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Developmental Biology 2

Differential susceptibility of midbrain and spinal cord patterning to

floor plate defects in the talpid2 mutant

Seema Agarwala a,*, Galina V. Aglyamova a, Amanda K. Marma b,

John F. Fallon c, Clifton W. Ragsdale b

a Section of Neurobiology, University of Texas at Austin, Austin, TX 78712-0248, USAb Department of Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, IL 60637-1431, USA

c Department of Anatomy, University of Wisconsin at Madison, Madison, WI 53706-1510, USA

Received for publication 12 July 2005, revised 13 September 2005, accepted 14 September 2005

Available online 21 October 2005

Abstract

The chick talpid2 mutant displays polydactylous digits attributed to defects of the Hedgehog (HH) signaling pathway. We examined the talpid2

neural tube and show that patterning defects in the spinal cord and the midbrain are distinct from each other and from the limb. Unlike the Sonic

Hedgehog (SHH) source in the limb, the SHH-rich floor plate (FP) is reduced in the talpid2 midbrain. This is accompanied by a severe depletion

of medial cell populations that encounter high concentrations of SHH, an expansion of lateral cell populations that experience low concentrations

of SHH and a broad deregulation of HH’s principal effectors (PTC1, GLI1, GLI2, GLI3). Together with the failure of SHH misexpression to

rescue the talpid2 phenotype, these results suggest that talpid2 is likely to have a tissue-autonomous, bidirectional (positive and negative) role in

HH signaling that cannot be attributed to the altered expression of several newly cloned HH pathway genes (SUFU, DZIP1, DISP1, BTRC).

Strikingly, FP defects in the spinal cord are accompanied by relatively normal patterning in the talpid2 mutant. We propose that this differential FP

dependence may be due to the prolonged apposition of the notochord to the spinal cord, but not the midbrain during development.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Sonic Hedgehog; Midbrain; Spinal cord; Floor plate; Motor neurons; Dopaminergic neurons; Notochord

Introduction

The role of the Hedgehog (HH) signaling cascade in

directing cellular diversity and patterning in the embryo is

well established. The HH pathway has been implicated in the

specification of a wide array of embryonic systems ranging

from segment patterning in Drosophila, limb development, the

establishment of left–right asymmetry and dorsoventral pat-

terning in the vertebrate neural tube (Ingham and McMahon,

2001). In humans, misregulation of the HH pathway causes

congenital defects (spina bifida, cyclopia, holoprosencephaly,

limb polydactyly) and can lead to various types of tumors of

the skin and brain (basal cell carcinomas, medulloblastomas,

glioblastomas; McMahon et al., 2003; Roessler et al., 1996;

Villavicencio et al., 2000). Given its importance in develop-

0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.ydbio.2005.09.034

* Corresponding author.

E-mail address: [email protected] (S. Agarwala).

ment and disease, HH signal transduction and its modulation

have received intense scrutiny.

The core HH signaling machinery appears to be conserved

across species and embryonic tissues (Aza-Blanc et al., 2000;

Hooper and Scott, 2005; Nybakken and Perrimon, 2002). The

HH ligand is bound by a 12-pass transmembrane receptor

protein, Patched (PTC; Marigo et al., 1996a,b; Stone et al.,

1996). In the absence of the HH ligand, PTC inhibits the

activity of the 7-pass membrane protein Smoothened (SMO),

blocking HH pathway activation (Akiyama et al., 1997; Alcedo

and Noll, 1997). The intracellular signal transduction beyond

these key events is complex and not fully understood, but the

activation of HH target genes is dependent upon a single

effector, cubitus interruptus (Ci) in flies, and upon three Ci

homologs in vertebrates, GLI1, GLI2 and GLI3 (Methot and

Basler, 2001).

In Drosophila, Ci forms a cytoplasmic complex with the

kinase Fused and Costal2 (Cos2) and is processed into a

truncated repressor when levels of HH signaling are low (Aza-

88 (2005) 206 – 220

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S. Agarwala et al. / Developmental Biology 288 (2005) 206–220 207

Blanc et al., 1997; Lum et al., 2003). This proteolytic step is

mediated sequentially by protein kinase A (PKA), glycogen

synthase kinase 3h and casein kinase 1, eventually leading to

the degradation of the carboxy-terminal end of Ci/GLI and the

formation of a Ci/GLI repressor (Jiang, 2002). In the presence

of HH, cleavage of Ci/GLI is inhibited, and the full-length

activator form of the Ci/GLI predominates (Ohlmeyer and

Kalderon, 1998). Modulation of the GLI gene family by

nucleo-cytoplasmic shuttling is also provided by Suppressor of

Fused (SUFU) and the newly identified zinc finger transcrip-

tion factor iguana (DZIP1; Kogerman et al., 1999; Sekimizu et

al., 2004; Wolff et al., 2004). Paradoxically, a number of genes

involved in the production of the Ci repressor (e.g., Cos2,

PKA) have additional roles as positive modulators of HH

signaling (Jia et al., 2004; Zhang et al., 2005). Recent mutant

analysis in the fly has revealed several new contributors to the

HH signaling cascade, a number of which (e.g., Slimb, Nedd8,

Cul1 and 3, Roc1) are dedicated to the proteolysis of GLI

proteins and the modulation of GLI function (Jiang, 2002).

Despite their importance in the HH signaling cascade,

vertebrate counterparts of these genes have yet to be described.

In this paper, we have examined the autosomal recessive

chick talpid2 mutant where defects in GLI protein modulation

are seen (Wang et al., 2000). Phenotypic defects in talpid2

include decreased interocular distance with occasional cyclo-

pia, defects in craniofacial development and, most strikingly,

the presence of polydactylous limbs that exhibit aberrant digit

size and identity (Dvorak and Fallon, 1991; Schneider et al.,

1999). Thus, although the molecular basis of the talpid2

mutation is as yet uncharacterized, the overall talpid2

phenotype is consistent with a defect in the Hedgehog (HH)

signaling cascade. Recent studies in the talpid2 limb have

further suggested a broad constitutive activation of the

Hedgehog signaling machinery (Caruccio et al., 1999).

