Supplementary Figures
Supplementary Figure S1 Motor column organization and identity along the rostrocaudal axis of
the spinal cord. a, Schematic representation of the segmentally restricted motor columns in a
stage 29 chick embryo. Lateral motor columns (LMC) are generated at brachial and lumbar levels
of the spinal cord, and at both levels contain lateral (orange) and medial (green) divisions.
Preganglionic autonomic neurons of the Column of Terni (CT) are generated at thoracic levels of
the spinal cord, and settle in a dorsomedial position. For simplicity, the medial and lateral
divisions of the median motor column (MMC) are not shown. b, Schematic of a cross section of
the brachial regions of a stage 29 chick spinal cord showing the position of the LMC. The medial
MMC is indicated by a dashed oval. c, Cross section of thoracic region of a stage 29 spinal cord
showing position of CT neurons. Lateral and medial MMC divisions are indicated by dashed
ovals. d, Expression of molecular markers of motor neuron columnar identity. RALDH2 is
expressed at brachial levels by all LMC motor neurons and Isl2 and Lim1 are co-expressed by
lateral LMC neurons (arrowhead). BMP5 is expressed at thoracic levels by CT neurons, prior to
and after their dorsomedial migration. Images (mirrored in some panels) are modified from Ref.
18 and Ref. 20.
Supplementary Figure S2 Spatial congruence in the expression of molecular markers for motor
columnar identity and Hox-c factors within motor neurons. a, Longitudinal section of stage 24
chick embryos demonstrating expression of RALDH2 in brachial-level LMC neurons. Numbers
to the right of the spinal cord indicate the approximate somite position. Arrows indicate rostral
and caudal expression boundaries. b, Hoxc6+, Isl1+ motor neurons overlap with RALDH2
expression in the brachial LMC. c, Hoxc8 expression intersects the caudal half of the brachial
LMC and extends into thoracic levels. d, e, Hoxc9+, Isl1+ motor neurons overlaps expression of
BMP5 in CT motor neurons. Caudal to somite 23 expression of BMP5 falls outside of the plane of
the section.
Supplementary Figure S3 Effects of elevated FGF signaling on the spinal cord. a-d, Expression
of FGF8 does not significantly change the normal pattern of Hox-c expression at mid-thoracic
levels of the spinal cord. e-h, Thoracic FGF8 expression does not affect motor columnar identity.
i, j, Lim3 and BMP5 expression at stage 29 after brachial FGF8 electroporation at stage 12 k,l ,
Adjacent sections of a brachial level stage 29 chick embryo showing loss of both Hoxc6 protein
and Hoxc6 transcripts after FGF8 expression. Lateral MMC neurons are also generated
selectively at thoracic levels and can be distinguished from other motor neuron columns by
expression of Isl1/2 in the absence of RALDH2 and Lim3. After brachial FGF8 expression,
many laterally positioned Isl1/2+ motor neurons lacked RALDH2 and Lim3 expression (panels
S3f and Fig. 2k), suggestive of the ectopic differentiation of lateral MMC neurons.
Supplementary Figure S4 Cell autonomy and temporal aspects of FGF signaling on Hox-c
profiles and motor columnar identity. a-d, Motor neuron columnar differentiation after brachial
expression of a constitutively active form of the FGF receptor 1 (FGFR1*). e-g, Detection of diphosphorylated of MAP kinase epitope in neural tube cells, but not in adjacent paraxial
mesoderm, 8 h after unilateral FGF8 electroporation of stage 12 brachial neural tube. h-k,
Comparison of effects of brachial FGF8 expression at stage 12 and stage 17, analyzed at stage 29.
Supplementary Figure S5 Expression of Hox6 paralogs and Hoxa9 in the spinal cord. a-b,
Hoxa6 is expressed at brachial levels of the ventral spinal cord (st27) but not at thoracic levels.
Expression of Hoxb6 was not detected in brachial or thoracic motor neurons. c-d, Hoxa9 is
expressed at thoracic levels of the ventral spinal cord (st28) but not at brachial levels. Expression
of Hoxb9 or Hoxd9 was not detected in brachial or thoracic CT motor neurons. c-e, Expression of
Hox6 paralogs in stage 15 chick embryos.
Supplementary Figure S6 Specificity and mimicry in Hox function in motor neuron columnar
specification. a-c, Expression of murine Hoxa9/GFP in brachial neural tube represses RALDH2
expression (a) and generates dorsomedially-positioned Hoxa9+, Isl1+ motor neurons that express
BMP5 (arrows in b and c). Brachial Hoxa9 expression did not induce ectopic Hoxc9 expression.
d, e, Expression of chick Hoxa6 in thoracic neural tube induces RALDH2 expression (arrow in
e). Thoracic Hoxa6 expression did not induce Hoxc6, but repressed Hoxc9 expression and CT
differentiation (data not shown). f, Expression of Hoxc6 in rostral thoracic neural tube does not
repress Hoxc8 expression in motor neurons. g, Expression of chick Hoxc8 at rostral brachial
levels does not affect the expression of Hoxc6. h, i, Expression of Hoxc8 in rostral brachial
neural tube does not repress RALDH2 expression or induce RALDH2 expression at thoracic
levels. j, Expression of Hoxc8 in rostral brachial neural tube represses Hoxc5 expression.
Supplementary Figure S7 Expression of EnR-Hoxc8 fails to alter the specification of brachial
LMC motor neurons.a, Expression of EnR-Hoxc8 fails to repress Hoxc6 expression. b,
Expression of EnR-Hoxc8 at rostral brachial levels fails to repress LMC identity as determined by
the presence of Hoxc8+/RALDH2+ cells. c, Expression of EnR-Hoxc8 at rostral brachial levels
represses Hoxc5 expression.
Supplementary Figure S8 Brachial motor neurons project to sympathetic chain ganglia (SCG)
after induction of Hoxc9 by FGF8. a, Brachial FGF8 expression generated dorsomedial Hoxc9+,
Isl1+ MNs. b, Orthograde labeling of motor axons by expression of Hb9-GFP revealing axonal
projections to SCG. c-d, Expression Hb-GFP alone does not generate dorsomedial Hoxc9+, Isl1+
cells, nor do axons project to SCG neurons. GFP expression within SCG represent innervation by
resident thoracic-levels MNs from more caudal levels.