Patterns &amp Phenotypes DOI10.1002/dvdy.172: Developmental Dynamics

Developmental Dynamics
PATTERNS & PHENOTYPES

DOI10.1002/dvdy.172
Histological Development and Integration of the Zebrafish Weberian Apparatus
Nathan C. Bird*, Selena S. Richardson, and Jeremy R. Abels

Department of Biology
McCollum Science Hall 107
University of Northern Iowa
Cedar Falls, IA 50614
*Author of Correspondence
Accepted Articles are accepted, unedited articles for future issues, temporarily published
online in advance of the final edited version.
© 2020 Wiley Periodicals, Inc.

Received: Aug 03, 2019;Revised: Dec 20, 2019;Accepted: Mar 23, 2020
This article is protected by copyright. All rights reserved.

Keywords: Danio rerio, Weberian ossicles, Inner Ear, Swim Bladder, Ontogeny,
Ossification, Soft Tissue
ABSTRACT
Background: The Weberian apparatus enhances hearing in otophysan fishes, including
zebrafish (Danio rerio). Several studies have examined aspects of morphological
development of the Weberian apparatus and hearing ability in zebrafish. A
comprehensive developmental description including both hard and soft tissues is
lacking. This information is critical for both interpretation of genetic developmental
analyses and to better understand the role of morphogenesis and integration on
changes in hearing ability.
Results: Histological development of hard and soft tissues of the Weberian apparatus,
including ossicles, ear, swim bladder, and ligaments are described from early larval
stages (3.8 mm NL) through adult. Results show a strong relationship in developmental
timing and maturation across all regions. All required auditory elements are present and
morphologically integrated early, by 6.5 mm SL. Dynamic ossification patterns and
changes in shape continue throughout the examined developmental period.
Conclusion: This study provides the first comprehensive histological description of

Weberian apparatus development in zebrafish. Morphological integration was found
early, before increases in hearing ability were detected in functional studies (>10 mm
TL), suggesting morphological integration precedes functional integration. Further
research is needed to examine the nature of the functional delay, and how maturation of
the Weberian apparatus influences functionality.

1. INTRODUCTION
The Weberian apparatus is a novel structure that allows for enhanced hearing in
otophysan fishes, a dominant freshwater clade with global distribution and over 10,000
species (Berra 2001, Nelson et al. 2016). The largest otophysan clade, Cypriniformes,
includes species such as minnows (including the zebrafish, Danio rerio), suckers, and
loaches. The Weberian apparatus allows for sound pressure detection, which increases
detectable frequency ranges and yields higher sensitivity at lower thresholds, increasing
overall hearing capabilities compared to fishes without hearing adaptations (Fay 1988,
Schellart & Popper, 1992, Fay & Simmons 1999).
The Weberian apparatus is a complex morphological structure comprised of
several modified elements. The elements of the Weberian apparatus span from the
posterior skull to the anterior trunk (Figure 1), and its components are morphologically
and functionally integrated (Rosen & Greenwood 1970, Chardon & Vandewalle 1997,
Bang et al. 2001, Bird & Mabee 2003). Three regions play vital roles in functionality of
the apparatus: swim bladder, vertebrae, and inner ear (Figure 1). The receptive organ is
the swim bladder, which pulsates in a sound field and captures sound pressure input.
This pulsation is transduced by the Weberian ossicles, a collection of modified vertebral
elements that, via rocking and ligamentous connection, transmit the signal anteriorly
toward in inner ear. The anteriormost ossicle, the scaphium, forms the lateral wall of the
posterior section of the fluid-filled sinus impar (sinus perilymphaticus). Lateral rocking of
the scaphium produces fluid motion in the sinus impar, which is then transferred
anteriorly to the transverse canal of the sinus endolymphaticus within the skull. The
transverse canal opens within the saccule, near the sagitta (otolith), causing differential
motion of the sagitta relative to the saccular macula. While no definitive study has
proven that the saccule is the singular endorgan for Weberian apparatus-mediated
sound pressure hearing, its fluted morphology, proximity to the transverse canal, and
stimulation at high frequencies suggest it is the likely endorgan for this type of reception

for many otophysans (Popper & Fay 1981, Ladich & Popper 2004, Popper and Schilt
2008, Schulz-Mirbach et al. 2013). However, the lagena may play a role in otophysic
hearing (particle motion, sound localization) in many otophysic species (Schellart and
Popper 1992, Popper & Fay 1999, Schulz-Mirbach & Ladich 2016)
The morphology of the Weberian apparatus within cypriniform fishes is highly
variable, exhibiting several different morphologies that likely correlate with environment
(Bird & Hernandez 2007). The acoustical environment likely plays a critical role in
hearing evolution, with several factors shaping both morphological structures related to
hearing, and hearing capabilities in general (Schulz-Mirbach & Ladich, 2016). The ability
to discern between important and non-important sounds (stream segregation of Popper
& Schilt 2008) would be an advantage in most environments. Many morphological
changes likely are necessary to compensate for noise created by changes in water flow
rate, as well as for a benthic lifestyle and its effect on the swim bladder and its primary
role with buoyancy. The relationship between hearing sensitivity and swim bladder size
and shape has been examined extensively in recent years, with smaller swim bladders
leading to reduced hearing ability in cichlids (Schulz-Mirbach et al. 2012) and catfishes
(Lechner & Ladich 2008, Zebedin & Ladich 2013, Ladich 2016). In addition, lower water
temperatures lead to decreased hearing functionality (Maiditsch & Ladich 2014).
1.1 Zebrafish Weberian apparatus development

While studies have been published regarding the developmental anatomy of the
zebrafish Weberian apparatus through ontogeny (e.g. Bird & Mabee 2003, Grande &
Young 2004), most research has been limited to specific regions (ear, vertebrae, and
swim bladder) or specific tissue types (skeleton). This narrow focus limits the ability to
assess the potential modularity and integration inherent to the system, as well as
limiting prediction of functionality through ontogeny.

