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Article:Elsy, Michael, Rowbotham, Abigail, Lord, Hannah et al. (2 more authors) (2019) Xenopus
laevis FGF16 activates the expression of genes coding for the transcription factors Sp5and Sp5l. International Journal of Developmental Biology. pp. 1-10. ISSN 1696-3547
https://doi.org/10.1387/ijdb.190113mp

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Int. J. Dev. Biol. 63: 631-639 (2019)https://doi.org/10.1387/ijdb.190113mp
www.intjdevbiol.com

Xenopus laevis FGF16 activates the expression of genes
coding for the transcription factors Sp5 and Sp5l
MICHAEL ELSY, ABIGAIL ROWBOTHAM, HANNAH LORD, HARRY V. ISAACS and MARY E. POWNALL*
Biology Department, University of York, UK

ABSTRACT Fibroblast growth factors (FGFs) comprise a family of signalling molecules with essential roles in early embryonic development across animal species. The role of FGFs in mesoderm
formation and patterning in Xenopus has been particularly well studied. However, little is known
about FGF16 in Xenopus. Using in situ hybridisation, we uncover the expression pattern of FGF16
during early Xenopus laevis development, which has not been previously described. We showthat the zygotic expression of FGF16 is activated in the mesoderm of the early gastrula as a ringaround the blastopore, with its first accumulation at the dorsal side of the embryo. Later, FGF16
expression is found in the otic vesicle, the branchial arches and the anterior pituitary, as well as in
the chordal neural hinge region of the tailbud. In addition, we show that FGF16 can activate the
MAPK pathway and expression of sp5 and sp5l. Like FGF16, sp5 is expressed in the otic vesicle
and the branchial arches, with all three of these genes being expressed in the tailbud. These data
provide evidence that FGF16 is present in the early mesoderm and can activate the expression ofdevelopmentally important transcription factors.

KEY WORDS: mesoderm, secreted growth factor, cell signaling, transcriptional regulation

Introduction
Fibroblast growth factors (FGFs) are small polypeptide signalling molecules defined by a conserved 120-140 amino acid core
and their high affinity for heparan sulfate (Goetz & Mohammadi,2013). FGFs signal through a family of tyrosine kinase receptors(Zhang et al., 2006) coded for by four distinct FGF receptor (FGFR)
genes that can be differentially spliced (Johnson et al., 1991).
This, together with multiple distinct ligands, leads to the impressive complexity underlying FGF signalling. Activation of the FGF
pathway involves the formation of a tripartite signalling complexof FGF ligand, heparan sulfate, and FGFR (Turnbull et al., 2001).
Receptor dimerization activates one of four downstream signaltransduction pathways: mitogen-activated protein kinase (MAPK),
phospholipase-C g1 (PLC-g1), phosphatidylinositol 3-kinase (PI-3kinase) or Janus kinase/signal transducer and activator of transcription (Jak/STAT) (Dorey & Amaya, 2010; Ornitz & Itoh, 2015).
The autophosphorylation of tyrosine residues within the FGFR
intracellular domain results in the phosphorylation of FRS2a, a
kinase substrate constitutively associated with the FGFR, and thesubsequent recruitment of the GRB2 adapter protein. GRB2 thenrecruits the guanine nucleotide exchange factor, son of sevenless(SOS) (Ong et al., 2000). For the activation of the MAPK path-way, SOS activates Ras GTPase, in turn activating Raf (a MAP
kinase kinase kinase), Mek (a MAP kinase kinase) and MAPKERK in a cascade of phosphorylation events. Phosphorylated
ERK translocates to the nucleus and modifies the activity of Ets
family transcription factors, resulting in effects on gene expression
downstream of FGF signalling (Randi et al., 2009).
There are 22 members of the FGF family, which are divided into
subfamilies: paracrine FGFs (FGF1-10, 16-18, 20, 22), intracellular
FGFs (FGF11-14), and endocrine FGFs (FGF15/19, 21,23). Most
FGFs require N-terminal signal peptides for secretion (Käll et al.,2004). However, the FGF9 group, including FGF 9, 16 and 20,
lack cleavable signal peptides (Itoh & Ornitz, 2004). The secretion of these FGFs relies upon un-cleavable bipartite signals in
the N-terminal and central hydrophobic region (Miyakawa et al.,
1999; Revest et al., 2000), with secretion, nonetheless, requiring the Golgi and the endoplasmic reticulum (ER) (Miyakawa &Imamura,2003).
FGF16 was originally identified in the rat heart (Miyake et al.,1998) and has known cardiogenic and cardioprotective roles
Abbreviations used in this paper: FGF, fibroblast growth factor; MAPK: mitogen-activatedprotein kinase (also called ERK); Sp5 and Sp5l, proteins related to the humantranscription factor Sp1; X.laevis, Xenopus laevis; X.tropicalis, Xenopus tropicalis.

