Hedgehog signaling regulates the amount of hypaxial muscle development during Xenopus myogenesis

Hedgehog (Hh) signaling is proposed to have different roles on differentiation of hypaxial myoblasts of amniotes. Within the somitic environment, Hh signals restrict hypaxial development and promote epaxial muscle formation. On the other hand, in the limb bud, Hh signaling represses hypaxial myoblast differentiation. This poses the question of whether differences in response to Hh signaling are due to variations in local environment or are intrinsic differences between pre- and post-migratory hypaxial myoblasts. We have approached this question by examining the role of Hh signaling on myoblast development in Xenopus laevis, which, due to its unique mode of hypaxial muscle development, allows us to examine myoblast development in vivo in the absence of the limb environment. Cyclopamine and sonic hedgehog (shh) mRNA overexpression were used to inhibit or activate the Hh pathway, respectively. We find that hypaxial myoblasts respond similarly to Hh manipulations regardless of their location, and that this response is the same for epaxial myoblasts. Overexpression of shh mRNA causes a premature differentiation of the dermomyotome, subsequently inhibiting all further growth of the epaxial and hypaxial myotome. Cyclopamine treatment has the opposite effect, causing an increase in dermomyotome and a shift in myoblast fate from epaxial to hypaxial, eventually leading to an excess of hypaxial body wall muscle. Cyclopamine treatment before stage 20 can rescue the effects of shh overexpression, indicating that early Hh signaling plays an essential role in maintaining the balance between epaxial and hypaxial muscle mass. After stage 20, the premature differentiation of the dermomyotome caused by shh overexpression cannot be rescued by cyclopamine, and no further embryonic muscle growth occurs.     

Postembryonic development of the posterior lateral line in zebrafish

We examine how the posterior lateral line of the zebrafish grows and evolves from the simple midbody line present at the end of embryogenesis into the complex adult pattern. Our results suggest that secondary neuromasts do not form through budding from the embryonic line, but rather new waves of neuromasts are added anteroposteriorly. We propose that the developmental module that builds the embryonic pattern of neuromasts is used repeatedly during postembryonic development and that additional (secondary) primordia generate the additional neuromasts. We show that differentiated neuromasts migrate ventrally, and eventually generate "stitches" by successive bisections. We also examine the repatterning of the terminal neuromasts, which anticipates the up-bending of the tail leading to the highly asymmetrical caudal fin of the adult (which develops exclusively from the ventral part of the tail). Because terminal repatterning affects all aspects of tail formation, including its sensory development, we speculate that terminal axis bending may have become intimately associated with the terminal Hox genes before the appearance of the tetrapod lineage.     

Regulation of hypaxial muscle development

The majority of skeletal muscles in higher vertebrates are hypaxial and stem from the lateral lip of the dermomyotomes. Various external signals converge on the dorsolateral quadrant of the somite to specify the hypaxial muscle precursors, to discriminate between migratory and non-migratory cells and to allow delamination of precursors destined for long-range migration. Within the somite, Pax3 acts as upstream regulator of hypaxial muscle development. Downstream targets are cMet and Lbx1, which may independently control identity, differentiation and motility of migratory muscle precursors. Received: 31 August 1998 / Accepted: 20 October 1998     

Chemokine signaling mediates self-organizing tissue migration in the zebrafish lateral line

The shape of most complex organ systems arises from the directed migration of cohesive groups of cells. Here, we dissect the role of the chemokine guidance receptor Cxcr4b in regulating the collective migration of one such cohesive tissue, the zebrafish lateral line primordium. Using in vivo imaging, we show that the shape and organization of the primordium is surprisingly labile, and that internal cell movements are uncoordinated in embryos with reduced Cxcr4b signaling. Genetic mosaic experiments reveal that single cxcr4b mutant cells can migrate in a directional manner when placed in wild-type primordia, but that they are specifically excluded from the leading edge. Moreover, a remarkably small number of SDF1a-responsive cells are able to organize an entire cxcr4b mutant primordium to restore migration and organogenesis in the lateral line. These results reveal a role for chemokine signaling in mediating the self-organizing migration of tissues during morphogenesis.     

