First published online February 18, 2004
doi: 10.1242/10.1242/dev.00986
Development 131, 983-995 (2004)
Published by The Company of Biologists 2004
Segmental development of reticulospinal and branchiomotor neurons in lamprey: insights into the evolution of the vertebrate hindbrain
Yasunori Murakami1,*,
Massimo Pasqualetti2,3,
Yoko Takio1,
Shigeki Hirano4,
Filippo M. Rijli2 and
Shigeru Kuratani1
1 Evolutionary Morphology Research Team, Center for Developmental Biology (CDB),
RIKEN, Kobe, Japan
2 Institut de Génétique et de Biologie Moléculaire et
Cellulaire, UMR 7104, CNRS/INSERM/ULP, BP 10142-67404 Illkirch Cedex, CU de
Strasbourg, France
3 Laboratori di Biologia Cellulare e dello Sviluppo, Università di Pisa,
Via G. Carducci 13, Pisa, Italy
4 Department of Medical Technology, School of Health Sciences, Faculty of
Medicine, Niigata University, Niigata 951-8518, Japan
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Fig. 1. Development of reticulospinal neurons in the lamprey. (A-D) Dextran
labeling of the reticular neurons. (A) Stage 23.5 embryo. Isthmic (I) and
medial inferior reticulospinal (mir) neurons are labeled, whereas bulbar (B)
and Mauthner (Mth) neurons are not yet detected. (B) Stage 24.5 embryo. Mth
neuron and the B neuron cluster are observed at the level of the otic vesicle
(OV). (C) The same embryo as in B, focused at a more dorsal hindbrain level.
Crossing axons of Mth neurons are visible (arrow). (D) Stage 28 larva. I and B
neurons have increased in number, and I1 neuron has appeared. Most of the
reticulospinal components of the lamprey are formed at this stage. Anterior is
towards the top.
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Fig. 2. Identification of rhombomeres in the lamprey. (A) LjKrox20
expression (blue) in relation to the localization of acetylated tubulin
(brown) in a stage 25 embryo (anterior is towards the left). (B) Cranial nerve
roots (arrowheads) and rhombomere boundaries (arrows) are visualized with the
anti-acetylated tubulin antibody. Note that LjKrox20 expression
domain corresponds well to the morphological rhombomere boundaries (compare
arrows in A and B). (C,D) LjPax6 expression (blue) and reticulospinal
neuron (brown) localization. (C) Dorsal view of a stage 26 embryo. Reticular
neurons including I1, B and mir have developed lateral to the LjPax6
expression domain at the hindbrain midline. (D) Lateral view of the same
embryo. Mth is located in r4, where LjPax6 is expressed at low
levels. B, bulbar neurons; I, isthmic reticular neurons; MHB, mid-hindbrain
boundary; mir, medial inferior reticulospinal neurons; Mth, Mauthner neuron;
Mth', auxiliary Mauthner neuron; V1, profundus ganglion; V2/3,
trigeminal ganglion; VII, facial ganglion
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Fig. 4. Expression of LjEphC and spatial patterning of reticulospinal
neurons. (A,B) Expression of LjEphC in a stage 25 lamprey embryo. (A)
Lateral view. LjEphC is expressed in the hindbrain with two clear
domains corresponding to r3 and r5. (B) Dorsal view of the same embryo. Note
that the otic vesicle is adjacent to an LjEphC-negative hindbrain
region, corresponding to r4 (arrows). (C-F) Positions of bulbar (B) and Mth
neurons in relation to the expression of LjEphC. Reticular neurons
are visualized by whole-mount in situ hybridization with a neurofilament
protein antisense riboprobe. (C) Lateral view of a stage 25 embryo. (D) Dorsal
view of the same embryo as in C. (E,F) Higher magnification of the embryo in
C. (E) Lateral view. (F) Dorsal view. Note that the B and Mth neurons are
located in the LjEphC-negative domain corresponding to r4.
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Fig. 3. Regulatory gene expression domains and positions of reticulospinal neurons.
(A,B) Neuronal labeling in combination with whole-mount in situ hybridization
using a LjKrox20 riboprobe. (A) Lateral view of a stage 25 embryo.
With respect to the LjKrox20 expression domains, the I4 neuron is
located in r3, Mth neuron in r4, and Mth' neuron in r5. (B) Lateral view
of a stage 26 embryo. Newly developed I3 neuron is located in putative r2.
Note that other labeled neurons are located at the same axial levels as in the
stage 25 embryo shown in A. (C,D) Reticulospinal neurons and LjHox3
expression. (C) Lateral view of a stage 25 embryo. (D) Ventral view of a stage
26 embryo. The position of the Mth neuron corresponds to the anterior
LjHox3 expression boundary (arrowheads).
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Fig. 5. Developmental patterns of lamprey branchial motoneurons and the
reticulospinal tract. (A-C) Confocal microphotographs of a stage 26 lamprey
larva. Lateral (A,C) and dorsal (B) views. In the hindbrain, the
rhodamine-labeled reticulospinal tract (purple) runs below the
fluorescein-labeled trigeminal (Vm) and facial (VII) motor nuclei (green).
