Integrative and Comparative Biology, volume 50, number 1, pp. 22–34
doi:10.1093/icb/icq048
SYMPOSIUM
Alternative Splicing in Development and Function of Chordate
Endocrine Systems: A Focus on Pax Genes
Linda Z. Holland1,* and Stephen Short†
*Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla,
CA 92093-0202, USA; †Institute of Marine Sciences, School of Biological Sciences, Portsmouth University, Portsmouth,
PO4 9LY, UK
From the symposium ‘‘Insights of Early Chordate Genomics: Endocrinology and Development in Amphioxus,
Tunicates and Lampreys’’ presented at the annual meeting of the Society for Integrative and Comparative Biology,
January 3–7, 2010, at Seattle, Washington.
1
Email: [email protected]
Synopsis Genome sequencing has facilitated an understanding of gene networks but has also shown that they are only a
small part of the answer to the question of how genes translate into a functional organism. Much of the answer lies in
epigenetics—heritable traits not directly encoded by the genome. One such phenomenon is alternative splicing, which
affects over 75% of protein coding genes and greatly amplifies the number of proteins. Although it was postulated that
alternative splicing and gene duplication are inversely proportional and, therefore, have similar effects on the size of the
proteome, for ancient duplications such as occurred in the Pax family of transcription factors, that is not necessarily so.
The importance of alternative splicing in development and physiology is only just coming to light. However, several
techniques for studying isoform functions both in vitro and in vivo have been recently developed. As examples of what is
known and what is yet to be discovered, this review focuses on the evolution and roles of the Pax family of transcription
factors in development and on alternative splicing of endocrine genes and the factors that regulate them.
Introduction
In the past 10 years, as the complete genome sequences of a wide range of organisms have become
available, a major focus of comparative genomics has
been on evolutionary changes in cis-regulation as a
driving force in animal evolution (Carroll et al. 2004;
Davidson and Erwin 2010). The best-studied gene
network is that mediating patterning of the sea
urchin endoderm (Peter and Davidson 2010).
Comparisons with the network patterning the endoderm in starfish have identified both conserved kernels of gene networks together with many diverged
connections (McCauley et al. 2010). Recently, comparative genomics has received a major boost from
‘‘next generation’’ sequencing, which provides much
shorter reads (18–400 or more bases) than the conventional Sanger sequencing, but provides high
throughput at a comparatively low cost. These
shorter reads can be assembled into a complete
genome sequence by matching to the genome of a
related organism (reference genome) or used to look
for conserved noncoding DNA or particular motifs
such as miRNAs or transposable elements. Data generated by these methods alone and in combination
with various types of gene chips/microarrays together
with high-throughput sequencing (Solexa/Illumina,
Solid) are giving insights into direct targets of individual transcription factors (ChIP on chip, ChIP-seq)
and alternative splice forms expressed at particular
times in development or under different physiological conditions (exon-microarrays, RNA-seq).
These new technologies are helping to show that
changes in cis-regulation, such as have occurred
repeatedly in the Pitx gene of stickleback fish, causing reduction of the pelvic girdle (Chan et al. 2010)
are just one of many driving forces in evolution.
Protein-coding sequences can also change,
and these changes can contribute to evolution of
development, reproductive isolation and speciation
[reviewed by Lynch and Wagner (2008) and
Advanced Access publication May 18, 2010
ß The Author 2010. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved.
For permissions please email: [email protected].
23
A focus on Pax genes
Palumbi (2008)]. For example, mutation of
C-terminal serine/threonine phosphorylation sites in
the homeodomain protein Ubx has been implicated
in evolution of a domain that represses formation of
abdominal appendages in six-legged arthropods
(Ronshaugen et al. 2002), while evolution of placental development in mammals correlates with positive
selection on the HoxA-11 protein, which is a direct
regulator of prolactin (Lynch et al. 2008). Moreover,
positive selection acting on gamete-interaction proteins has played a role in speciation of sea urchins
(Landry et al. 2003; Moy et al. 2008). However, in
addition to these genetic changes, a wide range of
epigenetic changes has been implicated as driving
evolutionary change. There are several definitions
of what constitutes epigenetics. The most common
one is heritable traits not directly encoded by the
genome. However, this definition is imperfect.
Some epigenetic traits such as DNA methylation
are often, but not always, inherited, while the proteins that mediate some epigenetic phenomena such
as alternative-splicing are encoded by the genome. In
addition, genomic elements such as transposons can
be subject to epigenetic modifications such as methylation, with the degree of methylation varying with
the location of the transposons in the genome and
among individuals (Reiss et al. 2010). In addition to
methylation, epigenetic mechanisms include a variety
of DNA modifications and posttranscriptional and
posttranslational modifications. The present review
concerns the evolution and the roles in development
and physiology of just one of these mechanisms,
alternative-splicing, with particular attention to the
Pax family of transcription factors and the role of
alternative splicing in endocrine systems.
Alternative splicing of pre-mRNAs is the
rule for eukaryotes
The organization of genes into exons and introns,
which are spliced out of pre-mRNAs to create the
mature mRNA, is ubiquitous in eukaryotes (Koonin
2009). Although some pre-mRNAs of multi-exon
genes are constitutively-spliced—that is, each intron
is spliced out and all the exons are incorporated into
the mature mRNA—it has been estimated that 490%
of human pre-mRNAs are alternatively spliced
(Blencowe et al. 2009). The more exons, the greater
is the number of potential splice forms. Thus, while
the number of protein-coding genes in the Bilateria
ranges from about 14,000 in Drosophila melanogaster
to 21,900 in the cephalochordate Branchiostoma floridae to 25,000–30,000 in humans (Claverie 2001;
Putnam et al. 2008), the proteome is typically
much larger. Splicing is mediated by a suite of five
small nuclear ribonucleoproteins (snRNPs) plus several proteins that recognize the sequences at splice
junctions [reviewed by Ritchie et al. (2009)]. The
canonical splice junction consists of AG/GTRAGT
and YYTTYYYYYNCAG/G at the donor (50 ) and acceptor (30 ) splice sites, respectively, where R is A or
G, Y is C or T, and N is any base. The canonical
GT–AG donor and acceptor sequences occur over
98% of the time (Burset et al. 2001). However,
there are exceptions such as GC-AG and AT-AC.
Alternative splicing can occur at both the canonical
and noncanonical sites (Rovescalli et al. 2000). Some
exons may have internal donor or acceptor
splice-sites that can mediate alternative splicing. A
database of alternative splice events and the isoform
splice patterns of genes from human and other species is available at http://www.ebi.ac.uk/asd/. The determinants of which alternative splice-sites are used
in a given cell types are very complex. They include
splice-site recognition by splicing factors, splicing silencers, inhibitors, and activators. The specific sequences that bind these factors and their distance
from the splice junction are important parameters
for regulation of alternative splicing. Up-to-date reviews on the splicing mechanisms and techniques for
identifying alternatively spliced exons are from Chen
and Manley (2009), Ritchie et al. (2009), and
Licatalosi and Darnell (2010).