*Address correspondence to: Mary E. Pownall. Biology Department University of York, York, YO10 5DD United Kingdom. Tel +44 (0) 1904 328692.
Fax +44 (0) 1904 328505. E-mail: betsy.pownall@york.ac.uk
Submitted: 19 June, 2019; Accepted: 11 September, 2019.

ISSN: Online 1696-3547, Print 0214-6282© 2019 UPV/EHU Press
Printed in Spain

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(Hotta et al., 2008; Lu et al., 2008; Wang et al., 2015), as well as
roles in the development of the chick inner ear (Chapman et al.,2006; Olaya-Sánchez et al., 2017), the formation of pectoral finbuds in zebrafish (Nomura et al., 2006), and the specification of
GABAergic neurons and oligodendrocytes in the zebrafish forebrain(Miyake et al., 2014). RNA-seq analysis shows FGF16 is expressed
during gastrulation in X.laevis and X.tropicalis embryos (Owens
et al., 2016; Session et al., 2016). For example, FGF16.L expression peaks at NF stage 12 and remains high during neurulationin X.laevis, before decreasing in early and late tailbud stages,
whereas FGF16.S is expressed at a much lower level, and is not
expressed after gastrulation (Owens et al., 2016; Session et al.,2016). Attempts to clone FGF16 from Xenopus tropicalis were
unsuccessful (Lea et al., 2009), and the spatial expression pattern

TABLE 1PRIMER SEQUENCES (5’ TO 3’)
cdx4

ForwardGGGAGGAATGGAACTCTTATGG

ReverseTGTACCGCAGAGTCACAAAG

Egr1

ForwardTCGATCACCTAACAGCAGATG

ReverseGGTAGTCTTTGAGACAGGGTATG

Xbra

ForwardGAGCCCACTGGATGAAAGAT

ReverseAGCATGTGAAAGAGACGAGTAG

rpl8
Sp5
Sp5l
FGF16

ForwardGGGCTRTCGACTTYGCTGAA

ReverseATACGACCACCWCCAGCAA

ForwardGCTGTCCTAAGGAATGACTCTC

ReverseCTGCACAGAGAGAACACACTT

Forward

ACCTGCATCTTCTGCATCTC

ReverseGCCTGAAGATGACAGATATGG

ForwardATGGCTGAGATTGGGAGCGTT

ReverseTCACCTATAGTGATAAAGATC

of FGF16 has not been previously described during the development of X.tropicalis or X.laevis.
Transcription downstream of FGF signalling has been investigated in Xenopus mesoderm (Branney et al., 2009) and the genetic
targets identified included sp5 and sp5-like (sp5l), which code forzinc finger transcription factors (Ossipova et al., 2002). Despite
this finding in Xenopus, most of the knowledge linking sp5 andsp5l to a role downstream of FGF signalling comes from work inzebrafish. Sp5 (bts1) expression in the neural plate was found to
be strongly reduced in response to FGF inhibition (Tallafub et al.,2001), and sp5l expression in the mesoderm is also dependent
on the presence of FGF signals (Zhao et al., 2003; Weidinger etal., 2005). Consistent with a role downstream of FGF signalling,
Sp5l can posteriorize the neuroectoderm, as it positively regulates
posterior neuroectodermal marker hoxb1b and represses the anterior markers fez and otx1 in whole embryos (Zhao et al., 2003).
These studies support the idea that sp5 and sp5l are downstream
targets of FGF signalling in zebrafish. Here we describe the cloning and characterisation of X. laevis FGF16 and identify sp5 andsp5l as transcriptional targets of FGF signalling during amphibian
development.

