XB-ART-24673Development August 1, 1991; 112 (4): 945-58.
Retinoic acid modifies the pattern of cell differentiation in the central nervous system of neurula stage Xenopus embryos.
Neural cell markers have been used to examine the effect of retinoic acid (RA) on the development of the central nervous system (CNS) of Xenopus embryos. RA treatment of neurula stage embryos resulted in a concentration-dependent perturbation of anterior CNS development leading to a reduction in the size of the forebrain, midbrain and hindbrain. In addition the overt segmental organization of the hindbrain was abolished by high concentrations of RA. The regional expression of two cell-specific markers, the homeobox protein Xhox3 and the neurotransmitter serotonin was also examined in embryos exposed to RA. Treatment with RA caused a concentration-dependent change in the pattern of expression of Xhox3 and serotonin and resulted in the ectopic appearance of immunoreactive neurons in anterior regions of the CNS, including the forebrain. Collectively, our results extend previous studies by showing that RA treatment of embryos at the neurula stage inhibits the development of anterior regions of the CNS while promoting the differentiation of more posterior cell types. The relevance of these findings to the possible role of endogenous retinoids in the determination of neural cell fate and axial patterning is discussed.
PubMed ID: 1682132
Article link: Development
Species referenced: Xenopus laevis
Genes referenced: b3gat1l evx1 ncam1 tbx2
Antibodies: B3gat1 Ab4 Evx1 Ab1 Ncam1 Ab10
Article Images: [+] show captions
|Fig. 1. Overall distribution of the four neural antigens examined in tadpole stage embryos. N-CAM-PSA (highly sialylated N-CAM) is recognized by the monoclonal antibody 5A5 (Dodd et al. 1988), and is expressed exclusively in the developing neural tube. The monoclonal antibody HNK-1 (Abo and Balch, 1981) recognizes a variety of neural and non-neural cell types, including Rohon-Beard sensory neurons in the dorsal region of the spinal cord (Nordlander, 1989). Antisera against the Xenopus homeobox gene Xhox3 recognize subsets of neurons in the midbrain, hindbrain and spinal cord (Ruiz i Altaba et al. 1991). Antisera against the neurotransmitter serotonin (5-HT) label a small population of cells in the ventral region of the anterior hindbrain (van Mier et al. 1986). Arrows point to relevant regions of antigen expression. In all cases, anterior is to the left and the dorsal side is upwards. Scale bar=lmm.|
|Fig. 2. Expression patterns of N-CAM-PSA in the CNS of normal and RA-treated embryos shown at the early (stage ~32-36; A-C) and late (stage —45; D, E) tadpole stages after application of RA at the early neurula stage. (A) Overall staining pattern of N-CAM-PSA showing the subdivisions of the developing CNS. Note the differential labelling within the major brain regions, especially the hindbrain where alternating levels of N-CAM-PSA in adjacent rhombomeres reveal the segmental organization of the hindbrain. The transition at the midbrain-hindbrain junction from cells expressing high levels of N-CAM-PSA to cells expressing low levels is also pronounced (arrow). (B) N-CAM-PSA immunoreactivity in the CNS of an embryo treated with an intermediate concentration of RA shows a reduction in the size of the anterior regions of the CNS including a compressed region with intense labelling which corresponds to the hindbrain (bracket). Fewer segmental subdivisions can be detected in the hindbrain. (C) Pattern of labelling of mAb 5A5 in an embryo treated with a high concentration of RA. Note the small anterior regions of the CNS and the reduced hindbrain (bracket). In all cases, the differential expression of N-CAM-PSA at the midbrain-hindbrain junction is maintained (arrows in panels A-C). (D) Normal expression of N-CAM-PSA in the anterior region of a late tadpole stage (stage ~45) embryo. Note the reduced expression of N-CAM-PSA in the rhombomeres and midbrain when compared with the embryo shown in panel A. At this stage, N-CAM-PSA is also expressed in