Fate map of the chick embryo neural tube

Develop. Growth Differ. (2009) 51, 145–165 doi: 10.1111/j.1440-169X.2009.01096.x

Blackwell Publishing AsiaReview

Fate map of the chick embryo neural tube

Raquel Garcia-Lopez,†1 Ana Pombero†1 and Salvador Martinez1*1Instituto de Neurociencias, Universidad Miguel Hernandez-Consejo Superior de Investigaciones Cientificas, Av. Ramon y Cajal s/n,San Juan de Alicante, 03550, Spain

Fate-map studies have provided important information in relation to the regional topology of brain areas in differentvertebrate species. Moreover, these studies have demonstrated that the distribution of presumptive territories inneural plate and neural tube are highly conserved in vertebrates. The aim of this review is to re-examine andcorrelate the distribution of presumptive neuroepithelial domains in the chick neural tube with molecular informationand discuss recent data. First, we review descriptive fate map studies of neural plate in different vertebrate speciesthat have been studied using diverse fate-mapping methods. Then, we summarize the available data on thelocalization of neuroepithelial progenitors for the brain subregions in the chick neural tube at stage HH10–11, themost used stage for experimental embryology. This analysis is mainly focused on experimental fate mappingresults using quail-chick chimeras.

Key words: brain, chicken, fate-map, neural plate, neural tube.

Fate maps of the central nervous system at the neural plate stage

By comparing patterns of molecular expression in manyvertebrate species, it was possible to identify the maincomponents of the central nervous system (Rubensteinet al. 1994; Shimamura et al. 1997; Puelles et al. 2000;Puelles and Rubenstein 2003). These studies providedevidence that the patterns of gene expression canbe related to primary morphogenetic processes thatorganize the histological primordia of the embryoniccentral nervous system into distinct domains. Neuralplate and neural tube are subdivided into longitudinaldomains: floor plate, basal plate, alar plate and roofplate. Signals produced by the axial mesendoderm andnon-neural ectoderm are able to generate consecutivelongitudinal domains of cell fate specification in theprogenitors of the embryonic central nervous system(Rubenstein et al. 1994, 1998; Shimamura et al. 1995;Tanabe and Jessel 1996; Dessaud et al. 2007). On theother hand, transverse domains (proneuromeres andneuromeres) expressing distinct combinations of genes

are present in the neural plate and neural tube (Rubensteinet al. 1994; Shimamura et al. 1997). These domainswere first observed by classic authors at the beginningof the last century and this idea was later recoveredin the prosomeric model (revised in Puelles et al. 1987and Puelles 1995). The prosomeric model proposesthat the brain is subdivided into longitudinal andtransversal segments (Bulfone et al. 1993; Puelles andRubenstein 1993, 2003; Rubenstein et al. 1994; Puelles1995, 2001a). These studies were mainly based on geneexpression patterns, but lacked information concerningthe cell lineage relationships between molecular domainsin the neuroepithelium and the neural formations in themantle layer of the central nervous system.

Fate-map studies contributed to solve this problemproviding important information in relation to the organ-ization of the brain in different vertebrate species (seeTable 1; axolotl: Jacobson 1959; Xenopus laevis: Eagle-son and Harris 1990; zebrafish: Woo et al. 1995; Staudtand Houart 2007; mouse: Inoue et al. 2000; chicken:Couly and Le Douarin 1985, 1987; Smith-Fernandezet al. 1998; Cobos et al. 2001b; Fernandez-Garre et al.2002; Garcia-Lopez et al. 2004; Pombero and Martinez2009). The fate-map technique consists of markingneuroepithelial fields in order to detect them during thesubsequent development, which allows us to relategene expression to the neuroepithelial origin of the neuralformations. Fate-map studies carried out in differentspecies have demonstrated that neural plate organizationis a highly conserved structure in vertebrates (Fig. 1).

*Author to whom all correspondence should be addressed.Email: [email protected]†These authors contributed equally to this workReceived 15 January 2009; revised 19 January 2009; accepted

20 January 2009.© 2009 The Authors Journal compilation © 2009 Japanese Society of

Developmental Biologists

146 R. Garcia-Lopez et al.

© 2009 The AuthorsJournal compilation © 2009 Japanese Society of Developmental Biologists

Fig. 1. Fate-maps of the neural plate in different vertebrate species: (a) axolotl (Ambistoma mexicanum) at stage 15 (modified fromJacobson 1959 and Rubenstein et al. 1998); (b) mouse (Mus musculus) at 5–7 somites stage (modified from Inoue et al. 2000); (c) Xenopus(Xenopus leavis) stage 15 (adapted from Eagleson & Harris 1990 and Rubenstein et al. 1998); (d) On the left-hand: Zebrafish (Danio renio)at 10 h gastrula stage (modified from Woo et al. 1995 and Rubenstein et al. 1998). On the right-hand: Zebrafish (Danio renio) fate-map ofthe diencephalon (modified from Staudt & Houart 2007); (e,f ) Chicken (Gallus gallus) at stage HH8 (adapted from Couly & Le Douarin 1987and Cobos et al. 2001b, respectively); (g) Chicken (Gallus gallus) at stage HH4 (adapted from Fernandez-Garre et al. 2002).

FPO

Fate map of the chick embryo neural tube 147


One of the best anatomically documented fate-maps of the neural plate was carried out in axolotl(Ambystoma mexicanum; Jacobson 1959). In this studythe telencephalic primordium was mapped to therostral edge of the neural plate, partially overlappingwith the anterior neural crest. When the medial regionof the neural crest was labeled, a patch of cells wasdetected in the medial region of the septum, just beforethe anterior commissure. Laterally to the anterior neuralcrest, the presumptive territory of the olfactory bulb andthe lateral, dorsal and medial cortex were mapped.Subpallial primordia were localized medial to theprospective pallium (Fig. 1a).

The fate-map at the neural plate stage in xenopus(Xenopus leavis) provided us with important anatomicalinformation about the diencephalon, establishing theprospective territories of the ventral thalamus, dorsalthalamus and pretectum in a rostro-caudal position(Eagleson and Harris 1990; Fig. 1c). In the same studyit was also observed that the anterior medial crestextended from the chiasmatic crest to the laminaterminalis and laterally, to the optic vesicles. In thepresent study, the lamina terminalis was considered asthe most rostral region of the neural plate, in agreementwith the fate-map carried out in axolotl (Jacobson1959).

A similar distribution of the neural primordia wasobserved in a fate-map carried out in zebra fish (Daniorenio) at the neural plate stage (Woo et al. 1995; Staudtand Houart 2007; Fig. 1c).

Inoue et al. (2000) studied the fate-map of the mouseneural plate by means of DiI labeling. Their experimentsshowed that the antero-medial region of the prosen-cephalic plate developed into the lamina terminalisand the chiasmatic plate, whereas the posterior partcontributed to the diencephalon. The prospectivetelencephalon was mapped around the chiasmatic plate.On the other hand, the hypothalamic primordium waslocated caudal to the chiasmatic plate (Fig. 1c).

Several fate-maps have been published in avianembryos (Fig. 1e–g). The quail-chick graft techniquewas used in most cases. This experiment involvescarrying out interspecific, homotopic and isochronictransplants using a quail embryo as the donor. Thequail tissue can be detected by the presence of a quailnucleolar marker (Le Douarin 1973) or using a quail-specific antiserum (Lance-Jones and Lagenaur 1987).This experimental approach eliminated one of the principalproblems of the methods based on cell labeling: themarker dilution. Chick (Gallus gallus) fate-map studiesshowed results in agreement with the conclusionsobtained in fate-map carried out in other vertebratespecies (Couly and Le Douarin 1985, 1987; Cobos et al.2001b). The first fate map studied in chick at the stage

of the neural plate (Couly and Le Douarin 1985, 1987)described the caudal position of the diencephalon inrelation to the telencephalon. Moreover, they observedthat topological relationships between the presumptivepallium and the subpallium primordium were maintainedas shown in other vertebrates. We carried out a mapat the neural plate stage (HH8) (Cobos et al. 2001b),using the quail-chick transplants as an experimentalapproach, which increased the level of information andresolution of the antero-posterior and medio-lateralorganization of the telencephalic primordium. Our resultsdemonstrated that the rostralmost alar regions arerepresented by the lamina terminalis, the optic chiasm,and the suprachiasmatic area. It was observed that thesubpallial telencephalon extended peripherally until thepallial commissure and the septal domain, and the pallio–subpallial boundary was placed nearly in a topologicallytransverse orientation. Laterally, the prospective territoriesof the pallium were found. According to the map, thecortex (medial and dorsal pallium) was peripheral inrelation to the dorsal ventricular ridge (DVR), and bothelements extended and converged in the olfactory bulb,in the anterior region, and in the amygdala, in theposterior region. The latero-posterior region containedthe prospective prethalamus and thalamus. Our resultswere largely consistent with the work of Couly and LeDouarin (1985, 1987), adding novel anatomical andtopological detail on forebrain subdivisions (Fig. 1e,f ).

