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Developmental Biology
journal homepage: www.elsevier.com/locate/developmentalbiology
Review article
Gene, cell, and organ multiplication drives inner ear evolution
Bernd Fritzsch1
, Karen L. Elliott
University of Iowa, Department of Biology, Iowa City, IA 52242, United States
A R T I C L E I N F O
Keywords:
Cell type evolution
Transcription factor multiplication
Diﬀerential expression regulation
A B S T R A C T
We review the development and evolution of the ear neurosensory cells, the aggregation of neurosensory cells
into an otic placode, the evolution of novel neurosensory structures dedicated to hearing and the evolution of
novel nuclei in the brain and their input dedicated to processing those novel auditory stimuli. The evolution of
the apparently novel auditory system lies in duplication and diversiﬁcation of cell fate transcription regulation
that allows variation at the cellular level [transforming a single neurosensory cell into a sensory cell connected
to its targets by a sensory neuron as well as diversifying hair cells], organ level [duplication of organ
development followed by diversiﬁcation and novel stimulus acquisition] and brain nuclear level [multiplication
of transcription factors to regulate various neuron and neuron aggregate fate to transform the spinal cord into
the unique hindbrain organization]. Tying cell fate changes driven by bHLH and other transcription factors into
cell and organ changes is at the moment tentative as not all relevant factors are known and their gene regulatory
network is only rudimentary understood. Future research can use the blueprint proposed here to provide both
the deeper molecular evolutionary understanding as well as a more detailed appreciation of developmental
networks. This understanding can reveal how an auditory system evolved through transformation of existing cell
fate determining networks and thus how neurosensory evolution occurred through molecular changes aﬀecting
cell fate decision processes. Appreciating the evolutionary cascade of developmental program changes could
allow identifying essential steps needed to restore cells and organs in the future.
1. Introduction
Ever since Ohno (Ohno, 2013) ﬁrst proposed in 1972 that gene
duplication might be a driving force for evolutionary change, multiple
examples of evolutionary innovation have been associated with gene
duplication: neo-functionalization (changes in the protein sequence)
and sub-functionalization [alteration in the expression of duplicated
genes (Lynch and Force, 2000; Peter and Davidson, 2015; Wagner,
2011)]. Many genes and enhancers originally discovered in multicellular
organism have now been detected in single-celled outgroups
suggesting that gene regulation evolution combined with gene multiplications
(Sebé-Pedrós et al., 2016) might drive derived traits of a
particular cell type [aka apomeres (Arendt et al., 2016)]. This process
forms the basis of multicellular evolution that, in turn, is the basis for
organ and thus organismal evolution . Among the many genes that
show duplication or multiplication in vertebrates relative to the
hypothetical single cell ancestor of all metazoans are cell fate decision
making genes such as those encoding basic Helix-Loop-Helix (bHLH)
transcription factors (Pan et al., 2012b). Speciﬁcally, current data
suggest multiplication of these factors occurred in evolution, resulting
in the formation of entire new classes showing properties not found in
single celled ancestors such as Class A bHLH transcription factors
(Fritzsch et al., 2015a). Such cellular decision making molecular
cascades were eventually modiﬁed by the evolution of novel signal
cascades such as the Wnt signaling (Booth and King, 2016) and
supplemented by the recruitment of other transcription factors coopted
from other organ development to generate unique multicellular
body parts such as sensory organs (Arendt et al., 2016) with dedicated
modiﬁcations of gene regulatory networks (Peter and Davidson, 2015).
Building on this general principle, we explore here how molecular
diversiﬁcation might have provided the material for major steps in
inner ear cellular development and evolution and how sensory organ
evolution might connect to the central nervous system evolution
through matched speciﬁc changes in developmental gene regulatory
networks:
A) The ancestral metazoan neurosensory cell likely was polarized into
a base and an apex similar to a vertebrate olfactory neurosensory
cell: one end of it was specialized to receive a stimulus and the
other end specialized to send the information decoded into an
action potential along its own axon to the central nervous system
for processing. An ancestral split generated two cell types out of this
http://dx.doi.org/10.1016/j.ydbio.2017.08.034
Received 9 December 2016; Received in revised form 27 April 2017; Accepted 25 August 2017
1
Address: 129 E Jeﬀerson, BB214, Iowa City 52242, USA.
E-mail address: bernd-fritzsch@uiowa.edu (B. Fritzsch).
Developmental Biology 431 (2017) 3–15
Available online 01 September 2017
0012-1606/ © 2017 Elsevier Inc. All rights reserved.
MARK
unicellular precursor: i). a sensory cell type specialized for sensory
stimulus reception but without an axon and ii). a sensory neuron
type specialized for conducting the information from the sensory
cell to the central nervous system (Fritzsch et al., 2007; Gasparini
et al., 2013; Patthey et al., 2014).
B) Aggregation of what might have initially been individually distributed
neurosensory cells and neurons into placodes led to the
formation of major cranial sensory organ formation such as the
ear, lateral line, electroreceptors, olfaction and taste system (Maier
et al., 2014; O'Neill et al., 2012; Schlosser et al., 2014). Some of
these simple organ systems eventually split into multiple organs
such as distinct lateral line neuromasts or distinct inner ear organs
(Fritzsch et al., 2002). Splitting of such organs, in combination with
the recruitment of novel genes, led to novel specializations that
enable diﬀerent mechanosensory organs to be tuned to speciﬁc
sensory stimuli (Fritzsch et al., 2013).
C) Analyzing various sensory information provided by the diﬀerent
kind of placodally-derived receptor organs requires that central
nervous system projections reach discrete areas of the brain
(Grothe et al., 2004) for segregated information processing enabled
by unique brain gene regulatory networks (GRN)-guided neuronal
development (Nothwang, 2016). Here we will develop a concept of
molecular transformation of a spinal cord like organization of
nuclei into a hindbrain organization (Hernandez-Miranda et al.,
2016) able to receive and process distinct information provided by
placodally-derived sensory cells (Elliott et al., 2017; Fritzsch et al.,
2005a, 2006b).
1.1. Evolution and development of vertebrate mechanosensory hair
cells and sensory neurons
Neurosensory cells, like all other cells of multicellular organisms,
evolved from a single celled ancestor, a choanoﬂagellate-like organism
(Booth and King, 2016; Fritzsch and Straka, 2014). These single celled
organisms were polarized into an apex and a base: they display on one
end a kinocilium surrounded by interconnected microvilli that function
as a basket to ﬁlter food propelled by the ﬂagellum; the opposite end
provides attachment to the substrate. Choanoﬂagellates thus show a
cellular polarity in line with the basal-apical cellular polarity in
epithelia (Boenigk, 2015; Leadbeater, 2015) as is so obvious in sponges
(Ueda et al., 2016). Development of mechanosensory cells apparently
recapitulates this process in that mechanosensory cells of vertebrates
develop ﬁrst an apical kinocilium surrounded by microvilli. The
kinocilium moves into an acentric position as mechanosensory cells
mature (Fig. 1). Eliminating kinocilia mediated apical reorganization
results in circular polarized hair cells (Jones et al., 2008). Beyond the
kinocilium as a signaling center, the apical acentric polarity development
relies on several regulatory factors, such as LGN and Gαi
(Tarchini et al., 2016) and Bbs8 and Ift20 (May-Simera et al., 2015)
to eliminate microvilli and move the kinocilium into an acentric
position and regulate the height and distribution of stereocilia to
properly polarize hair cells. The end product of this incompletely
understood process (Lu and Sipe, 2016; Schüler et al., 2013) is a highly
polarized cell that allows mechanical stimuli to exert changes on the
cell’s resting potential depending on the direction of the stimulation
(Fig. 1) through opening of channels attached to tip links [putative
candidates are TMC1/2 (Shibata et al., 2016; Wu and Müller, 2016)].
