Signals from trunk paraxial mesoderm induce pronephros formation in chick intermediate mesoderm

We used Pax-2 mRNA expression and Lim 1/2 antibody staining as markers for the conversion of chick intermediate mesoderm (IM) to pronephric tissue and Lmx-1 mRNA expression as a marker for mesonephros. Pronephric markers were strongly expressed caudal to the fifth somite by stage 9. To determine whether the pronephros was induced by adjacent tissues and, if so, to identify the inducing tissues and the timing of induction, we microsurgically dissected one side of chick embryos developing in culture and then incubated them for up to 3 days. The undisturbed contralateral side served as a control. Most embryos cut parallel to the rostrocaudal axis between the trunk paraxial mesoderm and IM before stage 8 developed a pronephros on the control side only. Embryos manipulated after stage 9 developed pronephric structures on both sides, but the caudal pronephric extension was attenuated on the cut side. These results suggest that a medial signal is required for pronephric development and show that the signal is propagated in a rostral to caudal sequence. In manipulated embryos cultured for 3 days in ovo, the mesonephros as well as the pronephros failed to develop on the experimental side. In contrast, embryos cut between the notochord and the trunk paraxial mesoderm formed pronephric structures on both sides, regardless of the stage at which the operation was performed, indicating that the signal arises from the paraxial mesoderm (PM) and not from axial mesoderm. This cut also served as a control for cuts between the PM and the IM and showed that signaling itself was blocked in the former experiments, not the migration of pronephric or mesonephric precursor cells from the primitive streak. Additional control experiments ruled out the need for signals from lateral plate mesoderm, ectoderm, or endoderm. To determine whether the trunk paraxial mesoderm caudal to the fifth somite maintains its inductive capacity in the absence of contact with more rostral tissue, embryos were transected. Those transected below the prospective level of the fifth somite expressed Pax-2 in both the rostral and the caudal isolates, whereas embryos transected rostral to this level expressed Pax-2 in the caudal isolate only. Thus, a rostral signal is not required to establish the normal pattern of Pax-2 expression and pronephros formation. To determine whether paraxial mesoderm is sufficient for pronephros induction, stage 7 or earlier chick lateral plate mesoderm was cocultured with caudal stage 8 or 9 quail somites in collagen gels. Pax-2 was expressed in chick tissues in 21 of 25 embryos. Isochronic transplantation of stage 4 or 5 quail node into caudal chick primitive streak resulted in the generation of ectopic somites. These somites induced ectopic pronephroi in lateral plate mesoderm, and the IM that received signals from both native and ectopic somites formed enlarged pronephroi with increased Pax-2 expression. We conclude that signals from a localized region of the trunk paraxial mesoderm are both required and sufficient for the induction of the pronephros from the chick IM. Studies to identify the molecular nature of the induction are in progress.     

Signals derived from the underlying mesoderm are dispensable for zebrafish neural crest induction

Signals from the non-neural ectoderm, the neural ectoderm, and the underlying mesoderm have all been implicated in the induction of neural crest. Bone morphogenetic protein (BMP) signaling in particular has an important role in this process; however, it is unclear whether this activity of BMP is due to its effects on patterning the underlying mesoderm, to its ability to establish a competent neural plate boundary zone, or to the direct specification of neural crest at intermediate levels of activity within a BMP gradient. We show neural crest induction occurs in zebrafish in the absence of involuted mesoderm, indicating that this tissue and signals derived from it are dispensable for the formation of neural crest. Dorsal-involuted mesoderm is a major source of secreted BMP antagonists, and the activity of BMP signaling is thought to depend on the presence of the opposing activity of these antagonists. We find that the three BMP antagonists known to be expressed during gastrulation in zebrafish, noggin1, follistatin, and chordin, are dispensable for neural crest induction. These results suggest that mechanisms for restricting the spatio-temporal pattern of BMP expression may compensate for the loss of secreted BMP antagonist activity in establishing dorso-ventral patterning, neural induction, and the neural crest.     

Patterns of cell motility in the organizer and dorsal mesoderm of Xenopus laevis.

