The cellular and molecular mechanisms that regulate regional specification of the forebrain are largely unknown. We studied the expression of transcription factors in neural plate explants to identify tissues, and the molecules produced by these tissues, that regulate medial-lateral and local patterning of the prosencephalic neural plate. Molecular properties of the medial neural plate are regulated by the prechordal plate perhaps through the action of Sonic Hedgehog. By contrast, gene expression in the lateral neural plate is regulated by non-neural ectoderm and bone morphogenetic proteins. This suggests that the forebrain employs the same medial-lateral (ventral-dorsal) patterning mechanisms present in the rest of the central nervous system. We have also found that the anterior neural ridge regulates patterning of the anterior neural plate, perhaps through a mechanism that is distinct from those that regulate general medial-lateral patterning. The anterior neural ridge is essential for expression of BF1, a gene encoding a transcription factor required for regionalization and growth of the telencephalic and optic vesicles.

In addition, the anterior neural ridge expresses Fgf8, and recombinant FGF8 protein is capable of inducing BF1, suggesting that FGF8 regulates the development of anterolateral neural plate derivatives. Furthermore, we provide evidence that the neural plate is subdivided into distinct anterior-posterior domains that have different responses to the inductive signals from the prechordal plate, Sonic Hedgehog, the anterior neural ridge and FGF8. In sum, these results suggest that regionalization of the forebrain primordia is established by several distinct patterning mechanisms: (1) anterior-posterior patterning creates transverse zones with differential competence within the neural plate, (2) patterning along the medial-lateral axis generates longitudinally aligned domains and (3) local inductive interactions, such as a signal(s) from the anterior neural ridge, further define the regional organization.

One of the central questions in developmental neurobiology is how regional diversity of the vertebrate central nervous system (CNS) is established. Available evidence suggests that patterning of the CNS primordium, the neural plate, is controlled by distinct mechanisms along the anterior-posterior (AP) and medial-lateral (ML; ventral-dorsal, VD, in the neural tube) axes (reviewed in Lumsden and Krumlauf, 1996). AP patterning may be regulated by several mechanisms, including vertical induction by tissues underlying the neural plate and planar induction (reviewed in Doniach, 1993; Ruiz i Altaba, 1994). ML regional identities within the posterior neural plate are specified in part by adjacent non-neural tissues. For instance, at the spinal cord level of the neural plate, medial cell fates are specified by the notochord (reviewed in Jessell and Dodd, 1992; Placzek, 1995; Tanabe and Jessell, 1996), whereas lateral cell fates are likely to be specified by the adjacent nonneural ectoderm (Dickinson et al., 1995; Liem et al. 1995). Furthermore, the molecular mechanisms underlying these inductive processes are being elucidated; medial patterning is regulated by Sonic Hedgehog (SHH) (Echelard et al., 1993; Roelink et al., 1994, 1995; Tanabe et al., 1995, Hynes et al., 1995b; Martí et al., 1995; Chiang et al., 1996; Ericson et al., 1996) and lateral signaling is likely to be regulated by members of the TGFβ superfamily (Basler et al., 1993; Liem et al., 1995, reviewed in Tanabe and Jessell, 1996).

Unlike the more posterior regions of the CNS, little is known about the molecular and cellular mechanisms underlying forebrain regionalization. The forebrain is a topographically complex structure that comprises tissues that are unique within the CNS, including the cerebral cortex, basal ganglia, eye, thalamus and hypothalamus. In addition, there is reason to believe that some aspects of forebrain patterning mechanisms may be distinct from those operating in other CNS regions. For instance, the notochord, which has a prominent role in ML pattern formation in the posterior CNS, does not underlie the forebrain. In addition, signals originating in specialized epithelial structures (e.g. placodes and Rathke’s pouch) are essential for the development of forebrain-specific structures, such as the olfactory bulb (Graziadei and Monti-Graziadei, 1992; Gong and Shipley, 1995), posterior pituitary (Daikoku et al., 1983) and retina (reviewed in Saha et al., 1992). Progress towards elucidating the control of embryonic forebrain development has been made through the identification of regionally expressed genes (reviewed in Rubenstein et al., 1994; Shimamura et al., 1995, 1997; Rubenstein and Shimamura, 1996). These expression patterns suggest that ML (VD) patterning of the forebrain is regulated by mechanisms that are also used in more posterior regions of the CNS (Shimamura et al., 1995). To test this hypothesis, and to search for other mechanisms that pattern the forebrain, we studied the regulation of regional gene expression in neural plate explants by non-neural tissues and the proteins produced by these tissues. We have studied the inductive and molecular properties of the prechordal plate, an axial mesendodermal tissue located rostral to the notochord, with regard to its ability to induce medial and repress lateral molecular characteristics in the anterior neural plate. In addition, we have tested whether the non-neural ectoderm adjacent to the forebrain regulates lateral (dorsal) patterning. The results of these experiments suggest that common ML (VD) patterning mechanisms are found in all CNS regions. Our findings have also revealed that different axial levels of the neural plate have distinct competence to respond to common medial patterning signals.

While the combination of AP and ML patterning generates a grid of molecularly distinct CNS domains, regional specification of the forebrain utilizes other patterning mechanisms to generate the extensive diversity of its tissues (e.g. induction of the posterior pituitary by Rathke’s pouch). Thus, we searched for localized organizing centers that regulate forebrain regional specification. We found that the anterior neural ridge, and a molecule expressed in it (FGF8), regulate expression of BF1, a transcription factor that is essential for telencephalon and eye development (Xuan et al., 1995).

Embryo dissection

Timed pregnant ICR mice were killed and embryos dissected from decidual tissue in chilled Hank’s solution. Fertilization is assumed to occur at the midpoint of the dark period. Staging of embryos used morphological criteria, such as the number of somites. The cephalic region of embryo was isolated and digested with 2.5% pancreatin and 0.5% trypsin in Ca2+/Mg2+-free Hepes-buffered saline (pH 7.4), followed by mechanical dissection with fine glass needles to separate the ectoderm, mesoderm and endoderm, which includes the prechordal and notochordal plate (Beddington, 1987) (see Fig. 1).

Fig. 1.

Schematic representation of the explant culture. (A-D) The cephalic region of mouse embryos ranging from the headfold to early somite stages (up to 5-somite) was processed to separate the neuroectoderm/ectoderm (C), head mesenchyme (mesoderm and neural crest), and embryonic endoderm (D). (A) Schematic cross section of an embryo at the level indicated in B. (C) A ventral view of isolated neuroectoderm/ectoderm. (D) A dorsal view of isolated endoderm that includes the prechordal and notochordal plates. The axial mesendoderm was dissected, as shown in a dotted box, when used for transplantation. Several types of manipulation with these tissues were performed; (E) culture of ectoderm/neuroectoderm with or without the axial mesendoderm, which was alternatively transplanted into an ectopic location (E′) or ectopically transplant after rotation by 180° (E′′); (F) or excision and/or transplantation of the anterior ectoderm that includes the anterior neural ridge (a junction between the neuroectoderm and non-neural ectoderm) indicated by a thick broken line. The explants were cultured for 24 hours. All of the explants described here do not contain head mesenchyme in order to achieve direct contact between the neural plate and transplanted tissues. The specimens were then examined with appropriate markers. Abbreviations for all figures: anr, anterior neural ridge; ec, ectoderm; en, endoderm; fg, foregut endoderm; hm, head mesenchyme; me, mesencephalon; nc, notochordal plate; ne, neuroectoderm; pcp, prechordal plate; pr, prosencephalon, rh, rhombencephalon. Bars, 100 μm.

Fig. 1.

Schematic representation of the explant culture. (A-D) The cephalic region of mouse embryos ranging from the headfold to early somite stages (up to 5-somite) was processed to separate the neuroectoderm/ectoderm (C), head mesenchyme (mesoderm and neural crest), and embryonic endoderm (D). (A) Schematic cross section of an embryo at the level indicated in B. (C) A ventral view of isolated neuroectoderm/ectoderm. (D) A dorsal view of isolated endoderm that includes the prechordal and notochordal plates. The axial mesendoderm was dissected, as shown in a dotted box, when used for transplantation. Several types of manipulation with these tissues were performed; (E) culture of ectoderm/neuroectoderm with or without the axial mesendoderm, which was alternatively transplanted into an ectopic location (E′) or ectopically transplant after rotation by 180° (E′′); (F) or excision and/or transplantation of the anterior ectoderm that includes the anterior neural ridge (a junction between the neuroectoderm and non-neural ectoderm) indicated by a thick broken line. The explants were cultured for 24 hours. All of the explants described here do not contain head mesenchyme in order to achieve direct contact between the neural plate and transplanted tissues. The specimens were then examined with appropriate markers. Abbreviations for all figures: anr, anterior neural ridge; ec, ectoderm; en, endoderm; fg, foregut endoderm; hm, head mesenchyme; me, mesencephalon; nc, notochordal plate; ne, neuroectoderm; pcp, prechordal plate; pr, prosencephalon, rh, rhombencephalon. Bars, 100 μm.