Many mechano-sensitive cells display a permutation of this common
scheme of kinocilia/microvilli. However, the variation of some nonvertebrate
lineages can be arranged into a graded set of changes toward
the vertebrate mechanosensory cell: a polarized organ pipe of stereocilia
with or without a kinocilium (Fig. 1). Future work needs to
determine how various proteins known to play part in hair cell polarity
formation were recruited. During development hair cells go through a
similar rearrangement (Fig. 1a) and certain mutations ‘freeze’ hair cells
at earlier developmental or evolutionary stages (Fig. 1b).
In addition to TMC1/2 and TMIE, Piezo channels, frequently found
in proprioceptors and Merkel cells, are also associated with normal
function of some hair cells, but are apparently not directly involved in
mechanotransduction (Wu et al., 2016). In addition, multiple functionally
important links between stereocilia and mechanotransduction
defects can be found in various mutations. For example, mutations in
stereocilin (Strc) cause the DFNb16 hearing loss, the most frequent
hair cell-related hearing loss (Francey et al., 2012; Moteki et al., 2016).
It would be important to establish how the stereocilin-mediated
connections of stereocilia molecularly relate to the microvilli connections
in choanoﬂagellates and sponges. This could help to reveal the
evolutionary origin of the leading candidates for the mechanosensory
transduction channel, TMC1/2 (Shibata et al., 2016) and TMHS/TMIE
(Wu et al., 2016; Wu and Müller, 2016).
Superimposed on these transformations in the apex of mechanosensory
cells that rearranges kinocilia and microvilli from a symmetric
non-sensory organization with a motile kinocilium in the center to an
asymmetric sensory system specialized for mechanotransduction, is a
partially overlapping set of changes that lead to the segregation of
mechanosensory cells into a sensory cell and a sensory neuron. This
transformation correlates with the multiplication of certain cell fate
determining transcription factors, the basic Helix-Loop-Helix transcription
factors (bHLH TFs; Fig. 2) and their binding sites (Fritzsch
et al., 2010; Fritzsch and Straka, 2014). Based on selective loss of
mechanosensory hair cell diﬀerentiation in mice null for Atoh1
(Bermingham et al., 1999) and of all sensory neurons but also some
hair cells in Neurog1 null mice (Ma et al., 2000) it was hypothesized
that some lineage relationship exists between sensory neurons and
mechanosensory hair cells (Fritzsch et al., 2000). Later, a third bHLH
TF, Neurod1, was identiﬁed that was originally claimed to also lead to
complete neuronal loss (Liu et al., 2000) but was later found to lead
only to partial loss, mostly of spiral ganglion neurons, due to other
partially co-expressed bHLH genes (Jahan et al., 2010a; Kim et al.,
2001). Subsequent work showed that there is a transient expression of
bHLH transcription factor Atoh1 in delaminating neurons and that
expression of markers common to hair cell and neuronal precursors
show a transient increase of hair cell precursor population, implying a
possible transformation of neurons into hair cells (Matei et al., 2005).
Further validation of this idea of a lineage relationship was provided
using a Neurog1-cre line for some sensory epithelia (Raft et al., 2007)
claiming a direct Neurog1-Atoh1 interaction (Fig. 2). Additional
analysis centered on the fate of diﬀerent cell types in the ear using a
combination of knock-out and knock-in approaches has modiﬁed this
model, mainly by adding the function of Neurod1 as a partially
understood mediator of Neurog1 and Atoh1 interactions (Fig. 2).
Neurog1 clearly forms such a negative feedback loop to Atoh1 in the
CNS but the role of Neurod1 was not clariﬁed in this process (Gowan
et al., 2001). Changes in neuronal or hair cell populations after
Neurog1 deletion (Matei et al., 2005) was also claimed to indicate
such a feedback loop in neurosensory precursors (Raft and Groves,
2015). However, a more complicated scenario of Neurog1 and Atoh1
being both variably cross-regulated by Neurod1 remains an alternative
possibility (Fig. 2) in light of more recent evidence of the existence of
such a complicated feed-forward and feed-back interactions between
these three bHLH TFs (Pan et al., 2012b). For example, a conditional
deletion model for Neurod1 showed that this transcription factor
suppresses Atoh1 expression in several systems such as the cerebellum
(Pan et al., 2009) and the ear (Jahan et al., 2010b) suggesting a
negative feedback loop for Atoh1 and Neurod1. Consistent with such a
role for Neurod1 is that loss of Neurod1 results in continued expression
of an otherwise transient Atoh1 expression in sensory neurons and
their transdiﬀerentiation into intraganglionic hair cells (Jahan et al.,
2010b). Such a potential negative feedback loop was also found in hair
cells that show a premature and more profound expression of Atoh1 in
the apex of Neurod1 CKO mice, resulting in a partial conversion of hair
cell types as a consequence of premature and elevated expression of
B. Fritzsch, K.L. Elliott Developmental Biology 431 (2017) 3–15
4
Atoh1 (Jahan et al., 2015a, 2010b, 2013). To formally test a more
direct interaction between Atoh1 and Neurog1 required replacement of
Atoh1 by Neurog1. In contrast to the eﬀective transformation of
neurons into hair cells in Neurod1 null mice, Neurog1 proved to be
unable to substitute for Atoh1 in hair cell precursors leading to the
rapid degeneration of such hair cell precursors (Jahan et al., 2012) in a
Fig. 1. Evolution and development of the apex from centric to acentric organization. Evolution(A): The kinocilia (red) shifts from a centric position (2) surrounded by
microvilli/stereocilia (green) found in choanoﬂagellates to an acentric position (a-d) in Deuterostomia, establishing cell polarity and allowing directional stimulation in vertebrates (3).
Protostomia show divergent specializations for mechanosensation (e,f). Movement direction is indicated by the arrow. Mutation (B): Various mutations that aﬀect the stereocilia.
Atoh1-cre, Atoh1kiNeurog1, Neurod1f/f Pax2-cre, Atoh1+kiNeurog1 and Atoh1ki/ki mice develop stereocilia aberrations suggesting that planar cell polarity is inﬂuenced by Atoh1
levels (Jahan et al., 2015a, 2015b; Pan et al., 2012a). Itf88 CKO mice can develop circular stereocilia in the absence of a kinocilia (Jones et al., 2008). Development(C): Hair cells
develop ﬁrst a central kinocilium surrounded by microvilli followed by shift into an acentric position and alteration of stereocilia diameters (bottom to top). Only organ of Corti hair cells
lose secondarily the kinocilium (top). Modiﬁed after (Fritzsch et al., 2007).
