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    Making A Neural Tube - Neural Induction and Neurulation

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    ‘Making a Neural Tube: Neural Induction and Neurulation Raj Ladher and Gary C. Schoenwolf INTRODUCTION As subsequent chapters will deseribe, the vertebrate nervous sys- tem is necessarily complex. However, this belies its humble beginnings, segregating relatively ealy'as a plate of cells in the dora stor ofthe embryo, iermed neural induction, occurs as a result of insiructive cues ‘win ibe enbryo andi sere is hater Oncendie. Once induced, the neural plate, in most vertebrates, rolls into a tube during a process known as neurulation. Tis tube i then later elaborated to form the central nervous system. In this chapter, we deseribe the model for how ectodermal cells become committed t0 a neural fate, and the studies that have led to this model, We will then review the mechanisms by which the induced neural ecto- deri ros up to form the neural tube SETTING THE SCENE In this section, we describe some of the ‘events that occur in embryogenesis prior to neural induction, We also introduce the main vertebrate model organisms used to investigate neural induction, and we discuss their strengths and appropriateness for various types of experimental studies. ‘Model Organisms ur verre model ssa hive ben sed exten inom | ol feeaedafeie Fig. 1). Two of these are classified as lower verthateszebratih and Xenopus and two ate lass fed a higher vertebrates hk and mouse Wo major fl thoes exist between loner and higher versa i ich was developed by higher vertebrate a an adaption I rest es lover versa are nannies hi hgh ‘rebates are amnotes,Seeond tue provth (ie, al dion lle by an incense in tplann teach dug ello “aout comparable othe parental cell in conta lenage, ‘recalls get progressnely sale wih son 8 mia thn morphogenesis in Tower eters, ba play an ee toe in morphogneis of higher vertebrates. nation to tase fifernces twee Tower an highs vere, anos major difference exists among the four model organisms: the Sip between te formate ells ofthe embryo and ter fod Gt Namely. enapudeags coin a lags eal store a {rs With lento ofthe ato for the spel Hast ths yolkisinrporated in the fring laomeres withthe vee tal blastomeres being much larger than, ‘in yee a i abate fia ick Bastoderm forms as» son top of the yolk tas ‘Finally, in the mouse egg, yolk is sparse; rather the embryc. ‘Neural induction, the process by which a subset of the ‘receives its nourishment from the mother, initially by simple ‘cioderm is instructed to follow a pathway leading to the forma- ‘nudity skal rod has been studied in model systems comprising four classes of vertebrates. Despite obvious = “ences in the geomeiry of the embryos of these classes (e.., the (e.g., the ‘carly frog embryo is spherical, whereas the ‘oveteminy, by and large their embryogenesis is comparable, and researchers can use the respective strengths of these models to address experimentally very specifie research questions, By syn- thesizing data that have emerged from these studies, a model has been formulated of how neural tssueis induced. ion an rg eta. Tse densi These diferencesin the amount and distribution of yolk in the ez ofthe four vertebrate models result in very different geometries inthe four organisms. ‘Thus, during the early developmental stages of eleavage, gasiru- Intion, neural induction, and neurulaton, the four model organ- isms appear very different from one another, ‘imechanisms a the tissue, cellular, and moleculat-genetic levels ‘are highly conserved. In Xenopusiand zebrafish, early development is directed ‘ternal products laid down during oogenesis; at the at the Raj Ladher # Laboratory of Sensory Development, RIKEN Center for Developmental Biology, Chuo-Ku, Kobe, Japan. Cay © Schoerwal Department of Neurobiology and Anatomy, and Children’s Health Research Center, University of Utah, Sal Lake City, UT, 84132, Developmental Neurobiology, Ath cet by Mahendra S. Rao and Marcus Jacobson. Kluwer Academic/Plenum Publishers, New York 208.1 2 Chapter 1 + Raj Ladher and Gary C.Schoenwolf mid-blastula transition, or MB, zygotic transcription com ences (Newport and Kirschner, 1982; Kane and Kimmel, 1993). Matetnally provided products are important ‘formation and germ layer identity. In chicks and mice, “MBI or ‘the onset of zygotic transcription, occurs soon after fertilization; thus, the exact role of maternal products in early development haas been difficult to decipher. ‘lag oly of Heiss on he develapmenfthe sap Saber ena te oat por Tg regarding the embryogenesis of the vertebrate nervous system— ‘wiht endl (Spemann and Mangold, 1924, 2001) and the Scmery of the tlt hernia of ea dace (Seat an De Rebar 197; Neuuop, 18 Westen snd ‘Hemmati-Brivanlou, 1999)—were obtained using amphibian embryos. These will be discussed later in tis chapter. The class itself can be split ilo the Anurans (fogs and toads) and the |Urodeles (newts and Salamanders), and despite some differences inthe details oftheir development, the many similarities make it possible to generalize the results and extend them to other onganisms, Although the Anuran, Xenopus is the model most used today, the starting point for most studies was the pivotal ‘work performed in Urodeles by Spemann and Mangold in the course of discovering the organizer (Spemann and Mangold, 2001). For a summary of the differences between Anurans and Urodeles, soe the excellent review by Malacinski et ai, (1997) For a schematic view of key phases of early Xenopus development, see Fig. 2. ‘The amphibian embryo is large, easily obtained, readily ‘accessible, and easily cultured in-a simple