Neuron,
Vol. 7, 239-247,
August,
1991, Copyright
0 1991 by Cell Press
Retinoic Acid Can Mimic Endogenous Signals Involved in Transformation of the Xenopus Nervous System Cohn R. Sharpe* Cancer Research Campaign Molecular Embryology Research Department of Zoology University of Cambridge Downing Street Cambridge CB2 3EJ England
Group
Summary In the frog Xenopus laevis, signals from the mesoderm divert part of the ectoderm from an epidermal to a neural fate. In the course of neural induction, the neurectoderm also acquires anterior-posterior polarity. In this report, the early expression of two genes, XIHbox6 and the neurofilament gene XIF6, is examined. The pattern of expression of the two genes seen in the tailbud embryo develops progressively over a 4 hr period following gastrulation. Physiological concentrations of retinoic acid can mimic this effect in isolated embryonic explants, consistent with the involvement of retinoic acid, or a closely related molecule, in localizing gene expression along the anterior-posterior axis of the neural tube. Introduction In Xenopus laevis, the nervous system forms as a result of interactions between mesoderm and ectoderm (Gimlich and Cooke, 1983; Jacobson and Rutishauser, 1986; Kintner and Melton, 1987; Sharpe et al., 1987). This process, neural induction, diverts a part of the ectoderm from an epidermal to a neural pathway of differentiation. Furthermore, neural induction results in the formation of a neural tube that displays distinct regional differences along its anterior-posterior axis, from forebrain to spinal cord (reviewed in Sharpe, 1990). Following a long history of research on the formation of neural tissue in Amphibia (reviewed in Hamburger, 1988), the challenge remains to understand the mechanism by which regional differentiation along the neural tube is achieved. Molecular genetic techniques have identified genes that are expressed solely in neural tissue (Kintner and Melton, 1987; Sharpe et al., 1987, 1989; Sharpe, 1988; Richter et al., 1988). Transcription from these genes can be used as aquantitative measure of neural induction. Using probes derived from three genes, expressed at different positions along the neural tube (Sharpe and’ Gurdon, 1990), it has been shown that quantitatively complete neural induction requires interactions between dor*Present address: Wellcome Trust, Cancer Research Campaign, Institute of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 IQR, England.
sal mesoderm and responding ectoderm until the late neural fold stage (stage 17; Nieuwkoop and Faber, 1967). Apart from the level of gene expression, an important feature of neural induction is the expression of neural genes in the correct position along the neural tube. The way in which gene expression becomes localized within the nervous system is analyzed in this paper. The currently favored model of neural induction is derived from the experiments of both Toivonen and Saxen (1955) and Nieuwkoop (1955,1958), reviewed in Saxen (1989), and postulates the progressive neuralization of the ectoderm. The first step of this two-step model is the activation of the ectoderm, presumably by a signal from the underlying mesoderm. In the absence of any further interaction the “activated” ectoderm forms solely anterior neural structures. In the second step, part of the activated ectoderm is transformed by a second signal that originates from the posterior region of the embryo. The response of the neurectoderm to the second signal results in the generation of the complete range of neural structures along the anterior-posterior axis. Evidence for this type of mechanism in Xenopus has recently been obtained. The anterior-posterior character of the neural tube, as assayed by histology, can be altered by culturing embryos in retinoic acid (RA) (Durston et al., 1989). RA-treated embryos have the same volume of neural tissue as untreated controls, but the tissue is of a more posterior character. Significantly, RA has been detected in early Xenopus embryos at a concentration of about 1.5 x IO-’ M (Durston et al., 1989). Whole embryos treated with RA have altered patterns of neural gene expression that correlate well with the observed morphology of the affected embryo, in that expression of anterior neural markers such as the engrailed antigen (Brivanlou and Harland, 1989) is reduced, while that of posterior neural markers such as XIHbox6 is elevated (Sive et al., 1990). Combined with the proposed role of RA as a diffusible morphogen in the chickwing bud (Tickle et al., 1982; Summerbell, 1983; Thaller and Eichele, 1987; Smith et al., 1989), it has been suggested (Durston et al., 1989) that RA is a good candidate for the agent of neural transformation. By examining the potential of dorsal regions of the embryo to express the neural marker genes XIHbox6 (Sharpe et al., 1987) and XIF6 (Sharpe, 1988), it has been possible to extend previous studies that have used RA in Xenopus (Durston et al., 1989; Sive et al., 1990; Cho and De Robertis, 1990) and show that RA applied to isolated regions can effectively localize the ability to express XIHbox6 and XIF6 to the correct part of the neural plate. It is suggested that RA, or a closely related molecule, can affect regional neural induction and may contribute to neural transformation in normal development.