Fig. 2. Relationship of the bHLH transcription factors and their E-boxes indicates likely multiplication and diversiﬁcation at molecular and cellular levels. (A)
Relationship between the bHLH transcription factors. (B) Relationship between the E-box binding sites of the various bHLH transcription factors. Note co-evolutionary changes in the
bHLH transcription factors (A) and the E-box sequences (B). (C) The presumed ancestral mechanosensory cell with an axon may have contained three bHLH transcription factors:
Atoh1, Neurod1, and Neurog1. Incomplete segregation of these three transcription factors result in a derived condition with separate hair cells expressing Atoh1 and Neurod1 and
sensory neurons expressing Neurod1 and Neurog1. Some experiments suggest that Neurog1 and Atoh1 are in a negative feedback loop [D (Raft et al., 2007)]. Knockin of Neurog1 into
Atoh1 and eﬀects of Neurod1 deletion suggest that this feedback loop is more complicated and at least in part mediated by Neurod1 (E). Modiﬁed after (Fritzsch et al., 2010; Pan et al.,
2012b).
B. Fritzsch, K.L. Elliott Developmental Biology 431 (2017) 3–15
5
pattern reminiscent of complete (Fritzsch et al., 2005b) or conditional
deletion of Atoh1 (Pan et al., 2011). These data clearly demonstrated
that Neurog1 cannot signal alone eﬀectively in postmitotic hair cell
precursors to initiate a diﬀerentiation as hair cells (Jahan et al., 2012).
This contrasts with the ﬂexibility of some neuronal precursors that
easily convert to intraganglionic hair cells upon elimination of Neurod1
(Jahan et al., 2010b). Importantly, mice heterozygotic for the
Atoh1tgNeurog1
knockin construct coexpressed both Atoh1 and
Neurog1 under Atoh1 promoter control with little negative eﬀects on
hair cell development or expression level of either gene. This suggested
that in diﬀerentiating hair cells there is no direct feedback loop of
Neurog1 on Atoh1 as previously claimed but a feedback loop of Atoh1
on Neurog1 remained a possibility (Raft and Groves, 2015).
It remained unclear whether this inability to rescue hair cell
development or to convert hair cells into neurons was related to
Neurog1 functional diﬀerences in activating Atoh1 speciﬁc e-boxes
(Fritzsch et al., 2010) or expression level diﬀerences. The latter
remained a possibility given the positive feedback loop Atoh1 protein
exerts on its own regulation via speciﬁc enhancer binding (Fritzsch
et al., 2006a; Helms et al., 2000). To distinguish between these two
possibilities, a mouse line was made that combined a delayed deletion
of Atoh1 (Pan et al., 2012a) with the misexpression of Neurog1 in the
second allele (Jahan et al., 2015b). These mice displayed a surprisingly
robust development of nearly all hair cells but in a disorganized organ
of Corti. Most importantly, the stereocilia phenotypes resembled those
observed in mutants of LGN and Gαi (Tarchini et al., 2016) and various
Myosin mutants (Quint and Steel, 2003). Combined these data suggest
that possibly the concentration and/or binding speciﬁcity of Atoh1 is
an essential aspect of normal stereocilia development. Further support
for the inﬂuence of Atoh1 on stereocilia development of hair cells
comes from mice in which Atoh1 expression level was manipulated
(Fig. 1) through Neurod1 mutations (Jahan et al., 2010b) or Sox2
manipulations (Dvorakova et al., 2016) or alterations in miRs (Soukup
et al., 2009).
The mechanosensory cell specializations to allow sensory cells to
develop this exquisite mechanosensation require a number of factors
for their normal development such as the basic Helix-Loop-Helix
transcription factors Atoh1 (Jahan et al., 2015b; Pan et al., 2012a) or
Neurod1 (Jahan et al., 2015a) that is tightly regulated in addition
through several other transcription factors such as Pou4f3 (Ikeda et al.,
2015; Xiang et al., 2003). Gﬁ1 (Hertzano et al., 2004) and Barlh1
(Chellappa et al., 2008) to name a few. Unfortunately, further work is
needed to narrow down how these multiple factors cooperate to
regulate the coordinated transformation of circular polarized cellular
apex found in choanoﬂagellates (Fritzsch and Straka, 2014) and certain
hair cell mutations (Jones et al., 2008) into a highly polarized hair cell
(Lu and Sipe, 2016). Additional information is needed to understand
the development of diﬀerent types of hair cells. Hair cells show variable
stereociliary diameters that clearly are aﬀected by bHLH genes (Jahan
et al., 2013, 2015b), but also show the unique stereocilia arrangements
of inner and outer hair cells that apparently evolved out of the much
simpler stereocilia bundle found in ancestral hair cells of the vestibular
type (Lewis et al., 1985). When and how factors that coordinate
polarity of hair cells across sensory epithelia (May-Simera et al.,
2015) evolved is unclear. Thus, while some connections between
Atoh1 level of expression and some of the downstream factors needed
for the apical development of the stereocilia bundle are likely (Taura
et al., 2016), we do not yet understand how vestibular and cochlear hair
cells molecularly evolved their unique stereocilia organization and loss
of a kinocilium. Atoh1 and other factors are also expressed in and
needed for Merkel cell development (Bermingham et al., 2001) and
Merkel cells share a mechanotransduction channel [Piezo2 (Woo et al.,
2014)] with some hair cells (Wu et al., 2016) indicating the possibility
that this highly conserved channel is directly tied into Atoh1 signaling,
possibly derived from an ancestral function in epidermal cells (Arendt
et al., 2016). Other channels associated with mechanosensory hair cells
have recently been identiﬁed as being part of the derived nonmechanosensory
‘hair cells’ of the electroreceptive organs, yet another
permutation of receptor cell development driven by a nearly identical
set of transcription factors (Bellono et al., 2017; Pierce et al., 2008).
In summary, we begin to understand how mechanosensory transduction
evolved in terms of the molecular machinery necessary to
develop the stereocilia arrangement and regulate mechanotransduction
channels, but details of this process still require additional work to
reveal the whole gene regulatory network.
Without doubt, the complete absence of any neuronal development
in the ear of mice null for Neurog1 demonstrates the essential nature of
this transcription factor for ear neurogenesis (Ma et al., 2000, 1998).
However, while there is an obvious interaction of neuronal precursor
formation with hair cell development (Matei et al., 2005), the detailed
nature of it remains unclear at a molecular and cellular level (Jahan
et al., 2015b; Raft and Groves, 2015). What is clear is the sequence of
activations of transcription factors that result in proliferation of
neuronal precursors and their diﬀerentiation (Goodrich, 2016).
Integral to this emerging perspective are several transcription factors
such as Neurod1 for normal diﬀerentiation and targeted projection to
the brain and the periphery (Jahan et al., 2010a), possibly through
regulation of the Pou-domain factor Brn3a (Pou4f1) and both regulate
expression of neurotrophin receptors (Huang et al., 2001; Kim et al.,
2001). The inner ear neurons are unique in that they co-express at
various levels the two neurotrophin receptors TrkB and TrkC whereas
hair cells and supporting cells express the two ligands, Bdnf and Ntf-3
(Fariñas et al., 2001; Fritzsch et al., 2016). These receptors and ligands
are essential for the viability of the neurons and participate in certain
aspects of guidance of neurons toward hair cells in response to the
neurotrophins released by hair cells and supporting cells (Yang et al.,
2011).
