Specific domains of FoxD4/5 activate and repress neural transcription factor genes to control the progression of immature neural ectoderm to differentiating neural plate.
ABSTRACT FoxD4/5, a forkhead transcription factor, plays a critical role in establishing and maintaining the embryonic neural ectoderm. It both up-regulates genes that maintain a proliferative, immature neural ectoderm and down-regulates genes that promote the transition to a differentiating neural plate. We constructed deletion and mutant versions of FoxD4/5 to determine which domains are functionally responsible for these opposite activities, which regulate the critical developmental transition of neural precursors to neural progenitors to differentiating neural plate cells. Our results show that up-regulation of genes that maintain immature neural precursors (gem, zic2) requires the Acidic blob (AB) region in the N-terminal portion of the protein, indicating that the AB is the transactivating domain. Additionally, down-regulation of those genes that promote the transition to neural progenitors (sox) and those that lead to neural differentiation (zic, irx) involves: 1) an interaction with the Groucho co-repressor at the Eh-1 motif in the C-terminus; and 2) sequence downstream of this motif. Finally, the ability of FoxD4/5 to induce the ectopic expression of neural precursor genes in the ventral ectoderm also involves both the AB region and the Eh-1 motif; FoxD4/5 accomplishes ectopic neural induction by both activating neural precursor genes and repressing BMP signaling and epidermal genes. This study identifies the specific, conserved domains of the FoxD4/5 protein that allow this single transcription factor to regulate a network of genes that controls the transition of a proliferative neural ectodermal population to a committed neural plate population poised to begin differentiation.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: The early steps of neural development in the vertebrate embryo are regulated by sets of transcription factors that control the induction of proliferative, pluripotent neural precursors, the expansion of neural plate stem cells, and their transition to differentiating neural progenitors. These early events are critical for producing a pool of multipotent cells capable of giving rise to the multitude of neurons and glia that form the central nervous system. In this review we summarize findings from gain- and loss-of-function studies in embryos that detail the gene regulatory network responsible for these early events. We discuss whether this information is likely to be similar in mammalian embryonic and induced pluripotent stem cells that are cultured according to protocols designed to produce neurons. The similarities and differences between the embryo and stem cells may provide important guidance to stem cell protocols designed to create immature neural cells for therapeutic uses.Molecules and cells. 09/2014;
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ABSTRACT: Cells in the developing neural tissue demonstrate an exquisite balance between proliferation and differentiation. Retinoic acid (RA) is required for neuronal differentiation by promoting expression of proneural and neurogenic genes. We show that RA acts early in the neurogenic pathway by inhibiting expression of neural progenitor markers Geminin and Foxd4l1, thereby promoting differentiation. Our screen for RA target genes in early Xenopus development identified Ets2 Repressor Factor (Erf) and the closely related ETS repressors Etv3 and Etv3-like (Etv3l). Erf and Etv3l are RA responsive and inhibit the action of ETS genes downstream of FGF signaling, placing them at the intersection of RA and growth factor signaling. We hypothesized that RA regulates primary neurogenesis by inducing Erf and Etv3l to antagonize proliferative signals. Loss-of-function analysis showed that Erf and Etv3l are required to inhibit proliferation of neural progenitors to allow differentiation, whereas overexpression of Erf led to an increase in the number of primary neurons. Therefore, these RA-induced ETS repressors are key components of the proliferation-differentiation switch during primary neurogenesis in vivo.Development 07/2013; · 6.60 Impact Factor
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ABSTRACT: THE EARLIEST STEPS OF EMBRYONIC NEURAL DEVELOPMENT ARE ORCHESTRATED BY SETS OF TRANSCRIPTION FACTORS THAT CONTROL AT LEAST THREE PROCESSES: the maintenance of proliferative, pluripotent precursors that expand the neural ectoderm; their transition to neurally committed stem cells comprising the neural plate; and the onset of differentiation of neural progenitors. The transition from one step to the next requires the sequential activation of each gene set and then its down-regulation at the correct developmental times. Herein, we review how these gene sets interact in a transcriptional network to regulate these early steps in neural development. A key gene in this regulatory network is FoxD4L1, a member of the forkhead box (Fox) family of transcription factors. Knock-down experiments in Xenopus embryos show that FoxD4L1 is required for the expression of the other neural transcription factors, whereas increased FoxD4L1 levels have three different effects on these genes: up-regulation of neural ectoderm precursor genes; transient down-regulation of neural plate stem cell genes; and down-regulation of neural progenitor differentiation genes. These different effects indicate that FoxD4L1 maintains neural ectodermal precursors in an immature, proliferative state, and counteracts premature neural stem cell and neural progenitor differentiation. Because it both up-regulates and down-regulates genes, we characterized the regions of the FoxD4L1 protein that are specifically involved in these transcriptional functions. We identified a transcriptional activation domain in the N-terminus and at least two domains in the C-terminus that are required for transcriptional repression. These functional domains are highly conserved in the mouse and human homologues. Preliminary studies of the related FoxD4 gene in cultured mouse embryonic stem cells indicate that it has a similar role in promoting immature neural ectodermal precursors and delaying neural progenitor differentiation. These studies in Xenopus embryos and mouse embryonic stem cells indicate that FoxD4L1/FoxD4 has the important function of regulating the balance between the genes that expand neural ectodermal precursors and those that promote neural stem/progenitor differentiation. Thus, regulating the level of expression of FoxD4 may be important in stem cell protocols designed to create immature neural cells for therapeutic uses.American journal of stem cells. 01/2013; 2(2):74-94.
Specific domains of FoxD4/5 activate and repress neural transcription factor genes to
control the progression of immature neural ectoderm to differentiating neural plate
Karen M. Neilsona, Steven L. Kleina, Pallavi Mhaskea, Kathy Moodb, Ira O. Daarb, Sally A. Moodya,⁎
aDepartment of Anatomy and Regenerative Biology, George Washington University School of Medicine and Health Sciences, 2300 I Street, NW, Washington DC, USA
bLaboratory of Cell and Developmental Signaling, NIH, NCI, Frederick National Laboratory, Building 560 - FCRDC, 22-3, 1050 Boyles Street, Frederick, MD, USA
a b s t r a c ta r t i c l e i n f o
Received for publication 7 September 2011
Revised 1 March 2012
Accepted 4 March 2012
Available online 10 March 2012
FoxD4/5, a forkhead transcription factor, plays a critical role in establishing and maintaining the embryonic
neural ectoderm. It both up-regulates genes that maintain a proliferative, immature neural ectoderm and
down-regulates genes that promote the transition to a differentiating neural plate. We constructed deletion
and mutant versions of FoxD4/5 to determine which domains are functionally responsible for these opposite
activities, which regulate the critical developmental transition of neural precursors to neural progenitors to
differentiating neural plate cells. Our results show that up-regulation of genes that maintain immature neural
precursors (gem, zic2) requires the Acidic blob (AB) region in the N-terminal portion of the protein, indicat-
ing that the AB is the transactivating domain. Additionally, down-regulation of those genes that promote the
transition to neural progenitors (sox) and those that lead to neural differentiation (zic, irx) involves: 1) an in-
teraction with the Groucho co-repressor at the Eh-1 motif in the C-terminus; and 2) sequence downstream of
this motif. Finally, the ability of FoxD4/5 to induce the ectopic expression of neural precursor genes in the
ventral ectoderm also involves both the AB region and the Eh-1 motif; FoxD4/5 accomplishes ectopic neural
induction by both activating neural precursor genes and repressing BMP signaling and epidermal genes. This
study identifies the specific, conserved domains of the FoxD4/5 protein that allow this single transcription
factor to regulate a network of genes that controls the transition of a proliferative neural ectodermal popula-
tion to a committed neural plate population poised to begin differentiation.
© 2012 Elsevier Inc. All rights reserved.
The vertebrate neural ectoderm is induced by antagonists of the
BMP and Wnt pathways that are secreted by cells in the Organizer re-
gion of the dorsal mesoderm (reviewed in De Robertis and Kuroda,
2004; Itoh and Sokol, 2007; Rogers et al., 2009a; Stern, 2005). These
antagonists enable the expression of a large number of transcription
factors in the dorsal ectoderm that in turn promote its conversion to
a neural ectodermal fate and prevent its reversion to a non-neural
fate. One of these transcription factors, FoxD4/5, acts very early in
the nascent neural ectoderm to promote the formation of the imma-
ture neural ectoderm, expand the neural plate and delay the onset of
neural differentiation (Fetka et al., 2000; Sölter et al., 1999; Sullivan et
al., 2001). It both up-regulates genes that maintain an immature, pro-
liferative neural ectoderm and down-regulates genes that promote
the transition to neural progenitors and lead to neural differentiation
(Sullivan et al., 2001; Yan et al., 2009, 2010). Determining how
FoxD4/5 both up-regulates and down-regulates its various target
genes is key to understanding the transcriptional network that regu-
lates the critical developmental transition of an immature, prolifera-
tive neural ectoderm to a definitive neural plate comprised of
neurally-committed, differentiating cells.
