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Border disputes: do boundaries play a role in growth-cone guidance?
Abstract
One of the earliest indications of regional patterning in the CNS is the spatially restricted expression of regulatory genes within the neuroepithelium. Many of these genes encode transcription factors and, although little is known of their downstream targets, it seems likely that they control the identity of cells in different regions of the CNS. This review discusses how the expression of these patterning genes might influence the location at which the first axon pathways in the CNS are pioneered. Evidence is described that suggests that the boundary regions between adjacent domains of regulatory gene expression influence where the first axons will extend.
... Mit dem Verlauf der frühen Axontrakte koinzidieren Grenzen zwischen den Expressionsdomänen regulatorischer Gene, die das Neuroepithel unterteilen und zur Ausbildung des geordneten Projektionsmusters der Axone beitragen (Wilson et al., 1993;Macdonald et al., 1994). Die growth cones später differenzierender Neurone verfügen über die Fähigkeit unabhängig von den bereits ausgebildeten Projektionen zu ihren Zielgebieten zu navigieren, was im letzten Abschnitt für die Wegfindung im retinotektalen Systems vorgestellt wird. ...
... Der embryonale Zebrafisch ist infolge seiner Transparenz, dem einfachen Zugang zum Nervensystem und einer detaillierten Beschreibung der Neuroanatomie zur in vivo Funktionsanalayse der molekularen Mechanismen der axonalen Wegfindung geeignet. Die Axone der primären Neurone wachsen als Pioniere zu ihren Zielgebieten (Chitnis und Kuwada 1990;Wilson et al., 1990;Wilson und Easter, 1991a, b;Ross et al., 1992), wobei die Expressionsmuster regulatorischer Gene das Neuroepithel in Domänen unterteilen, deren Grenzen die growth cones nach einer Hypothese von Wilson und Kollegen (1993) zur axonalen Wegfindung verwenden (Wilson et al., 1993;Macdonald et al., 1994). Während der ersten 24 hpf der Embryonalentwicklung wird somit ein stereotypes Axongrundgerüst im ZNS des Zebrafisches ausgebildet. ...
Zelladhäsionsmoleküle der Immunglobulin Superfamilie (IgSF-CAMs) vermitteln, neben Axonwachstum und Faszikulation, als Rezeptoren oder Teile von Rezeptorkomplexen auch wesentliche Funktionen bei der axonalen Wegfindung. Ziel dieser Arbeit war, die Funktionsanalyse der IgSF-CAMs Neurolin und E587-Antigen im Nervensystem des Gold- und des embryonalen Zebrafisches. Im Goldfisch stört eine Blockade von Neurolin durch polyklonale Antikörper die Wegfindung junger retinaler Ganglienzell-(RGZ) Axone zum Nervaustritt in der dorsalen Retina. Diese positionsspezifischen Effekte lassen vermuten, dass Neurolin als Rezeptor oder Teil eines Rezeptorkomplexes für eine Wegfindungskomponente fungiert. Zudem trägt Neurolin auch geringfügig zur dichten Bündelung der Axone im Faszikel bei. Eine Blockade von E587-Antigen durch polyklonale Antikörper führt zu einer ausgeprägten Defaszikulation, die ebenfalls verstärkt in der dorsalen Retina auftritt. E587Antigen ist somit entscheidend an der selektiven Faszikulation junger RGZ-Axone beteiligt und trägt im optischen Nerv zur Ausbildung einer Altersordnung bei. Eine Blockade von E587-Antigen im embryonalen Zebrafisch stört die Faszikulation spezifischer kommissuraler und longitudinaler Axontrakte im Vorder- und Hinterhirn. Während der Entwicklung des Zentralnervensystems im Zebrafisch ist das E587Antigen somit für die Ausbildung geordneter Axonprojektionen entlang eines Axon-Grundgerüstes erforderlich. Zur Funktionsanalyse von Neurolin wurde die Entwicklung der Projektionen sekundärer Motoraxone zu definierten Regionen des Myotoms untersucht. Eine Blockade von Neurolin führt zu Wegfindungsfehlern in allen Projektionen der sekundären Motoraxone und zu ihrer verzögerten Ausbildung sowie zur aufgelockerten Assoziation der Axone im Bündel. Im Zebrafisch ist Neurolin folglich für ein zielgerichtetes Wachstum und für korrekte Wegfindungsentscheidungen verantwortlich und fungiert dabei vermutlich als Rezeptor für eine Wegfindungsko Cell adhesion molecules (CAMs) of the immunoglobulin superfamily (IgSF) are involved in axon growth and fasciculation, and mediate essential functions as receptors or parts of receptor complexes during axon pathfinding. The aim of this work was to analyze the functions of the IgSF-CAMs Neurolin and E587-antigen in the nervous system of gold- and embryonic zebrafish. Blockage of Neurolin with polyclonal antibodies disturbs the pathfinding of young retinal ganglion cell (RGC) axons in the dorsal goldfish retina. These position specific defects suggests, that Neurolin functions as a receptor or part of a receptor complex for an axonal guidance component. Furthermore, Neurolin contributes slightly to the tight fasciculation of RGC axons. In contrast, blockage of E587-antigen through polyclonal antibodies causes a strong defasciculation of goldfish RGC axons, also most pronounced in the dorsal retina. E587-antigen is thus essentially involved in axon fasciculation and contributes to the creation of the age related order of RGC axons in the optic nerve. In embryonic zebrafish, blockage of E587-antigen disrupts the fasciculation of specific commissural and longitudinal axon trakts in the forebrain and hindbrain. Therefore E587-antigen is required for the creation of orderly axon projections along an axon-scaffold during the development of the zebrafish nervous system. Neurolin is only expressed on subsets of axons in embryonic zebrafish, e.g. the secondary motoraxons. Blockage of Neurolin causes pathfinding errors along all secondary motor axon projections, their delayed development and a less tight axon-association in fascicles. Thus, Neurolin is required for the directed growth and appropriate pathfinding decisions and most probably functions as a receptor for a guidance component in the path of secondary motor axons.
... An alternative explanation depends on the proximity to the boundary of regions of specific gene expression. Lumsden and Keynes (1989) first noted that axons in the hindbrain tended to grow in the interrhombomeric boundaries, and Wilson et al. (1993) have extended this idea to the prechordal brain of zebrafish, where regulatory genes are expressed in patches. The TPOC forms on the boundary of the pax-6 expression territory (Wilson et al., 1993); perhaps the same influences that caused the TPOC to appear there also cause the appearance of the pretract. ...
... Lumsden and Keynes (1989) first noted that axons in the hindbrain tended to grow in the interrhombomeric boundaries, and Wilson et al. (1993) have extended this idea to the prechordal brain of zebrafish, where regulatory genes are expressed in patches. The TPOC forms on the boundary of the pax-6 expression territory (Wilson et al., 1993); perhaps the same influences that caused the TPOC to appear there also cause the appearance of the pretract. A second alternative is that other axons already in place produce the pretract, perhaps by releasing diffusible signalling molecules. ...
The initial development of the optic tract was studied with light and electron microscopy in the zebrafish (Danio rerio). Intraocular injections of the fluorescent marker, 1,1'-dioctadecyl-3,3,3',3' tetramethylindocarbocyanine perchlorate (dil), labeled retinal axons and growth cones anterogradely, and injections of dil into the optic chiasm labeled retinal ganglion cells retrogradely. Labeled tissue was photoconverted and examined electron microscopically. The ventronasal retinal quadrant produced the first growth cones. They were the first growth cones in the optic stalk. The leading retinal growth cones, typically 4-10 in number, advanced alongside the tract of the postoptic commissure but rarely sent filopodia into it and never wrapped its axons. Instead, the retinal growth cones followed a pretract, a subpial region that was morphologically distinct from its surroundings and extended out in front of the leading growth cones, presaging the optic tract. Thus, the retinal growth cones, previously thought to be followers of preexisting axons, are actually cryptic pioneers whose proximity to the earlier axons masks their pioneering nature. We suggest that cryptic pioneers and pretracts are probably common elsewhere in the nervous system.
... Axons in con embryos that fail to exit the eye tend to form large fascicles that extend to the periphery of the eye along the equatorial region. It has been suggested that axons in the CNS grow along borders of gene expression domains to form the embryonic axon scaffold (Wilson et al., 1993 ). A similar phenomenon might be occurring for axons that are unable to exit the eye in con, revealing a functional border within the retina. ...
... What kind of cues might be responsible for determining the location of the optic nerve and postoptic commissure? As mentioned above, gene expression borders often correlate well with the positions of axon tracts in the developing CNS (Wilson et al., 1993 ). Several gene expression boundaries correspond with the position of the post-optic commissure and optic nerve in the developing zebrafish brain. ...
We have isolated mutants in the zebrafish Danio rerio that have defects in axonal connectivity between the retina and tectum. 5-day-old fish larvae were screened by labeling retinal ganglion cells with DiI and DiO and observing their axonal projections to and on the tectum.
82 mutations, representing 13 complementation groups and 6 single allele loci, were found that have defects in retinal ganglion cell axon pathfinding to the tectum. These pathfinding genes fall into five classes, based on the location of pathfinding errors between eye and tectum. In Class I mutant larvae (belladonna, detour, you-too, iguana, umleitung, blowout) axons grow directly to the ipsilateral tectal lobe after leaving the eye. Class II mutant larvae (chameleon, bashful) have ipsilaterally projecting axons and, in addition, pathfinding mistakes are seen within the eye. In Class III mutant larvae (esrom, tilsit, tofu) fewer axons than normal cross the midline, but some axons do reach the contralateral tectal lobe. Class IV mutant larvae (boxer, dackel, pinscher) have defects in axon sorting after the midline and retinal axons occasionally make further pathfinding errors upon reaching the contralateral tectal lobe. Finally, Class V mutant larvae (bashful, grumpy, sleepy, cyclops, astray) have anterior-posterior axon trajectory defects at or after the midline.
The analysis of these mutants supports several conclusions about the mechanisms of retinal axon pathfinding from eye to tectum. A series of sequential cues seems to guide retinal axons to the contralateral tectal lobe. Pre-existing axon tracts seem not to be necessary to guide axons across the midline. The midline itself seems to play a central role in guiding retinal axons. Axons in nearby regions of the brain seem to use different cues to cross the ventral midline. Mutant effects are not all- or-none, as misrouted axons may reach their target, and if they do, they project normally on the tectum. The retinotectal pathfinding mutants reveal important choice points encountered by neuronal growth cones as they navigate between eye and tectum.
... Analyses of expression domains of other genes from different families including the paired-domain-containing Pax family (Stoykova and Gruss, 1994) and the homeobox-containing Otx, Emx and Nkx families (Simeone et al., 1992a,b;Guazzi et al., 1990;Price et al., 1992;Shimamura et al., 1995) have provided further evidence for their hypothesis (see Boncinelli et al., 1993 andRubenstein et al., 1994 for reviews). Studies of chick and zebrafish embryos revealed that such gene expression boundaries partially restrict cell mixing and are correlated with the positions of early generated neurons and their axonal tracts (Figdor and Stern, 1993;Wilson et al., 1993;Macdonald et al., 1994;Guthrie, 1995). Although the functions of these regionspecific genes remain to be elucidated, some of them are expressed in specific populations of postmitotic neurons at later stages of development. ...
Although a number of genes have been found to have restricted expression domains in the embryonic forebrain and midbrain, it remains largely unknown how the expression of these genes is regulated at the cellular level. In this study, we explored the mechanisms for the differential expression of region-specific transcription factors in neuroepithelial cells by using both primary and immortalized neuroepithelial cells from the rat brain at embryonic day 11.5. We found that differential expression patterns of Pax-3, Pax-5, Pax-6, Dlx-1, Dlx-2, Emx2, Otx1 and Dbx observed in vivo were maintained even when the cells were isolated and cultured in vitro, free from environmental influences. Furthermore, in response to Sonic hedgehog, which is a major inductive signal from the environment for regional specification, neuroepithelial cells that maintain distinct regional identities expressed different sets of ventral-specific genes including Islet-1, Nkx-2.1 and Nkx-2.2. These results suggest that certain cell-autonomous mechanisms play important roles in regulating both environmental signal-dependent and -independent expression of region-specific genes. Thus, we propose that use of the in vitro culture systems we describe in this study facilitates the understanding of regulatory mechanisms of region-specific genes in neuroepithelial cells.
... This result suggests that the mechanisms guiding longitudinal axonal growth may be regulated by longitudinally arranged neuroepithelial domains. There is considerable interest in the possibility that boundary zones separating neuroepithelial domains may provide spatial information that direct the growth of axons (Wilson et al., 1993;Chien and Harris, 1994;MacDonald et al., 1994;Chédotal et al., 1995). Nkx-2.2 expression might define such a molecularly distinct longitudinal boundary zone. ...
Over the last century, several morphological models of forebrain organization have been proposed that hypothesize alternative topological solutions for the relationships of the histogenic primordia. Central to all of these models are their definitions of the longitudinal axis and the longitudinal organization of the neural plate and neural tube. To understand the longitudinal organization of the anterior brain, we have sought to identify molecular properties that are continuous along the entire longitudinal axis of the embryonic CNS. In this essay, we describe studies of the expression of several genes in the mouse between 7.5 (presomite stage) and 10.5 days post coitum (dpc) that provide evidence for the trajectory of the anteriorposterior axis and the longitudinal organization of the anterior CNS.
Specifically, we report that the expression of noggin, sonic hedgehog and Nkx-2.2 define longitudinal columns of cells that are present along the entire CNS axis. Within the forebrain, the expression of these genes, as well as that of Nkx-2.1 and BF-1, are in distinct longitudinal regions in the neural plate and tube. We demonstrate that the earliest longitudinal axon pathways of the forebrain are spatially correlated with the longitudinal domain defined by Nkx-2.2. Finally, expression of the former genes, and Otx-1 and Emx-2, suggests that the cephalic neural plate is organized into molecularly distinct domains delimited by longitudinal and transverse borders; these results provide a foundation for defining the mechanisms that pattern the neural plate.
... Recent observations suggest that early axonal tracts are established at the boundaries of gene expression domains (Wilson et a l, 1990;Macdonald et al, 1994. A number of models could explain how these regulatory genes act to guide axons at these boundaries (Fig 1.4; Wilson et al, 1993;Wilson et al, 1997). All of these models establish a complement of attractive and repulsive cues, which serve to push and pull the axons across a particular region. ...