In essence, a nearly parallel cascade of isoforms of transcription
factors that represent a permutation of a common gene regulatory
network lead to diﬀerentiation of two distinct neurosensory cell types.
Instead of a single neurosensory cell, vertebrates have two cell types
that are locked into a mutually dependent system of short term support
to maintain neurons via hair cells/supporting cells (Fritzsch et al.,
2016) and long term support of hair cells via unknown factors provided
by innervation (Kersigo and Fritzsch, 2015). It appears that the
evolutionary split of single neurosensory cells into sensory neurons
and sensory cells was possible by duplicating speciﬁc aspects of gene
activation sequence (Atoh1 and Pou4f3 for hair cells, Neurog1 and
Pou4f1 for neurons) with Neurod1 being expressed in both and
providing a negative feedback loop (Fig. 2) for both Neurog1 and
Atoh1 (Jahan et al., 2010b). The embryonic early expression of
neurotrophins allows to actually trace the fate of both delaminating
neuronal and hair cell precursors (Matei et al., 2005) as they are
expressed in both cell types. However, Neurod1 and Pou4f1 regulate
neurotrophin receptor expression whereas neurotrophin expression is
apparently regulated by factors expressed in precursors, such as Sox2,
that can regulate expression of neurotrophins in supporting cells and
undiﬀerentiated hair cells of Atoh1 null mice, and in addition by Atoh1
(Fritzsch et al., 2005b; Pan et al., 2011). Past work suggested that
neurotrophins and their receptors evolved only in vertebrates that also
had an ear (Hallböök et al., 2006). However, recent work suggests
neurotrophin and neurotrophin receptor presence in other deuterostomes
(Benito-Gutiérrez et al., 2006; Burke et al., 2006) and bilaterians
(Zhu et al., 2008). Given the likely ancestry of neurotrophins, it is
possible that the segregation of hair cell/neuronal precursors and the
neurotrophin expression in the common precursor for both is associated
with evolution of proliferative neurosensory precursor system as
an integral part of the evolution of a vertebrate placode (Fritzsch and
Beisel, 2003; Schlosser et al., 2014). Obviously, the trophically interacting
hair cell/neuronal system could only evolve after neurotrophins
and their receptors had evolved, much like eﬀerents reaching hair cells
could only evolve after the cholinergic receptor evolution (Fritzsch and
B. Fritzsch, K.L. Elliott Developmental Biology 431 (2017) 3–15
6
Elliott, 2017). Further work is needed to identify the enhancer
modiﬁcations that allowed the duplicated GRN to be expressed
selectively in either neuronal or sensory cell and how the GRN changes
were tied into the mechanotransduction and axonal pathﬁnding of hair
cells and neurons, respectively.
In summary, data presented here are in line with hypothesis of
cellular diversiﬁcation through transformation of entire cascades of
early cell fate deﬁning transcription factor regulatory networks to
generate parallel networks of sister genes that alter radically the cell
type of at least partially clonally related cells (Fritzsch et al., 2006a). In
essence, through duplication and modiﬁcation of an entire cascade of
transcription factors, evolution transformed a single, neurosensory cell
into two derived cells, in agreement with a recently proposed idea of
apomeric relationships of diversiﬁed cells (Arendt et al., 2016). Further
work is needed to detail how trans- and cis element variations enabled
the distinct functions associated with regulating sensory neurons and
hair cell diﬀerentiation. We have detailed here some aspects of this
segregation and will highlight the consequences for neuronal projections
in part C further below.
1.2. Evolution and development of a mechanosensory ear and the
transformation into hearing organs
Statocysts as complex sensory organs are widespread among
metazoans (Fritzsch and Beisel, 2003; Fritzsch and Straka, 2014).
Most of these mechanosensors have unique sensory cells that perceive
the translational forces such as gravity acting on a denser structure
such as otoconia or a mobile body part through diﬀerent specializations
of the apical sensory structures (Fig. 3). This widespread distribution of
mechanosensory organs able to monitor position in the earth gravity
ﬁeld, even in animals without a fully developed central nervous system
such as diploblastic coelenterates and comb jellies, indicates the
essential need for any mobile animal to monitor its position in space
(Fritzsch and Straka, 2014). Among the transcription factors associated
with development of these mechanosensory organs are the Pax2/5/8
factors (Jacobs et al., 2007; Matus et al., 2007). While Pax2/8 are
clearly necessary for normal ear development in mice (Bouchard et al.,
2010), using this transcription factor as an evolutionary marker is
complicated by the fact that it is also essential for kidney development.
In fact, the pronephros and otocyst are immediately adjacent in the Pax
2/8 expression domains in developing amphibians, albeit in mesoderm
and ectoderm (Buisson et al., 2015). Interestingly enough, several
other transcription factors that are essential for vertebrate ear development,
such as Eya1/Six1 (Xu et al., 1999; Zou et al., 2008), Gata3
(Duncan and Fritzsch, 2013; Karis et al., 2001) are also essential for
both kidney and ear development. In addition, several other transcription
factors are needed for ear development (Foxi3, Fgf3/10, Fgfr2,
Sox9, Tfap2a (Alsina and Streit, 2016; Birol et al., 2016; Khatri et al.,
2014; McMahon, 2016; Papadopoulos et al., 2016; Singh and Groves,
2016). Based on the evolution of statocysts in diploblasts, one could
argue that evolution of the proneural gene regulatory network (GRN),
involving possibly Eya1/Six1, Foxi3, Pax2/8 and Gata3 network of
interacting factors and its dependence on Fgfr2 signaling (Pauley et al.,
2003; Pirvola et al., 2000), evolved in ectodermal sensory placodes and
was co-opted for mesodermal kidney development in triploblasts.
Further evolutionary GRN analysis in diploblast statocyst development
is needed to validate or refute this suggestion.
Beyond these early transcription factors and their expression in
kidney and otocyst, the developing otic placode, but not the developing
kidney, more highly or exclusively expresses several genes that ensure
neurosensory development of the otic placode/ectoderm. Clearly, ear
neurosensory development depends on several Fgfs (3,8,10) and Fgfr2
for overall neurosensory and Fgfr3 for supporting cell development
(Pirvola et al., 2000; Puligilla et al., 2007). Kidney development also
depends on Fgf 7/10 ligands signaling mostly via the Fgfr1 receptor
(Bates, 2007; Rossini et al., 2005), but also Fgfr2 (McMahon, 2016).