the cranial nerves. (E) Expression of N-CAM-PSA in the CNS of a late tadpole (stage —45) embryo treated at the neurula stage with an intermediate concentration of RA, showing the absence of divisions and the reduced size of the brain. Cranial nerves appear to be absent, as judged by the lack of N-CAM-PSA labelling. C, cerebellar anlage; FB, forebrain; HB, hindbrain; MB, midbrain; N, normal untreated control embryos; o, otic vesicle; RA, embryos treated with RA at intermediate (I-RA) or high (H-RA) concentrations; SC, spinal cord; arabic numbers indicate the approximate positions of the different rhombomeres; roman numerals refer to the different cranial nerves; brackets depict domains of intense labelling corresponding to the hindbrain. Scale bar=0.5mm. Scale bars in panels A and D are valid for panels A-C and D-E, respectively.|
|Fig. 3. Expression patterns of the homeobox protein Xhox3 in control and RA-treated tadpole stage embryos. (A) At the early tadpole stage, Xhox3 is expressed in the dorsal-anterior hindbrain, mainly in rhombomere 1 and in a more ventral column of cells in the spinal cord and hindbrain that extends rostrally up to the boundary between rhombomeres 1 and 2. Within this ventral column, the expression of Xhox3 is highest in rhombomeres 5 and 6 (arrowhead in panel A). (B) Treatment of early neurula stage embryos with low concentrations of RA results in the reduction of the size of the hindbrain. This is manifest as a reduction in the distance between the eye and the otic vesicle (in focus), the eye and cells expressing Xhox3 dorsally in the anterior hindbrain and the eye and cells expressing Xhox3 at high levels normally found in rhombomeres 5 and 6 (slightly out of focus). (D) In older tadpoles, Xhox3 is also expressed in a small group of cells in the lateral midbrain. (C,E) Embryos treated with high concentrations of RA lack the expression of Xhox3 in the midbrain as well as any dorsal expression in the hindbrain but show ectopic expression of Xhox3 in the ventral anterior regions of the CNS, including the forebrain. Treatment with high concentrations of RA causes a reduction in the size of the CNS although the forebrain, midbrain and hindbrain sudbivisions can still be distinguished. High magnifications of the brains of control (D) and RA-treated embryos (E) show the changes in morphology and Xhox3 immunoreactivity in the anterior regions of the CNS. Only a few positive nuclei can be observed in panel E since at this magnification, few cells are in the plane of focus. E, eye; FB, forebrain; HB, hindbrain; MB, midbrain; N, untreated control embryos; o, otic vesicle; RA, embryos treated with low (L-RA) or high (H-RA) concentrations of RA; SC, spinal cord; arrowheads point to regions or cells showing nuclear Xhox3 labelling; numbers identify rhombomeres. Brackets depict the hindbrain in panels A-C. Scale bar=0.5mm. Scale bar in panels A and D are valid for panels A-C and D-E, respectively.|
|Fig. 4. Camera-lucida drawings which show the distribution of Xhox3 neurons in the CNS of representative normal and RA-treated embryos. Normal embryos (N; A, B) and embryos treated with intermediate (intermediate I-RA; C,D) or high (H-RA; E,F) concentrations of RA were labelled with Xhox3 antibodies. The pattern of Xhox3 expression was recorded by camera lucida. In each case, the contour of the anterior CNS (solid lines) is shown. Each dot represents the labelled nucleus of a single Xhox3 immunoreactive cell. The positions of the eye (e), otic vesicle (o) and notochord (n) are also indicated. All the drawings were obtained from embryos at the late tadpole stage (stage ~42) with the exception of that shown in A, which was obtained from a stage 34-36 tadpole. Note the absence of expression of Xhox3 in the dorsal hindbrain region in all RA-treated embryos (C-F), and the expansion of Xhox3-positive neurons into the anterior CNS in embryos treated with high concentrations of RA. The otic vesicle was absent in the embryos shown in panels C, E and F. FB, forebrain; HB, hindbrain; MB, midbrain; SC, spinal cord. Dotted line under the anterior regions of the CNS in A and C show the position of the notochord.|