salt souton. As all AS all cells ofthe embryo havea store of yok, pieces ofthe embryo and even single cells fom the early embryo (ie, (aiodnsnésisiin setahme nese A recent advantage in the use of| Xenopus is the ability to overexpress molecules of interest) Because early blastomeres are lrg, itis a simple matter to make RNA corresponding to a gene of interest and inject it into selected cells. The injected RNA is translated at high efficiency Be A & c { FIGURE 1. Photopraphs showing the locations ofthe neurestoerm a nerula sages in (A) Xenopus (ors view, immunohistoehemisty foe N-CAM at stage 15; couresyof Yoshiki Saal (B) zbraish (dorsal view n stu hybridization for So tal bu sage; couresyof Luca Caneparo and Covinne Howat); (C) cick (dora view in sition for Sbx-2at tage 6; courtesy of Susan Chapman}; and (D) mee (dorsolateral view st bybridiza tion for Son-Pat 8S dpe; courtesy of Ryan Anderson, Shannon Davis, and John Klingensmith). x é oe ip Oo c - D FIGURE 2. Ximopus development leading upto neurlation. Diagrams of embryos atthe (A) morula, (B) Bastla,(C) gastos, and (D) nourua stags of dovelopmea. Once the ees fertlizd. lenge occur, with the call ofthe animal Bemisphee darker and smaller han cc ofthe vegstal hemisphere, Atblastla tages, mesoderm is induce. In pateulat, dorsal mesoderm is speified and al gatola stages, this mesoderm stats ovo, forming the dor sal blastopeal lip and marking the site ofthe organizer. The osanzer induces neural tissu in tbe relying animal hemisphere. ap, animal poe; bl oral blastoporl ip mp, neal plate yp, vewtal pole. Modified from Nieuwkoop and Faber (1967), and is active, Indeed this technique has been used not only to assay a whole molecule, but also modified (ie, systematically and selectively mutated) versions of the gene. ‘As most developmental biology research in amphibians is performed on the Xenopusembryo, we will onsider its develop iment. Smith (1989) provides an excellent synthesis of the ‘embryological evens that occur prior to neural induction, ‘The Xenopustegg has an animal-veretal polar with the darker (ie, more heavily pigmented) animal hemisphere forming the ectoderm and mesoderm, and the lighter vegetal, yolktich ‘hemisphere forming the endoderm. ‘ional asymmetry on the egg, with the sperm entering the animal ‘seinasitmiiaThe sperm enlty point also determines the direction of rotation ofthe cortex of the egg in relation tothe eore eyto- plain, an this activates a spevific pathway leading ultimately to ‘the establishment of the dorsal pole of the embryo (Vincent and Gerhart, 1987; Moon and Kimelman, 1998). Specifically, the region ofthe vegetal hemisphere, the Nieuwkoop center, which is diametrically opposite the sperm entry point, is now conferred with the ability to induce the Spemann organizer in the adjacent animal hemisphere (Boterenbrood and Nieuwkoop, 1973). = ‘Spemann organizer has the ability to induce dorsal mesoderm and itern the rest ofthe mesoderm, as well as to direct the forma: ‘n(Geezuidn adele stg imlich and Cooke, 1983; Jacobson, 1984; see below and Box 1). Following fertilization, mesoderm is induced in the equa ‘orial region of the embryo, atthe junetion between the animal. ahha) Rg talan(Nieuwkoop, 1969). Amazingly, (has been experimentally recreated to great eect in later assays) {for both mesoderm-indusing signals and nearal-inducing signals) ‘When challenged with the appropriate signal, an isolated piece of Xenopus animal tissue, which would normally form epidermal Making a Neural Tube * Chaper 1 3 structures, will change its fate aceordingly This animal cap assay has, for years, provided researchers with @ powerful assay for induction. One important caveat must be noted here though. Barth (1941) found that the Ambystoma mexicanunand Rana pipiens, amongst others, auton ‘neuralizes: that is, the removal of the presumptive epidermis ‘from its normal environment actually changes is fate to neural, result supported and extended by Foltfieter (1944), who among other things showed that neural induction could oeeur even after the inducer had been killed (Holifteter, 1947) Ths result eould only be contextualized yeas later when the pathway for neural induction was worked out (see below). It should be noted here, however, that th eno} uio-neutalization; indeed as we discuss below, the Xeno animal eap is resistant to nonspecific neural induction by diverse agents (Kininer and Melton, 1987). This resistance to non- ‘specific neural induction strengthened the role of Xenopus ‘embryos inthe search for inducing signals. "Neural induction occurs dung he proses of gasulton ‘when the mesoderm and endoderm invaginate through the: ‘blastopore and, via a set of complex morphological movements: (see Keller and Winklbauer, 1992, for details ofthis process), are internalized. This results in the ectoderm remaining on’ the surface and forming the crust, and the mesoderm and endoderm coming to lie deep to the ectoderm, forming the core. A fuller description of neural induction is given below. he Zebrafish Embry “Tivo large-Seale ihutagenesis sereens propelled the zebra> {is embryo fo the foreront of developmental biology (Mullin (Mullins and Nusslein-Volhard, 1993; Driever, 1995). The combination of “The diseovery of th organize in 1924s one of the major milestones in developmental biology. This discovery has hada majr influence on our thinking about the mechanisms underlying nual induction (Speman and Mangold, 1924). The German sientiss, Hans Spemann and Hilda Mangold discovered that ezion ofthe amphibian gastul, the dorsal Tip of the blastopore, had the ability to direct formation ofthe neural plate (Fig, 3). By transplanting the dorsal ip from a donor embryo to the vent