BMP4 downregulation combined with Fgf expression is an essential
early step in neural induction (Fritzsch et al., 2006a; Meinhardt, 2015)
that results in an unclear proportion of BMP4/Fgf signaling (Singh and
Groves, 2016). This essential step of Bmp4 suppression and Fgf
Fig. 3. Inner ear evolution and development of sensory epithelia show multiplication and diversiﬁcation. Hagﬁsh have a simple torus as a labyrinth with only three
epithelia: a common macula for gravistatic orientation and 2 canal cristae for angular acceleration. Latimeria have multiplied and diversiﬁed these epithelia into three gravistatic organs
(saccule, utricle and lagena) three canal cristae (anterior, horizontal and poster crista; AC, HC, PC) and two organs of unknown function the neglected papilla (PN) and the basilar papilla
(BP). Mice have reduced the gravity sensing epithelia through the loss of the lagena to two (utricle, saccule), have three canals and associated cristae (AC,HC,PC) and have elongated the
lagena duct into a coiled cochlea containing the organ of Corti for hearing. The splitting of sensory epithelia can be traced during development in salamanders as a progressive
segregation of sensory primordia. Modiﬁed after (Fritzsch et al., 2002).
B. Fritzsch, K.L. Elliott Developmental Biology 431 (2017) 3–15
7
expression is not only essential for placode induction but also suﬃces
to induce stem cells into diﬀerentiation as ear organoids in culture (Liu
et al., 2016). How much of the developmental cascade of otic placode/
otocyst development is recapitulated in these in vitro conditions and
how closely the level of BMP4/Fgf protein signaling needs to be
regulated for this development to occur remains to be determined.
During neuronal development (most likely including the ear), the
expression of early transcription factors leads to the expression of
pro-proliferative and neural-fate stabilizing transcription factors, such
as Otx2, Gbx2, Geminin, and Sox2 (Janesick et al., 2013; Singh and
Groves, 2016; Yellajoshyula et al., 2011) and the Baf complex (Seo
et al., 2005a, 2005b), that ultimately regulates chromatin remodeling
and proneural bHLH gene expression. Level of bHLH gene expression
in turn depends on the action of the Baf complex variably supported by
Eya1/Six1, Pax2/8, Sox2, Foxi3 and Gata3. How all of these early
transcription factors interact to deﬁne the level and topology of bHLH
gene activation and how Wnt signaling ﬁts in to regulate the size of the
otic placode (Ohyama et al., 2007) remains to be determined experimentally.
Loss of several factors can result in either incomplete
invagination of the otic placode in mice mutant for Gata3 (Karis
et al., 2001) or Pax2/8 (Bouchard et al., 2010), complete suppression of
ear placode invagination such as in Foxi3 mutants (Birol et al., 2016;
Singh and Groves, 2016) or in frogs exposed to RA (Fritzsch et al.,
1998) or even incomplete formation of the ear following loss of Fgfr2
(Pirvola et al., 2000), indicating that several interactions are needed to
move an otic placode forward to form an otocyst.
Eya1/Six1 play not only a role in preplacodal speciﬁcation but also
in later bHLH gene regulation such as Atoh1, Neurog1 (Ahmed et al.,
2012a, 2012b) and several other transcriptional regulators of neuronal
development of the otic placode (Riddiford and Schlosser, 2016). Pax2/
8 as well as Foxi1/3 are chromatin remodeling factors that could enable
expression of many other genes (Sharma et al., 2015; Singh and
Groves, 2016). While these transcription factors can already be
assembled into a rudimentary GRN for early neurosensory fate
determination in developing otic region (Riddiford and Schlosser,
2016) and as initial aspects or otic placode enhancers begin to surface
(Chen and Streit, 2015), the details of this network require more work
to ensure guidance of otic development out of stem cells. Without a
doubt, some of the factors needed for neurosensory development in the
ear are not expressed or are very limitedly expressed in the developing
kidney but can later be associated with kidney tumors due to their proproliferative
signaling, such as Geminin (Dudderidge et al., 2005).
Assuming that a neurosensory ectodermal ‘placode’ to generate statocysts
evolved ﬁrst in diploblasts, the lack of mesoderm and thus kidney
formation in these animals suggests that a minimal network of Eya1,
Pax2/8, Gata3 and Foxi3 was complemented ﬁrst in the otocyst to
modify pro-proliferative placodal development toward neurogenesis
through Sox and bHLH gene expression regulation that are also tied
into the sensory organ development.
In essence, this new developmental GRN network (Riddiford and
Schlosser, 2016; Schlosser et al., 2014), that possibly evolved with early
statocysts already in diploblastic animals such as jelly ﬁsh, may have
co-opted already existing GRNs dedicated to diﬀerentiate sensory cells
(bHLH and Pou genes). Given that some of these factors are also
participating in other developmental GRNs, their evolution speciﬁcally
for neurosensory development regulation is less likely. For example,
Atoh1 is not only regulating hair cell development but many other cells
as well (Fritzsch et al., 2015a; Mulvaney and Dabdoub, 2012). This cooption
of a cellular diﬀerentiation program ensured that existing single
cell programs aggregate and integrate into a highly localized neurosensory
development program leading to otic placode and otocyst/
statocyst development. Some early stages of otic development in
chicken are accompanied by cellular migration to coalesce disperse
neurogenic pre-placodal cells into placodes (Steventon et al., 2016).
Unfortunately, the various morphogenetic movements in early placode
to otocyst formation (Alsina and Streit, 2016; Fritzsch et al., 1998)
make it diﬃcult to generalize from chicken development to otocyst
evolution. Molecular developmental programs of more diploblast
statocysts need to be analyzed to reveal the presence or absence of
the basic regulator network of localized transformation of ectoderm
into neurosensory ectoderm. It should be noted that several of the
genes of this putative network are conserved in eukaryotes or bilaterians
(Eya1, Six1, Pax2/8; Gata3, Sox2) and thus could indeed have
evolved into a GRN that was coopted for its current function in otic
development through altered downstream regulations and interactions
with neurosensory transcription factors.
Independent of these actively investigated issues on the evolution of
the molecular development of GRNs to make a placode, there is
documented evolutionary change in the otic placode-derived ear
(Lewis et al., 1985) which we like to explore next. For example, there
is the simple addition of sensory epithelia, some of which acquire novel
functions through association with speciﬁc structures to provide novel
mechanical stimulation to these multiplied sensory epithelia (Fritzsch
et al., 2002). In order to highlight this principle of duplication and
diversiﬁcation we will only discuss the molecular basis of the jawed
vertebrate lateral/horizontal canal evolution and the molecular basis of
the auditory organ evolution.
Lampreys and hagﬁsh are jawless vertebrates that have no lateral
(horizontal) canal with a uniquely associated lateral canal crista (Lewis
et al., 1985). However, while the ear of hagﬁsh is a simple torus with
two canal cristae and an elongated gravity sensing common macula
(Fig. 3), lampreys have evolved a complicated labyrinth with several
unique features including two medial canals that together seem to serve
the function of the single lateral canal of jawed vertebrates (Maklad
et al., 2014). It appears that several genes drive lateral canal development,
as a canal is missing in null mutants of these genes, Otx1
(Fritzsch et al., 2001; Morsli et al., 1999), N-Myc (Domínguez-Frutos
et al., 2011; Kopecky et al., 2011) and Lmx1a (Koo et al., 2009; Nichols
et al., 2008). In contrast to vertical canals where the Fgf10/BMP4
positive sensory epithelium drives canal formation (Chang et al., 2004;
Pauley et al., 2003), a horizontal canal forms even in the very transient
presence of a canal crista primordium in Foxg1 null mice (Hwang et al.,
2009; Pauley et al., 2006). Notably, truncating ear proliferation
through elimination of N-Myc seems to suﬃce to eliminate this
evolutionary late addition to the jawed vertebrate ear development
(Domínguez-Frutos et al., 2011; Kopecky et al., 2011) and proliferative
precursors segregation is apparently dependent on repressive interactions
between the notch signaling pathway (Daudet et al., 2007; Daudet
and Lewis, 2005) and Lmx1a (Nichols et al., 2008).
