_ - Castillo2010p7002Development.pdf

507

RESEARCH ARTICLE

INTRODUCTION

The vertebrate central nervous system (CNS) forms along the

embryonic anteroposterior (AP) axis from the neural plate, a sheet

of ectodermal cells that invaginates and transforms into a cylindrical

neural tube. Along the dorsoventral (DV) axis, the closed neural tube

divides into sensory and motor domains in dorsal and ventral

regions, respectively, whereas interneurons distribute between

sensory and motor regions.

Spinal cord (SC) DV patterning occurs through a balance of

Sonic hedgehog signals emanating ventrally from the notochord/

floor plate, together with dorsally derived bone morphogenetic

protein (BMP) and Wnt from the roof plate (RP). These opposing

influences activate homeodomain and basic helix-loop-helix

transcription factors that set in motion competing programs of

ventral and dorsal neuron specification. In this model, neural

progenitors obtain positional information according to the

strength of the ventralizing and dorsalizing signals they receive

(Wilson and Maden, 2005).

Signaling via vitamin A-derived retinoic acid (RA) is one of the

mechanisms that shape the vertebrate neural tube. RA signaling

participates in four major developmental programs of the CNS:

neural differentiation, AP patterning, specification of

motoneurons and DV organization (Diez del Corral et al., 2003;

Muhr et al., 1999; Nieuwkoop, 1952; Novitch et al., 2003;

Sockanathan and Jessell, 1998; Wilson et al., 2004). RA produced

by paraxial mesoderm controls early programs of neural

differentiation and AP patterning, whereas mesoderm, together

with ventral SC-derived RA, influence motoneuron specification

(Vermot et al., 2005). This contrasts with the much less well

understood roles in DV organization that are performed by the RA

that is produced inside the dorsal neural tube (Wilson et al., 2004).

In the dorsal neural tube, RA synthesis is associated with the RP

(Berggren et al., 1999), a dorsal-most group of glial cells that

plays a pivotal role in the specification of adjacent dorsal

progenitors of sensory neurons and dorsal interneurons (dIs)

(Chizhikov and Millen, 2004).

In vertebrates, retinoid signaling is triggered by the synthesis

of RA by two enzyme families.

raldh1-3

(

aldh1a1

) are paralogs

that belong to the Aldh1a family of all-trans and 9-cis

retinaldehyde dehydrogenases, whereas

raldh4

(

aldh8a1

) is a

member of the Aldh8 family, which represents enzymes with

preference for 9-cis retinaldehyde (Simões-Costa et al., 2008).

Among Raldh genes,

raldh2

plays the most important

developmental role. Raldh2 displays the highest affinity for

retinaldehyde, is the first Raldh to appear in mouse and chick

embryos in early development, and is regulated in patterns that

overlap with the activation of RA signaling (Blentic et al., 2003;

Lin et al., 2003; Moss et al., 1998; Niederreither et al., 1997;

Ulven et al., 2000; Wang et al., 1996; Xavier-Neto et al., 2000;

Zhao et al., 1996).

Development 137, 507-518 (2010) doi:10.1242/dev.043257

Laboratório de Genética e Cardiologia Molecular, InCor-FMUSP, 05403-000, São

Paulo, Brazil.

Departamento de Biologia Celular e do Desenvolvimento, ICB-USP,

05508-000, São Paulo, Brazil.

European Molecular Biology Laboratory Mouse

Biology Programme, via Ramarini 32, Monterotondo-Scalo (RM), Italy.

Instituto de

Ciências Biomédicas, UFRJ, 21941-590, Rio de Janeiro, Brazil.

Laboratory of

Experimental Ontogeny, Nucleus of Neural Morphogenesis, Anatomy and

Developmental Biology Program ICBM, Faculty of Medicine Universidad de Chile,

Clasificador 7 – Correo 7, Santiago, Chile.

Division of Biology, California Institute of

Technology, Pasadena, CA 91125, USA.

Department of Human Genetics, University

of Chicago, Chicago, IL 60637, USA.

*These authors contributed equally to this work

†

Author for correspondence (

xavier.neto@incor.usp.br

)

Accepted 4 December 2009

SUMMARY

Comparative studies of the tetrapod

raldh2

(

aldh1a2

) gene, which encodes a retinoic acid (RA) synthesis enzyme, have led to the

identification of a dorsal spinal cord enhancer. Enhancer activity is directed dorsally to the roof plate and dorsal-most (dI1)

interneurons through predicted Tcf- and Cdx-homeodomain binding sites and is repressed ventrally via predicted Tgif homeobox

and ventral Lim-homeodomain binding sites.

Raldh2

and

Math1/Cath1

expression in mouse and chicken highlights a novel,

transient, endogenous

Raldh2

expression domain in dI1 interneurons, which give rise to ascending circuits and intraspinal

commissural interneurons, suggesting roles for RA in the ontogeny of spinocerebellar and intraspinal proprioceptive circuits.

Consistent with expression of

raldh2

in the dorsal interneurons of tetrapods, we also found that

raldh2

is expressed in dorsal

interneurons throughout the agnathan spinal cord, suggesting ancestral roles for RA signaling in the ontogenesis of intraspinal

proprioception.

KEY WORDS: Retinoic acid, Spinal cord, Roof plate, Commissural interneurons, Proprioception, Paired fin loss, Mouse, Chicken,

Xenopus

Zebrafish, Lamprey, Medaka

Insights into the organization of dorsal spinal cord pathways

from an evolutionarily conserved

raldh2

intronic enhancer

Hozana A. Castillo

1,2,

*, Roberta M. Cravo

1,2,

*, Ana P. Azambuja

1,2

, Marcos S. Simões-Costa

1,2

Sylvia Sura-Trueba

, Jose Gonzalez

, Esfir Slonimsky

, Karla Almeida

, José G. Abreu

Marcio A. Afonso de Almeida

, Tiago P. Sobreira

, Saulo H. Pires de Oliveira

, Paulo S. Lopes de Oliveira

Iskra A. Signore

, Alicia Colombo

, Miguel L. Concha

, Tatjana S. Spengler

, Marianne Bronner-Fraser

Marcelo Nobrega

, Nadia Rosenthal

and José Xavier-Neto

1,†

DEVELOPMENT

508

Here we identify a conserved intronic enhancer that drives

raldh2

expression in the RP and dIs of the frog, mouse and

chicken dorsal SC, suggesting that DV programs of neural tube

development regulated by RA are encoded by evolutionarily

conserved cis-regulatory modules. The combined activation of

raldh2

in the RP and dIs by a single enhancer suggests that these

two RA signaling domains play related roles and this prompted us

to utilize a comparative approach to gain insight into these

functions. By investigating the patterns of

raldh2

expression in

the SC of agnathan, teleost and tetrapod embryos, we discovered

a novel, transient, field of RA synthesis in dI precursors. By

comparing the patterns of

raldh2

expression in agnathans and

teleosts with those of amniotes, we provide evidence that RA

signaling might be involved in the ontogeny of two specific SC

sensory functions: proprioception from vertebrate paired

appendages and modulation of the intrinsic SC locomotor

circuitry.