Results
Cloning of Xenopus laevis FGF16
The predicted sequence for X. laevis FGF16 on chromosome 8L
encodes a 202 amino acid protein that is highly homologous (86%amino acid identity) to human FGF16. The genomic organisationshows syntenic regions in Xenopus and human chromosomes,
providing confidence that the sequence (GenBank Accession No.
XM_018229763.1) encodes for the full-length X. laevis FGF16.L
protein. PCR primers were designed against the predicted cDNA
Fig. 1. Phylogenetic analysis of predicted FGF families. FGF familygroupings are represented by brackets. Bold red brackets indicate the FGF9
subfamily: FGF9, FGF16 and FGF20. Protein sequences were aligned using
MUSCLE. The evolutionary history was inferred by using the Maximum
Likelihood method based on the Jones-Taylor-Thornton (JTT) matrix-basedmodel (Jones et al., 1992). The percentage of trees in which the associated taxa clustered together is shown next to the branches (500 bootstrap
support). The tree is drawn to scale, with branch lengths measured in thenumber of substitutions per site. Labels are as follows: xl = Xenopus laevis;
gg = Gallus gallus; hs = Homo sapiens.

Xenopus FGF16 activates sp5 and sp5l 633
sequence (Table 1) and a 609bp product was amplified
from cDNA derived from X. laevis embryos (NF stage 11).
Analysis of gene expression at gastrula stages
DIG-labelled antisense RNA probes were used for in situ
hybridisation analysis of FGF16 gene expression (Figs 2
and 3). FGF16 expression is restricted to the dorsal blastopore lip during stage 10, and as gastrulation proceeds,
the expression of FGF16 extends to the mesoderm aroundthe whole of the blastopore by stage 11 and is seen in
the posterior mesoderm around the closed blastopore atstage 12. We have analysed the genes sp5 and sp5l as
potential targets of FGF signalling; at gastrula stages, both
sp5 and sp5l have much wider expression domains than
FGF16 (Fig. 2 D-E and G-H). At NF stage 12, FGF16, sp5
and sp5l are all co-expressed in the posterior mesoderm
around the closed blastopore (Fig. 2 C, F, and I; arrow).
Analysis of FGF16 at later stages
Using in situ hybridisation we have found that FGF16
is expressed in the posterior mesoderm of early tailbud
embryos, as well as the otic vesicle and anterior pituitary(Fig. 3A,B). FGF16 expression is detected in the branchialarches and mesoderm of later tailbuds (Fig. 3C,D). In thetail, FGF16 expression is restricted to the chordoneural
hinge and the posterior wall of the neuroenteric canal(Fig. 3D) (Tucker & Slack, 1995). FGF16 is expressedin the anterior pituitary during early tailbud development(Fig. 3B) in X.laevis, consistent with findings in zebrafish(Miyake et al., 2014).

A

B

C

D

E

F

G

H

I

Fig. 2. Expression analysis FGF16, sp5 and sp5l in gastrula stage X.laevis embryos. In situ hybridisation analysis was used to detect transcripts for FGF16 (A-C),
sp5 (D-F), and sp5l (G-I) during gastrulation. Stages 10 posterior/vegetal (A, D, G)
11 posterior/vegetal view (B, E, H) and 12 (C, F, I) posterior view. Arrow indicatesdorsal blastopore lip in all panels.