Other fate-maps have been carried out at early stages(late gastrula/early neurula stage). These fate-maps ofthe avian epiblast were generated through quail/chickgrafts (Garcia-Martinez et al. 1993) and fluorescentlylabeled grafts (Schoenwolf and Sheard 1990; Lopez-Sanchez et al. 2001). The fate-maps showed thesubdivisions of the prospective ectoderm, mesoderm,and endoderm, both within the epiblast prior to theiringression and within the primitive streak (Garcia-Martinez et al. 1993). Moreover, they explored the originof the cardiovascular system from the primitive streakat these stages and provided new information on theepiblast origin of the neural plate, heart and somites(Schoenwolf and Sheard 1990; Lopez-Sanchez et al.2001).

A new chick neural fate map at stage HH4 (late gastrula/early neurula stage) has been published (Fernandez-Garre et al. 2002; Fig. 1g). In this case, the epiblastarea was sampled with multiple overlapping grafts. Afixable intracellular carboxyfluorescein diacetate succin-imidyl ester (CFSE) marker was used for labeling thedonor embryos. In this work the contour of the neural/non-neural boundary was first explored, as well as theposition of the alar/basal limit (longitudinal subdivisions).They also observed that the prospective transversalsubdivisions separating the wedge-shape forebrain,



midbrain and hindbrain primordia, diverged uniformlyfrom the node, demonstrating that all of the presumptiveboundaries are oblique at stage HH4.

Fate-maps of central nervous system at neural tube stage

The quail-chick chimeric approach

The classic chick/quail transplant model introduced by LeDouarin (Le Douarin, 1969), involves grafting quail tissueto chick embryos (the interpretation of these experimentsis based on the assumption that grafts between thesetwo similar species are equivalent to grafts between asingle species). This technique allows one to follow aspecific group of cells (the graft) through a period ofdevelopment and to determine the fates and locationsof their progeny. The method is based on the occurrenceof condensed heterochomatin associated with thenucleoli of all quail cells. Because such a condensationdoes not exist in chick cells, the histochemical visualizationof DNA (the Feulgen’s method) has for years been thebest approach to distinguish quail cells from chick cellsin the chimeric embryos. Today, several immunomarkersof quail or chick cells have been obtained and thereforedouble labeling facilitates the further identification ofthe cells belonging to either of these two species.Alvarado-Mallart and Sotelo (1984) carried out thepioneering work to apply quail-chick transplants to thestudy of central nervous system development.

Boundaries of the neural tube

The formation of the earliest visible transverse boundariesoccurs as a gradual process after the closure of theneural tube in chicks, at HH9–10 (Hamburger andHamilton, 1992), and is caused by differential proliferationof neuroepithelial territories. Limits habitually appear asconstrictions (transversal neuroepithelial microzoneswith slower proliferation) that separate bulging neuralwall domains of various sizes. These bulges are calledsuccessively primary brain vesicles (forebrain, midbrain,hindbrain), proneuromeres (secondary vesicles) thatwill later subdivide, particularly in the hindbrain, andneuromeres (Vaage 1969; Bulfone et al. 1993; Puellesand Rubenstein 1993, 2003; Puelles 1995, 2001a;Rubenstein et al. 1998; Puelles et al. 2007).

At stage HH10 (Fig. 2 a,b), we can already distinguishin the chick neural tube three primary vesicles (forebrain,midbrain, and hindbrain). The anteror-posterior (AP)regionalization first causes the forebrain to becomesubdivided into rostral secondary prosencephalon(hypothalamo-telencephalic complex) and the caudaldiencephalon (Puelles et al. 1987, 2004; Puelles and

Rubenstein 2003). The diencephalic region next developsthree prosomeric transverse units, now known as pro-someres 1–3 (p1–p3; Puelles and Rubenstein 2003).While older published reports have usually attributedtwo neuromeres (mesomeres m1 and m2) to themidbrain primary vesicle (Vaage 1969), recent analysisof its morphogenesis in molecular perspective holdsthat the primary midbrain vesicle always retains itsproliferative field unity and therefore is not subdividedinto smaller vesicles (Puelles 2001a; Puelles et al. 2007).Anteroposterior subdivision of the hindbrain starts atHH9 +, simultaneously with the incipient establishmentof the isthmic organizer that controls further patterningin the midbrain and rostral hindbrain. Vaage (1969)thought that the isthmomesencephalic boundary (IMB)is continuosly dintinguishable from the primary three-vesicle stage onwards (HH10–11) due to the apparentexistence of an early constriction separating the primary‘midbrain’ and ‘hindbrain’ vesicles. For decades it wasindeed conventionally accepted that the prospectivefate of the early ‘midbrain’ vesicle at HH11 coincidedwith the adult midbrain structure. However, homotopicand isochronic quail-chick grafting experiments showedthat the caudal part of the ‘midbrain’ has a peculiarmorphogenesis and does not produce the caudalmostpart of the mesencephalon but a rostral part of theprospective isthmocerebellum (Martinez and Alvarado-Mallart 1989; Hallonet et al. 1990; Alvarez-Otero 1993;Hallonet and Le Douarin 1993; Marin and Puelles 1994;Millet et al. 1996).

Forebrain

Telencephalon. During the neurulation process, theanterior neural plate is an almost flat structure, whichfolds to form the neural tube. Later, the telencephalicfield bulges dorsally to generate the telencephalichemispheres, which are considered as a complexprotosegment not subdivided into prosomeres, whichexhibits patterning singularities (Puelles and Rubenstein2003). Several studies based on molecular expressionin many vertebrate species identified two homologouscomponents of the telencephalon: the pallium and thesubpallium (Smith-Fernandez et al. 1998 and Puelleset al. 2000). In mammals, the pallial area develops intothe cerebral cortex and the claustroamygdaloid complex(and corresponding parts in birds), while the subpalliumgive rise to the striatum and pallidum, as well asentopeduncular and preoptic areas.

Previous fate-map located the telencephalic primordiumin the anterior portion of the prosencephalic field(Couly and Le Douarin 1985, 1987; Balaban et al. 1988;Smith-Fernandez et al. 1998; Cobos et al. 2001b;Fernandez-Garre et al. 2002). The fate-map carried out



Fig. 2. The drawings on the top represent the different locations where the transplants were made. (a–l) Long survival experiments (HH35-40) fate mapping the alar and basal diencephalon. (a,b): Alar plate prosomere 1 grafts. Lateral sections of this case (R343) in which thequail cells mapped different structures inside the p1 domain (b) which was identified by cresyl violet staining in parallel series (a). (c,d): Basalplate grafts of prosomere 1 (R359). (e,f ): Alar plate p2 graft (R342). Lateral sections in which the quail cells mapped the different structuresof dorsal thalamus and caudal p3 nuclei (QCPN [Quail cell perinuclear ring] immunostaining: f; cresyl violet: e). (g,h): Example of prosomere2 basal plate graft (R354). (i,j): case of graft alar plate prosomere 3. Lateral sections of the case R382 in which the quail cells mappeddifferent p3 structures (QCPN immunostaining: j; cresyl violet: i). (k,l): Example of prosomere 3 basal plate graft (R368). For abbreviations,see abbreviation list.



at the neural tube stage by Smith-Fernandez et al.(1998) described three divisions in the telencephalon:the striatum, the dorsal ventricular ridge (DVR: ventraland lateral pallium) and the dorsal and medial pallium.We recently reported a fate-map of the telencephalonat the neural tube stage (Pombero and Martinez 2009)with a more detailed analysis of internal topologicalrelations among pallial and subpallial structures. Moreover,our results were in agreement with the previouslysuggested topographic arrangement of the maintelencephalic primordia, but added novel anatomicalinformation bearing upon current concepts of telen-cephalic regionalization in the context of the revisedprosomeric model (Puelles and Rubenstein 2003).

The subpallium. Our fate-map, which was restricted tothe portion of the dorsal forebrain that is visible andaccessible for in ovo grafting at stage HH10 (Figs 3,4a), showed that the presumptive subpallium derivesfrom the anterior-most region of the prosencephalicvesicle (Fig. 5a–d), which was in agreement with otherfate-maps in the neural plate (Cobos et al. 2001b;Fernandez-Garre et al. 2002) and neural tube (Smith-Fernandez et al. 1998). It was observed that at stage

HH10 the lamina terminalis, the anterior preoptic andentopeduncular areas primordia, were already ventrallypositioned (Figs 4a, 5a–d) in relation to the other sub-pallial derivates (pallidum and striatum). These resultssuggested that the morphogenetic movements producingthe cephalic flexure, that hides the anterior pole ofthe neural tube, have already commenced during earlyneurulation. This process shifts the lamina terminalis,anterior commissure and subpallium underneath pallialderivatives (Pombero and Martinez 2009). In this way, weobserved that anterior and antero-lateral prosencephalicgrafts in the neural tube were found to map to theprospective subpallial structures. These regions gaverise to the septum and adjacent pallidal and striatalregions (Fig. 5a–b). In agreement with the molecularregionalization reported by Puelles et al. (2000), thetransplanted areas largely corresponded to the subpallialseptum and the rostromedial areas of the pallidum andstriatum. The tissue located ventral to the anterior com-missure according to the neural plate fate map (Coboset al. 2001b) includes the territory of the preoptic areaand lamina terminalis.