Organ culture

In vitro tissue explant culture was carried out as described previously (Shimamura and Takeichi, 1992) with slight modifications. The isolated cephalic neuroectoderm was placed in a small drop of culture medium on a nuclepore filter (Costar, #110414) in combination with the isolated prechordal plate or the notochordal plate which was placed under the basal surface of the neuroectoderm. The explants were cultured on filters floating on DMEM supplemented 10% fetal bovine serum and 40% rat serum (Harlan, Indianapolis) in a CO2 incubator at 37°C for 24 hours. We found that these conditions are critical for consistent growth and morphogenesis of the explants. No significant necrosis or obvious abnormalities in morphogenesis were detected.

Recombinant proteins were kindly provided from Dr G. Martin (FGF4, 8), Dr P. Beachy (SHH-N) and Genetics Institute (BMP2, 4, 7). These proteins were applied to the explants either by their addition to the culture medium or by placing heparin-acrylic beads (Sigma, H5263) soaked in each reagent according to the method described by Crossley et al. (1996a).

RNA probes

Digoxigeninor fluorescein-labeled RNA probes were prepared according to the instruction of the manufacturer (BoehringerMannheim). A plasmid encoding Nkx2.1 is described in Shimamura et al. (1995). Additional plasmids were kindly provided from Drs R. Derynck (Bmp6), P. Gruss (Pax6), B. Hogan (HNF3β, Bmp4), G. Karsenty (Bmp7), E. Lai (BF1), G. Martin (Fgf2, 8), I. Mason (Fgf7), A. McMahon (Shh), M.-S. Qiu (Msx1), and A. Rosenthal (Fgf4, 6).

In situ hybridization

Whole-mount in situ hybridization was performed as described (Shimamura et al., 1994). For two-color staining, BM purple AP substrate and INT/BCIP (Boehringer-Mannheim) were used as chromogenic substrates for the alkaline phosphatase. Stained samples were observed and photographed with an Olympus SZH10 stereomicroscope or with a Nikon Optiphoto2 microscope under DIC optics when they are flat-mounted or sectioned with a cryostat.

Effect of the prechordal plate in ML patterning of the prosencephalic neural plate

ML regionalization of the prosencephalic neural plate is revealed by the expression of three mouse homeobox genes (Shimamura et al., 1995, 1997). Nkx2.1 is expressed medially, whereas Pax6 is expressed laterally (Fig. 2A,B). Each of these genes is essential for forebrain development: Nkx2.1 (also known as T/ebp and TTF1) is required for development of the basal plate of the forebrain (e.g. the tuberal hypothalamus) (Kimura et al., 1996), whereas Pax6 is essential for development of the alar plate of the forebrain (e.g. cerebral cortex) (Schmahl et al., 1993; Stoykova et al., 1996). We have used the expression of these genes as markers for patterning along the ML axis of the prosencephalic neural plate.

Fig. 2.

Expression patterns of medial-lateral markers in the forebrain neural plate and effects of the prechordal plate on the expression. (A) Nkx2.1 expression at the 3to 4-somite-stage of a mouse embryo. Nkx2.1 is expressed in a medial domain that ends at the level of cephalic flexure (arrow). (B) A frontal view of a 5-somite-stage embryo stained for Pax6. Pax6 is expressed in lateral regions of the prosencephalic neural plate, and is excluded from the medial region. (C) A transverse section of the anterior region of a 5-somitestage embryo, showing Shh (orange) and Nkx2.1 (purple) expression. Note that Nkx2.1 expression in the neuroectoderm is located in the vicinity of where the Shh-expressing prechordal plate (the dorsal roof of foregut) is juxtaposed to the medial neural plate (arrowhead). (D-J) Neural plate explants stained for regional markers: Nkx2.1 (purple, D-G; orange, H,I), Shh (orange, D,E,G,J) and Pax6 (purple, H-J). Shh expression was used to reveal the site of transplanted axial tissues. Locations of the transplanted tissues are indicated by a dotted line in G and J. (D) A headfold explant isolated from a 1-somite-stage embryo, cultured with intact axial mesendoderm. Nkx2.1 is expressed in the medial prosencephalic neural plate (posterior boundary is shown by arrow), while Shh is also expressed in the posterior part of the explant. Explants isolated from a 1somite-stage (E) or a 2to 3-somite-stage (F) embryo, cultured without the axial mesendoderm. Reduced or no expression of Nkx2.1 or Shh is detectable. (G) An explant derived from a 3-somite-stage embryo, in which the axial mesendoderm was left intact and an additional axial mesendoderm was transplanted under the left side of the explant. Ectopic expression of both Nkx2.1 and Shh are visible (arrowhead). (H) A cultured explant from 3-somite-stage embryo in which the axial mesendoderm was left intact. Bilateral stripes of Pax6 expression are seen in the rhombencephalon (arrow). (I) A similarly prepared explant except that the axial mesendoderm was removed. Note that the Pax6-expressing domain is expanded in the anterior end of the explant (asterisk), and the bilateral columns seen in I are fused (arrow). (J) An explant from a 3-somite-stage embryo in which the axial mesendoderm was left intact and an additional axial mesendoderm was transplanted under the left side of the explant. Pax6 expression is reduced around the site of the transplantation (compare level of expression indicated by the arrow on control side to the level of expression indicated by the arrowhead on the side with the ectopic axial mesendoderm). Bars, 0.1 mm (A,B,D; D-J, same magnification); 50 μm (C).

Fig. 2.

Expression patterns of medial-lateral markers in the forebrain neural plate and effects of the prechordal plate on the expression. (A) Nkx2.1 expression at the 3to 4-somite-stage of a mouse embryo. Nkx2.1 is expressed in a medial domain that ends at the level of cephalic flexure (arrow). (B) A frontal view of a 5-somite-stage embryo stained for Pax6. Pax6 is expressed in lateral regions of the prosencephalic neural plate, and is excluded from the medial region. (C) A transverse section of the anterior region of a 5-somitestage embryo, showing Shh (orange) and Nkx2.1 (purple) expression. Note that Nkx2.1 expression in the neuroectoderm is located in the vicinity of where the Shh-expressing prechordal plate (the dorsal roof of foregut) is juxtaposed to the medial neural plate (arrowhead). (D-J) Neural plate explants stained for regional markers: Nkx2.1 (purple, D-G; orange, H,I), Shh (orange, D,E,G,J) and Pax6 (purple, H-J). Shh expression was used to reveal the site of transplanted axial tissues. Locations of the transplanted tissues are indicated by a dotted line in G and J. (D) A headfold explant isolated from a 1-somite-stage embryo, cultured with intact axial mesendoderm. Nkx2.1 is expressed in the medial prosencephalic neural plate (posterior boundary is shown by arrow), while Shh is also expressed in the posterior part of the explant. Explants isolated from a 1somite-stage (E) or a 2to 3-somite-stage (F) embryo, cultured without the axial mesendoderm. Reduced or no expression of Nkx2.1 or Shh is detectable. (G) An explant derived from a 3-somite-stage embryo, in which the axial mesendoderm was left intact and an additional axial mesendoderm was transplanted under the left side of the explant. Ectopic expression of both Nkx2.1 and Shh are visible (arrowhead). (H) A cultured explant from 3-somite-stage embryo in which the axial mesendoderm was left intact. Bilateral stripes of Pax6 expression are seen in the rhombencephalon (arrow). (I) A similarly prepared explant except that the axial mesendoderm was removed. Note that the Pax6-expressing domain is expanded in the anterior end of the explant (asterisk), and the bilateral columns seen in I are fused (arrow). (J) An explant from a 3-somite-stage embryo in which the axial mesendoderm was left intact and an additional axial mesendoderm was transplanted under the left side of the explant. Pax6 expression is reduced around the site of the transplantation (compare level of expression indicated by the arrow on control side to the level of expression indicated by the arrowhead on the side with the ectopic axial mesendoderm). Bars, 0.1 mm (A,B,D; D-J, same magnification); 50 μm (C).

