Convergence and Extension Movements During Vertebrate Gastrulation,

Chunyue Yin, Brian Ciruna, Lilianna Solnica-Krezel

In: Thomas Lecuit, Editor(s), Current Topics in Developmental Biology, Academic Press, 2009, Volume 89, Pages 163-192, DOI: 10.1016/S0070-2153(09)89007-8.

During vertebrate gastrulation, coordinated cell movements shape the basic body plan. Key components of gastrulation are convergence and extension (C&E) movements, which narrow and lengthen the embryonic tissues, respectively. The rates of C&E movements differ significantly according to the position and the stage of gastrulation. Here, we review the distinct cellular behaviors that define the spatial and temporal patterns of C&E movements, with the special emphasis on zebrafish. We also summarize the molecular regulation of these cellular behaviors and the interplay between different signaling pathways that drive C&E. Finally, to ensure efficient C&E movements, cells must achieve mediolaterally-elongated cell morphology and polarize motile protrusions. We discuss the recent discoveries on the molecular and cellular mechanisms by which the mediolateral cell polarity is established.

Short- and long-range functions of Goosecoid in zebrafish axis formation are independent of Chordin, Noggin 1 and Follistatin-like 1b.

Dixon Fox M, Bruce AE.

Development 136, 1675-1685 (2009) doi: 10.1242/dev.031161

The organizer is essential for dorsal-ventral (DV) patterning in vertebrates. Goosecoid (Gsc), a transcriptional repressor found in the organizer, elicits partial secondary axes when expressed ventrally in Xenopus, similar to an organizer transplant. Although gsc is expressed in all vertebrate organizers examined, knockout studies in mouse suggested that it is not required for DV patterning. Moreover, experiments in Xenopus and zebrafish suggest a role in head formation, although a function in axial mesoderm formation is less clear. To clarify the role of Gsc in vertebrate development, we used gain- and loss-of-function approaches in zebrafish. Ventral injection of low doses of gsc produced incomplete secondary axes, which we propose results from short-range repression of BMP signaling. Higher gsc doses resulted in complete secondary axes and long-range signaling, correlating with repression of BMP and Wnt signals. In striking contrast to Xenopus, the BMP inhibitor Chordin (Chd) is not required for Gsc function. Gsc produced complete secondary axes in chd null mutant embryos and gsc-morpholino knockdown in chd mutants enhanced the mutant phenotype, suggesting that Gsc has Chd-independent functions in DV patterning. Even more striking was that Gsc elicited complete secondary axes in the absence of three secreted BMP antagonists, Chd, Follistatin-like 1b and Noggin 1, suggesting that Gsc functions in parallel with secreted BMP inhibitors. Our findings suggest that Gsc has dose dependent effects on axis induction and provide new insights into molecularly distinct short- and long-range signaling activities of the organizer.

Visualisation and Quantification of Morphogen Gradient Formation in the Zebrafish

Harvey SA, Smith JC (2009)

PLoS Biol 7(5): e1000101 doi:10.1371/journal.pbio.1000101

During embryonic development, signalling molecules known as morphogens act in a concentration-dependent manner to provide positional information to responding tissues. In the early zebrafish embryo, graded signalling by members of the nodal family induces the formation of mesoderm and endoderm, thereby patterning the embryo into three germ layers. Nodal signalling has also been implicated in the establishment of the dorso-ventral axis of the embryo. Although one can infer the existence of nodal gradients by comparing gene expression patterns in wild-type embryos and embryos in which nodal signalling is diminished or augmented, real understanding can only come from directly observing the gradients. One approach is to determine local ligand concentrations in the embryo, but this is technically challenging, and the presence of inhibitors might cause the effective concentration of a ligand to differ from its actual concentration. We have therefore taken two approaches to visualise a direct response to nodal signalling. In the first, we have used transgenic embryos to study the nuclear accumulation of a Smad2-Venus fusion protein, and in the second we have used bimolecular fluorescence complementation to visualise the formation of a complex between Smad2 and Smad4. This has allowed us to visualise, in living embryos, the formation of a graded distribution of nodal signalling activity. We have quantified the formation of the gradient in time and space, and our results not only confirm that nodal signalling patterns the embryo into three germ layers, but also shed light on its role in patterning the dorso-ventral axis and highlight unexpected complexities of mesodermal patterning.

One of the earliest events in vertebrate embryonic development is the patterning of the embryo into three germ layers: the ectoderm, mesoderm, and endoderm. Morphogens are signalling molecules that act in a concentration-dependent manner to induce the formation of different cell types. Members of the nodal family are thought to form a morphogen gradient in the developing zebrafish embryo and to be essential for pattern formation. Mesoderm and endoderm are believed to develop due to high levels of nodal signalling, while cells experiencing the lowest concentrations of nodal signalling become ectoderm. Although this idea is widely accepted, the formation of a nodal morphogen gradient has never been observed directly, and we have therefore used two different approaches to visualise the intensity of nodal signalling within individual cells. Our approaches have allowed us to visualise a gradient of nodal signalling activity in the developing zebrafish embryo. Quantification of the levels of nodal signalling experienced by individual cells confirms that nodal signalling patterns the animal-vegetal axis of the zebrafish embryo and, in contrast to previous studies, also suggests that it plays a role in patterning the dorso-ventral axis of the zebrafish embryo.