The apparent independence of canal formation from canal crista
formation in development of the horizontal canal suggests that GRNs
driving development of either were paired through independent events.
Comparison of lamprey ears with the sensory epithelia and their
innervation in Otx1 null mutants suggest that a sensory patch of
unknown function in lampreys may be the precursor for the horizontal
canal crista recruited to function in the novel angular acceleration
sensing system enabled through the development of a horizontal canal
(Fritzsch et al., 2002). If further work supports this, it would indicate
that formation of a separate sensory patch allowed implementation of a
novel level of angular acceleration sensing once the molecular alterations
in jawed vertebrates generated a horizontal canal. A similar
predisposition seem to be the case for the segregated patches of
neglected papilla that can under certain conditions evolve into the
amphibian papilla for middle frequency hearing (Fritzsch and Wake,
1988). Enough ﬂexibility in the boundary deﬁnitions of sensory
epithelia is needed to allow such segregation of distinct patches of
cells to occur and proliferation and Lmx1 seem to play a part in this
ﬂexibility (Fritzsch et al., 2013).
More complicated and contentious is the evolution of the basilar
papilla and its transformation into the organ of Corti of mammals.
There is agreement that a novel sensory patch had to form ﬁrst to be
later transformed in several steps into the auditory basilar papilla of
B. Fritzsch, K.L. Elliott Developmental Biology 431 (2017) 3–15
8
most tetrapods that eventually was transformed into the coiled
mammalian cochlea housing the organ of Corti (Fritzsch et al.,
2013). Part of the contention derives from the ﬁnding that some
aquatic sarcopterygian ﬁsh have such a unique sensory epithelium but
have no known sound pressure communication (Fritzsch, 1987, 1992).
Furthermore, the changing taxonomic positions of basal sarcopterygians
necessitating to invoke a loss of this sensory patch in lungﬁsh
(assuming the sensory organ in Latimeria is ancestral to the same
named organ in tetrapods), or the sensory epithelium in Latimeria
being a convergent evolution associated with an equally convergent
evolution of a lagena and a lagenar recess (Fritzsch et al., 2013). A
second area of contention is the structural dissimilarity of the organ of
Corti of all therians (Jahan et al., 2015a) and its evolution already in
monotremes in terms of inner and outer hair cells (Chen and Anderson,
1985; Ladhams and Pickles, 1996) and their prestin motor (Okoruwa
et al., 2008) separated by pillar cells with unusual molecular and
structural features not found in other supporting cell type of any
vertebrate (Jahan et al., 2015a). Importantly, in monotremes, the
basilar papilla/organ of Corti resides in the lagena recess that has a
lagena sensory epithelium at its tip that is entirely separated from the
sound conducting perilymphatic space connected to the round and oval
window (Schultz et al., 2016). Obviously, eutherian mammals lack a
lagena, have a coiled cochlea with the organ of Corti extending to the
apex, and have a continuous perilymphatic space (scala tympani, scala
vestibuli) that provides the means for sound pressure propagation from
the oval to the round window. Whether or not the lagena was lost or
incorporated into the expanding organ of Corti hinges currently on
evidence provided by mutations that truncate proliferation of hair cell
precursors (Fritzsch et al., 2013). In N-Myc mutant mice, the apex of
the continuous organ of Corti develops a vestibular type of epithelium
(Fig. 4) without any specialization of inner and outer hair cells
reminiscent of an organ of Corti (Kopecky et al., 2011). In addition,
much like in monotremes, the perilymphatic space loops around this
unusual apical epithelium that is in a shortened cochlea duct resembling
a monotreme lagena recess. Another similarity with monotremes
of the N-Myc mutants is the lack of a ductus reuniens allowing a broad
communication of the base of the cochlear/lagena recess with the
saccule. Most interesting is that many supposedly early evolutionary
markers for the ear such as Pax2 and Gata3 cause selective loss of the
organ of Corti in their absence (Bouchard et al., 2010; Duncan and
Fritzsch, 2013), indicating a much tighter control of the organ of Corti
development, compared to vestibular organs, by apparently ubiquitously-expressed
transcription factors. How early in organ of Corti/
basilar papilla evolution those transcription factors got involved in its
development remains to be shown across sarcopterygians.
The organ of Corti is the most sophisticatedly patterned sensory
organ of vertebrates with a highly conserved and extremely stable
cellular organization in all eutherian mammals (Jahan et al., 2015a). It
evolved out of a less organized organ found in monotremes through
reorganization of already speciﬁed inner and outer hair cells as a
consequence of coiling related elongation that rearranged multiple
rows of inner and outer hair cells of monotremes (Fritzsch et al., 2013;
Schultz et al., 2016) into the single row of inner and three rows of outer
hair cells of eutherian mammals (Lewis et al., 1985). Eliminating a
single gene in mouse development, Foxg1, can alter the organ of Corti
development to mimic that of monotremes in terms of shortening and
rearrangement of hair cells into multiple rows (Fritzsch et al., 2013;
Pauley et al., 2006) and similar eﬀects can be achieved by altering
convergent extension in PCP mutants (Jones et al., 2008).
Only the therian organ of Corti evolved pillar cells that allow a
rocking movement of the lateral part to enhance endolymph ﬂow across
the inner hair cells for stimulation (Richter et al., 2007). Molecularly,
this sophisticated arrangement into discrete rows of structurally and
functionally diﬀerent hair cells and supporting cells seems to depend
on the segregated expression of transcription factors that are coexpressed
in vestibular sensory epithelia but ﬂank the medial and
lateral aspect of the organ of Corti, Fgfs and BMP4 (Groves and Fekete,
2012; Pan et al., 2011). In addition, Wnt signaling plays a role in this
cellular assembly (Munnamalai and Fekete, 2016; Sienknecht et al.,
2014). Integrated into the diﬀusible factor signaling is the cell-cell
interaction mediated by lateral inhibition via the Delta-Notch signal
pathway (Korrapati et al., 2013) and diﬀerential eﬀects of IHC and
OHCs to mutations in the PCP pathway (Jones et al., 2008). This dual
diﬀusion/cell-cell interaction program ensures the highly stereotyped
organ of Corti development that requires both cell-cell interaction and
diﬀusible signal changes. If both are disrupted the Organ of Corti
shows a scrambled assembly of hair cells/supporting cells with limited
speciﬁcation of normally very distinct features (Jahan et al., 2015b)
and mice are deaf. Molecular analysis of embryonic development in
monotremes is needed to show how diﬀerentiation of two distinct hair
cell types with unique numerical ratios in the medial and lateral aspect
of the organ of Corti is guided. In essence, evolution of the organ of
Corti could be an excellent case of sub-functionalization where
diﬀerential expression regulation of diﬀusible factors (BMPs, Fgfs,
Wnts) drives an innovative cellular arrangement largely speciﬁed by
Delta/Notch interactions into a novel pattern enabling sound analysis
over a vast range of frequencies.