Forkhead/Fox genes constitute a large family of transcription fac-
tors that play key roles in numerous developmental processes in
nearly every tissue (Carlsson and Mahlapuu, 2002; Pohl and
Knochel, 2005; Wijchers et al., 2006). They all contain a highly con-
served winged-helix DNA-binding domain that defines the family.
However, sequences flanking this domain are so divergent, that the
family has been classified into 18 sub-families in vertebrates
(http://biology.pomona.edu/fox/). Some Fox proteins regulate tran-
scription by activation, some by repression, and a few by both,
depending upon the cell type, the developmental state or the avail-
ability of interacting proteins. In addition, some Fox proteins act as
“pioneer” transcription factors during development (Zaret, 2002;
Zaret et al., 2008). They stably bind to their recognition sites in chro-
matin domains of nuclear DNA that other factors cannot access, and
their binding then causes a conformational change to allow other
Developmental Biology 365 (2012) 363–375
⁎ Corresponding author at: Department of Anatomy and Regenerative Biology,
George Washington University, 2300 I Street, NW, Washington, DC, 20037, USA. Fax:
+1 202 994 8885.
E-mail address: email@example.com (S.A. Moody).
0012-1606/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/developmentalbiology
factors to engage the DNA (Cirillo et al., 2002). Because the DNA-
binding domain is so similar across the Fox family members, the se-
quences flanking it must account for their divergent activities.
It is important to establish which flanking regions of Fox proteins
account for these different kinds of transcriptional activities because
these proteins play critical roles in numerous developmental and dif-
ferentiation processes, and mutations or fusions of protein domains
underlie certain cancers (Carlsson and Mahlapuu, 2002; Pohl and
Knochel, 2005; Wijchers et al., 2006). Some Fox proteins contain acid-
ic domains, in either the amino (N)- or carboxy (C)-terminal regions,
that are thought to be involved in target gene activation (Ptashne,
1988; Schuddekopf et al., 1996); in Xenopus, only members of the
FoxD class contain an Acidic blob region (AB), which has yet to be
functionally characterized (Pohl and Knochel, 2005). The C-terminal
regions of some Fox proteins contain domains implicated in tran-
scriptional repression. These include a P/A/Q-rich region, a highly
charged Region II (R-II) and an Engrailed homology region-1 [Eh-1]
that can bind the well-known co-repressor protein, Groucho [Gro;
Grg in vertebrates; TLE in humans] (reviewed in Pohl and Knochel,
2005; Sullivan et al., 2001; Yaklichkin et al., 2007b). The Eh-1 motif
is conserved in about 50% of metazoan Fox proteins and in all FoxD
proteins (Yaklichkin et al., 2007b). Recently, the functional signifi-
cance of this motif in FoxD3, FoxA1 and FoxA2 was revealed: Gro/
Grg proteins bind to the Eh-1 motif and this interaction is required
for repression of downstream targets (Sekiya and Zaret, 2007;
Yaklichkin et al., 2007a).
Despite the key role demonstrated for FoxD4/5 in early neural de-
velopment in Xenopus (Fetka et al., 2000; Sölter et al., 1999; Sullivan
et al., 2001; Yan et al., 2009), little is known of its function in other
vertebrates. Homologues of FoxD4/5 have been identified across ver-
tebrates and they are expressed in the early nervous systems of zeb-
rafish, mouse and human (Freyaldenhoven et al., 2004; Kaestner et
al., 1995; Katoh and Katoh, 2004; Odenthal and Nusslein-Volhard,
1998; Pohl and Knochel, 2005; Suda et al., 1999; Tuteja and Kaestner,
2007; Yaklichkin et al., 2007b). Interestingly, this gene has been du-
plicated in primates; one gene (FoxD4) is most similar to mouse
foxd4 and one gene (FoxD4L1) is most similar to fish and frog foxD5.
Due to this homology, Xenopus foxD5 recently was reassigned the
name foxD4L1.1; since prior publications refer to it as foxD5, herein
we use the name foxD4/5.
We found that several vertebrate FoxD4/5 proteins contain many
of the aforementioned domains (Fig. 1). To identify which regions
are responsible for up-regulating and/or down-regulating several
known downstream targets, we made several deleted and mutated
versions of Xenopus FoxD4/5. We show that the ability of FoxD4/5
to up-regulate two genes that maintain an immature neural precursor
state (gem, zic2) requires the AB region, indicating that the AB is the
transactivating domain. Additionally, down-regulation of genes that
promote the transition to neural progenitors (sox) and of those that
lead to neural differentiation (zic, irx) involves: 1) an interaction
with the Gro/Grg4 co-repressor at the Eh-1 motif; and 2) sequence
C-terminal to this motif. Thus, FoxD4/5 contains both activating and
repressing domains, and can regulate the transition of an immature
neural ectoderm to a differentiating neural plate by up-regulating
some targets and down-regulating others. In addition, we show that
the previously demonstrated ability of FoxD4/5 to induce the ectopic
expression of neural genes in the ventral ectoderm (Yan et al., 2009)
involves both the AB region and the Eh-1 motif. We show that FoxD4/
5 accomplishes ectopic neural induction by both activating neural
genes and repressing BMP signaling and epidermal genes.
These results identify the specific domain that enables FoxD4/5 to
activate immature neural genes, and identify two domains that en-
able it to down-regulate neural progenitor and differentiation-
promoting genes, as well as epidermal genes. These findings illustrate
how this single transcription factor can regulate the transition of the
immature neural ectoderm, composed of proliferative precursor cells,
to neurally-committed progenitor cells, and then to definitive neural
plate cells that are beginning to differentiate.
Materials and methods
Creation of mutant FoxD4/D5 plasmids
The ΔN- and ΔC-FoxD4/5 plasmids were previously described
(Sullivan et al., 2001; Fig. 1A). We deleted and mutated additional
sites in Myc-tagged-foxD4/5 (Fig. 1A) in the pCS2+ vector using the
Quik-change mutagenesis kit (Stratagene). To delete the Acidic blob
sequence, the primer 5′-GAT TAT GCA GGA CTT TCT CCC TGC AGC
CTA AAG TCA C-3′ and its gene complement were used with an
annealing temperature of 55.0 °C for 1 min and extension was
performed at 68.0 °C for 10 min for 18 cycles. The ΔRII/Cterm-FoxD5
mutant was similarly constructed using the primer 5′-CCA TCC CAA
TTC ACA GAG CAA ATG TTG ATC TAG AAC TAT AGT GAG TCG-3′ and
its gene complement. The FSNIEI to AAAAAA mutant (A6-FoxD5)
was similarly constructed using the primer 5′-CCA TCC CAA TAA
TTC ACA GAG CAA ATG TTC AGC CGC TGC TGC GGC CGC CAT GAG
GAA ACC CAA GGA GCC-3′ and its gene complement. The FSNIEI to
ESNIEI point mutant (F>E-FoxD5) was similarly generated using
the primer 5′-CAG AGC AAA TGT TCA GAGA AGT ATT GAG AAC ATC
ATG AGG AAA CCC-3′ and its gene complement.
mRNA synthesis and injection
mRNAs encoding foxD4/5 wild-type and mutant proteins were
synthesized in vitro (Ambion, mMessage mMachine kit). These
mRNAs (100 pg/nl each) were mixed with nuclear-localized βgal
mRNA (100 pg/nl) as a lineage tracer. Embryos were obtained, cul-
tured and microinjected as previously described (Moody, 1999,
2000). One nanoliter of each mRNA mixture was microinjected into
a defined precursor of the neural ectoderm (blastomere D1.1;
Moody, 1987) on one side of the 16-cell embryo. This results in
FoxD4/5 protein expression in about 50% of the neural plate only on
the experimental side of the embryo, ensuring that the mutant pro-
teins do not disrupt earlier morphogenesis and avoiding non-
specific effects or embryonic lethal phenotypes. The uninjected side
of the embryo was used as an internal control.