When grafts included more caudal neuroepithelium,quail cells were detected in the medial striatum and

Fig. 3. Quail/chick chimeras: (a)Stage HH10 chick and quailembryos are used for thetransplantation process. Schematicrepresentation of chick and quailembryos in ovo after injection ofIndian ink underneath theblastoderm. (b) Schematic repre-sentation of the homotopic andisochronic quail-to-chick grafts inthe neural tube. The embryos areoperated at stage HH10, usingthe quail embryos as donors. At theworking magnification (40×), thedistance between each adjacentconcentric circumference in thegrid measured 40 μm.



the ventral part of the olfactory bulb (ventral pallium),as well as in the pallidum and the subpallial septum(Fig. 5c–d). Fate-maps carried out in the chick neuralplate stage (Couly and Le Douarin 1985, 1987; Coboset al. 2001b) placed the subpallial primordium at stage

HH8 in a medio-rostral area of the prosencephalicalar plate primordium, medial to the pallium. Duringneurulation, the telencephalic primordium progressivelyacquires a dorsal paramedian position in the neuraltube. Moreover, the axial bending process of the neural

Fig. 4. Schematic representation that resumes the fate map analysis of the neural tube. In (a) we represent the fate-map of the forebrain,midbrain and isthmus and the hindbrain fate-map is illustrated in (b). In both figures, on the left hand side there is the segmental subdivisionof the chick neural tube at the stage HH10. On the right hand table, the most important primordia of the forebrain (telencephalon anddiencephalon), midbrain and hindbrain (isthmus, rhombencephalon and medulla oblongata) are represented following their neural tubeposition. The different primordia have been placed according to a dorso-ventral and rostro-ventral display; that is, ventral is to the left androstral is at the top. Each region follows a color code. The limit of the telencephalon and diencephalon alar plate is reported according tothe diencephalon fate map developed by Garcia-Lopez et al. (2004). Boundaries between telencephalic subdomains have been drawnaccording to the fate-map of the telencephalon at stage HH10 (Pombero & Martinez 2009). The limit of the mesencephalic andmetencephalic alar plate is reported following the fate maps obtained by homotopic transplantation of various portions of these areas(Martinez & Alvarado-Mallart 1989). The subdivisions inside the hindbrain are reported according to the fate maps developed by Aroca &Puelles (2005), Marin & Puelles (1995) and Cambronero & Puelles (2000).



Fig. 4. Continued



Fig. 5. The drawings on the top represent the different locations where the transplants were made. (A) A representative drawing of thepresumptive neuroepithelium where the HEM organizer will develop, at the insertion of choridal plexus in the caudal edge of the medialpallium (the fimbria). (a–l) Analysis of the presencephalic grafts carried out in order to elaborate the telencephalic fate-map. All micrographiesare sagittal views of HH35 chick embryos (rostral to the left) detecting quail cells by anti-QCPN, cresyl violet shows the cytoarchitecture.(a–f ) Schematic representation of the subpallial grafts. (a,b) Results of antero-medial graft. Sections of HH35 fixed chimera (a,b) revealedthat the subpallial septum and pallidum had been grafted (anterior is to the left). (c–d) Case AQ184 presented a transplant that overlappedwith case AQ165, but it was extended caudally. At HH35, the analysis of this case, presented in sagittal sections, showed quail-derivedareas in the lateral subpallium: pallidum, striatum and subpallial septum, but also in the anterior part of the ventral pallium (rostral to theleft). (e,f ) Most lateral and caudal grafts revealed how preoptic and entopeduncular regions were included in the grafted area. (g–l) Analysisof pallial neuroepithelial grafts long survival time (HH35). (g,h) Results of experiments carried out in the most rostral tip in a HH10 neuraltube. Labeled cells are found in subpallial (anterior regions of septum, striatum and pallidum) and pallial areas (ventral and lateral pallium).(i,j) Example of experiments in which grafted tissue was caudal to the prospective subpallium. The graft derivatives are exclusively pallial(lateral and dorsal pallium). (k,l) Caudal grafts in the prospective secondary prosencephalon. The grafted territory contained the dorsal andmedial pallium, the fimbria and the choroid plexus.



tube pushes the telencephalic subpallium into anunderlying position (Fig. 4a). When the neural platecloses to become in a tube, the resulting midline willform the septal domain ending rostrally at the anteriorneuropore (Pombero and Martinez 2009).

Quail-chick transplants carried out in antero-lateralareas of the prosencephalic vesicle, gave rise to boththe lateral part of the striatum and the pallidum andthe entopeduncular and preoptic areas, which latergave rise to the peduncular subpallium (Fig. 5e–f ).Lateral grafts also showed quail derivatives in the retinafield, which in the chick neural plate, was locatedconcentrically to the subpallium (Cobos et al. 2001b).In our results we concluded that the growth anddorsal movement of the anterior alar plate to formthe roof of the prosencephalon, together with theevagination of the optic vesicle, resulted in the pre-sumptive retina moving from its central position in theneural plate to the lateral pole of the prosencephalicvesicle (Fig. 4a).

The pallium. The pallial territory is subdivided into fourmain subregions: medial, dorsal, lateral and ventralpallium. We have studied, by quail-chick grafts, thelimits between pallial subpallial domains and we haveidentified the intrapallial limits, as well as their extensioninto the complex longitudinal septal and amygdalinedevelopment regions (Fig. 4a; Cobos et al. 2001a, band Pombero and Martinez 2009).

Several overlapping grafts, which involved neural tissuefrom the tip of the neural tube to the center of the grid(Fig. 1), were analyzed to locate pallial primordium andthe different pallial subdomains (Fig. 5i–l). According toour data, the prospective pallial neuroepthelium occupiedthe central part of the dorsal prosencephalon, beinglocated in a caudal position regarding the subpalliumand medial to the retina (Fig. 4a).

The anterior and medial prosencephalic epitheliumgenerated the rostralmost pallial domains: the anteriorparts of the ventral pallium, including the olfactory bulb,as well as the medial regions of lateral and dorsalpallium. Cells in the prosencephalic dorsal midline

formed pallial septum and the commissural plate.The pallial septum, which is preceded rostrally bythe subpallial septal domain, contains the pallial andhippocampal commissure, and the anterior tip of thechoroidal plexus.

The fimbria is a longitudinal fiber tract that developsat the insertion of the choroidal tela (primordium of thechoroidal plexus) in the medial pallium (hippocampus).This structure has been proposed as a medial organizerregion known as the ‘cortical hem’ (Shimamura andRubenstein 1997; Rubenstein et al. 1998; Cheng et al.2006; Storm et al. 2006), and in fact, several genesare specifically expressed here: Fgf8 (Crossley and Martin1995; Crossley et al. 1996; Shimamura and Rubenstein1997), Noggin (Shimamura et al. 1995), BMP (Furutaet al. 1997; Pera and Kessel 1997; Golden et al. 1999;Streit and Stern 1999) and Wnt (Lindwall et al. 2007).In this organizer, a Wnt signal seems to be the principalmolecule involved in the normal development of themedial pallium (Garda et al. 2002; Shimogori et al.2004). When the telencephalic vesicle evaginates, thecaudal roof plate of the telencephalon extends rhom-boidally, resulting in a cerebellar-rombic-lip-like ‘corticalhem organizer’ (Fig. 5a). In both areas, hippocampus andcerebellum occur in extended periods of neurogenesis,suggesting an analogous morphogenetic mechanismat the cerebellar rhombic lip (reviewed by Chizhikovand Millen 2005) and at the hippocampal fimbria.

In the grafts of the caudal prosencephalic epithelium(Fig. 5k,l) the donor cells generated the dorsal, lateraland medial pallium in their lateral part; whereas in theroof plate, the donor epithelium included the caudalseptum, medial part of the fimbria and a part of thechoroidal plexus. Quail cells were detected forming thechoroid roof tissue (associated with the fimbria) solelywhen the caudal part of the pallium and the anteriordiencephalon (prethalamus; Puelles and Rubenstein2003) were included in the transplant. On the contrary,when slightly more rostral grafts were carried out,exclusively medial pallium was mapped in the septalmidline roof, with very few choroid derivatives. Accordingto these data, it was suggested that the roof plate of

Table 1. Neural plate fate maps according to species and to methodology employed

Species Authors Methodology

Ambystoma mexicano Jacobson, 1959 Albin-pigmented urodel graftsXenopus leavis Eagleson & Harris, 1990 Fluorescent labellingDanio renio Woo et al. 1995 Dextran labelling

Staudt and Houart, 2007 Cell grafts and ablationsMus musculus Inoue et al. 2000 DiI labellingGallus gallus Couly and Le Douarin, 1985, 1987 Quail-chick graftsGallus gallus Cobos et al. 2001b Quail-chick graftsGallus gallus Fernandez-Garre, 2002 Carboxyfluorescein diacetate succinimidyl ester (CFSE) marker



the telencephalon hardly has any choroid derivatives.Therefore, the prosencephalic choroid components aremostly situated caudally, in the adjacent diencephalicroof (Fig. 4a).