Neuron 240
A
neurecroderm \
Figure 1. The Organization toderm-Mesoderm \
,
,’
(A) Lateral view of a late gastrula embryo. The outline of the neural plate is beginning to appear. At the posterior end of the embryo is the yolk plug, the site of mesoderm invagination during gastrulation. (B) Transverse section through the neurectoderm-mesoderm of a late gastrula embryo showing the organization of the cell layers.Thedorsal mesoderm will segregate into notochord (along the midline) and somitic mesoderm in regions located more toward the posterior of the embryo. (C) Dorsal view of the isolated neurectoderm-mesoderm. A small amount of epidermal tissue may-flank the dissected explant. The location of the section in (B) is marked by the line T.S.
\ ANTERIOR
A prospective / ’ 0 ce”,enf gland region
POSTERIOR
--._ VENTRAL
yolk plug (remnant blastopore)
Results Patterns of Expression of Neural Marker Genes in Explants Taken from along the Anterior-Posterior Axis of the Embryo The time at which neural marker genes are expressed in the position corresponding to their final location along the anterior-posterior axis of the embryo is only poorly defined. To examine this question, a portion of the embryo, consisting of neurectoderm and underlying dorsal mesoderm (Figures IB and IC), was isolated from embryos at successive developmental stages following the end of gastrulation. The isolated neurectoderm-mesoderm was then divided into anterior, middle, and posterior pieces, and these were cultured as explants to the late tailbud stage (stage 28) when they were assayed for the regional neural markers, XIF3, XIHbox6, and XIF6 (Figure 2). By isolating these regions from along the anterior-posterior axis of the embryo, the regional inducing potential of the neurectoderm-mesoderm was effectively “frozen” at different stages of development. The suggestion is that if the explants taken at stage 12.5 express the same marker genes that are found in the equivalent regions of the tailbud embryo, then the neurectoderm and underlying mesoderm lying in register at stage 12.5 are sufficient to organize regional neural induction. However,thiswasfound notto bethecase. Each explant, isolated from along the anterior-posterior axis at stage 12.5, went on to express low levels oftheanteriorneural marker,XIF3(Sharpeetal.,1988). Explants removed 1 hr later at stage 13.5 showed that the ability to express XIF3, when grown to the tailbud stage, was now located mainlywithin the anterior portion. This pattern was similar to that found in the tailbud embryo itself, and did not change when explants were isolated at subsequent time points (Figure 3).
of the Neurec-
This suggests that the ability to express XIF3 in the correct location is organized by stage 13.5. However, for the posterior marker, XIHbox6, and the general neural marker, XIF6, removal at progressive times following gastrulation resulted in a progressive increase in the levels of XIHbox6 in the midregion and XIF6 in the anterior region (Figure 3). Explanted regions of the neurectoderm-mesoderm go on to express XIHbox6 and XIF6 in a pattern similar to that in the tailbud embryo only when isolated after stages 15 to 16. This suggests that interactions within the embryo, at least
MANIPULATION Isolate regions negrectoderm:mesoderm
TIMESCALE of
Hours
AMP
00 -I
Culture
Assay
for
to t?e ;aii,bud
XIF3
FlgureZ. Outlineof of Neural Markers plants
XIHbox6
Stage
*I
-
- -
13%
02
03 - I94 -
- -
14
-
16
-
28
12’12
15
stage
& XIF6
16
-
the Experiment Investigating the Expression in Isolated Neurectoderm-Mesoderm Ex-
Neurectoderm-mesoderm explants, removed at stage 12.5 as described in Figure 1, were dissected into anterior (A), middle (M), and posterior (P) portions and grown until control embryos had reachedthetailbud stage.Atthispointtheywereassayedfor the neural markers. This process was repeated at hourly intervals over a 4 hr period.
