l), encoded by the zebrafish ugly duckling (udu) locus, has previously been implicated in cell proliferation, cell survival, and specification of mesoderm-derived tissues including blood and somites, but its role in heart formation has not been studied. Here we report two distinct roles of Gon4l/Udu in heart development: regulation of cell proliferation and maintenance of ventricular identity. We show that zygotic loss of udu expression causes a significant reduction in cardiomyocyte number at one day post fertilization that becomes exacerbated during later development. We present evidence that the cardiomyocyte deficiency in udu mutants results from reduced cell proliferation, unlike hematopoietic deficiencies attributed to TP53-dependent apoptosis. We also demonstrate that expression of the G1/S-phase cell cycle regulator, cyclin E2 (ccne2), is reduced in udu mutant hearts, and that the Gon4l protein associates with regulatory regions of the ccne2 gene during early embryogenesis. Furthermore, udu mutant hearts exhibit a decrease in the proportion of ventricular cardiomyocytes compared to atrial cardiomyocytes, concomitant with progressive reduction of nkx2.5 expression. We further demonstrate that udu and nkx2.5 interact to maintain the proportion of ventricular cardiomyocytes during development. However, we find that ectopic expression of nkx2.5 is not sufficient to restore ventricular chamber identity suggesting that Gon4l regulates cardiac chamber patterning via multiple pathways. Together, our findings define a novel role for zygotically-expressed Gon4l in coordinating cardiomyocyte proliferation and chamber identity maintenance during cardiac development.

Stat3 Regulates Liver Progenitor Cell-Driven Liver Regeneration in Zebrafish.

After liver injury, regeneration manifests as either (1) hepatocytes proliferating to restore the lost hepatocyte mass or (2) if hepatocyte proliferation is compromised, biliary epithelial cells (BECs) dedifferentiating into liver progenitor cells (LPCs), which subsequently differentiate into hepatocytes. Following pharmacogenetic ablation of hepatocytes in Tg(fabp10a:CFP-NTR) zebrafish, resulting in severe liver injury, signal transducer and activator of transcription 3 (Stat3) and its target gene and negative regulator, socs3a, were upregulated in regenerating livers. Using either Stat3 inhibitors, JSI-124 and S3I-201, or stat3 zebrafish mutants, we investigated the role of Stat3 in LPC-driven liver regeneration. Although Stat3 suppression reduced the size of regenerating livers, BEC dedifferentiation into LPCs was unaffected. However, regenerating livers displayed a delay in LPC-to-hepatocyte differentiation and a significant reduction in the number of BECs. While no difference in cell death was detected, Stat3 inhibition significantly reduced LPC proliferation. Notably, stat3 mutants phenocopied the effects of Stat3 chemical inhibitors, although the mutant phenotype was incompletely penetrant. Intriguingly, a subset of socs3a mutants also displayed a lower number of BECs in regenerating livers. We conclude that the Stat3/Socs3a pathway is necessary for the proper timing of LPC-to-hepatocyte differentiation and establishing the proper number of BECs during LPC-driven liver regeneration.

Intracellular Golgi Complex organization reveals tissue specific polarity during zebrafish embryogenesis.

BACKGROUND: Cell polarity is essential for directed migration of mesenchymal cells and morphogenesis of epithelial tissues. Studies in cultured cells indicate that a condensed Golgi Complex (GC) is essential for directed protein trafficking to establish cell polarity underlying directed cell migration. Dynamic changes of the GC intracellular organization during early vertebrate development remain to be investigated. RESULTS: We used antibody labeling and fusion proteins in vivo to study the organization and intracellular placement of the GC during early zebrafish embryogenesis. We found that the GC was dispersed into several puncta containing cis- and trans-Golgi Complex proteins, presumably ministacks, until the end of the gastrula period. By early segmentation stages, the GC condensed in cells of the notochord, adaxial mesoderm, and neural plate, and its intracellular position became markedly polarized away from borders between these tissues. CONCLUSIONS: We find that GC is dispersed in early zebrafish cells, even when cells are engaged in massive gastrulation movements. The GC accumulates into patches in a stage and cell-type specific manner, and becomes polarized away from borders between the embryonic tissues. With respect to tissue borders, intracellular GC polarity in notochord is independent of mature apical/basal polarity, Wnt/PCP, or signals from adaxial mesoderm. Developmental Dynamics 245:678-691, 2016. © 2016 Wiley Periodicals, Inc.

Wnt/PCP signaling controls intracellular position of MTOCs during gastrulation convergence and extension movements.

