Chemotaxis or adhesion gradient? Pronephric duct elongation does not depend on distant sources of guidance information

Previously published experimental studies have led to conflicting interpretations concerning the mechanism guiding pronephric duct elongation in the urodele embryo. Although most studies have led to the conclusion that duct migration is directed by an adhesion gradient (haptotaxis), one set of experiments has been interpreted as supporting chemotactic guidance. We have resolved this conflict by conducting grafting experiments in Ambystoma embryos which distinguish between these two possible mechanisms of cell guidance. Our results provide an alternative explanation for the observations originally interpreted as supporting chemotaxis and add further evidence for adhesive guidance.     

Cell migration in the formation of the pronephric duct in Xenopus laevis

To determine if cell migration is involved in the formation of the pronephric duct in Xenopus, we used morphometry, ablation, and videomicroscopy of vitally stained cells to study duct formation. In St 23-24 (Nieuwkoop and Faber, 1956) embryos, a ridge of cells forms caudal to the pronephric rudiment. The ridge lengthens at approximately the same rate as the embryonic trunk from St 23 to St 31. Ablation experiments demonstrated that the ridge constitutes the pronephric duct rudiment (PDR); when the ridge was ablated at St 23-24, little or no duct formation occurred, whereas a duct formed when the pronephric rudiment was ablated and the ridge left intact. Vital dye injections showed that the PDR forms from the intermediate mesoderm ventral to myotomes IV-VIII. From St 29/30 to St 33/34, the PDR actively elongates along the ventral edge of the myotomes as far as myotome XIV, where it joins the cloaca as the pronephric duct. Videomicroscopy of vitally stained cells showed that the PDR elongates throughout its length and does not incorporate additional cells from the mesoderm over which it elongates. The results strengthen the case for a common mode of pronephric duct formation among amphibian species.     

Axolotl pronephric duct migration requires an epidermally derived, laminin 1-containing extracellular matrix and the integrin receptor alpha6beta1

The epidermis overlying the migrating axolotl pronephric duct is known to participate in duct guidance. This epidermis deposits an extracellular matrix onto the migrating duct and its pathway that is a potential source of directional guidance cues. The role of this matrix in pronephric duct guidance was assayed by presenting matrix deposited on microcarriers directly to migrating pronephric ducts in situ. We found that reorientation of extracellular-matrix-bearing carriers prior to their presentation to migrating ducts caused a corresponding reorientation of pronephric duct migration. Subepidermal microinjection of function-blocking antibodies against alpha6 integrin, beta1 integrin or the laminin-1/E8 domain recognized by alpha6beta1 integrin, all of which were detected and localized here, inhibited pronephric duct migration. Moreover, pre-exposure to anti-laminin-1/E8 function-blocking antibody prevented reoriented carriers of epidermally deposited matrix from reorienting pronephric duct migration. These results are incorporated into an integrated model of pronephric duct guidance consistent with all present evidence, proposing roles for the previously implicated glial cell-line derived neurotrophic factor and its receptor as well as for laminin 1 and alpha6beta1 integrin.     

Cranial neural crest cells exhibit directed migration on the pronephric duct pathway: further evidence for an in vivo adhesion gradient

Previous studies on the elongation of the Ambystoma pronephric duct provided evidence that this morphogenetic movement is adhesion directed. Through the use of a simple and rapid grafting technique that enables genetically marked donor and host cells to be distinguished in transplantation experiments, we demonstrate that cranial neural crest cells, which normally migrate concurrently with, but at a distance from, pronephric duct cells, are able to follow the pronephric duct guidance information. Utilizing neural crest cells as probes for adhesive properties of the lateral plate mesoderm, we extend our previous model of the formal properties of the pronephric duct guidance information. We propose that cells of the cranial neural crest, the pronephric duct primordium and the lateral plate mesoderm all exhibit molecular components of at least one shared cell adhesion system.     

