Ascidian prickle regulates both mediolateral and anterior-posterior cell polarity of notochord cells

The ascidian notochord follows a morphogenetic program that includes convergent extension (C/E), followed by anterior-posterior (A/P) elongation [1-4]. As described here, developing notochord cells show polarity first in the mediolateral (M/L) axis during C/E, and subsequently in the A/P axis during elongation. Previous embryological studies [3] have shown that contact with neighboring tissues is essential for directing M/L polarity of ascidian notochord cells. During C/E, the planar cell polarity (PCP) gene products prickle (pk) and dishevelled (dsh) show M/L polarization. pk and dsh colocalize at the notochord cell membranes, with the exception of those in contact with neighboring muscle cells. In the mutant aimless (aim), which carries a deletion in pk, notochord morphogenesis is disrupted, and cell polarization is lost. After C/E, there is a dynamic relocalization of PCP proteins in the notochord cells with dsh localized to the lateral edges of the membrane, and pk and strabismus (stbm) at the anterior edges. An A/P polarity is present in the extending notochord cells and is evident by the position of the nuclei, which in normal embryos are invariably found at the posterior edge of each cell. In the aim mutant, all appearances of A/P polarity in the notochord are lost.     

Eph regulates dorsoventral asymmetry of the notochord plate and convergent extension-mediated notochord formation

Background

The notochord is a signaling center required for the patterning of the vertebrate embryic midline, however, the molecular and cellular mechanisms involved in the formation of this essential embryonic tissue remain unclear. The urochordate Ciona intestinalis develops a simple notochord from 40 specific postmitotic mesodermal cells. The precursors intercalate mediolaterally and establish a single array of disk-shaped notochord cells along the midline. However, the role that notochord precursor polarization, particularly along the dorsoventral axis, plays in this morphogenetic process remains poorly understood.

Methodology/Principal Findings

Here we show that the notochord preferentially accumulates an apical cell polarity marker, aPKC, ventrally and a basement membrane marker, laminin, dorsally. This asymmetric accumulation of apicobasal cell polarity markers along the embryonic dorsoventral axis was sustained in notochord precursors during convergence and extension. Further, of several members of the Eph gene family implicated in cellular and tissue morphogenesis, only Ci-Eph4 was predominantly expressed in the notochord throughout cell intercalation. Introduction of a dominant-negative Ci-Eph4 to notochord precursors diminished asymmetric accumulation of apicobasal cell polarity markers, leading to defective intercalation. In contrast, misexpression of a dominant-negative mutant of a planar cell polarity gene Dishevelled preserved asymmetric accumulation of aPKC and laminin in notochord precursors, although their intercalation was incomplete.

Conclusions/Significance

Our data support a model in which in ascidian embryos Eph-dependent dorsoventral polarity of notochord precursors plays a crucial role in mediolateral cell intercalation and is required for proper notochord morphogenesis.     

A one-dimensional model of PCP signaling: Polarized cell behavior in the notochord of the ascidian Ciona

Despite its importance in development and physiology the planar cell polarity (PCP) pathway remains one of the most enigmatic signaling mechanisms. The notochord of the ascidian Ciona provides a unique model for investigating the PCP pathway. Interestingly, the notochord appears to be the only embryonic structure in Ciona activating the PCP pathway. Moreover, the Ciona notochord as a single-file array of forty polarized cells is a uniquely tractable system for the study of polarization dynamics and the transmission of the PCP pathway. Here, we test models for propagation of a polarizing signal, interrogating temporal, spatial and signaling requirements. A simple cell–cell relay cascading through the entire length of the notochord is not supported; instead a more complex mechanism is revealed, with interactions influencing polarity between neighboring cells, but not distant ones. Mechanisms coordinating notochord-wide polarity remain elusive, but appear to entrain general (i.e., global) polarity even while local interactions remain important. However, this global polarizer does not appear to act as a localized, spatially-restricted determinant. Coordination of polarity along the long axis of the notochord requires the PCP pathway, a role we demonstrate is temporally distinct from this pathway’s earlier role in convergent extension and intercalation. We also reveal polarity in the notochord to be dynamic: a cell’s polarity state can be changed and then restored, underscoring the Ciona notochord’s amenability for in vivo studies of PCP.     