The evolution of chordate Pax genes
Pax genes code for transcription factors with two
DNA-binding domains (the paired domain and the
homeodomain), a co-factor-interacting domain (the
octapeptide) and C-terminal activation and repression domains. Due to gene duplications in
pre-bilaterians, there were five Pax genes (Pax1/9,
Pax2/5/8, Pax3/7, Pax4/6, and Pox neuro) at the
base of the chordates. Pax1/9 genes have lost the
homeodomain, and Pax2/5/8 genes have only a partial homeodomain. Pox neuro is present in amphioxus, but has been lost from tunicates and vertebrates.
Early in the vertebrate lineage, the genome underwent two rounds of whole-genome duplication
with subsequent loss of many gene duplicates
(Putnam et al. 2008; van de Peer et al. 2009).
Thus, the basal chordate amphioxus has about
21,999 genes, while the human only has
25,000–30,000. However, duplicates of transcription
factors and genes in signaling pathways were preferentially retained (Putnam et al. 2008). Consequently,
there are nine Pax genes in mammals and, due to an
additional genome duplication at the base of the
24
teleosts (Meyer and Van de Peer 2005; Van de Peer
et al. 2009) there are 12 Pax genes in teleost fish.
Pax genes are interesting from the point of view of
chordate embryogenesis because they have key roles
in the development of such organs as the eye, muscles, central nervous system (CNS), kidney, pituitary,
the pharynx and its derivatives, and the gut and
related organs. Moreover, not only are Pax genes
involved in development of several endocrine
organs, they also help regulate endocrine genes.
Developmental expression of Pax genes is highly conserved in amphioxus and vertebrates. In both chordates, Pax1/9 genes are expressed in the pharyngeal
endoderm and developing muscles, and Pax2/5/8 in
the pharyngeal arches/forming gill slits in aquatic
vertebrates and amphioxus, the endostyle and/or its
homolog, the thyroid, the developing kidney, the
hindbrain and spinal cord, and pigment cells of the
L. Z. Holland and S. Short
eye in vertebrates and anterior photoreceptor in amphioxus (Heller and Brandli 1997, 1999; Kozmik
et al. 1999) (Figs. 1 and 2). Pax3/7 is expressed in
the edges of the neural plate and in developing
muscle in both amphioxus and vertebrates
(Goulding et al. 1991; Holland et al. 1999), while
Pax4/6 is expressed in the olfactory epithelium, anterior photoreceptor (amphioxus) or eye (vertebrates), the lamellar body (amphioxus) and its
homolog the pineal in vertebrates, and in the pituitary (vertebrates) or its homolog, Hatsheck’s diverticulum (amphioxus) (Glardon et al. 1998; Rath
et al. 2009). Pax3/7 is also expressed in the pharyngeal endoderm in amphioxus, but apparently not in
vertebrates (Holland et al. 1999).
Additional expression domains for Pax genes arose
in vertebrates subsequent to gene duplication and the
evolution of new structures. In mammals, Pax1 and
Fig. 1 Expression of Pax2/5/8 genes shows both evolutionary conservation and subfunctionalization and neofunctionalization
subsequent to gene duplication in vertebrates. (A) In amphioxus, the single Pax2/5/8 gene is broadly expressed in the hindbrain and
spinal cord (hb þ sc), pigment cells of the frontal eye (fe), the pronephric kidney (pk), endostyle (e), and around the forming gill slits
[branchial arches (ba)] as well as in Hatsheck’s anterior left diverticulum, which will fuse with the ciliated pit to become Hatsheck’s pit,
homologous to the vertebrate pituitary. (B–D) All but the last domain is conserved in one or more of the three Xenopus Pax2/5/8
genes. Pax2/5/8 genes have also acquired new domains in the vertebrate midbrain–hindbrain boundary (mhb) and in the otic placode
and vesicle (ov). Pax2 (B) has retained all of the presumed ancestral vertebrate domains, while Pax5 (C) has lost all the domains except
for that at the midbrain–hindbrain boundary and otic vesicle, and Pax8 (D) has lost all the domains except for those in the otic vesicle,
hindbrain and spinal cord and pronephric kidney. Thy, thyroid (homolog of the endostyle); os, optic stalk; va, visceral arches;
homologous to the branchial arches of amphioxus. Not shown is conserved expression of amphioxus Pax2/5/8 and Xenopus Pax2 in the
forming anus. Amphioxus data from Kozmik et al. (2001). Xenopus data from Heller and Brandli (1997, 1999).
25
A focus on Pax genes
and Pax6 are expressed in the pancreas and intestine
in vertebrates (Ritz-Laser et al. 2002). Pax-6 is required for development of a cells, which secrete glucagon, whereas Pax-4 is required for insulin secretion
and development of b and d cells, which produce
somatostatin (Ritz-Laser et al. 2002). Pax4 protein,
which acts as a transcriptional repressor, binds to a
regulatory element in the insulin promoter, and suppresses transcription (Campbell et al. 1999). Pax4
and Pax6, which is a transcriptional activator, also
bind to the glucagon promoter (Ritz-Laser et al.
2002), and it may be that they fine-tune transcription by competing with one another.
Alternative splicing of chordate
Pax genes
Fig. 2 Homology of the endostyle and thyroid as evidenced by
gene expression in the lamprey. Side views (A, C, and E) and
cross-sections at the level of arrows through the endostyle
(B, D, and F) of the anterior end of early lamprey embryos.
(A–C) In addition to expressing Pax2, The endostyle of the early
lamprey embryo expresses Ttf1, which is also expressed in the
amphioxus and tunicate endostyle and in the thyroid of jawed
vertebrates (McCauley and Bronner-Fraser, 2002; Kluge et al.
2005). Ttf1 is also expressed in the forebrain. (E and F) By stage
Tahara 29, thyroxine (T4 thyroid hormone) is secreted by the
larval lamprey endostyle as shown by immunostaining. T4 and
Pax2.1 are also expressed in the early zebrafish thyroid (Wendl
et al. 2002). The lamprey endostyle ultimately metamorphoses
into a thyroid gland (Marine, 1913).
Pax9 are expressed in the primordia of the thymus
and parathyroid, which are derivatives, in part, of the
pharyngeal endoderm (Liu et al. 2007). Pax1 is essential for development of the thymus (Wallin et al.
1996). In addition, Pax9 is expressed in mammalian
thyroid cell lines, although it does not mediate transcription from a 2-kb calcitonin (CT) promoter
(Suzuki et al. 2007) (see Table 1 for a list of endocrine organs and their secretions). In teleost fish,
Pax1 and Pax9 are also expressed in the untimobranchial gland, a pharyngeal derivative (Suzuki et al.
2005, 2007). Pax2 is also expressed in pancreatic
islets, where it transcriptionally activates glucagon
(Ritz-Laser et al. 2000). Both Pax2 and Pax5 are
expressed at the midbrain–hindbrain boundary
(Schwarz et al. 1997), where they are involved in
regulation of Engrailed 2 (Li Song and Joyner
2000), and Pax2, Pax5, Pax8, and Pax3 are expressed
in the otic and other neurogenic placodes (Heller
and Brandli 1999; Schlosser 2006; Nechiporuk et al.