Retinoic 241
Acid
and Neural
Transformation
XIHbox6
XIF6
XIF3 AMPAMPAMP I III1
I
III
STAGE Time (hours)
12%
Stage
13%~ -- 14
16
[RA] M 10 -7 -8 C -7 -8 C -7 -8 C C t I
IIIlIIIIlI
0
XIHboxG1
13%
2
14
3
15
5s RNA Figure 4. RA Can Stimulate gion Neurectoderm-Mesoderm
4
16
16
28
Figure 3. The Ability of the Neurectoderm-Mesoderm to Express Neural Marker Genes Develops Progressively
to the localization of expresalong the anterior-posterior
RA Affects the Expression of Both XIHbox6 and XIF6 in Neurectoderm-Mesoderm Explants One of two alternative mechanisms may contribute to the ability of the embryo to express XIHbox6 and XIF6 at the correct location. First, the ability may be conferred by an agent initially present in the posterior part of the embryo that becomes progressively available during neurulation either by diffusion or transport. Second, the observed effects could be due to the migration of either inducing or responding cells along the anterior-posterior axis. In this section the first
alternative
is considered,
by applying
of XIHbox6
in Midre-
RNAase protection assay of RNA derived from four explants per lane using probes for XIHbox6 and 5S RNA as a control for the recovery of RNA. Explants of midregion neurectoderm-mesoderm were isolated at stages 12.5, 13.5, and 14 and put into medium containing either lo-‘M RA, IO-@ M RA, or a control of 1 x MBS,I% DMSO(lanesC).Explantstakenatstage16wereput into control 1 x MBS, 1% DMSO.“t”indicates the negative control in which tRNA is used as the RNA source in the protection assay.
Explants
RNAase protection assays of explants taken at the times and stages shown in the left-hand column and according to the regime described in Figure 2. Five explants were taken per time point, and the RNAwas divided equally among the three probes. Autoradiography was for 16 hr (XIF3) and 3 days (XIHbox6 and XIF6).
up to stage 15, contribute sion of XIHbox6 and XIF6 axis of the neural tube.
the Expression Explants
a putative
agent, RA, to explants of neurectoderm-mesoderm. The possible contribution of cell migration is considered in the Discussion. It was tested whether RA, acting on explants taken from along the anterior-posterior axis of the neurectoderm-mesoderm at the end of gastrulation, can produce the same changes in expression of XIHbox6 and XIF3 that are seen in explants taken at later times dur-
ing neurulation.Todothis,the midregion explantwas isolated from embryos at stage 12.5 and then again from other embryos in the same batch at 1 and at 2 hr later. At each time point the removed midregion was grown in isolation from the rest of the embryo, in medium without RA, or medium containing lO-‘M or 1Om8 M RA, until control embryos had reached the tailbud stage (stage 28). Expression of the XIHbox6 gene was then determined by an RNAase protection assay. Incubation in medium containing IO-‘M RA resulted in the expression of XIHbox6 in the midregion explants taken at stage 12.5 (Figure4). Explants isolated at the same stage, but grown in control medium without RA, did not express XIHbox6. Therefore, RA at physiological concentrations can produce changes in the expression of XIHbox6 in midregion explants that are normally seen only when explants are isolated at later stages of development. These observations are consistent with the suggestion that XIHbox6 expression in the midregion explants taken during neurulation (Figure 3) depends on RA, or a related molecule, becoming progressivelyavailablefrom posteriortoanterior along the dorsal side of the embryo. Next it was tested whether RAcan produce the same striking changes that are seen in the expression of XIF6 when anterior neurectoderm-mesoderm explants are taken at intervals between stages 12.5 and 16 (Figure 3). Anterior explants were taken from the early neurula embryos and cultured in medium either without RA or at a concentration of IO-’ M or 1Om8 M RA. Again it was found that RA produces the same effect as isolating the equivalent explants 4 hr later, at
Neuron 242
STAGE
12’/2
[RA]M ,0-7-8 I
I
14
13%
C I
-7-8 I I
c I
-7-8 I
I
A
16 c I
c I
neurectoderm -____c
t I
5hr +RA
12hr culture
Assay
-dorsal mesoderm
B
Figure 5. RA Can Stimulate Neurectoderm-Mesoderm
the Expression Explants
RNAase protection assay of RNA isolated at stages 12.5, 13.5, and RA as described in Figure 4. One the stage 12.5 time point and two and 16 time points.