Analysis of sp5 and sp5l in X.tropicalis embryos
The expression of sp5 and sp5l has been described in
X. laevis, but not in X. tropicalis, which we report here (Ossipova
et al., 2002). Sp5 expression in X. tropicalis at neurula stage 18 isfound in the midbrain and the neural crest, as well as being faintly
expressed towards the posterior of the embryo (Fig. 4 A-B). Sp5l isexpressed along the neural folds and in the posterior region of theembryo (Fig. 4 D-F) and also expressed in migrating crest cells inthe branchial arch region (Fig. 4 D). At stage 25, early tailbud, sp5
has clear expression in the head, including the forebrain,
midbrain, midbrain-hindbrain barrier (MHB) and otic
placode, but also in a small domain in the tailbud (Fig. 4
C). Sp5l at stage 26 is expressed in the posterior of theneural tube and the tailbud, but also in a small domain
in the head (Fig. 4G-H). At stage 31, sp5 expression in
X. tropicalis is found in the forebrain, midbrain, MHB,
branchial arches (BA) 1-4 as previously described inX.laevis (Square et al., 2015), and dorsal to the otic
A
vesicles and in the tailbud (Fig. 4 I-K). At this stage, sp5l
Fig. 3. Developmental expression pattern of FGF16 in
X.laevis embryos. In situ hybridisation analysis was used todetect transcripts for FGF16 at several developmental stages in
X. laevis embryos. At least 15 embryos were analysed at eachstage (st). (A,B) St 26, lateral and anterior views respectively.
(C,D) St 35, lateral and magnified lateral tail views respectively.
Abbreviations are as follows: anterior pituitary (ap), otic vesicle(ov), plate mesoderm (pm), branchial arches (ba) and chordoneural hinge (cnh).

Cis only expressed in the tailbud (Fig. 4 L-M). These data revealthat the expression patterns of sp5 and sp5l are distinct throughout
development and have some overlapping regions of expression to
FGF16, as well as other FGF ligands (Lea et al., 2009).
Analysis of FGF16 signal transduction
The N-terminus of most FGF ligands has a region of high

B

D

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M. Elsy et al.

Fig. 4. Developmental expression pattern of sp5 and sp5l in X.tropicalis
embryos. Analysis of sp5 and sp5l expression by whole-mount in situ
hybridisation of X. tropicalis embryos; at least 15 embryos were analysedat each developmental stage for each probe. (A,B) sp5 expression atneurula st18, lateral and dorsal views respectively. White triangles show
faint expression in the posterior region of the embryos. (C) sp5 expression
at tailbud st25, lateral view. (D,E) sp5l expression at neurula st18, lateraland dorsal views respectively. (F) Transverse section of the embryo in (E) at the level of the dashed line. (G) sp5l expression at tailbud st26, lateralview. (H) Transverse section of embryo in (G) at the level of the dashed line. (I-K) sp5 expression at tailbud st31, lateral view. (L,M) sp5l expression
at tailbud st31, lateral view. Abbreviations: Midbrain (MB), Neural crest (NC), Forebrain (FB), Otic placode (OP), Midbrain Hindbrain Boundary (MHB),
Migrating crest cells (MCC) Neural fold (NF), Neural tube (NT), Branchial Arches (BA), Otic Vesicle (OV).

hydrophobicity called a signal sequence that is required for
secretion. However, vertebrate orthologues of FGF9, FGF16
and FGF20 have divergent N-termini and lack a signal sequence(Miyakawa & Imamura, 2003). These FGF ligands are characterised by conserved mid-regions and C-termini that allow
secretion (Katoh & Katoh, 2005). This is shown in Fig. 5, where
the low N-terminus hydrophobicity for the FGF9 subfamily iscompared to FGF4 (Fig. 5A, compared to Fig. 5 B-D). The
FGF9 subfamily members contain an internal hydrophobicity
region, which likely aids the proteins’ secretion out of the cell
(Miyakawa & Imamura, 2003).
The animal cap is a source of pluripotent cells which develops into atypical epidermis in the absence of additional signals(Green, 1999). Upon treatment with growth factors, including
FGFs, the animal cap can be diverted from this epidermal fate
to differentiate into different tissue types, such as mesoderm(Kimelman & Kirschner, 1987; Slack et al., 1987). Therefore, thisapproach provides a robust biological assay for FGF16 activity(Fig. 6). ERK, also known as MAPK, is the effector of the RasRaf-MEK-MAPK signalling pathway. The diphosphorylation of
ERK (dp-ERK) indicates that FGF16 can strongly activate the
MAPK signalling pathway in animal caps (Fig. 6A). FGF16 isshown to affect cell behaviour, due to untreated animal caps
forming round balls of atypical epidermis after 3 days (Fig. 6B),
whereas those expressing FGF16 form vesicles of mesoderm
including blood, mesothelium and muscle (Fig. 6C).
FGF signalling activates sp5 and sp5l expression
Previous research suggests that X.laevis sp5 and sp5l are
positively regulated targets of FGF signalling (Branney et al.,2009; Park et al., 2013). To further test this possibility, the effect of increasing FGF signalling on sp5 and sp5l expression
was investigated. X. laevis embryos were injected with 10pg
of mRNA coding for FGF4, known to be a potent mesoderm
inducer (Isaacs et al., 1994), or 50pg of mRNA coding for
FGF16. RT-PCR was performed on stage 12 whole embryos
and animal cap explants from injected embryos compared touninjected controls. Expression of sp5, sp5l and known FGF
targets, xbra, cdx4, and gr1, was analysed. Both FGF4 andFGF16 injection induced the FGF target genes as well as sp5and sp5l (Fig. 6D).
Collectively these results show that sp5 and sp5l are downstream targets of FGF signalling, with the identification of FGF16
as a novel ligand that has mesoderm inducing activity.