Biochemical studies have confirmed this idea by showing that

a disruption in the ratio of the GLI3 repressor and activator

across the limb may account for the observed supernumerary

preaxial digits seen in the talpid2 (Litingtung et al., 2002;

Wang et al., 2000). In this report, we have studied the effects of

the talpid2 mutation on neural tube development, with a focus

on neuronal patterning defects, and the expression of a battery

of HH pathway components including the HH ligand, GLI

transcription factors and a panel of mediators including PTC1,

SUFU, DZIP1, DISP1, HHIP and BTRC.

We have found that the talpid2 mutation produces a unique

and complex phenotype in the neural tube. Unlike the SHH-

rich zone of polarizing activity of the limb, the SHH-rich floor

plate (FP) is markedly reduced in size. In the midbrain, cell

types found close to the ventral midline are reduced in number,

and those arising at a distance from the FP are expanded and

inappropriately distributed. PCT1, GLI1, GLI2 and GLI3 are

all upregulated in a manner consistent with an increased range

and reduced magnitude of HH signaling. Taken together with

the craniofacial, eye and limb phenotypes, our results suggest

that talpid2 is likely to be a novel component of the vertebrate

HH signaling cascade capable of both positive and negative

regulation (Caruccio et al., 1999; Jia et al., 2004; Litingtung et

al., 2002; Park et al., 2000; Schneider et al., 1999; Wang et al.,

2000; Zhang et al., 2005).

Strikingly, we found profound rostrocaudal differences in

the effects of talpid2 on dorsoventral neuronal patterning: in

the spinal cord, although the FP is also compromised, neuronal

fate specification appears relatively normal. Our results thus

provide evidence that proper cell fate specification requires an

intact FP in the midbrain but not in the spinal cord, where the

proximity of the notochord may compensate for disrupted floor

plate development (Jeong and McMahon, 2005).

Materials and methods

Chick embryos

Fertilized eggs were obtained from talpid2 flocks maintained at the

University of Wisconsin, Madison and the University of California, Davis. All

eggs were incubated at 38-C in a humidified forced draft chamber and were

staged according to Hamburger and Hamilton (1951). Mutant embryos were

seen in Mendelian ratios and identified by the shape of their limb buds at

embryonic day 4 (E4)–E6. As we characterized the mutant midbrain, we

identified diagnostic gene expression defects that then allowed us to recognize

mutant embryos at earlier stages by in situ hybridization. A comparison of the

Wisconsin and Davis flocks with gene expression in the midbrain showed that,

in general, the talpid2 phenotype of the Wisconsin flock was slightly more

severe compared to the Davis flock. However, since the phenotype was

qualitatively similar, observations from both flocks were pooled. Figs. 1E, F

and 2B were drawn from in situ hybridizations done on Wisconsin embryos,

with the remaining data derived from embryos of the Davis flock.

In ovo electroporation

Protein-coated implants in a target tissue result in the delivery of variable

and declining protein concentrations over time, releasing >50% of their protein

within the first 24 h following implantation (Fallon et al., 1994). By contrast,

when delivered by electroporation, ectopic expression continues for at least 4

days (Agarwala and Ragsdale, 2002; Fig. 2). To obtain a prolonged and

continuous source of SHH, micro-electroporation was employed to introduce

the SHH-expressing plasmid pXeX-SHH into the midbrain of talpid2 embryos

and controls (Agarwala et al., 2001; Agarwala and Ragsdale, 2002; Momose et

al., 1999). Fifty to 250 nl of plasmid DNA solution (1 Ag/Al in 10 mM Tris, 1

mM EDTA, 0.02% Fast Green) was injected through a glass capillary needle

into the midbrain vesicle of stages 10–15 chick embryos displayed by

windowing. Electrodes were held at a distant of 1 mm, and 3 electric pulses (50

ms, 7 V) were delivered. The embryos were returned to the incubator for 3–4

days before harvesting. Our conclusions are based on an analyses of 41

successfully electroporated embryos (n = 34 control, n = 7 talpid2).

Cloning of HH pathway genes

Chicken HH pathways gene fragments for BTRC, DISP1, DZIP1, HHIP

and WNT11 were isolated by PCR from E7 chick ventral midbrain cDNA and

subcloned into pCR-TOPO (Invitrogen). Primers were designed with Mac

Vector 7.2 (Accelrys) based on sequences culled from BLAST searches of chick

EST and genomic databases (Boardman et al., 2002; Caldwell et al., 2005;

Hilier et al., 2004). The primer pairs successfully employed for our cDNA

cloning include BTRC-forward, 5V-TCTGAATGGGCACAAGCGTG-3V;BTRC-reverse, 5V-AGACGACCACTGAGAGCAGGAAAG-3V; DISP1-for-

ward, 5 V-GGACTCGTGGATTTCAAGTGAGC-3 V; DISP1-reverse,

5V-AAGGTCTGAGAGAAGGCATTACACC-3V; DZIP1-forward, 5V-CCAAGTCGGCTTCAGGATTGTAG-3V; DZIP1-reverse, 5V-TCAGGGC-TAAAAGACACAGGAGC-3V; HHIP-forward, 5V-AGAACGGTGGGC-

TATTGGACC-3V; HHIP-reverse, 5V-GCAGCATTTTCCTGTGGGAGTG-3V;WNT11-forward, 5V-AGACCTGCCGTAAAACTTTCTCAG-3V; WNT11 re-

verse, 5V-TGCGACCCCACTTTCTCATTC-3V.