Research into the development of the teleost ear and aquatic hearing long
predates the ascension of the zebrafish to model species status, and questions into
evolution and development of the otophysan ear have focused on other species (see
Platt & Popper 1981 and Popper & Platt, 1983 for comparative reviews). Most early
zebrafish research has focused on aspects of inner ear morphogenesis and partitioning,
as well as hair cell development and orientation. Embryonic development was first
documented by Kimmel et al. (1995), followed by detailed studies (including histology)
on early development by Haddon and Lewis (1996) and studies on mutation-based
effects on ear development (Malicki et al. 1996; Whitfield et al. 1996). Further detailed
analysis of the larval and later stages of inner ear development through 30 days post-
fertilization (dpf) were presented by Bever and Fekete (2002) and in specimens above
12 mm TL by Wang et al. (2015). Whole-mount development of the appropriate skull
region surrounding the pars inferior has been examined by Cubbage and Mabee (1996)
and in part by Grande and Young (2004).
Development of the zebrafish swim bladder has gained increased attention in
recent years. A definitive whole-mount description of the developing zebrafish swim
bladder was published by Robertson et al. (2007), which was in conjunction with an
earlier description of the structure and innervation of the adult zebrafish swim bladder
(Finney et al. 2006). Winata et al. (2009) added data on early development and
molecular markers during early (through 5 dpf) swim bladder formation and
development.
Vertebral development, with particular attention to the auditory ossicles, has
been studied in detail (Bird & Mabee 2003, Grande & Young 2004). Histological
analysis of the Weberian ossicles has a long history in close relatives of the zebrafish,
including Barbus barbus (Vandewalle et al. 1990), Carassius auratus (Watson 1939),
Labeo rohita (Kulshrestha 1977), Tribolodon hakonensis (Ichiyanagi et al. 1996), and
Zacco platypus (Ichiyanagi et al. 1996). Broader comparative studies have also been

carried out on varied cyprinid species (Alexander 1962, Bogutskaya 1991). Several
studies from the early 2000's documented the whole-mount development of the
zebrafish Weberian ossicles (Sanger & McCune 2002, Bird & Mabee 2003, Grande &
Young 2004), with a few including very limited histological detail (Bird & Mabee 2003,
Grande & Young 2004).
Functional testing of the hearing ability of the zebrafish has generally focused on
two stages: very early (embryonic to early larval) and late (juvenile to adult). These
studies have found conflicting results on the possible ontogenetic changes in hearing
ability of the zebrafish. Early studies by Zeddies and Fay (2005), Lu and DeSmidt
(2013), Bhandiwad et al. (2013), and Yao et al. (2016) examined hearing at 7 days post
fertilization and earlier, finding some increases in hearing capability during early larval
ontogeny, with their results likely varying due to differences in techniques. This is in
contrast to studies on later stage zebrafish by Higgs et al. (2001, 2003). In their studies,
only limited changes were seen from 10-47 mm TL, with one study finding no
differences in threshold or frequency detection (Higgs et al. 2001), while the other found
gradual increases using auditory brainstem response, but again no consistent difference
in frequency detection or amplitude were found across the ontogenetic stages
examined. This calls into question when the Weberian apparatus both begins to
function, and when it is fully operational. In an interesting comparative study, Monroe et
al. (2016) tested for hearing differences related to the genetic and transgenic
background in zebrafish lines, finding important differences between certain lines, and
underpinning the critical choice of appropriate line selection in hearing studies.
While whole-mount based development of the Weberian apparatus has been
presented in the research summarized above, little information is known about the soft
tissue components. Also, system-wide analysis on how these regions are integrating at
the tissue-level to form a functional apparatus is lacking. Precise connectivity between
elements and regions, accomplished primarily by ligaments, is crucial for functionality.

Histological data of all elements of the Weberian apparatus across an ontogenetic
series of zebrafish, spanning from early larval (3.8 mm Notochord Length) through adult
were used to reconstruct developmental timing and integration. For each length chosen,
the hard and soft tissue of the ear, vertebral column, and swim bladder were examined
for size, shape, ossification, connectivity, and integration. Data are presented in relation
to development within general developmental stages. While several studies have been
published with different proposed developmental stages based on various internal and
external features (Schilling 2002, Grande & Young 2004, Parichy et al. 2009), we
generally follow Schilling's (2002) simplified early larval, mid larval, late larval, and
juvenile stages.
Here we describe aspects of development (presence, growth, ossification,
composition, etc.) for elements of the following regions. In the ear, structures of the pars
inferior (the primary auditory region of the ear), including the bones lining the chambers
(exoccipital and basioccipital), the saccule and lagena, and the maculae and otolithic
membranes associated with each otolith (due to necessary decalcification, otoliths are
lost during processing, so otolithic membranes are used as a general proxy for
histology). Also described are the transverse canal (of the sinus endolymphaticus) and
the sinus impar (sinus perilymphaticus). In the vertebral region, the pars auditum is
described: scaphium (bony concha scaphium and cartilaginous articular process),
claustrum (cartilaginous corpus claustra and bony scutulum claustra), intercalarium
(bony manubrium and cartilaginous articular process), tripus (bony wing and
cartilaginous body, as well as the articular, anterior, posterior, and transformator
processes), and the elements of the fourth vertebra (cartilaginous parapophysis 4, bony
rib 4, and os suspensorium). Also described are the associated ligaments: interossicular
ligament (between scaphium and intercalarium, and between intercalarium and tripus),
the tripus-parapophysis 4 ligament, and the ligament joining parts of the tripus, os
suspensorium, and tunica externa of the swim bladder (herein referred to as the triple

ligament). The size and composition of the saccus paravertebralis surrounding the
ossicles are also described. Lastly, the swim bladder and its tunica layers (externa and
interna) are described.
The goals of this study are: 1) to describe the histological development of the
zebrafish Weberian apparatus, 2) to identify timing of development and morphological
integration across the apparatus, 3) to place our data in the context of previous
research on the zebrafish Weberian apparatus, and 4) to identify early barriers to
increased functionality in the earliest integrated apparatus. This study provides critical
new developmental data that give insight into how the Weberian apparatus forms,
matures, and becomes functional.
2. RESULTS
2.1 Early Larval Stages (3.8-5.0 mm NL; Figure 2)

Development of the Weberian apparatus within the early larval stage is unique
within each of the three main regions. Starting at 3.8 mm NL, there are no signs of
development of the vertebral ossicles or ligaments. Initial development of the otic
capsule (Haddon & Lewis 1996, Bever & Fekete 2002) and swim bladder has already
occurred. Both the anterior (lapillus) and posterior (sagitta) otoliths and their respective
maculae are present (otoliths noted in whole-mount). This initial modular independence
is indicative of the multiple roles both the ear and swim bladder play, and the necessity
to have those structures form early in development.
The ear is an open singular chamber at this stage. The distinct chambers of the
pars superior (semicircular canals and utricle) and pars inferior (saccule, lagena, and
macula neglecta) have begun to take shape, but both regions are open to one another.
Within the presumptive pars inferior, the dominant skeletal structure is the basioccipital,
which is cartilaginous. By 4.5 mm NL, substantial morphogenesis has occurred, with the
pars inferior becoming nearly separated from the rest of the inner ear. However, no