Chongmague reveals an essential role for laminin-mediated boundary formation in chordate convergence and extension movements

Although cell intercalation driven by non-canonical Wnt/planar cell polarity (PCP) pathway-dependent mediolateral cell polarity is important for notochord morphogenesis, it is likely that multiple mechanisms shape the notochord as it converges and extends. Here we show that the recessive short-tailed Ciona savignyi mutation chongmague (chm) has a novel defect in the formation of a morphological boundary around the developing notochord. chm notochord cells initiate intercalation normally, but then fail to maintain their polarized cell morphology and migrate inappropriately to become dispersed in the larval tail. This is unlike aimless (aim), a mutation in the PCP pathway component Prickle, which has a severe defect in early mediolateral intercalation but forms a robust notochord boundary. Positional cloning identifies chm as a mutation in the C. savignyi ortholog of the vertebrate alpha 3/4/5 family of laminins. Cs-lamalpha3/4/5 is highly expressed in the developing notochord, and Cs-lamalpha3/4/5 protein is specifically localized to the outer border of the notochord. Notochord convergence and extension, reduced but not absent in both chm and aim, are essentially abolished in the aim/aim; chm/chm double mutant, indicating that laminin-mediated boundary formation and PCP-dependent mediolateral intercalation are each able to drive a remarkable degree of tail morphogenesis in the absence of the other. These mechanisms therefore initially act in parallel, but we also find that PCP signaling has an important later role in maintaining the perinotochordal/intranotochordal polarity of Cs-lamalpha3/4/5 localization.     

Cell intercalation during notochord development in Xenopus laevis

Morphometric data from scanning electron micrographs (SEM) of cells in intact embryos and high-resolution time-lapse recordings of cell behavior in cultured explants were used to analyze the cellular events underlying the morphogenesis of the notochord during gastrulation and neurulation of Xenopus laevis. The notochord becomes longer, narrower, and thicker as it changes its shape and arrangement and as more cells are added at the posterior end. The events of notochord development fall into three phases. In the first phase, occurring in the late gastrula, the cells of the notochord become distinct from those of the somitic mesoderm on either side. Boundaries form between the two tissues, as motile activity at the boundary is replaced by stabilizing lamelliform protrusions in the plane of the boundary. In the second phase, spanning the late gastrula and early neurula, cell intercalation causes the notochord to narrow, thicken, and lengthen. Its cells elongate and align mediolaterally as they rearrange. Both protrusive activity and its effectiveness are biased: the anterioposterior (AP) margins of the cells advance and retract but produce much less translocation than the more active left and right ends. The cell surfaces composing the lateral boundaries of the notochord remain inactive. In the last phase, lasting from the mid- to late neurula stage, the increasingly flattened cells spread at all their interior margins, transforming the notochord into a cylindrical structure resembling a stack of pizza slices. The notochord is also lengthened by the addition of cells to its posterior end from the circumblastoporal ring of mesoderm. Our results show that directional cell movements underlie cell intercalation and raise specific questions about the cell polarity, contact behavior, and mechanics underlying these movements. They also demonstrate that the notochord is built by several distinct but carefully coordinated processes, each working within a well-defined geometric and mechanical environment.     

Notochord grafts do not suppress formation of neural crest cells or commissural neurons.

Grafting experiments previously have established that the notochord affects dorsoventral polarity of the neural tube by inducing the formation of ventral structures such as motor neurons and the floor plate. Here, we examine if the notochord inhibits formation of dorsal structures by grafting a notochord within or adjacent to the dorsal neural tube prior to or shortly after tube closure. In all cases, neural crest cells emigrated from the neural tube adjacent to the ectopic notochord. When analyzed at stages after ganglion formation, the dorsal root ganglia appeared reduced in size and shifted in position in embryos receiving grafts. Another dorsal cell type, commissural neurons, identified by CRABP and neurofilament immunoreactivity, differentiated in the vicinity of the ectopic notochord. Numerous neuronal cell bodies and axonal processes were observed within the induced, but not endogenous, floor plate 1 to 2 days after implantation but appeared to be cleared with time. These results suggest that dorsally implanted notochords cannot prevent the formation of neural crest cells or commissural neurons, but can alter the size and position of neural crest-derived dorsal root ganglia.     