The vertebrate forebrain is a highly complex structure containing millions of neurons that form highly ordered and precise connections. The mechanisms that give rise to this complexity are slowly being elucidated through work on a number of developmental systems. I have investigated the organisation of the zebrafish forebrain and the mechanisms by which a simple neuroepithelial sheet becomes patterned to the more complex adult forebrain structure. Initially, I examined the development of the zebrafish CNS during later stages of development (18 somites stage to 5 days postfertilisation) through studies on gene expression, axon pathfinding, morphology and cell proliferation. Using the prosomeric model as a basis, we can begin to understand how the regions demarcated by genes, such as Dlx2 and Emx1, relate to similar regions in other organisms, such as mice, chickens and turtles. In order to investigate the how different genes influence forebrain development, I conducted misexpression studies and have characterised zebrafish mutants. Analysis of the acerebellar mutant reveals that Ace/Fgf8.1 is responsible for correct specification of, and axon pathfinding within, the midline optic stalk territory. Furthermore Ace/Fgf8.1 is also responsible for neuronal differentiation in the dorsal forebrain and for correct specification of the olfactory bulb. Analysis of zebrafish embryos with mutations in both Ace/Fgf8.1 and Syu/Shh reveals that although patterning of the forebrain is not more severely affected in the double mutant, the growth of the ventral forebrain is greatly reduced. In zebrafish, the members of the Emx gene family are the earliest known markers for the presumptive telencephalon and loss of function studies in Drosophila and mice suggest an important role for Emx genes in development of anterior structures. To further investigate potential roles for Emx genes in forebrain development, the zebrafish homologues of Emx genes were misexpressed. However ectopic expression of Emx genes does not appear to have the capacity to promote ectopic telencephalic structures. In conclusion, my study has started to reveal how the zebrafish forebrain develops and the genes involved in growth, patterning, neurogenesis and axon pathfinding in the forebrain.
... An extraordinary example for the influence of site-specific extracellular cues is the guidance of axons by chemoattractants that are secreted by the target under the influence of boundary regions that separate specific domains of the developing CNS (Wilson et al., 1993). These cues can either be repulsive or attractive, acting over long or short distances. ...
... These regions correspond, respectively , to the pretectum (P1), the dorsal thalamus (P2), and the ventral thalamus (P3). Recent studies have suggested that boundaries between these presumptive domains provide scaffolds for the formation of early axonal tracts (Krauss et al., 1991; Wilson et al., 1993; Shimamura et al., 1995; Mastick and Easter, 1996; Ba-Charvet et al., 1998 ). One of these boundaries, the zona limitans intrathalamica , separates the dorsal thalamus from the ventral thalamus, where a strong hybridization signal for Pax-6 is found (Walther and Gruss, 1991; Stoykova and Gruss, 1994; Stoykova et al., 1996; Grindley at al., 1997; Warren and Price, 1997). ...
The mammillary bodies represent important relay stations for one of the major neuronal circuits in the brain: the limbic circuit. Mammillary projections traveling through the principal mammillary tract are established early during development, forming the mammillotegmental bundle, which appears fully developed by embryonic day 15 (E15). The mammillothalamic tract develops later, around E17–E18, forming a compact system of collateral fibers originating from the principal mammillary tract and reaching the thalamus by E20. The Pax-6 gene is expressed in various regions of the developing brain, among which the border separating the ventral thalamus from the dorsal thalamus, known as the zona limitans intrathalamica, is especially significant. In this report, the development of the efferent mammillary system of fibers was studied in wild type and Pax-6 mutant mice by using carbocyanine tracers and Golgi preparations. In mutant mice, the mammillotegmental bundle developed normally; however, the mammillothalamic tract was missing. By using anti-Pax-6 antibodies in wild type mice, the existence of an immunoreactive cell cluster is described surrounding the bifurcation point of the principal mammillary tract. The results of this study suggest that there is a correlation of these cells with a particular type of Golgi impregnated neuron. J. Comp. Neurol. 419:485–504, 2000. © 2000 Wiley-Liss, Inc.
... On the molecular level, many regulatory genes are expressed in a neuromere-related pattern. There is an apparent relationship between neuromeres or sites of regulatory gene expression and formation of axonal tracts (Krauss et al., 1991;Wilson et al., 1993Wilson et al., , 1997Boncinelli, 1994). In the hindbrain, the initial growth of dorsoventral axons are restricted to interneuromeric boundaries (Lumsden and Keynes, 1989). ...
In neural development, major tracts are often formed along the neuromere boundary regions, although the molecular mechanism underlying this formation remains to be clarified. In the diencephalon, axons from the habenular nucleus extend along the neuromere boundary region between p1 and p2. At embryonic days 13–15, among members of class 3 semaphorins, only semaphorin 3F (Sema3F) was expressed in the diencephalon. Sema3F, which was strongly expressed in the rostral p1, repulsed axons from habenular explants. While p2 explants did not exert a repulsive effect on axons from habenular explants at a distance, habenular axons did not grow into p2 explant. Explants from the ventral region of the caudal diencephalon where netrin-1 is expressed attracted the axons from habenular explants. The attractive effect was blocked by an antibody for DCC. These results suggest that the growth of axons from the habenular nucleus along the neuromere boundary region may be regulated by Sema3F from the rostral p1, and netrin-1 from the ventral region of the caudal diencephalon.
... In all vertebrates analyzed Nkx2.2 is expressed in the prosencephalon where it is known to be involved in the specification of progenitor domains and in establishing regionalization patterns. Moreover, it contributes to the differentiation of distinct areas and their compartmentalization, as well as to the acquisition of cellular identity and the regulation of the distribution of the earliest neurons in the brain (Wilson et al., 1993; Barth and Wilson, 1994; Vieira and Martínez, 2006; Vue et al., 2007). Thus, the analysis in anamniote and amniote vertebrates of the spatio-temporal distribution of this transcription factor is considered of relevance in the understanding of the establishment of the different subdivisions within the forebrain and in evaluating the degree of conservation across vertebrates (Price et al., 1992; Barth and Wilson, 1995; Holland et al., 1998; Schäfer et al., 2005; Vieira and Martínez, 2006; Vue et al., 2007; Ferran et al., 2009). ...
The expression of the Nkx2.2 gene is involved in the organization of the alar-basal boundary in the forebrain of vertebrates. Its expression in different diencephalic and telencephalic regions, helped to define distinct progenitor domains in mouse and chick. Here we investigated the pattern of Nkx2.2 protein distribution throughout the development of the forebrain of the anuran amphibian, Xenopus laevis. We used immunohistochemical and in situ hybridization techniques for its detection in combination with other essential territorial markers in the forebrain. No expression was observed in the telencephalon. In the alar hypothalamus, Nkx2.2 positive cells were scattered in the suprachiasmatic territory, but also in the supraopto-paraventricular area, as defined by the expression of the transcription factor Orthopedia (Otp) and the lack of xDll4. In the basal hypothalamus Nkx2.2 expressing cells were localized in the tuberal region, with the exception of the arcuate nucleus, rich in Otp expressing cells. In the diencephalon it was expressed in all three prosomeres (P1-P3) and not in the zona limitans intrathalamica. The presence of Nkx2.2 expressing cells in P3 was restricted to the alar portion, as well as in prosomere P2, whereas in P1 the Nkx2.2 expressing cells were located in the basal plate and identified the alar/basal boundary. These results showed that Nkx2.2 and Sonic hedgehog are expressed in parallel adjacent stripes along the anterior-posterior axis. The results of this study showed a conserved distribution pattern of Nkx2.2 among vertebrates, crucial to recognize subdivisions that are otherwise indistinct, and supported the relevance of this transcription factor in the organization of the forebrain, particularly in the delineation of the alar/basal boundary of the forebrain.
... While many studies have examined early diencephalon development in vertebrates (for review, Lim and Golden, 2007) as well as fiber tract formation in the forebrain and midbrain during a similar time period (for summary, BarreiroIglesias et al., 2008), the relationship between diencephalic partitioning and fiber tract formation has received comparatively less attention. Some experiments have indicated that fiber tracts form at diencephalic boundaries in a characteristic fashion ( Gribnau and Geijsberts, 1985;Figdor and Stern, 1993;Wilson et al. 1993;Macdonald et al., 1994) while other studies have not found this to be the case. Rather, fiber tracts form within diencephalic subdivisions themselves but not at their ...
The relationship between fiber tract formation and transverse and longitudinal borders of the diencephalon was investigated in Alligator embryos beginning when this structure was a single unit and continuing until internal subgroups were present within individual segments. At all stages of development, distinct bundles of fibers were not restricted to borders between morphological segments nor were they located at the alar/basal plate boundary. With the exception of a few fine fibers that occupied only a part of certain inter-diencephalic boundaries, fiber tracts were present within the parenchyma of respective subdivisions. In the process of this analysis, fiber tract formation was also documented in the telencephalon, secondary prosencephalon, and midbrain during this period of early development. Fiber tracts were classified into three groups based on orientation: transverse; longitudinal; and commissural. At early stages of development, similarities between Alligator and other species suggest that these bundles represent a primary scaffold for all vertebrates with two exceptions. One was the presence of the descending tract of the mesencephalic trigeminal nucleus in Alligator and other jawed animals but not in jawless vertebrates. The other was the absence of the dorsoventral diencephalic tract in Alligator which lacks a pineal gland.
... During embryonic development, enrichment of CSPG sugars is seen at the border regions where elongating axons and growth cones choose their direction by CSPG chains (Wilson et al. 1993;Wilson et al. 1997;Holt & Dickson 2005;Laabs et al. 2005). For example, CSPG sugars are rich in the boundaries between embryonic brain compartments (prosomeres) formed by homeoproteins (Nakamura & Sugiyama 2004), guiding the pioneer axons from early nerve nuclei to their ultimate destination (Nguyen Ba-Charvet et al. 1998). ...
The shaping of neuronal circuits is essential during postnatal brain development. A window of neuronal remodeling by sensory experience typically occurs during a unique time in early life. The many types of behavior and perception, like human language, birdsong, hearing and vision are refined by experience during these distinct 'critical periods'. The onset of critical periods for vision is delayed in animals that remain in complete darkness from birth. It is then predicted that a 'messenger' within the visual pathway signals the amount of sensory experience that has occurred. Our recent results indicate that Otx2 homeoprotein, an essential morphogen for embryonic head formation, is reused later in life as this 'messenger' for critical period plasticity. The homeoprotein is stimulated by visual experience to propagate into the visual cortex, where it is internalized by GABAergic interneurons, especially Parvalbumin-positive cells (PV-cells). Otx2 promotes the maturation of PV-cells, consequently activating critical period onset in the visual cortex. Here, we discuss recent data that are beginning to illuminate the physiological function of non-cell autonomous homeoproteins, as well as the restriction of their transfer to PV-cells in vivo.
... [Key words: optic chiasm, axon guidance, midline, radial glia, SEA-l, divergence] The advent of improved tract tracing techniques and antigen markers of early axons has heralded a resurgence in studies on the establishment of axonal pathways in the vertebrate brain (Chitnis and Kuwada, 1990;Wilson et al., 1990;Easter et al., 1993Easter et al., , 1994Chedotal et al., 1995). Aside from recent reports indicating that boundaries of gene expression may influence patterns of axon growth (Figdor and Stern, 1993;Wilson et al., 1993;Macdonald et al., 1994), little information exists on cells and molecules residing within axonal paths in the brain, in particular with respect to the first growing axons (Silver, 1984;Silver and Rutishauser, 1984;Wilson and Easter, 1991). In mammals, the retina sends both crossed and uncrossed projections to visual targets in the brain (Polyak, 1957;Guillery, 1982). ...
The retinofugal pathway is a useful model for axon guidance because fibers from each eye project to targets on both sides of the brain. Studies using static and real time analyses in mice at E15-17 demonstrated that uncrossed axons from ventrotemporal retina diverge from crossed axons in the optic chiasm, where specialized resident cells may direct divergence. Other studies, however, suggest that pioneering uncrossed retinal axons derive from a different retinal region, take a different course, and enter the ipsilateral optic tract independent of fiber-fiber interactions. We examine these differences by dye-labeling the earliest optic axons and immunocytochemically identifying cells in their path. The first optic axons arising from dorsocentral retina, enter the diencephalon at E12.5. All axons initially grow caudally, lateral to a radial glial palisade. In contrast to later growing axons, early uncrossed axons enter the ipsilateral optic tract directly. Crossed axons enter the glial palisade and course medially, then anteriorly, in a pathway corresponding to the border of an early neuronal population that expresses SSEA-1, CD44, and beta-tubulin. Axon patterning occurs independent of fiber-fiber interactions from both eyes, as the first uncrossed axons enter the optic tract before crossed ones from opposite eye. These analysis, in conjunction with our previous studies during the principal period of retinal axon growth in the diencephalon, suggest that the adult visual projection arises from age-dependent variations in the types and relative contribution of cues along the path through the emerging optic chiasm.
... Interestingly, we have recently shown thatfgf-3 transcripts become restricted to the boundaries between hindbrain rhombomeres in both chicken and mouse embryos and are also detected at the midbrain-hindbrain boundary ( [27] and R.M., I.M. and G. Morriss-Kay, manuscript submitted). This suggests that FGFs are involved in the regulation of properties characteristic of boundary regions within the developing brain, such as the preferential accumulation of axonal processes (reviewed in [28]). FGF-8 may also play a role in the regulation of the spatial organization of the midbrain, as it is a good candidate for a midbrain-hindbrain boundary signalling molecule, which has been implicated in posterior midbrain development and maintenance of En2 expression (for example, see [29]). ...
The outgrowth of the vertebrate limb bud is the result of a reciprocal interaction between the mesenchyme and a specialized region of the ectoderm, the apical ectodermal ridge (AER), which overlies it. Signals emanating from the AER act to maintain the underlying mesenchyme, called the progress zone, in a highly proliferative and undifferentiated state. Removal of the AER results in the cessation of limb bud growth, thus causing limb truncation. The best candidates for this AER-derived signal are members of the fibroblast growth factor (FGF) family, in particular FGF-4, which can maintain limb bud outgrowth following removal of the AER. However, FGF-4 is only expressed after considerable outgrowth has occurred and a well-developed limb bud has formed, and then only in the posterior part of the AER. Likewise, the other FGFs studied to date are not candidates for this activity.
We report evidence that a recently identified member of this family, FGF-8, is expressed in the ectoderm of the prospective limb territory prior to morphological outgrowth of the limb bud in both mouse and chick. Thereafter, expression is maintained throughout the AER during limb development. We have produced and purified the FGF-8 protein, and shown that it will substitute for the AER in maintaining limb bud outgrowth in mouse embryos from which the AER has been surgically removed. FGF-8 does not, however, maintain expression of the sonic hedgehog gene.
These results indicate that FGF-8 is an AER-derived mitogen that stimulates limb bud outgrowth. Moreover, our data suggest that FGF-8 may also be an ectodermally derived mitogen that stimulates the onset of limb bud outgrowth (budding) in the absence of a morphological AER, and indicate the possible involvement of FGF-8 in the establishment of the limb field.
... For instance, the nTPOC and nMLF are both positioned at the ventral boundary of expression of the receptor tyrosine kinase, rtk1, and the paired box transcription factor, pax6. These observations raised the possibility that cells at the interface between adjacent expression domains may have an identity distinct from that of either of the neighbouring domains (Wilson et al., 1993). nk2.2 is expressed in a band of cells at the interface where both the nTPOC and nMLF differentiate suggesting that this gene may be involved in the establishment or maintenance of the identity of cells at a zone of neuronal differentiation. ...