of XIF6
Probe
in Anterior
derived from anterior explants 14. Explants were treated with explant per lane was used for explants at the stage 13.5, 14,
stage 16 (Figure 5); that is, the early explants grown in IO-‘M RA now accumulate near normal levels of XIF6 transcripts. It is unlikely that explants of early anterior neurectoderm-mesoderm express little or no XIF6 because of a basic failure to make neural tissue, since the same explants express the well-characterized anterior neural marker XIF3 (Sharpe et al., 1990). XIF6 has previously been described as a general neural marker, because of the distribution of its transcripts in thetailbud embryo (Sharpe, 1988). This is not incompatible with the results presented here, which describe the mechanism by which the final patterns of gene expression are established. It appears likely that XIF6 is not distributed along the anterior-posterior axis following activation, but rather as a response to neural transformation. This may represent a modification of the previously proposed (Saxen, 1989) twostep model of neural induction in which general neural markers were considered to be stimulated in response to the first signal. To minimize the possibilty that these effects were due to toxicity, RA was used at concentrations equal to, or IO-fold below, the measured level in whole embryos (1.5 IO-’ M; Durston et al., 1989). Furthermore, Cho and De Robertis (1990) have reported that 10m5 M RA does not affect the viability of animal cap cells, some of which form neural tissue in normal development. It is also considered unlikely that RA causes premature differentiation of the neurectoderm since several groups have shown that RA cannot induce neural tissue in isolated animal caps from blastula or in early gastrula ectoderm (Sive et al., 1990; Cho and De Robertis, 1990), nor can RA stimulate XIF6 expression in neurectoderm or dorsal mesoderm isolated from the anterior neurectoderm-mesoderm explant
Probe
XIHbox6
cardiac actin
C Probe
XIF6
Figure Genes
6. RA Does in Isolated
“cytoskeletal” actin
Not Alter the Expression of the Neural Neurectoderm or Dorsal Mesoderm
Marker
(A) All but the very posterior part of the neurectoderm (for details of dissection see Sharpe and Gurdon, 1990) and the dorsal mesoderm were removed at the late gastrula stage and cultured separately. Samples were incubated either in 1 x MBS or in medium containing RA for 5 hr before being transferred to fresh media without RA. Samples were allowed to develop for a further 12 hr before the assay. (B) RNAase protection assay of the samples using the XlHbox6 probe. No transcripts were detected in any lane. (C) RNAase protection assay of the samples using the XIF6 probe. A low level of transcripts wasdetected in the neurectoderm only; but the level was unaffected by treatment with RA. (D) RNAase protection assay of the samples using the cardiac actin probe. Only the mesodermal samples express the cardiac actin. The reduced level in the R.&treated lane is probably due to the presence of less material, as shown by the cross-hybridizing actin bands, which probably represent the ubiquitously distributed cytoskeletal actins and which can be used as a measure of RNA recovery.