Ephrin-Eph signalling drives the asymmetric division of notochord/neural precursors in Ciona embryos

Asymmetric cell divisions produce two sibling cells with distinct fates, providing an important means of generating cell diversity in developing embryos. Many examples of such cell divisions have been described, but so far only a limited number of the underlying mechanisms have been elucidated. Here, we have uncovered a novel mechanism controlling an asymmetric cell division in the ascidian embryo. This division produces one notochord and one neural precursor. Differential activation of extracellular-signal-regulated kinase (ERK) between the sibling cells determines their distinct fates, with ERK activation promoting notochord fate. We first demonstrate that the segregation of notochord and neural fates is an autonomous property of the mother cell and that the mother cell acquires this functional polarity via interactions with neighbouring ectoderm precursors. We show that these cellular interactions are mediated by the ephrin-Eph signalling system, previously implicated in controlling cell movement and adhesion. Disruption of contacts with the signalling cells or inhibition of the ephrin-Eph signal results in the symmetric division of the mother cell, generating two notochord precursors. Finally, we demonstrate that the ephrin-Eph signal acts via attenuation of ERK activation in the neural-fated daughter cell. We propose a model whereby directional ephrin-Eph signals functionally polarise the notochord/neural mother cell, leading to asymmetric modulation of the FGF-Ras-ERK pathway between the daughter cells and, thus, to their differential fate specification.     

The Xenopus receptor tyrosine kinase Xror2 modulates morphogenetic movements of the axial mesoderm and neuroectoderm via Wnt signaling

The Spemann organizer plays a central role in neural induction, patterning of the neuroectoderm and mesoderm, and morphogenetic movements during early embryogenesis. By seeking genes whose expression is activated by the organizer-specific LIM homeobox gene Xlim-1 in Xenopus animal caps, we isolated the receptor tyrosine kinase Xror2. Xror2 is expressed initially in the dorsal marginal zone, then in the notochord and the neuroectoderm posterior to the midbrain-hindbrain boundary. mRNA injection experiments revealed that overexpression of Xror2 inhibits convergent extension of the dorsal mesoderm and neuroectoderm in whole embryos, as well as the elongation of animal caps treated with activin, whereas it does not appear to affect cell differentiation of neural tissue and notochord. Interestingly, mutant constructs in which the kinase domain was point-mutated or deleted (named Xror2-TM) also inhibited convergent extension, and did not counteract the wild-type, suggesting that the ectodomain of Xror2 per se has activities that may be modulated by the intracellular domain. In relation to Wnt signaling for planar cell polarity, we observed: (1) the Frizzled-like domain in the ectodomain is required for the activity of wild-type Xror2 and Xror2-TM; (2) co-expression of Xror2 with Xwnt11, Xfz7, or both, synergistically inhibits convergent extension in embryos; (3) inhibition of elongation by Xror2 in activin-treated animal caps is reversed by co-expression of a dominant negative form of Cdc42 that has been suggested to mediate the planar cell polarity pathway of Wnt; and (4) the ectodomain of Xror2 interacts with Xwnts in co-immunoprecipitation experiments. These results suggest that Xror2 cooperates with Wnts to regulate convergent extension of the axial mesoderm and neuroectoderm by modulating the planar cell polarity pathway of Wnt.     

Wnt5 is required for notochord cell intercalation in the ascidian Halocynthia roretzi

Background information. In the embryos of various animals, the body elongates after gastrulation by morphogenetic movements involving convergent extension. The Wnt/PCP (planar cell polarity) pathway plays roles in this process, particularly mediolateral polarization and intercalation of the embryonic cells. In ascidians, several factors in this pathway, including Wnt5, have been identified and found to be involved in the intercalation process of notochord cells. Results. In the present study, the role of the Wnt5 genes, Hr‐Wnt5α (Halocynthia roretzi Wnt5α) and Hr‐Wnt5β, in convergent extension was investigated in the ascidian H. roretzi by injecting antisense oligonucleotides and mRNAs into single precursor blastomeres of various tissues, including notochord, at the 64‐cell stage. Hr‐Wnt5α is expressed in developing notochord and was essential for notochord morphogenesis. Precise quantitative control of its expression level was crucial for proper cell intercalation. Overexpression of Wnt5 proteins in notochord and other tissues that surround the notochord indicated that Wnt5α plays a role within the notochord, and is unlikely to be the source of polarizing cues arising outside the notochord. Detailed mosaic analysis of the behaviour of individual notochord cells overexpressing Wnt5α indicated that a Wnt5α‐manipulated cell does not affect the behaviour of neighbouring notochord cells, suggesting that Wnt5α works in a cell‐autonomous manner. This is further supported by comparison of the results of Wnt5α and Dsh (Dishevelled) knockdown experiments. In addition, our results suggest that the Wnt/PCP pathway is also involved in mediolateral intercalation of cells of the ventral row of the nerve cord (floor plate) and the endodermal strand. Conclusion. The present study highlights the role of the Wnt5α signal in notochord convergent extension movements in ascidian embryos. Our results raise the novel possibility that Wnt5α functions in a cell‐autonomous manner in activation of the Wnt/PCP pathway to polarize the protrusive activity that drives convergent extension.     