We observed that the presumptive pallial subdomainsappeared to be located essentially transverse to thelongitudinal axis. The ventral pallium primordium wasthe rostralmost area in contact with the subpallium,whereas the lateral and the dorsal pallium occupiedposterior positions, preceding the medial pallium (Fig.4a). In addition, the medial pallium and the ventral palliumformed a continuous ring (the pallial ring; Puelles2001a) around a central island formed by the lateraland the dorsal pallium (Fig. 4a; Pombero and Martinez2009). On the other hand, the medial pallium appearedrostral to the future prethalamus (anterior diencephalicprosomeric region or p3; Puelles and Rubenstein2003), represented dorsally by the primordium of theprethalamic eminence. The telencephalic/diencephalicboundary was established at the roof plate just behindthe choroidal insertion, in the prospective fimbria andthe hippocampal commissure (Fig. 4a).

As a consequence of our data, the limits separatingthe intrapallial areas were represented as approximatelyparallel transverse limits (Fig. 4a). The topologicaltransverse character of the major pallial and subpallialdomains is highly significant for the understanding oftelencephalic anteroventral versus dorsoventral patterning,and their corresponding mechanisms. However, theinterpretation of pallial domains as the transversedisposition of the intrapallial domains needed to bequalified by the fact that the medial and the ventralpallium contact each other at the retrobulbar andamygdaloid meeting points, enclosing the dorsal andthe lateral pallium as an island (Bayer and Altman 1991;Puelles 2001a; Puelles et al. 2007; Pombero andMartinez 2009).

Both, cellular grafts (Pombero and Martinez 2009)and molecular regionalization (Puelles et al. 1999, 2000;Puelles and Rubenstein 2003) agree to identify sub-pallial and pallilal derivatives in the amygdala complex.Therefore, the primordium of the amygdala was drawnin the lateral part of the prosencephalic vesicle involvingthe lateral part of the pallial and subpallial primordia(Fig. 4a).

In conclusion, the reported fate maps predicted thepresumptive neuroepithelial progenitor regions of the maintelencephalic pallial and subpallial regions distributed inthe anterior prosencephalic vesicle, when the differentialmolecular characteristics or structurally visible landmarkshave not yet appeared (Fig. 4a).

The hypothalamus. The hypothalamus forms the non-telencephalic part of the secondary prosencephalon

(Puelles and Rubenstein 2003). While the roof and mostof the alar plates of this anterior region were includedin the telencephalon, the hypothalamus consists ofmore ventral alar, basal and floor plate components.Following novel classifications, we have includedlamina terminalis (lt) and preoptic area (POA) intothe subpallial domain of telencephalon (Puelles et al.2007).

The optic chiasma (oc) develops across the medianhypothalamic alar plate (Fig. 4a). Then the alar hypoth-alamus becomes divided into an upper optopeduncular(Op)-paraventricular (Pa) area, as well as a correspondingsuboptoeduncular (sOP) and subparaventricular (sPa)area, underneath (Puelles et al. 2008).

The basal hypothalamus has been subdivided intorostral and caudal halves (Puelles et al. 2008).The rostral hypothalamus contains the anlage of theantero-basal nucleus (ABa), which crosses the midlineof the basal plate (from left to right), and more ventralthan the ventromedial hypotamic nucleus (VMH), whilethe floor plate of this region contains the presumptiveterritories of the arcuate nucleus (Arc), median eminence(ME) and infundibulum/posterior pituitary (Pit) (Fig. 4a).The caudal hypothalamus is formed dorsally by thedorsomedial hypothalamic nucleus (DMH) and posteriorhypothalamic area (PH) and ventrally by the mammillary-retromammillary region (MM) with the retromammillarycommissure (xrm) (Fig. 4a).

Diencephalon. In the prosomeric model, the dien-cephalon proper represents the continuation of theforebrain caudal to the hypothalamus, down to theborder with the midbrain. Studies of gene expressionpatterns during vertebrate brain development demon-strated that the alar plate of the diencephalic epitheliumis a heterogeneous region, showing specific geneticexpression patterns that subdivide it into several moleculardomains (Rubenstein et al. 1994; Shimamura et al.1995; Yoon et al. 2000; Larsen et al. 2001; Puelles andRubenstein 2003). Most of these reports, have identifiedthree main transversal regions, which are, from caudalto rostral: the pretectal region (PT; synencephalon or thep1 alar plate), the thalamus (Th; posterior parencephalon,dorsal thalamus or the p2 alar plate) and the pretha-lamus (PTh; anterior parencephalon, ventral thalamusor the p3 alar plate). We recently produced a detailedfate map of presumptive territories in the diencephalonof a chick at the 10–11 somite stages (HH9–10), bymeans of homotopic and isochronic quail-chick grafts(Garcia-Lopez et al. 2004). The resulting fate mapdescribes the distribution of the presumptive diencephalicprosomeres in the neural tube, and demonstratestheir topologically conserved relationships throughoutthe neural development.



Prosomere 1 (p1) alar plate. Chimeric embryos withcaudal diencephalic grafts showed that the transplantedalar epithelium developed pretectal structures, such asthe dorsal components of each of the commissural,juxtacommissural and precommissural domains(Figs 2a,b, 4a) (Redies et al. 2000; Yoon et al. 2000;Garcia-Lopez et al. 2004; Puelles et al. 2007). Weobserved that the roof plate of the pretectal region (p1)contained the posterior commissure (pc) and rostrallyto it, the dorsal area of the precommissural region,which ended at the p2 roof plate derivatives (pineal [Pi]and diencephalic choroidal plexus [ch]). The limitbetween the diencephalon and the mesencephalon isidentifiable, even at stage HH8, by the caudal edge ofPax6 expression domain (Crossley et al. 2001; Lim andGolden 2002), and by the rostral edge of En1 expression(Araki and Nakamura 1999). The present fate maplocalized the diencephalic-mesencephalic limit at theanterior edge of the mesencephalic vesicle; themesencephalic-prosencephalic groove labels this limitexactly (Fig. 4a). We had neither observed cell migrationfrom the pretectal region to rostral p2 nor to caudalmesencephalic domains.

Prosomere 2 (p2) alar plate. Chimeric embryos withmore rostral grafts showed that anterior thalamic (p2)and prethalamic (p3) roof plates developed the choroidalplexus, which rostrally continues with the telencephalicchoroidal plexus. The caudal part of the p2 roof platedevelops the habenular commissure (xhb) and the pinealgland, at its caudal limit with p1 (Fig. 4a). The nucleiderived from the p2 alar epithelium were from dorsalto ventral: the habenular nucleu (Hb) in the epithalamus(ET); the dorso-lateral thalamic complex (DL), thenucleus rotundus (Rot), as well as the medial geniculatenucleus (MG) (Figs 2e,f, 4A). When the ZLI (Zona limitansintrathalamica) was included in the anterior pole of thegraft, some additional structures appeared to be formedby the quail cells: around the nucleus rotundus, boththe perirotundic area (PRot) and the subrotundusnucleus (sRot) were formed by donor cells, as well asthe intergeniculate leaflet (IGL) (Garcia-Lopez et al.2004). These nuclear structures formed an anterior andsuperficial belt around the nucleus rotundus. Dispersionof neuroepithelial transplanted cells over short distances(50–60 μm) in the anteroposterior or dorsoventral axeswere sometimes observed, when the graft partiallyincluded a segment inside p2 (Fig. 2b), suggestingsome restriction to cell movements inside the segment.Significant cell movements were not observed from thedorsal thalamus into the precommissural region (anteriorp1 derivatives, Fig. 2f ) or into the anterior thalamicnuclei, suggesting a significant restriction to cell move-ments across the interprosomeric limits (Fig. 2b).