Nkx2.1 is first detectable in the medial part of the mouse prosencephalic neural plate as early as the 3-somite stage (Fig. 2A) and Pax6 (Fig. 2B) is expressed more laterally at similar or slightly later stages. Transverse sections through the prosencephalic neural plate stained for Shh and Nkx2.1 provided a clue that the dorsal roof of the foregut endoderm may be the tissue responsible for ML forebrain patterning (Fig. 2C). Shh is expressed in the dorsal foregut which is juxtaposed to the Nkx2.1-expressing medial neural plate. The dorsal foregut is morphologically distinguishable from the lateral foregut endoderm (Fig. 2C). It also shares similar cellular morphologies and is continuous with the notochordal plate, the precursor of the notochord (Sulik et al., 1994). Herein, based on the studies of Sulik et al. (1994), and our observation, we refer to the dorsal foregut as the prechordal plate, although it is important to point out that this term has also been used to refer to mesodermal tissues underlying the anterior neural plate in amphibians (e.g. see Li et al., 1997). In addition, we define the prechordal and notochordal plates as the axial mesendoderm. To test whether the prechordal plate regulates Nkx2.1 expression, cephalic neural plate explants were isolated from various stages of mouse embryos, ranging from the headfold to the 5-somite-stage, and were cultured with or without the axial mesendoderm (see Fig. 1 and Materials and Methods). In the control neural plate explants that contained the underlying prechordal plate, Nkx2.1 was expressed in the expected location: the anteromedial neural plate (Fig. 2D); note its sharp posterior boundary (arrow in Fig. 2D). We then removed the prechordal plate at different stages of development and found graded effects on the expression of Nkx2.1: when it was removed at 0to 1-somite-stage, no expression of Nkx2.1 was detectable (Fig. 2E); removal at 1to 3-somite stage resulted in a greatly reduced level of expression (Fig. 2F); removal later than 4-somite-stage led to Nkx2.1 expression patterns (data not shown) that were indistinguishable from the controls (Fig. 2D). Moreover, when an ectopic anterior axial mesendoderm was transplanted (see Fig. 1D,E) laterally to the endogenous prechordal plate, ectopic expression of Nkx2.1 was induced in the neuroectoderm overlying the ectopic prechordal plate (Fig. 2G; n=8). We used Shh expression as a marker for the transplanted axial mesendoderm; Shh is also induced in the neural plate apposed to the axial mesendoderm (Fig. 2D,G).

Since the notochord can repress some dorsal characteristics of the spinal cord (Yamada et al., 1991; Basler et al., 1993; Goulding et al., 1993; Monsoro-Burq et al., 1995), we next examined whether the prechordal plate can repress dorsal (lateral) markers in the prosencephalic neural plate. We removed the prechordal plate at the 3-somite-stage, and found that Pax6 expression extends more medially (compare Fig. 2H and I; n=7/7). Moreover, a laterally placed ectopic prechordal plate reduced Pax6 expression in the lateral part of the explants (Fig. 2J; n=5).

These results demonstrate that the prechordal plate regulates ML patterning in prosencephalic neural plate explants. On the contrary, we found that neither the head mesenchyme (the sample in Fig. 2D lacks head mesenchyme) nor the lateral foregut endoderm had an obvious effect on ML patterning in the prosencephalon (data not shown). These results are consistent with the observation that Shh expression has not been detected in these tissues.

Distinct competence along the AP axis to signal(s) from the axial mesendoderm

Nkx2.1 expression is restricted to the prosencephalon both in vivo and in cultured explants (arrows in Fig. 2A,D). This raises the question of whether the restricted expression of Nkx2.1 is due to differences in the inductive/repressive properties of the prechordal plate and the notochord, or to regionally distinct competence within the neural plate. In order to address this question, we first examined whether the notochord can induce expression of Nkx2.1 in the prosencephalon. Notochordal plate isolated from the level of the spinal cord was transplanted beneath neural plate explants lacking underlying tissues, prepared from 0to 1somite-stage embryos. The notochord induced Nkx2.1 in the prosencephalic neural plate (Fig. 3A; n=6). To test whether the prechordal plate could induce anterior properties (Nkx2.1) in more posterior regions of the neural plate, the axial mesendoderm was transplanted in an inverted anteroposterior orientation, so that the prechordal plate underlay the mesencephalicrhombencephalic region and the notochord was located underneath the prosencephalon. We found that Nkx2.1 expression was not induced in the posterior neural plate (Fig. 3B; n=9/9).

Fig. 3.

Distinct competence in the neural plate along the AP axis leads forebrain-specific expression of Nkx2.1. (A) An explant made from a 1-somite-stage embryo in which the intact axial mesendoderm was removed and the notochordal plate devoid of the prechordal plate was transplanted under the prosencephalic neural plate. (B) A similarly prepared explant as A, except that the axial mesendoderm was transplanted in inverted anterior-posterior orientation, so that the prechordal plate is in contact with the posterior headfold. Note that induced Nkx2.1 expression is sharply delineated approximately at the presumptive prosencephalic-mesencephalic boundary, and the prospective mesencephalic region does not express Nkx2.1 in response to the prechordal plate (arrowhead). Explants isolated from 0-somite-stage embryos and cultured without (C) or with (D) recombinant N-terminal SHH protein (1.5 μg/ml), then double-stained for Nkx2.1 (purple) and HNF3β (red). Nkx2.1 is induced at the anterior pole of the explant (arrowhead), whereas HNF3β is also induced posteriorly. Bars, 0.1 mm (A,B; C,D, same magnification).

Fig. 3.

Distinct competence in the neural plate along the AP axis leads forebrain-specific expression of Nkx2.1. (A) An explant made from a 1-somite-stage embryo in which the intact axial mesendoderm was removed and the notochordal plate devoid of the prechordal plate was transplanted under the prosencephalic neural plate. (B) A similarly prepared explant as A, except that the axial mesendoderm was transplanted in inverted anterior-posterior orientation, so that the prechordal plate is in contact with the posterior headfold. Note that induced Nkx2.1 expression is sharply delineated approximately at the presumptive prosencephalic-mesencephalic boundary, and the prospective mesencephalic region does not express Nkx2.1 in response to the prechordal plate (arrowhead). Explants isolated from 0-somite-stage embryos and cultured without (C) or with (D) recombinant N-terminal SHH protein (1.5 μg/ml), then double-stained for Nkx2.1 (purple) and HNF3β (red). Nkx2.1 is induced at the anterior pole of the explant (arrowhead), whereas HNF3β is also induced posteriorly. Bars, 0.1 mm (A,B; C,D, same magnification).

SHH can induce expression of Nkx2.1 in explants isolated from the telencephalic and diencephalic regions of the chicken neural plate (Ericson et al., 1995). To further define the region(s) of distinct competence to SHH, we examined whether treatment of entire mouse cephalic neural plate explants with SHH results in regional expression of Nkx2.1. These explants were derived from headfold-stage embryos and their underlying tissues were removed. We found that Nkx2.1 was induced at the anterior pole of the explant by SHH. As a control for the ability of the posterior explant to respond to SHH, we assessed the expression of HNF3β (Fig. 3D). HNF3β is normally expressed in the posterior neural plate and is known to be regulated by SHH (Chiang et al., 1996; Ericson et al., 1996). We found that HNF3β, and not Nkx2.1, was expressed in posterior parts of the explants, demonstrating that this tissue responded to SHH and was not competent to express Nkx2.1. These results suggest that the neural plate is patterned along the AP axis, leading to distinct responses to signals from the axial organizers and SHH.

The anterior ectoderm regulates regionalization of the anterolateral neural plate

BF1 is a winged-helix transcription factor expressed in the anterolateral neural plate and eventually in a large part of the telencephalon and retina (Tao and Lai, 1992; Shimamura et al., 1995). BF1 expression is first detectable as early as the 3-somite-stage in the nonneural ectoderm underlying the anterior margin of the neural plate (Fig. 4A,C). By the 8-somitestage, the expression is also detectable in the anterolateral neural plate (Fig. 4B,D). This sequence of expression motivated us to address whether a signal(s) from the anterior ectoderm induces BF1 expression in the neural plate. Thus, we examined BF1 expression in neural plate explants with or without the anterior ectoderm (see Fig. 1F).