2007; McCabe and Bronner-Fraser 2008). Both Pax4
Pax genes are ideal for studies of alternative splicing
because they exhibit varying degrees of splicing,
roughly proportional to the number of exons, and
because they have several functional domains, each
of which can be altered by alternative-splicing. Using
exon-to-exon PCR with first-strand cDNAs from several embryonic stages and adults of amphioxus, we
identified two splicing events in Pax1/9, which has
5 exons, 12 splicing events for Pax2/5/8, which has
11 exons, five events for Pax3/7, which has 6 exons,
and 18 isoforms of Pax4/6, which has 13 exons
(Glardon et al. 1998; Short and Holland 2008)
(Fig. 3). These alternative splice forms include
exon-skipping, use of alternative 30 - and 50 -splice
sites and intron-inclusion.
Based on bioinformatic analyses of mammalian
EST sequences, it was proposed that alternative splicing and gene duplication are inversely proportional
with singleton genes having more splice forms than
duplicate ones (Su et al. 2006). To test this idea, we
compared alternative splicing of the Pax genes in
amphioxus with published data on vertebrates. We
found there was at least as much alternative splicing
in each of the vertebrate Pax genes as in their unduplicated amphioxus homologs (Short and Holland
2008). Moreover, although we found several evolutionarily conserved splice forms, including one of
Pax2/5/8 conserved with vertebrate Pax5 that would
be predicted to result in a truncated isoform lacking
the activation and repression domains, many isoforms were not conserved in amphioxus and
vertebrates.
The disparity between our results and results from
EST sequences may be due in part to the inability of
EST analyses to detect alternative splicing involving
the C-terminal half of the coding sequence, since
30 -ESTs typically only cover the 30 -UTR, and in
26
L. Z. Holland and S. Short
Table 1 Vertebrate endocrine hormones other than those secreted by the gonads.
Brain
Hypothalamus
Thyrotropin-releasing
hormone (TRH)
Somatostatin (SS)
Dopamine (DA)
Oxytocin
Vasopressin (ADH)
Pineal
Melatonin
Pituitary (neurohypophysis
þ adenohypophysis)
Growth hormone (GH)
Adrenocorticotropic hormone (ACTH)
Melanocyte-stimulating
hormone (MSH)
Growth hormone-releasing hormone
(GHRH)
Gonadotropin-releasing hormone
(GnRH)
Corticotrophin-releasing hormone
(CRH)
Follicle-stimulating
hormone (FSH)
Lutenizing hormone
(LH)
Throid-stimulating hormone (TSH)
Thyroxine (T4)
Calcitonin
Prolactin (PRL)
Pharyngeal derivatives
Thyroid
Triiodothyronine (T3)
Parathyroid
Parathyroid hormone
(PTH)
Gut and gut derivatives/associated organs
Stomach and intestine
Gastrin
Somatostatin
Secretin
Grelin
Histamine
Cholecystokinin
Neuropeptide Y (NPY)
Endothelin
Liver
Insulin-like growth factor
(IGF)
Angiotensin
Thrombopoietin
Pancreas
Insulin
Glucagon
Somatostatin
Erythropoietin
Calcitrol
Pancreatic poloypeptide
(PP)
Kidney
Renin
Thrombopoietin
Adrenal glands
Adrenaline
Glucocorticoids
Mineralocorticoids
Androgens
Noradrenaline
Dopamine
Enkephalin
In mammals, the ultimobranchial body, a derivative of the fourth pharyngeal pouch, fuses with the thyroid. In other vertebrates, the ultimobranchial body, which secretes calcitonin, remains as a separate organ.
part to the age of the duplications. Comparisons of
EST data from mouse and human, which diverged
96 million year ago (mya) (Nei et al. 2001),
showed that the amount of alternative splicing is
larger in small gene families and larger in duplicated
genes than in single ones (Jin et al. 2008). However,
while Su et al. (2006) agreed that for human genes,
smaller gene families have more duplicate genes, they
argued that shortly after gene duplication, splice
forms tended to be lost while Jin et al. (2008) concluded that newly duplicated genes have fewer constraints and, thus, are free to undergo more
alternative-splicing. Evidently, the relationship between alternative splicing and gene duplication depends on many variables such as the age of
duplicates, the size of the gene families and the organisms being compared. However, our study comparing amphioxus and vertebrates, whose lineages
split over 500 mya, concerned the whole-genome duplications giving rise to multiple Pax genes in the
vertebrate lineage that probably occurred 400–500
mya (Schubert et al. 2006). It may be that such old
gene duplicates have had time to evolve myriad
splice forms.
Alternatively spliced isoforms are
differentially expressed in development
Alternatively spliced isoforms of developmental genes
can be differentially expressed at various stages in
embryogenesis and in different tissues. However, relatively little is known about the functions of specific
isoforms in vivo. Perhaps the best examples of roles
of isoforms in development are in the central and
peripheral nervous systems. For example, in the vertebrate central nervous system, two isoforms of Fgf8
A focus on Pax genes
27
Fig. 3 Alternative splicing of the single amphioxus Pax2/5/8 pre-mRNA is extensive, but no more so than that of the separate
vertebrate Pax2, Pax5, and Pax8 mRNAs. (A) While several splice forms are evolutionarily conserved, others are not. Double vertical
lines indicate alternative stop codons. In the Xenopus genes, exon 4 is split into two exons, while Xenopus Pax5 has evolved exon 6.1,
and Xenopus Pax8 has evolved a new exon 2. Thus, exon 6 in amphioxus Pax2/5/8 is comparable to exon 7 in Xenopus Pax2 and Pax5
and to exon 8 in Xenopus Pax8. Alternative splicing of exons 7 and 8 in amphioxus Pax2/5/8 and exons 8 and 9 in Xenopus Pax2 results
in intron inclusion and a premature stop codon in the intron. Vertical stripes: paired box; open box: octapeptide; and diagonal stripes:
partial homeodomain. Data from Kozmik et al. (1993), Heller and Brandli (1997), and Short and Holland (2008). (B) Exon 8 in
amphioxus [Branchiostoma floridae (B. f)] is longer than the comparable exon in Xenopus laevis (X. l) and Danio rerio (D. r), but the core
sequence is highly conserved, suggesting that the function of this exon is also conserved.
(Fgf8a and Fgf8b) are expressed at the midbrain/
hindbrain boundary, together with Fgf17 and Fgf18,
which derived from duplication of an ancestral chordate Fgf8/17/18 gene. Of these four proteins, which
have different affinities for Fgf receptors, only Fgf8b
can induce Gbx2 expression in the hindbrain (Liu
et al. 2003; Olsen et al. 2006).
An extreme example of the roles of alternative
splice forms in neuronal development is the Down
syndrome cell adhesion molecule1 (Dscam1) in the immunoglobulin superfamily. Experiments have shown
that in Drosophila, thousands of isoforms of Dscam1
must be expressed during development for neurons
to have normal branching patterns (Hattori et al.