We have isolated zebrafish nk2.2, a member of the Nk-2 family of homeobox genes. nk2.2 is expressed in a continuous narrow band of cells along a boundary zone demarcating the location at which two of the earliest nuclei in the brain differentiate. This band of cells is located within a few cell diameters of cells expressing the signalling molecule sonic hedgehog/vertebrate hedgehog-1 (shh/vhh-1). Injection of shh/vhh-1 RNA results in ectopic expression of nk2.2 and concomitant abnormalities in the forebrain and eyes. Moreover, cyclops mutant embryos, which initially lack neurectodermal expression of shh/vhh-1, show a concomitant lack of nk2.2 expression. Together, these results suggest a requirement of shh/vhh-1 protein for the spatial regulation of nk2.2 expression.
... However, although fasciculation with its homologue is normal there are many anomalous branches that extend anteriorly towards the segment border. There is strong experimental support for a role of boundary regions, such as the segment border, in axon guidance (Wilson et al., 1993). Next, the AcP axon leaves its homologue to turn and grow anteriorly along the vMP2 fascicle. ...
This article describes the expression pattern and functional analysis of Lazarillo, a novel cell surface glycoprotein expressed in the embryonic grasshopper nervous system, and a member of the lipocalin family. Lazarillo is expressed by a subset of neuroblasts, ganglion mother cells and neurons of the central nervous system, by all sensory neurons of the peripheral nervous system, and by a subset of neurons of the enteric nervous system. It is also present in a few non neuronal cells associated mainly with the excretory system. A monoclonal antibody raised against Lazarillo perturbs the extent and direction of growth of identified commissural pioneer neurons. We propose that Lazarillo is the receptor for a midline morphogen involved in the outgrowth and guidance of these neurons.
... Three major neuronal clusters and their axon tracts are located near expression borders of svp [40]. The rostra1 expression domains of several zebrafish members of the eph, forkhead, pax and wnt gene families also demarcate the positions of early neuronal nuclei and axon pathways (Krauss et al., 1991a;Wilson et al., 1993;Macdonald et al., 1994). Accordingly, cells in the boundary zones of specific regional expression domains of both transcription factors (forkhead, pax) and signal transduction proteins (eph, wnt) are assumed to have distinct identities and cell surface properties that determine neuronal differentiation and axon guidance, respectively (Macdonald et al., 1994). ...
The protein encoded by the zebrafish gene svp[40] belongs to a distinct group within the steroid hormone receptor superfamily that includes Drosophila seven-up and several vertebrate orphan receptors. Svp[40] shares a particularly high degree of amino acid sequence identity (approximately 86%) with the mammalian transcription factors ARP-1 and COUP. The gene is expressed in specific regional and segmental domains within the developing brain. Correspondence between this expression pattern and early sites of neuronal differentiation and axonogenesis in the rostral brain may reflect an involvement in neural patterning. During the early embryonic stages when hindbrain rhombomeres are formed, a segmental expression pattern is established as a step gradient. The single steps of this gradient coincide directly with the four anteriormost segments suggesting a role in controlling rhombomere-specific expression of genes contributing to cell differentiation in the hindbrain. Since COUP/ARP-1 and retinoic acid receptors (RARs/RXRs) are known to have similar DNA-binding specificities, different levels of Svp[40] might modulate retinoid signaling through competition for binding to specific RAREs in the promoters of target genes. Treatment of zebrafish embryos with retinoic acid affects the svp[40] step gradient and causes an elimination of a regional expression domain in the retina. These observations are consistent with svp[40] being an integral part of the retinoid signaling network during hindbrain and eye development.
... It is not known whether this group of cells is terminally differentiated when, from about stage 16 of development, they begin to show these characteristics; nor has it been determined what the ultimate fate of these cells might be (for example, after morphological boundaries have disappeared). There is evidence in several developing systems including forebrain (Macdonald et al., 1994) and floorplate (see Wilson et al., 1993) as well as hindbrain, that the boundary at the interface of domains of gene expression forms a pathway for later axon tracts. The results presented here provide further evidence in support of the idea that boundaries contain a specialised population of cells and possibly a specific substrate pathway. ...
Hindbrain segments, rhombomeres, define distinct cellular and molecular domains which furnish the ground plan for important aspects of neural and cranial development. In this study, further evidence is presented that the interfaces between rhombomeres, rhombomere boundaries, contain both cells and extracellular matrix with specialised characteristics. Cells at rhombomere boundaries show temporally and spatially distinct expression patterns of developmentally important genes. Towards the end of the developmental period when rhombomeres are present, a fan-shaped array of cells at rhombomere boundaries, that constitute the ventricular ridge, shows decreased expression of two genes (Hoxb-1 and Krox-20), which earlier in development were expressed in all cells of specific rhombomeres. In contrast, these boundary cells show increased expression of another gene, Pax-6, which earlier in development has a rhombomere-specific expression pattern. A specialised identity for boundary cells is further suggested by increased labelling with an anti-vimentin antibody at rhombomere boundaries, indicating that at least some boundary cells are radial glia or glial precursors. In addition to distinct cellular properties, the extracellular domain at rhombomere boundaries is also specialised. Chondroitin sulphate proteoglycan (CSPG) immunoreactivity is increased and, as revealed by immuno-electron microscopy, localised to extracellular spaces. CSPG is also enriched in boundaries regenerated after ablation, or boundaries generated ectopically by rhombomere transplantation. We propose that rhombomere boundaries form their characteristic morphology at the interface between groups of cells with differing molecular characteristics, representing different cell states. A specialised band of cells then develops at the interface. Both the boundary cells and extracellur matrix have characteristics which could be important in later events of neural development such as axon guidance and cell migration.
... This result suggests that the mechanisms guiding longitudinal axonal growth may be regulated by longitudinally arranged neuroepithelial domains. There is considerable interest in the possibility that boundary zones separating neuroepithelial domains may provide spatial information that direct the growth of axons (Wilson et al., 1993; Chien and Harris, 1994; MacDonald et al., 1994; Chédotal et al., 1995). Nkx-2.2 expression might define such a molecularly distinct longitudinal boundary zone. ...
Over the last century, several morphological models of forebrain organization have been proposed that hypothesize alternative topological solutions for the relationships of the histogenic primordia. Central to all of these models are their definitions of the longitudinal axis and the longitudinal organization of the neural plate and neural tube. To understand the longitudinal organization of the anterior brain, we have sought to identify molecular properties that are continuous along the entire longitudinal axis of the embryonic CNS. In this essay, we describe studies of the expression of several genes in the mouse between 7.5 (presomite stage) and 10.5 days post coitum (dpc) that provide evidence for the trajectory of the anterior-posterior axis and the longitudinal organization of the anterior CNS. Specifically, we report that the expression of noggin, sonic hedgehog and Nkx-2.2 define longitudinal columns of cells that are present along the entire CNS axis. Within the forebrain, the expression of these genes, as well as that of Nkx-2.1 and BF-1, are in distinct longitudinal regions in the neural plate and tube. We demonstrate that the earliest longitudinal axon pathways of the forebrain are spatially correlated with the longitudinal domain defined by Nkx-2.2. Finally, expression of the former genes, and Otx-1 and Emx-2, suggests that the cephalic neural plate is organized into molecularly distinct domains delimited by longitudinal and transverse borders; these results provide a foundation for defining the mechanisms that pattern the neural plate.
... Analyses of expression domains of other genes from different families including the paired-domain-containing Pax family (Stoykova and Gruss, 1994) and the homeobox-containing Otx, Emx and Nkx families (Simeone et al., 1992a,b; Guazzi et al., 1990; Price et al., 1992; Shimamura et al., 1995) have provided further evidence for their hypothesis (see Boncinelli et al., 1993 and Rubenstein et al., 1994 for reviews). Studies of chick and zebrafish embryos revealed that such gene expression boundaries partially restrict cell mixing and are correlated with the positions of early generated neurons and their axonal tracts (Figdor and Stern, 1993; Wilson et al., 1993; Macdonald et al., 1994; Guthrie, 1995 ). Although the functions of these regionspecific genes remain to be elucidated, some of them are expressed in specific populations of postmitotic neurons at later stages of development. ...
Although a number of genes have been found to have restricted expression domains in the embryonic forebrain and midbrain, it remains largely unknown how the expression of these genes is regulated at the cellular level. In this study, we explored the mechanisms for the differential expression of region-specific transcription factors in neuroepithelial cells by using both primary and immortalized neuroepithelial cells from the rat brain at embryonic day 11.5. We found that differential expression patterns of Pax-3, Pax-5, Pax-6, Dlx-1, Dlx-2, Emx2, Otx1 and Dbx observed in vivo were maintained even when the cells were isolated and cultured in vitro, free from environmental influences. Furthermore, in response to Sonic hedgehog, which is a major inductive signal from the environment for regional specification, neuroepithelial cells that maintain distinct regional identities expressed different sets of ventral-specific genes including Islet-1, Nkx-2.1 and Nkx-2.2. These results suggest that certain cell-autonomous mechanisms play important roles in regulating both environmental signal-dependent and -independent expression of region-specific genes. Thus, we propose that use of the in vitro culture systems we describe in this study facilitates the understanding of regulatory mechanisms of region-specific genes in neuroepithelial cells.
... A variety of cell surface molecules have been shown to be vital for guiding a growth cone towards its eventual target including molecules which stimulate neurite outgrowth and molecules which inhibit or block axonal growth (Keynes and Cook, 1995). Inhibitory proteins are thought to play a role in channelling growing axons along the appropriate permissive tracts towards their eventual target (Wilson et al., 1993). In some cases they may act as a signal to demonstrate that a particular region of the nervous system is fully developed and further growth of axons in this region is not required. ...
We have previously identified a glycosylphosphatidylinositol-linked glycoprotein of 55 kDa (GP55) which inhibits neurite outgrowth. We now provide evidence that GP55, isolated from adult chick brain, consists of at least two bands, both of which are active, i.e., block outgrowth of neurites from chick dorsal root ganglion neurons. An antiserum raised against the adult proteins reverses the inhibition and preliminary experiments suggest that GP55 is restricted to the nervous system, increases during development from very low levels at embryonic day 10 and is most abundant after hatching. Immunofluorescence reveals that GP55 is expressed on neurons cultured from an embryonic day 14 chick brain but is barely detectable on embryonic day 10 dorsal root ganglion neurons or embryonic day 8 forebrain neurons; the neurons which respond to substrate-bound GP55. Peptide sequencing revealed considerable homology with OBCAM, a protein previously identified on the basis of binding opiates. Nested polymerase chain reaction using primers to the OBCAM sequence and internal primers to GP55 peptides produced two different polymerase chain reaction fragments with homology to OBCAM. A full length clone (E19S) corresponding to one polymerase chain reaction product and a partial length clone (E14S) corresponding to the second have been isolated from an embryonic chick brain library. Both are members of the immunoglobulin superfamily and have (or are expected to have) three C2 domains. E19S has 90% homology with LAMP at the amino acid level. This sequence only partially matches the peptides from the adult protein and hence is probably not a major component of the adult proteins. E14S (GP55-A) has 83% homology to OBCAM at the amino acid level over the region sequenced. The sequence matches several of the peptides from the adult protein and is hence likely to correspond to a major component of the adult proteins. Thus members of the GP55 family are related to OBCAM, neurotrimin, LAMP and a recently discovered chick protein CEPU-1. Our results suggest molecules within this family are capable of acting as cell adhesion molecules and inhibitors of neurite outgrowth.
... They are the site of local accumulations of circumferential axons in the hindbrain (Lumsden and Keynes, 1989; Heyman et al., 1993 Heyman et al., , 1995) and these accumulations are lost when boundaries are lost; however, the significance of this role in axonal organisation is not yet understood. Possible roles for boundary cells are either that they form a structural scaffold of specialised glial cells, which may be important for axon and neuronal patterning or that they constitute centres for the generation of particular cell types (Wilson et al., 1993; Heyman et al., 1995). The fate of boundary cells is not known. ...
Rhombomeres are segmental units of the hindbrain that are separated from each other by a specialised zone of boundary cells. Retinoic acid application to a recently segmented hindbrain leads to disappearance of posterior rhombomere boundaries. Boundary loss is preceded by changes in segmental expression of Krox-20 and Cek-8 and followed by alterations in Hox gene expression. The characteristic morphology of boundary cells, their expression of follistatin and the periodic accumulation of axons normally associated with boundaries are all lost. In the absence of boundaries, we detect no change in anteroposterior dispersal of precursor cells and, in most cases, no substantial cell mixing between former rhombomeric units. This is consistent with the idea that lineage restriction can be maintained by processes other than a mechanical barrier composed of boundary cells. Much of the early organisation of the motor nuclei appears normal despite the loss of boundaries and altered Hox expression.
... Considering the fact that the ventral and dorsal limits of Pax-6 expression corresponded to the exit points of SM and BM/VM nerves, respectively , it is likely that the Pax-6 domain defines the specific sites for different motor axons to leave the neural tube by controling some chemorepellent/attractant. This idea is supported by other developmental evidence in zebrafish showing that expression boundaries play crucial roles in growth cone guidance in the forebrain (Wilson et al., 1993). ...
Pax-6 is a member of the vertebrate Pax gene family, which is structurally related to the Drosophila pair-rule gene, paired. In mammals, Pax-6 is expressed in several discrete domains of the developing CNS and has been implicated in neural development, although its precise role remains elusive. We found a novel Small eye rat strain (rSey2) with phenotypes similar to mouse and rat Small eye. Analyses of the Pax-6 gene revealed one base (C) insertion in an exon encoding the region downstream of the paired box of the Pax-6 gene, resulting in generation of truncated protein due to the frame shift. To explore the roles of Pax-6 in neural development, we searched for abnormalities in the nervous system in rSey2 homozygous embryos. rSey2/rSey2 exhibited abnormal development of motor neurons in the hindbrain. The Islet-1-positive motor neurons were generated just ventral to the Pax-6-expressing domain both in the wild-type and mutant embryos. However, two somatic motor (SM) nerves, the abducent and hypoglossal nerves, were missing in homozygous embryos. By retrograde and anterograde labeling, we found no SM-type axonogenesis (ventrally growing) in the mutant postotic hindbrain, though branchiomotor and visceral motor (BM/VM)-type axons (dorsally growing) were observed within the neural tube. To discover whether the identity of these motor neuron subtypes was changed in the mutant, we examined expression of LIM homeobox genes, Islet-1, Islet-2 and Lim-3. At the postotic levels of the hindbrain, SM neurons expressed all the three LIM genes, whereas BM/VM-type neurons were marked by Islet-1 only. In the Pax-6 mutant hindbrain, Islet-2 expression was specifically missing, which resulted in the loss of the cells harboring the postotic hindbrain SM-type LIM code (Islet-1 + Islet-2 + Lim-3). Furthermore, we found that expression of Wnt-7b, which overlapped with Pax-6 in the ventrolateral domain of the neural tube, was also specifically missing in the mutant hindbrain, while it remained intact in the dorsal non-overlapping domain. These results strongly suggest that Pax-6 is involved in the specification of subtypes of hindbrain motor neurons, presumably through the regulation of Islet-2 and Wnt-7b expression.
