XB-ART-22797Development March 1, 1993; 117 (3): 961-75.
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Xenopus Distal-less related homeobox genes are expressed in the developing forebrain and are induced by planar signals.
The polymerase chain reaction (PCR) was used to isolated five Xenopus homeobox clones (X-dll1 to 5) that are related to the Drosophila Distal-less (Dll) gene and we propose a subdivision of the vertebrate distal-less gene family according to sequence similarities. cDNA clones were isolated for X-dll2, 3 and 4, and their expression was studied by RNase protection and in situ hybridization. X-dll2, which belongs to a separate subfamily than X-dll3 and 4, is not expressed in the neural ectoderm. X-dll3 and X-dll4, which belong to the same subfamily, have a similar but not identical pattern of expression that is restricted to anterior ectodermal derivatives, namely the ventral forebrain, the cranial neural crest and the cement gland. X-dll3 is also expressed in the olfactory and otic placodes while X-dll4 is expressed in the developing eye. X-dll3 differs from the other Xenopus genes and the previously isolated Dll-related mouse genes, in that localized expression can be detected by in situ hybridization very early in development, in the anterior-transverse ridge of the open neural plate. Based on that early expression pattern, we suggest that X-dll3 marks the rostral-most part of the neural plate, which gives rise to the ventral forebrain. Finally, we have used these Xenopus distal-less genes to show that the anterior neural plate can be induced by signals that spread within the plane of neural ectoderm, indicating that at least the initial steps of forebrain development do not require signals from underlying mesoderm.
PubMed ID: 8100768
Species referenced: Xenopus laevis
Genes referenced: acta1 actl6a dlc dll1 dll4 dlx2 dlx3 dlx5 eef1a1 en2 ncam1 por tbx2 twist1
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|Fig. 4. Developmental expression of X-dll4, 3 and 2 by whole- mount in situ hybridization. In all panels apart from these on the top row, anterior is to the leftand dorsal to the top. A, C, E, G, H, K and M show the ontogenesis of X-dll3 while F, I, J, L and N show the ontogenesis of X-dll4. (A) Frontal view of X-dll3 expression at the open neural plate stage (stage 16). Staining is observed in the cement gland and the anterior transverse rim of the neural plate. (B) Frontal view of X-twi expression in the premigratory cranial neural crest at the same stage (stage 16). InA and B, arrows indicate the lateral limits of expression. Note that X-twi and X-dll3 expression is not overlapping and if superimposed would label most of the anterior and anterolateral rim of the neural plate. (C) Frontal view of a non-cleared embryo showing the external expression of X-dll3 a few stages later, after neural tube closure. Expression externally is detected in a band of cells, probably representing the olfactory placode (arrow) and the cement gland (cg). (D) Rear view of the same embryo as inC, showing expression of X-dll3 around the closed blastopore lip, the prospective proctodeum (arrowhead). (E,F) Side views of cleared tailbud stage (stage 25) Xenopus, showing expression of X-dll3 (E) and X-dll4 (F). (G,I) Higher magnification of E and F. X-dll3 is expressed in the prosencephalon, olfactory placode, otic vesicle and cement gland (E,G) and X-dll4 is expressed in what appears to be the same region of the prosencephalon and, in addition, in the branchial arches and cement gland (F,I). The A-P limits of expression in the prosencephalon are delimited by arrows. The contour of the brain has been traced by a dashed line inG but is also visible in I. (H) Expression of X-dll3 in the brain of a cleared stage 33 embryo. (J) expression of X-dll4 at the same stage. Note that, in addition to the sites of expression at stage 25, X-dll3 is now also expressed in the branchial arches (H) and X-dll4 is also expressed in the eye (J). (K,L) Whole cleared tadpoles showing that X-dll3 (K) and X-dll4 (L) are expressed in the forebrain and all of the branchial arches. (M,N) Higher magnifications of X-dll3 and X-dll4 expression respectivelyat the same stage as in K and L. In M, small arrows point to the telencephalic-diencephalic boundary. In M and N, arrowheads point to the dorsal limit of expression in the telencephalon and diencephalon. Note that expression in the tadpole brain appears more extensive relative to expression at the tailbud stage (compare M and N with G and I; see text for details). The asterisk inN indicates a staining artifact. Abbreviations: ba, branchial arches; bl, blastopore; cg, cement gland; di, dienecephalon; dt, dorsal telencephalon; e, eye; fb, forebrain; op, olfactory placode; os, optic stalk; ov, otic vesicle; vt, ventral telencephalon.|