Whole embryo in situ hybridization
Embryos were cultured to stage 10.5 (nascent neural ectoderm),
stage 12 (transition to neural plate) and stage 14/15 (differentiating
neural plate), and processed for in situ hybridization (ISH) as previ-
ously described (Sive et al., 2000). Anti-sense Dig-labeled RNA probes
were synthesized as previously described (Yan et al., 2009). The ex-
pression patterns of gem, sox2, sox3, sox11, soxD, zic1–3, and irx1–3
were compared on the experimental and control sides of embryos de-
rived from at least three different clutches of eggs from different sets
of adult parents. The frequency at which embryos showed altered ex-
pression was compared to the frequency from wt-FoxD5-injected
samples using the Chi-squared statistic (pb0.001).
Anti-sense morpholino oligonucleotides directed against Xenopus
Gro/Grg4 (GroMO; GGTACATCTTGCTCAAGTCTCGAAT, Gene Tools,
LLC) were used to decrease the levels of endogenous Gro/Grg4.
Based on sequence analysis, GroMO will not bind to any other mem-
ber of the Xenopus Gro/TLE family. The effectiveness of GroMO to
block translation of an HA-tagged Gro/Grg4 protein was demonstrat-
ed by injecting Xenopus oocytes with 5 ng of in vitro transcribed
mRNA encoding either wild-type Gro/Grg4 or a mutant harboring 5
point mutations generated by PCR in the wobble codons of amino
K.M. Neilson et al. / Developmental Biology 365 (2012) 363–375
Fig. 1. Conserved domains in the FoxD4/5 proteins. (A) Wild-type (WT) and mutant constructs of Xenopus FoxD4/5. In ΔN, all of the amino acids upstream of a nuclear localization
signal (NLS, black) and winged helix (WH, red) DNA-binding domain were removed. In ΔC, all of the amino acids downstream of a second NLS and WH were removed. In ΔAB, only
the 14 amino acids constituting the Acidic blob were deleted. In ΔRII-Cterm, the entire R-II domain (dark green) as well as all of the amino acids downstream of it (light green) were
deleted. In A6, the amino acids constituting the Eh-1 motif (FSIENEM) within the R-II domain were mutated to AAAAAAM. In F>E, FSIENEM was mutated to ESIENIM.
(B) CLUSTALW alignment, viewed in ESPript (Gouet et al., 1999), of the N-terminal region of several vertebrate proteins in the FoxD4/5 family shows that within the AB domain
(underlined in yellow) there are several highly conserved residues. The black boxes highlight identical amino acids, the light boxes highlight conserved amino acids and the
bold letters indicate identical amino acids within a region of conserved amino acids. (UniProtKB/Swiss Prot Accession numbers are: human FoxD4, Q12950; human FoxD4L1,
Q9NU39; mouse FoxD4, Q60688; Danio FoxD4L1, O73784; Xenopus FoxD4L1.1, Q9PRJ8). (C) CLUSTALW alignment of the C-terminal regions of several vertebrate FoxD4/5 proteins
shows that the Eh-1 motif (light green line) is highly conserved. The positions of the A6 and F>E mutations are indicated. The dark green line indicates the sequence that was de-
leted in the ΔRII-Cterm construct. The C-terminal amino acids of the Danio (C) and Xenopus (Y) proteins are shown, whereas the mammalian proteins contain 3 (mouse), 9 (human
FoxD4L1) or 19 (human FoxD4) more amino acids that are not shown (indicated by: …). Note several highly conserved residues (boxes and bold as in Fig. 1B) downstream of the
Eh-1 motif, and the location of a predicted α-helical region near the C-terminus in the Xenopus protein (blue line).
K.M. Neilson et al. / Developmental Biology 365 (2012) 363–375
acids 2–6 of Gro/Grg4 (rescue mRNA). These mRNAs were injected
alone or in combination with 5 ng of GroMO, and the oocytes cultured
overnight at 21 °C. Lysates were prepared and Western analysis using
HA antibody was performed (Supplemental Fig. 1). In addition, the
reversal of the GroMO phenotype in whole embryos was demonstrat-
ed by co-injecting 60 pg of the rescue Gro/Grg4 mRNA.
In some experiments GroMO was injected into a single dorsal-
animal 8-cell blastomere (20 ng or 40 ng) and subsequently one of
the 16-cell daughters was injected with 1 nl of wt-foxD4/5 mRNA
(100 pg/nl or 50 pg/nl) plus nuclear-localized βgal mRNA (100 pg/
nl) as a lineage tracer. In other experiments GroMO (20 ng) was
injected into a single ventral-animal 8-cell blastomere and subse-
quently the equatorial 16-cell daughter was injected with 1 nl of
ΔAB-foxD4/5 mRNA (100 pg/nl) plus βgal mRNA or with ΔAB-foxD4/
5 mRNA (100 pg/nl) plus rescue Gro/Grg4 mRNA (60 pg) plus βgal
Western blots and Co-IPs
Oocyteswere injected with mRNAscodingfor wt-FoxD4/5, mutant
FoxD4/5 constructs and/or Gro/Grg4 and cultured, as above. For each
immunoprecipitation reaction, 150 μl of lysate (15 oocyte equiva-
lents) was mixed with 650 μl ice-cold TNSG lysis buffer and 1 μg of an-
tibody (raised against HA or Flag; Applied Biological Materials) and
incubated at 4 °C for 1–2 h, after which 25 μl protein A/G agarose
beads (Santa Cruz Biotechnology) were added to the reaction and ro-
tated in an orbital mixer overnight at 4 °C. Beads were briefly pelleted
at 4 °C and rinsed 3 times with ice-cold TNSG lysis buffer. All residual
buffer was removed with a flat pipette tip and beads were resus-
pended in 45 μl 1× RIPA sample buffer (RIPA Buffer: 150 mM NaCl,
1% NP40, 0.5% Na Deoxycholate, 0.1% SDS, 50 mM Tris (8.0); 4× sam-
plebuffer:4 mL10%SDS,2 mLglycerol,0.3086 gDTT,0.00001 gBrom-
phenol Blue; 4× sample buffer was diluted to 1× in RIPA buffer).
Samples were boiled at 100 °C for 10 min prior to loading on Tris-
glycine SDS-Polyacrylamide 10% gels. For expression checks, 15 μl
(1.5 oocyte equivalents) lysate was prepared with 4× sample buffer
and loaded on Tris-glycine SDS-Polyacrylamide 10% gels. Proteins
were resolved by SDS/PAGE, transferred to Immobilon-P transfer
membranes (Millipore) using standard methods, and blocked in
Tris-buffered saline (25 mM Tris)+0.2% Tween-20 (TBST)+5% non-
fat dry milk for at least 2 h to overnight at 4 °C. Whenever possible,
IP-Western blots were incubated with the following HRP-conjugated
primary antibodies to reduce background: anti-HA-HRP-conjugated
(Roche), and anti-Myc-HRP (Thermo Scientific). Following antibody
minescent HRP antibody detection reagent (Denville Scientific Inc.)
and exposed to film.
To demonstrate that mutant FoxD4/5 proteins had access to the
nucleus, dorsal blastomeres were injected with myc-tagged mRNAs
and embryos fixed in 4% paraformaldehyde at stage 11. Frozen sec-
tions were cut with a cryostat and subjected to standard immunoflu-
orescence staining protocols using an anti-Myc-tag primary antibody
(#9B11, Cell Signaling Tech., 1:2000), a goat anti-mouse IgG Alexa
Fluor 488 conjugated secondary antibody (#4408, Cell Signaling
Tech., 1:1000) followed by counterstaining of the nuclei with DAPI.
Images were collected using a Zeiss LSM 710 confocal system
equipped with 32-channel spectral photomultiplier. Thirty-two chan-
nel spectral stacks were collected at spectral resolution of 9.6 nm
within the range of 418–726 nm. To obtain the signature spectral
curves of autofluorescence, DAPI and Alexa Fluor 488 emissions, spec-
tral confocal images were taken with excitation of either the 405 nm
diode laser (DAPI and autofluorescence) or the argon 488 laser line
(Alexa Fluor 488); these spectral curves were then used to unmix
the DAPI, autofluorescence and Alexa Fluor 488 emissions registered
upon simultaneous excitation of the samples with 405 and 488 laser
lines (Supplemental Fig. 2).
To test whether C-terminal mutant FoxD4/5 proteins blocked BMP
signaling, ventral blastomeres were co-injected with non-tagged
mRNAs plus cytoplasm-localized βgal mRNA as a lineage tracer
(100 pg/nl each). Embryos were fixed in MEMPHA at stage 11 and
processed for whole mount immunostaining as previously described
(Yan et al., 2009) using an anti-Phospho-SMAD1,5,8 primary antibody
(#9511, Cell Signaling Tech., 1:100) and a goat anti-rabbit IgG HRP-
conjugated secondary antibody (#7074, Cell Signaling Tech., 1:250).