Fig. 1. Midbrain arcuate cell types are differentially affected in the talpid2 mutant. Left column: flock mate controls (wild type or heterozygote); right column:

talpid2 mutants. Images are of wholemounts (open book preparation) processed for one- or two-color in situ hybridization. Top is rostral, and the ventricular surface

faces the reader. (A, B) Loss of motor neurons (PHOX2A+, brown) and expansion of EVX1+ cells (blue) in the E5 talpid2 midbrain. (C, D) Reduction of TH+ (blue)

and PAX6+ cells (brown, arrowheads) in E6 midbrains. Note the increased cell dispersal in the talpid2 mutant (arrowheads, D). (E, F) Loss of the SHH+ (brown)

rostral floor plate (rFP) and the PHOX2A+ oculomotor neurons by E3 in the talpid2 mutant. 1: medial or 1st arc; MHB, midbrain–hindbrain boundary; rFP, rostral

floor plate.

S. Agarwala et al. / Developmental Biology 288 (2005) 206–220208

In situ hybridization

Embryos were harvested at E3 to E6 and immersion-fixed in 4%

paraformaldehyde in a phosphate-buffered saline solution. Digoxigenin

(DIG)- and fluorescein-labeled riboprobes were synthesized from chick cDNA

plasmids for BTRC/SLIMB , CLASS III b-TUBULIN, CHOX10, DISP1,

DZIP1, EN1, EVX1, FOXA2/HNF3b, GLI1, GLI2, GLI3, ISL1, LMX1B,

NKX2.2, OTX2, PAX6, PAX7, PHOX2A, PTC1, SHH, SIM1, SUFU, TH and

WNT11 (Agarwala et al., 2001; Agarwala and Ragsdale, 2002; Sanders et al.,

2002). High-stringency one and two-color wholemount and section in situ

hybridizations were carried out as described on brain, spinal cord and limbs

(Agarwala and Ragsdale, 2002).

Cell death assay

Wholemount cell death assays were performed with modifications of the

Yamamoto and Henderson (1999) method (Sanders, 2001). E5–E6 embryos

were harvested and immersion-fixed for 2–3 h in 4% paraformaldehyde

containing 0.1% Tween. Dissected midbrains were dehydrated and rehydrated

through graded ethanols and permeabilized with detergents. TUNEL

(Terminal deoxytransferase (Tdt)-mediated dUTP-digoxigenin nick end

labeling) was accomplished by incubation in a Tdt (Chemicon, CA) reaction

at 37-C for 40 min. Incorporation of DIG-labeled nucleotide allowed

detection as with non-radioactive in situ hybridization histochemistry.

Results

The SHH source and arc patterning are disrupted in the talpid2

mutant midbrain

In the midbrain, arcuate stripes of molecularly distinct cell

types (midbrain arcs) are arrayed bilateral to the midbrain or

rostral floor plate source of SHH (Fig. 1; Agarwala et al., 2001;

Kingsbury, 1922, 1930; Sanders et al., 2002). Previous work

has shown that an ectopic source of SHH can generate a full

complement of midbrain arcs in a size- and position-dependent

manner (Agarwala et al., 2001). In the limb, SHH-mediated

elements of pattern such as the size of the autopod, digit

number and digit identity are disrupted in the talpid2 mutant

(Caruccio et al., 1999). We asked whether the SHH-dependent

midbrain arcs are disrupted in the talpid2 mutant as well.

The anlagen of the oculomotor complex (OMC, PHOX2A+),

the red nucleus (RN, BRN3A+) and midbrain dopaminergic

neurons (MDN, TH+) arise from themost medial (1st) arc which

abuts the SHH source and thus constitutes cell fates specified by

Fig. 2. SHH misexpression does not restore the arcuate pattern of cell fates in the talpid2 midbrain. Orientation for this and subsequent midbrain wholemounts as in

Fig. 1. (A, B) Homeobox gene expression of PHOX2A, PAX6 and EVX1 (together referred to as HX; blue) at E6 confirms the downregulation of PHOX2A+ and

PAX6+ cell types closer to the SHH source (brown) compared to the massive upregulation of the more lateral EVX1+ cell types. Red arrow in panel A marks the

lateral edge of SHH gene expression in control midbrains. Note that the reduction of SHH gene expression in the talpid2 mutant (B) that was obvious at E3 (Fig. 1F)

continues at E6, being retained only along the ventral midline. (C, D) Unilateral (right side) overexpression of SHH at E5 following electroporation at E2. The left

half of the midbrain in each panel serves as the unelectroporated control. Since chick SHH was electroporated, SHH (brown) expression on the right in excess of the

left side represents the misexpressed SHH. (C) Ectopic induction of a SHH+ FP (brown, arrowhead) and an expansion of the arc-specific homeobox (HX), PHOX2A,

PAX6 and EVX1 (blue) genes at E5. (D) SHH overexpression in the talpid2 midbrain (right half) does not elicit an orderly pattern of arcuate territories when assayed

by HX gene expression at E5 nor does it produce a consolidated SHH source (brown, arrow and arrowheads) as seen in the controls (C). Instead, clumps of

ectopically expressed SHH (brown) surrounded by HX gene expression (blue) are seen throughout the electroporated region (arrowheads).


high concentrations of SHH (Agarwala and Ragsdale, 2002).

PAX6+ andEVX1+ arcuate stripes occupymore lateral positions

in ventral midbrain and are thought to encounter progressively

lower concentrations of SHH. In the talpid2midbrain, themedial

(OMC, RN and MDN) and intermediate (PAX6+) arcuate

populations are severely reduced, while lateral cell fates

(EVX1+) are expanded and expressed at ectopic locations (Figs.

1A–D, data not shown). In marked contrast with the normal

SHH source seen in the talpid2 and talpid3 limbs (Caruccio et

al., 1999; Francis-West et al., 1995; Lewis et al., 1999;

Schneider et al., 1999), the rostral floor plate (rFP) source of

SHH is severely disrupted in the midbrain by E3 (Figs. 1E, F).

Taken together, these data suggest that a reduced SHH

signaling source compromises medial cell fates normally

induced by high concentrations of SHH (‘‘high’’ HH fates)

but expands lateral cell fates that normally encounter low

concentrations of SHH (‘‘low’’ HH fates).