sie ofa host embryo, they found tha soeon axis can be initiated. The experiment was performed using salamander embryos not Xenopus, the current fvorite amphibian model. By using two species of salamander, one pigmented and the other unpigmented, Speman and Mango could identify which structures in the duplicated axis were derived from the donor and which were dived from the host. Careful analysis showed that whereas the sseondary notochord. and pars ofthe somites were derived from the donor dora lp, the neural plate and other regions of the somites within the secondary axis were ‘derived fom the host, As host tissues should have been fated to frm ‘ventral derivatives, such as lateral mestvdem and epidermal ectoderm, ‘Speman and Mangold reasoned thatthe ation of the donor dona t= ‘tc was not autonomous, and tat a nonautonomous action ined the surrounding tissues to take ona dorsal fate. By using a classical define ition of the word “induction"—the action of one Hssue on another to BOX 1. The Organizer change the latter’ fate, Spemann and Mangold defined new indetion in vertebrate embryos and localized its center of aetvty. As mentioned above, the action of an organizer is not just imited to amphibian embryos. A large number of studies have extended the Findings of Spemann and Mangold to embryos ofthe fish, bir, and ‘mammal (Waddington, 1934; Oppenheimer, 1936; Beddington, 1994; Fig. 38), Allof these studies have found that the organizer can induce the formation of @ sseondary axis. However, inthe mouse, there is an important diffrence. Whereas inthe fish, frog, and chick, trans- plantation of the organizer ean induce a secondary axis with all rostrocaudal levels (ie, from the forebrain to the caudal spinal cond), transplantation of the node in the mouse can induce only a super: rnumerary axis that begins rostral at the vel of the hindbrain (Beddington, 1994; Tam and Steiner, 199). Tis has led othe iden- {ifeation of @ second organizing centr, the anterior visceral endo- plex. The phosphorylated R-Smad can now associate withthe sceond class of Smads, the Co-Sma, wsually Sma, or additionally in Xenopus, Smadi0. The R-Smad'Co-Smad complex results in the BOX 2. The BMP Signaling Pathway nuclear translocation ofthese molecules (Lagnae al, 1996). Once in the eytoplasm, the Smads complex ats as cooeinatots fr the assem by ofa numberof temsription factors andl thereby modulates the tre sripion of spevifie gone. ‘The BMP signal trnsduction pathway is also subject to intr cellular antagonism, an aspect that provides negative feedback for BMP activity. As wel asthe R-Smads that are responsible fr activa ing BMP responsive genes, there are at last two inhibitory Smads (Smads), Smad6 and Smad, which associate with the type 1 oe=p- tor to prevent the binding ofthe RSmad/SARA complex (namara etal, 1997; Tsuneizumiet al, 1997; Inoue et al, 1998; Souchelnyskyi ‘etal, 1998) Itscems that the expression of [Smad is induced by BMP. activity itself (Nakaoer al, 1997; Afrakhteer a, 1998). Another intra cllular inhibitor is BMP and Activin Membrane Bound Inibitor (BAMBI, BAMBI shows considerable sequence homology to the BMP receptors, but lacks the intacelislar kis domain, making ita naturally oscurring dominant negative receptor (Onichichouk ef al. 1989), Homologues have been identified in mouse (Grotewold etal, 2001), humans (Degenet a, 1996), and zebrafish (Tsanget al. 2000), ‘The expression patter corelates well with the expression of BMP-2 and BMP-4, and indesd BAMBI is induced by BMP-4 expression and is lst n zebrafish mutant for brp-2 (Tsang eto, 2000). Another feature ofthe BMP pathway is its ability to intersect with ‘other signaling pathways (von Bubnoff and Cho, 2001) Particularly pertinent t this consideration of neural induction is the interaction, within the cel, with signaling from the fibroblast growth facto (FGF) {amily of molecules and the winglesstwnt group. Both can negatively influence BMP activity, and this is particularly germane tothe role of| these factors inthe induction ofthe nervous system in ammites. 10 Chapter 1 + Raj Ladher and Gary C.Schoenwolf oo _ ® Neural “t Epidermal 2 FIGURE 8. ‘The BMP sina tunsduction pathway. BMD activity pfs the etserm as epidermal its inhibition e.g by bing oa slubieinbibitorke chomin) fads to noua induction. Ligand binding induces tho type [and type IT repr to associate and causes the phosphorylation ofthe itso intermediate R-Smod el in place by the adaytor molecule SARA. R-Smad snow fee to asseite witha Co-Smad, casing taslcation into the miles, ‘where the complex partite inthe tansrptional modulation of mum of genes. Moir von Bubaol ad Cho (2001 that follistatin, an inhibitor of TGF signaling, was able to induce neural tissue suggested that inhibition of a pathway involving perhaps activin was responsible for the induction of neural ectoderm. These data were supported by studies using a truncated receptor fr activin. RNA encoding the activin receptor lacking the transducing, cytosolic domain but with the extracel- lular and transmembrane domains, ais asa dominant negative, that is, although ligand binding can occur, it is unable to elicit a response (Hemmati-Brivanlou and Melton, 1992). As this modi- fied molecule is present in far exeess ofthe wild-type molecule, it has the effect of sequestering the ligand. Animal eaps that express the dominant negative, truncated activin receptor Follow 4 neural pathway of differentiation (Hemmati-Brivanlow and Melton, 1994). ‘This led to somewhat of a paradox. Though it seemed that neural induction was @ result of activin inhibition, activin itself induced mesoderm and neural ectoderm, In actuality, the activin receptor used by Hemmati-Brivanlow and