FoxD4/5 proteins contain highly conserved Acidic blob and Eh-1 domains
We previously demonstrated that FoxD4/5 can both up-regulate
and down-regulate downstream targets (Sullivan et al., 2001; Yan
et al., 2009, 2010). To determine whether FoxD4/5 contains domains
indicative of both activating and repressing transcriptional activity,
vertebrate FoxD4/5 proteins (Fig. 1). Within the region N-terminal to
the winged-helix DNA-binding domain (WH) in Xenopus FoxD4/5
there is a 14 amino acid Acidic blob (AB) region (Fig. 1A). Within the
AB there are several residues that are highly conserved across verte-
brates (Fig. 1B). Within the region C-terminal to the WH in Xenopus
FoxD4/5 there is a P/A/Q-rich region, an R-II domain, and within the
R-II there is an Eh-1 motif (Fig. 1A). The Eh-1 motif and several down-
stream amino acids are highly conserved across vertebrates (Fig. 1C).
To determine whether any of these regions are specifically required
deletion constructs (Fig. 1A). We deleted: 1) the entire region (ΔN) up-
stream of a nuclear localization signal (NLS); 2) just the Acidic Blob
(ΔAB); 3) the entire region (ΔC) downstream of another NLS; or 4)
the R-II domain plus all the sequence C-terminal to it (ΔRII-Cterm).
Each deletion construct produces abundant protein that can access the
nucleus (Sullivan et al., 2001; Supplemental Fig. 3).
FoxD4/5 activates two neural precursor genes via an Acidic blob domain
in the N-terminus
We previously showed that wild-type (wt) FoxD4/5 up-regulates
the expression of gem and zic2 in both the nascent neural ectoderm
of the gastrulating embryo and in the neural plate, and that this oc-
curs via transcriptional activation (Yan et al., 2009). To identify the
region(s) of FoxD4/5 that are responsible for target gene activation,
we injected mRNA encoding each FoxD4/5 deletion construct
(Fig. 1A) into a dorsal blastomere that gives rise to clones in the me-
dial neural plate, and analyzed gem and zic2 expression within the
lineage-labeled clone by in situ hybridization (ISH). As previously
reported, β-Gal-tagged wt-FoxD4/5-positive cells express higher
levels of gem and zic2 compared to neighboring cells (Fig. 2A). For
both genes, the ΔN-FoxD4/5 construct caused a significant reduction
in the percentage of embryos showing up-regulated gene expression
within the labeled clone, whereas the ΔC-FoxD4/5 construct did not
(Fig. 2A). This result demonstrates that an activation domain likely
resides in the N-terminal part of the protein. Due to the acidity and
high level of sequence conservation in the AB, we tested this region
for activation activity. The percentage of embryos showing up-
regulated gem or zic2 expression was significantly reduced in the
ΔAB-expressing labeled clone, whereas deleting the ΔRII-Cterm re-
gion had no significant effect on gem or zic2 up-regulation (Fig. 3A).
Thus, the activation of gem and zic2 by FoxD4/5 requires the AB do-
main and is independent of the RII+Cterm region.
K.M. Neilson et al. / Developmental Biology 365 (2012) 363–375
Down-regulation of sox neural progenitor genes is affected by both
N-terminal and C-terminal domains
Over-expression of FoxD4/5 down-regulates the expression of
three neural progenitor genes (sox2, sox3, sox11) in the gastrula neu-
ral ectoderm (Yan et al., 2009). We found that the down-regulation of
each sox gene was altered by both N-terminal and C-terminal se-
quences. First, while deletion of the entire C-terminus did not alter
the percentage of embryos showing down-regulation of sox2, it
caused a significant reduction in down-regulation of sox3 and sox11
(Fig. 2B). Injection of the ΔRII-Cterm construct identified this region
as required for the down-regulation of sox3 and sox11 (Fig. 3B).
Fig. 2. N-terminal sequences are required for up-regulation, and C-terminal sequences are required for down-regulation of FoxD4/5 targets. (A) The graph presents the percentage
of embryos in which WT-, ΔN- or ΔC-FoxD4/5 caused up-regulation of gem, zic2 or sox11 (the latter at neural plate stages). Numbers above each bar indicates sample size; * indi-
cates significant difference from WT at the pb0.001 level. The images for gem expression are representative for zic2; examples of sox11 are presented in Fig. 4. Examples of endog-
enous expression patterns can be found in Supplemental Fig. 2. The FoxD4/5-expressing clones, marked by nuclear β-Gal (red or purple dots), are located in the neural ectoderm
and indicated by hatched lines. Boxed insets are higher magnifications of the clone, the position of which is indicated on the whole embryo by a bracket. In the insets for wt-FoxD4/5
and ΔC-FoxD4/5, the β-Gal labeled cells are more intensely stained than neighboring cells (e) that show the endogenous level of gem expression. The intense blue ISH label often
obscures the red-labeled nuclei in these cases. In the inset for ΔN-FoxD4/5, the β-Gal labeled cells stain for gem expression only slightly higher than the endogenous level in neigh-
boring cells (e). This example would be scored as a positive up-regulation in the graph, even though the level of up-regulation is much lower compared to wt-FoxD4/5 and ΔC-
FoxD4/5. (B) The graph presents the percentage of embryos in which WT-, ΔN- or ΔC-FoxD4/5 caused down-regulation of sox2, sox3 or sox11 at gastrulation stages. The images
for sox2 expression are representative for sox3; examples of sox11 are presented in Fig. 4. In the insets for wt-FoxD4/5 and ΔN-FoxD4/5, the β-Gal labeled cells are less intensely
stained than neighboring cells (e) that show the endogenous level of sox2 expression. Often with wt-FoxD4/5 the effect is not uniform throughout the clone. The extent of
down-regulation of sox2 is greater for ΔN-FoxD4/5. In the inset for ΔC-FoxD4/5, the β-Gal labeled cells stain for sox2 expression about the same as the endogenous level in neigh-
boring cells (e), which is the most frequent phenotype. (C) The graph presents the percentage of embryos in which WT-, ΔN- or ΔC-FoxD4/5 caused down-regulation of zic1, zic3 or
soxD. The images for zic1 expression are representative for the other two genes. In the insets for wt-FoxD4/5 and ΔN-FoxD4/5, nearly all of the β-Gal labeled cells are less intensely
stained than neighboring cells (e) that show the endogenous level of zic1 expression. In the inset for ΔC-FoxD4/5, the β-Gal labeled cells stain with the same intensity as neigh-
boring cells (e), indicating a lack of down-regulation. m, non-involuted mesoderm that does not normally express zic1. (D) The graph presents the percentage of embryos in
which WT-, ΔN- or ΔC-FoxD4/5 caused down-regulation of irx1, irx2 or irx3. The images for irx1 expression are representative for the other two genes. In the insets for wt-
FoxD4/5 and ΔN-FoxD4/5, nearly all of the β-Gal labeled cells are less intensely stained than neighboring cells (e) that show the endogenous expression levels of irx1. In the
inset for ΔC-FoxD4/5, the β-Gal labeled cells stain with the same intensity as neighboring cells (e), indicating a lack of down-regulation. All images are dorsal views with vegetal
pole to the bottom.
K.M. Neilson et al. / Developmental Biology 365 (2012) 363–375
Second, the N-terminal region also affected the expression levels of all
three sox genes. For both sox2 and sox3, deleting the entire N-terminus
showing down-regulation in the gastrula neural ectoderm and in-
creased the extent of the down-regulation within the clone (Figs. 2B,
3B). This result indicates that the AB domain normally ameliorates the
repression of sox2 and sox3 by wt-FoxD4/5. Either wt-FoxD4/5 directly
or it activates genes that repress sox gene repressors; distinguishing
between these possibilities requires further investigation. In con-
trast, deleting either the entire N-terminus or just the AB domain
caused sox11 to be up-regulated in the gastrula neural ectoderm
(ΔN: 95.7%, n=46; ΔAB: 74.6%, n=67; Fig. 4), suggesting that wt-
FoxD4/5 normally activates a gene that represses sox11. In fact, we
previously showed that sox11 expression is down-regulated by Zic2
(Yan et al., 2009). Consequently, we propose that deletion of the
AB domain, which causes a loss of zic2 up-regulation, also leads to a
de-repression of sox11.
In contrast to gastrula stage embryos, sox11 is up-regulated by wt-
FoxD4/5 at neural plate stages (Fig. 4), and this appears to be by di-
rect activation (Yan et al., 2009). Consistent with the results obtained
for gem and zic2, the neural plate stage up-regulation of sox11 re-
quires the N-terminus, and specifically the AB domain (Figs. 2A, 3A,
4). However, the up-regulation of sox11 in the neural plate also re-
quires the RII-Cterm domain, and specifically the Eh-1 motif in the
C-terminus (Figs. 3A, 4). These results indicate that the up-
Fig. 3. The Acidic blob domain is required for up-regulation, and the Eh-1 domain and the C-terminal region downstream of it are required for down-regulation of FoxD4/5 targets.