Control of intercalation is cell-autonomous in the notochord of Ciona intestinalis

Dishevelled signaling plays a critical role in the control of cell intercalation during convergent extension in vertebrates. This study presents evidence that Dishevelled serves a similar function in the Ciona notochord. Embryos transgenic for mutant Dishevelled fail to elongate their tails, and notochord cells fail to intercalate, though notochord cell fates are unaffected. Analysis of mosaic transgenics revealed that the effects of mutant Dishevelled on notochord intercalation are cell-autonomous in Ciona, though such defects have nonautonomous effects in Xenopus. Furthermore, our data indicate that notochord cell intercalation in Ciona does not require the progressive signals which coordinate cell intercalation in the Xenopus notochord, highlighting an important difference in how mediolateral cell intercalation is controlled in the two animals. Finally, this study establishes the Ciona embryo as an effective in vivo system for the study of the molecular control of morphogenetic cell movements in chordates.     

WGEF activates Rho in the Wnt-PCP pathway and controls convergent extension in Xenopus gastrulation

The Wnt-PCP (planar cell polarity, PCP) pathway regulates cell polarity and convergent extension movements during axis formation in vertebrates by activation of Rho and Rac, leading to the re-organization of the actin cytoskeleton. Rho and Rac activation require guanine nucleotide-exchange factors (GEFs), but the identity of the GEF involved in Wnt-PCP-mediated convergent extension is unknown. Here we report the identification of the weak-similarity GEF (WGEF) gene by a microarray-based screen for notochord enriched genes, and show that WGEF is involved in Wnt-regulated convergent extension. Overexpression of WGEF activated RhoA and rescued the suppression of convergent extension by dominant-negative Wnt-11, whereas depletion of WGEF led to suppression of convergent extension that could be rescued by RhoA or Rho-associated kinase activation. WGEF protein preferentially localized at the plasma membrane, and Frizzled-7 induced colocalization of Dishevelled and WGEF. WGEF protein can bind to Dishevelled and Daam-1, and deletion of the Dishevelled-binding domain generates a hyperactive from of WGEF. These results indicate that WGEF is a component of the Wnt-PCP pathway that connects Dishevelled to Rho activation.     

FGF8/17/18 functions together with FGF9/16/20 during formation of the notochord in Ciona embryos

Fibroblast growth factor (FGF) signalling has been implicated in the generation of mesoderm and neural fates in chordate embryos including ascidians and vertebrates. In Ciona, FGF9/16/20 has been implicated in both of these processes. However, in FGF9/16/20 knockdown embryos, notochord fate recovers during later development. It is thus not clear if FGF signalling is an essential requirement for notochord specification in Ciona embryos. We show that FGF-MEK-ERK signals act during two distinct phases to establish notochord fate. During the first phase, FGF signalling is required during an asymmetric cell division to promote notochord at the expense of neural identity. Consistently, ERK1/2 is specifically activated in the notochord precursors following this cell division. Sustained activation of ERK1/2 is then required to maintain notochord fate. We demonstrate that FGF9/16/20 acts solely during the initial induction step and that, subsequently, FGF8/17/18 together with FGF9/16/20 is involved in the following maintenance step. These results together with others' show that the formation of a large part of the mesoderm cell types in ascidian larvae is dependent on signalling events involving FGF ligands.     