noticeable division of the pars inferior into defined lagenar and saccular chambers has
occurred (Figure 2A).
The swim bladder remains fairly stable in size and shape during the early larval
stage. At 3.8 mm NL, the swim bladder is singular, and the tunica layers are difficult to
distinguish. The swim bladder is situated quite far anterior, located as rostral as the
second vertebral level, substantially more anterior than the adult position (vertebra 4).
No substantial changes are seen in the swim bladder at 4.0 or 4.5 mm NL, remaining
singular; however, the internal layers begin to become more defined.
The initial formation of the Weberian ossicles is seen histologically at 4.0 mm NL
as very small discrete mesenchymal condensations adjacent to the developing anterior
centra. The mesenchymal condensations become larger and more defined at 4.5 mm
NL (Figure 2B-E), but no clear matrix deposition is seen at this stage. In addition, no
sign of the sinus impar, ligaments, or saccus paravertebralis is seen. By 5.0 mm NL, the
initial signs of chondrification within the basidorsals and basiventrals of the Weberian
ossicles are present. Small areas of cartilage matrix are seen within the larger
mesenchymal mass of the presumptive articular processes of the scaphium,
intercalarium, tripus, and parapophysis 4. No ossification is seen within the ossicles at
this stage.
2.2 Mid-Larval Stages (5.5-7.0 mm SL, Figure 3)

The mid-larval stage is marked by rapid growth and ossification within the pars
auditum, full establishment of the pars inferior (including transverse canal of the sinus
endolymphaticus), formation of the anterior head of the swim bladder, and initial
integration of the apparatus. During this stage, the ossicles, remain surrounded by
substantial numbers of mesenchymal cells (likely osteoblasts and chondroblasts).
Pars Inferior (Figure 3A). By 5.5 mm SL, the pars inferior has become more
distinct from the rest of the ear. Within the pars inferior, the saccule and lagena are just

separating from each other, with the smaller saccule lying medial to the lagena near the
ventromedial corner of the chamber. At 6.0 mm SL, the pars inferior is still relatively
small and circular in shape. The lagena and saccule are now substantially separated
from the remaining ear. Dominated by the lagena, the saccule is limited to the
ventromedial corner in the posterior capsule. The majority of the exoccipital contribution
to the chamber walls is ossified at 6.0 mm SL, with the exception of a portion near the
midline where the two sides meet to form the dorsal wall of the sinus impar. All of the
basioccipital in this region remains cartilage, including the lateral walls of the sinus
impar. The lagenar macula is short, occupying close to half of the medial wall, while the
saccular macula is not nearly as expanded, but occupies most of the volume of the
saccule. The lagenar otolithic membrane is fairly large, occupying around a third of the
space of the lagena. The saccular otolithic membrane is quite small in comparison. The
saccule is very small and quite short, and only expands as the transverse canal is about
to enter, at which time the lagenar structures end and the saccule takes over the
remaining space of the pars inferior.
By 6.5 mm SL, the posterior ear is increasing in size, while remaining consistent
in overall shape and ossification (Figure 3A). The lagena (Figure 3A,l) is approximately
twice as tall as the saccule (Figure 3A, s), and approximately twice the volume. The
lagenar otolithic membrane (Figure 3A, lo) remains fairly large, occupying around a third
of the space of the lagena. The lagenar macula (Figure 3A, lm) occupies half of the
medial wall of the lagena, while the saccular macula (Figure 3A, sm) occupies nearly all
of the medial wall of the smaller saccule. The saccular otolithic membrane is quite small
(Figure 3A, so). No significant changes to shape or ossification were found at 7.0 mm
SL.
Sinus Impar (Figure 3A). At 5.5 mm SL, the slightest channel has begun to form
in the midline for the presumptive sinus impar, at the level of the anterior end of the
saccule. No transverse canal is present and the presumptive sinus impar does not

travel out of the skull or into the area of the developing scaphium. By 6.0 mm SL, the
sinus impar has developed into a small and rounded structure, and perilymph is now
present. The walls of the sinus impar are thin bone dorsally, but thick cartilage laterally.
The transverse canal is present and occupies a small portion of the impar anteriorly.
The sinus impar remains fairly small, more rounded anteriorly and becoming flatter as it
exits the skull and approaches the vertebrae. The impar remains very small as it
emerges from underneath the fibrocartilage pad and splits into the atria within the
scaphia. Perilymph is present within the atria, indicating a full connection has been
made between the ossicles and the ear. By 6.5 mm SL, the sinus impar continues to
increase in size within the skull and is a rounded triangle with a dorsomedial hump that
curves into the hindbrain (Figure 3A, si). The walls of the impar are thin and nearly
uniform in thickness. The transverse canal (Figure 3A, tc) continues to occupy a small
portion of the impar and is lined with a thin epithelium. At 7.0 mm SL, the sinus impar is
now a teardrop shape within the skull. The walls of the impar remain thin, and the
transverse canal occupies a small portion of the impar.
With the connection between the ear and scaphium present via the sinus impar,
it is important to note the dramatic shape changes that the structure undergoes while
traveling from posterior to anterior. Starting at the scaphium, as the atria of the sinus
impar progresses anteriorly it remains bounded ventrally by the fibrocartilage pad as
well as dense collagen fibers laterally along with the bony exoccipital. Progressing
anteriorly, the atria dive ventrally under the fibrocartilage pad, which has decreased in
size and lost its connection to centrum 1 and the basioccipital. The sinus impar typically
remains split for a short time by a small ridge of the fibrocartilage pad but quickly unifies
into a singular channel that is highly lobed and flat. As the impar enters the skull, it
remains flat but progressively loses its lobular appearance, followed by a significant
increase in size and variable shape, most highly influenced by the ossification state of
the basioccipital that makes up its lateral walls, which constrict the space while