Fig. 4.

Expression pattern of BF1 mRNA in the embryonic forebrain. (A) An oblique ventral view of a 5-somite-stage embryo, (B) A frontal view of a 7-somitestage embryo whole-mount stained for BF1. Sagittal sections of 4-somite-stage (C) and 8-somite-stage (D) embryos. BF1 expression is first detected in the non-neural ectoderm that underlies the anterior edge of neural plate (arrows in A,C,D), and later in the neuroectoderm (arrowhead in D). Additional abbreviation: he, heart primordium. Bars, 0.1 mm (C,D, same magnification).

Fig. 4.

Expression pattern of BF1 mRNA in the embryonic forebrain. (A) An oblique ventral view of a 5-somite-stage embryo, (B) A frontal view of a 7-somitestage embryo whole-mount stained for BF1. Sagittal sections of 4-somite-stage (C) and 8-somite-stage (D) embryos. BF1 expression is first detected in the non-neural ectoderm that underlies the anterior edge of neural plate (arrows in A,C,D), and later in the neuroectoderm (arrowhead in D). Additional abbreviation: he, heart primordium. Bars, 0.1 mm (C,D, same magnification).

The neural plate with its flanking ectoderm intact was isolated from the cephalic region of 3to 5-somite-stage embryos before neural expression of BF1 had begun. After the explant was in culture for 24 hours, BF1 expression was detected in the anterior neural plate (Fig. 5A; n=7). When the anterior ectoderm, including the anterior neural ridge (ANR), was unilaterally excised from the neural plate, BF1 expression on the operated side of the neural plate was not observed (Fig. 5B; n=17/17). The ANR is the junction between the anterior neural plate and anterior non-neural ectoderm. Placing the excised ectoderm underneath the anterior neural plate, restored BF1 expression (arrowhead in Fig. 5C,C′; n=5/7). Sectioning the explant confirmed that expression of BF1 is found in the anterior neuroectoderm when the anterior ectoderm was placed underneath it (Fig. 5C,C′, white arrowhead; see legend for details). These results provide evidence that the anterior ectoderm, which contains the ANR, is necessary and sufficient for BF1 expression in the neural plate.

Fig. 5.

The anterior ectoderm is necessary and sufficient for BF1 expression in the neural plate. (A) A neural plate explant from a 4somite-stage embryo in which both sides of the anterior ectoderm were left intact, has bilateral BF1-expressing neuroectodermal domains (purple) flanking the Nkx2.1-positive region (orange) which serves as a marker for the midline of the explants. (B) Same as A but with the left side of the anterior ectoderm including the anterior neural ridge excised; neural expression of BF1 is abolished on the operated side (arrowhead). (C) A specimen in which the excised anterior ectoderm from the left side was transplanted under neuroectoderm. BF1 expression was restored following transplantation of the anterior ectoderm (white arrowhead). Weaker staining in a more posterior domain of this sample (black arrow) corresponds to BF1 expression in the underlying tissue which is the transplanted anterior ectoderm underneath the neural plate. This result is best seen in a sagittal section of this explant in C′ (the section plate is indicated by the white bars). (D) Same as C, except that the transplanted ectoderm was derived from the trunk region. BF1 is not induced (arrowhead). (E) A control explant from a 4somite-stage embryo with no excision of the anterior ectoderm stained for Msx1. Msx1 is expressed symmetrically at the front of the explant (arrows). (F) A similarly operated explant as in B, but stained for Msx1. Msx1 expression is detectable on the control side (arrow), but is much reduced on the operated side (arrowhead). (G) An explant similar to D stained for Msx1, showing expression of Msx1 on the side with the posterior ectodermal transplant (arrowhead). This result is best seen in a sagittal section of this explant in G′ (the section plate is indicated by the white bars). A dotted line defines the outline of the unoperated side of the explant in G to enhance the ability to visualize it. Bars, 0.1 mm (A; A-G, same magnification); 50 μm (C′, G′)

Fig. 5.

The anterior ectoderm is necessary and sufficient for BF1 expression in the neural plate. (A) A neural plate explant from a 4somite-stage embryo in which both sides of the anterior ectoderm were left intact, has bilateral BF1-expressing neuroectodermal domains (purple) flanking the Nkx2.1-positive region (orange) which serves as a marker for the midline of the explants. (B) Same as A but with the left side of the anterior ectoderm including the anterior neural ridge excised; neural expression of BF1 is abolished on the operated side (arrowhead). (C) A specimen in which the excised anterior ectoderm from the left side was transplanted under neuroectoderm. BF1 expression was restored following transplantation of the anterior ectoderm (white arrowhead). Weaker staining in a more posterior domain of this sample (black arrow) corresponds to BF1 expression in the underlying tissue which is the transplanted anterior ectoderm underneath the neural plate. This result is best seen in a sagittal section of this explant in C′ (the section plate is indicated by the white bars). (D) Same as C, except that the transplanted ectoderm was derived from the trunk region. BF1 is not induced (arrowhead). (E) A control explant from a 4somite-stage embryo with no excision of the anterior ectoderm stained for Msx1. Msx1 is expressed symmetrically at the front of the explant (arrows). (F) A similarly operated explant as in B, but stained for Msx1. Msx1 expression is detectable on the control side (arrow), but is much reduced on the operated side (arrowhead). (G) An explant similar to D stained for Msx1, showing expression of Msx1 on the side with the posterior ectodermal transplant (arrowhead). This result is best seen in a sagittal section of this explant in G′ (the section plate is indicated by the white bars). A dotted line defines the outline of the unoperated side of the explant in G to enhance the ability to visualize it. Bars, 0.1 mm (A; A-G, same magnification); 50 μm (C′, G′)

In addition, we found that the ability of the anterior ectoderm to regulate BF1 expression is not present in more posterior ectodermal domains. Ectoderm, with or without the neural ridge, taken from the level of the spinal cord does not induce BF1 expression in anterior neural plate explants (Fig. 5D; n=27). On the contrary, these anterior neural plate explants, co-cultured with posterior ectoderm, express Msx1 (see arrowhead in Fig. 5G,G’; n=5). Msx1 is a dorsal CNS marker (roof plate and neural crest) that can be induced by non-neural ectoderm in the spinal cord (Dickinson et al., 1995; Liem et al., 1995). Furthermore, we provided evidence that the nonneural ectoderm is required for Msx1 expression in the prosencephalic neural plate. Removal of the non-neural ectoderm prior to Msx1 expression reduced Msx1 RNA levels in the neuroectoderm (arrowhead in Fig. 5F; n=5).

FGF8 induces BF1 expression in neural plate explants

Transplantation and ablation of the ectoderm that flanks the anterior neural plate suggests, but does not prove (in part due to an inability to unequivocally distinguish graft from host cells), that this tissue regulates the expression of BF1. To further investigate this issue, we searched for candidate signaling molecules that are expressed in the anterior ectoderm and tested whether they were sufficient for regulating BF1 expression. We found that members of the fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) gene families, Fgf8 and Bmp7, are expressed at the right time and place to be candidates to regulate BF1 expression, whereas other family members, including Fgf2, Fgf4, Fgf6, Fgf7, Bmp4 and Bmp6 are not (data not shown). The ectodermal expression of Fgf8 is restricted to a few locations, one of which is the ANR, where expression begins around the 4-somite-stage (Fig. 6A,B; Crossley and Martin, 1995), before BF1 expression in the neuroectoderm is detectable. In contrast, Bmp7 is expressed in most or all regions of the non-neural ectoderm adjacent to the neural plate (Fig. 6C,D; Liem et al., 1995).

Fig. 6.

Expression patterns of ectodermally expressed signaling factors in the cephalic region of the embryo. Frontal views of 5somite-stage embryos and sagittal sections of 7-somite-stage embryos whole-mount stained for Fgf8 (A, B) and for Bmp7 (C, D). Fgf8 is expressed at the anterior edge of the neural plate (arrowheads), while Bmp7 is expressed more widely in the nonneural ectoderm (arrowheads). Additional abbreviation: be, branchial ectoderm. Bars, 0.1 mm (A,C); (B,D).

Fig. 6.