During vertebrate gastrulation, convergence and extension cell movements are coordinated with the anteroposterior and mediolateral embryonic axes. Wnt planar cell polarity (Wnt/PCP) signaling polarizes the motile behaviors of cells with respect to the anteroposterior embryonic axis. Understanding how Wnt/PCP signaling mediates convergence and extension (C&E) movements requires analysis of the mechanisms employed to alter cell morphology and behavior with respect to embryonic polarity. Here, we examine the interactions between the microtubule cytoskeleton and Wnt/PCP signaling during zebrafish gastrulation. First, we assessed the location of the centrosome/microtubule organizing center (MTOC) relative to the cell nucleus and the body axes, as a marker of cell polarity. The intracellular position of MTOCs was polarized, perpendicular to the plane of the germ layers, independently of Wnt/PCP signaling. In addition, this position became biased posteriorly and medially within the plane of the germ layers at the transition from mid- to late gastrulation and from slow to fast C&E movements. This depends on intact Wnt/PCP signaling through Knypek (Glypican4/6) and Dishevelled components. Second, we tested whether microtubules are required for planar cell polarization. Once the planar cell polarity is established, microtubules are not required for accumulation of Prickle at the anterior cell edge. However, microtubules are needed for cell-cell contacts and initiation of its anterior localization. Reciprocal interactions occur between Wnt/PCP signaling and microtubule cytoskeleton during C&E gastrulation movements. Wnt/PCP signaling influences the polarity of the microtubule cytoskeleton and, conversely, microtubules are required for the asymmetric distribution of Wnt/PCP pathway components.

Hedgehog signaling induces arterial endothelial cell formation by repressing venous cell fate.

In vertebrate embryos, the dorsal aorta and the posterior cardinal vein form in the trunk to comprise the original circulatory loop. Previous studies implicate Hedgehog (Hh) signaling in the development of the dorsal aorta. However, the mechanism controlling specification of artery versus vein remains unclear. Here, we investigated the cell-autonomous mechanism of Hh signaling in angioblasts (endothelial progenitor cells) during arterial-venous specification utilizing zebrafish mutations in Smoothened (Smo), a G protein-coupled receptor essential for Hh signaling. smo mutants exhibit an absence of the dorsal aorta accompanied by a reciprocal expansion of the posterior cardinal vein. The increased number of venous cells is equivalent to the loss of arterial cells in embryos with loss of Smo function. Activation of Hh signaling expands the arterial cell population at the expense of venous cell fate. Time-lapse imaging reveals two sequential waves of migrating progenitor cells that contribute to the dorsal aorta and the posterior cardinal vein, respectively. Angioblasts deficient in Hh signaling fail to contribute to the arterial wave; instead, they all migrate medially as a single population to form the venous wave. Cell transplantation analyses demonstrate that Smo plays a cell-autonomous role in specifying angioblasts to become arterial cells, and Hh signaling-depleted angioblasts differentiate into venous cells instead. Collectively, these studies suggest that arterial endothelial cells are specified and formed via repressing venous cell fate at the lateral plate mesoderm by Hh signaling during vasculogenesis.

Myristoylated Naked2 antagonizes Wnt-beta-catenin activity by degrading Dishevelled-1 at the plasma membrane.

In Drosophila, naked cuticle is an inducible antagonist of the Wnt-beta-catenin pathway, likely acting at the level of Dishevelled (Dsh/Dvl), an essential component of this pathway. The mechanism by which naked cuticle and its two vertebrate orthologs, Naked1 (NKD1) and Naked2 (NKD2), inhibit Dvl function is unknown. NKD2 is myristoylated, a co-translational modification that leads to its plasma membrane localization. In contrast, myristoylation-deficient G2A NKD2 is cytoplasmic. Herein we show that the ability of Nkd2/NKD2 to antagonize Wnt-beta-catenin activity during zebrafish embryonic development and in mammalian HEK293 cells is myristoylation-dependent. NKD2 and Dvl-1 interact and co-localize at the lateral membrane of polarized epithelial cells. In reciprocal overexpression and siRNA knockdown experiments, NKD2 and Dvl-1 destabilize each other via enhanced polyubiquitylation; this effect is also dependent upon Naked2 myristoylation. Cell fractionation and ubiquitylation assays indicate that endogenous NKD2 interacts with a slower migrating, ubiquitylated form of Dvl-1 in plasma membrane fractions. These results provide a mechanism by which NKD2 antagonizes Wnt signaling: myristoylated NKD2 interacts with Dvl-1 at the plasma membrane, and this interaction leads to their mutual ubiquitin-mediated proteasomal degradation.