cartilaginous. In the later sizes of the mid-larval stage, the sinus impar achieves a rather
rectangular or mushroom shape due to the heavily cartilaginous basioccipital.
Scaphium (Figure 3B,G). At 5.5 mm SL, matrix deposition is occurring in all
portions of the scaphium. The articular and ascending processes are present as
cartilage, surrounded by mesenchymal cells. A very thin band of bone deposition is
seen in cross-section as the developing concha, which is more pronounced in horizontal
view. By 6.0 mm SL, the typical adult morphology of the scaphium is taking shape. The
scaphium remains small, with a laterally convex concha including only a shallow depth
for the atrium of the sinus impar. The concha is fully bone, thin and uniform in thickness,
with mesenchymal cells surrounding most surfaces. The articular process at this stage
is large and ovoid and is fully cartilage with no sign of ossification. A small, thin
fibrocartilage pad is developing at the level of the scaphium that lies ventral to the spinal
cord, dorsal to the centrum, and is flanked laterally by the growing atria of the sinus
impar. The fibrocartilage pad is composed of irregular organized fibers and rounder
nuclei, with no red staining collagen fibers.
At 6.5 mm SL, the scaphium is still fairly small, with increasing space for the atria
of the sinus impar (Figure 3B,G; asi), as well as a small indentation in the concha
(Figure 3B,G; con) just above the interossicular ligament (Figure 3B,G; iol) insertion
point. The concha remains thin, with the thickest section being the interossicular
ligament attachment site. The articular process remains large and ovoid and articulates
with the centrum surface (no socket or depression is present). The articular process
(Figure 3G, art) is still fully cartilage, with no sign of ossification. The thin fibrocartilage
pad (Figure 3B, fp) is slightly taller than the previous size but remains composed of
irregular organized fibers and rounder nuclei, with no red staining collagen fibers. By 7.0
mm SL, the concha has continued to increase in size and depth for the atria of the sinus
impar. The articular process maintains the same shape and is fully cartilaginous,

however, now sits in a clear depression in centrum 1. The fibrocartilage pad has
increased in size but has not changed in composition.
Claustrum (Figure 3B). From 5.5-6.0 mm SL, the claustrum is present as a
small cluster of chondrocytes surrounded by a larger mass of mesenchymal cells. The
mesenchymal condensation continues ventrally towards the concha scaphium as well
as dorsally to surround the developing cartilage of the neural complex. At 6.0 mm SL, a
sliver of direct-red positive bone is present at the ventral limit of the cellular mass.
By 6.5 mm SL, the claustrum is very short, composed almost exclusively of the corpus
(Figure 3B, cor), however, ossification has begun to develop ventrally. Perichondral
ossification progresses on the lateral surface of the corpus, as well as ventrally toward
the short scutulum that is now definitive. The dorsal tip of the corpus is surrounded by
mesenchymal cells that extend around the developing cartilages of the neural complex.
The scutulum (Figure 3B, scu) is limited to a small ventral nub that is born from the
corpus, and a small lateral bony protrusion that will eventually interact with the concha.
The claustrum and scaphium do not abut, but a thin layer of mesenchymal cells extends
from the tip of the concha to the claustrum in a continuous layer.
By 7.0 mm SL, the claustrum remains short, with the majority of the length of the
claustrum still formed from the corpus. The corpus is partially cartilaginous, particularly
dorsally, with perichondral ossification still progressing on its lateral surface and
ventrally toward the scutulum. Fibrous attachments connect the dorsal tip of the corpus
to the neural complex. The bony scutulum is short and thin and travels a quarter of the
height of the concha scaphium ventrally towards the articular process of the scaphium.
A very small lateral flange has formed at the midpoint of the claustrum and sits directly
ventral to the tip of the concha.
Intercalarium (Figure 3C,G). At 5.5 mm SL, the articular process of the
intercalarium is present as a small mass of matrix-positive cartilage. A small flange of
bone extends from the cartilage as the first sign the developing manubrium. The entire

structure is surrounded by mesenchymal cells. By 6.0 mm SL, the articular process is a
small round cartilage mass sitting in a minor depression in centrum 2. A clear
manubrium extends as bone from the distal tip of the articular cartilage. The manubrium
is thinner than the articular process and is fully ossified bone, and mesenchymal cells
(likely osteoblasts) can be seen laying down bone to extend the length of the
manubrium. At 6.5 mm SL, the articular process (Figure 3C, art) fits into a deeper
depression in the bony second centrum (Figure 3C, c). The articular process of the
intercalarium maintains its shape and remains fully cartilaginous. The manubrium
(Figure 3C,G; man) has become very elongate, is thinner than the articular process, and
is fully ossified bone. The distal tip of the manubrium is clearly embedded within the
interossicular ligament, which has become dense and fibrous. By 7.0 mm SL, the
articular process fits into a defined notch in centrum 2. A thin rim of perichondral
ossification has developed around the distal portion of the articular process, at the site
near its union with the manubrium. The entire distal third of the manubrium is embedded
within the interossicular ligament.
Tripus (Figure 3D,F,H). At 5.5 mm SL, the articular process of the tripus is
present as small cartilage mass within a large field of mesenchymal cells. Development
proceeds quickly in the tripus, and by 6.0 mm SL the articular process has quadrupled
in size, and bony extensions for the wing are present extending distally from the
cartilage mass. The articular process abuts neural arch 3 and centrum 3. Perichondral
ossification is present where the body tapers into the wing of the tripus. The thin wing of
the tripus is very short and ossified, smaller in length than the body/articular process.
No gaps or cavities are seen in the tripus. The wing of the tripus has short anterior and
posterior processes extending as bone from the main body, with a thick band of
mesenchyme surrounding the entire structure. The first indications of the transformator
process appear as sites of ossification within the tunica externa but have not yet
reached the midline.

At 6.5 mm SL, the rapid growth of the tripus has resulted in a shape reminiscent
of the adult form. The articular process (Figure 3D, art) still abuts neural arch 3 (Figure
3D, na) and centrum 3 (Figure 3D, c), and remains highly cartilaginous. Perichondral
ossification continues minimally in the body (Figure 3D, b) as it tapers into the wing
(Figure 3D,F; w). No gaps or cavities are seen in the tripus. The wing is slightly
narrower than the body of the tripus but has achieved substantial anterior-posterior
length to assume the wing shape indicative of the element. Both the anterior and
posterior processes of the tripus have increased in length, especially the posterior
process, which fully extends to the tunica externa (Figure 3F, te). The transformator
process continues ossifying within the tunica, with a sharp bend at the fusion site of the
posterior and transformator processes. By 7.0 mm SL, little overall change has occurred
from the previous size other than continued growth, especially at the posterior, anterior,
and transformator processes. Perichondral ossification remains limited to the tapering

end of the tripus body.
Parapophysis 4 (Figure 3E,H) and Os Suspensorium (Figure 3D-F). At 5.5
mm SL, parapophysis 4 is present as a small cartilage mass within a large field of
mesenchymal cells, similar to the articular process of the tripus. By 6.0 mm SL,
parapophysis 4 has expanded greatly, especially close to the centrum, and remains
cartilaginous. The parapophysis abuts centrum 4 and is continuous with neural arch 4 in
a narrow junction. Only the very distal tip of the cartilage mass shows ossification, at the
presumptive bifurcation site of rib 4 and the os suspensorium. Rib 4 was not detected at
this size. The presumptive os suspensorium is a short medial-projecting ossification
from the distal tip of the cartilage. The paired os suspensoria are narrow, short, and
thin, surrounded by mesenchyme, and do not interact with the swim bladder at this size.
By 6.5 mm SL, rib 4 is still not well defined (Figure 3E; rib), and the os suspensorium
(Figure. 2D-F; os) has increased in length slightly. The paired os suspensoria are
narrow, short, and thin, surrounded by connective tissue (Figure 3D). The midline