Cell fate polarization in ascidian mesenchyme/muscle precursors by directed FGF signaling and role for an additional ectodermal FGF antagonizing signal in notochord/nerve cord precursors

Asymmetric cell division plays a fundamental role in generating various types of embryonic cell. In ascidian embryos, asymmetric cell divisions occur in the vegetal hemisphere in a manner similar to those found in Caenorhabditis elegans. Early divisions in embryos of both species involve inductive events on a single mother cell that result in production of daughters with different cell fates. Here we show in the ascidian Halocynthia roretzi that polarity of muscle/mesenchyme mother precursors is determined solely by the direction from which the FGF9/16/20 signal is presented, a role similar to that of Wnt signaling in the EMS and T cell divisions in C. elegans. However, polarity of nerve cord/notochord mother precursors is determined by possible antagonistic action between the FGF signal and a signal from anterior ectoderm, providing a new mechanism underlying asymmetric cell division. The ectoderm signal suppresses MAPK activation and expression of Hr-FoxA, which encodes an intrinsic competence factor for notochord induction, in the nerve cord lineage.     

Chemical genetics suggests a critical role for lysyl oxidase in zebrafish notochord morphogenesis

As a result of a chemical genetic screen for modulators of metalloprotease activity, we report that 2-mercaptopyridine-N-oxide induces a conspicuous undulating notochord defect in zebrafish embryos, a phenocopy of the leviathan mutant. The location of the chemically-induced wavy notochord correlated with the timing of application, thus defining a narrow chemical sensitivity window during segmentation stages. Microscopic observations revealed that notochord undulations appeared during the phase of notochord cell vacuolation and notochord elongation. Notochord cells become swollen as well as disorganized, while electron microscopy revealed disrupted organization of collagen fibrils in the surrounding sheath. We demonstrate by assay in zebrafish extracts that 2-mercaptopyridine-N-oxide inhibits lysyl oxidase. Thus, we provide insight into notochord morphogenesis and reveal novel compounds for lysyl oxidase inhibition. Taken together, these data underline the utility of small molecules for elucidating the dynamic mechanisms of early morphogenesis and provide a potential explanation for the recently established role of copper in zebrafish notochord formation.     

Ciona intestinalis notochord as a new model to investigate the cellular and molecular mechanisms of tubulogenesis

Biological tubes are a prevalent structural design across living organisms. They provide essential functions during the development and adult life of an organism. Increasing progress has been made recently in delineating the cellular and molecular mechanisms underlying tubulogenesis. This review aims to introduce ascidian notochord morphogenesis as an interesting model system to study the cell biology of tube formation, to a wider cell and developmental biology community. We present fundamental morphological and cellular events involved in notochord morphogenesis, compare and contrast them with other more established tubulogenesis model systems, and point out some unique features, including bipolarity of the notochord cells, and using cell shape changes and cell rearrangement to connect lumens. We highlight some initial findings in the molecular mechanisms of notochord morphogenesis. Based on these findings, we present intriguing problems and put forth hypotheses that can be addressed in future studies.     

Tube formation by complex cellular processes in Ciona intestinalis notochord

In the course of embryogenesis multicellular structures and organs are assembled from constituent cells. One structural component common to many organs is the tube, which consists most simply of a luminal space surrounded by a single layer of epithelial cells. The notochord of ascidian Ciona forms a tube consisting of only 40 cells, and serves as a hydrostatic “skeleton” essential for swimming. While the early processes of convergent extension in ascidian notochord development have been extensively studied, the later phases of development, which include lumen formation, have not been well characterized. Here we used molecular markers and confocal imaging to describe tubulogenesis in the developing Ciona notochord. We found that during tubulogenesis each notochord cell established de novo apical domains, and underwent a mesenchymal–epithelial transition to become an unusual epithelial cell with two opposing apical domains. Concomitantly, extracellular luminal matrix was produced and deposited between notochord cells. Subsequently, each notochord cell simultaneously executed two types of crawling movements bi-directionally along the anterior/posterior axis on the inner surface of notochordal sheath. Lamellipodia-like protrusions resulted in cell lengthening along the anterior/posterior axis, while the retraction of trailing edges of the same cell led to the merging of the two apical domains. As a result, the notochord cells acquired endothelial-like shape and formed the wall of the central lumen. Inhibition of actin polymerization prevented the cell movement and tube formation. Ciona notochord tube formation utilized an assortment of common and fundamental cellular processes including cell shape change, apical membrane biogenesis, cell/cell adhesion remodeling, dynamic cell crawling, and lumen matrix secretion.     