MATERIALS AND METHODS

Bioinformatics

Aldh gene sequences were obtained from NCBI, the Ensembl genome

browser, JGI Eukaryotic Genomes and through the method of Sobreira and

Gruber (Sobreira and Gruber, 2008). Conserved non-coding elements

(CNEs) were identified using the ECR Browser and BLAST 2 sequences.

The CNE search was limited to between 30 kb upstream of the transcription

start site and 30 kb downstream of the stop codon. Transcription factor

binding site (TFBS) prediction was performed using a locally available

software that implements the MatInspector algorithm (Quandt et al., 1995)

with TFBS matrices from TRANSFAC 6.0 (Wingender et al., 1996).

TFBSs were accepted if at least 90% similar to a matrix core/whole matrix

score.

Phylogenetic analysis

Thirty Aldh protein sequences from six vertebrate species ranging from

agnathans to primates were aligned using MUSCLE (Edgar, 2004).

The alignment (available on request) consisted of 464 amino acid

positions, manually refined to eliminate gaps. Phylogenetic trees were

generated using neighbor-joining (NJ) (Saitou and Nei, 1987), maximum

parsimony (MP) (Swofford, 2000), maximum likelihood (ML) (Schmidt

et al., 2002) and Bayesian inference (BI) (Ronquist and Huelsenbeck,

2003) methods. The WAG model was selected by ProtTest (Abascal et al.,

2005). Node support was assessed in NJ and MP trees by 2000 bootstrap

replicates. ML was performed with 100,000 puzzling steps. For BI

we used two runs of 5,000,000 generations. Convergence was verified

and an appropriate burn-in period of 2000 was determined. Consensus

trees and posterior probabilities were calculated using the 50% majority

rule.

Plasmids and constructs

raldh2

intron 1G CNEs from mouse, chicken and

X. laevis

were PCR

amplified from genomic DNA and cloned into HSP68-lacZ and pTkeGFP

vectors (Kothary et al., 1989; Rossant et al., 1991).

Mutagenesis

Nested deletion mutants of the chicken

Raldh2

intron 1G enhancer with

734 bp, 450 bp, 355 bp and 149 bp fragments, as well as a 279 bp fragment

containing RP and interneuron activator elements (RPIE), plus

overlapping fragments A (62 bp), B (91 bp), C (92 bp) and D (114 bp),

were PCR amplified and cloned into pTkeGFP. For site-directed

mutagenesis we utilized QuickChange II (Stratagene, 200524).

Nucleotides belonging to a predicted binding site for the Lim-

homeodomain protein Lhx3 (116-126 bp) in the enhancer were substituted

by an

Asc

I restriction site. The Tcf/Cdx site (207-221 bp) was substituted

by an

Asc

I restriction site. A second Tcf/Cdx motif (353-367 bp) was

substituted by an

Fse

I restriction site. A Tcf/Tgif site (427-441 bp) was

substituted by an

Asc

I restriction site. All constructs were confirmed by

DNA sequencing.

Transgenic mice

The mouse

Raldh2

intron 1G CNE-HSP68-lacZ reporter was excised by

Sal

I digestion. Pronuclear injection was as described (Xavier-Neto et al.,

1999).

Electroporation

Enhancer-eGFP plasmid (2

m

g/ml) plus pCA



-RFP DNA (0.5-1.0

m

g/ml) in

0.5% Fast Green in water were injected into the lumen of the chicken neural

tube (HH17-18). In ovo electroporation was performed by applying six 60-

to 100-millisecond pulses at 15-18 V with a 0.5 mm platinum electrode.

Xenopus laevis

embryo injections

The

Xenopus

intronic enhancer, or the negative control pTkeGFP plasmid

(30 pg), were co-injected with



-galactosidase mRNA (250 pg total) at the

4-cell stage into both dorsal blastomeres. Injections were performed at the

animal pole ~45° from the top.

Staining and in situ hybridization

Stains were performed as described previously (Xavier-Neto et al., 1999)

using an anti-GFP rabbit polyclonal antibody (1:500; Abcam, ab6556) and

donkey anti-rabbit IgG conjugated with Alexa Fluor 488 (1:1000; Molecular

Probes, A21206). In situ hybridization (ISH) (Wilkinson, 1992) and double

ISH (Stern, 1998) were preformed as described previously, using mouse

(Zhao et al., 1996), chicken,

Xenopus

(Chen et al., 2001), zebrafish (via

Deborah Yelon, New York University School of Medicine, New York, USA)

and lamprey

raldh2

probes. Other probes included lamprey

zic-1

(Sauka-

Spengler et al., 2007), mouse

Math1

(Helms and Johnson, 1998) and chicken

Cath1

(Wilson and Wingate, 2006). Lamprey

raldh2

was cloned by low-

stringency hybridization with a

P-labeled zebrafish

raldh2

probe (Sauka-

Spengler et al., 2007). The lamprey

raldh2

sequence was deposited in

GenBank as FJ536260. An in situ probe for medaka

raldh2

was PCR

amplified using medaka cDNA.

RESULTS

raldh2

intron 1G is a conserved non-coding

element enriched with highly conserved

transcription factor binding sites

To identify conserved non-coding elements (CNEs) potentially

carrying

raldh2

regulatory sequences, we aligned

raldh2

orthologs

from vertebrates including fugu, zebrafish, frog, chick and mouse,

using human as baseline. This analysis revealed 72 CNEs

displaying more than 75% identity over 183±16 bp (range 32-905

bp) in 5

, intronic and 3

regions (Fig. 1). To identify sequence

modules that regulate

raldh2

expression, we screened vertebrate

raldh2

CNEs for enhancer function. We selected three CNEs: a 5

and a 3

CNE conserved in all species (Fig. 1, gray arrowheads),

plus

raldh2

intron 1G, the largest

raldh2

CNE (Fig. 1, red

arrowhead). Of those, only mouse

Raldh2

intron 1G displayed

enhancer activity in 10.5 days post-coitum (dpc) transient

transgenic mice.

raldh2

intron 1G is conserved in amphibians,

avians, rodents and primates and spans an average of 843±47 bp

(702-905 bp) (Fig. 1, red box; see Fig. S1 in the supplementary

material), but could not be detected in teleosts (see Fig. S2 in the

supplementary material).