between the paired os suspensoria is filled with mesenchyme, and they are not
connected. Near the ventral tip, the os suspensoria narrow to thin tips that insert in a
thick fibrous connective tissue pad. Parapophysis 4 (Figure 3E, pop) is large, especially
close to the centrum, abuts the centrum (Figure 3E, c), and is continuous with neural
arch 4 (Figure 3E, na); however, a thin synostosis is being laid down between the
structures. At 7.0 mm SL, the os suspensorium has continued to elongate, and rib 4 is
now present. Rib 4 is very short. The os suspensorium remains surrounded by
connective tissue, and the midline is filled with mesenchymal cells. The ventral tips of
the os suspensoria continue to insert in a thick fibrocartilage pad that is continuous with
the tunica externa. Parapophysis 4 continues to increase in size and remains
predominantly hyaline cartilage. It now fully interacts with neural arch 4 via a synostosis.
Thin perichondral ossification has begun around most of the perimeter of the
parapophysis.
Saccus paravertebralis (Figure 3B-D,G). The saccus paravertebralis is not
present at 5.5 mm SL. By 6.0 mm SL, as the ossicles develop rapidly, a small saccus
paravertebralis develops in concert. Very limited space is found around the perimeter of
the concha, and only a very narrow band of loose connective tissue is present lateral to
the presumptive claustrum. Minimal space is seen lateral and ventral to the manubrium,
and almost no space is found dorsally, where the spinal cord is in close proximity. Only
a thin layer of loose connective tissue surrounds the tripus. At 6.5 mm SL, the overall
volume of the saccus has increased only slightly. Very limited space is found around
concha (Figure 3B,G; sp), lateral to the corpus (Figure 3B, sp), and around the
intercalarium (Figure 3C, sp) and tripus (Figure 3D, sp). By 7.0 mm SL, the saccus
remains limited except for around the intercalarium, where substantial space is seen
both dorsal and ventral to the manubrium.
Ligaments and Swim Bladder (Figure 3D-H). At 5.5 mm SL, the initial
development of the ligaments has begun. Conspicuous parallel fiber arrangement can

be seen between the bodies of the tripus and parapophysis 4 (slight gray stain), while
the developing interossicular ligament can be seen on both sides of the developing
manubrium, and is taking up only a small amount of red color, indicating the initial
establishment of the collagen fibers. By 6.0 mm SL, both ligaments continue to increase
in fiber density, with the tripus-parapophysis ligament more substantially fibrous and
gray in color, while the interossicular ligament is denser with increased staining. By 6.5
mm SL, the tripus-parapophysis ligament has increased in fibrosity (Figure 3H; lig), and
is beginning to take up a bluish color. The interossicular ligament (Figure 3G) is now
quite thick and fibrous, and deeply red staining. In addition, the triple ligament (os
suspensorium-tripus-tunica externa) has begun to form as a triangular-shaped structure
posterior to the os suspensorium. At 7.0 mm SL, all three ligaments continue to
increase in size and fiber density and become deeper stained, particularly the
interossicular ligament and the triple ligament.

In the swim bladder, both layers are present at 5.5 mm SL, but no distinction in
staining and thickness, both being very thin and not extensively fibrous. The bladder
remains as far anterior as vertebra 2. By 6.0 mm SL, the swim bladder is quite large
relative to body size, with no clear differences in the tunica layers; however, some
indications of increased fiber density is seen in the area of tripus insertion. At 6.5 mm
SL, the tunica layers (Figure 3E-F; te, ti) remain quite similar overall, but clear increases
in thickness and fiber density are seen in the anterodorsal site of tripus insertion. By 7.0
mm SL, the insertion site has begun to take up red stain, indicating an increase in
specific collagen fiber.
2.3 Late Larval Stages (8.0-9.9 mm SL)

Development within the late larval stage is focused on overall growth maturation
of most structural elements. This includes the size and shape of the ossicles, the sinus
impar, and the ligaments.

Pars Inferior (Figure 4A). At 8.0 mm SL, the pars inferior is round, nearly
circular in shape. The chamber is dominated by the lagena, and the saccule is limited to
the ventromedial corner in the capsule, extending underneath a nook ventral to the
sinus impar, as seen in the previous stage. The chamber walls are ossified, except for a
large synchondrosis at the ventrolateral corner of the lagenar wall. The lagena is
approximately twice as tall as the saccule. The lagenar otolithic membrane is large,
occupying around a quarter of the space of the lagena, and the lagenar macula
occupies about a third of the medial wall. The saccular macula occupies nearly all its
medial wall, and the membrane of the saccular otolithic membrane remains quite small.
By 9.0 mm and 9.9 mm SL, the pars inferior has changed little. It remains rounded and
circular in shape (Figure 4A) and dominated by the lagena (Figure 4A,l). The large
synchondrosis at the ventrolateral corner of the lagenar wall remains present. The
dimensions of the lagenar (Figure 4A, lm) and saccular maculae (Figure 4A, sm) and
otoliths (Figure 4A; lo,so) have not changed.
Sinus Impar (Figure 4B). At 8.0 mm SL, the sinus impar is predominantly oval in
shape. The walls of the sinus impar are thin, with small syndesmoses or synchondroses
in its wall between the exoccipitals or between the exoccipital and basioccipital,
respectively. The transverse canal occupies a small portion of the impar and is lined
with a thin epithelium. The majority of the basioccipital remains cartilaginous, which
greatly affects the shape of the sinus impar, which shows rectangular and mushroom-
like forms as it progresses anteriorly. The series of shape changes seen in the mid-
larval stage are also seen here, but even more extensive given the increase in the
overall size of the sinus impar at the late larval stage. By 9.0 mm SL, the sinus impar
(Figure 4A, si) retains its shape, and the walls of the impar are still thin, but the
articulations are now all syndesmoses. The transverse canal (Figure 4A, tc) occupies a
larger portion of the impar more posteriorly than in 8.0 mm SL. The ventral cartilage of