|Fig. 7. Induction of gene expression by planar signals. Expression in tailbud stage embryos is shown on the left column (anterior to the left) and expression in age-matched Keller sandwiches is shown on the right column (anterior to the top). (A,B) X-dll3; (C,D) N- CAM; (E,F) En-2; (G,H) X-twi; (I,J) Actin. In A and B, arrows point to expression in the forebrain and arrowheads to expression in the cement gland. A higher magnification of A (Fig. 4G) shows that the level of X-dll3 expression in the prosencephalon is comparable to the level in cement gland and olfactory placode. Note that expression of X-dll3 in a Keller sandwich occurs around the edge of the non-elongated part of the neural ectoderm. In D and J, the limits of the mesoderm are denoted by arrowheads, illustrating that in a Keller sandwich the mesoderm forms away from the neural ectoderm. In D, the anterior limit of N-CAM hybridization is marked by an arrow and roughly the same level has been marked by arrows in B, F and H. In H, note that the X-twi hybridization is localized close to the base of the non-elongated part of the neural ectoderm (arrowheads), at about the same A-P level with en-2 (arrowheads in F). Both markers are clearly more posterior than X-dll3 (compare B with F and H). The staining denoted by an asterisk in F is an artifact.|
|Fig. 3. RNase protection showing the spatial distribution of transcripts of X-dll4, -3 and -2. Xenopus tadpoles were dissected as shown diagramatically on the right and RNA from the same number of tissue pieces were analyzed by RNase protection as shown on the left. Fragments 1, 2 and 3 (lanes 1, 2 and 3) represent the forebrain (telencephalon and diencephalon), midbrain and hindbrain, respectively. These samples were dissected free of associated structures such as eyes, epidermis and underlying mesoderm. Fragments 4 and 5 (lanes 4 and 5) represent the trunk and tail respectively including the skin, while fragment 6 (lane 6) represents the remaining of the embryo, including the eyes, branchial arches and cement gland. Since the same embryo equivalents were loaded for each lane (8 embryos/lane), the lanes corresponding to fragments 1, 2 and 3 appear underloaded when hybridized to eF1-a. To ensure the integrity of the RNA in these lanes, we have simultaneously analyzed the expression of the neural-specific marker N-CAM. The N-CAM signal in fragment 6 is presumably due to expression in the retina. Note that X-dll2 is not expressed in any part of the brain (lanes 1, 2 and 3) while X-dll4 and X-dll3 are specifically expressed in the forebrain (lane 1). The tadpole diagram was based on Nieuwkoop and Faber (1967).|
|Fig. 5. Expression pattern of X-dll3 and 4 in sections of Xenopus tadpoles, after whole-mount in situ hybridization. Serial sections through the brain of a stage 37 tadpole after in situ hybridization with a X-dll3 probe (A-E) or an X-dll4 probe (F-G). The plane of sectioning and the A-P level of the sections shown is approximately the same between A-E and F-G and is indicated by arrowheads in T, superimposed on whole-mount in situ hybridization of a stage 37 brain with X-dll3. For definition of the A-P and D-V axis see Fig. 6. In A and F, an arrowhead points to the dorsal limit of expression in the telencephalon. In B and C, arrowheads point to the ‘segment-like’ expression of x-dll3 in the ventral thalamus. The upper arrowhead marks the sulcus medius (unlabelled) that separates the dorsal from the ventral thalamus. Note however, that expression continues ventrally in more posterior sections, into the anterior hypothalamus (B,C and D for X-dll3, and G, H and I for X-dll4). In E and J, arrowheads point to restricted punctate expression in the posterior hypothalamus. (N,S) Sections