Prosomere 3 (p3) alar plate. The anterior diencephalondevelops complex morphogenetic transformationsrostrally to the ZLI due to the extensive growth of thetelencephalon and the optic vesicles. This differentialgrowth deforms dorsal areas of its alar and its roofplates to generate a pronounced ventricular ridgebetween the diencephalon and the telencephalon,known as the eminentia prethalami (PThE). Rostraldiencephalic grafts generated the anterior diencephalicregion, formed by the PThE (dorsally) and the pre-thalamus (PTh, ventrally) and in most of the cases alsocontained caudal telencephalic territories. Chimericembryos with these grafts showed that the oval pre-geniculate nucleus (OvPG), reticular thalamic nucleus (Rt),intercalated nucleus (ICl), nucleus of stria medullaris(sm) and more superficially, pregeniculate nucleus (PG)and subgeniculate nucleus (SubG; Fig. 2i,j) derivedfrom the prethalamic neuroepithelium (PTh).

Diencephalic basal plate. Basal plate grafts were carriedout throughout a dorsal window made from a roofplate section, and after alar plate lateralization (Garcia-Lopez et al. 2004). In relation to the three alar regionsdescribed, we transplanted the corresponding basalneural epithelium. Long-survival explants showed thatthe derived neural structures were localized in thepretectal tegmental region, known as the p1 tegmentum(p1Tg), where the interstitial nucleus of Cajal (InC), theperiventricular tegmental area (VTA) and the anteriorpart of substantia nigra (SNC) were formed by quailcells (Fig. 2c,d). Furthermore, we did not find massivemigratory movements of neuroepithelial grafted cellscrossing the predicted limits between the alar-basalplates (ventro-dorsal migration) or the interprosomericdomains.

The basal plate of the thalamus (p2), was alsomapped generating a narrow domain of the tegmentalstructures (Fig. 2g,h), known as p2 tegmentum (p2Tg)formed by: part of the periventricular tegmental area(VTA), the interstitial rostral nucleus (InR), the anteriorpole of substantia nigra (SNC) (Fig. 2g,h). Again we didnot identify significant cell movements crossing interfieldboundaries in any direction.

We explored the fate map of the prethalamic basalplate. These chimeras showed that quail cells formed thecaudal hypothalamus. In this fate-map we describedthat basal plate of p3 generated the retrommamillarytegmentum (RM) and subthalamic nucleus (STh) (theold anterior nucleus of the ansa lenticularis, Ala; Jiaoet al. 2000). STh cells migrated ventro-dorsally fromthe RM to more dorsal hypothalamus close to thetelencephalic peduncle (Fig. 2k,l).

Our grafts confirmed classical neuromeric descriptionsthat recognized the tegmental region between the



posterior hypothalamus and the mesencephalon, asthe basal plate corresponded to the thalamic andpretectal regions (see Puelles et al. 1987 for a review).We demonstrated that basal plate grafts of the corre-sponding diencephalic areas mapped different antero-posterior domains of p1–p3 tegmentum (p1–p3Tg),and do not generate basal hypothalamic structures.The developed fate-map agrees with the existence ofpseudo-longitudinal domains, which develops homologousstructures in each segment: the substantia nigra (SNC),ventral tegmental area (VTA), interstitial of Cajal nucleus(InC) together with the interstitial rostral nucleus (InR)in p1 and p2 domains. Rostrally, p3 basal plate retainsSNC and VTA components, but new cells populationslike STh and retromammillar tegmentum (RMM), repre-sent the transition to the more complex hypothalamicstructure.

Midbrain

The avian midbrain is wedged between prosomere 1and the isthmus, part of the hindbrain. Homotopic andisochronic quail-chick grafting experiments carried outin the late 1980s and the 1990s consistently showedthat the presumptive midbrain does not extend over thewhole length of the early vesicle classically conceivedas the ‘midbrain’ (Martinez and Alvarado-Mallart 1989;Hallonet et al. 1990; Alvarez-Otero 1993; Hallonet andLe Douarin 1993; Marin and Puelles 1994; Millet et al.1996; Puelles et al. 1996). The caudal portion of thisvesicle produces instead the rostralmost hindbrain,including at least the isthmus, with the trochlear motornucleus and the isthmic nuclei, and the median partof the presumptive cerebellum.

Its alar region is considerably enlarged rostrocaudallyand mediolaterally when compared with the basalregion, resulting in the so-called ‘optic lobe’, or optictectum (Tect), the homolog of the mammalian superiorcolliculus. The auditory structures that are fieldhomologous to the mammalian inferior colliculus arescarcely represented at the brain surface and buildinstead a massive intraventricular bulge, the torussemicircularis (ToS).

Alar plate. The alar region is divided into four differenthistogenetic domains, which form a rostrocaudal sequenceof topologically transverse complexes. Rostrally, there isthe tectal gray (TG), just behind the pretectum, followedby the optic tectum (Tect) and the torus semicircularis(ToS). Caudally, there is the preisthmic area (PreIs),wedged between the torus and the isthmus proper(Hidalgo-Sanchez et al. 2005; Puelles et al. 2007).

The tectal gray (TG) is a stratified midbrain structureattached to the rostral border of the optic tectum

(Garcia-Calero et al. 2002; Muller et al. 2004; Puelleset al. 2007).

The optic tectum (Tect). A study using quail/chickchimeric embryos with partial tectal transplants dem-onstrated important tangential migration of neurons intothe stratum griseum centrale (SGC) and the uppermostlaminae of the stratum griseum et fibrosum superficiale(SGFS) (Alvarado-Mallart and Sotelo 1984; Senut andAlvarado-Mallart 1987; Martinez et al. 1992).

The torus semicircularis (ToS) (Puelles et al. 1994) inthe chicken was mapped in structural subdivisions byPuelles et al. (1994). The torus semicircularis was foundto consist of three main divisions: intercollicular area,toral nucleus, and toral superficial area complex.

The preisthmic area (PreIs) of the midbrain is a recentconcept (Hidalgo-Sanchez et al. 2005). Conventionally,the toral midbrain domain was thought to contact thehindbrain isthmus, so that an outstanding nucleusfound in this transitional area (called ‘nucleus isthmimagnocellularis). Recently, the nucleus isthmi magno-cellularis was renamed magnocellular preisthmic nucleus(MCPI; Puelles et al. 2007). However, MCPI representsonly the superficial stratum of the preistmic area, adeep appearing preisthmic part of the midbrain (PreIs,Puelles et al. 2007).

Liminar zone. This zone is represented by a longitudinaldomain at the limit between the alar and basal plates.It is formed rostrally, under the tectal gray, by thenucleus of the tectothalamic tract (TTh; Puelles et al.2007). More caudally, under the tectum and torus,there is the intercollicular area (ICo; Puelles et al. 1994;Puelles et al. 2007).

Basal and floor plates. The midbrain tegmentum (mTg)is larger than the diverse diencephalic tegmental regions,but shows some continuity with them (Puelles et al.2007). A characteristic midbrain tegmental element isthe oculomotor complex (including the Edinger-Westphal nucleus) (IIIm). The intermediate tegmentalstratum is represented by the midbrain reticular formation(mRt), the magnocellular red nucleus (RMC), themesencephalic part of the pararubral nucleus (mPaR).The ventral tegmental area and the substantia nigracompact (VTA, SNC) lies within the PreIs tegmental area(Puelles et al. 2007). The reticular part of the substantianigra (SNR) has been identified only in the midbrain ina central part of the tegmentum (Puelles et al. 2007).

Hindbrain

Early in development, transverse intersegmental boundariesdivide the central part of the hindbrain (rhombencephalon)into neuromeres, called ‘rhombomeres’ (Vaage 1969;



Lumsden 1990; Gilland and Baker 1993). Thesemorphological units are polyclonal neuroepithelialcompartments (Frase et al. 1990; Birgbauer and Fraser1994), which are mutually bound by characteristicinterrhombomeric cell populations (Layer and Alber 1990;Martinez et al. 1992; Heyman et al. 1993). Rhombomeresexpress restricted combinations of developmentalgenes, which are presumed to define the respectiveidentity and histogenetic fate of each hindbrain segment(reviews by Krumlauf et al. 1993 and Wilkinson 1993).Fate-map studies of the chicken rostral hindbrainaddressed exclusively rhombomeres 2–6 (Tan and LeDouarin 1991; Marin and Puelles 1995; Wingate andLumsden 1996). However, the hindbrain territory rostralto the rhombomere 2 (r2) is divided into an isthmusproper (r0) and an r1 proper (Martinez and Alvarado-Mallart 1989; Hallonet et al. 1990; Alvarado-Mallart 1993;Alvarez-Otero 1993; Hallonet and Le Douarin 1993;Millet et al. 1996; Wingate and Hatten 1999; Wingate2001; Gilthorpe et al. 2002; Aroca et al. 2006). These tworegions include the cerebellum as a dorsal derivative(‘isthmocerebellum’; Rubenstein and Puelles 1994).Patterning and neuronal differentiation within thisterritory are controlled by the isthmic organizer (Crossleyet al. 1996; Martinez 2001; Ye et al. 2001; Sato et al.2004; Aroca and Puelles 2005; Nakamura and Watanabe2005; Nakamura et al. 2005). Studies centered on therole of Hox and other regulatory genes confirmed thesegmental nature of the hindbrain (revised by Krumlaufet al. 1993; Kmita and Duboule 2003; Deschamps andvas Nes 2005; Iimura and Pourquie 2007).