at the end of gastrulation (Figure 6). Although RA has teratogenic effects on the formation of the neural tube, this is not incompatible with a function in normal development. Indeed, in the experiments reported here, it is emphasized that the application of RA results in establishment of the normal patterns of gene expression seen during development. In summary, the exogenous application of RA to dorsal explants taken from along the anterior-posterior
Retinoic 243
Acid
and Neural
Transformation
STAGE [RA]
13% M
10
-’ I
XIHbox6
-8 I
14 C I
-’ I
-8 I
16 C I
A I
M I
P I
5hr
t I
-
k RA
*
m
- 12hr culture-
Assay
Vol. 7, 239-247,
August,
1991, Copyright
0 1991 by Cell Press
Retinoic Acid Can Mimic Endogenous Signals Involved in Transformation of the Xenopus Nervous System Cohn R. Sharpe* Cancer Research Campaign Molecular Embryology Research Department of Zoology University of Cambridge Downing Street Cambridge CB2 3EJ England
Group
Summary In the frog Xenopus laevis, signals from the mesoderm divert part of the ectoderm from an epidermal to a neural fate. In the course of neural induction, the neurectoderm also acquires anterior-posterior polarity. In this report, the early expression of two genes, XIHbox6 and the neurofilament gene XIF6, is examined. The pattern of expression of the two genes seen in the tailbud embryo develops progressively over a 4 hr period following gastrulation. Physiological concentrations of retinoic acid can mimic this effect in isolated embryonic explants, consistent with the involvement of retinoic acid, or a closely related molecule, in localizing gene expression along the anterior-posterior axis of the neural tube. Introduction In Xenopus laevis, the nervous system forms as a result of interactions between mesoderm and ectoderm (Gimlich and Cooke, 1983; Jacobson and Rutishauser, 1986; Kintner and Melton, 1987; Sharpe et al., 1987). This process, neural induction, diverts a part of the ectoderm from an epidermal to a neural pathway of differentiation. Furthermore, neural induction results in the formation of a neural tube that displays distinct regional differences along its anterior-posterior axis, from forebrain to spinal cord (reviewed in Sharpe, 1990). Following a long history of research on the formation of neural tissue in Amphibia (reviewed in Hamburger, 1988), the challenge remains to understand the mechanism by which regional differentiation along the neural tube is achieved. Molecular genetic techniques have identified genes that are expressed solely in neural tissue (Kintner and Melton, 1987; Sharpe et al., 1987, 1989; Sharpe, 1988; Richter et al., 1988). Transcription from these genes can be used as aquantitative measure of neural induction. Using probes derived from three genes, expressed at different positions along the neural tube (Sharpe and’ Gurdon, 1990), it has been shown that quantitatively complete neural induction requires interactions between dor*Present address: Wellcome Trust, Cancer Research Campaign, Institute of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 IQR, England.
sal mesoderm and responding ectoderm until the late neural fold stage (stage 17; Nieuwkoop and Faber, 1967). Apart from the level of gene expression, an important feature of neural induction is the expression of neural genes in the correct position along the neural tube. The way in which gene expression becomes localized within the nervous system is analyzed in this paper. The currently favored model of neural induction is derived from the experiments of both Toivonen and Saxen (1955) and Nieuwkoop (1955,1958), reviewed in Saxen (1989), and postulates the progressive neuralization of the ectoderm. The first step of this two-step model is the activation of the ectoderm, presumably by a signal from the underlying mesoderm. In the absence of any further interaction the “activated” ectoderm forms solely anterior neural structures. In the second step, part of the activated ectoderm is transformed by a second signal that originates from the posterior region of the embryo. The response of the neurectoderm to the second signal results in the generation of the complete range of neural structures along the anterior-posterior axis. Evidence for this type of mechanism in Xenopus has recently been obtained. The anterior-posterior character of the neural tube, as assayed by histology, can be altered by culturing embryos in retinoic acid (RA) (Durston et al., 1989). RA-treated embryos have the same volume of neural tissue as untreated controls, but the tissue is of a more posterior character. Significantly, RA has been detected in early Xenopus embryos at a concentration of about 1.5 x IO-’ M (Durston et al., 1989). Whole embryos treated with RA have altered patterns of neural gene expression that correlate well with the observed morphology of the affected embryo, in that expression of anterior neural markers such as the engrailed antigen (Brivanlou and Harland, 1989) is reduced, while that of posterior neural markers such as XIHbox6 is elevated (Sive et al., 1990). Combined with the proposed role of RA as a diffusible morphogen in the chickwing bud (Tickle et al., 1982; Summerbell, 1983; Thaller and Eichele, 1987; Smith et al., 1989), it has been suggested (Durston et al., 1989) that RA is a good candidate for the agent of neural transformation. By examining the potential of dorsal regions of the embryo to express the neural marker genes XIHbox6 (Sharpe et al., 1987) and XIF6 (Sharpe, 1988), it has been possible to extend previous studies that have used RA in Xenopus (Durston et al., 1989; Sive et al., 1990; Cho and De Robertis, 1990) and show that RA applied to isolated regions can effectively localize the ability to express XIHbox6 and XIF6 to the correct part of the neural plate. It is suggested that RA, or a closely related molecule, can affect regional neural induction and may contribute to neural transformation in normal development.