Melton was not specific for activin; rather it recognized other members of the ‘TGF superfamily (Hemmati-Brivanlou and Melton, 1994). As the truncated receptor also induced dorsal mesoderm, rather than recognizing activin, another TGF- family member setive on the ‘ventral side of the embryo could be the native ligand. BMP-2 and BMP-4, members of the TGF-B superfamily, are both expressed in the ventral part of the embryo (Daler aly 1992; Jones er al, 1992). Consequently, their potential role in neural induction was placed under scrutiny, which grew more intense with the discovery of chordin, another seereted molecule capable of inducing neural tissue. Chondin was discovered by virtue of its expression in Spemann's organizer. Late, itis expressed in the axial tissue of the prechordal mesoderm and ‘notochord al structures capable of neural induction (Sasa a, 1994), Examination ofthe primary sequence of chordin provided further insight into the mechanism of neural induetion. It was found that chordin shows considerable homology tothe fui fly Drosophila gene short of gastrulation (sos). Genetic analysis in Drosophilaad already shown that sog acted as an antagonist to another gene, decapentaplegicdpp), which is homologous to the vertebrate genes BMP-2 and BMP-4. The similarities with flies are not limited to the sequence (Holley er ai, 1998). In flies, eliminating dpp converts the epidermal eells of the fly into neuroectoderm. Overexpression of dpp changes the fate of neuroectodermal cells into epidermal (Biehser ai, 1996). In the atmphibian, BMP-4is also expressed in the non-neural ectoderm, consistent with it being an epidermal inducer. Moreover, when BMP-4is added to dissociated animal cap cells, neural induction is prevented regardless of how long reassociation is delayed (Wilson and Hemmat-Brivanlou, 1995). Overexpressing BMP-4 RNA on te dorsal side ofthe embryo results in an embryo with 4 loss of neural ectoderm, However, it should be noted that dorsal mesoderm, the primary neural-inducing tissue, is also inissing (Dale er ai, 1992; Jones era, 1992). The data pointed to neural induction deeurring by inhibition of the BMP pathway, and indicated that perhaps not only chordin, lke its Drosophila counterpart sog, but also noggin and follistatin acted as antago- nists of BMP activity. Indeed chordin, noggin, and follistatin bind to BMP-4 and the closely related BMP-2 (Piccolo et ai, 1996; Zimmermanet ai, 1996; lemura et ai, 1998), and from genetic ‘analysis in Drosophila, where chordin or noggin were ectopically expressed in various fly mutants in components ofthe BMP path= ‘way, te site of action ofchordin and noggin was placed upstream of the receptor, inthe extracellular matrix (Holley et al, 1995, 1996). An additional number of extracellular, secreted antazonists of BMP activity have been found. These’ molecules, such as Cerberus, Gremlin, and Xnr-3 Xenopus nodal related), all induce neural fates in the animal cap of the Xenopus embryo (Smither al, 1995; Bouwmeesteret al, 1996; Hsuet ai, 1998) Further support for the idea that BMP inhibition is ger- ‘ane tothe induction of neural tissue eame from inhibiting the inacellular components of the BMP signal-transduetion path- ‘way (Box 2). As well asthe truncated activin receptors, ating as dominant negative forms of the endogenous receptor, which have been shown to bind BMP-2 and BMP-4, negative forms of the Smad molecules have been shown to promote neural dfferentia- tion in the animal cap (Li et a, 1996; Bhushan et a, 1998). Indeed, even negative forms ofthe transcription factors that form the nuclear response to BMP signaling have been shown to neu- ralize the animal cap (Onichtchouk et ai, 1998; Trindade et al, 1999), Many of these experiments have been repeated in. the zebrafish embryo, with simila,ifnot identical, results (e.g, Imai eral, 2000). Complexities and Questions ‘That BMP inhibition, emanating ftom the organizer, is responsible for neural induction has been well demonstrated in anamniote (ish and frog) embryos. However, the data from the chick and mouse are confusing and challenge this idea Is the Organizer Responsible for Neural Induction? ‘The tole of the chick and mouse equivalents of the corganizer—Hensen’s node and the node, respectively—in neural induction has been questioned over the years In the chick, neural induction can occur even after the node is surgically ablated (Waddington, 1932; Abercrombie and Bellars, 1954). This result ‘was interpreted as showing that Hensen’s node, though sufficient for neural induction, was not necessary. However, subsequent studies have shown that after extirpation, the node is reconsti- tuted quickly owing toa series of complex inductive interactions (Yuan e7 al, 1995; Psychoyos and Stern, 1996; Yuan and Schoenwolf, 1998, 1999; Joubin and Stern, 1999). Genetic abla- tion of the node and notochord in the mouse and fish also has it the effect on the induction of neural tissue (Gritsmaner at, 1999; Klingensmith er ai, 1999). Recently, it has become clear that ‘neural induction in all vertebrates occurs earlier than previously thought, beginning before the appearance of a morphologically distinct organizer. For example, in chick, neural induction begins before the appearance of Hensen’s node, as determined by the Making Neural Tube * Chaper 1. 