Receptor protein tyrosine kinases are important for the regulation of cell growth, differentiation and pattern formation. To date 14 subfamilies are known, of which the Eph subfamily is the largest. In this thesis I examine the regulation of expression of the Eph-related Cek-8 gene during limb development and hindbrain segmentation. Cek-8 was found to be expressed in the mesenchyme at the tip of chick limb buds with high levels of transcripts posteriorly and apically but fading out anteriorly. Expression of Cek-8 in distal mesenchyme was found to be regulated by signals from the apical ridge, which could be substituted by FGF, and also by signals from the polarising region and by retinoic acid. Cek-8 expression was uniform across the antero-posterior axis of limb buds in talpid3, a chick mutant with polydactylous limbs with up to 7-8 morphologically similar digits. These findings indicate that Cek-8 expression responds to regulatory signals during limb patterning and suggest that this receptor tyrosine kinase may have a role in coordinating responses to signals in the progress zone of early buds. Later on in limb development, Cek-8 expression was found to be associated with cell condensations that form tendons and their attachments to cartilage rudiments and then in developing feather buds. Cek-8 is also expressed in the developing hindbrain in rhombomeres 3 and 5. Local treatment with retinoic acid led to a partly unsegmented hindbrain and this loss of segmentation (or boundaries) was found to be preceded by changes in segmental gene expression of Cek-8. Loss of boundaries was always correlated with changes in Cek-8 expression. Changes in Hox (homeobox containing genes) gene expression were only apparent after morphological changes occurred and did not seem to impose a new identity on neural precursor cells. Normally, cells from adjacent rhombomeres do not mix and no substantial cell mixing could be detected even in the absence of a physical boundary. Possible roles of Cek-8 during hindbrain segmentation are discussed.
The central nervous system of vertebrates exhibits a bewildering diversity of cell types, yet the organization of the tissue is remarkably ordered. In particular, both the positions of diverse classes of neurons and the trajectories of their interconnections are precisely orchestrated, yielding classifiable neuroanatomical patterns. The mechanisms which might govern the emergence of this highly complex assembly have been a subject of fascination and reflection for developmental biologists over many decades, but have remained largely elusive. Only with the availability of new cellular and molecular techniques and tools has it been possible to uncover novel aspects of the processes involved. The present chapter will discuss some of these developments and attempt to describe the conceptual framework of current tendencies in developmental neurobiology.
The neural plate and early neural tube are formed by a single layer of columnar cells, the neuroepithelium (Fig. 5.1a). As this layer thickens it gradually acquires the configuration of a pseudostratified epithelium; that is to say, its nuclei become arranged in more and more layers, but all elements remain in contact with the external and internal surface (Figs. 5.1b, 5.2, 5.3a). Mitotic figures are found exclusively along the ventricular surface (Fig. 5.1). His (1889) believed that these mitoses belong to cells which form a ventricular layer of germinal cells (Keimzellen), and that the more peripherally located cells represent spongioblasts, primordial glial elements forming a syncytial meshwork (Markgerüst). Neuroblasts produced by the germinal cells were supposed to migrate peripherally in the intercellular spaces of this meshwork.
Early studies of glial boundaries, which are composed of immature astrocytes and extracellular matrix mol ecules (which they express), initially offered insight into the partitioning that occurs in the developing nervous system. More recently, however, it has been suggested that similar "boundaries" may have important roles in other processes occurring in the brain, including repair after traumatic brain injury. As more is understood about the expression and function of boundary molecules and glia, their potential importance is becoming apparent in numerous neuropathological conditions, including neurodegeneration and neuroregeneration in Alzheimer's and Huntington's diseases as well as in brain neoplasms. Furthermore, before we can hope to fully understand and facilitate regeneration in the compromised brain, our knowledge of the glial boundary, both during development and in the adult, must be more complete. The Neuroscientist 1:142-154, 1995
The vertebrate central nervous system is characterized by regional specialization, which arises during early development and contributes to patterning the emerging central nervous system (CNS). In the hindbrain, rhombomeres demarcate nonoverlapping regions of the CNS that give rise to distinct neural structures. The cellular structures that define boundaries between adjacent rhombomeres are as yet unclear. However, in certain species the boundary regions between discrete CNS regions appear to be defined by specialized glial cells. Here, we show that in developing Xenopus, DMγ, a membrane protein of the proteolipid protein family, is expressed in a subset of radial glia. During development, DMγ transcripts are first expressed in presumptive glial cells throughout the hindbrain, but later become confined to the ventricular zone at rhombomere centers, whereas the protein is exclusively expressed in radial glial cell processes that occupy the rhombomere boundary regions. Likewise, early in development vimentin and glial fibrillary acidic protein are extensively coexpressed in hindbrain radial glia but subsequently define distinct rhombomere domains: vimentin remains localized in radial glia at the rhombomere boundary regions, whereas expression of glial fibrillary acidic protein becomes restricted to the centers. Moreover, radial glial processes at the boundary region are distinguishable from those at the center region; the processes of the boundary region radial glia extend upward in a fan-shaped arrangement and are encircled by the processes from the center glia. These data suggest that an early event in determining rhombomere topology is the specification of both morphologically and biochemically distinct subsets of radial glia. J. Comp. Neurol. 424:47–57, 2000. © 2000 Wiley-Liss, Inc.
Numerous studies of the past decade have illuminated the importance of intercellular adhesion events for neural pattern formation. It has been documented that members of the Ig and cadherin gene superfamilies, that glycoproteins and, probably to some extent, proteoglycans of the extracellular matrix play a role in this context. Recent observations suggest that, in addition to adhesive interactions, repulsive and/or inhibitory phenoma are also of importance in regulating neural pattern formation. Several molecules are under study which are cosidered possible mediators of inhibitory interactions in the nervous system. The hypothesis has been advanced that some of these might be partially responsible for restrictive, boundary-like properties ascribed to glial cells in developing and regenerating tissues. The current review summarizes these studies and focusses on molecular aspects of boundary and compartmentation phenomena. © 1995 Wiley-Liss, Inc.
This study reports the spatio-temporal pattern of BEN expression (a molecule of the immunoglobulin superfamily) during early stages of the first axonal tract formation, in the fore- and midbrain of chick embryos [Hamburger and Hamilton (HH) stages 12–22]. The expression of BEN has been analysed using immunohistochemistry and non-radioactive in situ hybridization. Furthermore, double labelling experiments (combining anti-class III β-tubulin, a pan-neuronal marker, and anti-BEN antibodies) have been carried out to determine whether BEN is expressed by all first axonal tracts. The first neurons expressing BEN appear around stage HH13–14, in the caudal diencephalon. They belong to the interstitial nucleus of Cajal, and their axons are the first components of the medial longitudinal fasciculus. By HH14, two other early axonal tracts appear: the tract of the postoptic commissure and the descending root of the mesencephalic nucleus of the trigeminal nerve. Only the latter expresses BEN. At later stages of development numerous new axonal tracts appear in the telencephalic, diencephalic and mesencephalic domains. Only a few of them (the fourth nerve, the lemniscus lateralis, the tectobulbar and habenulopeduncular tracts) express BEN. In all BEN positive systems, the cell bodies, axons and growth cones are uniformly labelled by the antibody. We have found that none of the early axonal tracts grows preferentially at interneuromeric boundaries. Moreover, each tract is formed by several thin fascicles rather than a single one. The expression of BEN is transient and disappears shortly before hatching. These results suggest that BEN may serve to promote axonal outgrowth of precise neuronal systems involved in ‘axonal scaffolding’.
We examine the role of the Engrailed homeobox gene in establishment of local tectal topography. In the mesencephalon, a gradient of Engrailed appears early and defines the rostrocaudal axis of the tectum. Various experiments that cause ectopic Engrailed expression cause predictable readjustments of the retinotectal map. The newly discovered ‘realisators’ of the retinotopic map, such as receptor tyrosine kinase ligands ELF-I and RAGS could be controlled directly by Engrailed. Indeed, recent results show that Engrailed regulates the expression of these ligands. The Engrailed gradient itself appears to be set up by signals including FGF8 and WNTI, allowing us to begin to trace the molecular cascade that is responsible for the correct wiring of the visual projection back into the early embryo. Trends Neurosci. (1996) 19, 542–546
Early embryonic development of the nervous system of a lamprey, Lampetra japonica, was studied by using immunohistochemical techniques and by scanning electron microscopy. The earliest appearance of axons was detected at Tahara's stage 21-, when dorsolateral and ventral longitudinal fasciculi were present in the hindbrain and spinal cord regions. The branchiomeric nerve roots began to appear at stage 22; the fibers were joined to the dorsolateral fasciculus proximally and also extended distally into each pharyngeal arch. The anterior neural tube was divided into several neuromeres: the mid-hindbrain sulcus became apparent first, then the portion rostral to this sulcus was subdivided into two portions by the syn-parencephalic boundary. In the hindbrain around stage 23, rhombomeres developed transiently, of which, rhombomere 4 was the most distinctive. Putative crest cells forming the octavofacial nerve root anlage were selectively adhering to rhombomere 4, whereas no crest cells were found on rhombomere 3. The assignment of the crest-derived nerve anlage to rhombomeres is conserved between gnathostomes and L. japonica. The neuromerical scheme of the neural tube of L. japonica is also mostly in accordance with that in gnathostomes, sharing the basic developmental patterning of axon bundles at early developmental stages. The most distinct difference between these two groups is the topographical relationships between the hindbrain neuraxis and pharyngeal arches, as well as the otic placode.
We designed a protocol to identify cell surface molecules expressed in restricted spatial patterns in the developing central nervous system (CNS) that might be regulated by regionally restricted transcription factors. The immunogen was a membrane fraction from NT2/D1 embryocarcinoma cells that were induced to differentiate into neurons and upregulate Hox gene expression in response to retinoic acid. One monoclonal antibody (mAb), FORSE-1, specifically labels the rostral rat CNS from the earliest stages. Staining is observed in the rostral but not caudal neural folds of the embryo prior to neural tube closure. Staining is enriched in the forebrain as compared to the rest of the CNS, until E18. Between E11.5 and E13.5, only certain areas of the telencephalon and diencephalon are labeled. Later, up to E17.5, FORSE-1 labeling is specifically restricted to the telencephalon, where a correlation with mitotic activity is apparent: the ventricular zone labels with FORSE-1, while the cortical plate is negative. The staining of the neuroepithelium is intensified by acetone fixation, which also reveals, between E11.5 and E13.5, a dorsoventrally restricted, FORSE-1-positive region of the spinal cord. After E18, the entire CNS is labeled, through adulthood. The mAb labels the surfaces of dissociated, living cells. Other, non-CNS areas of FORSE-1 labeling are nasal and otic placodes, nasal epithelium, nasal glands, and early (E9.5-10.5) endoderm. mAb FORSE-1 recognizes an epitope present on both a high-molecular-weight (> 200 kDa) proteoglycan from embryonic and early postnatal brain, and on a 80 kDa doublet that is restricted to the CNS in the adult. These findings suggest the FORSE-1 antigen as a candidate cell surface molecule for mediating regional specification from the earliest stages of CNS development.
A crucial phase of development in the vertebrate rhombencephalon involves transient organization into segments. Recent studies on the diencephalon and telencephalon pose the question of whether segmentation might also play a role in the development of more rostral brain regions. Criteria for segmentation formulated for the hindbrain might be met by the diencephalon, although there is disagreement as to the number and arrangement of segmental units. In contrast to the hindbrain, these segments appear when neurogenesis has begun, and might represent definitive functional units. Regarding the telencephalon, it is at present unclear whether domains of gene expression are associated with other features that are characteristic of segmental development, or whether other mechanisms control specification of this region.
The primordial neuroepithelium of the vertebrate forebrain consists of transverse and longitudinal morphogenetic compartments ("neuromeres"). During development, neurons born in the ventricular zone of each neuromere migrate outward to the mantle zone. Here, neuroblasts gradually accumulate and aggregate either into sheets ("laminae") or into roundish structures ("nuclei"). As brain architecture matures, sets of nuclei and laminae derived from several neuromeres become connected by fiber tracts to form functional circuits. We show by immunostaining and in situ hybridization techniques that, in the E3-E5 chicken embryo, the cell adhesion molecule R-cadherin is expressed in several stripes and patches in the forebrain neuroepithelium. This expression pattern reflects, at least in part, the neuromeric organization of the forebrain. For example, in both the ventral and dorsal thalamus, R-cadherin expression has a sharp border at the respective caudal neuromere boundary. Moreover, focusing on the mid-hypothalamic region, we demonstrate that a subset of postmitotic neuroblasts in the ventricular zone express R-cadherin during their migration to the mantle zone, where they aggregate into particular nuclei. In the mantle zone, R-cadherin-expressing neuroblasts accumulate in parallel with neuroblasts expressing another cadherin, N-cadherin. The two types of cells segregate from each other to form adjacent nuclei. Some of the R- and the N-cadherin-positive nuclei form parts of particular functional circuits in the mature brain. In conclusion, our results suggest that cadherins play a role in the formation of brain nuclei and in the developmental transformation from neuromeric to functional organization in the vertebrate forebrain.
Cadherins are a family of cell surface glycoproteins which mediate cell-cell adhesion by a Ca(2+)-dependent mechanism. Results from in vitro studies with cadherin-transfected cell lines show that cadherins preferentially bind to each other in a homophilic fashion. In the developing vertebrate brain, at least 10 cadherins are found. Some of these cadherins are expressed in a restricted fashion in particular developing brain nuclei and neural circuits. Based on these results, specific morphogenetic roles for cadherins during CNS development have been proposed. This review focuses on the possible role of cadherin-mediated sorting and aggregation of early neurons and neurites in the formation of brain nuclei, fiber tracts, and neural circuits. Moreover, at least 1 cadherin is also expressed in a segmental ("neuromeric") fashion in the early chicken forebrain, suggesting that this cadherin regulates developmental processes involved in the transformation from the neuromeric organization of the early neuroepithelium to the functional organization of the mature brain.
Zebrafish pax-6 (pax[zf-a]) and its murine homologue are structurally and functionally related to the Drosophila paired box gene eyeless, a master control gene for eye development. This report details the zebrafish pax-6 embryonic expression pattern both at the mRNA and protein level. Transcripts are first detected in the presumptive forebrain and hindbrain regions of the neural plate. After formation of the neural keel, Pax-6 protein accumulates within the same two domains. Expression is also observed in the optic vesicles and lens placodes, confirming that the Pax-6 protein is expressed in those areas of the eye where it is assumed to control differentiation. The relative DNA-binding affinity of the zebrafish Pax-6 protein to different categories of Pax recognition sites is shared with the murine homologue.