Isthmus (r0). The isthmus is properly viewed as therostralmost part of the hindbrain. Its morphogeneticand structural peculiarities compared with the caudalmidbrain and hindbrain can be attributed to its pro-ximity to the isthmic secondary organizer, active duringdevelopment at the istmo-mesencephalic junction(Martinez and Alvarado-Mallart 1989; Hallonet et al.1990; Alvarado-Mallart 1993; Hallonet and Le Douarin1993; Marin and Puelles 1994; Puelles et al. 1996; Hal-lonet and Alvarado-Mallart 1997; Martinez 2001; Yeet al. 2001; Sato et al. 2004; Aroca and Puelles 2005;Nakamura and Watanabe 2005; Nakamura et al. 2005).Using quail/chick chimeras, it was demostrated thatthe transplanted quail mesencephalic alar plate givesrise to metencephalic derivatives that were the isthmicregion and portion of the rostral cerebellum, in additionto the already described mesencephalic derivatives:optic tectum and mesencephalic grisea (Alvarado-Mallart and Sotelo 1984; Senut and Alvarado-Mallart1987; Martinez and Alvarado-Mallart 1989).

The alar plate of r0 largely forms a part of an indistinctisthmic reticular formation, the main isthmic nuclei

represented by the isthmic parvicellular nucleus (IsPC),and the isthmooptic nucleus (IsO) (Puelles and Martinez-de-la-Torre, 1987; Martinez and Puelles 1989; Arocaand Puelles 2005). A caudodorsal part of the r0 alarplate was shown to give rise to the entire medianvermal sector of the cerebellum, extending all the waydown to the rhombic lip and the choroidal roof tissueat the back of the cerebellum (Aroca and Puelles 2005).

Isthmic basal plate generates the cochlear motornucleus (IVm) and reticular formation (IsRt). The paramedialand floor plate generate the dorsal raphe nucleus (DR)and the caudal pole of VTA and SNC (Fig. 4b).

Rhombomere 1 (r1). Fate-map analysis (Aroca et al.2006) showed that the major cholinergic nucleus in r1was the semilunar nucleus (SLu; found lateral andsuperficial, just caudal to the IsPC). Other derivativesof the r1 alar plate are the ventral isthmic nucleus (IsV;ventrocaudal relative to the ventral horn of IsPC), thelaterodorsal tegmental nucleus (LDT; within the peri-ventricular stratum), the pedunculopontine nucleus (PP)and the so-called nucleus profundus mesencephalic(pM). Caudodorsal to all of these nuclei, the alar deriv-atives of r1 expanded into the rostralmost part of thesuperior vestibular nucleus and paramedian sector ofthe cerebellum. Also, alar r1 produced the major partof the largest nor-adrenergic cell population in the isth-mocerebellum, the locus coeruleus (LoC) plus theassociated intracerebellar A4 cell group (Aroca et al.2006). The experimental results confirmed that the LoCoriginates in the alar plate throughout the rostrocaudalextent of r1 and ruled out a rostrocaudal translocation.LoC cells migrate from their origin in the alar plate to afinal position in the basal plate. Other heterogeneousnuclei formed in alar r1 included the entire parabrachialcomplex, the dorsal nucleus of the lateral lemniscus,the main sensory trigeminal nucleus, another part ofthe superior vestibular nucleus, and the major lateraland auricular parts of the cerebellum (Vaage 1973;Feirabend 1990; Hallonet and Le Douarin 1993; Marinand Puelles 1995; Wingate and Hatten 1999; Arocaet al. 2006).

Concerning the origin of the cerebellum, the chimericapproach has been extremely useful in determining thelocation and extent of the cerebellar primordium in theavian neural tube (Martinez and Alvarado-Mallart 1989;Hallonet et al. 1990). The cerebellum arises fromboth mesencephalic and rhombencephalic vesicles, incontrast to the classical view of its exclusive rhomben-cephalic origin. The chimeric approach has alsodetermined that the cerebellum primordium extendedfrom the caudal third of the mesencephalic vesicle tothe anterior part of r2, as the entire r1 and most ante-rior part of r2 provide cells for the medial and most



lateral parts of the cerebellum, respectively (Martinezand Alvarado-Mallart 1989; Hallonet et al. 1990; Marinand Puelles 1995; Sotelo 2004).

Rhombomeres. Previous fate map of the avian hindbrainconsisted of grafts of the entire length of the rhomben-cephalon caudal to the cerebellum (r3–r8) with emphasison the analysis of different dorsoventral wall sectorsand cell migrations (Tan and Le Douarin 1991). Later,quail rhombomeres two to six (r2–r6) were individuallygrafted homotopically into the hindbrain of chickembryos (Marin and Puelles 1995).

The principal results, obtained in the fate-map of therostral hindbrain (Fig. 4b), elaborated by Marin and Puelles(1995), are described in the following paragraphs.

Chochlear nuclei. The cochlear longitudinal columnconsists conventionally of the two cochleo-recipientnuclei angularis (cha) and magnocellularis (chmc).Other functionally related acoustic centers include thenucleus laminaris, the superior olive and the diverse laterallemniscal nuclei (Cajal 1908b; Boord and Rasmussen1963; Parks and Rubel 1978; Arends and Zeigler 1986).In the chimeric embryos, Marin and Puelles (1995)observed that nucleus angularis (a) was formed acrossr3 and r4. Also they observed that nucleus angularisis normally covered superficially by a parvicellular stratumcalled the ‘angularis shell’ (as) formation, which isextended across r3 to r5 (Fig. 4b). The nucleus magno-cellularis (mc) was unlabeled in r5 grafts and only itsrostral part appeared labeled in r6 grafts. The nucleuslaminaris (la) derived from r5 and r6 and nucleus olivarissuperior (os) from r5. The avian lemniscus showsseveral caudorostrally ordered interstitial nuclei (Arendsand Zeigler 1986). The caudalmost one (the ventralnucleus, llv) appeared within the r3-derived domain.However, this nucleus contained numerous neuronsthat had migrated from r4 and r5. Rostrodorsal to itappears the intermediate nucleus of the lateral lemniscus(lli), which formed within the r2-derived domain.

Vestibular nuclei. This column lies just ventral to thecochlear one (Hugosson 1957). Marin and Puelles(1995) found that all of the commonly accepted sub-divisions of the vestibular column (Wold 1976) wereoriginated across two or more rhombomeres, with theexception of the Deiters ventralis nucleus (Dv), whichis restricted to r4. They observed that the rostral com-plex of the vestibular column consists of a superficialcomponent (nucleus quadrangularis), an intermediatecomponent (nucleus superior sensu stricto) and adeep or medial component (nucleus A of Wold 1976)extended across r1 to r3. The nucleus vestibularisdesdendens (d) extends caudalwards from the ventral

Deiters nucleus, stretching across r5 to r7. In theirchimeras, the nucleus tangentialis (tg) and the VIII nerveroot placed across the r4/r5 limit. The nucleus vestib-ularis medialis (m) cross r4 to r7 and the nucleus ofDeiters dorsalis (Dd) is formed by r3, r4 and r5 cellsbut their grafts of r2 also showed that some cellsmigrated into this formation. Finally, the superficialvestibular population (B) known as cell group B (Wold1976) was placed at the boundary between the vestibularand trigeminal columns and they appeared labeled intheir r4 and r5 chimeras.

Trigeminal column. This column is placed superficially,ventral to the vestibular nuclei and is related to theascending and descending tracts of the trigeminalnerve. Marin and Puelles (1995) showed that grafts ofr1 formed the well-defined nucleus principalis trigeminalis(p) and the descending trigeminal column (Vd) extendedbetween r2 and the spinal dorsal horn.

Motor nuclei. The rhombomeric origin of the motornuclei, which derive initially from a medial longitudinalcolumn, were observed in the basal plate. The principaltrigeminal motor nucleus (V) formed within r2 andthe caudal ones were found to belong to r3, with theexception of a small rostralmost cell group, whichappeared related to the host r1 in r2 grafts. The dorsaltrigeminal motor nucleus appeared in more medialsections, clearly within r3 (dV). The dorsal facial motornucleus was formed from r4 and r5 (VII, Fig. 4b) whilethe superficial facial motor nucleus was also formedfrom r4 and r5 (sVII), the abducens motor nucleus(VI) and the accessory abducens motor nucleus wereformed from r5 and r6 portions, the dorsal motorglossopharyngeal nucleus appeared divided into r6 and r7(IX) and the retrofacial nucleus of the glossopharyngeusderived from r6 (rIX).

Reticular formation. Marin and Puelles (1995) identifiedprecisely only the nucleus reticularis pontis caudalis parsgigantocellularis, which developed within r3 and r4 (gc).