SHH misexpression cannot rescue the talpid2 midbrain

phenotype: talpid2 is required autonomously in HH-responsive

tissue

The dramatic reduction in size of the midbrain FP, which is

apparent at E3 (Fig. 1F) and maintained through E6 (Figs. 2A,

B), is sufficient to explain a loss of medial cell fates that are

induced by high HH concentrations. To address the mechan-

isms of the impaired talpid2 FP development, we took

advantage of our earlier finding that misexpression of SHH

by electroporation is able to induce an ectopic floor plate along

with a complete ectopic arc pattern (Agarwala et al., 2001). Our

prediction was, if talpid2 FP development is compromised

because of a deficit in the inductive signals from axial

mesoderm, ectopic SHH should rescue the FP development

program (Placzek et al., 1993). By contrast, a failure of ectopic

SHH to elicit an ectopic FP and ectopic arcs would demonstrate

an HH signaling defect intrinsic to the HH-responsive cells of

the midbrain.

We found in control experiments that, as expected, unilateral

overexpression of SHH elicited an expanded, uninterrupted

SHH-rich FP, surrounded by ectopic arcs (Fig. 2C; Agarwala et

al., 2001). In the talpid2 midbrain, however, SHH over-

expression is unable to elicit a coherent SHH-rich source (Fig.

2D). Patches of arc-specific gene expression were seen around

the ectopic SHH+ patches (Fig. 2D), demonstrating that,

although the talpid2 midbrain can respond to HH signaling,

this signaling is defective and results in the inability of ectopic

SHH to produce the FP expansion and the organized arc pattern

elicited in controls (Fig. 2C).

Talpid2 limbs grafted to wild type limbs self-differentiate

and form talpid2 digits (Dvorak and Fallon, 1992; MacCabe

and Abbott, 1974). Furthermore, talpid2, but not wild type

limbs, can regenerate the resected 2/3rd of the posterior limb

Fig. 3. The principal effectors of HH are upregulated in the talpid2 midbrain. (A–H) Gene expression of PTC1 (A, B), GLI1 (C, D), GLI2 (E, F) and GLI3 (G, H) is

greatly upregulated in the E5 talpid2 midbrain (B, D, F, H) in comparison with controls (A, C, E, G). Note that, for all four genes, the talpid2 ventral midline remains

unlabeled and that, particularly for GLI3, the expansion is very patterned (also seen in Figs. 8E, F). (I, J) Talpid2 midbraiin showing a strictly segregated mosaicism

of SHH (brown) and GLI3 (blue) mRNA expression wherever the two genes are co-expressed. (I) Unilateral SHH misexpression on the right side of the embryo with

the left side serving as the unelectroporated control. As with Fig. 2, SHH (brown) expression on the right in excess of the left side represents misexpressed chick

SHH. Clumps of ectopic SHH+ tissue seen beyond the domain of ectopic GLI3 expression (arrowheads, I) indicate that the ectopic upregulation of GLI3 cannot

explain the failure in talpid2 midbrains of ectopic SHH deposits to coalesce. Panel J illustrates at high power the boxed region in panel I.



bud in the absence of detectable SHH mRNA expression

(Caruccio et al., 1999). Taken together with our SHH

misexpression experiments (Figs. 2C, D), these results indicate

that talpid2 function is likely to be cell-autonomous in multiple

embryonic systems. Thus, the FP defect and the consequent arc

patterning defects are likely to result from incorrect HH signal

transduction in the HH-responsive cells of the midbrain such

that FP induction by axial mesoderm and the homeogenetic

induction of FP by itself are compromised (Placzek et al.,

1993).

Deregulation of HH effector genes in the talpid2 mutant

midbrain

To further characterize the nature of the defect in the

talpid2 mutant, we next examined gene expression of the

principal effectors of HH signal transduction. As in the

talpid2 limb, gene expression of PTC1, a receptor and

negative regulator of HH signaling, is massively upregulated

in the mutant midbrain (Figs. 3A, B). Notably, the dynamic

spatial modulation of PTC1 seen along the mediolateral axis

in controls is replaced by uniformly high expression that only

avoids the midline of rFP (Figs. 3A, B). Gene expression of

PTC1 is a reliable readout of the range of HH signaling

(Goodrich et al., 1997). The increased PTC1 gene expression

thus suggests that a broader than normal expanse of the

talpid2 midbrain behaves as if responding to HH signaling

(Caruccio et al., 1999).

GLI transcription factors are required for HH signal

transduction (Bai et al., 2004; Krishnan et al., 1997; Methot

and Basler, 2001). In the talpid2 limb, Caruccio et al. (1999)

found that GLI1 gene expression is increased. We found in the

talpid2 midbrain that expression levels of all three GLI genes

are raised (Figs. 3C–H). The domains of GLI1 and GLI2

occupy most of midbrain with a notable lack of any GLI

expression in the PTC1-poor ventral midline (Figs. 3C–F).

Although GLI3 expression also avoids the midline, its ectopic

upregulation is more restrained, being concentrated along the

midbrain–hindbrain junction (MHB), the dorsoventral (DV)

border of the midbrain, and, most prominently, flanking the

ventral midline where ectopic GLI3 expression forms a non-

overlapping mosaic pattern with SHH (Figs. 3G–J; see also

Figs. 8E, F). Interestingly, we also saw this mosaicism between

SHH and GLI3 expression patterns when we misexpressed

SHH in the talpid2 midbrain by electroporation (Figs. 3I, J).

Misexpression of SHH, however, elicits clumps of ectopic

SHH-expressing cells even outside the regions expressing

ectopic GLI3, indicating that the failure of the talpid2 midbrain

to support normal rFP development is unlikely to be due

simply to interference from pools of GLI3-rich cells (Fig. 3I).

Not all HH pathway genes are disrupted in the talpid2 mutant

Our observations suggest that the talpid2 mutation is likely

to involve the HH transport, reception or signal transduction

machinery. We therefore isolated cDNAs for chicken Dis-

patched (DISP1) and Hedgehog Interacting Protein (HHIP),

which are known to regulate HH transport (Ingham and

McMahon, 2001; Jeong and McMahon, 2005; Tian et al.,

2004). Gene expression for HHIP1, a glycoprotein that binds

HH and attenuates the range and magnitude of HH signaling

(Chuang and McMahon, 1999; Jeong and McMahon, 2005), is

lost from the talpid2 midbrain (Figs. 4A, B). DISP1 is required

for cholesterol-modified HH to be secreted from the HH-

producing cells, and lowering the levels of DISP produces a

dose-dependent reduction in HH signaling (Tian et al., 2004).