Identification of common and unique modifiers of zebrafish midline bifurcation and cyclopia

Wuhong Pei and Benjamin Feldman

Developmental Biology Volume 326, Issue 1, 1 February 2009, Pages 201-211 doi:10.1016/j.ydbio.2008.11.008

Loss of the zebrafish Nodal-related protein Squint causes a spectrum of phenotypes including cyclopia and midline bifurcations (MB). Here we examine MBs and their relation to cyclopia in maternal-zygotic squint (MZsqt) mutants. There is a concordance of MB with cyclopia in MZsqt embryos. Heat treatment and depletion of Hsp90a are “common” risk factors, each of which increases the incidence of both phenotypes. Midline identity is specified on both sides of MBs, and deep-layer cells are initially lacking within bifurcations, whereas enveloping layer cells are intact. Bifurcations do not appear until the completion of gastrulation and are preceded by gaps in the expression of wnt5b, an essential regulator of dorsal convergence. The incidence of early MBs and wnt5b expression defects in heated MZsqt embryos is high, but there is also substantial recovery. Wnt5b depletion increases the incidence of MB, but not cyclopia, and as such Wnt5b is a “unique” risk factor for MB. Reciprocally, depletion of Wnt11 or Hsp90b increases cyclopia only. In summary, we find that MB arises after gastrulation in regions that fail to express wnt5b, and we show that two complex dysmorphologies – MB and cyclopia – can be promoted by either common or unique risk factors.

Cooperation of polarized cell intercalations drives convergence and extension of presomitic mesoderm during zebrafish gastrulation

Chunyue Yin, Maria Kiskowski, Philippe-Alexandre Pouille, Emmanuel Farge, and Lilianna Solnica-Krezel

The Journal of Cell Biology, Vol. 180, No. 1, 221-232 doi:10.1083/jcb.200704150

During vertebrate gastrulation, convergence and extension (C&E) movements narrow and lengthen the embryonic tissues, respectively. In zebrafish, regional differences of C&E movements have been observed; however, the underlying cell behaviors are poorly understood. Using time-lapse analyses and computational modeling, we demonstrate that C&E of the medial presomitic mesoderm is achieved by cooperation of planar and radial cell intercalations. Radial intercalations preferentially separate anterior and posterior neighbors to promote extension. In knypek;trilobite noncanonical Wnt mutants, the frequencies of cell intercalations are altered and the anteroposterior bias of radial intercalations is lost. This provides evidence for noncanonical Wnt signaling polarizing cell movements between different mesodermal cell layers. We further show using fluorescent fusion proteins that during dorsal mesoderm C&E, the noncanonical Wnt component Prickle localizes at the anterior cell edge, whereas Dishevelled is enriched posteriorly. Asymmetrical localization of Prickle and Dishevelled to the opposite cell edges in zebrafish gastrula parallels their distribution in fly, and suggests that noncanonical Wnt signaling defines distinct anterior and posterior cell properties to bias cell intercalations.

Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy

Philipp J. Keller, Annette D. Schmidt, Joachim Wittbrodt, Ernst H. K. Stelzer

Published Online October 9, 2008 Science DOI: 10.1126/science.1162493

A long-standing goal of biology is to map the behavior of all cells during vertebrate embryogenesis. We developed digital scanned laser light sheet fluorescence microscopy and recorded nuclei localization and movement in entire wild-type and mutant zebrafish embryos over the first 24 hours of development. Multiview in vivo imaging at 1.5 billion voxels per minute provides “digital embryos” (i.e., comprehensive databases of cell positions, divisions, and migratory tracks). Our analysis of global cell division patterns reveals a maternally defined initial morphodynamic symmetry break, which identifies the embryonic body axis. We further derive a model of germ layer formation and show that the mesendoderm forms from one-third of the embryo’s cells in a single event. Our digital embryos, with 55 million nucleus entries, are provided as a resource.

In vivo analysis of choroid plexus morphogenesis in zebrafish.

García-Lecea M, Kondrychyn I, Fong SH, Ye ZR, Korzh V.

PLoS ONE. 2008 Sep 1;3(9):e3090.

BACKGROUND: The choroid plexus (ChP), a component of the blood-brain barrier (BBB), produces the cerebrospinal fluid (CSF) and as a result plays a role in (i) protecting and nurturing the brain as well as (ii) in coordinating neuronal migration during neurodevelopment. Until now ChP development was not analyzed in living vertebrates due to technical problems.
METHODOLOGY/PRINCIPAL FINDINGS: We have analyzed the formation of the fourth ventricle ChP of zebrafish in the GFP-tagged enhancer trap transgenic line SqET33-E20 (Gateways) by a combination of in vivo imaging, histology and mutant analysis. This process includes the formation of the tela choroidea (TC), the recruitment of cells from rhombic lips and, finally, the coalescence of TC resulting in formation of ChP. In Notch-deficient mib mutants the first phase of this process is affected with premature GFP expression, deficient cell recruitment into TC and abnormal patterning of ChP. In Hedgehog-deficient smu mutants the second phase of the ChP morphogenesis lacks cell recruitment and TC cells undergo apoptosis.
CONCLUSIONS/SIGNIFICANCE: This study is the first to demonstrate the formation of ChP in vivo revealing a role of Notch and Hedgehog signalling pathways during different developmental phases of this process.