In a companion paper (Shih, J. and Keller, R. (1992) Development 116, 901-914), we described a sequence of cell behaviors, called mediolateral intercalation behavior (MIB), that produces mediolateral cell intercalation, the process that drives convergence and extension of the axial and paraxial mesoderm of Xenopus. In this paper, we describe the pattern of expression of MIB in the mesoderm during gastrulation, using video image processing and recording of cell behavior in 'shaved', open-faced explants of the marginal zone. At midgastrula stage (10.5), MIB begins at two dorsolateral sites in the prospective anterior mesoderm and progresses medially along two arcs that lengthen toward and meet at the midline to form a single arc of cells expressing MIB, called the vegetal alignment zone (VgAZ). The notochordal-somitic mesodermal boundary forms within the VgAZ at stage 11, and then progresses animally and laterally, along the prospective anterior-posterior axis, eventually bounding a trapezoidal area the shape of the fate-mapped notochord. Meanwhile, from its origin in the VgAZ, MIB spreads in the prospective posterior direction along the lateral boundaries of both the notochordal and somitic mesoderm. From there it spreads medially in both tissues. Subsequently, vacuolation of notochord cells, and segmentation and expression of a somite-specific marker repeat the progression of mediolateral intercalation behavior. Thus cells in the posterior, medial regions of the notochordal and the somitic territories are the last to express mediolateral intercalation behavior and subsequent tissue differentiations. In explants that do not converge, these cells neither express mediolateral intercalation behavior nor differentiate. These facts suggest that progressions of MIB in the anterior-posterior and lateral-medial directions may be organized by signals emanating from the lateral somitic and notochordal boundaries. These signals may have limited range and may be dependent on convergence, driven by mediolateral cell intercalation, to bring cells within their range. In the embryo, the posterior progression of MIB results in arcs of convergence, anchored in the vegetal endoderm at each end, acting on the inside of the blastoporal lip to produce involution of the IMZ.     

Axis-inducing activities and cell fates of the zebrafish organizer

We have investigated axis-inducing activities and cellular fates of the zebrafish organizer using a new method of transplantation that allows the transfer of both deep and superficial organizer tissues. Previous studies have demonstrated that the zebrafish embryonic shield possesses classically defined dorsal organizer activity. When we remove the morphologically defined embryonic shield, embryos recover and are completely normal by 24 hours post-fertilization. We find that removal of the morphological shield does not remove all goosecoid- and floating head-expressing cells, suggesting that the morphological shield does not comprise the entire organizer region. Complete removal of the embryonic shield and adjacent marginal tissue, however, leads to a loss of both prechordal plate and notochord. In addition, these embryos are cyclopean, show a significant loss of floor plate and primary motorneurons and display disrupted somite patterning. Motivated by apparent discrepancies in the literature we sought to test the axis-inducing activity of the embryonic shield. A previous study suggested that the shield is capable of only partial axis induction, specifically being unable to induce the most anterior neural tissues. Contrary to this study, we find shields can induce complete secondary axes when transplanted into host ventral germ-ring. In induced secondary axes donor tissue contributes to notochord, prechordal plate and floor plate. When explanted shields are divided into deep and superficial fragments and separately transplanted we find that deep tissue is able to induce the formation of ectopic axes with heads but lacking posterior tissues. We conclude that the deep tissue included in our transplants is important for proper head formation.     

Xenopus frizzled 4 is a maternal mRNA and its zygotic expression is localized to the neuroectoderm and trunk lateral plate mesoderm

We describe the identification and expression pattern of Xenopus frizzled 4 (Xfz4) gene during early development. Xfz4 protein presents characteristic features of a frizzled family member. The mature protein sequence of Xfz4 is 93% identical to murine Mfz4. Xfz4 is a maternal mRNA, its expression level remains constant during early development. The mRNA is first localized during gastrulation to the dorsal presumptive neuroectoderm. At the end of gastrulation, Xfz4 mRNA is detected in the dorso-anterior neuroectoderm. During neurulation, Xfz4 mRNA is expressed as a band on both side of the forebrain, and in the trunk lateral plate mesoderm. As development proceeds, expression of Xfz4 mRNA in the trunk lateral plate mesoderm decreases but persists in the forebrain. It is also expressed in the posterior unsegmented somitic mesoderm from late tail-bud stage onward.     

Removal of vegetal yolk causes dorsal deficencies and impairs dorsal-inducing ability of the yolk cell in zebrafish.