Normal levels of DISP1 transcripts are distributed along the

ventral midline in both the control and mutant midbrain,

suggesting that the talpid2 mutation does not interfere with

DISP1 expression (Figs. 4C, D).

Biochemical evidence from the talpid2 limb suggests that

one consequence of the mutation is the misregulation of GLI3

such that excess full-length GLI3 (presumed activator) is

produced and abnormally distributed in the anterior (preaxial)

limb bud, while the amount of GLI3 repressor is reduced

(Litingtung et al., 2002; Wang et al., 2000). To examine GLI

gene modulation in the talpid2 midbrain, we isolated from

chickens the cDNAs for several genes involved in GLI gene

processing. We found that both SUFU, whose protein product

is involved in the cytoplasmic retention of GLI proteins, and

BTRC/SLIMB, which is involved in targeting hyperphosphory-

lated Ci/GLI proteins for degradation (Jiang, 2002; Kogerman

et al., 1999), are expressed normally in the talpid2 mutant

midbrain (Figs. 4E–H).

A recently identified member of the HH family is the

zinc finger transcription factor DZIP1 which may participate

in the nuclear-cytoplasmic shuttling of GLI proteins, thereby

regulating the ratio of GLI activator and repressor (Sekimizu

et al., 2004; Vokes and McMahon, 2004; Wolff et al.,

2004). In the zebrafish dzip1 mutant iguana, cell fates

triggered by high levels of HH signaling are compromised,

while those promoted by low levels of HH are expanded, a

feature shared by the talpid2 mutant (Sekimizu et al., 2004).

In addition, the lateral floor plate is absent in the igu mutant

(Karlstrom et al., 1996; Odenthal et al., 2000). Given the

similarities between the iguana and the talpid2 phenotypes,

we asked if chick DZIP1 gene expression was misregulated

in the talpid2 embryo. We found that DZIP1 expression was

not perturbed in the talpid2 midbrain, spinal cord or limb

bud (Figs. 4I, J, data not shown).

Cell death, tissue size and thickness are compromised in the

talpid2 mutant

The loss of the rFP and medial midbrain cell populations

could be due to increased cell death, possibly triggered by

upregulation of PTC1 expression in the talpid2 mutant

(Thibert et al., 2003). We found increased cell death in the

midbrain of the talpid2 mutant with TUNEL labeling (Figs.

5A, B). Unlike control midbrains, where cell death was

largely confined to the ventricular layer (Fig. 5A, data not

shown), apoptotic cells in the talpid2 midbrain were spread

across the ventricular and mantle layer and concentrated in

the FP along the midline (Fig. 5B, data not shown).

Fig. 4. Expression of key modulators of HH function in the talpid2 midbrain. (A, B) Hedgehog-interacting protein (HHIP) gene expression is lost from the midbrain

of the talpid2 mutant, although it can be seen in the forebrain (arrowhead). (C–J) Expression patterns of DISPATCHED 1 (DISP1), SUPPRESSOR OF FUSED

(SUFU), BTRC/SLIMB and DZIP1/IGUANA are largely unaffected by the talpid2 mutation. E4 (A, B, I, J) and E5 (C–H) midbrains harvested from control (A, C, E,

G, I) and talpid2 (B, D, F, H, J) embryos. Identification of talpid2 mutants in I, J was based on SHH expression since they could not be distinguished by their limb

phenotype at this stage. III: 3rd ventricle. rFP: rostral floor plate, MHB: midbrain–hindbrain junction.


Fig. 5. Cell death, tissue size and thickness are compromised in the talpid2 midbrain. (A, B) TUNEL staining demonstrates increased numbers of apoptotic cells in

the E5 talpid2 midbrain (B) over those of control (A). (C, D) Increased size of the talpid2 ventral midbrain at E5 (D) photographed at the same magnification as an

E5 control (C). Midbrain dorsoventral and midbrain– forebrain boundaries are identified by gene expression of GLI3 (blue) in tectum (tec) and PAX6 (brown) in

caudal forebrain (FB). (E, F) CLASS III b-TUBULIN labeling demonstrates largely normal neuronal differentiation in the E5 talpid2 midbrain (F) as compared with

controls (E). (G–H) Transverse sections of the wholemounts shown in panels E and F demonstrate the reduced thickness of the talpid2 midbrain. Images of the

controls and the corresponding talpid2 midbrains are taken at the same magnification. III: 3rd ventricle; FB: forebrain; tec: tectum or dorsal midbrain.


Intriguingly, this increased cell death was accompanied by

an increase in the width (DV axis) of the midbrain (Figs.

5C, D). An increase in tissue size is also reported to occur

along the anteroposterior (AP) axis of the talpid2 and the

talpid3 limbs (Caruccio et al., 1999; Lewis et al., 1999).

CLASS III b-TUBULIN gene expression, which identifies

differentiated neurons of the mantle layer (Figs. 5E–H),

demonstrated a marked reduction in the thickness of

midbrain tissue in cross-sections (Figs. 5G, H). These

results indicate that, although the talpid2 midbrain is wider

compared to the controls, the mantle layer is thinner.