Intercellular communication of notochord cells during their differentiation in Cynops orientalis

Intercellular communication of notochord cells during their differentiation was studied by microinjection of a fluorescent dye.Lucifer Yellow,Close correlation existed between the incidences of dye coupling and quantitative evaluation of gap junctions.high incidences of dye coupling and of gap junctions occurred at a stage when notochord cells were active in the change of cell shape and cell arrangement.With the subsidence of cell movements,both dye coupling and gap junctions were reduced to lower levels.It was,therefore,Suggested that intercellular communication via gap junctions played an important role in the coordination of notochord cell movements.Gap Junctions of altered configuration occurred in notochord cells in late taibud stage.The comparison of incidences of dye coupling at this stage with those at other stages strongly suggested that the gap junctions of altered configuration functioned just as those of generalized type.     

Programmed cell death in the ascidian embryo: modulation by FoxA5 and Manx and roles in the evolution of larval development

Programmed cell death (PCD) has been discounted in the ascidian embryo because the descendants of every embryonic cell appear to be present in the tadpole larva. Here we show that apoptotic PCD is initiated in the epidermis and central nervous system (CNS) but not in the endoderm, mesenchyme, muscle, and notochord cells during embryogenesis in molgulid ascidians. However, the affected cells do not actually die until the beginning of metamorphosis. Although specific patterns of PCD were different in distantly related ascidian species, the results suggest that removal of CNS cells by apoptosis is a urchordate feature predating the origin of the vertebrates. Certain molgulid ascidian species have evolved an anural (tailless) larva in which notochord cells fail to undergo the morphogenetic movements culminating in tail development. These anural species include Molgula occulta, the sister species of the urodele (tailed) species Molgula oculata. We show that PCD in the notochord cell lineage precedes the arrest of tail development in M. occulta and other independently evolved anural species. The notochord cells are rescued from PCD and a tail develops in hybrid embryos produced by fertilizing M. occulta eggs with M. oculata sperm, implying that apoptosis is controlled zygotically. Antisense inhibition experiments show that zygotic expression of the FoxA5 and Manx genes is required to prevent notochord PCD in urodele species and hybrids with restored tails. The results provide the first indication of PCD in the ascidian embryo and suggest that apoptosis modulated by FoxA5 and Manx is involved in notochord and tail regression during anural development. Differences in PCD that occur between ascidian species suggest that diversity in programming apoptosis may explain differences in larval form.     

Left-right asymmetry in BMP4 signalling pathway during chick gastrulation

Determination of the left-right (L-R) axis was shown recently to implicate several genes, among which TGFbeta-related molecules such as Activin betaB, lefty1 and 2 and Nodal. We show here that Bmp4 and its signal transduction pathway partners BMPR IA and Smad1 are transiently expressed on the right side of Hensen's node, when L-R polarity is being established. Moreover, Smad1 is expressed asymmetrically in the nascent notochord. These observations suggest a role for a BMP4-dependent autocrine or paracrine mechanism during early L-R determination.     

Tissue interactions affecting the migration and differentiation of neural crest cells in the chick embryo.

A series of microsurgical operations was performed in chick embryos to study the factors that control the polarity, position and differentiation of the sympathetic and dorsal root ganglion cells developing from the neural crest. The neural tube, with or without the notochord, was rotated by 180 degrees dorsoventrally to cause the neural crest cells to emerge ventrally. In some embryos, the notochord was ablated, and in others a second notochord was implanted. Sympathetic differentiation was assessed by catecholamine fluorescence after aldehyde fixation. Neural crest cells emerging from an inverted neural tube migrate in a ventral-to-dorsal direction through the sclerotome, where they become segmented by being restricted to the rostral half of each sclerotome. Both motor axons and neural crest cells avoid the notochord and the extracellular matrix that surrounds it, but motor axons appear also to be attracted to the notochord until they reach its immediate vicinity. The dorsal root ganglia always form adjacent to the neural tube and their dorsoventral orientation follows the direction of migration of the neural crest cells. Differentiation of catecholaminergic cells only occurs near the aorta/mesonephros and in addition requires the proximity of either the ventral neural tube (floor plate/ventral root region) or the notochord. Prior migration of presumptive catecholaminergic cells through the sclerotome, however, is neither required nor sufficient for their adrenergic differentiation.