the basioccipital restricts the shape of the impar ventrally and creates a mushroom
shape through a long stretch of the sinus impar.
At 9.9 mm SL, the sinus impar retains most of its shape, but a large hump at the
dorsal midpoint interacts with the hindbrain. The walls of the impar are variable in
thickness, with the most robust thickness found in the dorsolateral corners of the
structure. The small articulations are syndesmosis between the exoccipitals and
variable composition between the exoccipitals and the basioccipital (connective tissue
with slight alcian-blue positivity). The transverse canal occupies a large portion of the
impar.
Scaphium (Figure 4B,G). At 8.0 mm SL, the scaphium has obtained its adult
form. The concha is laterally convex and cup-shaped. It has a small indentation at the
midpoint near the interossicular ligament insertion. The concha wall is thin, with a slight
thickening at the interossicular ligament attachment site. There is broad space medial to
the concha, allowing for substantial space for the atria of the sinus impar to expand. The
articular process is large and ovoid and sits in a large socket in centrum 1. The articular
process is fully cartilage, with no sign of ossification. The fibrocartilage pad remains
thin, and is composed primarily of irregular organized fibers and rounder nuclei.
However, the dorsal section has changed staining profile, and a narrow layer of
collagen fibers near the spinal cord are staining red, while the bulk of the pad remains
gray-blue. By 9.0 mm SL, the scaphium has changed little from 8.0 mm SL. The concha
(Figure 4B,G; con) shows the same shape and composition, while the articular process
(Figure 4B,G; art) has begun to show minimal perichondral staining near the connection
with the concha. The bulk of the articular process remains cartilaginous. The
fibrocartilage pad (Figure 4B, fp) has increased in height and maintains a gradient of red
staining collagen fibers dorsally, and gray-blue staining fibers ventrally. The overall
morphology of the elements of the scaphium and the fibrocartilage pad remain constant

at 9.9 mm SL, with the exception of the formation of a layer of fibrous connective tissue
between the articular process and the centrum.
Claustrum (Figure 4B). At 8.0 mm SL, the claustrum is short, with the bulk of
the element formed by the corpus (Figure 4B, cor). The corpus is partially cartilaginous,
particularly dorsally, with perichondral ossification progressing on its lateral surface and
ventrally toward the scutulum. Fibrous attachments connect the dorsal tip of the corpus
to the neural complex. The scutulum (Figure 4B, scu) is short, thin, and fully ossified,
traveling half of the distance of the concha ventrally towards the articular process of the
scaphium. A small lateral flange extends from the midpoint of the claustrum, the tip of
which sits directly ventral to the tip of the concha, enclosing the atrium of the sinus
impar (Figure 4B,G; asi). The claustrum remains constant in size, shape, and
composition through both 9.0 and 9.9 mm SL.
Intercalarium (Figure 4C,G). At 8.0 mm SL, the articular process of the

intercalarium fits into a notch in the bony centrum 2 (Figure 4C,G; c). The articular
process is minimally ossifying, with only a small rim of perichondral ossification (as it
becomes continuous with the bony manubrium). The manubrium is as thick as the
articular process and is fully ossified bone. At both 9.0 and 9.9 mm SL, the articular
process (Figure 4C,G; art) of the intercalarium continues to be minimally ossified, and
the manubrium (Figure 4C,G; man) remains constant. As seen with the scaphium, a
layer of fibrous connective tissue has formed between the articular process and the
centrum, deep within the socket.
Tripus (Figure 4D-F,H). At 8.0 mm SL, the articular process of the tripus
continues to abut neural arch 3 and centrum 3. The main body of the tripus remains a
large mass of cartilage, with perichondral ossification still limited to the tapering of the
body into the wing. The thin wing of the tripus is fully ossified and minimally surrounds
the distal portion of the body as it tapers into the wing. No gaps or cavities are seen in
the tripus. The bony wing continues into the anterior and posterior processes, the

anterior of which is now highly defined. The transformator process is clearly seen within
the tunica externa. By 9.0 mm SL, the size and shape of the tripus change little in the
articular process (Figure 4D, art), with further growth focused on the anterior and
posterior ends of the wing (Figure 4D,F; w), and with little change in ossification within
the body (Figure 4D, b).
Parapophysis 4 and Os Suspensorium (Figure 4E,F). At 8.0 mm SL,
parapophysis 4 remains large and cartilaginous. The parapophysis abuts the centrum
and neural arch 4 via a synostosis. The body of the parapophysis is hyaline cartilage
with thin perichondral bone lining the perimeter. As the body tapers distally, the cartilage
is surrounded by bone into the bifurcation site. The os suspensorium and rib 4 are fully
ossified and continuous with parapophysis 4. Both structures start as narrow extensions
of bone that widen as they extend away from the parapophysis. Rib 4 is half the length
of the os suspensorium, continues laterally from the parapophysis, and is bounded

laterally by the musculature and medially by adipose tissue. The os suspensorium
travels ventromedially to near the midline where it meets the opposite side os
suspensorium. The paired os suspensoria are narrow and quickly taper, surrounded by
connective tissue, and are connected at the midline by a syndesmosis. Near the ventral
tip, the os suspensoria further narrow to thin tips that insert in a thick fibrocartilage pad
that is continuous with the tunica externa. By 9.0 mm SL, both rib 4 (Figure 4E, rib) and
the os suspensorium (Figure 4E,F; os) continue ossifying ventrally, while the cartilage
parapophysis 4 (Figure 4E, pop) remains consistent in shape and ossification. By 9.9
mm SL, the tapering body of parapophysis 4 has begun to lose cartilage matrix and is
being replaced with adipose tissue, creating a small cavity at the branching site of rib 4
and the os suspensorium. Limited endochondral ossification has begun in the cartilage
adjacent to the growing cavity.
Saccus paravertebralis (Figure 4B-D,G). At 8.0, the saccus paravertebralis
remains quite small. No space is available dorsolateral to the scaphium, but a small