For a list of transcription factor binding sites (TFBSs) that display

matrix identities to the matrix core that are higher than 90% and that

are conserved across amphibian, avian, marsupial, rodent and primate

raldh2

intron 1G enhancers, see Fig. S3 in the supplementary material.

There is deep conservation for TFBSs associated with Wnt signaling

(i.e. Tcf binding sites), for homeodomain and Lim-homeodomain

factors, and for factors such as Pax, Pou and basic helix-loop-helix.

Some predicted sites for Forkhead factors, Vsx2 (Chx10), Lhx3,

Pou3f1 and Klf4 are so rare that their presence in the enhancer reflects

a statistically significant event when compared with their occurrence

in a billion-bp random set.

RESEARCH ARTICLE

Development 137 (3)

DEVELOPMENT

The mouse

Raldh2

intron 1G CNE directs a dorsal

subset of the

Raldh2

expression domain in

transgenic mice

Transient transgenic mice revealed transcription driven by the

mouse

Raldh2

intron 1G CNE in embryonic tissues within the

endogenous domains of

Raldh2

expression (compare Fig. 2A-D

with 2E).



-galactosidase activity was observed in the dorsal neural

tube, running from the brachial level down to the tail bud of 10.5 dpc

founder (F0) embryos (Fig. 2A-D). Age-matched embryos

processed for

Raldh2

in situ hybridization (ISH) indicated that the

intron 1G CNE directs a dorsal-most subset of the endogenous

Raldh2

expression pattern in the posterior embryonic quarter (Fig.

2E). Two stable transgenic lines, #191 and #583, confirmed that the

neural tube is a prime target for this mouse

Raldh2

intron 1G

enhancer.

The intron 1G enhancer mirrors

Raldh2

expression

in the mouse dorsal spinal cord, but some

features of its activity differ from the

endogenous gene

The

Raldh2

intron 1G enhancer regulates subsets of the endogenous

Raldh2

expression domain, and some aspects of its activity differ

from the endogenous gene. For example, stable intron 1G transgenic

mouse lines display an early

lacZ

domain at the midbrain/hindbrain

boundary (see Fig. S4 in the supplementary material). The midbrain/

hindbrain region contains cerebellum and tectum progenitors that

normally activate

Raldh2

, but only from 15.5 dpc onwards (Zhang

et al., 2003). Thus, it is possible that the two stable transgenic mouse

lines (#191 and #583) display heterochronic acceleration of an

endogenous

Raldh2

domain (see Fig. S4G-I in the supplementary

material), suggesting that some important

Raldh2

repressors are

missing from the intron 1G CNE. Therefore, the intronic enhancer

is not the sole regulator of

Raldh2

expression in all tissues, but drives

Raldh2

in the developing dorsal SC, which we validated as a natural

domain of mouse

Raldh2

expression (see below).

The mouse

Raldh2

intron 1G enhancer activates

expression in the roof plate and dorsal

interneurons of the spinal cord

The mouse

Raldh2

intron 1G enhancer activates

lacZ

in the neural

tube of transgenic mouse embryos (Fig. 2A-D). The major territory of

enhancer activation is a broad domain of the dorsal SC (Fig. 2F).

Brachial sections of 10.5-11.5 dpc mouse embryos revealed intense



-galactosidase activity in the RP (

Fig. 3B,F, arrowhead). From the

RP, a second field of



-galactosidase was found in dI1 interneuron

progenitors (Fig. 3B,F, arrow). These progenitors migrate ventrally,

giving rise to neurons that receive proprioceptive afference and project

to the cerebellum and to commissural interneurons (CIs), which cross

the floor plate and project to the contralateral SC (Bermingham et al.,

2001; Helms and Johnson, 1998; Lewis, 2006). A trail of diffuse



galactosidase activity in the mantle region highlighted the ventral

migration of dIs (Fig. 3B-F, asterisk), similar to the expression of a

Math1

(

Atoh1

) transgene and of

Dcc

and

Cntn2

(

Tag-1

) (Bermingham

et al., 2001; Dodd et al., 1988; Furley et al., 1990; Helms and Johnson,

1998; Keino-Masu et al., 1996). Ventrally,



-galactosidase activity

was found in CI axonal tracts (Fig. 3B-F, white arrows), reminiscent

Math1

-expressing CIs (Altman and Bayer, 1997; Helms and

Johnson, 1998).

Roof plate and interneuron functions are

conserved in tetrapod enhancers

To determine whether RP and dI enhancer functions are conserved

in amniotes and amphibians, we cloned chicken and

X. laevis raldh2

intron 1G CNEs. The chicken CNE drove eGFP expression in the

RP and dI1 (Fig. 3J,L). Expression of eGFP was also observed in dIs

migrating towards the ventral thoracic SC (Fig. 3J,L), as well as in

ventral axonal projections of CIs (Fig. 3L), reminiscent of

lacZ

patterns in transgenic mice (compare Fig. 3F with 3L). Injection of

the

X. laevis

enhancer reporter construct into

X. laevis

dorsal

blastomeres produced restricted eGFP expression in the RP and dIs

(Fig. 4A,B), indicating that the dorsal SC function associated with

the enhancer has been conserved for at least 370 million years since

the divergence of amphibians and amniotes (Hedges and Kumar,

2003).

509

RESEARCH ARTICLE

Spinal cord organization from an

raldh2

enhancer

Fig. 1. Evolutionary conservation of

raldh2

Five

raldh2

orthologs

(fugu, zebrafish, frog, chick and mouse) were aligned using

Homo

sapiens

as baseline. Vertical bars, conserved non-coding elements

(CNEs). The red box highlights

raldh2

intron 1G conservation.

Arrowheads indicate the three CNEs chosen for transient transgenesis.

Exons, blue bars. Introns, gray bars. 5

and 3

regions, violet bars. The

asterisk indicates the CNE conserved between chicken and human, but

lost from mouse.

Fig. 2. Mouse

Raldh2

intron 1G CNE activity in transient

transgenic mice.

(

A-D

)



The CNE drives

lacZ

expression (blue) at the

posterior dorsum in all (4/4) transient transgenic mice harvested at 10.5

dpc. (

)



This



-galactosidase field is a subset of the endogenous

Raldh2

expression domain, as indicated by

Raldh2

in situ hybridization (ISH).