11 stage at which explants of prospective neural ectoderm first express neural markers (Darnell eta, 1999; Wilson a, 2000) In Xenopus, neural induction is initiated before gastrulation ‘Using the clearance ofthe expression of components of the BMP ignaling pathway as a marker for when neural induction is occurring it has been shown that neural induetion occurs during Inte blastula stages of Xenopus embryogenesis (Hemmati- Brivanlou and Thomsen, 1995; Faureet al, 2000), In fish containing the mutation one-eyed-pinhead (oep), the embryonic shield and dorsal mesoderm do not form. Despite this, these mutants still express chordin, indicating that some neural-inducing activity stil persists (Gritsmanet a, 1999). The uation in the mouse HNF-38 mutant is more striking, Even in the absence of a node and axial mesoderm, and despite the lack of expression of many markers of the mouse organizer, the rostral streak, from which the node derives, is still capable of neural induction (Klingensmither a, 1999). Js BMP Inhibition Sufficient for Neural Induction? Experiments again in the chick first questioned the hypothesis that BMP inhibition mediates neural induction. Steit and coworkers showed that neural tissue could not be induced by clumps of noggin- or chordin-expressing cells, even though rafts of Hensen’s node in parallel experiments induced neural tissue (Streit eral, 1998). Inthe same study, Streit er ai. (1998) showed that cells expressing BMP-2 or BMP-7 failed to inhibit neural plate formation. However, Wilson and coworkers showed that BMP-4 was able to induce epidermis in explants ofthe chick embryos fated to become neural ectoderm (Wilsonet al, 2000). ‘The difference between these sets of data seem tobe the sage at Which the experiments were performed, with the experiments using expressing eels being done at mid-gastrula stages, and the explant-induction experiments being done at blastula to early- sstrula stages. In the mouse, null mutants of BMP-2 (Zhang and Bradley, 1996), BMP-4 (Winer et ai, 1995), and BMP-7 (Dudley et ai, 1995) do not alter their pattern of neural induc- tion. However, there is probably functional redundancy between these molecules, with one compensating forthe loss of another (Dudley and Robertson, 1997). Compound mutants have not yet been established to address this issue ‘The expression patterns in the chick ofthe BMP inhibitors noggin, follistatin, and chordin are not strictly correlated with tis- sues that contain neural-inducing ability (Connollyet ai, 1995, 1997; Stet er a, 1998). Taken with the data from mice doubly mutant for noggin and chordin, which stil have neural tissue (Bachilleret ai, 2000), this seems to indicate that BMP inhibi- tion isnot required for neural induction in amniotes, However, as discussed above, there are other inhibitors of BMP signaling, both extracellular and intracellular, which may account for neural induction (von Bubnoff and Cho, 2001; Mufioz-Sanjuan and Hemmati-Brivanlou, 2002). For example, support for the idea that BMP inhibition induces neural character in te chick embryo comes from an inspection ofthe localization of phosphorylated Smadl, -S, and -8. Using an antibody that recognizes the activated form of these Smads as an indication of BMP signaling, 12 Chapter 1 + Raj Ladher and Gary C.Schoenwolf Faure et ai (2002) showed that there is no BMP signaling activity in the forming neural plate, An argument has also beet made that BMP inhibition merely stabilizes and reinforces neural cel fates, and that other families of signaling molecules are the primary neural inducers (Streit and Stern, 1999). Until the fall ‘complement of molecules that ean induce neural tissue is known, and a fll understanding of the signaling networks is understood, this question will not be fully resolved. The Role of Other Signals in Neural Induction Fibroblast Growth Factors (FGF) Both the FGF family and the wnt family have been shown, to play a role inthe induction of neural tissue. This role is distinct from their roles in patteming of the neural tube, which are dis- cussed in the subsequent chapter. In Xenopus, FGF ean actually induce neuralization of animal cap cells that have undergone brief dissociation, a procedure that diminishes the amount of BMP activity (Kengaku and Okamoto, 1993). Furthermore, blocking FGF signaling using a truncated FGF receptor makes the animal cap reffactory to neuralization by low amounts of chordin (Launay e ai, 1996). In chick, the role of FGF in neural induction has received considerable attention, Streit et ai. (2000) reported that an FGF-sesponsive gene, Early Response to Neural Induction (ERND, marks the territory inthe chick epiblast fated to become neural, and it rapidly induced FGF expression. By using an FGF receptor antagonist, SUS402, Wilson er a. (2000) showed that neural differentiation could be blocked in chick epiblast explants normally fated to become neural ectoderm. ‘The exaet role ofthe FGF pathway in neural induction is unclear, Some ofthe data point fo a role for FGF signaling in aiding the clearance of BMP activity from the neural plate; indeed, down- stream effectors ofthe FGF pathway have been shown to inhibit the nuclear accumulation of the R-Smad/Co-Smad complex (Kretzschmar et al, 1997, 1999). FGF may also induce neural tissue by a mechanism independent of BMP inhibition, An inves- tigation of Smadi0, a Co-Smad, in Xenopus, has yielded some relevant data (LeSeuret a, 2002), Smad0, a component of the BMP signaling pathway actually induces neural tissue within the animal cap. More surprisingly, by removing Smad10 protein Using antisense oligonucleotides, neural tissue is never formed in the affected embryos. Using co-injction studies, it has been found that Smadi0 cannot inhibit the BMP pathway, indicating some ‘ther mechanism for its function. One such mechanism is the identification ofa stein the Smad 10 protein that becomes phos- phorylaed and activated as a result of FGF signaling (LeSeur et ai, 2002) ‘An altemative view suggests that FGF signaling provides the ectoderm with competence to become defined as