In summary, once the ﬁrst step of aggregation of neurosensory cell
development into a discrete region was accomplished through either
development of or co-option of a cellular GRN, further evolution
followed the principles established for molecular and cellular evolution:
multiply and diversify. Even such structurally and functionally
diﬀerent organs, such as the organ of Corti and the saccule, seem to
form from a single anlage that needs to segregate properly (Daudet
et al., 2007; Kopecky et al., 2011; Nichols et al., 2008) to enable normal
regulation of expression of diﬀusible factors that drive the unusual
cellular distribution and specializations of this most complicated
cellular assembly in the vertebrate body.
1.3. Evolution of central projections and auditory nuclei
Processing of novel information requires that sensory organs not
only are tuned to such novel stimuli but also transmit this information
separately to the brain where it needs to be processed by a distinct set
of neurons as a discrete sensory information. A prime example is the
evolution of the basilar papilla/organ of Corti (Fig. 5) that projects in
all tetrapods with a sound ampliﬁcation capable middle ear (Manley
and Sienknecht, 2013) to recognizable auditory nuclei of the brainstem
(Grothe et al., 2004) using a dedicated set of sensory neurons (spiral
ganglion neurons in eutherian mammals). In principle, there are three
possible scenarios how this interconnected system of sound reception,
information transmission and information processing could have
evolved:
1) Outside-in with sensory organ evolution followed by sensory
neuron evolution followed by auditory nuclei evolution
2) Inside-out starting from cochlear nuclei going backward to the
auditory periphery.
3) Starting with the evolution of sensory neurons followed by sequential
or simultaneous evolution of the sensory organ in the ear and
the sensory nuclei in the brain.
Current information of trophic dependency strongly supports
assumption 1. Sensory neurons depend on the peripheral target for
viability (Fritzsch et al., 2016) and cochlear nuclei neurons depend on
innervation for viability and diﬀerentiation much like other brainstem
second order sensory neurons (Elliott et al., 2015; Rubel and Fritzsch,
2002). Conversely, lack of parts of auditory nuclei has only a limited
eﬀect on sensory neuron viability (Maricich et al., 2009) and hair cells
depend on innervation only with a long delay (Kersigo and Fritzsch,
2015). During development, spiral ganglion neurons exit the cell cycle
before hair cells and cochlear nucleus neurons (Altman and Bayer,
B. Fritzsch, K.L. Elliott Developmental Biology 431 (2017) 3–15
9
1980; Matei et al., 2005) and project both centrally and peripherally
before their target cells start to diﬀerentiate (Fritzsch et al., 2005a,
2005b). In fact, correct targeting of auditory aﬀerents is possible
without either hair cells or auditory nuclei indicating that other
mechanisms guide aﬀerents (Elliott et al., 2017). This aspect of
development implies support for scenario 3, but might also represent
a heterochronic shift in the developmental timetable.
Irrespective to this question, aﬀerents need to respond to inner ear
and CNS guidance cues to allow segregated projections to a unique
inner ear sensory organ dedicated to sound perception as well as a
unique central target able to process sound without vestibular stimulus
mixing (Fig. 5). Salamanders lack a tympanic ear and show limited
sensitivity to sound (Zeyl and Johnston, 2016) but have a basilar
papilla with separate sensory projection within the CNS though they
lack morphologically-distinct auditory nuclei (Herrick, 1948;
Nieuwenhuys et al., 1998; Will and Fritzsch, 1988). Similar segregated
projections in the absence of nuclei were found in the visual system
(Fritzsch, 1980) and might indicate a more general evolutionary
principle, segregated neuropils appear before segregated nuclei
(Herrick, 1948). Details of the molecular basis of aﬀerent segregation
in the ear and to the CNS remain obscure but may be related to several
known pathﬁnding molecules such Ephrins (Cramer and Gabriele,
2014; Siddiqui and Cramer, 2005), semaphorins, and other diﬀusible
factors released from the hindbrain such as Wnts and Bmps
(Henríquez and Osses, 2016; Seiradake et al., 2016) as well as cellular
interactions. Indeed, downstream factors of the Wnt signlaing pathway
aﬀect the topology of cochlear projections (Yang et al., 2017). Clearly,
Gata3 and Neurod1 are critical for this segregated projection from the
ear to the cochlear nuclei (Fritzsch et al., 2006b; Goodrich, 2016;
Jahan et al., 2010a) but exactly when the relevant genes were ﬁrst
expressed in the auditory inner ear neurons and how they tied into the
novel pathﬁnding selection process to connect a novel periphery with a
novel central target remains to be shown (Fritzsch et al., 2015b).
Consistent with the ancestral split of a neurosensory cell with an axon
into a hair cell and its sensory neuron is that central projections of
sensory neurons do not depend on neural crest derived Schwann cells
(Mao et al., 2014) whereas the peripheral projection is highly disorganized
and many aﬀerents miss the hair cells and sensory epithelia
(Fig. 5). Moreover, at least in frogs, transplanted ears near the
hindbrain can project directly to or reroute to the vestibular nuclei
(Elliott et al., 2014), strongly supporting the notion that diﬀusible
factors released from the hindbrain (Fig. 5) play a role in this process
as substrate interactions are out of the question in these transplants. In
contrast, inner ear aﬀerents of ears transplanted to the orbit, enter
Fig. 4. Loss of N-Myc results in loss of the horizontal canal and the formation of a “lagena” at the apex of the cochlea. Compared with a control inner ear (A), loss of Nmyc
results in a truncated cochlea (B) that is in broad continuation with the saccule. Furthermore, there is loss of the horizontal canal and at the enlarged apex of the cochlea (B), there is
a structure reminiscent of the lagena found in monotremes, including a vestibular-like organization of hair cells (black arrow in D) instead of one row if inner and three rows of outer hair
cells found in the control organ of Corti (white arrow in C,D). Modiﬁed after (Kopecky et al., 2011).
B. Fritzsch, K.L. Elliott Developmental Biology 431 (2017) 3–15
10
randomly the midbrain (Elliott et al., 2013).