(

)



Thoracic transverse section depicts dorsal spinal cord (SC) and dorsal

root ganglia enhancer activation. (

)



Brachial transverse section shows

Raldh2

expression in the roof plate (RP) and motoneurons (mn). Dashed

lines in A and E indicate the plane of the sections in F and G,

respectively.

DEVELOPMENT

510

In heterologous assays in chicken embryos, the mouse and

laevis

enhancers activated reporter transcription in the RP of the SC

(Fig. 4E-H), albeit with differences in domain extension when

compared with the chicken enhancer (Fig. 4C,D). Neither the mouse

nor the

X. laevis

enhancer was activated in chicken dIs (Fig. 4E-H),

suggesting that the RP function, rather than the dI role, has been

conserved across tetrapod enhancers.

The enhancer is activated by three

Tcf-homeodomain sites and is repressed by

Lim-homeodomain and Tgif sites

We generated deletion mutants of the chicken

Raldh2

intron 1G

enhancer and electroporated constructs into the chicken SC (Fig.

5A). Removal of the 5

144 bp did not interfere with RP or dI

expression, but led to eGFP expression throughout the SC,

indicating that repression of ventral interneuron expression was lost

in the 734 bp construct (Fig. 5B). Because this 5

144 bp region

contains a predicted Lim-homeodomain binding site that is

conserved from amphibians to primates, we used site-specific

mutagenesis to modify this sequence in the context of the full intron

1G enhancer. Electroporation of this Lim mutant resulted in eGFP

expression in SC domains ventral to the RP and dI1, indicating that

this sequence is one of the repressors in the 5

144 bp fragment (Fig.

5G).

Further removal of 279 bp from the 734 bp sequence abrogated

RP and interneuron expression in the 450 bp construct (Fig. 5C),

indicating that the cis-regulatory elements directing RP and

interneuron expression are contained in a 279 bp fragment. This

fragment, termed the RP/interneuron activator element (RPIE), was

isolated and shown to drive RP and interneuron expression

throughout the SC (Fig. 5H). The RPIE was divided into four

consecutive overlapping fragments, A-D, and we determined that

RP and interneuron activities reside in fragments B and D (Fig.

5J,L). To identify the cis-elements controlling RP and interneuron

expression, we aligned chicken and mouse B and D regions to detect

sequences displaying full conservation. These were then altered by

site-directed mutagenesis, creating one mutant for fragment B and

two mutants for fragment D (D1 and D2). Sites B and D1 contain

overlapping nested binding sites for the Wnt pathway transcription

factor Tcf and for the Cdx-homeodomain protein. These sites display

matrix identities to the matrix core that range between 86 and 90%

and are conserved across amphibian, avian, marsupial, rodent and

primate enhancers. Site D2 contains another, overlapping Tcf-

homeodomain binding site, this time for the repressor homeodomain

RESEARCH ARTICLE

Development 137 (3)

Fig. 3. The

Raldh2

intron 1G enhancer is a roof plate and

dorsal interneuron enhancer in amniotes.

(

) Dorsal views of

stable transgenic mice. (

)



Transverse sections at the brachial SC of

the stable transgenic mice shown in A and E, respectively. The

enhancer is active in the RP (arrowhead), in dorsal interneurons (DI)

(black arrow) and in ventrally migrating dorsal interneurons (DIm)

(asterisk).



-galactosidase expression is also observed in axons of

commissural interneurons (CI ax) (white arrow). (

) ISH shows

Raldh2

expression in the 10.5-11.5 dpc mouse brachial RP.

(C,G)



Dorsal views. (D,H)



Transverse sections. (

I-O

)



Enhancer activity is

conserved in chicken. (I)



Chicken thoracic SC 48 hours after

Raldh2

intron 1G electroporation. (J)



Section of the embryo in I showing RP

(arrowhead), dorsal interneuron (black arrow) and migrating dorsal

interneuron expression (asterisk). (L)



Chicken thoracic SC 72 hours

after

Raldh2

intron 1G electroporation showing enhancer activation

in the RP (arrowhead), dorsal interneuron (black arrow) and

migrating dorsal interneurons (asterisk), as well as reporter

expression in CI ax (white arrow). (K,O)



RFP expression (red) driven by

the chicken beta-actin promoter (positive control). (M,N)



eGFP

expression driven by the minimal

promoter. Dashed lines

indicate plane of section in adjacent panels. (

)



Scheme of enhancer-

driven expression in mouse and chicken embryonic SC.

mn, motoneurons.

Fig. 4. The

raldh2

intron 1G enhancer is a tetrapod dorsal spinal

cord enhancer.

(

)



Injection of the

Xenopus raldh2

intron 1G CNE

into

Xenopus laevis

blastomeres activates eGFP expression in the RP

(arrowhead) and in dIs (arrow) at NF25. (

C-H

)



Electroporation of chicken

embryos. Frog (E,F) and mouse (G,H) enhancers retain RP, but not

interneuron, activity in the electroporated chicken thoracic SC at HH25,

contrasting with robust activity of the chicken enhancer in the chicken

RP (arrowhead) and dIs (arrow) (C,D). The neural tube boundary is

outlined.

DEVELOPMENT

511

RESEARCH ARTICLE

Spinal cord organization from an

raldh2

enhancer

Fig. 5. Dissection of the chicken

Raldh2

intron 1G enhancer.

(

)



The

wild-type chicken enhancer drives RP and

dorsal-most interneuron expression.

(

)



Removal of the 5

144 bp expands

expression throughout the SC,

highlighting the presence of strong

inhibitors of ventral interneuron

expression. (

)



Further removal of 279 bp

abrogates RP and interneuron

expression, indicating that this fragment

contains a RP and interneuron activator

element (RPIE). (

)



Further removal of

95 bp (D) and 207 bp (E) does not reveal

relevant cis regulators. (

)



A minimal

promoter does not drive significant eGFP

expression. (

)



Mutation of a Lim-

homeodomain site within the 5

144 bp

repressor releases interneuron

expression. (

)



The isolated RPIE drives RP

and interneuron expression. (

I-L

)



RPIE

dissection into four overlapping

fragments (A-D) indicates that RP and

interneuron activities reside in fragments

B and D (J,L). (

)



Mutation of a double

Tcf/Cdx site in fragment B abolishes RP

and interneuron expression. (

)



Mutation

of a double Tcf/Cdx site in D1 abrogates

RP and interneuron expression.