Xenopus FGF16 activates sp5 and sp5l 635

Fig. 5. Hydrophobicity plots for
FGF9 family. Kyte and Doolittlehydrophobicity plots for (A) FGF4and FGF9 sub-family members, (B)FGF9, (C) FGF16, and (D) FGF20.
Scores above 0 (represented by thedotted line) indicate hydrophobic
amino acids; sites of high hydrophobicity are depicted as shaded
areas. FGF4 has a “classic” signal
sequence (SS) for co-translationalsecretion, but the members of the
FGF9 subfamily have an internalhydrophobicity region which is associated with secretion.

A

B

C

D

Discussion
FGF16 signal transduction
In this study, FGF16 was found to be able to activate MAPK
signalling and induce mesoderm. The Ras-Raf-MEK-ERK (MAPK)pathway has a well-defined role in regulating mesoderm induction
in response to FGF signalling (Cornell & Kimelman, 1994; LaBonneet al., 1995; LaBonne & Whitman, 1994; Umbhauer et al., 1995;
Whitman & Melton, 1992). FGF16 has also been shown to signal
through the MAPK pathway in human ovarian cancer cells (Basuet al., 2014), and here we demonstrate FGF16-mediated MAPK
activity to be observed in the mesoderm induction assay. The
role of FGF9, FGF16 and FGF20 in mesoderm induction has notbeen well characterised. However, FGF9/16/20, an ancestral formof the vertebrate FGF9 subfamily, has been shown to induce
mesenchyme formation in Ciona intestinalis embryos (Imai et al.,
2002; Tokuoka et al., 2004). The presence and activity of this classof FGF ligand in a distant chordate relative supports the notionthat FG9/16/20 is a conserved factor for mesoderm specification(Davidson et al., 2006). Furthermore, an RNA-seq screen of X.
laevis gastrula expression in dorsal and ventral lip tissue identified FGF16.S and FGF16.L as having moderate positive Pearson
correlation coefficients of 0.48 and 0.25 respectively to chordin,
indicating the two genes may share transcriptional regulation
mechanisms and that FGF16 may be involved in dorsal-ventral
patterning (Ding et al., 2017).
The paracrine FGFs signal through the four different FGFRs,
triggering multiple downstream signalling pathways to result in
the regulation of transcription factors required for controlling manydifferent developmental processes, such as mesoderm induction(reviewed in Ornitz and Itoh, 2015). The alternative splicing of