Expression patterns of ectodermally expressed signaling factors in the cephalic region of the embryo. Frontal views of 5somite-stage embryos and sagittal sections of 7-somite-stage embryos whole-mount stained for Fgf8 (A, B) and for Bmp7 (C, D). Fgf8 is expressed at the anterior edge of the neural plate (arrowheads), while Bmp7 is expressed more widely in the nonneural ectoderm (arrowheads). Additional abbreviation: be, branchial ectoderm. Bars, 0.1 mm (A,C); (B,D).

We examined the capability of FGF8 and BMP7 to induce BF1 expression in neural plate explants using heparin-acrylic beads (Sigma, H-5263) soaked with the recombinant proteins (Crossley et al., 1996a). The anterior ectoderm including the ANR was unilaterally excised from the neural plate and beads were placed underneath the neural plate on the operated side. These explants were derived from 3to 5-somite-stage embryos and also lacked their mesoderm and endoderm. Following 24 hours of culture, we found extensive BF1 expression in the vicinity of the FGF8-beads (Fig. 7B; n=25/25), whereas none of the PBS-soaked control or BMP7beads induced BF1 (Fig. 7A,C; n=11/11 and 15/15, respectively). Instead, BMP7-beads induced Msx1 in the anterior neural plate (Fig. 7D; n=7/7). BMP2 and BMP4 produced results indistinguishable from BMP7 (n=5 and 9; data not shown). FGF8 did not induce Msx1 (n=3; data not shown). We found that FGF4 can also induce BF1 (n=9/9; data not shown), although expression of this gene has not been detected in the anterior neural ridge or the prosencephalic neural plate (data not shown; Niswander and Martin, 1992).

Fig. 7.

Effects of ectodermally expressed factors (FGF8 and BMP7) on gene expression in the anterior neural plate. Heparin-acrylic beads soaked with PBS (A), recombinant FGF8 (0.25-0.5 mg/ml) (B,E,F) or BMP7 (0.3 μg/ml) (C,D) proteins were placed under the left side of neural plate explants where the anterior ectoderm was removed. Following 24 hours in culture, expression of BF1 (purple) (A-C,E,F) and Nkx2.1 (orange) (A,B,E), or Msx1 (D) was detected using in situ RNA hybridization. FGF8 induced BF1 in the neural plate, while BMP7 did not induce BF1 but did induce Msx1. The locations of beads are indicated by arrowheads. The outline of the explants is marked by a dotted line. (E,F) Explants with two FGF8-beads, one in the anterior (arrowhead) and the other in the posterior (arrow) part of the explant. (E) BF1 is only induced by the anterior FGF8-bead and the domain of induced expression is delineated by a sharp boundary. (F) Moreover, while the anterior bead induced BF1 (purple), the posterior one induced En2 (orange), indicating distinct regions of competence in the neural plate along the anterior-posterior axis. Bar, 0.1 mm

Fig. 7.

Effects of ectodermally expressed factors (FGF8 and BMP7) on gene expression in the anterior neural plate. Heparin-acrylic beads soaked with PBS (A), recombinant FGF8 (0.25-0.5 mg/ml) (B,E,F) or BMP7 (0.3 μg/ml) (C,D) proteins were placed under the left side of neural plate explants where the anterior ectoderm was removed. Following 24 hours in culture, expression of BF1 (purple) (A-C,E,F) and Nkx2.1 (orange) (A,B,E), or Msx1 (D) was detected using in situ RNA hybridization. FGF8 induced BF1 in the neural plate, while BMP7 did not induce BF1 but did induce Msx1. The locations of beads are indicated by arrowheads. The outline of the explants is marked by a dotted line. (E,F) Explants with two FGF8-beads, one in the anterior (arrowhead) and the other in the posterior (arrow) part of the explant. (E) BF1 is only induced by the anterior FGF8-bead and the domain of induced expression is delineated by a sharp boundary. (F) Moreover, while the anterior bead induced BF1 (purple), the posterior one induced En2 (orange), indicating distinct regions of competence in the neural plate along the anterior-posterior axis. Bar, 0.1 mm

Crossley et al. (1996b) have demonstrated that FGF8 can induce En2 expression and midbrain development when it is ectopically introduced into the posterior forebrain. We found that FGF8 induces BF1 in the anterior neural plate, raising the possibility that cells located at different axial positions within the neural plate have distinct responses to FGF8. We addressed this issue by placing FGF8-beads at different axial locations and assayed for the induction of BF1 and En2. We found that BF1 expression was detected only around the anterior bead (Fig. 7E, F), whereas expression of En2 was induced by posterior beads (Fig. 7F). The induced BF1-expressing domain is delineated posteriorly by a sharp boundary which may be orthogonal to the long axis of the explants (Fig. 7E). Interestingly, the posterior boundary of BF1 and the anterior boundary of En2 are nearly adjacent (Fig. 7F).

We have provided evidence for the tissues, and proteins produced by them, that can regulate regionalization of the prosencephalic neural plate. Our results suggest that ML (VD) patterning of the prosencephalon is regulated by mechanisms that also pattern the rest of the CNS: medial specification is regulated by SHH produced by the axial mesendoderm (prechordal plate and notochord), and lateral specification is regulated by BMPs produced by the non-neural ectoderm. We also found that the ectoderm along the edge of the neural plate (the neural ridge) is regionalized with respect to its inductive properties; in particular the FGF8-expressing anterior neural ridge regulates regionalization of the anterolateral neural plate. While these results indicate how gene expression within the anterior neural plate is regulated, they do not directly address how these processes are involved in regionally distinct histogenesis within the forebrain. To overcome this shortcoming, we chose to study the expression of transcription factors whose function is essential for the development of various regions of the forebrain. For instance, Nkx2.1 is essential for development of the mammillary and tuberal hypothalamus (Kimura et al., 1996) and BF1 for regionalization and growth of the telencephalic and optic vesicles (Xuan et al., 1995). Thus, the use of these particular molecular markers provides a link between our gene expression analyses in neural plate explants and the development of specific forebrain structures.

The axial organizers: prechordal plate and notochord

Because the notochord does not underlie the forebrain, it has been unclear whether patterning of the medial (ventral) forebrain is regulated by mechanisms distinct from more posterior CNS regions. On the contrary, several lines of molecular and genetic evidence now suggest that patterning of the medial (ventral) forebrain is regulated by mechanisms related to medial (ventral) specification in the posterior CNS. Morphological and molecular analysis of the zebrafish cyclops mutant revealed defects in the entire ventral CNS (Hatta et al., 1991, 1994). Gene expression patterns of Nkx2.2, Shh and HNF3β are continuous along the entire medial (ventral) embryonic CNS (Sasaki and Hogan, 1993; Echelard et al., 1993; Shimamura et al., 1995). Furthermore, Echelard et al. (1993) found that Shh is expressed in the dorsal foregut underlying the forebrain, suggesting that SHH could be a signal for patterning the medial (ventral) forebrain. In fact, Ericson et al. (1995) demonstrated that SHH can induce Nkx2.1 in forebrain neural plate explants. Recently, mice lacking a functional Shh gene have been analyzed; the phenotype of these mutants demonstrates that SHH is essential for medial patterning of the entire CNS (Chiang et al., 1996). In the above studies, the tissue that is required for initial patterning of the medial prosencephalic neural plate was not explicitly demonstrated. Our results directly show that the prechordal plate (dorsal foregut), which underlies the midline of the prosencephalic neural plate, induces medial (ventral) properties in prosencephalic explants, which is consistent with the role of axial tissue underlying the prosencephalon in patterning the medial eye field in Xenopus and chicken (Li et al., 1997). Furthermore, other tissues underlying the anterior neural plate in 0to 1-somite-stage embryos [mesenchyme (mesoderm and cranial neural crest) and lateral foregut] do not regulate Nkx2.1 in the explant induction assays (data not shown), providing evidence that the prechordal plate functions alone in the initial specification of the medial prosencephalon.

In addition to sharing similar inductive properties, the prechordal and notochordal plates have some similar morphological and molecular characteristics during gastrulation. For instance, scanning electron microscopy showed that the cells in the prechordal and notochordal plates display very similar morphology (Sulik et al., 1994), and those tissues express some of the same regulatory genes, such as Shh, Chordin and HNF3β (Echelard et al., 1993; Sasai et al., 1994; Sasaki and Hogan, 1993). In addition, fate mapping during gastrulation demonstrates a similar origin for the prechordal and notochordal plates (Psychoyos and Stern, 1996).