that also show the restricted expression of X-dll3 (N) and X-dll4 (S) in the posterior hypothalamus at a level more posterior to that shown in E and J, and at slightly older embryos (stage 39). The sections shown in E, J, N and S, pass though the punctate ‘line’ of expression that is indicated by an arrow in T and by arrowheads in O. (O) Parasagittal section though a stage 37 brain hybridized with x-dll3 (the same result was obtained with X-dll4; data not shown) In O also note that there is non-expressing ‘band’ of tissue between the telencephalon and diencephalon (unlabelled), which is presumably the tel-diencephalic boundary. Although Xdll3 and X-dll4 appear to have the same A-P and D-V limits of expression within the neural ectoderm, they differ in that X-dll3 is expressed more medially than Xdll4, as can be seen by comparing the mediolateral limit of expression (arrow) between B (X-dll3) and G (X-dll4), or between C (X-dll3) and H (X-dll4). They also differ in their expression in the sense organs in that X-dll3 is expressed in the olfactory placode (A and B), the otic vesicle (K) but not in the eye (L), while X-dll4 is not expressed in the olfactory placode (F,G), and the otic vesicle (P), but is expressed in the eye (arrowhead in Q). Both genes are expressed in the branchial arch neural crest, shown in M for X-dll3 and R for X-dll4, but X-dll3 is expressed strongly only in the distal part and shows a sharp proximodistal boundary (indicated by arrows in M). Note that the neural crest envelopes a core of nonexpressing mesodermally derived muscle cells. Abbreviations: ch, optic chiasma; cg, cement gland; ddi, dorsal diencephalon; di, diencephalon; dt, dorsal telencephalon; Et, epithalamus; e, eye; Ht, hypothalamus; hy, hypophysis; Tv, ventral thalamus; Td, dorsal thalamus; me, mesencephalon; m, muscle; nc, neural crest; on, optic nerve; op, olfactory placode; os, optic stalk; ov, otic vesicle; ph, pharynx; vdi, ventral diencephalon; vt, ventral telencephalon.|
|Fig. 6. Schematic summary of expression of X-dll3 at neurula (upper left), and of X-dll3 and X-dll4 at tailbud (upper right) and tadpole stage (lower middle). Only expression in the neural ectoderm is shown (shaded), based on whole-mount in situ hybridization data.The A-P and D-V axis for the tadpole stages have been defined in relation to the tadpole as a whole, in contrast to other descriptions that unroll and stretch the neural tube. Therefore, the A-P and D-V nomenclature that we use reflects the position of structures in the tadpole instead of their ontogenetic relationship. The ontogenetic relationship of the tadpole’s ventral and dorsal forebrain is demonstrated in the neural plate fate map (upper left) which is based on Eagleson and Harris, (1989) and Eagleson (personal communication). This map shows that part of the prospective ventral forebrain originates in a more anterior position than prospective dorsal forebrain. However, during neurulation the forebrain undergoes a rotation movement that brings rostral material in a more ventral and posterior position. Also note that the dorsal telencephalon occupies a very small area of the fate map at this stage. After closure of the neural plate, the expression pattern of X-dll3 and Xdll4 evolves as is shown at the tailbud (upper right) and tadpole stage (lower middle) diagram, based on tracings of Fig. 4G,I for the tailbud stage and Fig. 4M,N and Fig. 5T for the tadpole stage. The tadpole brain diagram is based on Kuhhlenbeck, 1973, vol.3, Fig. 112. The position where the sulcus limitans ends rostrally is controversial (see Kuhlenbeck, 1973), but the present interpretation is also consistent with the rostral extent of the basal plate as this is defined in Puelles et al. (1987). Abbreviations: cr, chiasmatic ridge; ddi, dorsal diencephalon; di, diencephalon; dt, dorsal telencephalon; hy, hypophysis; inh, infudibular hypothalamus; me, mesencephalon (midbrain); nt, notochord; op, olfactory placode; os, optic stalk; ov, otic vesicle; por, preoptic recess; ppr, posterior prosencephalon; pr, prosencephalon; prv, prosencephalic ventricle; rh, rhombencephalon (hindbrain); sc, spinal cord; sl, sulcus limitans; sm, sulcus medius; tdb, tel-diencephalic boundary; vdi, ventral diencephalon; vt, ventral telencephalon.|