Axolotl pronephric duct cell migration is sensitive to phosphatidylinositol-specific phospholipase C

On the basis of its distribution pattern in embryos of the axolotl (Ambystoma mexicanum), we recently identified alkaline phosphatase as a molecule potentially involved in guiding the migration of the pronephric duct. Alkaline phosphatase is a cell surface protein anchored to cell membranes via a covalent linkage to a phosphatidylinositol glycan (PI-G). The enzyme phosphatidylinositol-specific phospholipase C (PIPLC) specifically releases from cell surfaces molecules anchored by the PI-G linkage. In order to test the possibility that a PI-G anchored protein is involved in directing pronephric duct cell migration, PIPLC was applied to axolotl embryos. The enzyme was introduced into embryos through the use of a novel slow-release bead material, hydrolysed polyacrylamide. PIPLC blocked pronephric duct cell migration without interfering with somite fissure formation, a concurrent, neighbouring morphogenetic cell rearrangement which occurs with little if any alkaline phosphatase present. In addition, alkaline phosphatase activity was markedly diminished in the vicinity of the implanted beads. These observations suggest that at least one protein anchored to the cell membrane by a PI-G linkage, possibly alkaline phosphatase, is involved in guiding or promoting pronephric duct cell migration.     

Requirement of Wnt/β-catenin signaling in pronephric kidney development

The pronephric kidney controls water and electrolyte balance during early fish and amphibian embryogenesis. Many Wnt signaling components have been implicated in kidney development. Specifically, in Xenopus pronephric development as well as the murine metanephroi, the secreted glycoprotein Wnt-4 has been shown to be essential for renal tubule formation. Despite the importance of Wnt signals in kidney organogenesis, little is known of the definitive downstream signaling pathway(s) that mediate their effects. Here we report that inhibition of Wnt/β-catenin signaling within the pronephric field of Xenopus results in significant losses to kidney epithelial tubulogenesis with little or no effect on adjoining axis or somite development. We find that the requirement for Wnt/β-catenin signaling extends throughout the pronephric primordium and is essential for the development of proximal and distal tubules of the pronephros as well as for the development of the duct and glomus. Although less pronounced than effects upon later pronephric tubule differentiation, inhibition of the Wnt/β-catenin pathway decreased expression of early pronephric mesenchymal markers indicating it is also needed in early pronephric patterning. We find that upstream inhibition of Wnt/β-catenin signals in zebrafish likewise reduces pronephric epithelial tubulogenesis. We also find that exogenous activation of Wnt/β-catenin signaling within the Xenopus pronephric field results in significant tubulogenic losses. Together, we propose Wnt/β-catenin signaling is required for pronephric tubule, duct and glomus formation in Xenopus laevis, and this requirement is conserved in zebrafish pronephric tubule formation.     

Experimental evidence for a proteinaceous presegmental wave required for morphogenesis of axolotl mesoderm

Mesoderm of axolotl embryos at various developmental stages was briefly exposed to a calcium-free 0.01% trypsin solution by temporary removal of the epidermis. This treatment was found to disrupt somite segmentation in a localized region and the pronephric duct was unable to migrate through this region. The affected area, consisting of 3.91 +/- 1.04 somites, traveled through the embryo in synchrony with, and 3.55 +/- 0.69-somite widths ahead of segmentation. Trypsinization in the presence of 340 microM calcium resulted in normal duct migration while somite segmentation was still affected. These results demonstrate the existence of a trypsin-sensitive region in the somitic mesoderm and the lateral mesoderm of the duct path that travels in advance of somite segmentation and in synchrony with it. In addition, the trypsin sensitivity of the duct path is calcium dependent whereas that of the somitic mesoderm is not.     

Parallel early development of zebrafish interrenal glands and pronephros: differential control by wt1 and ff1b

Steroids are synthesized mainly from the adrenal cortex. Adrenal deficiencies are often associated with problems related to its development, which is not fully understood. To better understand adrenocortical development, we studied zebrafish because of the ease of embryo manipulation. The adrenocortical equivalent in zebrafish is called the interrenal, because it is embedded in the kidney. We find that interrenal development parallels that of the embryonic kidney (pronephros). Primordial interrenal cells first appear as bilateral intermediate mesoderm expressing ff1b in a region ventral to the third somite. These cells then migrate toward the axial midline and fuse together. The pronephric primordia are wt1-expressing cells located next to the interrenal. They also migrate to the axial midline and fuse to become glomeruli at later developmental stages. Our gene knockdown experiments indicate that wt1 is required for its initial restricted expression in pronephric primordia, pronephric cell migration and fusion. wt1 also appears to be involved in interrenal development and ff1b expression. Similarly, ff1b is required for interrenal differentiation and activation of the differentiated gene, cyp11a1. Our results show that the zebrafish interrenal and pronephros are situated close together and go through parallel developmental processes but are governed by different signaling events.     