Irrespective to the sequence of formation of central projections, the
spinal cord-like organization of the hindbrain has to be transformed
into a hindbrain nuclear organization, necessitating alterations in
functional longitudinal columns that extend from the spinal cord to
the midbrain/hindbrain boundary (Albuixech-Crespo et al., 2017;
Fritzsch and Glover, 2006; Nieuwenhuys and Puelles, 2015). Since
vestibular, cochlear, mechanosensory, and electrosensory lateral line
aﬀerents are in all vertebrates restricted to the hindbrain (Fig. 5), the
basic patterning of the spinal cord needs to be altered through the
addition of those nuclei and/or transformation of existing spinal cord
nuclei into nuclei able to receive and process this information
distinctly. As it turns out, some of the transcription factors ensuring
diﬀerentiation of the most dorsal spinal cord and hindbrain second
order sensory nuclei development appear to be conserved from spinal
cord to the cerebellum. Most dorsal in either is the expression of Atoh1,
Neurog1/2 and Ascl1 (Bermingham et al., 2001; Fritzsch et al., 2006b)
with Neurog1/2 being only transiently expressed. However, more
recent data show subdivisions in these populations through the nested
expression of various transcription factors that likely are driven by the
intricate pattern generated by ventralizing Shh signal emitted from the
ﬂoor plate and the dorsalizing Wnt/BMP signal emitted from the roof
plate (Fritzsch et al., 2006b; Lai et al., 2016). The basic pattern of
bHLH genes is supplemented by addition of other bHLH and homeobox
factors (Hernandez-Miranda et al., 2016) and shows local,
rhombomere-speciﬁc variation in the hindbrain. Most importantly,
bHLH genes such as Ptf1a are both highly conserved in their expression
(Sugahara et al., 2016) but also elicit a local rearrangement of
nuclear development when mutated (Iskusnykh et al., 2016). How the
GRNs of spinal cord cellular population are both systematically and
rhombomere-speciﬁcally inﬂuenced to give rise to the four above
mentioned nuclei dedicated to processing the information from the
ear in vestibular and cochlear nuclei as well as the mechanosensory and
electrosensory lateral line nuclei remains to be determined in the light
of an apparently rather stable Hox code (Krumlauf, 2016; Murakami
et al., 2004; Parker et al., 2016). What is beginning to emerge is that
speciﬁc transcription factors can deﬁne speciﬁc subsets of brainstem
nuclei such as the solitary tract (Qian et al., 2001) or auditory nuclei
(Fritzsch et al., 2006b; Wang et al., 2005).
More recent data suggest that bHLH gene interactions and crossregulation
may be essential for diﬀerent compartment speciﬁc alar
plate diﬀerentiation. Chief among those is Ptf1a, a bHLH transcription
Fig. 5. Scheme of diﬀerences in vestibular, auditory, mechanoreception, and electroreception organ projections in the hindbrain as revealed by tracing
experiments. The various peripheral sensory organs [vestibular (VN), auditory (AN), mechanosensory lateral line (LL), or electrosensory ampullary organs (ELL)] each have unique
central projections that terminate in distinct, non-overlapping regions in the hindbrain. The transition from aquatic, anamniotic vertebrates to terrestrial amniotic vertebrates was
accompanied by the loss of all lateral line organs and the gain of auditory sound pressure receivers projecting via newly evolved auditory sensory neurons of the ear (AN) to apparently
new auditory nuclei. In addition, eﬀerent cells lose connections to mechanosensory lateral line and evolve new connections of a segregated population to auditory epithelia. Formation of
central nuclei is mediated in part by bHLH transcription factors that also are important in ear neurosensory development (Atoh1, Neurog1) and their expression is speciﬁed by the
dorso-ventral patterning morphogen gradients in the hindbrain (BMPs, Wnts and Shh). Aﬀerent projection of diﬀerent ganglia related to lateral line organs or other sensory modalities
(A) develop progressively over time (A′, A′′, A′′′, A′′′′). Likewise, diﬀerent inner ear organs (B) project directly to their distinct target nuclei, possibly guided by morphogen gradients
(indicated as colored wedges representing ligands in the brain and receptors in the ganglia). Elimination of Schwann cells through conditional deletion of Sox10 does not alter central
projections (C,D) but partially disables aﬀerents from ﬁnding the organ of Corti (E). Tectorial membrane, TM; auditory epithelia, AE; vestibular epithelia, AN, auditory neurons; VE,
vestibular epithelia; VN, vestibular neurons; PLS, perilymphatic space. Modiﬁed after (Fritzsch et al., 2005a, 2015b, 2006b; Mao et al., 2014; Sienknecht et al., 2014).
B. Fritzsch, K.L. Elliott Developmental Biology 431 (2017) 3–15
11
factor that is essential for cerebellar (Pascual et al., 2007) (Millen et al.,
2014) and dorsal cochlear nucleus development (Fujiyama et al., 2009)
but other bHLH genes play a role as well (Cai et al., 2016). In addition,
to be relevant for the formation of nuclei or brain areas, Ptf1a also
plays an essential role in cell fate determination within a given nucleus
deﬁning excitatory and inhibitory neurons (Iskusnykh et al., 2016)
Beyond formation of entire nuclei, several genes are now emerging that
interact to deﬁne diﬀerent cell types in diﬀerent brainstem nuclei
important for their normal function (Nothwang, 2016). Above we
outlined how multiplication of existing cell fate determining bHLH
genes provides the molecular start of distinct cell fate evolution (Arendt
et al., 2016; Fritzsch et al., 2000) for neurosensory evolution of the ear.
Here we suggest that the evolution of various rhombomere-speciﬁc
hindbrain nuclei is a consequence of bHLH transcription factors that
are variably tied into the Hox code regulation to achieve local cell fate
changes and, by logical extension, formation of novel nuclei.
While cell fate switching during metamorphosis from lateral line to
auditory (Herrick, 1948) was a reasonable idea in analogy to the wellestablished
Reichert-Gaupp hypothesis of middle ear ossicle transformation
(Maier and Ruf, 2016), it rather appears now that these nuclei
actually die by apoptosis in frogs losing the lateral line (Fritzsch et al.,
1988; Wahnschaﬀe et al., 1987) or co-exist in those retaining the
lateral line. Formation of new nuclei is more likely to reﬂect assembly
of novel interactions of the increasingly complex transcriptional landscape
of the hindbrain that drives both expansion of cell populations
through proliferation regulation and acquisition of novel phenotypes by
regulatory changes of already complicated GRNs that evolved in the
spinal cord to guide sophisticated, yet diﬀerent, cell assemblies (Lai
et al., 2016). In this context it is remarkable that some molecular
conservation exists in peripheral pain receptors in skin and ear
[Piezo1,2 (Wu et al., 2016)] and that loud sound causes pain sensation
comparable to the pain perceived in the spinal cord.
In summary, it appears that gene regulatory network evolution is
underlying both auditory neuron and auditory nuclei evolution. How
the already established gene regulatory networks for inner ear neuron
formation and hindbrain nuclear formation was nearly simultaneously
modiﬁed to yield novel projections to process a novel stimulus acquired
by a modiﬁed sensory epithelium dedicated for sound processing to be
processed by a highly modiﬁed spinal cord neuronal network forming
apparently novel auditory nuclei remains to be worked out.
2. Conclusion and outlook
This review summarizes evolutionary changes in molecular developmental
gene regulatory networks that highlights speciﬁc, likely steps
taken in the assembly of mechanosensory systems starting from a
single celled organism ending with an integrated circuit consisting of
an interactive neuronal connectome for auditory signal processing.
While the broad outline of such changes appear to be highly supported
by descriptive and experimental data, details in particular of the gene
expression regulations underlying the functional diversiﬁcation of
multiplied GRNS remain obscure for the time being and require more
work.
Acknowledgements
This work was supported by NIH R01 DC005590 to BF and R03
DC015333 to KE.
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