This chapter summarizes the recent work in tract formation in the vertebrate brain. The biggest step forward in the study of early axonogenesis came with the introduction of fluorescent tracers: the procion dyes Individual cells were injected with fluorescent tracers directly and the outgrowth of the axons and dendrites were monitored over time. This is perhaps the ideal way to study tract development, particularly because these fluorescent tracers can often be made electron-dense, allowing electron microscopic examination of identified processes. Some investigators have been successful in injecting individual cells in the vertebrate CNS but most of the recent reports on axonogenesis have employed markers applied extracellularly, either horseradish ∼ peroxidase (HRP) or carbocyanine dyes applied locally or antibodies against molecules associated with early axons applied more broadly. The development of the first tracts is investigated in the presumptive fore- and mid-brains in zebra fish. Embryonic chick and mouse brains are also examined with methods similar to those used on zebrafish and frog; i.e. fixing them lightly in formaldehyde and reacting them with neuron-specific antibodies in whole-mounts and sections. In the mouse, the work has shown that the first TuJ1-positive cells appear in the neural plate on E8.5, prior to neural tube closure, and are almost certainly the precursors of the mesencephalic nucleus of the trigeminal nerve.
One of the earliest events in the development of the central nervous system is the establishment of positional identity along the anteroposterior (A-P) axis of the neuroepithelium. In recent years, regulatory genes with regionally restricted expression in the neuroepithelium have been identified which are believed to specify its developmental fate. We have previously described Brain Factor-1 (BF-1), a winged helix (WH) transcription factor expressed in the telencephalic neuroepithelium (Tao and Lai, 1992) Neuron 8:957-966. Here we report the cloning of the mouse cDNA for a novel WH protein, BF-2. We show that BF-2 is a sequence-specific DNA binding protein with a binding specificity distinct from BF-1. Its expression in the CNS during embryogenesis is restricted to the rostral diencephalic neuroepithelium. The caudal boundary of BF-2 expression is at the zona limitans intrathalamica. Rostrally, the BF-2 expression domain is adjacent to that of BF-1. The expression domains of these two factors define a boundary within the developing forebrain neuroepithelium. The BF-1/BF-2 boundary also extends laterally to divide the optic stalk and the retina into nasal (anterior) and temporal (posterior) domains. These observations suggest that in addition to playing a role in the subdivision of the forebrain, these two WH factors may also function to establish positional information in the retinal neuroepithelium.
The FORSE-1 monoclonal antibody (mAb) was generated using a strategy designed to produce mAbs against neuronal cell surface antigens that might be regulated by regionally restricted transcription factors in the developing CNS. To determine whether FORSE-1 has a labeling pattern similar to that of known transcription factors, the expression of BF-1 and Dlx-2 was examined by in situ hybridization on sections serial to those labeled with FORSE-1. We find a striking overlap between BF-1 and FORSE-1 in the telencephalon; both are expressed in the lateral but not the medial walls of the telencephalon, and the boundaries of expression are apparently identical. FORSE-1 staining is detected prior to BF-1 expression in the neural tube, however. FORSE-1 and Dlx-2 have very different patterns of expression in the forebrain, suggesting that regulation by Dlx-2 cannot by itself explain the distribution of FORSE-1. However, they share some sharp boundaries in the diencephalon. In addition, FORSE-1 identifies some previously unknown boundaries in the developing forebrain. These results indicate that a new cell surface marker can be used to subdivide the embryonic telencephalon and diencephalon into regions smaller than previously described, providing necessary complexity to the developmental patterning in the forebrain.
Embryonic cholinesterases are assigned important functions during morphogenesis. Here we describe the expression of butyrylcholinesterase and acetylcholinesterase, and the binding of peanut agglutinin, and relate the results to mitotic activity in chick wing and leg buds from embryonic day 4 to embryonic day 9. During early stages, butyrylcholinesterase is elevated in cells under the apical ectodermal ridge and around invading motoraxons, while acetylcholinesterase is found in the chondrogenic core, on motoraxons and along the ectoderm. Peanut agglutinin binds to the apical ectodermal ridge and most prominently to the chondrogenic core. Measurements of thymidine incorporation and enzyme activities were consistent with our histological findings. Butyrylcholinesterase is concentrated near proliferative zones and periods, while acetylcholinesterase is associated with low proliferative activity. At late stages of limb development, acetylcholinesterase is concentrated in muscles and nonexistent within bones, while butyrylcholinesterase shows an inverse pattern. Thus, as in other systems, in limb formation butyrylcholinesterase is a transmitotic marker preceding differentiation, acetylcholinesterase is found on navigating axons, while peanut agglutinin appears in non-invaded regions. These data suggest roles for cholinesterases as positive regulators and peanut-agglutinin-binding proteins as negative regulators of neural differentiation.
To analyze the molecular mechanism of pattern formation in the anteriormost regions of the zebrafish embryo, we isolated two zebrafish sequences, zOtx1 and zOtx2, related to the Drosophila orthodenticle (otd) and two murine Otx genes. zOtx1 and zOtx2 encode predicted gene products which are 82% and 94% identical to the corresponding mouse proteins. Transcripts of both zebrafish genes appear abruptly at high levels in a triangular patch at the animal pole of the mid-gastrula, a region which contains cells fated to become midbrain and forebrain. Between 9 and 14 h of development, zOtx transcripts disappear from forebrain regions in a manner characteristic for each gene, and from 14 to 24 h, particular regions of the forebrain and midbrain express one or both genes. The posterior limit of expression of both genes in 10-30-h embryos forms a sharp boundary at the posterior border of the midbrain. As in the mouse, the early expression patterns of the zOtx genes are consistent with a role in defining midbrain and forebrain territories. However, there are a number of interesting differences between the forebrain and midbrain regions which express the genes in the two species.
During development of the zebrafish forebrain, a simple scaffold of axon pathways is pioneered by a small number of neurons. We show that boundaries of expression domains of members of the eph, forkhead, pax, and wnt gene families correlate with the positions at which these neurons differentiate and extend axons. Analysis of genetically or experimentally altered forebrains indicates that if a boundary is maintained, there is appropriate neural differentiation with respect to the boundary. Conversely, in the absence of a boundary, there is concomitant disruption of neural patterning. We also show that a strip of cells within the dorsal diencephalon shares features with ventral midline cells. This strip of cells fails to develop in mutant fish in which specification of the ventral CNS is disrupted, suggesting that its development may be regulated by the same inductive pathways that pattern the ventral midline.
Axonal tracts in the vertebrate brain seem to respect domains of homeobox gene expression. To test the role of engrailed in tract formation in the midbrain, we inhibited its expression using antisense (AS) oligonucleotides. Phosphorothioate-modified AS oligos caused navigational errors in both the optic projection (OP) and the intertectal commissure (ITC). These oligos, however, also inhibited bFGF binding to the brain. To determine whether these tract phenotypes were due to inhibition of bFGF function or engrailed expression, we used partially phosphorothioate-modified (pp) oligos, which inhibit engrailed expression but do not affect bFGF binding. These ppAS oligos caused the ITC phenotype but had no effect on the OP. Thus, interference with bFGF function correlates with the OP phenotype, while the ITC phenotype is directly related to engrailed expression.
We investigated the potential role of rostral-caudal and dorsal-ventral subdivisions of the early rostral brain by relating these subdivisions to the early patterning of neuron cell bodies and their axon projections. The earliest neurons were mapped using the lipophilic axon tracers diI and diO on embryos fixed on embryonic days 9.5-10.5 (E9.5-E10.5); neuromeric boundaries were marked by diO. The tracts were small in number, were organized orthogonally (2 dorsal-ventral and 4 rostral-caudal), and originated from groups of cell bodies which we term "sources." Two parallel longitudinal axon systems, one dorsal (the tract of the postoptic commissure and the mesencephalic tract of the trigeminal nerve) and one ventral (the mammillotegmental tract and the medial longitudinal fasciculus), projected caudally from the prosencephalon into the rhombencephalon. We argue that the dorsal longitudinal pathway marked the boundary between the alar and basal plates along the entire neuraxis. The dorsal-ventral axons coursed circumferentially and either crossed the midline (forming the posterior and ventral tegmental commissures) or turned caudally without crossing the midline. The dorsal-ventral axons were not generally restricted to the interneuromeric boundaries, as others have suggested. Earlier, all neighboring neurons projected their axons together; later, nearby neurons projected into different pathways. Some tracts originated in single neuromeres, while other tracts had origins in two or more neuromeres. The dorsal longitudinal axons altered course at several of the borders, but the ventral longitudinal axons did not. In summary, the early subdivisions appeared to influence some, but not all, aspects of tract formation.
In this review, we describe general features of the expression of cadherins in the developing central nervous system (CNS) of vertebrates. In the early neuroepithelium, the expression of several cadherins is restricted to specific regions corresponding to segmental domains. Segmental boundaries often coincide with changes in cadherin expression, subdividing the primordial CNS into different adhesive domains. In the different neuromeric domains, early neurons are generated which differentially express cadherins. In the mantle layer, these early neurons seem to sort out according to which cadherin they express, and they aggregate into various gray matter regions (brain nuclei and cortical lamina and regions). The gray matter structures expressing a given cadherin become connected to one another to form parts of particular functional systems or neuronal circuits. Together, these findings show that cadherins provide a molecular system reflecting both early embryonic and mature nervous system architecture. The possible roles of cadherins in the formation and maintenance of segmental and functional nervous system structures are discussed.
Axonin-1/TAG-1, a member of the immunoglobulin (Ig) superfamily of adhesion molecules, has been shown to be selectively expressed by a subset of neurons and fiber tracts in the developing nervous system of vertebrates. Axonin-1/TAG-1 is thought to play a role in the outgrowth, guidance, and fasciculation of neurites. In the present study, we map the expression of axonin-1 in the diencephalon of the chicken brain at early and intermediate stages of development [2-8 days of incubation; embryonic day (E)2-E8] by immunohistochemical methods. Results show that axonin-1 is first expressed at about E2.5 by postmitotic neurons scattered throughout most of the diencephalon. During the neuromeric stage of brain development (about E3-E5), axonin-1+ nerve cell bodies are predominantly found in two neuromeric subdivisions: 1) in the alar plate of the precommissural pretectum and dorsal thalamus and 2) in the posterior preoptic region of the hypothalamus. The axonin-1+ fiber bundles emerging from these areas grow across segmental boundaries. For example, axonin-1+ neurites originating in the dorsal thalamus cross the zona limitans intrathalamica at a right angle to project to the striatum. Later, the axonin-1+ neuromere areas give rise to particular axonin-1+ gray and white matter structures. Most of these structures correspond to the structures described to express TAG-1 in rodents. In conclusion, axonin-1 can be used as a marker to study aspects of the transition from the early neuromeric structure to the mature anatomy of the chicken brain.
The Pax-6 gene encodes a transcription factor that is expressed in regionally restricted patterns in the developing brain and eye. Here we describe Pax-6 expression in the early forebrain (prosencephalon) on embryonic day 9.5 (E9.5) to E10.5 using both whole-mount in situ hybridization and antibody labeling. We find close correlations between Pax-6+ domains and initial neural patterning, and identify corresponding defects in embryos homozygous for the Pax-6 allele, Small eye (Sey). Pax-6 expression defines the prosencephalon-mesencephalon boundary, and mutant embryos lack this morphological boundary. Markers of the caudal prosencephalon are lost (Pax-6, Lim-1, Gsh-1) and a marker for mesencephalon is expanded rostrally into the prosencephalon (Dbx). We conclude that the caudal prosencephalon (prosomere 1) is at least partially transformed to a mesencephalic fate. This transformation results in a specific deficit of posterior commissure axons. Sey/Sey embryos also exhibit an axon pathfinding defect specific to the first longitudinal tract in the prosencephalon (tpoc, tract of the postoptic commissure). In wild type, tpoc axons fan out upon coming in contact with a superficial patch of Pax-6+ neuron cell bodies. In the mutant, the tpoc axons have normal initial projections, but make dramatic errors where they contact the neuron cell bodies, and fail to pioneer this first tract. Thus Pax-6 is required for local navigational information used by axons passing through its domain of expression.
We conclude that Pax-6 plays multiple roles in forebrain patterning, including boundary formation, regional patterning, neuron specification and axon guidance.
Nucleotide sequences of most vertebrate genes will be available in the next few years as ESTs and genomic sequences. This information will allow the deduction of encoded proteins, possibly providing clues to their functions in vivo. To determine unequivocally the physiological roles of all genes and in particular those of medical importance, mutational analyses must be performed. Large-scale random mutagenesis screens, which allow the systematic mutation of all genes, are a valuable approach to this task1, 2, 3, 4, 5, 6, 7, 8, 9. To complement the sequencing efforts, we established a consortium generating a reference library of gene-trap sequence tags (GTST) which eventually will represent insertional mutations for most mammalian genes expressed in mouse embryonic stem (ES) cells.
Pattern formation in the hindbrain and paraxial mesoderm of vertebrates occurs by the formation of a series of repeated segments. These processes of segmentation appear different at the morphological level, since hindbrain segments, the rhombomeres, form by the subdivision of the neural epithelium into compartments, whereas the mesodermal somites form by the sequential aggregation of mesenchymal cells into epithelial balls. Previous studies have implicated genes encoding transcription factors in the development of hindbrain segments, but nothing is known of genes involved in the formation of somites. Cellular interactions and signal transduction must be an important aspect of hindbrain segmentation, so we have screened for tyrosine kinases expressed in rhombomere-restricted patterns in the developing mouse embryo. We have identified a receptor protein tyrosine kinase, Sek, that has high relative levels of expression in rhombomeres 3 and 5. This alternating pattern is established coincidentally, both spatially and temporally, with the expression of Krox-20, a zinc-finger gene expressed prior to the morphological formation of rhombomeres. In addition, Sek expression occurs in several other developing tissues, including a dynamic regulation in the developing forebrain, spinal cord, early mesoderm and anterior presomitic mesoderm (segmental plate). The latter expression occurs in two stripes that correlate with, and presage, the formation of somites. Sek expression initially occurs throughout the presumptive somite, then becomes restricted anteriorly, and finally is down-regulated as the definitive somite is formed. These data suggest that despite the morphological differences in the segmentation of the hindbrain and mesoderm, Sek is involved in the segmental patterning of both of these tissues.
Two retrovirus promoter trap vectors (U3His and U3Neo) have been used to disrupt genes expressed in totipotent murine embryonal stem (ES) cells. Selection in L-histidinol or G418 produced clones in which the coding sequences for histidinol-dehydrogenase or neomycin-phosphotransferase were fused to sequences in or near the 5' exons of expressed genes, including one in the developmentally regulated REX-1 gene. Five of seven histidinol-resistant clones and three of three G418-resistant clones generated germ-line chimeras. A total of four disrupted genes have been passed to the germ line, of which two resulted in embryonic lethalities when bred to homozygosity. The ability to screen large numbers of recombinant ES cell clones for significant mutations, both in vitro and in vivo, circumvents genetic limitations imposed by the size and long generation time of mice and will facilitate a functional analysis of the mouse genome.