Neuron 240
A
neurecroderm \
Figure 1. The Organization toderm-Mesoderm \
,
,’
(A) Lateral view of a late gastrula embryo. The outline of the neural plate is beginning to appear. At the posterior end of the embryo is the yolk plug, the site of mesoderm invagination during gastrulation. (B) Transverse section through the neurectoderm-mesoderm of a late gastrula embryo showing the organization of the cell layers.Thedorsal mesoderm will segregate into notochord (along the midline) and somitic mesoderm in regions located more toward the posterior of the embryo. (C) Dorsal view of the isolated neurectoderm-mesoderm. A small amount of epidermal tissue may-flank the dissected explant. The location of the section in (B) is marked by the line T.S.
\ ANTERIOR
A prospective / ’ 0 ce”,enf gland region
POSTERIOR
--._ VENTRAL
yolk plug (remnant blastopore)
Results Patterns of Expression of Neural Marker Genes in Explants Taken from along the Anterior-Posterior Axis of the Embryo The time at which neural marker genes are expressed in the position corresponding to their final location along the anterior-posterior axis of the embryo is only poorly defined. To examine this question, a portion of the embryo, consisting of neurectoderm and underlying dorsal mesoderm (Figures IB and IC), was isolated from embryos at successive developmental stages following the end of gastrulation. The isolated neurectoderm-mesoderm was then divided into anterior, middle, and posterior pieces, and these were cultured as explants to the late tailbud stage (stage 28) when they were assayed for the regional neural markers, XIF3, XIHbox6, and XIF6 (Figure 2). By isolating these regions from along the anterior-posterior axis of the embryo, the regional inducing potential of the neurectoderm-mesoderm was effectively “frozen” at different stages of development. The suggestion is that if the explants taken at stage 12.5 express the same marker genes that are found in the equivalent regions of the tailbud embryo, then the neurectoderm and underlying mesoderm lying in register at stage 12.5 are sufficient to organize regional neural induction. However,thiswasfound notto bethecase. Each explant, isolated from along the anterior-posterior axis at stage 12.5, went on to express low levels oftheanteriorneural marker,XIF3(Sharpeetal.,1988). Explants removed 1 hr later at stage 13.5 showed that the ability to express XIF3, when grown to the tailbud stage, was now located mainlywithin the anterior portion. This pattern was similar to that found in the tailbud embryo itself, and did not change when explants were isolated at subsequent time points (Figure 3).
of the Neurec-
This suggests that the ability to express XIF3 in the correct location is organized by stage 13.5. However, for the posterior marker, XIHbox6, and the general neural marker, XIF6, removal at progressive times following gastrulation resulted in a progressive increase in the levels of XIHbox6 in the midregion and XIF6 in the anterior region (Figure 3). Explanted regions of the neurectoderm-mesoderm go on to express XIHbox6 and XIF6 in a pattern similar to that in the tailbud embryo only when isolated after stages 15 to 16. This suggests that interactions within the embryo, at least
MANIPULATION Isolate regions negrectoderm:mesoderm
TIMESCALE of
Hours
AMP
00 -I
Culture
Assay
for
to t?e ;aii,bud
XIF3
FlgureZ. Outlineof of Neural Markers plants
XIHbox6
Stage
*I
-
- -
13%
02
03 - I94 -
- -
14
-
16
-
28
12’12
15
stage
& XIF6
16
-
the Experiment Investigating the Expression in Isolated Neurectoderm-Mesoderm Ex-
Neurectoderm-mesoderm explants, removed at stage 12.5 as described in Figure 1, were dissected into anterior (A), middle (M), and posterior (P) portions and grown until control embryos had reachedthetailbud stage.Atthispointtheywereassayedfor the neural markers. This process was repeated at hourly intervals over a 4 hr period.