FGFR receptors greatly increases ligand binding specificity, particularly through the generation of two isoforms of Ig-like domain
III (epithelial b splice forms or mesenchymal c splice forms) (Yehet al., 2003). FGF9 subfamily members have similar FGFR binding
affinities (Zhang et al., 2006). For example, mouse homologue
FGF9 preferentially binds to FGFR2 and FGFR3 c splice variants,
displaying greatest affinity for FGFR3, but does not bind FGFR1 or
FGFR4 (Hecht et al., 1995; Ornitz et al., 1996; Santos-Ocampo etal., 1996). Zhang et al., (2006) confirm a strong binding of FGF9,
16 and 20 to FGFR3c, FGFR3b and FGFR2c when cataloguingthe receptor binding of all FGF ligands in the murine Baf3 cellline. FGF20 binding matches the other FGF9 subfamily members,
although it has higher affinity for FGFR2b (Zhang et al., 2006).
Murine FGF16 appears to have the highest affinity for FGFR3c,
followed by FGFR3b and FGFR2c, but does not bind to FGFR2b
or FGFR1b and only weakly binds FGFR4 (Zhang et al., 2006).
The same binding profile for FGF16 is revealed using the Baf3
assay, with very weak FGFR1c affinity demonstrated (Lavine etal., 2005; Lu et al., 2008). Furthermore, FGF16-mediated invasion in a human ovarian cancer model persists upon additionof a selective FGFR1 inhibitor, PD 173074 (Mohammadi et al.,1998), confirming FGF16 to not preferentially signal throughFGFR1 (Basu et al., 2014). Konishi et al., (2000) also report that
FGF16 only binds the extracellular domain of FGFR4, showing noaffinity for FGFR1c or FGFR2c, during embryonic brown adipose
tissue development, suggesting altered FGF16 ligand specificity
for different developmental stages or tissue types.
FGF16 secretion
Phylogenetic analysis of sequences obtained from Xenbase
matched the consensus arrangement of the 22 FGF members into

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M. Elsy et al.

7 different subfamilies (Fig. 1) (Itoh & Ornitz, 2004).
FGF9 subfamily members are highly homologous
in structure and have similar receptor bindingsites (Itoh & Ornitz, 2004; Zhang et al., 2006). For
example, they have a conserved C-terminus andcentral hydrophobic region, which is required for
secretion (Miyakawa & Imamura, 2003). Despite
this knowledge, the mechanism of FGF16 secretion
remains to be elucidated.
Most FGFs require N-terminal signal peptides for
secretion (Kapp et al., 2009). However, FGF1, FGF2,
FGF9, FGF16 and FGF20 lack cleavable signal
peptides. FGF1 and 2 can be released in response
to damage via an ER-Golgi independent exocytotic
secretory pathway (Itoh & Ornitz, 2004; Mignatti
et al., 1992). Instead, FGF2 directly translocatesacross the plasma membrane for secretion (Nickel,
2010; Zehe et al., 2006). Although FGF9 subfamily
members are secreted, they reply upon uncleavable bipartite signals including the N-terminal andcentral hydrophobic region (Miyakawa et al., 1999;
Revest et al., 2000). Interestingly, Miyakawa &Imamura (2003) showed that FGF16 is secreted bya process requiring the endoplasmic reticulum and
Golgi; retrograde Golgi transport of FGF16 was firstinhibited by using brefeldin A and N-glycosylation of
the N-terminus was separately observed, indicating that FGF16 must have progressed to the ER
for processing.

Fig. 6. FGF16 activates the MAPK pathway and activates sp5 and sp5l expression.
(A) Western blot to detect dpERK in uninjected control animal caps compared and animal
caps injected with 50 pg of mRNA coding for FGF16 mRNA. Antibodies to diphospho-ERK(dp-ERK) and total ERK were incubated on the same blot sequentially after stripping. (B)Control animal caps after 3 days culture. (C) Animal caps expressing FGF16 from siblingembryos to those shown in (B). (D) RT-PCR on cDNA derived from whole embryos atstage 12 and on animal cap explants cultured to stage 12 that were either uninjected
control explants or explants expressing FGF4 or FGF16. Expression of known FGF targets,
xbra (27 cycles), egr1 (27 cycles) and cdx4 (27 cycles), as well as sp5 (25 cycles), sp5l
and rpl8 loading control. Water, in which H2O replaced cDNA, and - RT, where no reverse
transcriptase was used, functioned as negative controls.