Postembryonic development of the posterior lateral line in the zebrafish

SUMMARY The posterior lateral line (PLL) of zebrafish comprises seven to eight sense organs at the end of embryogenesis, arranged in a single antero-posterior line that extends along the horizontal myoseptum from the ear to the tip of the tail. At the end of larval life, four antero-posterior lines extend on the trunk and tail, comprising together around 60 sense organs. The embryonic pattern is largely conserved among teleosts, although adult patterns are very diverse. Here we describe the transition from embryonic to juvenile pattern in the zebrafish, to provide a framework for understanding how the diversity of adult patterns comes about. We show that the four lines that extend over the adult body originate from latent precursors laid down by migrating primordia that arise during embryogenesis. We conclude that, in zebrafish, the entire development of the PLL system up to adulthood can be traced back to events that took place during the first 2 days of life. We also show that the transition from embryonic to adult pattern involves few distinct operations, suggesting that the diversity of patterns among adult teleosts may be due to differential control of these few operations acting upon common embryonic precursors.     

Delta-Notch signaling and lateral inhibition in zebrafish spinal cord development

Background  

Vertebrate neural development requires precise coordination of cell proliferation and cell specification to guide orderly transition of mitotically active precursor cells into different types of post-mitotic neurons and glia. Lateral inhibition, mediated by the Delta-Notch signaling pathway, may provide a mechanism to regulate proliferation and specification in the vertebrate nervous system. We examined delta and notch gene expression in zebrafish embryos and tested the role of lateral inhibition in spinal cord patterning by ablating cells and genetically disrupting Delta-Notch signaling.     

Dynamic Fgf signaling couples morphogenesis and migration in the zebrafish lateral line primordium

The collective migration of cells in the form of cohesive tissues is a hallmark of both morphogenesis and repair. The extrinsic cues that direct these complex migrations usually act by regulating the dynamics of a specific subset of cells, those at the leading edge. Given that normally the function of tissue migration is to lay down multicellular structures, such as branched epithelial networks or sensory organs, it is surprising how little is known about the mechanisms that organize cells behind the leading edge. Cells of the zebrafish lateral line primordium switch from mesenchyme-like leader cells to epithelial rosettes that develop into mechanosensory organs. Here, we show that this transition is regulated by an Fgf signaling circuit that is active within the migrating primordium. Point sources of Fgf ligand drive surrounding cells towards a ;non-leader' fate by increasing their epithelial character, a prerequisite for rosette formation. We demonstrate that the dynamic expression of Fgf ligands determines the spatiotemporal pattern of epithelialization underlying sensory organ formation in the lateral line. Furthermore, this work uncovers a surprising link between internal tissue organization and collective migration.     

Role of zebrafish lbx2 in embryonic lateral line development

Background

The zebrafish ladybird homeobox homologous gene 2 (lbx2) has been suggested to play a key role in the regulation of hypaxial myogenic precursor cell migration. Unlike their lbx counterparts in mammals, the function of teleost lbx genes beyond myogenesis during embryonic development remains unexplored.

Principal Findings

Abrogation of lbx2 function using a specific independent morpholino oligonucleotide (MO) or truncated lbx2 mRNA with an engrailed domain deletion (lbx2^eh-) resulted in defective formation of the zebrafish posterior lateral line (PLL). Migration of the PLL primordium was altered and accompanied by increased cell death in the primordium of lbx2-MO-injected embryos. A decreased number of muscle pioneer cells and impaired expression pattern of sdf1a in the horizontal myoseptum was observed in lbx2 morphants.

Significance

Injection of lbx2 MO or lbx2^eh- mRNA resulted in defective PPL formation and altered sdf1a expression, confirming an important function for lbx2 in sdf1a-dependent migration. In addition, the disassociation of PPL nerve extension with PLL primordial migration in some lbx2 morphants suggests that pathfinding of the PLL primordium and the lateral line nerve may be regulated independently.     

Development of the zebrafish lateral line

The lateral line system is simple (comprising six cell types), its sense organs form according to a defined and reproducible pattern, and its neurons are easily visualized. In the zebrafish, these advantages can be combined with a wealth of genetic tools, making this system ideally suited to a combined molecular, cellular and genetic analysis. Recent progress has taken advantage of these various qualities to elucidate the mechanism that drives the migration from head to tail of the sense organ precursor cells, and to approach the questions surrounding axonal guidance and target recognition.     

Building the posterior lateral line system in zebrafish

The posterior lateral line (pLL) in zebrafish has emerged as an excellent system to study how a sensory organ system develops. Here we review recent studies that illustrate how interactions between multiple signaling pathways coordinate cell fate,morphogenesis, and collective migration of cells in the posterior lateral line primordium. These studies also illustrate how the pLL system is contributing much more broadly to our understanding of mechanisms operating during the growth, regeneration, and self-organization of other organ systems during development and disease.     