Medulla oblongata. The medulla oblongata, which con-tains sensory, motor, and reticular reflex centers, is thehindbrain caudal region. The caudal hindbrain wasconsidered as a non-segmented region, according tocriteria based on gross morphologic observations(Tanaka et al. 1987). The fact that the medullo–spinalboundary cannot be detected without fate-maps studiessupported the non-segmental interpretation. Therefore,the subregions that constitute the medulla oblongatawere called pseudo-rhombomeres, which implied certainsimilarity of pseudorhombomeres to rhombomeres, butdoes not assume a segmental nature (Lumsden 1990;



Cambronero and Puelles 2000). This region seemed toshare cellular and morphological features with thespinal cord. On the other hand, the results obtainedfrom Hox gene expression studies in the rostral hindbrain(revised by Nolte and Krumlauf 2005) and the caudalhindbrain (Marin et al. 2008) showed that there wereimportant differences in gene expression patterning inboth areas.

The fate-map of the medulla oblongata (Cambroneroand Puelles 2000) together with the correlation betweenthe pseudorhombomeres and the Hox gene expression(Marin et al. 2008), largely supported the hypothesis ofa hidden metameric organization present in the avianmedulla oblongata (Cambronero and Puelles 2000;Marin et al. 2008).

The fate-map of the caudal hindbrain at stage HH10-11 (Cambronero and Puelles 2000), which comple-mented the Marin and Puelles (1995) rhombomere fatemap, threw much light on the histogenesis of thenon-segmented medullary hindbrain portion and itsneuronal aggregates, and it also helped to understandthe boundary patterns of the medullary nuclear sub-divisions. The methodology used implied grafting neuraltube pieces, taking the centers of contiguous somitesas the limits between adjacent pseudorhombomeres(somitic-antimeric neural tube portions; Vaage 1969).This allowed them to map the caudal hindbrain with aresolution similar to that used before in the r2–r6domain (Marin and Puelles 1995). Consecutive numberswere assigned to the rhombomere series and quotationmarks (‘r7’–‘r11’) were used to emphasize their atypicalcharacter.

Longitudinal functional columns were describedclassically in the hindbrain, resulting in a multisegmentalorganization of several nuclei across r1–r6 (Hugosson1955; Marin and Puelles 1995; Diaz et al. 1998) thatwas also observed in the pseudorhombomeric domainstudied through the fate-map elaborated by Cambroneroand Puelles (2000). An important conclusion of thefate-map, however, was that most nuclei had limits thatcoincided with the interpseudorhombomeric limits,which constituted an evidence of a latent organizing ofthe chicken medulla oblongata in relation to the planesparameric to intersomitic limits. Moreover, they observedthat the number of columns was diminished at thelimit between spinal cord and hindbrain, suggesting anintertagmatic nature of this boundary.

According to the data obtained from this fate-map,the rombospinal boundary lies between ‘r11’ and ‘my1’, atthe center of the fifth somite (Fig. 4b). The fate–mappedmedullospinal boundary was found slightly caudally tothe obex and connected to the end of the choroidaltissues at the roof plate (ch). The complete solitarynucleus, the vestibulocochlear column, the interpolar

trigeminal sensory subnucleus, and the hypoglossal,ambiguus and vagal nuclei as well as the whole inferiorolive and raphe nuclear complexes were found in themedullar segment; whereas the dorsal column nucleiand the spinal sensory trigeminal subnucleus mainly fellwithin the rostralmost spinal cord.

The principal results obtained in the fate-map of themedulla oblongata (Fig. 4b), elaborated by Cambroneroand Puelles (2000), are described in following paragraphs.

Alar plate

Choroidal plexus of the roof plate. The choroidal roof (ch)of the hindbrain starts at the rostral to r1 (Marin andPuelles 1995) and end caudally in ‘r11’ according with thefate-map elaborated by Cambronero and Puelles (2000).

Chochlear column. This formation gives rise to thecerebellum (cb) and the cochlear and vestibular nuclei.The choclear column extended from r3 (Marin andPuelles 1995) to ‘r8’ just caudal to the cerebellum,whereas the choclear nucleus magnocellularis (mc)involved the portion comprising from r6 to ‘r8’. Theyobserved that the medial vestibular nucleus (m), thecaudal portion of which was positive for Hox4 genes(Marin et al. 2008), ended at the ‘r8’/‘r9’ limit. The inferiorvestibular nucleus was traced ending into ‘r10’ (r5-‘r10’).Therefore, the whole vestibular column extended fromr1 (Marin and Puelles 1995) to ‘r10’.

Somatosensory and viscerosensory columns. Somato-sensory nuclei develop ventral to the statoaccousticcolumn and in a dorsal position in relation to viscero-sensory nuclei, which occupy a periventricular position.

It was observed that the descending trigeminal nucleus(t) was first located ventrally in the vestibular column,but its caudal continuation was progressively moredorsomedial as the vestibular (d) and solitary nuclei (s)disappeared in the caudal direction. All rhombomeres(Marin and Puelles 1995) and pseudorhombomerescontributed to this nucleus, which continued with thedorsal horn (dh) of the spinal cord (r1-‘r11’). Thus, thesomatosensory region was mapped starting at r1(Marin and Puelles 1995) and ending at ‘r11’, into thedorsal horn (dh) of the spinal cord (Tan and Le Douarin1991; Cambronero and Puelles 2000).

The solitary column (s), which it appeared locatedventral to the medial vestibular nucleus (m), was enclosedinto four pseudorhombomeric (‘r8’–‘r11’): ‘r8’ contributedto the rostral part, ‘r9’ to the lateral and medial sub-divisions and ‘r10’ and ‘r11’ to the caudal region.

Inferior olive, Pontine nuclei and Raphe nuclei. Theseventral hindbrain nuclei are characterized by the



presence of cells from the rhombic lip in their compo-sition. These cells, from the rhombic lip (rhl), tangentiallymigrate into the ventral hindbrain.

The inferior olive (oi) was found mapping in the ventralmedulla oblongata, from the caudal part of ‘r8’ to therostralmost part of ‘r11’. On the other hand, the lateral(pl) and the medial pontine nucleus (pm) extendedfrom the caudal end of r1 (Marin and Puelles 1995) to‘r8’. Additionally, an extensive tangential migration wasobserved from r6 (Marin and Puelles 1995), ‘r7’ and‘r8’ to rostral rhombomeres as r2 and r1.

It was proposed that the Raphe nuclei, which wasmapped from r6 (Marin and Puelles 1995) to ‘r11’, wasat least partially formed by cells from the rhombic lip(Harkmark 1954; Tan and Le Douarin 1991). In this fate-map, two raphe cell groups with the same longitudinalextent (r6–‘r11’) were detected: the nucleus rapheobscures (ro) and, ventrally, the nucleus raphe pallidus (rp).

Basal plate. The medullary basal plate was found ventrally,in a position adjacent to the raphe and the somaticmotor column. The basal medulla is represented by thehypoglossal nucleus (XII), the glossopharyngeus motornucleus (IX), the dorsal motor nucleus of the vagus (X),the ambiguous (amb) and the reticular formation.

The hypoglossal nucleus. This nucleus, which waslocated ventrally to the dorsal vagal nucleus, is subdividedinto two components: pars lingualis (XIIl), anterior part,and pars tracheosyringealis (XIIts), posterior region(Nottebohm et al. 1976). The boundary between its twoelements was described approximately at the ‘r10’/‘r11’ limit, whereas the pars lingualis was originated in‘r8’. The pars tracheosyringealis extended caudallyinto the spinal cord.

The vagal motor nucleus (X) and the glossopharyngeusnucleus (IX). The glossopharyngeus nucleus (IX) wasmapped from r6 to ‘r8’ and was followed by the vagalmotor nucleus (X), which was found extended from ‘r8’to the limit ‘r11’/‘my1’.

Ambiguus nucleus. It was observed that the ambiguousnucleus (amb) started just caudally to the retrofacialpart of the glossopharingeal nucleus (r6 and r7; Medinaand Reiner 1994; Marin and Puelles 1995) and continueduntil the pseudorhombomere ‘r11’. In addition, largemotor neurons derived from ‘r9’ were detected tangen-tially migrating to ‘r8’ and, less importantly, to ‘r10’.

Reticular formation. The grafts carried out from ‘r7’ to‘r11’ labeled the parvicellular reticular nucleus (rpc),located laterally. The gigantocellular reticular nucleus(rgc), which is included in the medial reticular formation,

was found between ‘r7’ and ‘r8’, whereas the centralreticular nucleus (rc), which is also integrated in themedial reticular formation, extended from ‘r9’ to ‘r11’caudally to the gigantocellular reticular nucleus (rgc).