Retinoic 241
Acid
and Neural
Transformation
XIHbox6
XIF6
XIF3 AMPAMPAMP I III1
I
III
STAGE Time (hours)
12%
Stage
13%~ -- 14
16
[RA] M 10 -7 -8 C -7 -8 C -7 -8 C C t I
IIIlIIIIlI
0
XIHboxG1
13%
2
14
3
15
5s RNA Figure 4. RA Can Stimulate gion Neurectoderm-Mesoderm
4
16
16
28
Figure 3. The Ability of the Neurectoderm-Mesoderm to Express Neural Marker Genes Develops Progressively
to the localization of expresalong the anterior-posterior
RA Affects the Expression of Both XIHbox6 and XIF6 in Neurectoderm-Mesoderm Explants One of two alternative mechanisms may contribute to the ability of the embryo to express XIHbox6 and XIF6 at the correct location. First, the ability may be conferred by an agent initially present in the posterior part of the embryo that becomes progressively available during neurulation either by diffusion or transport. Second, the observed effects could be due to the migration of either inducing or responding cells along the anterior-posterior axis. In this section the first
alternative
is considered,
by applying
of XIHbox6
in Midre-
RNAase protection assay of RNA derived from four explants per lane using probes for XIHbox6 and 5S RNA as a control for the recovery of RNA. Explants of midregion neurectoderm-mesoderm were isolated at stages 12.5, 13.5, and 14 and put into medium containing either lo-‘M RA, IO-@ M RA, or a control of 1 x MBS,I% DMSO(lanesC).Explantstakenatstage16wereput into control 1 x MBS, 1% DMSO.“t”indicates the negative control in which tRNA is used as the RNA source in the protection assay.
Explants
RNAase protection assays of explants taken at the times and stages shown in the left-hand column and according to the regime described in Figure 2. Five explants were taken per time point, and the RNAwas divided equally among the three probes. Autoradiography was for 16 hr (XIF3) and 3 days (XIHbox6 and XIF6).
up to stage 15, contribute sion of XIHbox6 and XIF6 axis of the neural tube.
the Expression Explants
a putative
agent, RA, to explants of neurectoderm-mesoderm. The possible contribution of cell migration is considered in the Discussion. It was tested whether RA, acting on explants taken from along the anterior-posterior axis of the neurectoderm-mesoderm at the end of gastrulation, can produce the same changes in expression of XIHbox6 and XIF3 that are seen in explants taken at later times dur-
ing neurulation.Todothis,the midregion explantwas isolated from embryos at stage 12.5 and then again from other embryos in the same batch at 1 and at 2 hr later. At each time point the removed midregion was grown in isolation from the rest of the embryo, in medium without RA, or medium containing lO-‘M or 1Om8 M RA, until control embryos had reached the tailbud stage (stage 28). Expression of the XIHbox6 gene was then determined by an RNAase protection assay. Incubation in medium containing IO-‘M RA resulted in the expression of XIHbox6 in the midregion explants taken at stage 12.5 (Figure4). Explants isolated at the same stage, but grown in control medium without RA, did not express XIHbox6. Therefore, RA at physiological concentrations can produce changes in the expression of XIHbox6 in midregion explants that are normally seen only when explants are isolated at later stages of development. These observations are consistent with the suggestion that XIHbox6 expression in the midregion explants taken during neurulation (Figure 3) depends on RA, or a related molecule, becoming progressivelyavailablefrom posteriortoanterior along the dorsal side of the embryo. Next it was tested whether RAcan produce the same striking changes that are seen in the expression of XIF6 when anterior neurectoderm-mesoderm explants are taken at intervals between stages 12.5 and 16 (Figure 3). Anterior explants were taken from the early neurula embryos and cultured in medium either without RA or at a concentration of IO-’ M or 1Om8 M RA. Again it was found that RA produces the same effect as isolating the equivalent explants 4 hr later, at
Neuron 242
STAGE
12’/2
[RA]M ,0-7-8 I
I
14
13%
C I
-7-8 I I
c I
-7-8 I
I
A
16 c I
c I
neurectoderm -____c
t I
5hr +RA
12hr culture
Assay
-dorsal mesoderm
B
Figure 5. RA Can Stimulate Neurectoderm-Mesoderm
the Expression Explants
RNAase protection assay of RNA isolated at stages 12.5, 13.5, and RA as described in Figure 4. One the stage 12.5 time point and two and 16 time points.