(

)



Mutation of a double Tcf/Tgif site

(D2) does not change RP expression, but

expands expression into more-ventral

interneurons (arrows). (

P-R

)



Single

mutations in double Tcf/homeodomain

sites in the context of the full chicken

Raldh2

intron 1G enhancer. (P)



Tcf/Cdx

mutation limits RP expression and

restricts interneuron expression. (Q)



Tcf/Cdx mutation limits RP expression

and eliminates interneuron expression.

(R)



Tcf/Tgif mutation derepresses ventral

interneuron expression. (

S-U

)



Double

mutants B+D1, B+D2 and D1+D2 do not

eliminate RP expression. (

)



Dorsal SC

expression is eliminated in the triple

mutant. (

)



Model of

Raldh2

intron 1G

regulation in the SC. HD, homeodomain.

DEVELOPMENT

512

transcription factor 5

-TG-3

interacting factor (Tgif; also known as

TGFB-induced factor homeobox 1) (Knepper et al., 2006). These

binding sites display matrix identities of 90% and are conserved in

amphibian, avian, marsupial, rodent and primate enhancers. We

showed that RP and interneuron expression was eliminated when

fragments B and D were mutated at sites B and D1 (Fig. 5M,N),

indicating that they contain RP and dI activators. By contrast,

mutation of fragment D at site D2 (Fig. 5O) expanded expression

into more-ventral interneurons (Fig. 5O, arrows), suggesting that it

contains a repressor element.

Next, we mutated sites B, D1 and D2 in the context of the full

intron 1G enhancer. RP expression was not eliminated by individual

mutations (Fig. 5P-R), although dI expression was sharply restricted

in B and D1 mutants (Fig. 5P,Q). Interestingly, D2 mutants displayed

increased ventral interneuron expression, confirming that the

mutated Tcf/Tgif sequence contains an interneuron-specific

repressor (Fig. 5R). Complete inhibition of RP expression was not

observed in double mutants (Fig. 5S-U), but was achieved in the

triple mutant (B, D1 and D2), highlighting redundant mechanisms

of RP expression (Fig. 5V). Thus, the dorsal SC activity of the

chicken

Raldh2

intron 1G enhancer is achieved by three elements

that induce activation in the RP and dorsal-most interneurons, as

well as by the combined inhibition of ventral expression by at least

two repressors: a Lim-homeodomain site in the 5

144 bp fragment

and a Tcf/Tgif motif in D2 (Fig. 5W).

Enhancer activation in the spinal cord reveals a

novel transient domain of endogenous

Raldh2

expression in amniotes

Prompted by the expression patterns driven by the

Raldh2

intron 1G

enhancer, we re-examined the profiles of

Raldh2

expression in the

amniote SC. Besides being strongly expressed in the mouse RP, the

endogenous

Raldh2

domain was seen to extend ventrally in the 9.0-

10.5 dpc lumbar SC, reaching DV levels occupied by dI1 precursors

(Fig. 6E,G,H; Fig. 7R; see Fig. S5C in the supplementary material).

This novel

Raldh2

domain might have previously escaped detection

because of its transient nature. Indeed, at 11.5 dpc,

Raldh2

expression rapidly receded from interneuron precursors, remaining

highest in the RP (see Fig. S5E in the supplementary material,

arrowhead), although residual expression was observed as a faint

labeling in dI precursors (see Fig. S5E in the supplementary

material, bracket). In chicken,

Raldh2

expression initiated bilaterally

in dIs of the brachial SC (Fig. 6N). Later, expression receded in dIs,

becoming restricted to the RP (Fig. 6J, arrowhead; Fig. 7N,O,

arrowhead).

To directly demonstrate that

Raldh2

is expressed in SC dIs we

performed single and double ISH for

Raldh2

and

Math1/Cath1

(

Cath1

is the chicken homolog of mouse

Math1

), a dI1 marker. Fig.

6A,B display

Raldh2

and

Math1

interneuron domains along the

mouse SC and Fig. 6C shows that these two domains overlap at the

hindlimb level. Sections of the lumbar SC showed that

Raldh2

expression extends from the RP, overlaps with

Math1

expression

and extends beyond it, reaching interneurons ventral to the

Math1

domain (Fig. 6G,H and see Fig. S5D in the supplementary material).

In chicken,

Cath1

was expressed in dIs at the same AP level as those

that express

Raldh2

(Fig. 6J,K,N,O) and double-marker analysis

indicated that

Raldh2

and

Cath1

overlap in dI1 and that the

Raldh2

domain constitutes a medial, ventricular subset of the

Cath1

territory

(Fig. 6L,P,Q).

RA signaling is an ancestral mechanism associated

with dorsal interneuron ontogenesis in the

vertebrate spinal cord

To establish whether expression of

raldh2

in the RP and in dIs is

a tetrapod feature or an ancestral vertebrate trait, we cloned

raldh2

from the agnathan lamprey

Petromyzon marinus

(Fig. 7,

Fig. 8).

The

raldh2

identity of the lamprey clone was confirmed

by phylogenetic and expression analyses (Fig. 8) (M.S.S.-C.,

RESEARCH ARTICLE

Development 137 (3)

Fig. 6. A novel spinal cord

Raldh2

domain in dorsal interneurons.

(

)



Bilateral

Raldh2

expression in dIs of 10.5 dpc mouse (A) and chicken (J)

embryos. Arrows and arrowheads point to interneuron and RP domains, respectively.

Raldh2

expression in the chicken dorsal SC shifts from bilateral

interneuron fields at brachial levels to a single midline RP domain at cervical levels. (

)



Mouse

Math1

/chicken

Cath1

expression marks dorsal-most

(dI1) interneurons. (

)



Double ISH for

Raldh2

and

Math1/Cath1

indicates overlapping

Raldh2

and

Math1/Cath1

domains in dI1. (

)



Diagrams

depicting embryo position in A-C and J-L and section levels (dashed lines) in E-H and N-Q. (

)



Raldh2

expression in the 10.5 dpc mouse lumbar SC

from the RP (arrowhead) to adjacent dIs (arrow). (

)



Math1

expression labels mouse dI1 (arrow). Note the lack of staining in the RP (open

arrowhead). (

)



Double ISH for

Raldh2

(light blue) and

Math1

(dark blue). (

)



Enlargement of the boxed region in G.

Raldh2

expression spreads

from the RP (arrowhead) through the

Math1

domain (dark-blue arrow), emerging ventral to dI1 (light-blue arrow). (

)



Scheme of

Raldh2

and

Math1

expression in the mouse dorsal lumbar SC. (

)



Raldh2

expression in the HH18 chicken brachial SC.