2009). In vertebrates, a nervous-system-specific splicing factor (nSR100) was found that regulates splicing
of brain-specific alternative exons of many genes
(Calarco et al. 2009). Mutation of nSR100 inhibits
differentiation of nerve cells in both cell culture and
in the zebrafish brain. The human mid-fetal brain
expresses 76% of the total genes, of which 28%
showed region-specific expression of specific splice
forms (Johnson et al. 2009). Interestingly, these alternatively spliced genes did not include DSCAM or
the related DSCAML1.
Differential expression of isoforms of a number of
genes in the CNS and peripheral nervous systems of
other mammals has also been described. These include transcription factors (e.g., Prrxl1, which regulates processing of nociceptive information),
cell-adhesion structural proteins (protocaderin-a
genes involved in projections of serotonergic neurons), and signaling molecules (e.g., GSK3b involved
in Wnt/b-catenin signaling) (Katori et al. 2009;
Rebelo et al. 2009; Wood-Kaczmar et al. 2009).
For Pax genes, the two isoforms of amphioxus
Pax1/9, which use alternative 50 -splice sites in the
C-terminal exon, one of which causes a frame shift
and premature stop codon, are differentially expressed in development. The isoform with the premature stop codon is dominant at all developmental
stages, while the other is expressed at a low level at
28
the early larval stage only, when expression in the
muscle is at its highest (Short and Holland 2008).
Similarly, Pax2 isoforms are temporally expressed
differentially during development in Xenopus
(Heller and Brandli 1997). In addition, two isoforms
of Pax2 are expressed in the vertebrate pancreas. One
of these, Pax2B, activates transcription at a higher
level than the other, Pax2A. Both of these bind to
the G3 regulatory element in the glucagon gene and,
with lower affinity to the G1 element, which has a
high affinity for Pax6 (Ritz-Laser et al. 2000). A third
Pax2 isoform (Pax2D2) is also expressed in cell-lines
that produce insulin; however, Pax2 does not regulate transcription from the insulin gene. In chordates,
Pax4/6 genes are expressed in the developing pineal
gland in vertebrates (Estivill-Torrús et al. 2001) and
in its homolog, the lamellar body in amphioxus
(Glardon et al. 1998). Pax6 is required for development of the pituitary (Estivill-Torrús et al. 2001).
Amphioxus Pax4/6 has at least six isoforms, while
for vertebrate Pax4 and Pax6 genes 5 and 13 splicing
events, respectively, have been identified (Bandah
et al. 2007; Short and Holland 2008). Only two of
four isoforms of Pax4 identified in the mouse are
expressed in the developing pituitary (Rath et al.
2009), indicative of the roles that differential expression of isoforms play in development.
Unique constellations of isoforms of Pax
genes can be expressed in cancers and
genetic disorders
Considerable information on alternative splice forms
has come from cancers and genetic defects. Mutants
of all vertebrate Pax genes, except for Pax4, have
been associated both with cancers and birth defects
(reviewed by Wang et al. 2008). While many of these
involve point mutations resulting in truncated proteins, several involve splice forms. For example, in
pancreatic cancer cell lines, an alternatively-spliced
isoform of Pax6, which has a 14-amino acid insertion in the paired domain, is expressed at higher
levels than is the isoform lacking the insertion
(Mascarenhas et al. 2009), while in a multiplemyeloma cell line, a Pax5 isoform with a variant
transactivation domain is expressed at lower levels
than in normal B-lymphocytes (Borson et al. 2006).
For Pax5, many isoforms have been found expressed
in B-lymphocytes, which are produced in the bone
marrow and migrate to the thymus (Arseneau et al.
2009). Most of these isoforms are also expressed in
lymphomas. Two of them code for isoforms lacking
the DNA-binding domains, octapeptide and activation and repression domains. Two have the paired
L. Z. Holland and S. Short
domain and octapeptide, but not the homeodomain
and the others are C-terminal variants.
In vertebrates, congenital defects caused by
mis-splicing are known for Pax6 and Pax3. Pax6
produces at many isoforms (41 in the pigeon), several of which are expressed in specific regions of the
retina (Bandah et al. 2007). Three of these isoforms,
one of which lacks a paired domain, are produced by
use of an alternative promoter and ATG start
codons. A second contains a 14 amino-acid insertion
at the N-terminal end of the paired domain.
Overexpression of the second isoform in the mammalian eye results in extreme micropthalmia (Kim
and Lauderdale, 2008). Moreover, in humans, a mutation at a splice junction that removes exon 4 resulting in an altered paired-domain with 13 extra
amino acids congenital nystagmus (involuntary eye
movements) and various defects of the iris (Vincent
et al. 2004). Similarly, in the mouse Sey (small eye)
mutant, a point mutation results in intron retention
and a premature stop codon C-terminal of the
homeodomain of Pax4 (Stuart et al. 1993). The original Splotch mouse, which has defective neural tube
closure, has an A to T transversion at the 30 -AG
splice acceptor of intron 3 of Pax3, which prevents
normal splicing of that intron, thereby creating four
mis-spliced mRNAs. Due to use of cryptic 30 -splice
sites in the downstream exon, two of these have deletions that cause an altered reading frame. The other
two result in retention of intron 3 and deletion of
exon 4, respectively (Epstein et al. 1993). Mutants of
other Pax genes with defective splicing have not yet
been described.
Alternative splicing and
endocrine systems
Alternative splicing affects pre-mRNAs of hormones,
hormone receptors and proteins that regulate their
expression. Although many endocrine hormones are
not subject to alternative-splicing, several are. An alternative splice form of thyroid stimulating hormone
b-subunit occurs in the human pituitary and in leukocytes in the peripheral blood and thyroid, whereas
expression of the major isoform is limited to the
pituitary (Schaefer and Klein 2009). Moreover, several peptide hormones can be derived from a single
pre-mRNA by alternative splicing. An example is the
Calca gene, which encodes two peptides, CT and
a-CT gene-related peptide (a-CGRP) by alternative
splicing of pre-mRNA. This gene has six exons.
Inclusion of exon 4, which results in a premature
stop codon and a polyA signal at the 30 -end of the
exon, creates CT, while skipping of exon 4 and
29
A focus on Pax genes
inclusion of exons 5 and 6 generates a-CGRP (Chew
1997). Splicing is tissue-specific, with CT, which regulates bone resorption, being produced mainly by
the thyroid (although there is some expression in
motor neurons), while a-CGRP, which is a neuropeptide produced by the cells in both the central and
peripheral nervous systems, regulates vascular properties (Terrado et al. 1998; Huebner et al. 2008).
Not only does Calca mRNA undergo alternative
splicing, but so does the CT receptor, which has at
least seven different isoforms with three alternate
promoters, which show tissue-specific expression
(Anusaksathien et al. 2001). All seven are expressed
in osteoclasts, but not at equal levels, while only
some of the isoforms are expressed in kidney and
brain.
Another example of alternative-splicing of primary
transcripts for encoding peptide hormones is the proglucagon gene. The proglucagon mRNA is cleaved into
several peptide hormones. The 29-amino-acid glucagon is secreted from pancreatic islet A-cells and helps
regulate metabolism. Mammalian glucagon-like peptide 1 (GLP-1) is secreted from L-cells in the intestine and potentiates insulin secretion from pancreatic
islet B-cells. Glucagon-like peptide 2 (GLP-2) is
known only from mammals, where it can promote
growth of intestinal cells (reviewed by Irwin 2001).