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.

A missense variant in SLC39A8 is associated with severe idiopathic scoliosis.

Genetic factors predictive of severe adolescent idiopathic scoliosis (AIS) are largely unknown. To identify genetic variation associated with severe AIS, we performed an exome-wide association study of 457 severe AIS cases and 987 controls. We find a missense SNP in SLC39A8 (p.Ala391Thr, rs13107325) associated with severe AIS (P = 1.60 × 10-7, OR = 2.01, CI = 1.54-2.62). This pleiotropic SNP was previously associated with BMI, blood pressure, cholesterol, and blood manganese level. We replicate the association in a second cohort (841 cases and 1095 controls) resulting in a combined P = 7.02 × 10-14, OR = 1.94, CI = 1.63-2.34. Clinically, the minor allele of rs13107325 is associated with greater spinal curvature, decreased height, increased BMI and lower plasma manganese in our AIS cohort. Functional studies demonstrate reduced manganese influx mediated by the SLC39A8 p.Ala391Thr variant and vertebral abnormalities, impaired growth, and decreased motor activity in slc39a8 mutant zebrafish. Our results suggest the possibility that scoliosis may be amenable to dietary intervention.

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.

Axis formation--beta-catenin catches a Wnt.

In this issue of Cell, the Heasman group implicates Wnt11 as a component of the canonical Wnt signaling pathway that specifies Xenopus laevis axis formation (Tao et al., 2005). This important work not only identifies a long-sought-after dorsalizing factor but also highlights the pivotal role of extracellular cofactors in specifying the activation of either canonical or noncanonical Wnt pathways.

Initiation of convergence and extension movements of lateral mesoderm during zebrafish gastrulation.

Embryonic morphogenesis is accomplished by cellular movements, rearrangements, and cell fate inductions. Vertebrate gastrulation entails morphogenetic processes that generate three germ layers, endoderm, mesoderm, and ectoderm, shaped into head, trunk, and tail. To understand how cell migration mechanistically contributes to tissue shaping during gastrulation, we examined migration of lateral mesoderm in the zebrafish. Our results illustrate that cell behaviors, different from mediolaterally oriented cell intercalation, also promote convergence and extension (C&E). During early gastrulation, upon internalization, individually migrating mesendodermal cells contribute to the elongation of the mesoderm by moving animally, without dorsal movement. Convergence toward dorsal starts later, by 70% epiboly (7.7 hpf). Depending on location along the Animal-Vegetal axis, an animal or vegetal bias is added to the dorsalward movement, so that paths fan out and the lateral mesoderm both converges and extends. Onset of convergence is independent of noncanonical Wnt signaling but is delayed when Stat3 signaling is compromised. To understand which aspects of motility are controlled by guidance cues, we measured turning behavior of lateral mesodermal cells. We show that cells exhibit directional preference, directionally-regulated speed, and turn toward dorsal when off-course. We estimate that ectoderm could supply from a fraction to all the dorsalward displacement seen in mesoderm cells. Using mathematical modeling, we demonstrate that directional preference is sufficient to account for mesoderm convergence and extension, and that, at minimum, two sources of guidance cues could orient cell paths realistically if located in the dorsal midline.

Visualizing morphogenesis in transgenic zebrafish embryos using BODIPY TR methyl ester dye as a vital counterstain for GFP.

Green fluorescent protein (GFP) technology is rapidly advancing the study of morphogenesis, by allowing researchers to specifically focus on a subset of labeled cells within the living embryo. However, when imaging GFP-labeled cells using confocal microscopy, it is often essential to simultaneously visualize all of the cells in the embryo using dual-channel fluorescence to provide an embryological context for the cells expressing GFP. Although various counterstains are available, part of their fluorescence overlaps with the GFP emission spectra, making it difficult to clearly identify the cells expressing GFP. In this study, we report that a new fluorophore, BODIPY TR methyl ester dye, serves as a versatile vital counterstain for visualizing the cellular dynamics of morphogenesis within living GFP transgenic zebrafish embryos. The fluorescence of this photostable synthetic dye is spectrally separate from GFP fluorescence, allowing dual-channel, three-dimensional (3D) and four-dimensional (4D) confocal image data sets of living specimens to be easily acquired. These image data sets can be rendered subsequently into uniquely informative 3D and 4D visualizations using computer-assisted visualization software. We discuss a variety of immediate and potential applications of BODIPY TR methyl ester dye as a vital visualization counterstain for GFP in transgenic zebrafish embryos.