neural ‘There is precedence for this; Corelle al. (1995) have shown that FGF signaling acts to define the competence of tissue to respond to mesoderm induction by TGF signals in Xenopus, the very same tissue that ean respond to neural-inducing signals. In fact, itis likely that both a competence-defining role carly in development and a later neura-stabilizing role will be shown for the FGF family. However, like many of the controver- sies surrounding neural induction, we will have to wait until all the players and the way they interact are known before adequate resolution can be achieved Wits ‘The role of the wnt family of molecules has also been investigated during the induction of neural ectoderm. In the chick, wnt overexpression converts the epiblast fated to become neural to become epidermal (Wilson et al, 2001). Conversely in presumptive epidermal tissue fated to form epidermis, wnt ini- bition causes the explant to take on a neural fate. In adlition, at a subthreshold concentration of wnt inhibitors, below the level required for neural induction inthe epidermal epiblast explants, BMP inhibition and FGF signaling were able to induce newal ectoderm. One proposed mechanism is that wnt signaling causes an upregulation of BMP expression (Wilson et al, 2001), and thereby induces epidermal fate, although in Xenopus, additional dia suggest that wnt expression downregulates BMP expression Baker et al, 1999; Gomez-Skarmeta et ai, 2001). However, \wnt signaling may also regulate the strength of the transduced BMP signal via activation of the ealmodulin’C#* pathway Gisnmerman et ai, 1998; Scherer and Graff, 2000). This may explain why BMP inhibition cannot induce neural tissue in epidermal epiblat explants. Ifthe level of abrogation of BMP signaling is not complete the sensitized transduction pathway can sill receive an input, resulting in epidermal cell fates. I however, wnt signaling is ls inhibited, reception is desensitized and when combined with BMP inhibition ean lead to neural cell fates Interestingly, two naturally occurring inhibitors of wnt sig- naling, Fr2B and Strp-2, are expressed in the presumptive neural plate at round the stages that neural induction has been proposed to be occurring (Ladher eta, 2000), Insulin-Like Growth Factor ‘The insulin-like growth factor (IGF) family can also neu- ralize the Xenopus animal cap (Pera et al, 2001). The necessity for IGF signaling bas also been shown using a truncated IGF receptor In these embryos, neural induction mediated by noggin is inhibited. The authors propose thatthe IGF pathway may act downstream of BMP inhibition during neural induction, and that aswell asa passive role for BMP inhibition, neural induction nay not be a default as previously thought. Instead, it may aso require an active signal, induced asa result of BMP inhibition Summary of the Molecular Events of Neural Induction As discussed above, the main mechanism by which the neural ectoderm is induced is via the inhibition of the BMP pathway. Other ftors do play ole, namely the FGF family and ‘the wnt family. As yet itis unclear what the exact roles of these molecules are, whether they are required as competence factors ‘or whether they act to aid the clearing of BMP signals and their reception from the neural plate. Once induced, the neural ectoderm—also known at this Jiuneture as the neuroepithelium—stil has a daunting journey ahead oft to form the central nervous system: it must rol up into 2 tube, which is subsequently patterned. We will describe in the next section the mechanism by which the specified neural ecto- der becomes a tube; other chapters later in this book deal with the elaboration of the neural tube into the adult central nervous system, NEURULATION ‘The process of neural induction results in a plate of cells running along the rostrocaudal length ofthe embryo. The medial part of the neural plate will eventually form te ventral part ofthe neural tube, and the ateromost edges will be brought together to form the dorsal part ofthe tube during the process of neurulation ‘The end result of neurulation isa hollow nerve cord ‘Neurulation ean be subdivided into a number of events, each requiring diferent interactions. First, neurlation occurs in two phases called primary and secondary neuruation. When one speaks of neurulation, they are typically referring to primary neurulation, a process that occurs in four stages defined as for- ‘mation, shaping, and bending ofthe neural plate, and closure of the neural groove (Figs. 9 and 10). Each stage willbe described in turn. This diseusson focuses primarily on the chick embryo, as most of the mechanistic studies have been performed on this embryo, Fora more in-depth discussion, the reader is directed t0 several reviews (Schoenwolf and Smith, 1990; Smith and Schoemwolf, 1997; Colas and Schoenwolf, 001) Formation of the Neural Plate ‘The neural plate is a thickened region of the ectoderm located medially within the embryo. The thickening forms by an apicobasal elongation of ectodermal cells, an action known as cell palisading. The thickening ofthe neural plate is not a result ‘of an inerease in the number of cel layers; the neuroepithelium remains pseudostatified (See Fig. 9A). It has been shown that thickening of the neural plate isan intrinsic propery ofthe ecto- dermal cells once they have been induced as neural (Sehoenolf, 1988) Shaping of the Neural Plate Daring shaping, diferent cell behaviors convert the neural plate from a relatively short (in the rostrocaudal axis) and squat (wide in the mediolateral plane) structure to one that is long and narrow (see Fig. 10) This results from a combination of contin- ‘ued cell elongation, convergent extension, and cell division, as well as the caudalvard regression of the primitive streak (Schoenwolf and Alvarez, 1989; Schoenwolf er ai, 1989) Neuroepithelial cells continue their apicobssal elongation during shaping, a process initiated shortly after neural induction and resulting in formation of the neural plate. AS a result of ‘Makinga Neural Tube * Chaper 1. 