There is no evidence for alternative splicing of proglucagon mRNA in lampreys, although in teleost fish,
there is apparently alternative splicing as the processed mRNA in the pancreas codes only for
GLP-1, not GLP-2, whereas the reverse is true of
mRNAs in the intestine (Irwin and Wong 1995).
The situation in the chicken and frog is similar
(Yeung and Chow 2001).
The enzymes that cleave endocrine peptides from
their precursor proteins are also alternatively spliced.
The major gene family involved in cleaving these
peptides includes the subtilisin-like proprotein convertases of which there are seven members in mammals—SPC1-7 (reviewed by Steiner 1998). The
primary neurohormone processers are SPC3
(PC1/PC3) and SPC2 (PC2). Moreover, SPC6
(PC5/PC6) is expressed in the brain, the adrenal
gland and the intestine and is alternatively spliced,
as is SP1 (PACE4), which is expressed in the testis
(Rouillé et al. 1995; Steiner 1998). These enzymes
typically cleave the hormone precursors at lysine/
argine or arginine/arginine boundaries. The family
radiated early in metazoan evolution. Blast searches
indicate that these proprotein convertases are present
in chordates other than vertebrates, but only a few
have been studied. The basal chordate amphioxus
(Branchiostoma) has homologs of PC2 and
PC1/PC3 as well as PC6 (Oliva et al. 1995, 2000).
PC6 is alternatively spliced to yield three cDNAs differing in the C-terminal. However, there is no evidence for alternative splicing of SPC2 and SPC3.
Receptors for neuroendocrine peptides are also
typically alternatively spliced with different splice
forms expressed in different cells. For example, isoforms of the lutenizing hormone receptor (LH-R) are
differentially expressed in cumulus cells and theca
cells in the cow ovary whereas all isoforms are expressed in granulose cells, although the levels of expression vary depending on cell size (Robert et al.
2003). Similarly, splice forms of the estrogen related
receptor b (ERRb), only one of which can increase
transcription of estrogen receptor a (ERa), are differentially expressed in the human endometrium
(Bombail et al. 2008, 2010).
For deuterostomes other than jawed vertebrates,
there is comparative little information concerning
alternative splicing of hormone receptors and the
pre-mRNAs of hormones. However, in the sea lamprey, the GnRH-1 pre-mRNA produces three mature
mRNAs by alternative splicing of the 30 -end (Suzuki
et al. 2000). All three of the translated proteins are
cleaved to yield the same 10-amino-acid GNRH-1
peptide. However, the carboxy terminal half of
each protein is cleaved to yield a different
GNRH-associated peptide (GAP). The function of
these three GAP peptides from the lamprey
GNRH-1 gene is unknown, but the single GAP peptide cleaved from the human GNRH precursor protein can inhibit secretion of lutenizing hormone
(LH), follicle stimulating hormone (FSH), and prolactin from cultures of rat pituitary (Nicolics et al.
1985).
Future directions
Thanks to the sequencing of numerous eukaryotic
genomes and the development of new technologies
such as exon microarrays and high throughput sequencing of cDNAs (RNA-seq), the pervasiveness of
epigenetic phenomena has come to light. However,
the roles of epigenetic phenomena in development
and homeostasis have only begun to be appreciated.
Much of the recent focus on epigenetics has been on
the roles of miRNAs in posttranscriptional gene silencing. With the realization that over 75% of all
genes are alternatively spliced, comes an appreciation
that the emerging picture of the most studied gene
networks, such as that for the sea urchin endoderm,
as complicated as it is, is only a rough framework.
Nearly every gene in the network is really represented
30
by several proteins, expression of which is tightly
regulated in time and space within an organism.
Techniques for investigating the function of particular isoforms include in vitro analyses.
Transactivation and DNA-binding assays can be
used for transcription factors. In transactivation
assays, the activation or repression domains of transcription factors are linked to a DNA binding
domain (typically Gal4) and transfected into
tissue-culture cells together with a reporter plasmid
with Gal4 binding sites and the luciferase coding sequence. The amount of luminescence produced when
the cell lysate is mixed with ATP, and luciferin is
proportional to the ability of the protein to activate
transcription (Hsieh and Hayward 2001). These
assays depend on the particular cell line having the
requisite co-factors for transactivation. Not all cell
lines are adequate for all transcription factors. In
addition, binding assays can be used to determine
the relative ability of isoforms with alternatively
spliced DNA-binding domains to interact with
target sequences, while chromatin immunoprecipitation (ChIP) can reveal the proteins that bind to a
particular DNA-binding domain (Collas 2010). There
are various modification of ChIP such as ChIP on
chip microarrays and ChIP-seq that allow binding
sites of particular DNA-binding proteins to be determined. Arrays for mammals are commercially available, but could be constructed for any organism for
which the genome has been sequenced.
There is a wide range of techniques for studying
protein–protein interactions (Phizicky and Fields
1995; Li and Wu 2009), which theoretically could
be applied to individual isoforms. Some methods
such as protein-affinity chromatography may
require a relatively large amount of protein, while
others such as affinity blotting, similar to western
blotting except that a labeled protein rather than
an antibody is used, require smaller amounts.
Immunoprecipitation is used to isolate a protein
complex. Recently, peptide arrays have been developed for identification of proteins that bind particular modular domains (Li and Wu 2009).
It is more difficult to study protein functions
in vivo. However, in vivo function of some isoforms
can be studied by overexpression or knock-down of
particular isoforms. For organisms with genetics such
as the mouse, it can be possible to engineer mutants
defective in splicing of particular sequences. For example, in the mouse, Fgf8 produces two major isoforms, Fgf8a and Fgf8b, which differ in that due to
use of an alternative 50 -splice site in exon 4, Fgf8a is
11 amino acids shorter than Fgf8b, resulting in different affinities of the two for the FGF receptors
L. Z. Holland and S. Short
(MacArthur et al. 1995). A mutation that eliminates
all isoforms containing the longer exon 4 has the
same effect as null mutation of Fgf8 in that the midbrain and cerebellum are defective. However, a mutation that eliminates only isoforms with the short
exon 4 has no apparent effect on development of the
mouse (Guo et al. 2010). For organisms without genetics such as amphioxus, splice-blocking antisense
morpholino oligonucleotides (morpholinos) promise
to
allow
knockdown of
some
isoforms.
UV-light-activated morpholinos offer the opportunity to inject morpholinos into early embryos but
delay the effects until later in development
(Tomasini et al. 2009). This technique can be used
with splice-blocking morpholinos. Techniques have
also been developed for morpholino-knock-downs
in adult animals (Moulton and Jiang 2009); these
could facilitate an investigations of the roles of
splice forms of endocrine peptides and their
receptors.
Finally, oligonucleotide probes synthesized with
locked nucleic acids, which have a methylene
bridge between the 20 O and the 40 C on the ribose
ring, have the potential for demonstrating
tissue-specific expression of particular isoforms. To
date, locked nucleic acid probes have been extensively used to identify tissue-specific expression of
miRNAs and for fluorescent in situ hybridization to
determine the location of particular genes on chromosomes. However, the technique has recently been
extended to identify tissue-specific expression of alternatively splice exons (Darnell et al. 2010).