However, the prechordal plate and notochord do have some different properties. For instance, two homeobox genes, goosecoid (gsc) and cNot1 are differentially expressed in these tissues; gsc in the prechordal plate and cNot1 in the notochord (Stein and Kessel, 1995). Thus, the prechordal plate and notochord possess distinct properties which may endow them with specific inductive abilities (Placzek et al., 1993).

Evidence for local patterning of the prosencephalic neural plate by FGF8 in the anterior neural ridge

Specific ectodermal domains lateral to the neural plate are known to regulate brain development. For instance, there is evidence for the role of ectoderm-derived placodal tissues in regulating development of the olfactory bulbs, optic vesicles and posterior pituitary (Jacobson, 1963; Graziadei and MontiGraziadei, 1992; Webb and Noden, 1993; Byrd and Burd, 1993; Saha et al., 1992; Daikoku et al., 1983).

Here we provided evidence for a localized signaling center in the anterior neural ridge (ANR) that regulates gene expression in the anterolateral neural plate. The ANR is a morphologically defined structure at the junction of the anterior neural plate and the non-neural ectoderm (Couly and Le Douarin, 1988; Eagleson et al., 1995). This structure is also characterized by the expression of Fgf8 (Fig. 6A,C). We found that the ANR is required for the induction and/or maintenance of BF1 expression. In addition, FGF8 can substitute for the function of the ANR, with respect to regulation of BF1 expression. The onset of Fgf8 expression in the ANR is around the 4-somite stage, whereas BF1 expression in the neuroectoderm is detectable by 7to 8-somite stage. This temporal relationship strongly suggests that FGF8 produced from the ANR induces BF1 in the neuroectoderm.

Since inactivation of BF1 causes hypoplasia of the cerebral hemispheres (Xuan et al., 1995), our results provide evidence that a signal(s) from the anterior neural ridge, such as FGF8, regulates growth of the telencephalon. Furthermore, this signal(s) may also regulate regional specification within the forebrain, since BF1 mutants lack molecular properties (e.g. Dlx2 expression) which are thought to define the basal telencephalon (Xuan et al., 1995). In addition, an ectopic expression study provided evidence that BF1 can regulate regional identity within the retina (Yuasa et al., 1996). Thus, BF1 induction by FGF8 produced by the ANR may be essential for both growth and regional specification within the forebrain. This would also provide a molecular explanation for embryological studies that have suggested a role for ANR-related tissues in regulating telencephalic development (Graziadei and Monti-Graziadei, 1992; Byrd and Burd, 1993).

Although the BF1-inducing signal appears to arise from ectoderm, and ectoderm has been implicated in lateral (dorsal) patterning in the CNS (Dickinson et al., 1995; Liem et al., 1995), we suggest that the BF1-inducing mechanism is distinguishable from general lateral (dorsal) patterning. We demonstrated that BF1 was not induced by the posterior ectoderm or by BMP7 (Figs 5D, 7C). On the contrary, BMP7, as well as the ectoderm isolated from both spinal cord and forebrain levels, induced Msx1 in the anterior neural plate (Figs 5E,G, 7D).

Thus, the anterior neural ridge may be an example of a local organizing center. The isthmus, which also expresses Fgf8 and patterns the midbrain and cerebellar territories, may be another local organizer (Martinez et al., 1991; Marín and Puelles, 1994; Crossley et al., 1996b). As development proceeds, additional local patterning sources are probably generated that progressively define the regional organization of the brain. For instance, local patterning sources might be localized to boundaries, such as the zona limitans intrathalamica where Shh is expressed, or to local sources of SHH such as in the medial ganglionic eminence (Rubenstein and Shimamura, 1996).

Differential competence along the AP axis of the neural plate contributes to regional complexity

While signals from local organizing centers can regulate regional gene expression, distinct competence within the neural plate also contributes to generating regional differences (Yamada et al., 1991; Ericson et al., 1995; Hynes et al., 1995a; Simon et al., 1995). For instance, we found that only the anterior neural plate is competent to express Nkx2.1 in response to the axial mesendoderm and SHH (Fig. 3). In addition, only the anterior neural plate can express BF1 in response to FGF8 (Fig. 7E,F), whereas we and others have shown that midbrain levels of the neural plate express En2 in response to FGF8 (Fig. 7F; Crossley et al., 1996b).

Although we were not able to precisely define the location of these boundaries of distinct competence, we hypothesize that they may correspond to a single boundary which becomes the zona limitans intrathalamica (ZL). The ZL is a transverse boundary that separates two subdivisions (prosomeres) in the diencephalon (Rubenstein et al., 1994). Three observations suggest that the ZL primordium is this boundary of competence for Nkx2.1, BF1 and En2 : (1) the posterior boundary of Nkx2.1 expression in the hypothalamus is close to the ZL (Shimamura et al., 1995); (2) induction of En2 by FGF8 abuts the ZL (Crossley et al., 1996b); (3) the FGF8-induced BF1and En2expressing domains appear to be nearly adjacent in the neural plate explants (Fig. 7F).

Although it is not known how these zones of distinct competence are generated, it is likely that some aspects of AP patterning are involved. For instance, vertical induction from the underlying mesendodermal tissues may specify transverse domains within the neural plate (Ang et al., 1994). This process might be regulated by factors such as Cerberus, a headinducing secreted protein that is expressed in the anterior endoderm (Bouwmeester et al., 1996).

In conclusion, regionalization of the prosencephalon appears to result from the superposition of multiple distinct patterning mechanisms (Fig. 8). AP patterning creates transverse zones with differential competence within the neural plate (Yamada et al., 1991; 1993; Hynes et al., 1995a, Simon et al., 1995; reviewed in Lumsden and Krumlauf, 1996), and patterning along the ML axis generates longitudinally aligned domains (reviewed in Shimamura et al., 1995; Tanabe and Jessell, 1996). The combination of ML (VD) and AP patterning generates a grid-like organization of distinct histogenic forebrain primordia as proposed in the Prosomeric model (Puelles and Rubenstein, 1993; Rubenstein et al., 1994; Rubenstein and Shimamura, 1996). Additional levels of regional complexity are generated by local inductive sources, such as the ANR.

Fig. 8.

Model of patterning mechanisms that regionalize the prosencephalic neural plate. AP patterning mechanisms generate transverse subdivisions (e.g. indicated by the thick broken line between prosencephalon and mesencephalon) that have distinct responses to medial and local signals. Medial (ventral) patterning signals (e.g. Sonic Hedgehog; black arrows), which induce the primordia of the basal plate (bp) (e.g. Nkx2.1 expression in the hypothalamic anlage), arise from the axial mesendoderm (prechordal plate, pcp). Lateral (dorsal) patterning signals (e.g. BMPs; gray arrows), which arise from the non-neural ectoderm (ec) flanking the neural plate, induce expression of Msx1 and pattern the primordia of the alar plate (ap). Local signals, arising from the ANR (thick black line) (e.g. FGF8; white arrows) induce expression of BF1, which regulates development of specific forebrain structures (telencephalic and optic vesicles). Additional abbreviations: A, anterior; D, dorsal; L, lateral; M, medial; P, posterior; V, ventral.

Fig. 8.

Model of patterning mechanisms that regionalize the prosencephalic neural plate. AP patterning mechanisms generate transverse subdivisions (e.g. indicated by the thick broken line between prosencephalon and mesencephalon) that have distinct responses to medial and local signals. Medial (ventral) patterning signals (e.g. Sonic Hedgehog; black arrows), which induce the primordia of the basal plate (bp) (e.g. Nkx2.1 expression in the hypothalamic anlage), arise from the axial mesendoderm (prechordal plate, pcp). Lateral (dorsal) patterning signals (e.g. BMPs; gray arrows), which arise from the non-neural ectoderm (ec) flanking the neural plate, induce expression of Msx1 and pattern the primordia of the alar plate (ap). Local signals, arising from the ANR (thick black line) (e.g. FGF8; white arrows) induce expression of BF1, which regulates development of specific forebrain structures (telencephalic and optic vesicles). Additional abbreviations: A, anterior; D, dorsal; L, lateral; M, medial; P, posterior; V, ventral.