To examine the nature of cytoplasm determinants for dorsal specification in zebrafish, we have developed a method in which we remove the vegetal yolk hemisphere of early fertilized eggs (vegetal removed embryos). When the vegetal yolk mass was removed at the 1-cell stage, the embryos frequently exhibited typical ventralized phenotypes: no axial structures developed. The frequency of dorsal defects decreased when the operation was performed at later stages. Furthermore, the yolk cell obtained from the vegetal-removed embryos lost the ability to induce goosecoid in normal blastomeres while the normal yolk cell frequently did so in normal and vegetal-removed embryos. These results suggested that the vegetal yolk cell mass contains the dorsal determinants, and that the dorsal-inducing ability of the yolk cell is dependent on the determinants.     

A strategy for immortalizing lines committed to endoderm, neuroectoderm or mesoderm from mouse teratocarcinoma

With the aim of immortalizing embryonic cells fixed at early embryonic stages, various plasmids carrying the SV40 early region were introduced into the mouse embryonal carcinomas (EC) F9 and 1003. Only the construction PK4, in which the SV40 oncogenes are placed under the control of the adenovirus E1A promoter, led to the immortalization of the cells at the onset of differentiation. Clones corresponding to committed precursors of each embryonic lineage (neuroectoderm, mesoderm and endoderm) were then selected with high efficiency according to the following strategy: selection of immature cells which: have lost EC cell markers, keep a stable phenotype, are immortalized by the expression of the SV40 oncogenes and are still able to differentiate along a restricted lineage in vitro or in vivo. Examples of an endodermal precursor (H7) which differentiates into extraembryonic and embryonic endoderm, of a neuroectodermic clone (ICII) committed to a serotoninergic differentiation, and of a mesodermal osteogenic clone (CI) which gives rise to bone in vivo and in vitro, are given.     

Follistatin possesses trunk and tail organizer activity and lacks head organizer activity.

In this report the organizer activity of follistatin was examined by transplantation of pieces of the animal cap, isolated from embryos injected with follistatin mRNA, into the blastocoele of an early host blastula (Einsteck explants). Host embryos developed a secondary axis consisting of myotomes, notochord and neural tube of the trunk or tail character. Secondary structures that are characteristic of a head, such as cement glands or brain and eyes, did not develop in these experiments. These findings suggested that follistatin may have the trunk and tail organizer activity, while it was not possible to reconstitute its head organizer activity.     

Nodal/Bozozok-independent induction of the dorsal organizer by zebrafish cell lines

Formation of the dorsal organizer (Spemann organizer) is an important process in early vertebrate development. In zebrafish, two molecular cascades—Bozozok/Dharma (Boz) and Nodal signaling—act in parallel to induce the dorsal organizer. However, the complete molecular mechanism regulating this event remains unclear. Here we report that zebrafish cell lines derived from various developmental stages can induce a secondary axis when they are implanted into the mid-blastula but not the early gastrula. The implanted cellsthemselves did not differentiate, but instead induced ectopic expression of dorsal organizer markers incells around the implanted cells and induced notochord formation in the secondary axis. These results indicate that cultured cell lines have the ability to induce a secondary axis through the initiation of dorsal organizer activity. However, ectopic expression of boz and sqt were not observed in cultured cells. In addition, implanted cell lines could induce the dorsal organizer even in maternal-zygotic one-eyed pinhead mutants, which are not responsive to Nodal signaling. Finally, the Nodal signaling pathway was not activatedfollowing implantation of cultured cells. Collectively, these data suggest that zebrafish cell lines induce the dorsal organizer independent of the boz and Nodal signaling pathways.     

Collagen XV, a novel factor in zebrafish notochord differentiation and muscle development

Muscle cells are surrounded by extracellular matrix, the components of which play an important role in signalling mechanisms involved in their development. In mice, loss of collagen XV, a component of basement membranes expressed primarily in skeletal muscles, results in a mild skeletal myopathy. We have determined the complete zebrafish collagen XV primary sequence and analysed its expression and function in embryogenesis. During the segmentation period, expression of the Col15a1 gene is mainly found in the notochord and its protein product is deposited exclusively in the peri-notochordal basement membrane. Morpholino mediated knock-down of Col15a1 causes defects in notochord differentiation and in fast and slow muscle formation as shown by persistence of axial mesodermal marker gene expression, disorganization of the peri-notochodal basement membrane and myofibrils, and a U-shape myotome. In addition, the number of medial fast-twitch muscle fibers was substantially increased, suggesting that the signalling by notochord derived Hh proteins is enhanced by loss of collagen XV. Consistent with this, there is a concomitant expansion of patched-1 expression in the myotome of morphant embryos. Together, these results indicate that collagen XV is required for notochord differentiation and muscle development in the zebrafish embryo and that it interplays with Shh signalling.     