In summary, there are many techniques for investigating protein functions both in vitro and in vivo
that are at least theoretically applicable to specific
isoforms. Several of these techniques have been developed only recently. With the realization that multiple isoforms are the rule rather than the exception,
it can be anticipated that our understanding of the
roles of isoforms in modulating development and
physiology will increase exponentially in the next
few years.
Funding
This work was supported by the National Science
Foundation (grants IOS07-43485 and MCB06-20019
to L.Z.H.).
References
Anusaksathien O, Laplace C, Li X, Ren Y, Peng L,
Goldring SR, Galson DL. 2001. Tissue-specific and ubiquitous promoters direct the expression of alternatively spliced
transcripts from the calcitonin receptor gene. J Biol Chem
276:22663–74.
A focus on Pax genes
Arseneau J-R, Laflamme M, Lewis SM, Maı̈cas E, Ouellette RJ.
2009. Multiple isoforms of PAX5 are expressed in both
lymphomas and normal B-cells. Br J Haematol 147:328–38.
Bandah D, Swissa T, Ben-Shlomo G, Banin E, Ofri R,
Sharon D. 2007. A complex expression pattern of Pax6 in
the pigeon retina. Invest Ophthalmol Vis Sci 48:2503–9.
Blencowe BJ, Ahman S, Lee LJ. 2009. Current-generation
high-throughput sequencing: deepening insights into mammalian transcriptomes. Genes Dev 23:1379–86.
Bombail V, Collins F, Brown P, Saunders PTK. 2010.
Modulation of ERRa transcriptional activity by the
orphan nuclear receptor ERRb and evidence for differential
effects of long- and short-form splice variants. Mol Cell
Endocrinol 314:53–61.
31
spliced mRNA transcripts in the splotch (Sp) mouse
mutant. Proc Natl Acad Sci USA 90:532–6.
Estivill-Torrús G, Vitalis T, Fernández-Llebrez P, Price DJ.
2001. The transcription factor Pax6 is required for development of the diencephalic dorsal midline secretory radial
glia that form the subcommissural organ. Mech Dev
109:215–24.
Glardon S, Holland LZ, Gehring WJ, Holland ND. 1998.
Isolation and developmental expression of the amphioxus
Pax-6 gene (AmphiPax-6): insights into eye and photoreceptor evolution. Development 125:2701–10.
Goulding MD, Chalepakis G, Deutsch U, Erselius JR, Grüss P.
1991. Pax-3, a novel murine DNA binding protein
expressed during early neurogenesis. EMBO J 10:1135–47.
Bombail V, MacPherson S, Critchley HOD, Saunders PTK.
2008. Estrogen receptor related beta is expressed in
human endometrium throughout the normal menstrual
cycle. Hum Reprod 23:2782–90.
Guo Q, Li K, Sunmonu NA, Li JYH. 2010. Fgf8b-containing
spliceforms, but not Fgf8a, are essential for Fgf8 function
during development of the midbrain and cerebellum. Dev
Biol 338:183–92.
Borson N, Lacy M, Wettstein P. 2006. Expression of mRNA
for a newly identified Pax5 exon is reduced in multiple
myeloma. Mamm Genome 17:248–56.
Hattori D, Chen Y, Matthews BJ, Salwinski L, Sabatti C,
Grueber WB, Zipursky SL. 2009. Robust discrimination
between self and non-self neurites requires thousands of
Dscam1 isoforms. Nature 461:644–8.
Burset M, Seledtsov IA, Solovyev VV. 2001. SpliceDB:database
of canonical and non-canonical mammalian splice sites.
Nucleic Acids Res 29:255–59.
Calarco JA, et al. 2009. Regulation of vertebrate nervous
system alternative splicing and development by an SRRelated protein. Cell 138:898–910.
Campbell SC, Cragg H, Elrick LJ, Macfarlane WM,
Shennan KIJ, Docherty K. 1999. Inhibitory effect of Pax4
on the human insulin and islet amyloid polypeptide (IAPP)
promoters. FEBS Lett 463:53–7.
Carroll SB, Grenier JK, Weatherbee SD. 2004.
In: Hoboken NJ, editor. From DNA to diversity: molecular
genetics and the evolution of animal diversity. USA: WileyBlackwell.
Chan YF, et al. 2010. Adaptive evolution of pelvic reduction
in sticklebacks by recurrent deletion of a Pitx1 Enhancer.
Science 327:302–5.
Chen M, Manley JL. 2009. Mechanisms of alternative splicing
regulation: Insights from molecular and genomics
approaches. Nature Rev Mol Cell Biol 10:741–54.
Heller N, Brandli A. 1997. Xenopus Pax-2 displays multiple
splice forms during embryogenesis and pronephric kidney
development. Mech Dev 69:83–104.
Heller N, Brandli A. 1999. Xenopus Pax2/5/8 orthologues:
novel insights into Pax gene evolution and identification
of Pax-8 as the earliest marker for otic and pronephric
cell lineages. Dev Genet 24:208–19.
Holland LZ, Schubert M, Kozmik Z, Holland ND. 1999.
AmphiPax3/7, an amphioxus paired box gene: Insights
into chordate myogenesis, neurogenesis, and the possible
evolutionary precursor of definitive vertebrate neural
crest. Evol Dev 1:153–65.
Hsieh JJ-D, Hayward SD. 2001. Identification of transactivation, repression, and protein-protein interacting domains
using GAL4-fusion proteins. Methods Mol Biol 174:259–69.
Huebner AK, Keller J, Catala-Lehnen P, Perkovic S, Streichert T,
Emeson RB, Amling M, Schinke T. 2008. The role of calcitonin and a-calcitonin gene-related peptide in bone formation.
Arch Biochem Biophys 473:210–7.
Chew SL. 1997. Alternative splicing of mRNA as a mode of
endocrine regulation. Trends Endocrinol Metabol 8:405–13.
Irwin D, Wong J. 1995. Trout and chicken proglucagon: alternative splicing generates mRNA transcripts encoding glucagon-like peptide 2. Mol Endocrinol 9:267–77.
Claverie J-M. 2001. Gene number: what if there are only
30,000 human genes? Science 291:1255–7.
Irwin DM. 2001. Molecular evolution of proglucagon. Regul
Pept 98:1–12.
Collas P. 2010. The current state of chromatin immunoprecipitation. Mol Biotechnol 45:87–100.
Jin L, Kryukov K, Clemente JC, Komiyama T, Suzuki Y,
Imanishi T, Ikeo K, Gojobori T. 2008. The evolutionary
relationship between gene duplication and alternative splicing. Gene 427:19–31.
Darnell DK, Stanislaw S, Kauer S, Antin PB. 2010. Whole
mount in situ hybridization detection of mRNAs using
short LNA containing DNA oligonucleotide probes. RNA
16:632–7.
Davidson EH, Erwin DH. 2010. Evolutionary innovation and
stability in animal gene networks. J Exp Zool B Mol Dev
Evol 314:182–6.