(A) The graph presents the percentage of embryos in which WT- and mutant FoxD4/5 caused up-regulation of gem, zic2 or sox11 (the latter at neural plate stages). Numbers above
each bar indicates sample size; * indicates significant difference from WT at the pb0.001 level. The images for zic2 expression are representative for gem; examples of sox11 are
presented in Fig. 4. The FoxD4/5-expressing clones, marked by nuclear β-Gal (red or purple dots), are located in the neural ectoderm and indicated by hatched lines. Boxed insets
are higher magnifications of the clone, the position of which is indicated on the whole embryo by a bracket. For WT-FoxD4/5, ΔRII-Cterm, A6 and F>E, the β-Gal labeled cells are
more intensely stained than neighboring cells (e), that show the endogenous level of gem expression. The intense blue ISH label often obscures the red-labeled nuclei in these cases.
For ΔAB-FoxD4/5, the β-Gal labeled cells stain at the same level as endogenous (e), and thus do not show up-regulation. (B) The graph presents the percentage of embryos in which
WT- and mutant FoxD4/5 caused an initial down-regulation of sox2, sox3 or sox11 at gastrulation stages. The images for sox3 expression are representative for sox2; examples of
sox11 are presented in Fig. 4. For WT- and ΔAB-FoxD4/5, the β-Gal labeled cells are more weakly stained than neighboring cells (e) that show the endogenous level of sox3 expres-
sion. For the other mutants, the β-Gal labeled cells stain at levels similar to the endogenous expression in neighboring cells (e). (C) The graph presents the percentage of embryos in
which WT- and mutant FoxD4/5 caused down-regulation of zic1, zic3 or soxD. The images for zic3 expression are representative for the other two genes. For WT-, ΔAB and F>E-
FoxD4/5, the β-Gal labeled cells are less intensely stained than neighboring cells (e) that show the endogenous level of zic3 expression. For ΔRII-Cterm and A6-FoxD4/5 the β-Gal
labeled cells stain at levels similar to the endogenous expression in neighboring cells (e). *, indicates that the ΔRII-Cterm construct represses significantly less frequently than the
A6 construct (pb0.001). (D) The graph presents the percentage of embryos in which WT- and mutant FoxD4/5 caused down-regulation of irx1, irx2 or irx3. The images for irx2
expression are representative for the other two genes. For WT-, ΔAB and F>E-FoxD4/5, the β-Gal labeled cells are less intensely stained than neighboring cells (e) that show
the endogenous level of irx2 expression. For ΔRII-Cterm and A6-FoxD4/5, the β-Gal labeled cells stain at levels similar to the endogenous expression in neighboring cells (e). *, in-
dicates that the ΔRII-Cterm construct represses significantly less frequently than the A6 construct (pb0.001). All images are dorsal views with vegetal pole to the bottom.
K.M. Neilson et al. / Developmental Biology 365 (2012) 363–375
regulation of sox11 by FoxD4/5 can be achieved directly by activation
mediated by the AB domain and indirectly by repressing other target
genes mediated by C-terminal sequences. It should be noted that
while the deletion of the entire C-terminus significantly reduces up-
regulation of sox11 in the neural plate (Fig. 2A) the phenotype is
less frequent than for the more discreet deletions/mutations made
within the C-terminus (Fig. 3A). This requires further experimental
investigation, but may indicate involvement of the P/A/Q domain
(Fig. 1A) or reflect a conformational change that affects function
when such a drastic alteration to the protein is imposed.
SoxD is a member of the SoxG family that is unique to amphibians
and appears to act downstream of Sox2 to expand neural progenitors
(Rogers et al., 2009a). Its expression in the neural plate is down-
regulated by wt-FoxD4/5 (Yan et al., 2009). We found that the per-
centage of embryos in which soxD is down-regulated in the neural
plate is moderately, but significantly reduced by deletion of the AB
domain and eliminated by deletion of the RII-Cterm domain
(Fig. 3C). As for the other sox genes, these results indicate that wt-
FoxD4/5 affects soxD transcription by both activation and repression
most likely by involving intermediate genes.
FoxD4/5 represses genes that promote neural differentiation via the
Wt-FoxD4/5 strongly down-regulates five other neural transcrip-
tion factors that promote the expression of the bHLH neural differen-
tiation genes (zic1, zic3, irx1–3), and this appears to be mediated by
transcriptional repression (Yan et al., 2009). We found that both the
ΔN and ΔAB constructs caused repression of all five genes at frequen-
cies equivalent to wt-FoxD4/5 (Figs. 2C, D, 3C, D). In contrast, both the
ΔC and ΔRII-Cterm constructs failed to repress their expression, indi-
cating that the RII-Cterm domain is required for transcriptional re-
pression. Unlike the sox genes, the N-terminal portion of the FoxD4/
5 protein does not have a role in the repression of these neural
The role of Groucho binding in FoxD4/5-mediated transcriptional
Groucho (Gro) is a well-studied transcriptional co-repressor that
can bind to the Eh-1 motif and thereby mediate the repressive effects
of some Fox proteins (Sekiya and Zaret, 2007; Yaklichkin et al., 2007a,
2007b). Because the FoxD4/5 protein in a number of vertebrates also
contains an Eh-1 motif within the R-II domain (Fig. 1C), we tested
whether Gro/Grg4 is responsible for the ability of FoxD4/5 to repress
Fig. 4. Effects of FoxD4/5 constructs on the expression of sox11 at gastrula and neural plate stages. Top panel: At gastrulation stages, wt-FoxD4/5 causes β-Gal labeled cells (clone is
outlined) to express sox11 at lower levels than neighboring cells (e) that show the endogenous level of sox11 expression. In contrast, each mutant construct caused β-Gal labeled
cells to express sox11 at higher levels, indicated by darker staining compared to neighboring cells (e). All images are dorsal views with vegetal pole to the bottom. Bottom panel: At
neural plate stages, wt-FoxD4/5 causes β-Gal labeled cells (outlined within the normal expression domain in the neural plate) to express sox11 at higher levels than neighboring
cells (e). The same effect is observed with the ΔC-FoxD4/5 mutant, but in significantly fewer embryos compared to wtr-FoxD4/5 (see Fig. 2A). However, for all other mutant clones
(outlined only within the neural plate) the sox11 expression levels are similar to those of the neighboring cells (e) that show endogenous levels. Note that the neural plate is
broader on the injected side (red arrow) in embryos expressing N-terminal mutants (ΔN, ΔAB) but not in embryos expressing C-terminal mutants (ΔC, ΔRII, A6, F>E), consistent
with a previous report that the C-terminal domain is required for neural plate expansion (Sullivan et al., 2001). The ΔC image is a dorsal view with anterior to the bottom; all other
images are anterior views with dorsal to the top.
Fig. 5. Gro/Grg4 binds to the Eh-1 motif of FoxD4/5. (A-D) Myc-tagged versions of
(A6, F>E) or deleted for the Eh-1 domain (ΔRII) in FoxD4/5 were expressed in Xenopus
oocytes along with HA-tagged wild-type Gro/Grg4 (Grg4). Co-immunoprecipitation (IP)
and Western blot (WB) analyses of Xenopus oocyte lysates expressing HA- and Myc-
tagged constructs are indicated. (A) Although all constructs are equivalently expressed,
only full-length FoxD4/5 effectively binds with Gro/Grg4. The control panels (B–D)
show that the IPs each contain similar levels of FoxD4/5 wild-type and mutant proteins
(B), as do the direct lysates (C). Gro/Grg4 expressing lysates also show similar levels of
this protein (D).