sp5 and sp5l expression and regulation
Our data indicate that sp5 and sp5l are candidates for regulation by FGF16. There is overlap of
their expression domains in the early mesoderm
and FGF16 is able to activate the expression ofboth sp5 and sp5l in a mesoderm induction assay.
Compared to its relatives, sp5 is unique having two paralogs in
the Xenopus and teleost lineages (Ossipova et al., 2002; Zhao
et al., 2003, Tallafub et al., 2001), whilst there is only one locus
present in mammals (Harrison et al., 2000). Phylogeny suggeststhat these genes were duplicated from the same ancestor, but
one copy was lost subsequently from the mammalian and bird
lineages in evolution (Zhao et al., 2003; Pei and Grishin, 2015).
In Xenopus these paralogs are referred to as sp5 and sp5l.
The Sp1-like transcription factors are a family of proteins with
important regulatory roles in development (Reviewed by Zhao andMeng., 2005). The family comprises of Sp1 along with a numberof structurally similar transcription factors which share defining
features such as a triple C2H2 zinc finger domain in the C-terminal
region and a preceding buttonhead (Btd) box (Reviewed by Zhao
and Meng, 2005; Ossipova et al., 2002). Sp1-like transcription
factors have been identified in a variety of species, including
Xenopus, which has 10 members (Presnell et al., 2015). Thesetranscription factors regulate the expression of target genes byacting as transcriptional activators or repressors in a contextdependent manner (Fujimura et al., 2007; Hagen et al., 1995;
Birnbaum et al., 1995; Phan et al., 2004; Majello et al., 1997).
The DNA binding specificity of these factors is similar across thefamily, with the conserved triple zinc finger domain recognising

GC-rich sequences in the promoter regions of genes (Kadonaga
et al., 1987). Differences in key residues of the zinc finger motifs
modulate this DNA binding specificity between family members(Reviewed by Kaczynski et al., 2003). Although this family sharescommonality in structure and DNA binding, different members
appear to play distinct roles in embryonic development, which isdemonstrated by their dynamic expression patterns (Reviewed
by Zhao and Meng, 2005). In situ hybridisation in Xenopus species showed that sp5 and sp5l have distinct expression patterns
throughout development.
Sp5 and sp5l clearly have differential expression patterns
throughout development, indicating they likely have different
functional roles. Presence of sp5 in the neural crest and related
structures, the branchial arches and otic vesicle, suggests a rolein neural crest formation. Supporting this hypothesis, knockdownof sp5 results in defective neural crest structures and sp5 overexpression or loss causes up- or down-regulation of neural crest
markers sox10, sox9 and slug (Park et al., 2013). Sp5l expression
is similar to posterior factors cdx4 and hoxA7, with expression in
the posterior neural tube and tailbud (Northrop and Kimelman,
1993; Pownall et al., 1998) suggesting a role in posterior patterning.
In comparison to Xenopus, murine Sp5 is expressed in theprimitive streak throughout gastrulation and is subsequently inXenopus FGF16 activates sp5 and sp5l 637
the midbrain, MHB, neural tube, somites, pharyngeal region
and the tailbud (Harrison et al., 2000). Xenopus sp5 and sp5l
recapitulate different aspects of mammalian Sp5 patterning andpossibly function. Murine Sp5 and Xenopus sp5 share expression
in the midbrain, MHB and pharyngeal region, whereas Xenopus
sp5l and murine Sp5 share expression in the neural tube. Both
Xenopus paralogs are expressed in the tailbud, like murine Sp5.
Mammals do not possess Sp5l, hence mammalian Sp5 may
perform the roles of both Xenopus sp5 and sp5l. This suggests
a divergence of function in Xenopus Sp5 and Sp5l proteins after
their duplication event, whilst mammals lost their Sp5l locus andSp5 retained all function.