Johnson MB, Kawasawa YI, Mason CE, Krsnik Z, Coppola G,
Bogdanovic D, Geschwind DH, Mane SM, State MW,
Sestan N. 2009. Functional and evolutionary insights into
human brain development through global transcriptome
analysis. Neuron 62:494–509.
Epstein DJ, Vogan KJ, Trasler DG, Gros P. 1993. A mutation
within intron 3 of the Pax-3 gene produces aberrantly
Katori S, Hamada S, Noguchi Y, Fukuda E, Yamamoto T,
Yamamoto H, Hasegawa S, Yagi T. 2009. Protocadherin-a
32
family is required for serotonergic projections to
appropriately innervate target brain areas. J Neurosci
29:9137–47.
Kim J, Lauderdale JD. 2008. Overexpression of pairedless
Pax6 in the retina disrupts corneal development and affects
lens cell survival. Dev Biol 313:434–54.
Kluge B, Renault N, Rohr KB. 2005. Anatomical and molecular reinvestigation of lamprey endostyle development
provides new insight into thyroid gland evolution. Dev
Genes Evol 215:32–40.
Koonin EV. 2009. Intron-dominated genomes of early ancestors of eukaryotes. J Hered 100:618–23.
Kozmik Z, Holland LZ, Schubert M, Lacalli TC, Kreslova J,
Vlcek C, Holland ND. 2001. Characterization of amphioxus
AmphiVent, an evolutionarily conserved marker for chordate ventral mesoderm. Genesis 29:172–9.
Kozmik Z, Holland ND, Kalousova A, Paces J, Schubert M,
Holland LZ. 1999. Characterization of an amphioxus paired
box gene, AmphiPax2/5/8: developmental expression
paterns in optic support cells, nephidium, thyroid-like
structures and pharyngeal gill slits, but not in the midbrain-hindbrain boundary region. Development 126:
1295–304.
Kozmik Z, Kurzbauer R, Dorfler P, Busslinger M. 1993.
Alternative splicing of Pax-8 gene transcripts is developmentally regulated and generates isoforms with different
transactivation properties. Mol Cell Biol 13:6024–35.
Landry C, Geyer LB, Arakaki Y, Uehara T, Palumbi SR. 2003.
Recent speciation in the Indo West Pacific: rapid evolution
of gamete recognition and sperm morphology in cryptic
species of sea urchin. Proc Roy Soc London Biol Sci
270:1839–47.
Li Song D, Joyner AL. 2000. Two Pax2/5/8-binding sites in
Engrailed2 are required for proper initiation of endogenous
mid-hindbrain expression. Mech Dev 90:155–65.
Li SS-C, Wu C. 2009. Using peptide array to identify binding
motifs and interaction networks for modular domains.
Methods Mol Biol 570:67–76.
Licatalosi DD, Darnell RB. 2010. RNA processing and its
regulation: global insights into biological networks.
Nature Rev Genet 11:75–87.
Liu A, Li JYH, Bromleigh C, Lao Z, Niswander LA,
Joyner AL. 2003. FGF17b and FGF18 have different midbrain regulatory properties from FGF8b or activated FGF
receptors. Development 130:6175–85.
Liu Z, Yu S, Manley NR. 2007. Gcm2 is required for
the differentiation and survival of parathyroid precursor
cells in the parathyroid/thymus primordia. Dev Biol
305:333–46.
Lynch VJ, Tanzer A, Wang Y, Leung FC, Gellersen B,
Emera D, Wagner GP. 2008. Adaptive changes in the transcription factor HoxA-11 are essential for the evolution of
pregnancy in mammals. Proc Natl Acad Sci USA
105:14928–33.
Lynch VJ, Wagner GP. 2008. Resurrecting the role of transcription factor change in developmental evolution.
Evolution 62:2131–54.
L. Z. Holland and S. Short
MacArthur CA, Lawshe A, Xu J, Santos-Ocampo S,
Heikinheimo M, Chellaiah AT, Ornitz DM. 1995. FGF-8
isoforms activate receptor splice forms that are expressed
in mesenchymal regions of mouse development.
Development 121:3603–13.
Marine D. 1913. The metamorphosis of the endostyle (thyroid
gland) of ammocoetes branchialis (larval land-locked
Petromyzon marinus (Jordan) or Petromyzon dorsatus
(Wilder)). J Exp Med 17:379–95.
Mascarenhas JB, Young KP, Littlejohn EL, Yoo BK, Salgia R,
Lang D. 2009. PAX6 is expressed in pancreatic cancer and
actively participates in cancer progression through activation of the MET tyrosine kinase receptor gene. J Biol Chem
284:27524–32.
McCabe KL, Bronner-Fraser M. 2008. Essential role for PDGF
signaling in ophthalmic trigeminal placode induction.
Development 135:1863–74.
McCauley BS, Weideman EP, Hinman VF. 2010. A conserved
gene regulatory network subcircuit drives different developmental fates in the vegetal pole of highly divergent echinoderm embryos. Dev Biol 340:200–8.
McCauley DW, Bronner-Fraser M. 2002. Conservation of Pax
gene expression in ectodermal placodes of the lamprey.
Gene 287:129–39.
Meyer A, Van de Peer Y. 2005. From 2R to 3R: evidence for a
fish specific genome duplication (FSGD). Bioessays
27:937–45.
Moulton J, Jiang S. 2009. Gene knockdowns in adult animals:
PPMOs and vivo-morpholinos. Molecules 14:1304–23.
Moy GW, Springer SA, Adams SL, Swanson WJ,
Vacquier VD. 2008. Extraordinary intraspecific diversity
in oyster sperm bindin. Proc Natl Acad Sci USA
105:1993–8.
Nechiporuk A, Linbo T, Poss KD, Raible DW. 2007.
Specification of epibranchial placodes in zebrafish.
Development 134:611–23.
Nei M, Xu P, Glazko GV. 2001. Estimation of divergence
times from multiprotein sequences for a few mammalian
species and several distantly related organisms. Proc Natl
Acad Sci USA 98:2497–502.
Nicolics K, Mason AJ, Szonyi E, Ramachandran J,
Seeburg PH. 1985. A prolactin-inhibiting factor within
the precursor for human gonadotropin-releasing hormone.
Nature 316:511–7.
Oliva AA, Chan SJ, Steiner DF. 2000. Evolution of the prohormone convertases: identification of a homologue of PC6
in the protochordate amphioxus. Biochim Biophysi Acta.
Protein Struct Mol Enzymol 1477:338–48.
Oliva AA, Steiner DF, Chain SJ. 1995. Proprotein convertases
in amphioxus: Predicted structure and expression of
proteases SPC2 and SPC3. Proc Natl Acad Sci USA
92:3591–5.
Olsen SK, Li JYH, Bromleigh C, Eliseenkova AV,
Ibrahimi OA, Lao Z, Zhang F, Linhardt RJ, Joyner AL,
Mohammadi M. 2006. Structural basis by which alternative
splicing modulates the orgtanizer activity of FGF8 in the
brain. Genes Dev 20:185–98.