K.M. Neilson et al. / Developmental Biology 365 (2012) 363–375
downstream genes. In Xenopus, Gro/Grg4 is widely expressed
throughout the neural ectoderm from the earliest stages (Molenaar
et al., 1997; Neilson et al., 2010). To assess whether FoxD4/5 and
Gro/Grg4 can interact, we conducted an immunoprecipitation (IP)
analysis of lysates from Xenopus oocytes co-expressing either wt-
FoxD4/5 or C-terminal deletion or amino acid substitution constructs
of FoxD4/5 along with Gro/Grg4. Co-IP analysis demonstrates that
Gro/Grg4 is found in the FoxD4/5 immunoprecipitates (Fig. 5A), indi-
cating that these two proteins interact in an in vivo Xenopus expres-
To test whether this interaction has a role in repressing down-
stream targets, we made the same mutations in the Eh-1 motif
that Yaklichkin et al. (2007a) showed prevents Gro/Grg4 binding
to Xenopus FoxD3. In one construct (A6), the first six amino acids
of the Eh-1 motif (FSIENIM) were changed to alanine (AAAAAAM),
and in the second construct (F>E), the first amino acid was changed
to glutamic acid (ESIENIM) (Fig. 1C). Both Eh-1 mutant constructs
are abundantly expressed in oocytes in the presence of Gro/Grg4,
but they do not interact with Gro/Grg4 in a co-IP assay (Fig. 5A). It
should be noted that the RII-Cterm deletion construct also does not
interact with Gro/Grg4 (Fig. 5A), which is expected because the en-
tire Eh-1 motif plus downstream sequence is deleted. The controls
show that the IPs contain similar levels of FoxD4/5 wild-type and
mutant proteins and that expression of the proteins was similar
among oocytes lysates (Fig. 5B–D).
We next tested whether the A6 or F>E mutants would fail to
down-regulate the sox, zic or irx genes, and thus implicate the require-
ment for Gro/Grg4 binding to the Eh-1 motif. The down-regulated tar-
get genes fell into two groups. Some target genes were repressed by
the A6 mutant at a frequency indistinguishable from the ΔRII-Cterm
deletion, suggesting that the binding of Gro/Grg4 to the Eh-1 motif is
responsible for repression (sox3, sox11, soxD, irx2) (Figs. 3B, C, 4).
There also was no significant difference between the ΔRII-Cterm and
A6 constructs for sox2 (Fig. 3B), but since neither these nor the ΔC-
FoxD4/5 construct altered the frequency of sox2 repression compared
to wt-FoxD4/5, we cannot with confidence conclude that binding of
Gro/Grg4 to the Eh-1 motif is involved. Other target genes were re-
pressed by the A6 mutant at a significantly lower frequency than the
ΔRII-Cterm construct (zic1, zic3, irx1, irx3) (Figs. 3C, D). This latter re-
sult indicates that there are additional regions in the RII-Cterm do-
main that are needed for full repression of the zic and irx genes. This
is supported by the observation that the point mutation, F>E, which
does not bind Gro/Grg4 in a co-IP oocyte assay (Fig. 5A), nonetheless
represses sox2, sox3, zic1, zic3, and irx1–3 at a frequency indistinguish-
able from that of wt-FoxD4/5 (Figs. 3B–D).
We next tested whether an interaction between FoxD4/5 and Gro/
Grg4 contributes to repressing those genes (zic1, zic3, irx1) for which
down-regulation requires additional C-terminal sequence. First, we
injected either foxD4/5 or gro/grg4 mRNAs at several concentrations
to find a dose of each that is not effective at repressing downstream
genes. For zic1, zic3, and irx1, 100 pg of foxD4/5 mRNA per 16-cell
blastomere caused repression in the majority of embryos (Yan et al.,
2009), whereas 10 pg caused repression in only a few embryos
(Fig. 6). Likewise, 10 pg of gro/grg4 mRNA per blastomere caused re-
pression of these genes in less than half of the embryos (Fig. 6). Next,
we co-injected these sub-optimal doses (10 pg foxD4/5+10 pg gro/
grg4 mRNAs); repression occurred at significantly higher frequencies
than either mRNA alone, nearly equivalent to the optimal 100 pg dose
of wt-foxD4/5 mRNA alone (Fig. 6). Thus, FoxD4/5 and Gro/Grg4 can
cooperatively repress these three neural genes in a dose dependent
manner. However, depleting endogenous Grg4 with a specific MO
(GroMO) did not reduce the ability of exogenous FoxD4/5 to efficient-
ly repress zic1, zic3, or irx1. Co-injecting embryos with a combination
of 100 pg wt-foxD4/5 mRNA+20 ng GroMO or with 50 pg wt-foxD4/5
mRNA+40 ng GroMO did not significantly reduce the repression of
these genes compared to mRNA injection alone. These results indicate
that Gro/Grg4 is not required for FoxD4/5 to repress zic1, zic3 or irx1.
It is possible that endogenous levels of other Groucho family mem-
bers that are expressed in the neural ectoderm (Molenaar et al.,
1997; Neilson et al., 2010) and are not targeted by the GroMO may
have substituted for Gro/Grg4 under these experimental conditions.
Alternatively, since binding site affinity can affect transcription factor
occupancy and activation versus repressive function (Essien et al.,
2009), Gro/Grg4 may facilitate FoxD4/5 repression of these genes
when the concentration/occupancy of FoxD4/5 is low, and not be re-
quired when the concentration/occupancy of FoxD4/5 is higher.
Ectopic induction of neural genes in the ventral ectoderm requires both
the AB and Eh-1 domains
Injection of FoxD4/5 mRNA into the ventral epidermal lineage in-
duces the ectopic expression of gem, zic2 and sox11, and down-
regulates BMP signaling and subsequent expression of epidermal
genes (Yan et al., 2009, 2010). We hypothesized that both gene acti-
vation and gene repression would be involved since ectodermal cells
must switch from an epidermal to a neural fate. In fact, deleting either
the N-terminus or the C-terminus reduced, but did not eliminate ec-
topic ventral induction of gem, zic2 and sox11 (data not shown), sug-
gesting that both domains, and thereby transcriptional activities, are
involved. This was confirmed by injecting the ΔAB or ΔRII-Cterm con-
structs; each significantly reduced, but did not eliminate, the frequen-
cy of ectopic expression (Fig. 7A). Also, the number of ventral
ectodermal cells expressing neural genes and the intensity of that ex-
pression was reduced compared to wt-FoxD4/5 (Fig. 7B). A role for
Gro/Grg4 is implicated because the A6 mutant caused a similar reduc-
tion in ectopic ventral induction of each gene. Because both activation
and repression are involved, we hypothesized that for optimal ectopic
Fig. 6. Gro/Grg4 and FoxD4/5 co-operate to cause transcriptional repression. The per-
centages of embryos showing decreased expression of zic1, zic3, or irx1 after injection
of: 100 pg wt-FoxD4/5 mRNA (Fox-100), 10 pg wt-FoxD4/5 mRNA (Fox-10), 10 pg
Gro/Grg4 mRNA (Grg4-10), 10 pg FoxD4/5 mRNA plus 10 pg Gro/Grg4 mRNA (Fox-
10+Grg4-10), 100 pg wt-FoxD4/5 mRNA plus 20 ng GroMO (Fox-100+GroMO),
50 pg wt-FoxD4/5 mRNA (Fox-50), or 50 pg wt-FoxD4/5 mRNA plus 40 ng GroMO
(Fox-50+2X GroMO). A low dose (10 pg) of either FoxD4/5 or Gro/Grg4 down-
regulates the expression of these three genes in many fewer embryos than a higher
dose of FoxD4/5 alone (100 pg). However, low doses of FoxD4/5 plus Gro/Grgr4 act
synergistically to restore down-regulation at a level significantly higher than either
mRNA alone (*, pb0.001), and at a level approaching 100 pg FoxD4/5 alone. However,
MO knock-down of endogenous Gro/Grg4 expression does not reduce the ability of
FoxD4/5 to cause down-regulation. At either a high (100 pg, black bar) or lower
(50 pg, dark purple bar) dose of FoxD4/5, addition of GroMO did not significantly
change the frequency of down-regulation of these genes (cf. to black bar to grey bar
and dark purple bar to light purple bar). Numbers above bars indicate sample sizes.