Materials and Methods
Embryos
X. laevis and Xenopus tropicalis embryos were obtained using artificial
fertilisation. X.laevis embryos were microinjected in 0.3 x normal amphibian medium (NAM) + 5% ficoll and cultured in 0.1 x NAM between temperatures of 14°C – 23°C. X.tropicalis embryos were cultured in MRS/20
between temperatures 20°C – 24°C. All embryos were staged according
to Nieuwkoop & Faber (1967).
Multiple sequence alignments for FGF16
Xenbase was used to obtain the available X. laevis FGF amino acidand coding sequences (Karimi et al., 2018). BLAST (Basic Local Alignment Search Tool) searches identified the Gallus gallus (chick) and Homosapiens (human) FGF sequences (Priyam et al., 2015). X. laevis FGF16
is unavailable on Xenbase; the human FGF16 amino acid sequence wasused to identify the X. laevis FGF16 genomic sequence. The apparent
evolutionary relationship between all members of the X. laevis, Gallus gallus and Homo sapiens FGF families was examined. A maximum-likelihood
phylogenetic tree was created using Mega7 software and alignments
performed for amino acid sequences using MUSCLE (Edgar, 2004; Kumar
et al., 2016). FGF9, FGF16 and FGF20 alignments were created using
GeneDoc software (Nicholas, 1997).
Cloning of Xenopus laevis FGF16
RNA was extracted from stage 11, 17, 20 and 25 X. laevis embryos
via Trizol (Invitrogen) extraction and the Zymo-SpinTM IC column RNA
preparation procedure (Zymo Research), according to the manufacturer’s
instructions. Extracted RNA was used to generate cDNA using the SuperScript® IV First-Strand Synthesis System (Invitrogen). Stage 11 cDNA wasamplified by polymerase chain reaction (PCR) with GoTaq® Green MasterMix (Promega) and specific FGF16 primers (Table 1). PCR products wereligated into the pGEM-T Easy DNA vector (Promega), and subsequently
subcloned into pCS2+ using EcoR1.
In vitro transcription of DIG-labelled probes
pGEM-FGF16 was linearized using Sph1; pCS107 containing Sp5(GenBank Accession: AAH62500) using NcoI, and pCS107 containing
Sp5l (GenBank Accession: AAI54679) using HindIII before transcription
using the DIG RNA Labelling Kit (Roche). T7 RNA polymerase was used
for sp5 and sp5l DIG transcription and SP6 RNA polymerase for FGF16.
In situ hybridisation
Embryos were collected at the appropriate stages and fixed in MEMFA(0.1M MOPS, 2mM EDTA, 1mM MgSO4, 3.7% formaldehyde). In situ hybridisations were performed following the procedure described in Fisher
et al., (2002). Hybridisation was carried out overnight at 60°C with 1mg/
ml of FGF8- and FGF16- DIG probes. Embryos were then incubatedovernight at 4°C with a 1/2000 dilution of affinity-purified sheep anti-DIG
antibody coupled to alkaline phosphatase (AP) in blocking solution. Colour
reactions were subsequently performed overnight using the BM purple

precipitating AP detection system (Roche). Pigment was removed using
5% H2O2 solution in PBS.
Photography
Images were taken using the SPOT insight 4 MP CCD colour camera
attached to a Leica MZFLIII microscope.
Generating synthetic mRNA
pCS2+-FGF16 was linearized using NotI and Capped FGF16 mRNA
was generated using the mMessage mMachine SP6 transcription kit (Invitrogen), following the manufacturer’s instructions.
Western blot analysis
X. laevis animal cap explants were dissected at NF stage 8 and cultured
until NF stage 12 when they were collected and homogenised in sample
buffer and centrifuged (Keenan et al., 2006). Supernates were loaded
onto an acrylamide gel for SDS-PAGE and protein electro-transferred onto
Immobilon-P PDVF membranes (Millipore). Membranes were blocked in 5%
milk/PBSAT. Primary antibody dilutions were: mouse a-dp-ERK (Sigma),
1/4000; a-total-ERK, 1/500,000; a-pP38, 1/2000; a-pAKT, 1/5000. Secondary antibody dilutions were: dpERK a-mouse HRP, 1/4000; total ERK,
pP38 and pAKT a-rabbit, 1/2000. BM chemiluminescence blotting substrate(Roche) was used for peroxidase activity detection.
Semi-quantitative RT-PCR
5 embryos, or 10 animal cap (ectodermal) explants, were flash-frozen
on dry ice, and RNA was extracted using TRIzol Reagent® (Sigma). cDNA
was synthesised from 1mg of total RNA using SuperScript® IV Reverse
Transcriptase (Invitrogen). In short, total RNA was incubated for 5mins
at 65℃ with 50mM random hexamer primers and then incubated withreverse transcriptase at 23℃ for 10mins, 55℃ for 10mins and 80℃ for
10mins. To check for any genomic contamination, control RNA was also
processed without reverse transcriptase. PCR amplification was performed
using primers in Table 1, including those for ribosomal protein L8 (rpl8) asa ubiquitously expressed loading control.
Acknowledgements
ME, AR and HL undertook this work as part of their undergraduate
research projects at the University of York.

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