|Fig. 5. Xhox3-immunoreactive cells in normal and RA-treated embryos. Panels A-C show cross sections of a normal tadpole stage (stage —36) embryo at the forebrain (A), hindbrain (B) and spinal cord (C) levels. Panels D-F show cross sections at the forebrain (D), hindbrain (E) and spinal cord (F) levels of an embryo treated with a high concentration of RA. Xhox3 immunoreactivity in cell nuclei is denoted by arrowheads. Note the absence of Xhox3 expressing cells in the dorsal hindbrain (E) and the ectopic expression of this homeobox gene in the forebrain (D) in RA treated embryos. FB, forebrain; HB, hindbrain; N, normal embryo; RA, embryo treated with a high concentration of RA (H-RA); SC, spinal cord. Scale bar=10/um.|
|Fig. 6. Expression of serotonin (5-HT) in normal and RA-treated embryos. All embryos shown are at the tadpole stage (stage ~32-34). Panel A shows the localization of serotonergic neurons in untreated embryos. Small groups of cells in the skin are also labelled. This labelling is not detected in the skin of the forehead. The axons of these neurons project both anteriorly and posteriorly. Panel B shows a high magnification view of the normal pattern of seroronin expression in the brain of tadpole stage embryos. The arrow denotes the midbrain-hindbrain junction. Early neurula stage embryos treated with low concentrations of RA show the presence of additional serotonergic neurons in more anterior regions of the CNS (C-E). Panel E shows a high magnification of the embryo shown in D. At intermediate concentrations of RA, the embryos display a reduced number of serotonergic cells (F, G). There is also a lack of axons projecting anteriorly (F). Embryos treated with high concentrations of RA show a complete absence of serotonergic cells in the CNS (H). In all cases anterior is to the left and dorsal side is upwards. E, eye; FB, forebrain; HB, hindbrain; MB, midbrain; N, normal untreated control embryos;. o, otic vesicle; RA, embryos treated with low (L-RA), intermediate (I-RA) or high (H-RA) concentrations of RA; SC, spinal cord; arrows point to sites of staining; brackets depict the A-P extent of the region containing serotonergic neurons; numbers identify rhombomeres. Scale bar in panel A=0.5mm, valid for panels A, C-D and F-G. Scale bars in panels B and E=0.1mm.|
|Fig. 7. Expression of HNK-1 by Rohon-Beard neurons in normal and RA-treated embryos. Panel A shows the pattern of expression of the HNK-1 antigen in wholemount preparations of tadpole stage (stage —38-40) embryos. mAb HNK-1 labels a variety of structures in the CNS including the cell bodies of Rohon-Beard neurons in the dorsal spinal cord (arrowheads). Panel B shows the labelling pattern of HNK-1 in the spinal cord of an embryo treated with high concentrations of RA. The inhibition of normal anterior development is not accompanied by an anterior extension of the domain over which Rohon-Beard neurons appear (bracket). Cg, cement gland; HB, hindbrain; o, otic vesicle; RA, embryo treated with high (H-RA) concentration of RA; SC, spinal cord. Scale bar=0.5mm.|
|Fig. 8. Histological sections of normal and RA-treated embryos showing the location of HNK-1-labelled Rohon-Beard cells. Panels A and B show cross sections of a normal embryo at the tadpole stage (stage ~36) at the level of the hindbrain (A) and spinal cord (B). Panels C and D show cross sections at the level of the hindbrain (C) and spinal cord (D) of an embryo at the tadpole stage (stage ~36) treated with a high concentration of RA. In normal embryos, HNK-1 labels the cell body and axonal membranes of Rohon-Beard cells and also a small compact region within the cell body (B). The dorsal position of Rohon-Beard neurons in the spinal cord and the absence of Rohon-Beard neurons in the hindbrain are not altered by RA treatment. Arrowheads point to Rohon-Beard neurons. Fp, floor plate; N, normal embryo; No, notochord; RA, embryo treated with a high (H-RA) concentration of RA; S, somites. Scale bar=10/an.|