33
A focus on Pax genes
Palumbi SR. 2008. Speciation and the evolution of gamete
recognition genes: pattern and process. Heredity 102:66–76.
Schlosser G. 2006. Induction and specification of cranial placodes. Dev Biol 294:303–51.
Peter IS, Davidson EH. 2010. The endoderm gene regulatory
network in sea urchin embryos up to mid-blastula stage.
Dev Biol 340:188–99.
Schubert M, Escriva H, Xavier-Neto J, Laudet V. 2006.
Amphioxus and tunicates as evolutionary model systems.
Trends Ecol Evol 21:269–77.
Phizicky EM, Fields S. 1995. Protein-protein interactions:
methods for detection and analysis. Microbiol Rev
59:94–123.
Schwarz M, Alvarez-Bolado G, Urbanek P, Busslinger M,
Gruss P. 1997. Conserved biological function between
Pax-2 and Pax-5 in midbrain and cerebellum development:
evidence from targeted mutations. Proc Natl Acad Sci USA
94:14518–23.
Putnam NH, et al. 2008. The amphioxus genome and
the evolution of the chordate karyotype. Nature 453:
1964–2072.
Rath MF, Bailey MJ, Kim J-S, Ho AK, Gaildrat P, Coon SL,
Moller M, Klein DC. 2009. Developmental and diurnal
dynamics of Pax4 expression in the mammalian pineal
gland: nocturnal down-regulation is mediated by adrenergic-cyclic adenosine 30 ,50 -monophosphate signaling.
Endocrinology 150:803–11.
Rebelo S, Lopes C, Lima D, Reguenga C. 2009. Expression of
a Prrxl1 alternative splice variant during the
development of the mouse nociceptive system. Int J Biol
Sci 53:1089–95.
Reiss D, Zhang Y, Rouhi A, Reuter M, Mager DL. 2010.
Variable DNA methylation of transposable elements.
Epigenetics 5:1–12.
Ritchie DB, Schellenberg MJ, MacMillan AM. 2009.
Spliceosome structure: piece by piece. Biochim Biophys
Acta 1789:624–33.
Ritz-Laser B, Estreicher A, Gauthier B, Philippe J.
The paired homeodomain transcription factor
Is expressed in the endocrine pancreas and
activates the glucagon gene promoter. J Biol
275:32708–15.
2000.
Pax-2
transChem
Ritz-Laser B, Estreicher A, Gauthier BR, Mamin A, Edlund H,
Philippe J. 2002. The pancreatic beta-cell-specific transcription factor Pax-4 inhibits glucagon gene expression through
Pax-6. Diabetologia 45:97–107.
Robert C, Gagné D, Lussier JG, Bousquet D, Barnes FL,
Sirad M-A. 2003. Presence of LH receptor mRNA in granulosa cells as a potential marker of oocyte developmental
competence and characterization of the bovine splicing isoforms. Reproduction 125:437–46.
Ronshaugen M, McGinnis N, McGinnis W. 2002. Hox protein mutation and macroevolution of the insect body plan.
Nature 415:914–7.
Rouillé Y, Duguay SJ, Lund K, Furuta M, Gong Q, Lipkind G,
Oliva AA, Chan SJ, Steiner DF. 1995. Proteolytic processing
mechanisms in the biosynthesis of neuroendocrine peptides: the subtilisin-like proprotein convertases. Frontiers
Neuroendocrinol 16:322–61.
Short S, Holland LZ. 2008. The evolution of alternative splicing in the Pax family: The view from the basal chordate
amphioxus. J Mol Evol 66:605–20.
Steiner DF. 1998. The proprotein convertases. Curr Op Chem
Biol 2:31–9.
Stuart ET, Kioussi C, Gruss P. 1993. Mammalian Pax genes.
Annu Rev Genet 27:219–36.
Su Z, Wang J, Yu J, Huang X, Gu X. 2006. Evolution of
alternative splicing after gene duplication. Genome Res
16:182–9.
Suzuki K, Gamble R, Sower S. 2000. Multiple transcripts
encoding lamprey gonadotropin-releasing hormone-I precursors. J Mol Endocrinol 24:365–76.
Suzuki M, Hidaka Y, Uemae Y, Tanaka S. 2005. Molecular
evoltuion and subfunctionalization of Nkx2.1 in the rainbow trout. Ann NY Acad Sci 1040:479–82.
Suzuki M, Katagiri N, Ueda M, Tanaka S. 2007. Functional
analysis of Nkx2.1 and Pax9 for calcitonin gene transcription. Gen Comp Endocrinol 152:259–66.
Terrado J, Gerrikagoitia I, Raldúa D, Sorribas V, Martı́nezMillán L, Sarasa M. 1998. The two mature transcripts of
the chick calcitonin gene are expressed within the
central nervous system during embryogenesis. Mech Dev
77:81–4.
Tomasini AJ, Schuler AD, Zebala JA, Mayer AN. 2009.
PhotoMorphs: A novel light-activated reagent for controlling gene expression in zebrafish. Genesis 47:736–43.
Van de Peer Y, Maere S, Meyer A. 2009. The evolutionary
significance of ancient genome duplications. Nature Rev
Genetics 10:725–732.
Vincent M-C, Gallai R, Olivier D, Speeg-Schatz C, Flament J,
Calvas P, Dollfus H. 2004. Variable phenotype related to a
novel PAX 6 mutation (IVS4þ5G4C) in a family presenting congenital nystagmus and foveal hypoplasia. Am J
Ophthalmol 138:1016–21.
Wallin J, Eibel H, Neubuser A, Wilting J, Koseki H, Balling R.
1996. Pax1 is expressed during development of the thymus
epithelium and is required for normal T-cell maturation.
Development 122:23–30.
Rovescalli AC, Cinquanta M, Ferrante J, Kozaki CA,
Nierenberg M. 2000. The mouse Nkx-1.2 homeobox gene:
Alternative RNA splicing at canonical and noncanonical
splice sites. Proc Natl Acad Sci USA 97:1982–7.
Wang Q, Fang W-H, Krupinski J, Kumar S, Slevin M,
Kumar P. 2008. Pax genes in embryogenesis and oncogenesis. J Cell Mol Med 12:2281–94.
Schaefer JS, Klein JR. 2009. A novel thyroid stimulating hormone [beta]-subunit isoform in human pituitary, peripheral blood leukocytes, and thyroid. Gen Comp Endocrinol
162:241–4.
Wendl T, Lun K, Mione M, Favor J, Brand M, Wilson S,
Rohr K. 2002. Pax2.1 is required for the development of thyroid follicles in zebrafish. Development
129:3751–60.
34
Wood-Kaczmar A, Kraus M,
Gordon-Weeks PR. 2009.
form of glycogen synthase
growing neurites and growth
42:184–94.
L. Z. Holland and S. Short
Ishiguro K, Philpott KL,
An alternatively spliced
kinase-3b is targeted to
cones. Mol Cell Neurosci
Yeung C-M, Chow BKC. 2001. Identification of a
proglucagon cDNA from Rana tigrina rugulosa that
encodes two GLP-1s and that is alternatively spliced
in a tissue-specific manner. Gen Comp Endocrinol
124:144–51.