of XIF6
Probe
in Anterior
derived from anterior explants 14. Explants were treated with explant per lane was used for explants at the stage 13.5, 14,
stage 16 (Figure 5); that is, the early explants grown in IO-‘M RA now accumulate near normal levels of XIF6 transcripts. It is unlikely that explants of early anterior neurectoderm-mesoderm express little or no XIF6 because of a basic failure to make neural tissue, since the same explants express the well-characterized anterior neural marker XIF3 (Sharpe et al., 1990). XIF6 has previously been described as a general neural marker, because of the distribution of its transcripts in thetailbud embryo (Sharpe, 1988). This is not incompatible with the results presented here, which describe the mechanism by which the final patterns of gene expression are established. It appears likely that XIF6 is not distributed along the anterior-posterior axis following activation, but rather as a response to neural transformation. This may represent a modification of the previously proposed (Saxen, 1989) twostep model of neural induction in which general neural markers were considered to be stimulated in response to the first signal. To minimize the possibilty that these effects were due to toxicity, RA was used at concentrations equal to, or IO-fold below, the measured level in whole embryos (1.5 IO-’ M; Durston et al., 1989). Furthermore, Cho and De Robertis (1990) have reported that 10m5 M RA does not affect the viability of animal cap cells, some of which form neural tissue in normal development. It is also considered unlikely that RA causes premature differentiation of the neurectoderm since several groups have shown that RA cannot induce neural tissue in isolated animal caps from blastula or in early gastrula ectoderm (Sive et al., 1990; Cho and De Robertis, 1990), nor can RA stimulate XIF6 expression in neurectoderm or dorsal mesoderm isolated from the anterior neurectoderm-mesoderm explant
Probe
XIHbox6
cardiac actin
C Probe
XIF6
Figure Genes
6. RA Does in Isolated
“cytoskeletal” actin
Not Alter the Expression of the Neural Neurectoderm or Dorsal Mesoderm
Marker
(A) All but the very posterior part of the neurectoderm (for details of dissection see Sharpe and Gurdon, 1990) and the dorsal mesoderm were removed at the late gastrula stage and cultured separately. Samples were incubated either in 1 x MBS or in medium containing RA for 5 hr before being transferred to fresh media without RA. Samples were allowed to develop for a further 12 hr before the assay. (B) RNAase protection assay of the samples using the XlHbox6 probe. No transcripts were detected in any lane. (C) RNAase protection assay of the samples using the XIF6 probe. A low level of transcripts wasdetected in the neurectoderm only; but the level was unaffected by treatment with RA. (D) RNAase protection assay of the samples using the cardiac actin probe. Only the mesodermal samples express the cardiac actin. The reduced level in the R.&treated lane is probably due to the presence of less material, as shown by the cross-hybridizing actin bands, which probably represent the ubiquitously distributed cytoskeletal actins and which can be used as a measure of RNA recovery.
at the end of gastrulation (Figure 6). Although RA has teratogenic effects on the formation of the neural tube, this is not incompatible with a function in normal development. Indeed, in the experiments reported here, it is emphasized that the application of RA results in establishment of the normal patterns of gene expression seen during development. In summary, the exogenous application of RA to dorsal explants taken from along the anterior-posterior
Retinoic 243
Acid
and Neural
Transformation
STAGE [RA]
13% M
10
-’ I
XIHbox6
-8 I
14 C I
-’ I
-8 I
16 C I
A I
M I
P I
5hr
t I
-
k RA
*
m
- 12hr culture-
Assay