In vitro analysis of sympathetic neuron differentiation from chick neural crest cells

Sympathetic neuron differentiation was studied using a fluorescence histochemical assay to detect the appearance of cell-bound catecholamines. Results from in vitro organ cultures indicate that chick neural crest cells must interact with both ventral neural tube (defined throughout as the ventral neural tube plus the notochord) and somitic mesenchyme in order to differentiate into sympathoblasts. Somite, ventral neural tube, and crest were cultured transfilter in various combinations to define these tissue interactions more precisely. Results from these experiments indicate that neural crest cells must be contiguous to somite in order to differentiate into sympathoblasts, but ventral neural tube may act across a Millipore filter membrane (type TH, 25 μm thick) either on somite, crest, or both. To distinguish among these possibilities, somite was cultured transfilter to ventral tube for a short period, after which ventral tube was removed and fresh crest was added to the somite. The results from this and other experiments support the hypothesis that the ventral tube does not act directly on crest cells, but elicits a developmental change in somitic mesenchyme, which then promotes sympathoblast differentiation. To study the relationship of nerve growth factor (NGF) to the differentiation of sympathetic neurons, cultures of somite + crest were temporarily exposed transfilter to ventral tube, in the presence or the absence of exogenous NGF. The results of these and other experiments are consistent with the hypothesis that the continued presence of ventral tube is required to ensure the survival of the differentiating sympathetic neurons. With respect to this second function, ventral tube can be replaced by exogenous NGF.     

Fibronectin visualized by scanning electron microscopy immunocytochemistry on the substratum for cell migration in Xenopus laevis gastrulae

In amphibian gastrulae, scanning electron microscopy (SEM) has shown the presence of a network of extracellular fibrils on the inner aspect of the ectoderm layer, which serves as the substratum for migration by the presumptive mesoderm cells. In vitro experiments have shown that the fibril network promotes attachment and migration by mesoderm cells, and probably guides the migration by contact guidance. Filopodia of the migrating cells showed preferential attachment to the fibrils. Use of a colloidal gold probe for SEM immunocytochemistry has shown that fibrils observed by SEM contain fibronectin, probably as a major component. This provides direct evidence that the extracellular matrix containing fibronectin provides the substratum and guides cell migration in morphogenetic movement.     

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.     

Contact behaviour exhibited by migrating neural crest cells in confrontation culture with somitic cells

Summary When grown in confrontation culture on a planar substratum, avian neural crest cells and somite cells display both homotypic and heterotypic contact inhibition of movement as judged by analysis of time-lapse video recordings of locomotory and contact behaviour, and by use of a nuclear overlap assay. It is therefore unlikely that migration of neural crest cells within the embryo, and within embryonic tissues, can be explained on the basis of a lack of contact inhibition. The results are discussed in the general context of cell invasiveness.     

Dynamics of pigment pattern formation in the zebrafish, Brachydanio rerio. III. Effect of anteroposterior location of three-day lateral line melanophores on colonization by the second wave of melanophores

The mode of colonization of the lateral line melanophore band of the zebrafish, Brachydanio rerio, by the second wave of melanophores has been investigated. This stripe forms in two consecutive stages. First, there is an initial migration and reorientation of pigment cells in an anteroposterior wave into the site to form an interrupted stripe. Following this, a round of melanophores differentiates directly at the site and fills in the gaps between the initial cells. An analysis of the distributions of initial and second wave melanophores along the stripe site has shown that both groups of cells are selective as to localization. Initial wave melanophores colonize more anterior somite areas than do second wave melanophores. However, both groups of cells exhibit preferential colonization of the same anterior sites. It is suggested that second wave melanophores attempt to colonize the same somite areas of the stripe as the initial wave of melanophores but are forced to move to more posterior locations due to the presence of initial wave melanophores anteriorly. Observations were also made on later stages of development of the lateral line melanophore band. These melanophores retain the ability to migrate. Some of them reorient out onto the flank and contribute to the juvenile flank pigment pattern.     