13, cell elongation, a concomitant narrowing of the neural plate occurs, as neural plate ces maintain their individual volumes. Convergent extension movements further exaggerate the narrowing of the neural plate; that is, cells of the neural plate intecalate in the mediolateral plate, effectively causing the neural plate to lengthen rostrocaudally while narrowing simultaneously, Cell division also contributes to the lengthening of the neuro- epithelium: about half of the division planes are oriented such that they place the daughter eels into the length of the neural plate rather than adding to its width (Sausedo ef ai, 1997). Isolation experiments have shown that the cll behaviors causing shaping ofthe neural plate are autonomous to the neural plate. In ther words, sch changes in cell behavior within the neural plate erierateintrnsieforees fr its shaping. However, for the shaping ofthe neural plate to occur completely normally, noma gastru- Inkion movements also must occur, as the axis develops in the wake of the regressing Hensens node Bending of the Neural Plate Bending involves the establishment of localized deforma- tions ofthe cells of the neuroeitelium and the subsequent ele- vation of the two flanks of the neuroepthelium, converting it from the neural plate to the neural groove. Bending is actually deiven by two distinct types of movement: furrowing and folding (Colas and Schoenwolt, 2001). Furrowing i a behavior intrinsic to the hinge points within the neuroepthelium. There are three hinge points within the neural plate: a single median hinge point, found along the neuroaxis (except atthe future forebrain level) and coincident with the floor plate of the neuroepithelium; and ‘the paired (right and left) dorsolateral hinge points, found pri- marily at levels where the brain will form (Fig. 11; Schoenwolf and Franks, 1984). Neuroepitheial cells within the hinge points undergo wedging, that is, apical consrietion with a concomitant basal expansion, driven in part by the basalvard interkinetic movement of the nucleus (see Fig. 11; Smith and Schoenwolf, 1987, 1988). This acts not only to deform the neuroepithelium, creating a furrow, but it also provides points around which the neural plate can rotate during folding; that is to say, true to their nomenclature, the hinge points do act like hinges during neurulation Folding is a more complicated process and is driven by the nnon-neural ectoderm, The net result is rather like closing & pair of calipers. The easiest way to close calipers isto apply a force Interally atthe tp of the calipers, and eventually the tips will meet, folding around the hinge. Like calipers, the neural plate elevates and folds by forces generated laterally in the non-neural ectoderm, This force results, in part, from cell shape changes in the non-neural ectoderm (Alvarez. and Schoenwolt, 1992; Sausedo et al, 1997). These cells undergo apicobasal flattening, thus effectively increasing their surface area, Folding itself can be divided into thre distinet events (Fig. 12), occurring while the lateral epithelium provides a medialward force (Lawson ef aly 2001). The frst is epithelial kinking, where cells atthe interface between the neural and non-neural ectoderm deform, with each 14 Chapter 1 Raj Ladher and Gary C. Schoenwolf FIGURE 9. Whole mounts (for orientation; transverse lines indicate levels of eros-tctions iene by long arows) and seaming electon micrograph «ros-setions ofthe neuroepitlinm ding neulatin, Showa ae change in the netroepithium that oer dng the (A) frmation, (BC) shaping, md (AC) bending ofthe noua pate, and closure ofthe neural groove (CD). Dats ae previa inthe text. hp, dosolator hinge poi; e, endoderm; e, epidermal estar i foregut hn, head mesenchyme rp, median hinge point, nolosbon af, wc od: at neal ube aeows (D), nea rest el Modified from Schoeawolf 2001), ‘Making Neural Tube * Chaper 1. 15 FIGURE 10, Whole mount embeyos viewed fom thir dorsal side during ncurlation A-E indicate progressively olds, yt patllyovesaping. sages of neuultion, begining with (A) formation ofthe neural plate, (B-E) shaping of the neural pate, (B-E) bending ofthe neural pa, and (DE) elosure ofthe oul roo, The acuospithslinm atthe time ofits formntion ea relatively short and st structs, os sen in surface view Homever, ding coversent extension movements tat commence concomitant wih repression of Hensen's nod, the neural plate lengthens resrocaually and ares mediolateral. Hensen’s node: nf neural fol: ng, neural groove: np, neural plat: ps, primitive streak: dashed lines, lateral borders of neural plate, Moi from Smith and Schoenwolf (199%). FIGURE 11. Cell behavior nthe neural plat during its bending. Shown is 2 diagram ofa cross-section through the ner tube during bending. Highlighted (darker shang are the thre hinge poins the median hinge psn (asters), indent with the floor plate of the neural tobe, and the doeslateral hinge Pits (double asarisks), Found in the future tain love of the neuraxs cpidermal ectoderm: n, atochord; arrows, directions of expansion of the epidermal ecloderm. Modified fom Schoenwof and Smith (1980), forming an inverted wedge that i apically expanded and basally constricted. The next step is epithelial delamination, This involves the deposition of extracellular matrix atthe neural fold interface (ie, the space between the two ectodermal