Insight into the genetic control of the identity of specific regions along the body axis of vertebrates has resulted primarily from the study of vertebrate homologues of regulatory genes operating in the Drosophila trunk, but little is known about the development of most anterior regions of the body either in flies or vertebrates. Three Drosophila genes have been identified that are important in controlling the development of the head, two of which, empty spiracles and orthodenticle, have been cloned and shown to contain a homeobox. We previously cloned and characterized Emx1 and Emx2, two mouse genes related to empty spiracles that are expressed in restricted regions of the developing forebrain, including the presumptive cerebral cortex and olfactory bulbs. Here we report the identification of Otx1 and Otx2, which are related to orthodenticle. We have compared the expression domains of the four genes in the developing rostral brain of mouse embryos at a developmental stage, day 10 post coitum, when they are all expressed. Otx2 is expressed in every dorsal and most ventral regions of telencephalon, diencephalon and mesencephalon. The Otx1 expression domain is similar to that of Otx2, but contained within it. The Emx2 expression domain is comprised of dorsal telencephalon and small diencephalic regions, both dorsally and ventrally. Finally, Emx1 expression is exclusively confined to the dorsal telencephalon. Thus at the time when regional specification of major brain regions takes place, the expression domains of the four genes seem to be continuous regions contained within each other in the sequence Emx1 less than Emx2 less than Otx1 less than Otx2.
The homeotic genes of Drosophila encode transcription factors that specify morphological differences between segments. To identify the genes that they control, we developed a chromatin immunopurification approach designed to isolate in vivo binding sites for the products of the homeotic gene Ultrabithorax. Here, we report the analysis of one immunopurified binding site. This 110 bp fragment maps within a regulatory region of a gene under homeotic control, connectin. A 4 kb DNA fragment, including the immunopurified binding site, is sufficient to reproduce the appropriate homeotic control within a subset of the full tissue distribution of connectin. Analysis of the role of the 110 bp binding site indicates that it mediates transcriptional controls by Ultrabithorax and other homeotic genes. This is the first report of a functional in vivo binding site isolated using the chromatin immunopurification method. We also show that the protein product of the connectin gene is predicted to be a cell-surface molecule containing leucine-rich repeats. The protein, connectin, can mediate cell-cell adhesion thus suggesting a direct link between homeotic gene function and processes of cell-cell recognition.
The wnt gene family codes for a group of cysteine-rich, secreted proteins, which are differentially expressed in the developing embryo and are possibly involved in cellular communication. Here, we describe the polymerase chain reaction based cloning and embryonic expression patterns of four zebrafish wnt-related sequences; wnt[a], wnt[b], wnt[c] and wnt[d]. One of these genes, wnt[a], is a potential homologue of murine Wnt-3, while the other three genes most likely represent new members of the vertebrate wnt gene family. In zebrafish embryos, transcripts of wnt[a] are confined to the dorsal diencephalon, the dorsal midbrain, the rhombic lips and the dorsal portions of the spinal cord. wnt[b] is expressed in the tail bud and at considerably lower levels in the mesoderm of the head. wnt[c] transcripts are present within the diencephalon and the posterior midbrain whereas wnt[d] shows a surprisingly similar expression pattern to zebrafish wnt-1. By analogy to wnt-1, it is likely that the members of the zebrafish wnt gene family play an important role in cell-to-cell signalling during pattern formation in the neural tube and the tail bud.
Each abdominal hemisegment in the Drosophila embryo contains a stereotyped array of 30 muscles, each specifically innervated by one or a few motoneurons. We screened 11,000 enhancer trap lines, isolated several expressing beta-galactosidase in small subsets of muscle fibers prior to innervation, and identified two of these as inserts in connectin and Toll, members of the leucine-rich repeat gene family. Connectin contains a signal sequence, ten leucine-rich repeats, and a putative phosphatidylinositol membrane linkage; in S2 cells, connectin can mediate homophilic cell adhesion. Connectin is expressed on the surface of eight muscles, the motoneurons that innervate them, and several glial cells along the pathways leading to them. During synapse formation, the protein localizes to synaptic sites; afterward, it largely disappears. Thus, connectin is a novel cell adhesion molecule whose expression suggests a role in target recognition.
A general strategy for selecting insertion mutations in mice has been devised. Constructs lacking a promoter and including a beta-galactosidase gene, or a reporter gene encoding a protein with both beta-galactosidase and neomycin phosphotransferase activity, were designed so that activation of the reporter gene depends on its insertion within an active transcription unit. Such insertion events create a mutation in the tagged gene and allow its expression to be followed by beta-galactosidase activity. Introduction of promoter trap constructs into embryonic stem (ES) cells by electroporation or retroviral infection has led to the derivation of transgenic lines that show a variety of beta-galactosidase expression patterns. Intercrossing of heterozygotes from 24 strains that express beta-galactosidase identified 9 strains in which homozygosity leads to an embryonic lethality. Because no overt phenotype was detected in the remaining strains, these results suggest that a substantial proportion of mammalian genes identified by this approach are not essential for development.
Small eye (Sey) in mouse is a semidominant mutation which in the homozygous condition results in the complete lack of eyes and nasal primordia. On the basis of comparative mapping studies and on phenotypic similarities, Sey has been suggested to be homologous to congenital aniridia (lack of iris) in human. A candidate gene for the aniridia (AN) locus at 11p13 has been isolated by positional cloning and its sequence and that of the mouse homologue has been established (C.T., manuscript in preparation). This gene belongs to the paired-like class of developmental genes first described in Drosophila which contain two highly conserved motifs, the paired box and the homeobox. In vertebrates, genes which encode the single paired domain as well as those which express both motifs have been described as the Pax multigene family. A Pax gene recently described as Pax-6 is identical to the mouse homologue of the candidate aniridia gene. Here we report the analysis of three independent Sey alleles and show that indeed this gene is mutated and that the mutations would predictably interrupt gene function.
We have previously shown that one of two chicken engrailed-like genes, chick En-2, is expressed in a restricted region of the early chick embryo brain: the mes/metencephalon (Gardner et al. 1988). In this study, we examine the role of the cellular environment in regulation of engrailed-like (En) protein expression in quail-chick chimeric embryos. Two types of transplant surgery were performed at the 9-15 somite stage to produce chimeric embryos. In the first, the mid-mesencephalic vesicle or caudal mesencephalic vesicle alar plate (which is En protein-positive) was transplanted from a quail embryo into an En protein-negative region of chick neuroepithelium, the prosencephalon (mMP and cMP grafts, respectively). In the second reciprocal surgery, prosencephalic alar plate which is En protein-negative, was transplanted into the En protein-positive mesencephalic vesicle (PM grafts). A polyclonal antiserum, aEnhb-1, which recognizes chick
En proteins (Davis et al. 1991) was used to identify En-positive cells 48 h after surgery. In mMP embryos, 71 % of integrated grafts had lost En expression (n=17). In contrast, in cMP grafts, 93% of integrated grafts continued to stain with the antiserum (n=14). In addition, in 86 % of these embryos, the graft induced adjacent chick host diencephalic cells to become En protein-positive as well. All PM grafts contained aEnhb-1 -positive cells; such cells never expressed this protein in their normal environment. These early changes in En protein expression correlate well with the morphological changes observed in similar graft surgeries assayed later in development. Thus, our results are consistent with the hypothesis that En genes play a role in the regionalization of the early cranial neuroepithelium.
To learn how neural segments are structured in a simple vertebrate, we have characterized the embryonic zebrafish hindbrain with a library of monoclonal antibodies. Two regions repeat in an alternating pattern along a series of seven segments. One, the neuromere centers, contains the first basal plate neurons to develop and the first neuropil. The other region, surrounding the segment boundaries, contains the first neurons to develop in the alar plate. The projection patterns of these neurons differ: those in the segment centers have descending axons, while those in the border regions form ventral commissures. A row of glial fiber bundles forms a curtain-like structure between each center and border region. Specific features of the individual hindbrain segments in the series arise within this general framework. We suggest that a cryptic simplicity underlies the eventual complex structure that develops from this region of the CNS.
Gene trapping offers an alternative method to create random insertional mutations that are immediately accessible to molecular characterization. By eliminating the time-consuming step of constructing targeting vectors for each gene of interest, the rate at which new mutations may be introduced in the germ line by gene trapping far exceeds that of conventional gene targeting. Thus, gene trapping represents a rapid and cost-efficient method for the identification and functional analysis of new genes in mice. Gene trap vectors are activated through the production of a reporter gene fusion transcript following insertions of the vector within endogenous transcription units. Given the time and resources required for the phenotypic analysis of mutant mice, it is advantageous to tailor screens toward specific genes of interest. A variety of criteria may be used to preselect insertional mutants prior to germ line transmission and phenotype analysis. These include the sequence of the target gene, the expression profile of the target gene, and the subcellular localization of the fusion product. This chapter focuses on a strategy developed to select insertional mutations specifically in genes encoding cell surface proteins based on the subcellular localization of the fusion protein products in embryonic stem (ES) cells. An overview of this technology is presented in the chapter followed by a detailed description of the experimental procedures.
1.
InDysdercus the abdominal segments are isolated from each other by an intersegmental region which can be distinguished on the basis of morphological and physiological criteria.
2.
The intersegmental region (ISR) consists of the visible segment border and narrow strips of cells anterior and posterior to it.
3.
The anterior strip (w-ISR) is white and merges with the white segment region (wS) in front of it, and the posterior strip (r-ISR) is red and merges posteriorly with the red segment region (rS). The wS and the rS meet in the middle of the segment; together they form the segment proper.
4.
Grafts from the ISR have been transplanted to various positions within a segment. The reactions of graft and host, respectively, can be distinguished in combinations involving a colour mutant and/or individuals of different sexes.
5.
The results show that cells of the r-ISR and the w-ISR each have some adhesiveness towards those tissues which they border in situ, and less adhesiveness towards other tissues. That is, the w-ISR is adhesive towards the r-ISR and wS and is usually rejected by tissue of the rS, whereas the r-ISR is adhesive towards tissue of the w-ISR and rS, but is rejected by tissue of the wS.
6.
The role which the ISR plays as a barrier between adjacent segments can essentially be interpreted on the basis of differences in cell adhesiveness.
7.
Besides these adhesiveness properties the two parts of the ISR show a long-range influence on polarity and pigment synthesis in surrounding segment tissue.
8.
The adhesiveness properties of the r-ISR and w-ISR can explain why the segment boundary forms such a straight line and why the ISR tends to grow between tissues from non-contiguous segment levels. This property can explain the hitherto not understood healing capacity of the ISR which even after wounding prevents cellular interactions between adjacent segments so effectively.
The homeobox gene en, homologous to the gene engrailed of Drosophila, is expressed in the metencephalic-mesencephalic segment of the vertebrate neural tube. Using quail-chick chimeras, an antibody against en proteins, and cytoarchitectonic techniques, we demonstrate that metencephalon transplanted to prosencephalon, at E2, maintains a high level of en proteins and its presumptive cerebellar fate. The ectopic metencephalon induces in the contiguous host prosencephalon the expression of en and, subsequently, a mesencephalic phenotype. These related genetic and phenotypic expressions indicate that the transcriptional regulatory en gene is involved in cerebellar and mesencephalic cytodifferentiation. The expression of en can also be induced in chick prosencephalon by a mammalian metencephalic graft, indicating that the factors regulating the transcription of en are phylogenetically well conserved.
The neural cell adhesion molecule (NCAM), probably the best characterized and most abundant cell adhesion molecule on neurons, is thought to be a major regulator of axonal growth and pathfinding. Here we present a detailed analysis of these processes in mice deficient for all NCAM isoforms, generated by gene targeting. The hippocampal mossy fiber tract shows prominent expression of polysialylated NCAM and the generation of new axonal projections throughout life. Focusing on this important intrahippocampal connection, we demonstrate that in the absence of NCAM, fasciculation and pathfinding of these axons are strongly affected. In addition we show alterations in the distribution of mossy fiber terminals. The phenotype is more severe in adult than in young animals, suggesting an essential role for NCAM in the maintenance of plasticity in the mature nervous system.
Since its initial identification in mouse mammary tumors, the proto-oncogene Wnt-1 has been shown to encode a member of a family of putative signalling molecules. Recent work has implicated Wnt-1, and other members of the Wnt gene family, in regulation of a number of basic developmental processes in Drosophila, Xenopus and the mouse. These studies are leading to a new appreciation of the normal and oncogenic actions of Wnt genes.
Abstract
As the human genome project approaches completion, the challenge for mammalian geneticists is to develop approaches for the systematic determination of mammalian gene function. Mouse mutagenesis will be a key element of studies of gene function. Phenotype-driven approaches using the chemical mutagen ethylnitrosourea (ENU) represent a potentially efficient route for the generation of large numbers of mutant mice that can be screened for novel phenotypes. The advantage of this approach is that, in assessing gene function, no a priori assumptions are made about the genes involved in any pathway. Phenotype-driven mutagenesis is thus an effective method for the identification of novel genes and pathways. We have undertaken a genome-wide, phenotype-driven screen for dominant mutations in the mouse. We generated and screened over 26,000 mice, and recovered some 500 new mouse mutants. Our work, along with the programme reported in the accompanying paper, has led to a substantial increase in the mouse mutant resource and represents a first step towards systematic studies of gene function in mammalian genetics.
In our previous studies on studies on spinal cord regeneration in the adult lizard and the newt, we observed that the radial processes of the regenerating ependyma form between them channels which are subsequently invaded by growing neurites. In the present study we compare embryogenesis of the newt spinal cord with regeneration in the adult. Except for minor differences, we observed that the germinal neuroepithelium of the embryo and larva patterns the longitudinal neural tracts in a similar manner. With these facts in mind we propose the blueprint hypothesis which asserts that inherent in the primitive germinal neuroepithelium and its derivative primitive glia is the pattern of the primary neuronal pathways which is expressed in neurogenesis as formed channels or spaces between the processes of the epithelial cells, the surfaces of which contain trace pathways which the growing neurites follow toward their destination. The trace pathways are envisoned as mechanical-chemical itineraries which the neurities follow according to their individual affinities. The hypothesis is compared to extant theories and the limitations in central nervous regeneration of vertebrates is compared.
The developing axons of retinal ganglion cells follow a stereotyped trajectory through the diencephalon to the optic tectum. In Xenopus, this trajectory closely parallels that of a preexisting fiber tract, the tract of the postoptic commissure (TPOC). This tract comprises part of the early CNS scaffold and has been proposed to play a critical role in guiding the later growing optic axons. We have tested this possibility using heterochronic and xenoplastic transplants of eye primordia to force optic axons to enter the brain before scaffold tracts have arisen in the forebrain. We show that optic axons can navigate appropriately in the absence of the TPOC or any other axons, indicating that axonal pathfinding cues are present in the axonless neuroepithelial sheet. We suggest that molecularly distinct heterogeneities within the neuroepithelium are used for pathfinding by early and late developing axons alike in normal development.