Raldh2

is expressed in the ventricular zone of dIs,

but not in the RP (open arrowhead). (

)



Cath1

expression labels dI1 (arrow). Note the lack of staining in the RP (open arrowhead). (

)



Double ISH

for

Raldh2

(light blue) and

Cath1

(dark blue). Note the dorsal expression of

Raldh2

and

Cath1

and ventral expression of

Raldh2

in motoneurons.

(

)



Enlargement of the boxed region in P.

Raldh2

is expressed in a ventricular (medial) subset of the

Cath1

interneuron (dI1) domain. (

)



Scheme of

Raldh2

and

Cath1

expression.

DEVELOPMENT

unpublished). Lamprey embryos displayed strong

raldh2

activation throughout the early dorsal SC (Fig. 7A, Fig. 8C),

similar to tetrapods, which exhibit expression along most of the

dorsal SC (Fig. 7J,K,N,Q). Although regional AP activation of

raldh2

in the dorsal neural tube was similar in lampreys and

tetrapods (Fig. 7A,J,N,Q), there were DV differences. In

lampreys, activation of

raldh2

was observed in dIs, but not in the

RP (Fig. 7C; Fig. 8B,D,F). This selective activation of lamprey

raldh2

in dIs contrasts with expression of the dorsal marker

zic-1

in the lamprey RP and dIs, indicating that a RP is present in this

agnathan (Fig. 8G-L). Thus, the absence of

raldh2

expression in

the RP of limbless lampreys contrasts with

raldh2

expression in

the RP and, transiently, in the dIs of tetrapods (Fig. 7J-R). In

connection with this, we identified a short sequence homologous

to the tetrapod

raldh2

intron 1G enhancer among lamprey genome

traces (see Fig. S1 in the supplementary material). This sequence

(gnl|ti|1386265511) aligns to a stretch of 230 bp in the 3

region

of the

raldh2

intron 1G enhancer with 69.1% identity, and a

reciprocal BLAST search against the non-redundant database

identified the

H. sapiens RALDH2

intron 1G enhancer as a high-

score match for the lamprey sequence (score 60.8, E-value

3.0

–6

), supporting the idea that activation of RA signaling in

dIs is an ancestral vertebrate feature.

Comparative ontogeny of

raldh2

expression

suggests that RA functions in the innervation of

vertebrate appendages

In contrast to the conserved AP patterns of

raldh2

expression in

the agnathan and tetrapod SC, the teleost

D. rerio

exhibits RP

expression of

raldh2

only in a small region between the caudal

hindbrain and SC, adjacent to somites 2 to 3 (Skromne et al.,

2007). This restricted cranial domain of zebrafish

raldh2

expression corresponds to the AP levels at which the pectoral fin

buds form in the lateral mesoderm (Fig. 7E,F). Thus,

raldh2

expression in the zebrafish RP is restricted to this cranial area,

indicating posterior truncation of most of the SC domain (Fig.

7E,F). To establish whether this

raldh2

pattern is specific for

rerio

, or is a general teleost characteristic, we cloned medaka

(

Oryzias latipes

)

raldh2

and characterized its expression pattern

in carefully stage-matched embryos (Signore et al., 2009). As

shown in Fig. 7G-I, medaka embryos also display the restricted

cranial domain of

raldh2

expression at the level of the pectoral fin

buds. Therefore, the divergent patterns of

raldh2

expression in the

dorsal neural tube displayed by

D. rerio

and

O. latipes

are

consistent with our inability to find an ortholog of the tetrapod

raldh2

intron 1G enhancer in any of the sequenced teleost

genomes. Perhaps, another regulatory sequence drives the

divergent teleost

raldh2

domain. However, there is an important

parallel between the onset of

raldh2

expression in the dorsal SC

of chicken, zebrafish and medaka. In chicken and teleost embryos,

dorsal SC expression of

raldh2

begins in restricted AP domains

at the level of the forelimb/pectoral fin buds (Fig. 7E,H,M),

suggesting that

raldh2

expression in the dorsal SC is functionally

related to the innervation of vertebrate forelimbs/fins.

DISCUSSION

We describe an intronic enhancer of the

raldh2

gene that is activated

in the RP of the SC and adjacent dIs, consistent with activation of

endogenous

raldh2

in the RP and, transiently, in the amniote dI1

domain. Enhancer analysis suggests a model for

raldh2

regulation

in the developing neural tube whereby

raldh2

expression in the

caudal SC results from the interaction of positive influences,

513

RESEARCH ARTICLE

Spinal cord organization from an

raldh2

enhancer

Fig. 7. Ontogeny of

raldh2

expression in the vertebrate dorsal

spinal cord.

(

A-C

)



In the lamprey (

Petromyzon marinus

) embryo,

raldh2

is expressed in dIs (C, arrow), but not in the RP. (

D-F

)



raldh2

expression

in zebrafish embryos. (D) No

raldh2

expression is detected in the

zebrafish neural tube 24 hours post-fertilization (hpf), but at 31 hpf

raldh2

expression is detected in a small domain in the hindbrain/SC

transition at the pectoral fin bud level (E, asterisk; E,F, arrowheads).

(

G-I

)



raldh2

ISH in stage-matched medaka (

Oryzias latipes

) embryos

indicates onset of expression in the same hindbrain/SC domain as in

zebrafish (H,I). (

J-R

)



Frog (J-L), chicken (M-O) and mouse (P-R) embryos

express

raldh2

in the RP (arrowheads) and in dIs (arrows). (M-O)



Raldh2

expression starts in chicken interneurons at HH18, at the

forelimb bud level (M, arrows).

Raldh2

expression is restricted to the RP

in HH32 chicken SC (M-O). In frogs (J-L) and mice (P-R),

raldh2

initially expressed in RP and in dIs, but is subsequently restricted to the

RP. Insets show whole embryos, with plane of section indicated (dashed

line). Inset in M shows

Raldh2

staining (arrows) in chicken brachial

sections. Expression patterns are represented schematically on the right.

DEVELOPMENT

514

represented by an interaction with Cdx and Wnt signaling, and

inhibitory influences, represented by Lim-homeodomain and Tgif

factors. In this model, the intersection between Cdx and Wnt family

activation focuses

raldh2

expression to the dorsal SC and this dorsal

expression domain is refined through inhibition by Lim-

homeodomain and Tgif factors that are expressed ventral to the

dorsal-most interneuron domains (i.e. dI1), restricting

raldh2

expression to the RP and dI1 (Fig. 9) (Wine-Lee et al., 2004; Zhou

et al., 2003; Iulianella et al., 2003; Knepper et al., 2006; Sheng et al.,

1997).