Anteroposterior patterning is preserved in talpid2 midbrain,

but dorsoventral territory boundaries are no longer sharp

AP and DV patterning

The midbrain phenotype observed in the talpid2 mutant

could be a consequence of defects in other non-HH-mediated

signaling cascades originating along the boundary regions of

the ventral midbrain. The orthodenticle homolog OTX2 is

expressed throughout anterior neural tube (forebrain and

midbrain), and its caudal limit coincides with the midbrain–

hindbrain boundary (Fig. 6A). We found that OTX2 expression

Fig. 6. The midbrain–hindbrain boundary remains sharp, but the tectotegmental (DV) boundary becomes irregular in the talpid2 midbrain. (A, B) OTX2 gene

expression demonstrates that the sharp and smooth contours of the midbrain–hindbrain boundary (A) are retained in the E5 talpid2 mutant (B). (C, D) PAX7

expression distinguishes the dorsal midbrain (tec, tectum) from the ventral midbrain and documents that the talpid2 dorsoventral boundary is broad and disrupted

(arrowheads, D) compared to that of controls (C). MHB: midbrain–hindbrain boundary; tec: tectum.


is appropriately regionalized along the AP axis of the neural

tube, with its caudal expression terminating sharply at the

MHB (Figs. 6A, B). We also observed the normal localization

of BMP7 (rostral) and FGF8 (caudal) expression at the

midbrain–hindbrain boundary (MHB; data not shown). We

next examined the tectotegmental (DV) boundary of the

midbrain with PAX7 gene expression identifying the dorsal

midbrain or tectum. In controls as well as the mutants, PAX7

gene expression remained restricted to the tectum, suggesting

that the talpid2 midbrain was divided as usual into dorsal and

ventral domains (Figs. 6C, D). The confinement of PAX3/PAX7

to dorsal neural tube is thought to be accomplished by low

levels of HH signaling, confirming yet again that low level HH

signaling can occur in the talpid2 midbrain (Ericson et al.,

1996) and that the principal effects of the talpid2 mutation are

confined to the ventral midbrain. Interestingly, unlike the

caudal midbrain boundary, which remained sharp and regular

in the talpid2 mutant, the DV boundary presented a ‘‘wiggly’’,

irregular border (Figs. 6C, D; data not shown; Lawrence,

1997). Along with the lack of coherent arcs and the inability of

ectopic SHH to assemble into a coalesced morphogen source

(Figs. 1A–D and 2C, D), these results suggest changes in

midbrain cells’ adhesive properties or increased cell death of

HH target cells or both (Figs. 5A, B; Jarov et al., 2003;

Lawrence et al., 1999; Rodriguez and Basler, 1997).

Cell fate specification is normal in the talpid2 spinal cord

In the spinal cord, motor neurons and multiple classes of

ventral interneurons (V3–V0) are specified in response to

graded HH signaling (Ericson et al., 1996; Roelink et al.,

1995). We found that all ventral spinal neurons including motor

neurons (ISL1+) as well as the V3 (SIM1+), V2 (CHOX10+),

V1 (EN1+) and VO (EVX1+) interneurons were specified in

near-normal numbers along the dorsoventral axis of the talpid2

mutant with only a modest disturbance in their spatial

patterning (Figs. 7A–J). Furthermore, an SHH-rich FP and

notochord were readily detected in the talpid2 mutant (Figs.

7K, L) and were accompanied by a normal distribution of

PTC1 and GLI3 transcripts in the spinal cord (Figs. 7M–P).

Thus, unlike the midbrain, neuronal specification proceeds

normally in the talpid2 spinal cord, although, as shown below,

the caudal talpid2 FP is compromised in a manner similar to

that of midbrain FP.

Floor plate defects in the talpid2 midbrain and spinal cord

By gene expression and embryonic origin, the avian floor

plate has been partitioned into medial (node-derived, SHH+,

NKX2.2�) and lateral (neural-plate-derived, SHH+, NKX2.2+)

subdivisions at spinal cord levels (Charrier et al., 2002). We

cloned out chick WNT11, a marker of medial-most FP at spinal

cord levels, and found that, though slightly reduced in extent, it

was retained in the talpid2 ventral midline (Figs. 8A, B; see

also Figs. 7K, L). The NKX2.2+ lateral floor plate region

(LFP), however, was severely reduced in the talpid2 caudal

spinal cord (Figs. 8C, D). This was accompanied by an

increased overlap between SHH and NKX2.2 expression more

medially, suggesting a defect in LFP induction, maintenance or

segregation from the medial FP (MFP; Figs. 8C, D). In

zebrafish and rats, a midline HH signal is required for the

proper homeogenetic induction of lateral regions of FP and for

Fig. 7. Cell fate specification in the talpid2 spinal cord. Transverse sections demonstrating the relatively normal specification of SIM1+ V3 interneurons (A, B),

ISL1+ motor neurons (C, D), CHOX10+ V2 (E, F), EN1+ V1 (G, H) and EVX1+ VO (I, J) interneurons in control (columns 1 and 3) and talpid2 spinal cords

(columns 2 and 4). (K, L) SHH gene expression demonstrates that the floor plate (FP) and notochord (nc) are retained in the talpid2 mutant. (M, N) PTC1 expression

appears normal in the talpid2 spinal cord. (O, P) Normal relationship between GLI3 (blue) and SHH (brown) domains in the talpid2 spinal cord (P), relative to

controls (O). CHX10: CHOX10; DR: dorsal root ganglion; MN, motor neurons. A, B, E–H: E5 spinal cords; C, D, I–N: E4 spinal cords; O, P: E3 spinal cords.


the survival of cells within the medial regions of FP (Le

Douarin and Halpern, 2000; Odenthal et al., 2000; Placzek et

al., 1993). Defective HH signaling in the talpid2 may therefore

prevent the proper induction of the spinal cord LFP by MFP or

its segregation from MFP. In addition, it may result in the

observed reduction of MFP due to increased cell death (Figs.

7K, L and 8A, B; see also Figs. 5A, B).