This fate-map proposed a possible latent segmentationof the caudal hindbrain that was supported by Hox geneexpression analysis in this region, at the midgestationstage (Marin et al. 2008). The latter showed that Hoxgenes transversal boundaries coincided with mostpseudorhombomeric limits (Cambronero and Puelles2000): the Hoxd4 gene arrived at r6/‘r7’, Hox5 genesended at ‘r8’/‘r9’, Hox6 genes at ‘r9’/‘r10’ and Hox7genes at ‘r10’/‘r11’. Although the ‘r7’/‘r8’ boundary didnot present a specific Hox genes group to define thislimit, variations in the level of expression of the Hox4genes overlapped with this border (Marin et al. 2008).According to these results, medullary longitudinalcolumns appeared regionalized consistently with Hoxgene expression, as it was described in the rostralhindbrain (r2–r6; revised by Nolte and Krumlauf 2005),suggesting a common mechanism of segmentation inthe whole hindbrain. Gene expression variations werealso observed in some caudal hindbrain structures,suggesting that Hox genes may be involved in the internalpatterning of these structures (Marin et al. 2008).

Conclusion

Development of fate maps represents the best approachto analyze cellular mechanisms underlying brain mor-phogenesis. The establishment of clonal relationshipsbetween neuroepithelium and matle layer brain structuresis the necessary platform to adequately design embry-ology experiments to determine the relation betweenmolecular expression and cell fate in the nervous system.Sequential analysis using this approach at differentdevelopmental stages also seems necessary to appro-priately determine topographical modifications and cellmovements during brain development. The modernmethodologies to apply experiments during the earlystages of vertebrate development (experiments in ovo andin utero) necessarily will require these kinds of studies.

Acknowledgments

We would like to thank our technicians M. Rodenas,C. Redondo, A. Torregrosa and O. Bahamonde for tech-nical assistance in this study. We also express ourthanks to the URGASA farm (Lleida) for kindly providingquail eggs used for this work. This work was supportedby Spanish Grants BFU2008-00588 and IP from EULSHG-CT-2004-512003. A. Pombero was funded bythe Spanish MEC and R. Garcia-Lopez by the EuropeanUnion Grant: EU LSHG-CT-2004-512003.



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Abbreviations

A, amygdale; a, nucleus angularis; ABa, antero-basal nucleus of the hipothalamus; ac, anterior commissure; AEP, entopeduncular area;amb, ambiguus motor nucleus; AP, alar plate; ap, area postrema; APT, anterior pretectal nucleus; ar, alar reticular nucleus arc, arcuatenucleus; Avc, arcuate nucleus of the hypothalamus; aVI, accessory abducens motor nucleus; B, vestibular cell group B; br, basalreticular nucleus; cb, cerebellum; ce, external cuneate nucleus; ch, choroid plexus; cha, choclear nucleus angularis; chas, coclearnucleus angularis shell; chd, choclear nucleus leminaris; chmc, choclear nucleus magnocelularis; cho, nucleus olivary superior; cp,commissural plate, pallial commissure; d, descending vestibular nucleus; dcn, dorsal column nuclei; Dd, nucleus Deiters dorsalis; DF,dorsofrontal nucleus; dh, dorsal horn; Di, diencephalon; DL, dorsolateral anterior nucleus of the thalamus; DLL, dorsal nucleus of thelateral lemniscus; DM, dorsomedial anterior nucleus of the thalamus; DMH, dorsomedial hypothalamic nucleus; DP, dorsal pallium;DPT, diffuse pretectal area; DR, dorsal raphe nucleus; dV, dorsal trigeminal motor nucleus; Dv, nucleus Deiters ventralis; dVr, rostralpart of dorsal trigeminal motor nucleus; Eth, thalamic eminence; FB, forebrain; Hb, habenular nucleus; HB, hindbrain; ICl, intercalatednucleus; ICo, intercollicular area; IGL, intergeniculate leaflet; IIIm, oculomotor nerve; IIIv, third ventricle; InC, interstitial nucleus of Cajal;InR, rostral interstitial nucleus; IP, interpeduncular nucleus; IPT, intermediate pretectal nucleus; Is, isthmus; IsO, isthmooptic nucleus;IsPC, isthmic parvocellular nucleus; isRt, isthmic reticular formation; IsV, ventral isthmic nucleus; IVm, pathetic nucleus; IX, glossophar-ingeal motor nucleus; la, nucleus laminaris; lld, nucleus lemnisci lateralis, pars dorsalis; lli, nucleus lemnisci lateralis, pars intermedia;llv, nucleus lemnisci lateralis, pars ventralis; LoC, locus ceruleus; LP, lateral pallium; lt, lamina terminalis; m, medial vestibular nucleus; MB,midbrain; MCPI, magnocellular preisthmic nucleus; ME, medial eminence; Mes, mesencephalon; MG, medial geniculate nucleus;MM, mammillary/retromammillary region; MP, medial pallium; mRt, mesencephalic reticular formation; OB, olfactory bulb; och, opticchiasm; oid, inferior olivary nucleus pars dorsalis; oiv, inferior olivary nucleus pars ventralis; Op, optopeduncular area; os, optic stalk;OvPG, oval pregeniculate nucleus; p, principal sensory nucleus of the trigeminal column; pA, pallial amygdala; PA, pallidum; Pa,paraventricular area; Pal, pallium; pc, posterior commissure; PG, pregeniculate nucleus; PH, posterior hypothalamic area; Pi, pinealgland; Pit, infundibulum/posterior pituitary; pl, lateral pontine nucleus; pm, medial pontine nucleus; pM, mesencephalic profundusnucleus; POA, preoptic area; PRT, prerubral tegmentum; PreIs, preisthmic region of the midbrain; PRot, perirotundic area; PrPT, principalpretectal nucleus of the commissural pretectum; pSe, pallial septum; psp, pallial–subpallial boundary; PT, pretectum; PTh, prethalamus;PThE, prethalamic eminence; p1Rt, reticular formation of p1; p1Tg, pretectal tegmentum; p2Rt, reticular formation of p2; p2Tg, thalamictegmentum; p3, reticular formation of p3; R, retina; rc, central reticular nucleus; rgc, gigantocellular reticular nucleus; Rh, rhomben-cephalon; rIX, retrofacialglossopharyngeal motor nucleus; rl, lateral reticular nucleus; rhl, rhombic lip:; rhl(oi), inferior olivary nucleus;rhl(pl), lateral pontine nucleus; rhl(pm), medial pontine nucleus; rhl(egl), external granular layer; RM, retromammillary nucleus; RMC, rednucleus, magnocellular part; RMM, retromammillar tegmentum; ro, nucleus raphe obscurus; Rot, rotundus nucleus; rp, nucleus raphepallidus; RP, roof plate; rpc, parvicellular reticular nucleus; RPC, red nucleus, parvicellular part; rpgc, nucleus reticularis ponti/giganto-cellularis; Rt, reticular nucleus; s, nucleus of the tractus solitarius; Se, septum; SLu, nucleus semilunaris; SNC, substantia nigra, compactpart; SNR, substantia nigra, reticular part; sOp, suboptopeduncular area; SP, secondary prosencephalon; spA, subpallial amygdalesPa, subparaventricular area; sPal, subpallium spd, dorsal supraespinal motor nucleus; SpL, lateral spiriform nucleus; SpM, medialspiriform nucleus; spSe, subpallial septum; spv, ventral supraespinal motor nucleus; SRot, subrotundus nucleus of the thalamus; ST,striatum; STh, subthalamic nucleus; SubG, subgeniculate nucleus of the prethalamus; sVII, superficial facial motor nucleus; Tect, optictectum; TG, tectal gray formation; Th, thalamus; ToS, torus semicircularis; TPP, tegmento pedunculopontine nucleus; TTh, nucleus ofthe tectothalamic tract; V, trigeminal motor nucleus; vA, vestibular cell group A; Vd, descending trigeminal nucleus; vh, ventral horn ofspinal cord; VMH, ventromedial hypothalamic nucleus; vn, vestibular nuclei:; vn(i), inferior vestibular nucleus.; vnA, nucleus A of Wold;vnB, vestibular cell group B; vnd, vestibular descendens nucleus; vnDd, Deiters dorsalis nucleus; vnDv, Deiters ventralis nucleus; vnm,vestibular medial nucleus; vntg, nucleus tangentialis; vnVs, vestibular superior nucleus; Vp, principal trigeminal nucleus; Vd, descendenttrigeminal nucleus; VI, abducens motor nucleus; VII, medial facial motor nucleus; VP, ventral pallium; Vs, superior vestibular nucleus;VTA, ventral tegmental area; X, vagal motor nucleus; XI, spinal accessory nerve nucleus; XIIL, hypoglossal motor nucleus, pars lingualis;XIIts, hypoglossal motor nucleus, pars tracheosyringealis; xa, anterior commissure; xcb, cerebellar commissure; xhb, habenular com-missure; xIV, pathetic commissure; xp, pallial commissure; xrm, retromammillary comissure; xs, spinal commissure; xtect, tectalcommissure; zi, zona intermedia.

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Fate map of the chick embryo neural tube