layers of each neural fold) and a re-orientation of neural and non-neural cells around the interface, such that their basal surfaces abut ‘The final step is epithelial apposition, which oocurs in the brain region. This is essentially @ rapid expansion of the neural folds, with extension of the area of epithelial delamination in the mediolateral plane and further deposition of extrcelular matrix along the expanding width of the neural fold interface ‘Additionally, the non-neural ectoderm intercalates and undergoes rinted cell division, thereby contributing to the mediolateral forees generated in the epidermal ectoderm ‘Tissue isolation experiments have been used to identi the cell types responsible for generating the forces of folding (Schoenwolf, 1988). Removal ofthe lateral, non-neural ectoderm resuls inthe loss of folding, but futrowing of the neural pate within the hinge points stil occurs Hackett er a, 1997). Ifthe mesoderm and endoderm lateral to the neural plate are removed, but leaving the non-neural eciodermal layer intact, both folding and furrowing occur (Alvarez and Schoenwolf, 1992), Thus, the non-neural ectoderm is both necessary and sufficient for folding to occur. Closure of the Neural Groove Bending brings the tips of the neural folds into close contact at the site ofthe dorsal midline of the embryo. During closure, the two tips altach and fuse. Eaeh component ofthe tip must se correctly, such that the non-neural epithelium. forms 4 continuous sheet overlying the newly formed root plate of the neural tube and the associated neural crest, The exact mechanism of this concluding step of neurulation is not well undersiood, and the molecules that mediate adhesion, epithelial breakdown, and sion are not known, 16 Chapter 1 + Raj Ladher and Gary C.Schoenwolf Epithelial Kinking A af np, B Epithelial Delamination at alnp. ma np, e Epithelial Apposition FIGURE 12. Formation ofthe neutl folds. The seaming clecton micrographs and accompanying digrams highlight the formation aod morphogcasss of the aura folds, in particular, (A) epithelial kinking, (B) delamination, and (©) nthe rain region) apposition. dp, donot hinge point, pidermal ston: af, aural flde 9p, noua pi Secondary Neurulation At caudal levels of the neuraxis of birds and. mammals (eg. the lumbar and sacral regions), the neural tube develops in ‘a manner distinet from more rostral regions. Caudal neural tube formation occurs through a process known as secondary neurt- Tation, Rather than the rolling up of a fat plate of cell, as isthe case in primary neurulation, secondary neurulation consists of the cavitation of a solid epithelial cord of cells inthe tail of the embryo. Secondary neurulation begins when cells within the tail bud condense to form an epithelial cord of cells, known a the medullary cord (Sehoenwolf, 1979, 1984; Schoenwolf and DeLongo, 1980). The outer cells of the medullary cord then undergo elongation, forming a pseudostatified columnar epithe- lium similar to that of the neural plate during primary neurula- tion. This pseudostratified epithelium then becomes polarized, resulting in the formation and fusion of small lumina at the apices of the outer layer, around a central core of mesenchymal cells. These inner cells are removed during cavitation, by cell rearrangements and perhaps limited apoptosis. Cavitation results inthe formation of a singe, secondary lumen, whieh wall join ‘with the primary lumen ofthe rostral neural tube shod lie. ntrace between the tw etoderl lye o curl fold, Modified from Lawsonet at (2001), SUMMARY ‘The future central nervous system is derived from an ‘unspecified sheet of ectoderm, with fate being instructed by sig- nals emanating, in the main, from a specialized region of the early embryo, the organizer. The organizer secretes signals that hhave the net effect of inhibiting the BMP pathy, be it by extra- cellular antagonism or by intracellular modulation of the ability ofthe cel to perceive BMP signals. Other factors also play a role in neural induction, for example, the FOF family of molecules, but their exact roe in neural induction remain unknown, As more players are identified in what undoubtedly will be a signaling network leading to neural induction, the exaet molecular mecha nism of neural induction can be established Once induced, the neuroepitheliui rolls into the neural tube. One model, and one that has gained widespread acceptance, is the hinge point mode. In this model, both extrinsic (i, out- side the neural plate) and intrinsic forces cooperate and synergize in bending the neural plate. Although the cellular behaviors of snuch of this process have been well characterized the molecular bases for these behaviors have so far proved elusive. The relationship between induction of the neuroepithelium and its subsequent morphological movements is of particular interest to the developmental neurobiologist ACKNOWLEDGMENTS RKLL. acknowledges the support of MEXT and the leading Projects of Japan. Results described from the Schoenwolt laboratory were obiained with support by grants from the National Institutes of Heath. Support was als gratefully received from the Ministry of Science and Education of Japan and the Leading Projects. We thank past and present members of the Schoenwolf laboratory for their contributions. REFERENCES, Abercrombie, M and Bellas, R., 1954, The effets in chick blastoderm of replacing the primitive noe bya gral of posterior primitive streak. LB nbs. Exp. Morph. 235-2 Afiakite, M, More, A. Jessan, 8, Itoh, S. Sampath, K,, Wedermark, B al, 1998, Induction of nitty Smadé al Smad mRNA by TOF- ‘ata family members, Biochem. Bios. Res. 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