We thank Dr P. Crossley for help in preparation of FGF8-beads and Dr L. Sussel for critical reading of the manuscript. We also thank Drs P. Beachy, R. Derynck, P. Gruss, B. Hogan, G. Karsenty, D. Kingsley, E. Lai, G. Martin, I. Mason, A. McMahon and A. Simeone, and the Genetics Institute for probes and/or recombinant proteins. This work was supported by research grants to: K. S. from JSPS, and J. L. R. R. from March of Dimes, NARSAD, the John Merck Fund, Human Frontiers Science Program and NIMH RO1 MH49428-01, RO1 MH51561-01A1 and K02 MH01046-01.18.

Ang
,
S.-L.
,
Conlon
,
R. A.
,
Jin
,
O.
and
Rossant
,
J.
(
1994
).
Positive and negative signals from mesoderm regulate the expression of mouse Otx2 in ectoderm explants
.
Development
120
,
2979
2989
.
Basler
,
K.
,
Edlund
,
T.
,
Jessell
,
T. M.
and
Yamada
,
T.
(
1993
).
Control of cell pattern in the neural tube: regulation of cell differentiation by dorsalin-1, a novel TGFβ family member
.
Cell
73
,
687
702
.
Beddington
,
R. S. P.
(
1987
).
Isolation, culture and manipulation of post implantation mouse embryos
.
In Mammalian Development: a Practical Approach
(ed.
M.
Monk
), pp.
43
69
.
Oxford
:
IRL Press
.
Bouwmeester
,
T.
,
Kim
,
S.-H.
,
Sasai
,
Y.
,
Lu
,
B.
and
De Robertis
,
E.
(
1996
).
Cerberus is a head-inducing secreted factor expressed in the anterior endoderm of Spemann’s organizer
.
Nature
382
,
595
601
.
Byrd
,
C. A.
and
Burd
,
G. D.
(
1993
).
The quantitative relationship between olfactory axons and mitral/tufted cells in developing Xenopus with partially deafferented olfactory bulbs
.
J. Neurobiol
.
24
,
1229
1242
.
Chiang
,
C.
,
Litingtung
,
Y.
,
Lee
,
E.
,
Young
,
K. E.
,
Corden
,
J. L.
,
Westphal
,
H.
and
Beachy
,
P. A.
(
1996
).
Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function
.
Nature
383
,
407
413
.
Couly
,
G.
and
Le Douarin
,
N. M.
(
1988
).
The fate map of the cephalic neural primordium at the presomitic to the 3-somite stage in the avian embryo
.
Development
103
Supplement
,
101
113
.
Crossley
,
P. H.
and
Martin
,
G. R.
(
1995
).
The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo
.
Development
121
,
439
451
.
Crossley
,
P. H.
,
Minowada
,
G.
,
MacArthur
,
C. A.
and
Martin
,
G. R.
(
1996a
).
Roles for FGF8 in the induction, initiation, and maintenance of chick limb development
.
Cell
84
,
127
136
.
Crossley
,
P. H.
,
Martinez
,
S.
and
Martin
,
G. R.
(
1996b
).
Midbrain development induced by FGF8 in the chick embryo
.
Nature
380
,
66
68
.
Daikoku
,
S.
,
Chikamori
,
M.
,
Adachi
,
T.
,
Okamura
,
Y.
,
Nishiyama
,
T.
and
Tsuruo
,
Y.
(
1983
).
Ontogenesis of hypothalamic immunoreactive ACTH cells in vivo and in vitro: role of Rathke’s pouch
.
Dev. Biol
.
97
,
81
88
.
Dickinson
,
M. E.
,
Selleck
,
M. A. J.
,
McMahon
,
A. P.
and
Bronner-Fraser
,
M.
(
1995
).
Dorsalization of the neural tube by the non-neural ectoderm
.
Development
121
,
2099
2106
.
Doniach
,
T.
(
1993
).
Planar and vertical induction of anteroposterior pattern during the development of the amphibian central nervous system
.
J. Neurobiol
.
24
,
1256
1275
.
Eagleson
,
G.
,
Ferreiro
,
B.
and
Harris
,
W. A.
(
1995
).
Fate of the anterior neural ridge and the morphogenesis of the Xenopus forebrain
.
J. Neurobiol
.
28
,
146
158
.
Echelard
,
Y.
,
Epstein
,
D. J.
,
St-Jacques
,
B.
,
Shen
,
L.
,
Mphler
,
J.
,
McMahon
,
J. A.
and
McMahon
,
A. P.
(
1993
).
Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity
.
Cell
75
,
1417
1430
.
Ericson
,
J.
,
Muhr
,
J.
,
Placzek
,
M.
,
Lints
,
T.
,
Jessell
,
T. M.
and
Edlund
,
T.
(
1995
).
Sonic hedgehog induces the differentiation of ventral forebrain neurons: a common signal for ventral patterning within the neural tube
.
Cell
81
,
747
756
.
Ericson
,
J.
,
Morton
,
S.
,
Kawakami
,
A.
,
Roelink
,
H.
and
Jessell
,
T. M.
(
1996
).
Two critical periods of Sonic Hedgehog signaling required for the specification of motor neuron identity
.
Cell
87
,
661
673
.
Gong
,
Q.
and
Shipley
,
M. T.
(
1995
).
Evidence that pioneer olfactory axons regulate telencephalon cell cycle kinetics to induce the formation of the olfactory bulb
.
Neuron
14
,
91
101
.
Goulding
,
M. D.
,
Lumsden
,
A.
and
Gruss
,
P.
(
1993
).
Signals from the notochord and floor plate regulate the region-specific expression of two Pax genes in the developing spinal cord
.
Development
117
,
1001
1016
.
Graziadei
,
P. P. C.
and
Monti-Graziadei
,
A. G.
(
1992
).
The influence of the olfactory placode on the development of the telencephalon in Xenopus laevis
.
Neurosci
.
46
,
617
629
.
Hatta
,
K.
,
Kimmel
,
C. B.
,
Ho
,
R. K.
and
Walker
,
C.
(
1991
).
The cyclops mutation blocks specification of the floor plate of the zebrafish central nervous system
.
Nature
350
,
339
341
.
Hatta
,
K.
,
Puschel
,
A. W.
and
Kimmel
,
C. B.
(
1994
).
Midline signalling in the primordium of the zebrafish anterior central nervous system
.
Proc. Natl. Acad. Sci. USA
91
,
2061
2065
.
Hynes
,
M.
,
Poulsen
,
K.
,
Tessier-Levigne
,
M.
and
Rosenthal
,
A.
(
1995a
).
Control of neuronal diversity by the floor plate: contact-mediated induction of midbrain dopaminergic neurons
.
Cell
80
,
95
102
.
Hynes
,
M.
,
Porter
,
J. A.
,
Chiang
,
C.
,
Chang
,
D.
,
Tessier-Lavigne
,
M.
,
Beachy
,
P. A.
and
Rosenthal
,
A.
(
1995b
).
Induction of midbrain dopaminergic neurons by Sonic Hedgehog
.
Neuron
15
,
35
44
.
Jacobson
,
A. G.
(
1963
).
The determination and positioning of the nose, lens and ear. I. Interactions within the ectoderm, and between the ectoderm and underlying tissues
.
J. Exp. Zool
.
154
,
273
284
.
Jessell
,
T. M.
and
Dodd
,
J.
(
1992
).
Floor plate-derived signals and the control of neural cell pattern in vertebrates
.
Harvey Lecture
89
,
87
128
.
Kimura
,
S.
,
Hara
,
Y.
,
Pineau
,
T.
,
Fernandez-Salguero
,
P.
,
Fox
,
C. H.
,
Ward
,
J. M.
and
Gonzalez
,
F. J.
(
1996
).
The T/ebp null mouse: thyroid-specific enhancer-binding protein is essential for the organogenesis of the thyroid, lung, ventral forebrain, and pituitary
.
Genes Dev
.
10
,
60
69
.
Li
,
H.-s.
,
Tierney
,
C.
,
Wen
,
L.
,
Wu
,
J. Y.
and
Rao
,
Y.
(
1997
).
A single morphogenetic field gives rise to two retina primordia under the influence of the prechordal plate
.
Development
124
,
603
615
.
Liem
,
K. F.
,
Tremml
,
G.
,
Roelink
,
H.
and
Jessell
,
T. M.
(
1995
).
Dorsal differentiation of neural plate cells induced by BMP-mediated signals from epidermal ectoderm
.
Cell
82
,
969
979