The enormous lateral expansion of midbrain FP along with

its distinctive mode of induction and embryonic origin

precluded the use of medial MFP and LFP definitions derived

from spinal cord studies (Kingsbury, 1922, 1930; Placzek and

Briscoe, 2005). However, based on high and low levels of SHH

expression, clear medial and lateral divisions could be

discerned in the E3 midbrain (Figs. 8E, F). The lateral region,

Fig. 8. Disruption of lateral floor plate (LFP) in the spinal cord and midbrain of the talpid2 mutant. (A, B) Cross-section of E5 spinal cord demonstrating that the

medial-most region of the medial FP (MFP) stained by WNT11 in the control (A) is retained in the talpid2 spinal cord (B). (C, D) Cross-sections through E5 spinal

cord demonstrating a reduced LFP (NKX2.2+, brown) in the talpid2 mutant, while the MFP (SHH+) is retained. (E, F) E3 midbrain wholemounts demonstrate

downregulation of SHH (brown) in LFP in the talpid2 mutant (F) concomitant to the ectopic expression of GLI3 (blue) in this region. (G, H) Expression of FOXA2

(brown), which extends lateral to the SHH domain in E5 control midbrains (G), is repressed in the talpid2 midbrain (H). (I, J) LMX1B staining of E5 midbrain

demonstrates a retained MFP in the talpid2 midbrain (J). LFP: lateral floor plate; MFP: medial floor plate; MHB: midbrain–hindbrain junction.


marked by the loss of SHH and ectopic GLI3 expression, was

severely disrupted in the talpid2 mutant (Figs. 8E, F, see also

Figs. 4C, D; I, J). By E5, the enormous growth and complex

subarchitecture of the rFP foreclosed any simple analysis of the

midbrain FP into lateral and medial subdivisions. Even so, the

talpid2 E5 rFP resembled in quality the talpid2 spinal cord FP:

a disruption of lateral FP regions with FP tissue along the

ventral midline remaining relatively intact, displaying only a

modest increase in cell death (Figs. 8G, H; Figs. 5A, B).

LMX1B and DISP1 gene expression corroborated the retention

of medial-most rFP at E5 (Figs. 8I, J and 4C, D). In the

zebrafish as well as in the rat, LFP specification and MFP cell

survival require proper HH signaling (Le Douarin and Halpern,

2000; Odenthal et al., 2000; Placzek et al., 1993). The failure of

LFP to be correctly specified in the midbrain and spinal cord

and the increased cell death in the medial regions of the FP

together suggest that talpid2 may function as a component of

the HH signaling cascade without which the lateral regions of


FP cannot be correctly specified and the medial regions of FP

suffer increased cell death.

Discussion

In this study, we show that talpid2 is likely to be a novel

component of the avian HH signal transduction pathway, its

misregulation resulting in dramatically different phenotypes in

the limb, spinal cord and the midbrain. In the limb, the readout

is loss of growth control and polydactyly with indeterminate

digit identity (Caruccio et al., 1999). In the spinal cord, the

principal defect is a disruption of floor plate development,

while cell fate specification of motor and interneurons remains

fairly normal. In the midbrain, by contrast, multiple facets of

patterning associated with HH signaling are disrupted. These

disruptions include reduced cell survival, loss of size regula-

tion, deregulation of cellular affinities and, most profoundly, a

disruption in cell fate specification.

Defective HH signaling in the talpid2 mutant

HH signaling is sufficient for normal midbrain patterning

and for the segregation of ventral (PAX7�) from dorsal

(PAX7+) neural tube (Agarwala et al., 2001; Ericson et al.,

1996). In the talpid2 midbrain, the correct dorsal (tectal)

localization of PAX7 and the residual presence of HH target cell

fates suggest that some HH signaling does indeed occur in the

midbrain (Figs. 1, 2, 5, 6; Ericson et al., 1996). However, HH

signaling in the talpid2 is fundamentally defective as demon-

strated by the autonomous defects in HH signal transduction

within the HH-responsive tissue (Fig. 2; Caruccio et al., 1999;

Dvorak and Fallon, 1992; MacCabe and Abbott, 1974), the

ligand-independent regulation of several HH effector genes

(PTC, GLIs), by the upregulation of ‘‘low’’ HH cell fates and

by the reduction of ‘‘high’’ HH cell fates. Several features of

the talpid2 mutant could account for this phenotype.

Upregulation of PTC1 and the loss of HHIP

HH signal transduction requires the function of its receptor

PTC1 (Ingham et al., 1991). A product of this signal

transduction is the transcriptional upregulation of PTC1

expression, making PTC1 expression a reliable readout of the

occurrence of HH signaling (Marigo et al., 1996b; Goodrich et

al., 1997). Paradoxically, PTC1 also sequesters HH and

attenuates HH signaling (Marigo et al., 1996b; Goodrich et

al., 1997). The glycoprotein HHIP, also an HH binding protein,

a transcriptional target of HH and a negative regulator of HH

signaling, serves much the same function in vertebrates as does

PTC1 (Chuang and McMahon, 1999; Jeong and McMahon,

2005). The simultaneous removal of PTC and HHIP in mice

results in a massively ventralized neural tube and in a loss of

size regulation (Jeong and McMahon, 2005). In the mouse

Ptc1�/� mutant, the absence of PTC1 function results in

increased HH signaling and consequently in increased levels of

ptc1 expression (Goodrich et al., 1997). The upregulated PTC1

mRNA levels in the talpid2 midbrain may therefore reflect

lowered PTC function and, together with the loss of HHIP

expression, suggests an increased range and magnitude of HH

signaling in the midbrain. These findings fit well with the

ectopic upregulation of low HH fates (EVX1+; Fig. 1) and the

loss of size regulation seen in the midbrain (Figs. 5C, D). But,

they cannot readily account for the loss of ‘‘high’’ HH fates in

the talpid2 midbrain and are not concordant with the lowered

levels of SHH observed (Fig. 1). These results suggest instead

that the upregulation of PTC1 expression in the talpid2 may

occur in a ligand-independent manner as suggested in the

talpid2 limb (Caruccio et al., 1999). However, as discussed

below, additional explanation is required to explain the full

talpid2 midbrain phenotype.

Defective GLI modulation

The talpid2 midbrain exhibits lowered levels of HH gene

expression along with the upregulation of all three GLI genes

(Fig. 3). This is accompanied by a complex midbrain

phenotype with the widespread upregulation of ‘‘low’’ HH

fates (e.g., EVX1+ cells) and a depletion of ‘‘high’’ HH fates

such as the FP, oculomotor and dopaminergic neurons (Fig.