K.M. Neilson et al. / Developmental Biology 365 (2012) 363–375
induction the neural genes need to be up-regulated via an intact AB
domain and a second set of genes needs to be down-regulated via
binding of Gro/Grg4 to the Eh-1 motif. Two experiments support
this idea. First, co-injection of the ΔAB and A6 constructs completely
restored the frequency and intensity of ectopic ventral induction
(Figs. 7A, B). Since we assume that these constructs bind to DNA inde-
pendently, because only FoxP proteins have been shown to form di-
mers (Carlsson and Mahlapuu, 2002; Li et al., 2004; Wijchers et al.,
2006), they are likely to affect at least two different targets, activating
some (the A6 mutant contains an intact AB domain) and repressing
others (the ΔAB construct has an intact Eh-1 motif). Second, simulta-
neously preventing either activation or repression by co-injecting the
ΔAB construct with GroMO significantly reduced ventral ectopic in-
duction; this effect was rescued for gem (13/15) and zic2 (12/15;
Fig. 7B) by co-injecting a morpholino-insensitive version of Gro/
Grg4 (rescue; Supplemental Fig. 1). Together, these data demonstrate
that the ability of FoxD4/5 to ectopically induce neural genes in the
ventral epidermis requires both activation of the neural genes, medi-
ated via the AB domain, and repression, mediated via the Eh-1 motif
binding to Gro/Grg4. We predicted that the likely targets of repres-
sion are BMP signaling and epidermal genes because both are signif-
icantly repressed by wt-FoxD4/5 (Yan et al., 2009, 2010). This was
confirmed by showing: 1) neither the ΔRII-Cterm nor A6 construct
prevented the nuclear localization of phosphorylated Smad1/5/8,
Fig. 7. Both the Acidic blob and the Eh-1 motif are required for ectopic expression of gem, zic2 and sox11 in the ventral epidermis. (A) The percentages of embryos showing ectopic
ventral expression of gem, zic2, and sox11 after injection of wt- and mutant FoxD4/5 mRNAs. Although the AB and C-terminal mutants significantly reduced the frequency of ectopic
ventral expression compared to wt-FoxD4/5 (*, pb0.001), none eliminated it, indicating that both activating and repressing activities are required. Providing both activating and
repressing activities, by co-expressing both ΔAB and A6 mutants (ΔAB+A6), restored the frequency of ectopic ventral expression to wt levels. Conversely, eliminating both acti-
vating and repressing activities, by co-expressing both ΔAB and GroMOs (ΔAB+GroMO), significantly (pb0.001) reduced the frequency of ectopic ventral expression of all three
genes, compared to wt (*), ΔAB (#) or ΔAB+A6 ($). Numbers above bars indicate sample sizes. (B) Examples of the ventral ectopic expression of gem, zic2 and sox11 after injection
of each mutant mRNA (plus βgal, indicated by red or purple dots) into an epidermal precursor blastomere. In wt and ΔAB+A6 clones, most cells exhibit a high level of expression
(blue stain). In ΔRII, A6 and ΔAB+GroMO clones, fewer cells express the gene and expression is only faintly detectable. Co-expressing a morpholino-insensitive Grg4 mRNA with
ΔAB+GroMO rescued the high level of gem and zic2 ectopic ventral expression; surprisingly, this was not observed for sox11 (n=21).
K.M. Neilson et al. / Developmental Biology 365 (2012) 363–375
which indicates intact BMP signaling; and 2) neither repressed the
expression of two epidermis specific genes (Fig. 8).
FoxD4/5 contains both activating and repressing domains
FoxD4/5 has been shown to play a key role in early neural devel-
opment in Xenopus (Fetka et al., 2000; Sölter et al., 1999; Sullivan et
al., 2001; Yan et al., 2009). FoxD4/5 both up-regulates genes that
are involved in maintaining an immature neural ectoderm and
down-regulates genes that promote neural differentiation in the neu-
ral plate. To understand how this transcription factor regulates its nu-
merous targets, it is important to establish which regions of a Fox
protein outside the DNA-binding domain account for these different
kinds of transcriptional activities. Therefore, we sought to identify
the specific domains that are responsible for transcriptional activa-
tion and transcription repression.
Our results demonstrate that within the N-terminus the AB domain
is required for the up-regulation of gem and zic2 in both the neural ec-
toderm (Fig. 2) and the ventral epidermis (Fig. 7). In contrast, deletions
and mutations in the C-terminal part of the protein have a minimal ef-
fectontheir expression in theneural ectoderm.Since the up-regulation
of gem and zic2 occurs in the absence of protein synthesis, indicating
that FoxD4/5 directly activates these two genes, and is mimicked by a
VP16-FoxD4/5 activating construct (Yan et al., 2009), we conclude
that the AB comprises the transactivation domain of FoxD4/5. This is
scription factors (Ptashne, 1988; Schuddekopf et al., 1996).
Our studies also show that the Eh-1 motif is responsible for the
down-regulation of the sox genes and irx2 (Fig. 3). The Eh-1 motif
has been shown in Xenopus FoxD3 (Yaklichkin et al., 2007a), and
now in FoxD4/5, to bind the Gro/Grg4 co-repressor. In Xenopus, gro/
grg4 is expressed at low levels ubiquitously (Molenaar et al., 1997),
and is enhanced in the neural ectoderm and neural plate regions
(Neilson et al., 2010). Thus, it is endogenously available to interact
with FoxD4/5. Our experiments demonstrate that FoxD4/5 and Gro/
Grg4 can interact via the Eh-1 motif, and that Gro/Grg4 binding en-
hances the ability of low concentrations of FoxD4/5 to repress zic
and irx genes.
However, while the A6 mutant showed that the Eh-1 motif was
sufficient to account for the repression of some downstream neural
genes, this motif does not account for all of the repressive activity of
FoxD4/5. Repression of zic1, zic3, irx1 and irx3 requires additional se-
quence downstream of the Eh-1 motif (Figs. 3, 6). Analysis of the
FoxD4/5aminoacid sequenceusing a varietyof bioinformaticsservers
to predict secondary structure and identify additional functional do-
mains (PSIPRED: Jones, 1999; McGuffin et al., 2000; GlobPlot:
Linding et al., 2003; Porter: Pollastri and McLysaght, 2005) showed
be flexible and dynamic, forming multiple meta-stable conformations
that enable the protein to bind multiple targets (protein and/or DNA),
causing the protein to undergo transitions to more structured states
(Dyson and Wright, 2005). While no known functional domains or
protein interaction domains were identified, our sequence analyses
predicted a short α-helical region close to the C-terminus of the pro-
tein (Fig. 1C, blue bar) adjacent to a region that is highly conserved
across species. Interestingly, our analyses showed that mouse and
human FoxD4 proteins are also predicted to have a short α-helical re-
gion in this area. We propose that this conserved C-terminal region
with predicted secondary structure may influence the efficacy of tran-
scriptional repression by FoxD4/5, either by strengthening the inter-
action with Gro/Grg at the Eh-1 motif or by interacting with other
proteins. The fact that this region is intact in the F>E point mutant
may account for this construct's near wild-type ability to repress tar-
getgenes(Fig.3). WefoundthatmouseFoxA1andFoxA2alsoare pre-
dicted to a have short α-helical regions, but they are located in close
proximity to the Eh-1 motif rather than near the C-terminus. Because
the ability of these two proteinsto repress target genes relies on an in-
teraction with Gro/Grg that subsequently binds to acetylated histone
to compact nucleosomes (Sekiya and Zaret, 2007), we speculate that
the secondary structure of FoxD4/5 may participate in interactions of
Gro/Grg4 with other proteins.
FoxD4/5 regulates the transition of an immature neural ectoderm to a
differentiating neural plate by both gene activation and repression
Gene regulatory networks define the transcription factors in-
volved in a developmental process, the hierarchy of their functional
Fig. 8. C-terminal domains are required to prevent BMP signaling and repress epidermal gene expression in ventral ectodermal lineages. Wt-FoxD4/5 prevents nuclear accumula-
tion of phospho-Smad1/5/8 (see inset) and expression of epidermal genes (Yan et al., 2009), but C-terminal mutants do not. Left column: ventral ectodermal cells expressing either
the ΔRII-Cterm or A6-FoxD4/5 mutant protein (blue cytoplasm) are positive for nuclear-localized phospho-Smad1/5/8 (brown nuclei), indicating a response to BMP signaling
(ΔRII-Cterm: 100%, n=19; A6: 85.7%, n=21). For comparison, inset shows a wt-FoxD4/5 clone within the hatched lines (blue cytoplasm) in which phospho-Smad 1/5/8 staining
is not detected in most nuclei, whereas all nuclei outside the clone are stained. Middle and right columns: ventral ectodermal cells expressing either the ΔRII-Cterm or A6-FoxD4/5
mutant protein (red or purple nuclei within the hatched lines) express normal levels of epidermal genes (ΔRII-Cterm: AP2, 100%, n=45; Epi-ker, 100%, n=60; A6: AP2, 100%,
n=34; Epi-ker, 100%, n=62).