The activity of the

raldh2

intron 1G enhancer

suggests a role for RA signaling in the

organization of dorsal spinal cord pathways

The

raldh2

intron 1G enhancer activates transcription in the dorsal

SC when dI identities are being determined and when the

ventralizing effects of vitamin A deprivation are pronounced

(Berggren et al., 1999; Wilson et al., 2004; Wilson and Maden,

2005). This suggests that the roles of retinoid signaling in DV

organization of the SC are played by transient, autocrine RA

signaling in dI progenitors and by ventral diffusion of the RP-

derived RA. This is consistent with the fact that dIs are within range

of activation by the RA diffusing from the RP or dI1, as established

by expansion of ventral SC markers in vitamin A-deprived avian

embryos (Wilson et al., 2004).

raldh2

expression suggests that paracrine RA

from the roof plate plays roles in spinocerebellar

proprioception

The onset of

raldh2

expression in the dorsal SC in register with

forelimb buds in chicken, and the restricted RP and dI expression of

raldh2

at the teleost pectoral fin level, suggest that

raldh2

expression

in the dorsal SC is associated with the formation of circuits that

convey sensory information from vertebrate fins/limbs. Because the

RP is glial and does not take part in neural circuits, it is likely that

RP-derived RA signaling plays a role in the development of the

RESEARCH ARTICLE

Development 137 (3)

Fig. 8.

raldh2

expression in the lamprey spinal cord.

(

A-F

)



Time course of

raldh2

SC expression in the lamprey embryo. SC

raldh2

expression

starts at embryonic day (ED) 6.5 in dIs (arrow in B), in a small anterior domain (arrow in A). At ED 8.5,

raldh2

expression extends to encompass the

whole AP axis of the embryonic SC (C, arrows), where it is restricted to dIs (D, arrow). Later (ED 10.5),

raldh2

expression is restricted to dIs of the

anterior SC domain (E,F, arrows). (

G-L

)



Expression of the dorsal marker

zic-1

labels RP and dIs throughout the lamprey neural tube, indicating that a

RP is present in lamprey (arrowhead) and highlighting the restriction of lamprey

raldh2

expression to dIs. (

M-P

)



Phylogenetic trees constructed from

the alignment of vertebrate Aldh protein sequences. Nodes with bootstraps inferior to 80%, 80%, 80% and 90% were collapsed in n

eighbor-

joining (NJ), maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) trees, respectively.

DEVELOPMENT

adjacent dI1 progenitors, which give rise to SC circuits and

ascending pathways (Bermingham et al., 2001; Helms and Johnson,

1998). Activation of the mouse

Raldh2

intron 1G enhancer and of

Raldh2

expression in the RP, coupled with transient

Raldh2

expression in dIs, suggests that RA signaling is involved in dI1

development. Math1-expressing dI1 interneurons migrate to ventral

locations in the gray matter, coming to rest between laminae VI and

VIII to form integrative centers that receive proprioceptive input

from fore and hindlimbs, as well as from trunk, neck and thorax,

relaying it to the cerebellum via dorsal and ventral spinocerebellar

tracts, which are depleted in

Math1

-null mice (Bermingham et al.,

2001; Bloedel and Courville, 1981; Brodal, 1981; Helms and

Johnson, 1998; Lewis, 2006). The integrative SC center that serves

the dorsal spinocerebellar system is Clarke’s nucleus. These

interneuron cells receive afferents from lower trunk and hindlimbs

and are concentrated in lamina VII, which also receives information

from posterior trunk, hindlimbs and forelimbs and projects to the

cerebellum via ventral spinocerebellar and rostral spinocerebellar

tracts (Bloedel and Courville, 1981; Brodal, 1981). Therefore, RP-

derived RA is poised to influence dI1 interneurons, which give rise

to proprioceptive spinocerebellar circuits.

raldh2

expression suggests that autocrine RA in

dorsal interneurons plays roles in crossed spinal

cord proprioception

The exclusive expression of

raldh2

in interneurons of the

developing lamprey SC provides clues to

raldh2

function in tetrapod

SC interneurons. The lamprey SC contains neurons that are

responsible for locomotion (Grillner, 2003). The basic SC locomotor

network is modulated by sensory signals from spinal stretch

receptors. During lamprey locomotion, segmental muscular

contractions on one side activate intraspinal stretch receptors on the

contralateral, distended side. These glycinergic stretch receptors

send axons to synapse with, and inhibit, pattern generator neurons

on the active side (Grillner et al., 1984). The regular activity of these

crossed inhibitory interneurons is crucial for motor coordination

during locomotion and it is likely that crossed circuits of this sort are

present in most vertebrates (Grillner, 2003). The expression patterns

directed by the

raldh2

intron 1G enhancer in the tetrapod dorsal SC

suggest that RA signaling serves a regulatory network that supports

the development of CIs belonging to sensory pathways that are akin

to the crossed inhibitory pathway of lampreys. A subset of these CIs

descends from

Math1

-expressing dI1 progenitors that are

underrepresented in the SC of

Math1

-null mice (Bermingham et al.,

2001). As demonstrated in Figs 3 and 6 and Fig. S5 in the

supplementary material, activation of the

Raldh2

intron 1G enhancer

and of

Raldh2

at the early stages of dI development supports a role

for RA signaling in dI1 ontogeny. Moreover,



-galactosidase activity

driven by the mouse

Raldh2

intron 1G enhancer specifically labels

CIs, indicating that the enhancer is also activated in commissural

progeny of dI1 (Fig. 3F), consistent with demonstrations of cellular

RA-binding protein (Crabp) expression in these cells (Colbert et al.,

1995; Maden et al., 1989).

Proprioceptive pathways from vertebrate

fins/limbs: RA signaling and the simplification of

teleost fins

When contrasted with agnathan and tetrapod dorsal SC domains,

the diminutive

raldh2

cranial domain of zebrafish and medaka

suggests that the absence of the

raldh2

intron 1G enhancer in

teleosts is secondary to regulatory simplifications associated with

anatomic reductions/losses of fins in these actinopterygians

(Davis et al., 2007; Freitas et al., 2007; Santini and Tyler, 2003;

Shapiro et al., 2004; Tanaka et al., 2005). Zebrafish fins are filled

with dermal rays and have only small proximal radials, which

provide support for actuation by only two muscles that adduct or

abduct the fin (Thorsen and Hale, 2007). Moreover, teleost

pelvic fins are lost in freshwater populations of stickleback

(

Gasterosteus aculeatus

), or lost outright in

Tetraodon

and fugu,

contrasting with the fins/limbs of basal actinopterygeans,

sarcopterygeans and tetrapods that display complex endochondral

bone elements, tendons and articular surfaces innervated with

afferents conveying information about position, muscular

contraction and tendon tension to the SC (Kandel et al., 1991).