William McGinnis* and Robb Krumiauft *Department of Molecular Biophysics and Biochemistry Yale University New Haven, Connecticut 06511 tNationai institute for Medical Research Mill Hill London NW7 1AA England Animal embryos need to specify different cell types, and the genetic functions that provide such controls are easy to comprehend since they result in the formation of overtly differentiated tissues such as muscle, bone, and nerves. Embryos (and other fields of developing ceils) also need a more abstract system of genetic controls to respond to signals that establish asymmetry during early embryogen- esis (reviewed by St. Johnston and Niissiein-Volhard, 1992 [this issue of Ce//)), and transform those signals into different positional fates. On the anterior-posterior (A-P) axis of most embryos, such a system provides ceils with specific positional identities that eventually result in the development of structures appropriate to the position within the field, i.e., head structures from the anterior part of the field and so on. A fascinating surprise of recent years is that a Drosophila genetic system that specifies A-P positional identities is conserved in many other animals, where it apparently serves a similar function. The core of this system consists of a set of structurally similar genes that were orginaiiy discovered in Drosophila through the homeotic transformations (duplicated A-P positional vai- ues) that resulted from their mutation (reviewed by Lewis, 1976; Sanchez-Herreroet al., 1965; Kaufman et al., 1990). The proteins encoded by these homeotic selector genes are related at the structural level by their conservation of similar versions of the homeodomain motif. Over the past seven years, the term “homeodomain” has evolved to define a class of protein domains that have recognizable similarity to a 60 amino acid motif (encoded by 160 bp homeobox sequences) originally recognized in three Drosophila homeotic and segmentation proteins (reviewed by Scott et al., 1969). Those homeodomains that have been tested contain sequence-specific DNA binding activities and are part of larger proteins that function as transcriptional regulators (reviewed by Levine and Hoey, 1968). Recent nuclear magnetic resonance and crystaiio- graphic studies on two homeodomains have shown their structures to be related to the helix-turn-helix motif of pro- karyotic DNA-binding proteins (Otting et al., 1999; Kis- singeret al., 1990). Using amino acid sequence similarities within the homeodomain and in flanking protein sequence, it is possible to divide homeodomain proteins and the genes that encode them arbitrarily into various classes. One such is the Antp class, which includes homeodomains that share 69% or more identity with the Antennapedia
Rostrocaudal polarity of the tectum is first detectable at embryonic day 2 (E2) in the chick by the rostrocaudal gradient of en expression. When ectopic tectum is produced by heterotopic transplantation at E2 in the diencephalon, the gradient of en expression is inverted from the host polarity by environmental influences. Here we report that the retinal fibers project to the ectopic tectum in a topographic order in accord with the inverted gradient of en expression. Moreover, if ectopic tectum was produced at E3, when the en pattern was preserved, the retinotectal projection pattern was also in accord with the en pattern, suggesting that rostrocaudal polarity of retinotectal order follows en expression patterns.
To determine the role of the floor plate (FP) in CNS development, I have used labeling techniques, including immunolabeling, to analyze cyclops mutant embryos, which lack the FP. Except for the anterior brain, the mutant phenotype is almost exclusively confined to the vicinity of the ventral CNS midline. In the midbrain, the number of ventral neurons is reduced and cell patterning is disturbed. In contrast, the neuronal arrangement in the spinal cord is almost normal, including in particular both primary and secondary motoneurons. Longitudinal axonal bundles are disorganized in both the brain and spinal cord. Laser ablating the FP in wild-type embryos locally phenocopies cyclops axonal disturbances, and transplanting wild-type FP precursor cells into mutants locally rescues the disturbances. These results demonstrate a significant role for the FP in pathfinding and fasciculation by axons in situ, especially during their longitudinal courses.
The extracellular matrix component, s-laminin, is a homologue of the B1 subunit of laminin. S-laminin is concentrated in the synaptic cleft at the neuromuscular junction and contains a site that is adhesive for motor neurons, suggesting that it may influence neuromuscular development. To ascertain whether s-laminin may also play roles in the genesis of the central nervous system, we have examined its expression in the brain and spinal cord of embryonic and postnatal rats. S-laminin was not detectable in synapse-rich areas of adults. However, s-laminin was present in discrete subsets of three laminin-containing structures: (1) In the developing cerebral cortex, laminin and s-laminin were expressed in the subplate, a transient layer through which neuroblasts migrate and cortical afferents grow. Both laminin and s-laminin disappeared as embryogenesis proceeded; however, laminin was more widely distributed and present longer than s-laminin. (2) In the developing spinal cord, laminin was present throughout the pia. In contrast, s-laminin was concentrated in the pia that overlies the floor plate, a region in which extracellular cues have been postulated to guide growing axons. (3) In central capillaries, s-laminin appeared perinatally, an interval during which the blood-brain barrier matures. In contrast, laminin was present in capillary walls of both embryos and adults.
The role of the midline floor plate cells in the neuronal differentiation of the spinal cord was examined by comparing putative GABAergic neurons in wildtype zebrafish embryos with those in cyc-1 mutant embryos. The mutation produces a pleiotropic recessive lethal phenotype and is severe in rostral brain regions, but its direct effect in the caudal hindbrain and the spinal cord is apparently restricted to the depletion of the midline floor plate cells. In wildtype embryos, an antibody against the neurotransmitter GABA labeled the cell bodies, axons, and growth cones of three classes of previously identified neurons; dorsal longitudinal neurons (DoLA), commissural secondary ascending neurons (CoSA), and ventral longitudinal neurons (VeLD). A novel ventral cell type, Kolmer-Agduhr (KA) neurons, was also labeled. In the cyc-1 mutant, abnormalities were observed in some, but not all, of the GABAreactive CoSA, VeLD, and KA axons, while the axonal trajectories of DoLA neurons were not affected. Furthermore, the number of KA cells was reduced in the mutant while the numbers of the other GABAreactive cells were unperturbed. These observations corroborate our earlier hypothesis that the floor plate cells are one of several guidance cues that direct axonal outgrowth near the ventral midline of the spinal cord. They also suggest that the floor plate cells may play a role in the cellular differentiation of the spinal cord of zebrafish embryos.
We have identified a novel frog gene, Pintallavis (the Catalan for lipstick), that is related to the fly fork head and rat HNF-3 genes. Pintallavis is expressed in the organizer region of gastrula embryos as a direct zygotic response to dorsal mesodermal induction. Subsequently, Pintallavis is expressed in axial midline cells of all three germ layers. In axial mesoderm expression is graded with highest levels posteriorly. Midline neural plate cells that give rise to the floor plate transiently express Pintallavis, apparently in response to induction by the notochord. Overexpression of Pintallavis perturbs the development of the neural axis, suppressing the differentiation of anterior and dorsal neural cell types but causing an expansion of the posterior neural tube. Our results suggest that Pintallavis functions in the induction and patterning of the neural axis.
The floor plate is a cell group implicated in the control of neural cell pattern and axonal growth in the developing vertebrate nervous system. To identify molecules that might mediate the functions of the floor plate, we have used subtractive hybridization techniques to isolate floor plate-enriched cDNA clones. One such clone encodes a novel secreted protein, F-spondin, which is expressed at high levels in the floor plate. The C-terminal half of the protein contains six repeats identified previously in thrombospondin and other proteins implicated in cell adhesion. F-spondin is expressed in the floor plate at the time that axons first extend and at lower levels in the peripheral nerve. Recombinant F-spondin promotes the attachment of spinal cord and sensory neuron cells and the outgrowth of neurites in vitro. F-spondin may contribute to the growth and guidance of axons in both the spinal cord and the PNS.
We previously documented a greater than 100-fold rostrocaudal gradient of chloramphenicol acetyltransferase (CAT) expression in the muscles of adult mice that bear a myosin light chain-CAT transgene: successively more caudal muscles express successively higher levels of CAT. Here we studied the development and maintenance of this positional information in vitro. CAT levels reflect the rostrocaudal positions of the muscles from which the cells are derived in cultures established from adult muscles, in clones derived from individual adult myogenic (satellite) cells, in cultures prepared from embryonic myoblasts, and in cell lines derived by retrovirus-mediated transfer of an oncogene to satellite cells. Our results suggest that myoblasts bear a positional memory that is established in embryos, retained in adults, cell autonomous, heritable, stable to transformation, and accessible to study in clonal cell lines.
The spinal cord of early zebrafish embryos contains a small number of neuronal classes whose growth cones all follow stereotyped, cell-specific pathways to their targets. Two classes of spinal neurons make cell-specific turns at or near the ventral midline of the spinal cord, which is occupied by a single row of midline floor plate cells. We tested whether these cells guide the growth cones by examining embryos missing the midline floor plate cells due either to laser ablation of the cells or to a mutation (cyc-1). In these embryos the growth cones followed both normal and aberrant pathways once near the ventral midline. This suggests that normally the midline floor plate cells do provide guidance cues, but that these cues are not obligatory.
We have confirmed that the gene trap vector pGT4.5 creates spliced fusion transcripts with endogenous genes and prevents the synthesis of normal transcripts at the site of integration. cDNA was prepared to the lacZ fusion transcript in three ES cell lines to recover endogenous exon sequences upstream of lacZ. Each of the clones detected a unique-sized endogenous transcript, as well as the fusion transcript in the ES cell line from which the clone was derived. Sequence analysis of these clones and larger clones isolated from a random-primed cDNA library showed that the splice acceptor was used properly. For two insertions, the expression patterns of the lacZ reporter and the associated endogenous gene were compared in situ at three embryonic stages and were found to be similar. Three gene trap insertions were transmitted into the germ line, and abnormalities were observed with two of the three insertions in the homozygous state. RNA obtained from mice homozygous for the two mutant gene trap insertions was analyzed for normal endogenous transcripts and negligible amounts were detected, indicating that little splicing around the gene trap insertion occurred. This work demonstrates the capacity of the gene trap vector to generate lacZ fusion transcripts, to accurately report endogenous gene expression, and to mutate the endogenous gene at the site of integration.
In vertebrates the developing hindbrain is organized in segmental units. These units provide the primary grid for differentiation and axonal outgrowth. In the more anterior regions of the brain, however, the subdivisions remain more controversial. Cellular and molecular studies of the embryonic brain in lower vertebrates such as the zebrafish, Brachydanio rerio, may reveal remnants of such subdivisions. We have isolated complementary DNA clones for two zebrafish pax genes related to Drosophila and mouse paired-box-containing segmentation genes. The expression of these two genes is confined to specific regions in the embryonic forebrain and midbrain. Strikingly, the borders of expression of the two pax genes coincide with morphological landmarks corresponding to the primary axon tracts that are generated in the embryonic brain a few hours after the initiation of expression of these genes.
A multigene family of paired-box-containing genes (Pax genes) has been identified in the mouse. In this report, we describe the expression pattern of Pax-6 during embryogenesis and the isolation of cDNA clones spanning the entire coding region. The Pax-6 protein consists of 422 amino acids as deduced from the longest open reading frame and contains, in addition to the paired domain, a paired-type homeodomain. Beginning with day 8 of gestation, Pax-6 is expressed in discrete regions of the forebrain and the hindbrain. In the neural tube, expression is mainly confined to mitotic active cells in the ventral ventricular zone along the entire anteroposterior axis starting at day 8.5 of development. Pax-6 is also expressed in the developing eye, the pituitary and the nasal epithelium.
Certain types of glial structures, located at strategic positions along axon pathways, may provide the mechanical and/or chemical elements for the construction of barriers which can grossly direct the elongation of axons during development. The roof plate, a putative axon barrier, is located along the dorsal midline of the developing spinal cord and may be important for the guidance of the commissural and dorsal column axons. We examined the roof plate to determine the developmental morphology of the region and to determine which molecules were correlated with the barrier function when axons were growing nearby. Light and electron microscopic observations of the roof plate revealed that this glial domain undergoes a dramatic change in shape from a "wedge" with large extracellular spaces between the cell apices at E12.5 to a thin, dense septum with reduced extracellular space at E15.5. Immunocytochemical techniques demonstrated that highly sialylated neural cell adhesion molecule (N-CAM), the carbohydrate recognized by L2 monoclonal antibody, cholinesterase, stage-specific embryonic antigen 1, and a ligand that binds tetragonolobus purpureas agglutinin are expressed by the roof plate. These molecules, however, were also found in other regions of the spinal cord which are permissive or attractive to axon growth. A molecule which is unique to the roof plate when axons grow close to, but do not cross, the dorsal midline is a glycosaminoglycan (GAG), keratan sulfate. Keratan sulfate is also present in the tectal midline and in other noninnervated regions such as the outer epidermis and developing cartilage. Our data suggest that keratan sulfate, alone or in combination with other molecules expressed by the roof plate, may be responsible, in part, for the inhibition of axon elongation through the roof plate in the embryonic spinal cord.
Differentiation of individual rhombomeres of the chicken hindbrain directly follows the emergence of primary brain vesicles. Immediately after the constric tion of the prosencephalon at HH9, a series of vesicles of decreasing size is established almost simultaneously between HH9 and HH10, including mesencephalon, four preotic (R2–R5) and one postotic (R6/R7) rhombo meres. Thereby, the cranial neural tube is ventrally embedded in a mesodermal PNA-binding matrix that particularly accumulates underneath vesicular constric tion sites, as demonstrated for the segregation of the prosencephalon at HH9 and the cerebellar rhombomere R1 from R2 at HH13. The subsequent period of hind brain differentiation is analyzed by cholinesterase (AChE, BChE) and peanut lectin histochemistry, by the BrdU and the neurite-specific G4 antibodies. Preotically, differentiation of two pairs of rhombomeres (R4+R5, R2+R3) starts in R4, immediately followed by R2. The caudal rhombomeres of both pairs are delayed (R5, R3). Then the postotic rhombomere is subdivided, whereby R7 differentiates before R6. Thus, the development in the direct vicinity of the otic vesicle is delayed (R5, R6). R7 is the last rhombomere that is demarcated caudally.
Based on these findings, we postulate two processes that may regulate rhombomere formation in the chicken embryo: (a) an early rostrocaudal wave establishing the major brain vesicles, (b) a superimposed pairwise seg mentation emanating rostrally and caudally from the otic vesicle. The segregation of the cerebellar rhombo mere is a late step.
Abbreviations: AChE, acetylcholinesterase (E.C. 3.1.1.7);
BChE, butyrylcholinesterase (E.C. 3.1.1.8);
BrdU, bromodesoxyuridine;
D, diencephalon;
E, eye stalk;
G4, neurite-specific antigen;
M, mesencephalon;
NP, neuropore;
OV, otic vesicle;
PNA, peanut agglutinin;
P, prosencephalon;
Rl, rhombomere No. 1;
R, rhombencephalon; T, telencephalon.