K.M. Neilson et al. / Developmental Biology 365 (2012) 363–375
interactions, and the regulatory loops that maintain a particular cellu-
lar state, and thereby elucidate the molecular regulation of a develop-
mental process (Levine and Davidson, 2005). We experimentally
defined the general epistatic relations between several early neural
transcription factor genes by gain-of-function and loss-of-function
studies in whole embryos, and showed that FoxD4/5 is a critical up-
stream component of this network (Yan et al., 2009). Increasing the
level of FoxD4/5 in the neural ectoderm differentially affected the ex-
pression levels of 11 other early neural genes (Fig. 9): two that are
known to promote a proliferative, immature neural ectoderm were
up-regulated by direct transcriptional activation; three that are
known to promote a neural progenitor state were down-regulated
transiently in the neural ectoderm of the gastrula (but not neural
plate); and six that are known to promote the expression of the
bHLHneuraldifferentiationgenes weredown-regulated via transcrip-
tional repression. Because these results indicated that FoxD4/5 can act
as both a transcriptional activator and repressor, we sought to identify
those protein domains that influence these various genes that togeth-
er regulate the transition of an immature, proliferative neural ecto-
derm to a neurally-committed neural plate whose cells are
beginning to differentiate (Fig. 9). Three functional regions of the pro-
tein were revealed: the AB, Eh-1 and RII+C-terminal domains.
When FoxD4/5 levels are high in the immature neural ectoderm,
gem and zic2 are up-regulated by transcriptional activation depen-
dent upon the AB domain. Like FoxD4/5, Gem and Zic2 both promote
a proliferative neural ectoderm and suppress neural differentiation
(Rogers et al., 2009a). gem blocks bHLH neural differentiation gene
transcription by regulating SWI/SNF chromatin-remodeling proteins
and keeping cells in the cell cycle (Kroll, 2007; Kroll et al., 1998;
Seo and Kroll, 2006; Seo et al., 2005). Zic2 represses bHLH neural dif-
ferentiation genes and counteracts the formation of ectopic neurons
produced by over-expression of Ngnr1 (Brewster et al., 1998). Our
previous work showed that downstream of FoxD4/5, Gem and Zic2
up-regulate each other and down-regulate zic1, zic3 and irx1–3 (Yan
et al., 2009). Therefore, we propose that at the initiation of neural
ectoderm formation, FoxD4/5 is expressed at high levels to coordi-
nately activate these two genes via the AB (Fig. 9), and together
they subsequently maintain an immature neural ectoderm and pre-
vent premature neural differentiation.
When FoxD4/5 levels are high in the immature neural ectoderm,
three sox genes are down-regulated by activities in both the AB and
Eh-1 domains (Fig. 9). sox2 and sox3 are transcriptional activators
thought to counteract the ability of SoxB2 repressor proteins to in-
duce neuronal differentiation (Bylund et al., 2003; Graham et al.,
2003; Uchikawa et al., 1999). Sox2 and Sox3 are required for the ex-
pression of bHLH neural differentiation genes, but when their levels
are experimentally increased in the embryo, there is a delay in
bHLH gene expression (Dee et al., 2008; Graham et al., 2003; Kishi
et al., 2000; Rogers et al., 2009b; Schlosser et al., 2008). This indicates
that when levels of Sox2/Sox3 are high they hold cells in an interme-
diate neural progenitor state. Fewer functional studies are available
for sox11, but it also appears to be involved in the transition of an im-
mature neural progenitor to an early differentiating state (Bergsland
et al., 2006; Hyodo-Miura et al., 2002; Uwanogho et al., 1995). Be-
cause FoxD4/5 delays the onset of bHLH neural differentiation gene
expression (Sullivan et al., 2001), we hypothesized that it delays the
transition to a neural progenitor state by down-regulating the expres-
sion of the sox genes during gastrulation (Rogers et al., 2009a; Yan et
al., 2009). Herein, we show that the early down-regulation of sox2,
sox3 and sox11 by FoxD4/5 in the neural ectoderm is affected by
both the AB and the Eh-1 domains (Fig. 9). These results suggest sev-
eral possible regulatory mechanisms including: 1) FoxD4/5 may both
activate and repress sox genes, dependent upon the concentration of
the protein, the affinities of the Fox binding sites in their enhancers,
or interactions with other factors at adjacent binding sites; 2)
FoxD4/5 may indirectly cause the down-regulation of sox genes by ac-
tivating and/or repressing intermediate genes; or 3) FoxD4/5 may ac-
tivate both sox genes and repressors of sox genes. For sox11, we favor
the last explanation based on two previous observations: 1) in the ab-
sence of protein synthesis, FoxD4/5 can directly activate sox11; and 2)
when Zic2 levels are increased, sox11 expression is down-regulated
(Yan et al., 2009). In this model FoxD4/5 likely activates both zic2
and sox11, but in those cells in which Zic2 achieves high levels,
sox11 would be repressed. However, FoxD4/5 repression of another
gene at neural plate stages also must be involved in sox11 up-
regulation, since deletions and mutations in the C-terminal region
prevent this phenotype. Distinguishing between these possibilities
requires further investigation.
High levels of FoxD4/5 down-regulate the expression of zic and irx
genes via the R-II plus downstream C-terminal sequence (Fig. 3).
These genes are effectors of initiating the gene program that activates
the bHLH neural differentiation genes (Rogers et al., 2009a); they ex-
pand the expression of bHLH neural differentiation genes in whole
embryo and explant assays, and the irx genes are required for the ex-
pression of the bHLH genes (Bellefroid et al., 1998; Gomez-Skarmeta
et al., 1998; Mizuseki et al., 1998; Nakata et al., 1997). Together
with the observation that the EnR-FoxD4/5 construct down-
regulates the zic and irx genes as effectively as wt-FoxD4/5 (Yan et
al., 2009), we conclude that these genes are directly repressed by
FoxD4/5 when levels of this protein are high. As endogenous levels
of FoxD4/5 drop during the formation of the neural plate, these
differentiation-promoting genes would be released from repression
from both FoxD4/5 and the transitional sox genes (Fig. 9).
We also provide evidence that the Eh-1 domain has an additional
involvement in repressing BMP signaling, which in turn prevents ex-
pression of epidermal fate (Figs. 8, 9). In this manner, FoxD4/5 pro-
vides a signaling environment that allows the formation of neural
ectoderm, even in ectopic locations. It will be critical to determine ex-
actly how the C-terminal regions interact with Gro/Grg4 to repress
the BMP pathway and to detail which specific parts of the BMP path-
ways are affected.
Fig. 9. Fox D4/5 plays a critical role in regulating a gene network that controls the tran-
sition of an immature neural ectoderm to neural progenitors, and then to a differenti-
ating neural plate. Wild-type FoxD4/5 affects the expression of three classes of neural
transcription factors affiliated with each of these phases of neural development, and
does so via different functional regions of the protein. First, the ability of FoxD4/5 to
up-regulate two genes that maintain a proliferative, immature neural ectodermal
state (gem, zic2) requires the AB activation domain. Second, the ability of FoxD4/5 to
down-regulate during gastrulation three sox genes that promote the transition to neu-
ral progenitors involves both repression via the RII-Cterm region and activation via the
AB domain. It is possible that the AB domain directly activates sox genes, or activates an
unknown factor (X), which in turn represses another gene (Y) that represses sox ex-
pression. Third, the ability of FoxD4/5 to down-regulate genes that promote neural dif-
ferentiation (zic, irx) requires the RII-Cterm region. FoxD4/5 additionally inhibits BMP
signaling, dependent upon the Eh-1 domain within the RII-Cterm region that leads to
repression of epidermal fate. Approximate timeline in Nieuwkoop and Faber (1967)
stages is given below.
K.M. Neilson et al. / Developmental Biology 365 (2012) 363–375
The identified functional domains are highly conserved across
These studies define for the first time several functional domains
in the FoxD4/5 protein that enable it to both activate and repress
transcription of downstream targets that play critical roles in expand-
ing the nascent neural ectoderm and regulating the onset of neural
differentiation. While it is notable that most of the target neural
genes have been studied in many other animals, only in Xenopus
have they been studied in relation to FoxD4/5. In fact, to our knowl-
edge there are no functional studies of the activity of FoxD4/5 in the
nervous system of any other vertebrate. Since these important func-
tional domains are highly conserved across vertebrates (Fig. 1), we
predict that the results reported herein are likely to apply to the func-
tion of the FoxD4/5 protein in many other animals, including humans.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ydbio.2012.03.004.
This work was supported in part by NSF grant IOS-0817902, funds
from the George Washington University Medical Center, and the In-
tramural Research Program of the NIH, National Cancer Institute. Con-
focal microscopy was performed at the GWU Center for Microscopy
and Image Analysis with support from NIH grants S10 RR025565
and P30 HD040677 (IDDRC at CNMC). We thank Dr. Anastas Popratil-
off (GWUMC) for confocal microscopy, Dr. John Orban (Univ. Mary-
land) for advice on protein structure analyses and Ms. Rebecca He
for immunostaining. Dr. Klein's efforts were supported by the Nation-
al Science Foundation while working at the Foundation. Any opinion,
finding, and conclusions or recommendations expressed in this mate-
rial are those of the authors and do not necessarily reflect the views of
the National Science Foundation.
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