.
Lumsden
,
A.
and
Krumlauf
,
R.
(
1996
).
Patterning the vertebrate neuraxis
.
Science
274
,
1109
1115
.
Marin
,
F.
and
Puelles
,
L.
(
1994
).
Patterning of the embryonic avian midbrain after experimental inversions: a polarizing activity from the isthmus
.
Dev. Biol
.
163
,
19
37
.
Martinez
,
S.
,
Wassef
,
M.
and
Alvarado-Mallart
,
R.-M.
(
1991
).
Induction of a mesencephalic phenotype in the 2-day-old chick prosencephalon is preceded by the early expression of the homeobox gene en
.
Neuron
6
,
971981
.
Marti
,
E.
,
Bumcrot
,
D. A.
,
Takada
,
R.
and
McMahon
,
A. P.
(
1995
).
Requirement of 19K form of Sonic hedgehog for induction of distinct ventral cell types in CNS explants
.
Nature
375
,
322
325
.
Monsoro-Burq
,
A.-H.
,
Bontoux
,
M.
,
Vincent
,
C.
and
Le Douarin
,
N. M.
(
1995
).
The developmental relationships of the neural tube and the notochord: short and long term effects of the notochord on the dorsal spinal cord
.
Mech. Dev
.
53
,
157
170
.
Niswander
,
L.
and
Martin
,
G. R.
(
1992
).
Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse
.
Development
114
,
755
768
.
Placzek
,
M.
,
Jessell
,
T. M.
and
Dodd
,
J.
(
1993
).
Induction of floor plate differentiation by contact-dependent, homeogenetic signals
.
Development
117
,
205
218
.
Placzek
,
M.
(
1995
).
The role of the notochord and floor plate in inductive interactions
.
Curr. Opin. Genet. Dev
.
5
,
499
506
.
Psychoyos
,
D.
and
Stern
,
C. D.
(
1996
).
Fates and migratory routes of primitive streak cells in the chick embryo
.
Development
122
,
1523
1534
.
Puelles
,
L.
and
Rubenstein
,
J. L. R.
(
1993
).
Expression patterns of homeobox and other putative regulatory genes in the embryonic mouse forebrain suggest a neuromeric organization
.
Trends Neurosci
.
16
,
472
479
.
Roelink
,
H.
,
Augsburger
,
A.
,
Heemskerk
,
J.
,
Korzh
,
V.
,
Norlin
,
S.
,
Ruiz i Altaba
,
A.
,
Tanabe
,
Y.
,
Placzek
,
M.
,
Edlund
,
T.
,
Jessell
,
T. M.
and
Dodd
,
J.
(
1994
).
Floor plate and motor neuron induction by vhh-1, a vertebrate homolog of hedgehog expressed by the notochord
.
Cell
76
,
761
775
.
Roelink
,
H.
,
Porter
,
J. A.
,
Chiang
,
C.
,
Tanabe
,
Y.
,
Chang
,
D. T.
,
Beachy
,
P. A.
and
Jessell
,
T. M.
(
1995
).
Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis
.
Cell
81
,
445
455
.
Rubenstein
,
J. L. R.
,
Martinez
,
S.
,
Shimamura
,
K.
and
Puelles
,
L.
(
1994
).
The embryonic vertebrate forebrain: the prosomeric model
.
Science
266
,
578
580
.
Rubenstein
,
J. L. R.
and
Shimamura
,
K.
(
1996
).
Regulation of patterning and differentiation in the embryonic vertebrate forebrain
.
In Molecular and Cellular Approaches to Neuronal Development
. (ed.
W. M.
Cowan
,
T. M.
Jessell
and
S. L.
Zipurski
),
Oxford
:
Oxford University Press
, in press.
Ruiz i Altaba
,
A
. (
1994
).
Pattern formation in the vertebrate neural plate
.
Trends Neurosci
.
17
,
233
243
.
Saha
,
M. S.
,
Servetnick
,
M.
and
Grainger
,
R. M.
(
1992
).
Vertebrate eye development
.
Curr. Opin. Genet. Dev
.
2
,
582
588
.
Sasai
,
Y.
,
Lu
,
B.
,
Steinbeisser
,
H.
,
Geissert
,
D.
,
Gont
,
L. K.
and
De Robertis
,
E. M.
(
1994
).
Xenopus chordin: a novel dorsalizing factor activated by organizer-specific homeobox genes
.
Cell
79
,
779
790
.
Sasaki
,
H.
and
Hogan
,
B. L. M.
(
1993
).
Differential expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo
.
Development
118
,
47
59
.
Schmahl
,
W.
,
Knoedlseder
,
M.
,
Favor
,
J.
and
Davidson
,
D.
(
1993
).
Defects of neuronal migration and the pathogenesis of cortical malformations are associated with small eye (sey) in the mouse, a point mutation at the Pax-6 locus
.
Acta Neuropathol
.
86
,
126
135
.
Shimamura
,
K.
and
Takeichi
,
M.
(
1992
).
Local and transient expression of Ecadherin involved in mouse embryonic brain morphogenesis
.
Development
116
,
1011
1019
.
Shimamura
,
K.
,
Hirano
,
S.
,
McMahon
,
A. P.
and
Takeichi
,
M.
(
1994
).
Wnt1-dependent regulation of local E-cadherin and αN-catenin expression in the embryonic mouse brain
.
Development
120
,
2225
2234
.
Shimamura
,
K.
,
Hartigan
,
D. J.
,
Martinez
,
S.
,
Puelles
,
L.
and
Rubenstein
,
J. L. R.
(
1995
).
Longitudinal organization of the anterior neural plate and neural tube
.
Development
121
,
3923
3933
.
Shimamura
,
K.
,
Martinez
,
S.
,
Puelles
,
L.
, and
Rubenstein
,
J. L. R.
(
1997
).
Patterns of gene expression in the neural plate and neural tube subdivide the embryonic forebrain into transverse and longitudinal domains
.
Dev. Neurosci
.
19
,
88
96
.
Simon
,
H.
,
Hornbruch
,
A.
and
Lumsden
,
A.
(
1995
).
Independent assignment of antero-posterior and dorso-ventral positional values in the developing chick hindbrain
.
Curr. Biol
.
5
,
205
214
.
Stein
,
S.
and
Kessel
,
M.
(
1995
).
A homeobox gene involved in node, notochord and neural plate formation of chick embryos
.
Mech. Dev
.
49
,
37
48
.
Stoykova
,
A.
,
Fritsch
,
R.
,
Walther
,
C.
and
Gruss
,
P.
(
1996
).
Forebrain patterning defects in Small eye mutant mice
.
Development
122
,
3453
3465
.
Sulik
,
K.
,
Dehart
,
D. B.
,
Inagaki
,
T.
,
Carson
,
J. L.
,
Vrablic
,
T.
,
Gesteland
,
K.
and
Schoenwolf
,
G. C.
(
1994
).
Morphogenesis of the murine node and notochordal plate
.
Dev. Dyn
.
201
,
260
278
.
Tanabe
,
Y.
,
Roelink
,
H.
and
Jessell
,
T. M.
(
1995
).
Induction of motor neurons by Sonic hedgehog is independent of floor plate differentiation
.
Curr. Biol
.
5
,
651
658
.
Tanabe
,
Y.
and
Jessell
,
T. M.
(
1996
).
Diversity and pattern in the developing spinal cord
.
Science
274
,
1115
1123
.
Tao
,
W.
and
Lai
,
E.
(
1992
).
Telencephalon-restricted expression of BF-1, a new member of the HNF-3/folk head gene family in the developing rat brain
.
Neuron
8
,
957
966
.
Webb
,
J. F.
and
Noden
,
D. M.
(
1993
).
Ectodermal placodes: contributions to the development of the vertebrate head
.
Amer. Zool
.
33
,
434
447
.
Xuan
,
S.
,
Baptista
,
C. A.
,
Balas
,
G.
,
Tao
,
W.
,
Soares
,
V. C.
and
Lai
,
E.
(
1995
).
Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres
.
Neuron
14
,
1141
1152
.
Yamada
,
T.
,
Placzek
,
M.
,
Tanaka
,
H.
,
Dodd
,
J.
and
Jessell
,
T. M.
(
1991
).
Control of cell pattern in the developing nervous system: polarizing activity of the floor plate and notochord
.
Cell
64
,
635
647
.
Yuasa
,
J.
,
Hirano
,
S.
,
Yamagata
,
M.
and
Noda
,
M.
(
1996
).
Visual projection map specified by topographic expression of transcription factors in the retina
.
Nature
382
,
632
635
.