Rhombomeres are regarded as the manifestation of innate segmentation within the vertebrate CNS. To investigate developmental changes occurring in the CNS and PNS, a series of chick embryos were immunostained with several monoclonal antibodies. The HNK-1-immunoreactivity (IR) appeared in rhombomeres (r) 3 and r5 around stage 15, when r2 and r4 were not stained. This alternate pattern is similar to the Krox-20 gene expression in the mouse embryo. At levels of r2 and r4, HNK-1+ neural crest cell masses were attached to the CNS forming cranial sensory ganglia. In these rhombomeres, an accumulation of neuroepithelial cells near the cranial nerve root and early development of neuroblasts in the basal plate were observed. The above observations seem to suggest that the alternate HNK-1-IR in rhombomeres might be related to the expression of cell adhesion molecules, and therefore also to the adhesion of the cranial ganglion precursors to the CNS, which takes place every other rhombomere in the preotic region. Thus, the alternate pattern of the HNK-1-IR seems to be related to the morphogenesis of preotic branchial nerves.
A century has elapsed since Ramón y Cajal proposed his chemotropic theory of axon guidance, i.e. the attraction of developing axons by diffusible molecules emanating from their targets. Although the precise contribution of axonal chemoattractants to guidance in vivo remains to be established, two lines of investigation have provided evidence for their existence and importance. First, concentration gradients of nerve growth factor (NGF) have been shown to orient the growth of regenerating sensory axons in vitro. Although NGF does not appear to guide axons during development, these studies show that growth cones can orient in gradients of diffusible molecules. Second, the cellular targets of several different classes of developing neurons have been shown to secrete as yet unidentified diffusible factors that can orient axons. We review these studies and discuss the potential contribution of chemotropism to the establishment of axonal projection patterns in vertebrates.
Early in its development, the chick embryo hindbrain manifests an axial series of bulges, termed rhombomeres. Rhombomeres are units of cell lineage restriction, and both they and their intervening boundaries form a series that reiterates various features of neuronal differentiation, cytoarchitecture, and molecular character. The segmented nature of hindbrain morphology and cellular development may be related to early patterns of cell division. These were explored by labeling with BrdU to reveal S-phase nuclei, and staining with basic fuchsin to visualise mitotic cells. Whereas within rhombomeres, S-phase nuclei were located predominantly toward the pial surface of the neuroepithelium, at rhombomere boundaries S-phase nuclei were significantly closer to the ventricular surface. The density of mitotic figures was greater toward the centres of rhombomeres than in boundary regions. Mitotic cells did not show any consistent bias in the orientation of division, either in the centres of rhombomeres, or near boundaries. Our results are consistent with the idea that rhombomeres are centres of cell proliferation, while boundaries contain populations of relatively static cells with reduced rates of cell division.
Spatial gradients of axon guiding molecules have long been suspected to provide positional and directional cues for retinal
ganglion cell axons growing within the optic tectum. With the identification of a guiding activity from tectal cell membranes,
it has become possible to investigate the potential physiological significance of molecular gradients for retinal growth cone
behavior in vitro. A subset of retinal growth cones, those from the temporal half, were highly sensitive to small concentration
changes of the guiding component. The degree of response was correlated with the strength of the gradient. These findings
demonstrate that the neural growth cone can read gradients of surface-associated information.
Development in the chick hindbrain is founded on a segmented pattern. Groups of cells are allocated to particular segmental levels early in development, the cells of each segment (rhombomere) mixing freely with each other, but not with those of adjacent segments. After rhombomere formation, cells in the boundary regions become increasingly specialised. Rhombomeres are thus separate territories that will ultimately pursue different developmental fates. We are investigating the mechanisms that establish and maintain the pattern of rhombomeres and their boundaries. Donor-to-host transplantation experiments were used to confront tissue from different axial levels within the hindbrain. The frequency of boundary regeneration and patterning in the hindbrain was then assessed, based on gross morphology, arrangement of motor neurons and immunohistochemistry. We found that when rhombomeres from adjacent positions or positions three rhombomeres distant from one another were confronted, a normal boundary was invariably reconstructed. Juxtaposition of rhombomere 5 with 7 also yielded a new boundary. By contrast, donor and host tissue of the same positional origin combined without forming a boundary. The same result was obtained in combinations of rhombomeres 3 and 5. Confrontation of tissue from even-numbered rhombomeres 4 with 6 or 2 with 4 also failed to regenerate a boundary in the majority of cases. These results suggest that cell surface properties vary according to rhombomeric level in the hindbrain, and may support the idea of a two-segment periodicity.
The floor plate of the vertebrate nervous system has been implicated in the guidance of commissural axons at the ventral midline. Experiments in chick have also suggested that at earlier stages of development the floor plate induces the differentiation of motor neurons and other neurons of the ventral spinal cord. Here we have examined the development of the spinal cord in a mouse mutant, Danforth's short-tail, in which the floor plate is absent from caudal regions of the neuraxis. In affected regions of the spinal cord, commissural axons exhibited aberrant projection patterns as they reached and crossed the ventral midline. In addition, motor neurons were absent or markedly reduced in number in regions of the spinal cord lacking a floor plate. Our results suggest that the floor plate is indeed an intermediate target in the projection of commissural axons and support the idea that several different mechanisms operate in concert in the guidance of axons to their cellular targets in the developing nervous system. In addition, these experiments suggest that the mechanisms that govern the differentiation of the floor plate and other ventral cell types in the neural tube are common to mammals and lower vertebrates.
Notochordless Xenopus embryos were produced by u.v. irradiation of the uncleaved fertilized egg. The spinal cords were examined using intermediate filament staining for glial cells, retrograde HRP staining for neuronal morphology and an anti-glycinergic antibody to reveal commissural cells and axons. The floorplate cells of the normal cord appear to be absent and their position along the ventral midline of the cord is occupied by motor neurones, Kolmer-Agduhr cells, radial glial cells and a ventrally placed marginal zone containing the longitudinal axons. Motor neurone number is reduced to 15% of control values, and the sensory extramedullary cell number is increased twentyfold. Commissural axons are still able to cross the ventral cord but do so at abnormal angles and some commissural axons continue to grow circumferentially up the contralateral side of the cord rather than turning to grow longitudinally. Extracellular electrophysiological recordings from motor axons reveal that the normal alternation of locomotor activity on the left and right side of the embryo is lost in notochordless animals. These results suggest that the notochord and/or the normal floor plate structure are important for the development of the laterality of spinal cord connections and may influence motor neurone proliferation or differentiation.
In the E4 (embryonic day 4) chick tectal primordium, engrailed expression is strong at the caudal end and gradually weakens toward the rostral end. We used quail-chick chimeric tecta to investigate how the caudorostral gradient of engrailed expression is established and whether it is correlated with the subsequent rostrocaudal polarity of tectal development.
To examine the positional value of the tectal primordium, we produced ectopic tecta in the diencephalon by transplanting a part of the mesencephalic alar plate heterotopically. In the ectopic tectum, the gradient of the engrailed expression reversed and the strength of the expression was dependent on the distance from the mes-diencephalon junction; the nearer the ectopic tectum was to the junction, the weaker the expression was. Consequently, the pattern of the engrailed expression in the host and ectopic tecta was nearly a mirror image, suggesting the existence of a repressive influence around the mes-diencephalon junction on the engrailed expression.
We examined cytoarchitectonie development in the ectopic tecta, which normally proceeds in a gradient along the rostrocaudal axis; the rostral shows more advanced lamination than the caudal. In contrast, the caudal part of the ectopic tecta (near to the mesdiencephalon junction) showed more advanced lamination than the rostral. In both the host and ectopic tecta, advanced lamination was observed where the engrailed expression was repressed, and vice versa.
Next we studied the correlation between engrailed expression and retinotectal projection from a view of plasticity and rigidity of rostrocaudal polarity in the tectum. We produced ectopic tecta by anisochronal transplantations between E3 host and E2 donor, and showed that there is little repressive influence at E3 around the mes-diencephalon junction. We then made chimeric double-rostral tectum (caudal half of it was replaced by rostral half of the donor tectum) or double-caudal tectum at E3. The transplants kept their original staining pattern in hosts. Consequently, the chimeric tecta showed wholly negative or positive staining of engrailed protein on the grafted side. In such tecta retinotectal projection pattern was disturbed as if the transplants retained their original position-specific characters.
We propose from these heterotopic and anisochronal experiments that the engrailed expression can be a marker for subsequent rostrocaudal polarity in the tectum, both as regards cytoarchitectonie development and retinotectal projection.
SC1, an integral membrane glycoprotein of 100 kd, is uniquely and transiently expressed on spinal cord motoneurons early in development and appears in peripheral neurons and several other tissues during development. SC1 has been purified by immunoaffinity techniques, and SC1 cDNA clones have been obtained by screening an E4 chick embryo phage expression library with a rabbit polyclonal antibody produced against purified SC1. The deduced protein sequence of 588 amino acids consists of a signal peptide, five immunoglobulin-like domains, a transmembrane region, and a short cytoplasmic tail. The sequence is most similar to MUC18, reported as a tumor progression marker in human melanoma. Transfection of SC1 cDNA into mammalian cells leads to cell surface expression of SC1 antigen and a subsequent increase in cell-cell adhesion. SC1 molecules bind to each other via a homophilic adhesion mechanism, independently of calcium or magnesium ions. SC1 may have a role in lateral motor column formation or neurite growth or fasciculation.
Individual classes of neural cells differentiate at distinct locations in the developing vertebrate nervous system. We provide evidence that the pattern of cell differentiation along the dorsoventral axis of the chick neural tube is regulated by signals derived from two ventral midline cell groups, the notochord and floor plate. Grafting an additional notochord or floor plate to ectopic positions, or deleting both cell groups, resulted in changes in the fate and position of neural cell types, defined by expression of specific antigens. These results suggest that the differentiation of neural cells is controlled, in part, by their position with respect to the notochord and floor plate.
Cadherins are a family of cell adhesion receptors that are crucial for the mutual association of vertebrate cells. Through
their homophilic binding interactions, cadherins play a role in cell-sorting mechanisms, conferring adhesion specificities
on cells. The regulated expression of cadherins also controls cell polarity and tissue morphology. Cadherins are thus considered
to be important regulators of morphogenesis. Moreover, pathological examinations suggest that the down-regulation of cadherin
expression is associated with the invasiveness of tumor cells.
Neurite outgrowth promoting properties of neural cell surface proteins can be assessed by immobilizing isolated membrane proteins on nitrocellulose-coated petri dishes. Using this method, we have identified a unique cell surface antigen, designated P84, as a new neural cell adhesion molecule. Immunoaffinity purified P84 contains three polypeptides with molecular weights of 167, 85, and 66 kDa. When spotted onto nitrocellulose-coated plates, P84 supports adhesion of mouse cerebellar neurons and neurite outgrowth. Glial cell attachment was also observed. Intact monoclonal antibodies directed against P84 inhibit adhesion and outgrowth on a P84 substrate. This antigen is found on the surfaces of neurons in cultures of cerebellar cells. It is also found on a subclass of unidentified flat cells. P84 is not found on oligodendrocytes or GFAP-positive astrocytes. As early as E9, P84 could be detected in the floor plate region of the spinal cord. This pattern persists throughout embryonic development. Postnatally, widespread expression of P84 is observed in a variety of CNS tissues.
Three known genes guide circumferential migrations of pioneer axons and mesodermal cells on the nematode body wall. unc-5 affects dorsal migrations, unc-40 primarily affects ventral migrations, and unc-6 affects migrations in both directions. Circumferential movements still occur, but are misdirected whereas longitudinal movements are normal in these mutants. Pioneer growth cones migrating directly on the epidermis are affected; growth cones migrating along established axon fascicles are normal. Thus these genes affect cell guidance and not cell motility per se. We propose that two opposite, adhesive gradients guide circumferential migrations on the epidermis. unc-5, unc-6, and unc-40 may encode these adhesion molecules or their cellular receptors. Neurons have access to the basal lamina and the basolateral surfaces of the epidermis, but mesodermal cells contact only the basal lamina. These genes probably identify molecular cues on the basal lamina that guide mesodermal migrations. The same basal lamina cues, or perhaps related molecules on the epidermal cell surfaces, guide pioneer neurons.
We have examined neuronal differentiation and the formation of axon tracts in the embryonic forebrain and midbrain of the zebrafish, between 1 and 2 days postfertilisation. Axons were visualised with three techniques; immunocytochemistry (using HNK-1 and antiacetylated tubulin antibodies) and horseradish peroxidase (HRP) labelling in whole-mounted brains, and transmission electron microscopy. Differentiation was monitored by histochemical staining for acetylcholinesterase (AChE).
These independent methods demonstrated that a simple grid of tracts and commissures forms the initial axon scaffold of the brain. At 1 day, the olfactory nerve, four commissures, their associated tracts and three other non-commissural tracts are present. By 2 days, these tracts and commissures have all greatly enlarged and, in addition, the optic nerve and tract, and three new commissures and their associated tracts have been added.
Small applications of HRP at various sites revealed the origins and projections of some of these earliest axons. Retrogradely labelled cell bodies originated from regions that were also positive for AChE activity. At 1 day, HRP-labelled axons were traced: (1) from the olfactory placode through the olfactory nerve to the dorsal telencephalon; (2) from the telencephalon into the tract of the anterior commissure and also to the postoptic region of the diencephalon; (3) from the hindbrain through the ventral midbrain and diencephalon to the postoptic commissure; (4) from the dorsal diencephalon (in or near the epiphysis) to the tract of the postoptic commissure; (5) from ventral and rostral midbrain through the posterior commissure. Three new projections were demonstrated at 2 days: (1) from the retina through the tract of the postoptic commissure to the tectum; (2) from the telencephalon to the contralateral diencephalon; and (3) from the telencephalon to the ventral flexure.
These results show that at 1 day, the zebrafish brain is impressively simple, with a few small, well-separated tracts but by 2 days the brain is already considerably more complex. Most of the additional axons added onto pre-existent tracts rather than pioneered new ones supporting the notion that other axons play a crucial role in the guidance of early central nervous system (CNS) axons.
A strategy was devised for identifying regions of the mouse genome that are transcriptionally active in a temporally and spatially restricted manner during development. The approach is based on the introduction into embryonic stem cells of two types of lacZ reporter constructs that can be activated by flanking mouse genomic sequences. Embryonic stem cells containing the lacZ constructs were used to produce chimaeric mice. Developmental regulation of lacZ expression occurred at a high frequency. Molecular cloning of the flanking endogenous genes and introduction of these potential insertional mutations into the mouse germ line should provide an efficient means of identifying and mutating novel genes important for the control of mammalian development.
Identification of specific neuronal populations and their projections in the developing hindbrain reveals a segmental organization in which pairs of metameric epithelial units cooperate to generate the repeating sequence of cranial branchiomotor nerves. Neurogenesis also follows a two-segment repeat, suggesting parallels with insect pattern formation.