Note that the Mauthner (Mth) neuron is located between Vm and VII. (D) Stage
30 embryo. The Mth neuron is at the posterior margin of Vm (purple) as in
stage 26. (E) A stage 28 larva in which facial (VII), glossopharyngeal (IX)
and vagus (X) nerves have been labeled. Branchial motor nuclei (green) are
arranged along the anteroposterior axis, as seen in the adult. Note that the
Mth neuron (purple) appears in the rostral hindbrain and adjacent to the VIIth
nucleus as in the stage 26 larva.
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Fig. 6. Comparison of branchial motor neurons and regulatory gene expression
domains. Branchial motor neurons (brown) are labeled in combination with
LjKrox20 (A-C) or LjHox3 (D) expression domains (blue). (A)
Lateral view of a stage 26 larva. (B) Dorsal view of the same larva. Note that
the trigeminal (Vm) nucleus expands posteriorly into the
LjKrox20-negative region, i.e. the presumptive r4 (white arrows). The
facial (VIIm) nucleus also partially maps in the presumptive r4 domain (black
arrow). (C) The glossopharyngeal motor (IXm) nucleus is just posterior to the
r5 domain of LjKrox20. (D) The rostral boundary of LjHox3
expression maps between the Vm and VIIm nuclei. The inset clearly shows that
the trigeminal motor nucleus is located just rostral to the LjHox3
expression domain.
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Fig. 8. Evolution of reticular and branchiomotor neurons in the hindbrain of the
vertebrate embryo. The hypothetical rhombomeric organization of the lamprey
brain is based on LjPax6, LjEphC, LjKrox20 and LjHox3
expression data. LjHox3 is strongly expressed up to the mid r4 level
in this scheme. (Top) Hypothetical schema for the evolution of reticular
neurons. Based on this architecture, lamprey I3 is positioned in r2, and I4 in
r3. Mth and B neurons are localized in r4, and Mth' appears in r5. Thus,
the developmental pattern of the lamprey reticular neurons is segmental.
(Bottom) Branchiomotor patterning through phylogeny. Branchiomotor neurons,
including trigeminal (V), facial (VII), glossopharyngeal (IX) and vagus (X)
motor nuclei, are shown in different colors. The lamprey trigeminal motor
nucleus extends posteriorly into r4, which is not seen in gnathostomes. Note
that the boundary between the trigeminal and facial nuclei maps in the middle
of r4 correspond to the strong LjHox3 expression boundary, and not to
the r4/r5 boundary as in gnathostomes.
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Fig. 7. Effect of exogenous RA on the development of the reticulospinal and
branchiomotor neurons. (A-C) Confocal micrographs of reticulospinal neurons in
stage 26 lamprey larvae that had been treated with 0 µM (A), 0.01 µM
(B), 0.1 µM (C) all-trans RA. (D-F) Positions of the Mth neuron of stage 25
embryos in relation to the expression of LjKrox20 after treatment
with RA. The Mth neuron is visualized by whole-mount in situ hybridization
with a neurofilament protein antisense riboprobe. Note that the Mth neuron is
always located at the middle of r4. (G,H) Positions of Mth neurons in stage 25
embryos in relation to the expression of LjHox3 in the control (G)
and embryos treated with 0.1 µM RA (H). LjHox3 domain is shifted
anteriorly in the RA-treated larvae (compare arrows in G and H). (I,J)
Expression of Lj Fgf8/17 in the control (I) and 0.1 µM RA-treated
(J) stage 26 larvae. (K-N) Confocal micrographs of stage 26 larvae. (K,M)
Control larvae in which trigeminal (Vm), facial (VIIm), glossopharyngeal (IXm)
and vagus (Xm) neurons are clustered along the anteroposterior axis (K), and
the trigeminal motor nuclei positioned posterior to the mid-hindbrain boundary
(MHB: M). In 0.1 µM RA-treated larvae (L,N), boundaries between the
branchiomotor nuclei (BM) have become unclear (L), and the presumptive
trigeminal nucleus extends rostrally beyond the MHB (N).
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Fig. 9. Hypothetical scenario for the evolution of the vertebrate hindbrain. In the
hypothetical common ancestor of the chordates, the neural tube is assumed to
be regionalized along the anteroposterior axis by the nested expression
patterns of the Hox genes. In the common ancestor of vertebrates, segmental
patterns of reticular neuron development are established in the hindbrain
region. The appearance of hindbrain segmentation results in repeated sets of
serially homologous reticular neurons. In support of this hypothesis,
reticular-like neurons are already present in amphioxus
(Fritzsch, 1996 ), although
this organism does not possess hindbrain segmentation. The anteroposterior
specification of branchiomotor neurons is already under the control of a Hox
code in the common ancestor. In vertebrates, the registering of hindbrain
segmentation and Hox code regulation appears in the gnathostome lineage, as
suggested by the analysis of the lamprey branchiomotor neuron spatial pattern
and Hox regulation. In amniotes, large interneurons have been lost, together
with the overt serial homology of these neurons. This scenario of vertebrate
hindbrain evolution postulates independent mechanisms for neuronal patterning,
established as evolutionary events distinct from hindbrain segmentation
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© The Company of Biologists Ltd 2004