Prioritising guidance cues: directional migration induced by substratum contours and electrical gradients is controlled by a rho/cdc42 switch

Coordinated cell migration is a fundamental feature of embryogenesis but the intracellular mechanism by which cells integrate co-existing extracellular cues to yield appropriate vectoral migration is unknown. Cells in the cornea are guided by a naturally occurring DC electric field (EF) (electrotaxis) as they navigate non-planar substrata but the relative potencies of electrotaxis and guidance by substratum shape (contact guidance) have never been determined. We tested the hypothesis that vectoral migration was controlled by selective activation of rac, cdc42 or rho in response to a 150 mV/mm EF or to a series of parallel substratum nanogrooves (NGs) 130 nm deep. EFs and NGs were presented singly or in combination. Electrotaxis of dissociated bovine corneal epithelial cells (CECs) on planar quartz required signalling by cdc42 and rho but not rac. Contact guidance by substratum NGs required rho but not cdc42 or rac activities. When an EF and NGs were superimposed in parallel, cathodal electrotaxis along NGs was enhanced compared to that on planar quartz but when they were superimposed orthogonally (vertical NGs with horizontal EF) cells were recruited from contact guidance to electrotaxis, suggesting that the EF was more potent. However, increasing the EF to 250 mV/mm was insufficient to recruit the majority to electrotaxis. Consistent for the cues in isolation, when an EF (150 mV/mm) and NGs were superimposed orthogonally, rac activity was not essential for either contact guidance or electrotaxis. However, attenuation of cdc42 signalling abolished electrotaxis and enhanced contact guidance relative to controls (no drug), whereas inhibiting rho signalling enhanced electrotaxis and rho stimulation enhanced contact guidance. Our data are consistent with the idea that migrating CECs use a cdc42/rho “switch” to sort vectoral cues, with cdc42 controlling electrotaxis and rho controlling contact guidance.     

Precardiac cell migration: fibronectin localization at mesoderm-endoderm interface during directional movement

The pathway of directional movement of chick precardiac mesoderm cells was studied by indirect immunofluorescence and by scanning electron microscopy. Directional movement of the precardiac cells begins at stage 6 from the lateral sides of the embryo at the level of Hensen's node. The cells move anteriorly in an arc to the embryo's midline. By stage 8 the cells arrive at the lateral sides of the anterior intestinal portal and movement ceases. The interval of this directional movement is approximately 10 hr. During migration the precardiac cells are in close association with the underlying endoderm. As migration proceeds, the cells encounter increasing amounts of fibrils in the substratum at the mesoderm-endoderm interface. Concomitant with increasing fibril formation there is an increase in fibronectin (FN) in the heart-forming region. During stage 5 FN first appears in the lateral heart-forming regions and increases in amount during the period of cell migration. By stage 7 a concentration difference of FN is apparent in the lateral regions with more FN cephalad and decreasing amounts caudad. At stages 7 and 8 large amounts of extracellular FN-associated fibrils are observed at the lateral sides of the anterior intestinal portal where the cells stop moving. The precardiac cells moving into this region are oriented perpendicular to the anterior intestinal portal and in close association with these fibrils. There is no evidence that the fibrillar meshwork forming the substratum of the precardiac mesoderm cells is physically oriented as a guide for directional movement. The correlations between FN distribution at the mesoderm-endoderm interface and directional cell movement suggest that the precardiac cells may migrate by haptotaxis, i.e., by moving along the substratum toward areas of greater adhesiveness.     

Zebrafish Tbx16 regulates intermediate mesoderm cell fate by attenuating Fgf activity

Progenitors of the zebrafish pronephros, red blood and trunk endothelium all originate from the ventral mesoderm and often share lineage with one another, suggesting that their initial patterning is linked. Previous studies have shown that spadetail (spt) mutant embryos, defective in tbx16 gene function, fail to produce red blood cells, but retain the normal number of endothelial and pronephric cells. We report here that spt mutants are deficient in all the types of early blood, have fewer endothelial cells as well as far more pronephric cells compared to wildtype. In vivo cell tracing experiments reveal that blood and endothelium originate in spt mutants almost exclusive from the dorsal mesoderm whereas, pronephros and tail originate from both dorsal and ventral mesoderm. Together these findings suggest possible defects in posterior patterning. In accord with this, gene expression analysis shows that mesodermal derivatives within the trunk and tail of spt mutants have acquired more posterior identity. Secreted signaling molecules belonging to the Fgf, Wnt and Bmp families have been implicated as patterning factors of the posterior mesoderm. Further investigation demonstrates that Fgf and Wnt signaling are elevated throughout the nonaxial region of the spt gastrula. By manipulating Fgf signaling we show that Fgfs both promote pronephric fate and repress blood and endothelial fate. We conclude that Tbx16 plays an important role in regulating the balance of intermediate mesoderm fates by attenuating Fgf activity.     