Molecular dissection of the migrating posterior lateral line primordium during early development in zebrafish

Background  

Development of the posterior lateral line (PLL) system in zebrafish involves cell migration, proliferation and differentiation of mechanosensory cells. The PLL forms when cranial placodal cells delaminate and become a coherent, migratory primordium that traverses the length of the fish to form this sensory system. As it migrates, the primordium deposits groups of cells called neuromasts, the specialized organs that contain the mechanosensory hair cells. Therefore the primordium provides both a model for studying collective directional cell migration and the differentiation of sensory cells from multipotent progenitor cells.     

Neuronal differences prefigure somatotopy in the zebrafish lateral line

The central projection of the fish lateral line displays somatotopic ordering. In order to know when and how this ordering is established, we have labelled single sensory neurones and followed the growth of their neurites. We show that the neuromast cells and the corresponding neurones are not related by a fixed lineage, and also that somatotopic differences between anterior and posterior line neurones, and among neurones of the posterior line, are present before innervation of the sense organs. We propose that the position of the central projection defines the peripheral position that the neurone will innervate.     

Pattern formation in the lateral line of zebrafish.

The lateral line of fish and amphibians is a sensory system that comprises a number of individual sense organs, the neuromasts, arranged in a defined pattern on the surface of the body. A conspicuous part of the system is a line of organs that extends along each flank (and which gave the system its name). At the end of zebrafish embryogenesis, this line comprises 7-8 neuromasts regularly spaced between the ear and the tip of the tail. The neuromasts are deposited by a migrating primordium that originates from the otic region. Here, we follow the development of this pattern and show that heterogeneities within the migrating primordium prefigure neuromast formation.     

An enhancer trap zebrafish line for lateral line development and regulation of six2b expression

Zebrafish lateral line system which is derived from neurogenic placodes has become a popular model for developmental biology since its formation involves cell migration, pattern formation, organogenesis, and hair cell regeneration. Transgenic lines play a crucial role in lateral line system study. Here, we identified an enhancer trap transgenic zebrafish line Et(gata2a:EGFP)189b (ET189b for short), which expressed enhanced green fluorescent protein (EGFP) in the pituitary, otic, and lateral line placodes and their derivatives. Especially, in neuromast, the accessory cells rather than hair cells were labeled by EGFP. Furthermore, we found the Tol2 transposon construct is integrated at the proximal upstream region of six2b gene locus. And EGFP expression of ET189b closely reflects the expression of endogenous six2b during development and after dkk1b over-expression. Taken together, our results indicated that ET189b is an ideal line for research on lateral line development and regulation of six2b expression.     

Role of lbx2 in the noncanonical Wnt signaling pathway for convergence and extension movements and hypaxial myogenesis in zebrafish

It has been suggested that mouse lbx1 is essential for directing hypaxial myogenic precursor cell migration. In zebrafish, the expression of lbx1a, lbx1b, and lbx2 has been observed in pectoral fin buds. It has also been shown that knocking down endogenous lbx2 in zebrafish embryos diminishes myoD expression in the pectoral fin bud. However, downstream lbxs signals remain largely unexplored. Here, we describe a previously unknown function of zebrafish lbx2 (lbx2) during convergent extension (CE) movements. The abrogation of the lbx2 function by two non-overlapping morpholino oligonucleotides (MOs) resulted in the defective convergence and extension movements in morphants during gastrulation. Our transplantation studies further demonstrated that the overexpression of lbx2 autonomously promotes CE movements. Expression of wnt5b is significantly reduced in lbx2 morphants. We have demonstrated that application of the wnt5b MO, a dominant-negative form of disheveled (Dvl) and a chemical inhibitor of Rho-associated kinase Y27632 in zebrafish embryos have effects reminiscent that are of the CE and hypaxial myogenesis defects observed in lbx2 morphants. Moreover, the CE and hypaxial mesoderm defects seen in lbx2 morphants can be rescued by co-injection with wnt5b or RhoA mRNA. However, this reduced level of active RhoA and hypaxial myogenesis defects in the embryos injected with the dominant-negative form of Dvl mRNA cannot be effectively restored by co-injection with lbx2 mRNA. Our results suggest that the key noncanonical Wnt signaling components Wnt5, Dvl, and RhoA are downstream effectors involved in the regulative roles of lbx2 in CE movement and hypaxial myogenesis during zebrafish embryogenesis.