1). In all embryonic systems examined, GLI1 ubiquitously

exhibits activator function, and GLI3 protein can serve as an

activator or a repressor depending on the presence or absence

of HH signaling respectively (Ohlmeyer and Kalderon, 1998;

Dai et al., 1999). The increased GLI1 and PTC1 expression is

thus in conflict with the low levels of SHH expression

observed in the talpid2 midbrain. Furthermore, the readout of

GLI1 would be in conflict with the increased GLI3 levels,

which would be presumably be predominantly of the

repressor variety due to the lowered levels of HH. What

could the function of talpid2 be to produce a phenotype

consistent with both positive and negative regulation of HH

signaling?

To our knowledge, DZIP1/iguana, which modulates GLI

activity, is the only other HH pathway gene in vertebrates,

whose defect is known to result in the differential regulation of

‘‘high’’ (neural tube) and ‘‘low’’ (somites) HH target cell fates

(Sekimizu et al., 2004). However, the gene expression of

DZIP1 in the midbrain, spinal cord and limb of the talpid2

mutant appears to be normal, although a point mutation or a

mis-sense mutation cannot be ruled out (Figs. 4I, J; data not

shown). Unlike vertebrates, several components of the Dro-

sophila HH pathway required for Ci/GLI processing including

Cos2 and PKA display a similar dual (positive and negative)

regulation of the HH signaling cascade. Mutations of such Ci-

modulating genes result not only in the derepression of the HH

pathway, but also attenuate ‘‘high’’ HH activity (e.g., Jia et al.,

2004). For example, PKA, which is required for the phosphor-

ylation and proteolysis of Ci into its repressor form, also

phosphorylates and targets SMO to the cell surface, an event

essential for the upregulation of ‘‘high’’ HH cell fates (Jia et al.,

2004). Thus, the differential regulation of high vs. low HH

fates, the misregulation of GLI gene expression and the

incorrect processing of GLI3 protein in the talpid2 limb

together lead us to predict that the talpid2 is likely to be a

novel component of the vertebrate HH signaling cascade that

can modulate HH-GLI activity bidirectionally.


Floor plate specification is defective in the talpid2 neural tube

A major finding of this study is the selective disruption of

the lateral regions of floor plate in the midbrain and spinal cord

(Fig. 8). A subdivision of the FP into medial and lateral regions

is well established in the teleosts and has also been proposed in

the chick and other amniotes (Ang and Rossant, 1994; Charrier

et al., 2002; Placzek and Briscoe, 2005; Odenthal et al., 2000;

Placzek et al., 1991; Roelink et al., 1995; Sasaki and Hogan,

1993; Yamada et al., 1991). Studies involving quail–chick

chimeras suggest that, as with the zebrafish, the avian MFP

(SHH+, NKX2.2�) is derived from the node (embryonic shield

in fish), while LFP (SHH+, NKX2.2+) has its origin within the

neurectoderm and requires a midline HH signal for its

induction (Charrier et al., 2002; Melby et al., 1996; Odenthal

et al., 2000; Placzek et al., 1993; Shih and Fraser, 1996). Our

studies in control animals suggest that SHH misexpression can

indeed induce an ectopic FP in the midbrain that expands

through homeogenetic induction (Fig. 2; Agarwala et al., 2001;

Placzek et al., 1993). However, similar misexpression of SHH

in the talpid2 midbrain cannot sustain such an FP expansion,

consistent with the idea that SHH signaling is defective in the

talpid2 mutant and that the induction of LFP requires SHH

signaling (Placzek and Briscoe, 2005).

The above discussion suggests that defective HH signaling

leads to a disruption of LFP specification or maintenance or

both, but it does not explain why the MFP is relatively spared

in the talpid2 mutant. It is possible that the residual HH

signaling (Figs. 1, 2, 6) that does remain in the talpid2 mutant

is talpid2-independent and is sufficient for the development of

the largely intact MFP. It is equally possible, that as in

anamniotes, HH-independent mechanisms play a role in the

induction of the avian MFP (Strahle et al., 2004). Likely

candidates for this induction are Nodal, other TGFh family

members and their antagonists and the Notch–Delta signaling

cascades (Lopez et al., 2005; Strahle et al., 2004; Placzek and

Briscoe, 2005).

The distinct regional effects of talpid2 may be due to the

differential proximity of the midbrain and spinal cord to the

notochord

What could account for the differential severity of the loss

of talpid2 function in midbrain and spinal cord patterning? One

explanation may be the proximity of each region to the

subjacent notochord and its relative importance in neuronal cell

fate specification. By E3, the notochord has already regressed

from the midbrain, which likely leaves the FP as the principal

ventral source of patterning signals at this AP level. By

contrast, the notochord remains apposed to the spinal cord for a

considerably longer period of time, at least as late as E4 (Figs.

7K, L). Recent observations in the mouse have suggested that

the allocation of cell fates in the spinal cord may well depend

upon notochord-derived signals rather than the FP (Jeong and

McMahon, 2005). Such signals could be HH-dependent, HH-

independent or both. The midbrain, by contrast, cannot depend

upon the notochord due to its early rostral regression and must

rely on patterning signals from the rFP which is severely

disrupted in the talpid2 mutant. As a result, many aspects of

patterning including LFP expansion, cell fate specification,

cell-adhesion, size control and cell survival are affected in the

talpid2 midbrain.

Acknowledgments

We thank R. Bayly, J. Fogel, T. Sanders and J. Wallingford

for critical comments, Jacqueline Pisenti at UC Davis for her

help in supplying us with talpid2 fertilized eggs and P. Beachy,

P. Brickell, C. Cepko, D. Cleveland, G. Eichele, C. Fan, C.

Goridis, M. Goulding, B. Houston, T. Jessell, A. Leutz, A.

McMahon, G. Martin, C. Tabin and M. Wassef for DNA

reagents. This research was supported by grants from the

March of Dimes and the NIH (C.W.R.; J.F.F.) and from UT-

Austin (S.A.).

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