GDNF and GFRalpha-1 are components of the axolotl pronephric duct guidance system

In mammals, secretion of GDNF by the metanephrogenic mesenchyme is essential for branching morphogenesis of the ureteric bud and, thus, metanephric development. However, the expression pattern of GDNF and its receptor complex-the GPI-linked ligand-binding protein, GFRalpha-1, and the Ret tyrosine kinase signaling protein-indicates that it could operate at early steps in kidney development as well. Furthermore, the developing nephric systems of fish and amphibian embryos express components of the GDNF signaling system even though they do not make a metanephros. We provide evidence that GDNF signaling through GFRalpha-1 is sufficient to direct pathfinding of migrating pronephric duct cells in axolotl embryos by: (1) demonstrating that application of soluble GFRalpha-1 to an embryo lacking all GPI-linked proteins rescues PND migration in a dose-dependent fashion, (2) showing that application of excess soluble GFRalpha-1 to a normal embryo inhibits migration and that inhibition is dependent upon GDNF-binding activity, and (3) showing that the PND will migrate toward a GDNF-soaked bead in vivo, but will fail to migrate when GDNF is applied uniformly to the flank. These data suggest that PND pathfinding is accomplished by migration up a gradient of GDNF.     

The Delayed Entry of Thoracic Neural Crest Cells into the Dorsolateral Path Is a Consequence of the Late Emigration of Melanogenic Neural Crest Cells from the Neural Tube

Neural crest cells migrate along two pathways in the trunk: the ventral path, between the neural tube and somite, and the dorsolateral path, between the somite and overlying ectoderm. In avian embryos, ventral migration precedes dorsolateral migration by nearly 24 h, and the onset of dorsolateral migration coincides with the cessation of ventral migration. Neural crest cells in the ventral path differentiate predominantly as neurons and glial cells of the peripheral nervous system, whereas those in the dorsolateral path give rise to the melanocytes of the skin. Thus, early- and late-migrating neural crest cells exhibit unique morphogenetic behaviors and give rise to different subsets of neural crest derivatives. Here we present evidence that these differences reflect the appearance of specified melanocyte precursors, or melanoblasts, from late- but not early-migrating neural crest cells. We demonstrate that serum from Smyth line (SL) chickens specifically immunolabels melanocyte precursors, or melanoblasts. Using SL serum as a marker, we first detect melanoblasts immediately dorsal and lateral to the neural tube beginning at stage 18, which is prior to the onset of dorsolateral migration. At later stages every neural crest cell in the dorsolateral path is SL-positive, demonstrating that only melanoblasts migrate dorsolaterally. Thus, melanoblast specification precedes dorsolateral migration, and only melanoblasts migrate dorsolaterally at the thoracic level. Together with previous work (Erickson, C. A., and Goins, T. L.,Development121, 915–924, 1995), these data argue that specification as a melanoblast is a prerequisite for dorsolateral migration. This conclusion suggested that the delay in dorsolateral migration (relative to ventral migration) may reflect a delay in the emigration of melanogenic neural crest cells from the neural tube. Several experiments support this hypothesis. There are no melanoblasts in the ventral path, as revealed by the absence of SL-positive cells in the ventral path, and neural crest cells isolated from the ventral path do not give rise to melanocytes when explanted in culture, suggesting that early, ventrally migrating neural crest cells are limited in their ability to differentiate as melanocytes. Similarly, neural crest cells that emigrate from the neural tubein vitroduring the first 6 h fail to give rise to any melanocytes or SL-positive melanoblasts, whereas neural crest cells that emigrate at progressively later times show a dramatic increase in melanogenesis under identical culture conditions. Thus, the timing of dorsolateral migration at the thoracic level is ultimately controlled by the late emigration of melanogenic neural crest cells from the neural tube.