515

RESEARCH ARTICLE

Spinal cord organization from an

raldh2

enhancer

Fig. 9. Model for dorsal spinal cord

raldh2

regulation.

(

)



Cdx genes are expressed throughout the posterior SC. (

)



Wnt genes are

expressed in the RP throughout the anterior neural tube and the SC. (

)



Combined expression of Lim-homeodomain genes, such as Lhx1-3,9 and

Tgif, define a SC domain ventral to the RP and dorsal-most interneurons. (

)



Intersection of positive (Cdx and Wnt) and negative Lim-

homeodomain and Tgif regulators of the

raldh2

intron 1G enhancer in the embryo (G) and SC (H). (

)



raldh2

intron 1G enhancer and endogenous

raldh2

domains in the SC are defined by a positive layer of regulation due to Cdx and by autocrine and paracrine activation by the Wnt

pathway in

the RP and adjacent dIs. Enhancer and gene expression domains are refined through inhibition via Tgif homeoboxes and ventrally

expressed

repressors such as Lim-homeodomain (HD) transcription factors. The dashed line in the top row indicates the plane of section in

the bottom row.

DEVELOPMENT

516

The complex traffic of motor and sensory information to and from

the muscular limbs of cartilaginous fish, basal actinopterygeans

and sarcopterygeans is associated with a higher number of nerves

servicing these appendages than in the simpler fins of

D. rerio

(Thorsen and Hale, 2007). Therefore, it is possible that the

volume of information from sarcopterygean limbs requires a

denser network of SC interneuron circuits and ascending fibers

than is required for the comparatively simpler teleost fins.

Afferent information from amniote limbs is routed to SC

segments level with the emergence of fore/hindlimbs, where they

are grouped as Clarke’s nucleus. However, a great deal of limb

afferent information is also distributed to SC segments above or

below the limb buds (Brodal, 1981). As such, amniotes display

an extensive SC interneuron column, termed Clarke’s column,

which is distributed along neck, trunk and lumbar segments. The

restriction of RP

raldh2

expression to a cranial SC domain that

is level with teleost pectoral fin buds brings into question the

existence of a teleost homolog of the amniote Clarke’s column.

It is possible that the relatively small amount of afferent

information from teleost fins is handled exclusively by SC

segments in register with, or adjacent to, the fins, and for this

reason no structure homologous to the Clarke’s column has been

reported in teleosts. The distribution of SC nuclei is indeed

plastic in vertebrates and new spinal nuclei/columns are formed

when sensory input from peripheral receptors increases after the

emergence of complex peripheral structures, or novel sensory

capabilities, as indicated by the development of specific SC

nuclei/columns associated with the evolution of chemosensation

in the pectoral fins of the teleost northern sea robin (

Prionotus

carolinus

) (Finger, 2000). In summary, the absence of an

raldh2

intron 1G enhancer and of

raldh2

expression throughout most of

the SC in zebrafish and medaka are consistent with the loss of

cis and trans factor components of regulatory networks

associated with the simplification of teleost fins (Hildebrand and

Goslow, 1998; Shapiro et al., 2004; Tanaka et al., 2005). This is

further supported by the absence of expression of

raldh

paralogs

in the embryonic teleost SC (Liang et al., 2008; Pittlik et al.,

2008).

The ontogeny and phylogeny of RA signaling in

the dorsal spinal cord

The

raldh2

intron 1G enhancer is a module that controls RA

signaling in dIs and RP. This module is ontogenetically and

phylogenetically associated with the development of two

proprioception modes: one launched by the early activation of

autocrine RA signaling in dI progenitors and linked to the

emergence of intraspinal proprioceptive circuits responsible for

motor coordination across both sides of the SC during locomotion

(Fetcho, 1992); and another represented by a later paracrine

signaling from the RP to a subset of interneuron progenitors that

is linked to the development of spinocerebellar neural circuits

conveying fin/limb proprioception. The first mode is probably

older and might trace back to the ancestral chordate, presumably

a finless cephalochordate-like animal capable of bending its body

to swim, whereas the second mode probably evolved with the

appearance of vertebrate paired appendages. Thus, RA signaling

is an ancestral mechanism of DV organization of the SC that

seems to be plastic, allowing for changes in genetic regulation

associated with the evolution of the diverse locomotor patterns

and morphologies of vertebrate paired appendages (Goulding,

2009). In this sense, it is likely that the lack of

raldh2

expression

in the lamprey RP is a derived feature. Although lampreys

display ancestral vertebrate characteristics,

Haikouichthys

Myllokunmingia

and other agnathan fossils indicate that primitive

vertebrates already had prototypes of bilateral fins represented by

a pair of continuous mediolateral fin-folds spanning the AP axis,

implying that fin absence in extant agnathans is a derived feature

of lampreys (Forey, 1995). Thus, it is possible that

raldh2

expression was lost from the RP owing to a secondary loss of

paired fins in lampreys (Forey, 1995).

A comparative approach to understanding

signaling in spinal cord development and

evolution

Combining comparative genomic and developmental methods is a

fruitful approach to investigating the ontogeny/phylogeny of

developmental mechanisms that are controlled by signaling

systems in the vertebrate CNS. A considerable amount of non-

coding sequence conservation between distantly related

vertebrates is represented around genes that play essential roles in

CNS and heart development (Woolfe et al., 2005), organs that

harbor key vertebrate-specific innovations (Gans and Northcutt,

1983; Simoes-Costa et al., 2005; Xavier-Neto et al., 2007). Thus,

it is feasible to search for core, conserved vertebrate

developmental gene regulatory networks that can be used, in

selected cases, to infer how specific vertebrate adaptions have

emerged and to understand how class-specific vertebrate body

plans differ from each other.

Acknowledgements

We are indebted to Ursula Dräger, Peter McCaffery and Marcus Vinicius Baldo

for comments and suggestions; to Richard Behringer and Wellington Cardoso

for comments on the manuscript; to Masanori Uchikawa and Jane Johnson for

reagents; and to the Faculty of Medicine of the University of São Paulo for

access to its high-performance computing cluster. This work was supported by

grants from FAPESP (02/11340-2; 04/11569-5; 04/15704-4; 05/60637-6;

06/50843-0; 06/61317-8), CNPq 305260/2007-3 and by a Development

Travelling Fellowship from The Company of Biologists.

Competing interests statement

The authors declare no competing financial interests.

Supplementary material

Supplementary material for this article is available at

http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.043257/-/DC1

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