space is present ventrally and ventrolaterally. A small pocket of connective tissue is
present lateral to the corpus claustri. Substantial space is seen both dorsal and ventral
to the intercalarium, with more limited space seen laterally as it approaches the
musculature. Thin layers of adipose tissue surround the tripus both dorsal and ventral to
the structure; however, the kidney is in very close proximity ventrally. No space is
present lateral to the wing, which at points nearly inserts into the musculature. This
limited space continues through 9.0 (Figure 4B-D,G; sp) and 9.9 mm SL.
Ligaments and Swim Bladder (Figure 4B-C,E-H). From 8.0-9.9 mm SL, all
ligaments are present and highly developed. The tripus-parapophysis 4 ligament
undergoes the most variability in this stage, changing in fiber composition to present a
blue-green color at 8.0 mm SL, to gray at 9.0 mm SL (Figure 4H, lig), and finally
predominantly blue at 9.9 mm. At each size, the ligament remains highly cellular, and
the fibers appear loosely organized. Both the interossicular ligament (Figure 4B-C,G;
iol) and the tripus-os suspensorium-tunica externa ligaments remain largely unchanged.
Both ligaments remain highly fibrous, reduced cellularity, and deeply red collagen
staining.
The swim bladder in this stage also remains largely unchanged, continuing to be
large and round, limited anteriorly by the os suspensorium. At 8.0 mm SL, the tunica
layers are similar in thickness, and the tunica externa fibers have begun to slightly
increase in red staining collagen fibers (especially near the tripus and os suspensorium)
while the tunica interna remains gray in color and less fibrous. By 9.0 mm SL, the tunica
externa (Figure 4E,F; te) has begun to pick up red stain across the majority of the layer,
while the tunica interna (Figure 4E, ti) remains gray. By 9.9 mm SL, the staining
intensity and thickness of the tunica externa has increased substantially in the region of
the tripus insertion.
2.4. Juvenile-Adult Stages (12.3-17.1 mm SL, Adult (30.1-33.7 mm TL)

Pars Inferior (Figure 5A). At 12.4 mm SL, the otic capsule at the pars inferior is
rounded and nearly circular in shape. The overall volume remains dominated by the
lagena, with the saccule remaining limited to the ventromedial corner in the capsule,
partially ventral to the sinus impar. The chamber walls are ossified except for the
synchondrosis at the ventrolateral corner of the lagenar wall. The lagena remains
approximately twice as tall as the saccule. The lagenar otolithic membrane is large,
occupying around a third of the space of the lagena. The lagenar macula occupies
about half of the medial wall of the lagena, while the saccular macula continues to
occupy nearly all of the medial wall of the saccule. The saccular otolithic membrane
remains quite small. By 15.1 mm SL, the otic capsule is rounded (Figure 5A), but overall
taller than wide rather than circular. As such, the lagena (Figure 5A, l) is now nearly
three times taller than the saccule (Figure 5A, s). The lagenar otolithic membrane
(Figure 5A, lo) is quite large, occupying around a third of the space of the lagena, and
the lagenar macula (Figure 5A, lm) continues to occupy about half of the medial wall of
the lagena. The saccular macula (Figure 5A, sm) occupies nearly all of the medial wall
of the saccule, and the saccular otolithic membrane remains limited in size. At 17.1 mm
SL, the otic capsule has returned to a more rounded shape. No other changes in shape,
ossification, or morphology were found at 15.1 mm SL. This morphology continues in
the adult sizes examined.
Sinus Impar (Figure 5A). At 12.4 mm SL, the sinus impar is tall and a rounded
teardrop in shape, with a large hump at the dorsal midpoint that interacts with the
hindbrain. The walls of the impar are variable in thickness, with the most robust
thickness found in the dorsolateral corners of the structure. All articulations within the
impar walls are syndesmoses. The transverse canal is relatively small with a thin
epithelium. By 15.1 mm SL, the sinus impar (Figure 5A, si) remains tall and rounded
and retains the pronounced dorsal hump. The walls of the impar are thin, with very
small syndesmoses at the three articulation sites in the impar walls. The transverse

canal (Figure 5A, tc) remains relatively small with a thin lining. At 17.1 mm SL, the impar
has returned to a rounded teardrop shape. The walls are fully ossified, with ossified
synostoses between the bones making up its walls. The transverse canal is relatively
small compared to the maximum size of the impar. In the adult, the sinus impar is
teardrop-shaped, and all articulations within its walls remain synostoses.
Scaphium (Figure 5B,J). At 12.4 mm SL, the scaphium has become large and
deep cup-shaped and may have a slight medial indentation in the middle of the concha.
The concha is thin dorsally but thickens slightly in the region where the interossicular
ligament inserts. The articular process is large and circular and sits in a deep socket in
centrum 1. Most of the articular process is remains cartilage, however small sites of
endochondral ossification are present, especially the area nearest to the concha. The
fibrocartilage pad has grown quite large, with two distinct regions. The dorsal third is
composed of red staining fibers with flat cell nuclei interspersed among the fibers. The
ventral two-thirds retains the morphology seen in earlier stages, with irregular bluish
fibers interspersed with round cell nuclei. As seen in earlier stages, the fibrocartilage
pad reduces in size and separates from centrum 1 and the basioccipital to allow the
sinus impar to proceed into the skull.
By 15.1 mm SL, the scaphium retains its typical shape and continues to ossify. A
large area of endochondral ossification is seen in the dorsolateral third of the articular
process (Figure 5B,J-K; art) that is continuous with the concha (Figure 5B,J; con) as
well as perichondral ossification around the lateral edges of the process. The
fibrocartilage pad (Figure 5B, fp) continues its transition to a more fibrous form, with
now the dorsal half exhibiting dense red staining fibers with flat nuclei. The densest
fibers are directly ventral to the spinal cord. At 17.1 mm SL, several small but noticeable
changes have occurred. While the scaphium retains its same shape, the concha is
significantly thicker at the site of interossicular ligament insertion, more than twice the
thickness of other regions of the concha. The articular cartilage continues to ossify, with

perichondral ossification around its margin, while endochondral ossification is seen
across the majority of the process. The large median fibrocartilage pad has become
fully fibrous in nature, with deep red staining fibers and flattened nuclei found
throughout. In the adult, the scaphium retains the same shape and morphology as
found in juvenile stages. The rounded articular process is quite small within a deep
depression in centrum 1, and remains a mix of endochondrally ossifying cartilage. The
fibrocartilage pad remains fibrous; however, the median region of the pad becomes
nearly devoid of cells, while the lateral edges appear less fibrous and nearly cellular
again.
Claustrum (Figure 5B). At 12.4 mm SL, the claustrum remains fairly short and
thin. The corpus is heavily ossified with small areas of alcian positive cartilage in its
dorsal half, with fibrous attachments still connecting the claustrum to the neural
complex. The bony scutulum is short and thin and travels a third of the distance
ventrally towards the articular process of the scaphium. The small flange remains in the
midpoint of the claustrum, still projecting laterally to abut the concha and close the
atrium of the sinus impar. By 15.1 mm SL, the claustrum has elongated due to an
increase in the length of the bony scutulum (Figure 5B, scu). The corpus (Figure 5B,
cor) is nearly fully ossified, very thin, with only the corpus-scutulum junction showing
alcian positive tissue. The scutulum is thin and travels ventrally half the distance
towards the articular process of the scaphium. The lateral flange fits just underneath the
concha such that the concha overlaps with the edge of the claustrum to enclose the
atrium of the sinus impar (Figure 5B,J; asi), suggesting variability in this interaction
between individuals.
By 17.1 mm SL, the claustrum has further elongated, as the scutulum now
extends nearly to the articular process of the scaphium. The small medial flange again
abuts the concha to seal the dorsal border of the atrium of the sinus impar. The corpus
is nearly fully ossified, with only a couple of cells remaining alcian positive. In the adult,

