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The ancestry of dopaminergic (DA) neurons in the Xenopus laevis hypothalamus was investigated by combining intracellular lineage dye injections of 16- and 32-cell blastomeres with the immunofluorescent detection of tyrosine hydroxylase at tadpole stages. At these stages, DA neurons in the hypothalamus comprise a discrete nucleus that contains from 22 to 45 cells on each side [mean = 32.6 +/- 6.6 (SD)]. The DA nucleus descends from only four of the 16-cell blastomeres. The two dorsal midline blastomeres (D1.1) are the major progenitors, and in all embryos studied they contributed to the DA nucleus. The two dorsal lateral blastomeres (D1.2) contribute to the DA nucleus in only about half of the embryos. Thus, the DA nucleus descends only from a discrete group of progenitors, and the participation of some of the progenitors in the DA lineage is only probabilistic. The number of DA neurons generated by the same blastomere varied greatly in different animals. This variation in cell number correlated with the degree of coherence and the density of the clone in the hypothalamus, rather than with clonal ancestry. Bilateral deletion of the major 32-cell progenitor (D1.1.1) resulted in a nearly complete restitution of the DA nucleus in 74% of the embryos that successfully completed gastrulation and neurulation. In the rest, the hypothalamus was smaller than normal or missing, and the DA nucleus was significantly reduced in size or absent. These results show that the DA nucleus can be restored after its normal lineage is deleted, but complete regulation is not always accomplished. Several blastomere progenitors dramatically altered their contribution to the DA nucleus after D1.1.1 ablation, including two blastomeres that normally do not contribute to the DA lineage. Thus, the fate to produce DA neurons is not determined at cleavage stages.
Figure 1. Outline of the experimental procedure. The nomenclature of 16-cell blastomeres is given on the left and of 32-cell blastomeres is given
on the right of the cleavage stage embryo. Only those blastomeres that were injected in this experiment are labeled. The big solid circle represents
TRA injected into one blastomere. In the fur right diagram, which represents a frontal section of the diencephalon, solid circles indicate the location
of members of the TRA-labeled clone, open circles indicate TH-positive cells, and dense-cored circles indicate double-labeled (DA-TRA) cells.
Figure 2. A pair of photomicrographs showing both TH immunofluorescence and TRA lineage dye. A, TH-positive neurons labeled with AMCA
fluoresce blue under UV excitation. B, A double-exposed micrograph showing blue TH-positive cells and red cells that are members of a clone
descended from D 1.2. One cell (arrowhead) is double labeled. Asterisk indicates the TRA-labeled axon tract. Scale bar, 25 um.
Figure 3. A, Camera lucida drawings of serial frontal sections showing the distribution of DA neurons (small open circles) in the hypothalamus
(Hy) and infundibulum (In) of a stage 44 embryo. From rostra1 (top left) to caudal (bottom right), the DA neurons form a coherent, continuous
cell cluster. B-D, Examples of DA neurons at different regions of the hypothalamus. B, Photomicrograph of a rostral DA neuron (e.g., in the first
section of the top row in A) located in the intermediate zone. Note the laterally oriented axon. C, DA neurons in the middle region of the
hypothalamus (e.g., the last section of the top row in A) form a coherent cell cluster. D, Caudally in the infundibulum (e.g., the last sections in the
bottom row of A) DA neurons are restricted to the dorsolateral margin. Scale bars: A, 100 pm; B-D, 25 um.
Figure 4. Transverse sections illustrating the different distribution patterns of TRA-labeled cells in the stage 43 forebrain, including the infundibulum (In). Asterisks indicate the labeled axon tracts. A, A typical distribution of Dl. 1.1 clone with dense ventral labeling and many contralateral cells. B, In other cases of D 1.1.1 clones, the TM-labeled cells are more dorsally located and scattered in small numbers in the hypothalamus. C, Usually, the descendants of Dl.2 and its daughters are located in the dorsal and intermediate forebrain. D, In some cases, the Dl.2 clone shifts ventrally and enters the hypothalamus. Scale bars, 100 um.
Figure 6. Regression line indicates a significant correlation between
the density of the TKA clone in the hypothalamus and the percentage
of DA neurons that are double labeled. The clones used for quantification
descended from D 1.1.1 (squares) and D 1.1.2 (triangles). These
results indicate that the number of DA cells produced by each blastomere
is related to the density of progeny in the hypothalamus rather
than to ancestry.
Figure 7. A, Camera lucida drawing
of frontal sections of the forebrain of
an embryo in which Dl.l.1 had been
deleted bilaterally. The brain is small
and the infundibulum is missing (compare
to Fig. 3). The DA neurons (solid
circles) are fewer in number and are distributed
along the ventralmost surface
of the brain. Hy, hypothalamus. B, A
photomicrograph of the ectopically located
DA neurons. Arrowheads point to
the ventral border of the forebrain. The
arrow points to an autofluorescent cell.
Scale bars: A, 100 um; B, 25 um.
Figure 8. Histogram showing the contribution of the 32-cell stage
blastomeres to DA nucleus in normal embryos (shaded) and in embryos
in which the major DA progenitor (Dl. 1.1) was deleted bilaterally at
the 32-cell stage (hatched). Note that the lineages of four blastomeres
change significantly (**, p < 0.01; *, p < 0.1). Blastomeres Vl.2.2,
Vl.1.2, D2.1.2, and D2.2.2 also were tested, and none of them contributed
DA neurons.