Snrk-1 is involved in multiple steps of angioblast development and acts via notch signaling pathway in artery-vein specification in vertebrates

Chang Z Chun, Sukhbir Kaur, Ganesh V Samant, Ling Wang, Kallal Pramanik, Maija K Garnaas, Keguo Li, Lyndsay Field, Debabrata Mukhopadhyay and Ramani Ramchandran

Prepublished online Aug 22, 2008; doi:10.1182/blood-2008-06-162156

In vertebrates, molecular mechanisms dictate angioblasts’ migration and subsequent differentiation into arteries and veins. In this study, we used a microarray screen to identify a novel member of the sucrose non-fermenting related kinase (snrk-1) family of serine/threonine kinases expressed specifically in the embryonic zebrafish vasculature and investigated its function in vivo. Using gain and loss of function studies in vivo, we show that Snrk-1 plays an essential role in the migration, maintenance and differentiation of angioblasts. The kinase function of Snrk-1 is critical for migration and maintenance, but not for the differentiation of angioblasts. In vitro, snrk-1 knockdown endothelial cells show only defects in migration. The snrk-1 gene acts downstream or parallel to notch and upstream of gridlock during artery-vein specification, and the human gene compensates for zebrafish snrk-1 knockdown, suggesting evolutionary conservation of function.

From the discussion :

Angioblasts require specific molecular cues to target them to the midline, and during migration, they must maintain enough numbers to develop into a mature vessel. Our study implies that Snrk-1 plays a critical role in two steps in this process. In the first step where specified angioblasts migrate to the midline, Snrk-1 and its kinase activity are important for angioblast maintenance and migration. In the second step of angioblast specification to arteries or veins, snrk-1 works upstream of grl and downstream or parallel to notch in the signaling cascade involved in A/V specification. Whether snrk-1 performs similar functions in mammalian development is not known and is currently under active investigation.

Tri-phasic expression of posterior Hox genes during development of pectoral fins in zebrafish: Implications for the evolution of vertebrate paired appendages

Ahn D, Ho RK

Dev Biol (2008 ) Article in press

“D’un coup d’un seul” hein ?

During development of the limbs, Hox genes belonging to the paralogous groups 9–13 are expressed in three distinct phases, which play key roles in the segmental patterning of limb skeletons. In teleost fishes, which have a very different organization in their fin skeletons, it is not clear whether a similar patterning mechanism is at work. To determine whether Hox genes are also expressed in several distinct phases during teleost paired fin development, we re-analyzed the expression patterns of hox9–13 genes during development of pectoral fins in zebrafish. We found that, similar to tetrapod Hox genes, expression of hoxa/d genes in zebrafish pectoral fins occurs in three distinct phases, in which the most distal/third phase is correlated with the development of the most distal structure of the fin, the fin blade. Like in tetrapods, hox gene expression in zebrafish pectoral fins during the distal/third phase is dependent upon sonic hedgehog signaling (hoxa and hoxd genes) and the presence of a long-range enhancer (hoxa genes), which indicates that the regulatory mechanisms underlying tri-phasic expression of Hox genes have remained relatively unchanged during evolution. Our results suggest that, although simpler in organization, teleost fins do have a distal structure that might be considered comparable to the autopod region of limbs.

Liu L, Zhu S, Gong Z, Low BC (2008 ) K-ras/PI3K-Akt Signaling Is Essential for Zebrafish Hematopoiesis and Angiogenesis. PLoS ONE 3(8): e2850. doi:10.1371/journal.pone.0002850

The RAS small GTPases orchestrate multiple cellular processes. Studies on knock-out mice showed the essential and sufficient role of K-RAS, but not N-RAS and H-RAS in embryonic development. However, many physiological functions of K-RAS in vivo remain unclear. Using wild-type and fli1:GFP transgenic zebrafish, we showed that K-ras-knockdown resulted in specific hematopoietic and angiogenic defects, including the impaired expression of erythroid-specific gene gata1 and ße3-hemoglobin, reduced blood circulation and disorganized blood vessels. Expression of either K-rasC40 that links to phosphoinositide 3-kinase (PI3K) activation, or Akt2 that acts downstream of PI3K, could rescue both hematopoietic and angiogenic defects in the K-ras knockdown. Consistently, the functional rescue by k-ras mRNA was significantly suppressed by wortmannin, a PI3K-specific inhibitor. Our results provide direct evidence that PI3K-Akt plays a crucial role in mediating K-ras signaling during hematopoiesis and angiogenesis in vivo, thus offering new targets and alternative vertebrate model for studying these processes and their related diseases.