Lineage restriction maintains a stable organizer cell population at the zebrafish midbrain-hindbrain boundary

The vertebrate hindbrain is subdivided into segments, termed neuromeres, that are units of gene expression, cell differentiation and behavior. A key property of such segments is that cells show a restricted ability to mix across segment borders -- termed lineage restriction. In order to address segmentation in the midbrain-hindbrain boundary (mhb) region, we have analyzed single cell behavior in the living embryo by acquiring time-lapse movies of the developing mhb region in a transgenic zebrafish line. We traced the movement of hundreds of nuclei, and by matching their position with the expression of a midbrain marker, we demonstrate that midbrain and hindbrain cells arise from two distinct cell populations. Single cell labeling and analysis of the distribution of their progeny shows that lineage restriction is probably established during late gastrulation stages. Our findings suggest that segmentation as an organizing principle in early brain development can be extended to the mhb region. We argue that lineage restriction serves to constrain the position of the mhb organizer cell population.     

Developing reagents and conditions to induce mesoderm subsets from ES cells

Embryonic stem (ES) cell differentiation can serve as a model to investigate early stages of development. Nishikawa and colleagues, in a recent issue of Stem Cells (Era et al., 2007), have used selectable markers to detect lineage-specific gene expression and dissect the induction of mesoderm subsets in ES cell cultures.     

Transient infection of the zebrafish notochord with E. coli induces chronic inflammation

Zebrafish embryos and larvae are now well-established models in which to study infectious diseases. Infections with non-pathogenic Gram-negative Escherichia coli induce a strong and reproducible inflammatory response. Here, we study the cellular response of zebrafish larvae when E. coli bacteria are injected into the notochord and describe the effects. First, we provide direct evidence that the notochord is a unique organ that is inaccessible to leukocytes (macrophages and neutrophils) during the early stages of inflammation. Second, we show that notochord infection induces a host response that is characterised by rapid clearance of the bacteria, strong leukocyte recruitment around the notochord and prolonged inflammation that lasts several days after bacteria clearance. During this inflammatory response, il1b is first expressed in macrophages and subsequently at high levels in neutrophils. Moreover, knock down of il1b alters the recruitment of neutrophils to the notochord, demonstrating the important role of this cytokine in the maintenance of inflammation in the notochord. Eventually, infection of the notochord induces severe defects of the notochord that correlate with neutrophil degranulation occurring around this tissue. This is the first in vivo evidence that neutrophils can degranulate in the absence of a direct encounter with a pathogen. Persistent inflammation, neutrophil infiltration and restructuring of the extracellular matrix are defects that resemble those seen in bone infection and in some chondropathies. As the notochord is a transient embryonic structure that is closely related to cartilage and bone and that contributes to vertebral column formation, we propose infection of the notochord in zebrafish larvae as a new model to study the cellular and molecular mechanisms underlying cartilage and bone inflammation.KEY WORDS: Zebrafish, Neutrophils, Inflammation, Interleukin-1β     

Directional mesoderm cell migration in the Xenopus gastrula.

The movement of the dorsal mesoderm across the blastocoel roof of the Xenopus gastrula is examined. We show that different parts of the mesoderm which can be distinguished by their morphogenetic behavior in the embryo are all able to migrate independently on the inner surface of the blastocoel roof. The direction of mesoderm cell migration is determined by guidance cues in the extracellular matrix of the blastocoel roof and by an intrinsic tissue polarity of the mesoderm. The mesodermal polarity shows the same orientation as the external guidance cues and is strongly expressed in the more posterior mesoderm. The guidance cues of the extracellular matrix are recognized by all parts of the dorsal mesoderm and even by nonmesodermal cells from other regions of the embryo. The extracellular matrix consists of a network of fibronectin-containing fibrils. The adhesiveness of this matrix does not vary along the axis of mesoderm movement, excluding haptotaxis as a guidance mechanism in this system. However, an intact fibronectin fibril structure is necessary for directional mesoderm cell migration. When the assembly of fibronectin into fibrils is inhibited, mesoderm explants still migrate on the amorphous extracellular matrix, but no longer directionally. It is proposed that polarized extracellular matrix fibrils may normally guide the migrating mesoderm to its target region.