the claustrum is fully ossified, with no sign of lingering cartilage present. The lateral
flange is more pronounced and maintains its interaction with the concha scaphium to
enclose the sinus impar. The main body serves as a dorsal cap on the atria of the sinus
impar. The corpus of the claustrum narrows and tapers dorsally as it nears supraneural
2.
Intercalarium (Figure 5B,C,J-L). At 12.4 mm SL, the articular process of the
intercalarium fits into a notch in centrum 2. The articular process is minimally ossified,
with only a small rim of perichondral ossification. The bony manubrium remains thin and
long, with a significant portion of the distal manubrium embedded within the
interossicular ligament. By 15.1 mm SL, the proximal half of the articular process
(Figure 5C,J; art) is hyaline cartilage, while the distal half is a mix of endochondral
ossification and perichondral ossification (as it becomes continuous with the bony
manubrium). The manubrium (Figure 5C,J-L; man) is equal in thickness to the articular
process.
At 17.1 mm SL, the majority of the articular process is undergoing ossification,
with a limited region of hyaline cartilage remaining deep within the element. The most
proximal portion of the articular process also appears to be undergoing a significant
transition to fibrocartilage in the region that directly articulates with the centrum. The
manubrium is slightly thicker than the articular process at this stage. In the adult, the
proximal region of the articular process remains cartilage, although most are in the
process of endochondral ossification. The most proximal portion next to the centrum
appears to have fully transitioned to a more ligamentous or fibrocartilage nature. The
manubrium remains thick bone, with the distal tip embedded within the interossicular
ligament.
Tripus (Figure 5D-F,H). At 12.4 mm SL, the tripus articulates with centrum 3 in a
slight depression in the lateral centrum surface, as well as abutting neural arch 3. The
main body remains very large and predominantly hyaline cartilage. Multiple sites of

endochondral ossification are present throughout the body of the tripus, especially in its
middle portion, and perichondral ossification is present around most of the structure.
The bony wing of the tripus has grown in thickness, and minimally surrounds the distal
portion of the body as it tapers into the wing. No gaps or cavities are seen in the tripus
at this stage. The anterior, posterior, and transformator processes are fully formed, and
the full wingspan of the tripus is present. The transformator process remains deeply
embedded within the tunica externa.
Through the remaining juvenile sizes, only minor developmental changes have
occurred in addition to standard growth. A pronounced ventral bend is present near the
midpoint of the wing (Figure 5E-F, w) at 15.1 mm SL. By 17.1 mm SL, the body of the
tripus is undergoing substantial ossification, with the proximal half undergoing both
perichondral and endochondral ossification. The distal half of the body is still deeply
staining hyaline cartilage, with no sign of endochondral ossification. However,

perichondral ossification has occurred around its perimeter and is continuous with the
bony wing. In the adult, the large bony wing maintains the ventral bend, which due to
differential growth is close to the distal margin. The main body is now almost completely
devoid of cartilage; rather, it is filled with adipose tissue. Only a small region of cartilage
is present in the most proximal location, acting as a synchondrosis with the centrum in a
small groove.
Parapophysis 4 (Figure 5E) and Os suspensorium (Figure 5E-G). At 12.4 mm
SL, parapophysis 4 remains large and predominantly cartilaginous and continues to
abut the centrum and neural arch 4 via synostoses. The body of the parapophysis is
predominantly hyaline cartilage proximally, with thin perichondral bone lining the
parapophysis. The small area near the bifurcation point that was transitioning to adipose
tissue now houses a large fat-filled cavity, while cartilage remains in the proximal
portion of the body. Limited endochondral ossification is present in the cartilage nearest
the adipose tissue. The tapering body is now almost fully ossified bone. The os

suspensorium and rib 4 have continued to elongate. The os suspensorium has
broadened dorsally, and a thick fibrous syndesmosis connects the os suspensoria in the
midline. Near the ventral tip, the thin narrow tips of the os suspensoria remain
embedded in the thick fibrocartilage pad (which remains continuous with the tunica
externa).
The overall morphology of the structures of the fourth vertebra remains constant
at 15.1 mm SL (other than normal continued size changes due to growth). At 17.1 mm
SL, the most notable change is in the form of the distal tip of the os suspensorium,
which has flared laterally to resemble a blade, and the fibrocartilage par surrounding it
has become thick fibrous tissue. In the parapophysis, the fat-filled distal region has
expanded in size, limiting the cartilage to the middle and proximal regions, with
increasing endochondral ossification distally. In the adult, the os suspensorium remains
long and broad that still tapers ventral to embed into a thick fibrous mass. Most of the
syndesmosis connecting the right and left sides has ossified into a tight synostosis. In
parapophysis 4, the majority of the internal space is filled with adipose tissue, which
closely resembles the main body and articular process of the tripus, with a limited
hyaline cartilage band sequestered proximally and serving as a synchondrosis with the
centrum.
Saccus paravertebralis (Figure 5B-D,H,J-L). At 12.4 mm SL, the overall extent
of the saccus paravertebralis is largely unchanged. Around the scaphium, no space is
found dorsolateral to the concha, but some fat-filled space is present ventrally and
ventrolaterally. A large pocket of adipose tissue is present lateral to the corpus claustri.
Substantial space is seen both dorsal and ventral to the intercalarium, with more limited
space seen laterally. Narrow layers of adipose tissue surround the tripus both dorsally
and ventrally; however, no space is present lateral to the wing. The limited space
continues throughout the remaining juvenile sizes examined. In the adult, space is
limited around the scaphium to only ventral areas. Significant space remains ventral and

dorsal to the manubrium; however, lateral space has become very limited. No space is
found lateral to the tripus (muscle abuts), but extensive space remains both dorsal and
ventral to the wing.
Ligaments and Swim Bladder (Figure 5B-C,E-G,I-J,L). At juvenile and adult
stages, the ligaments are relatively stable in shape and composition and are increasing
in size relative to overall body growth. The interossicular ligament (Figure 5B,C,J-L; iol)
and the triple ligament both remain very fibrous, dense, and deeply red collagen
staining. The tripus-parapophysis 4 ligament (Figure 5I, lig) continues to vary in stain
between blue to gray but remains highly cellular with loose fibers.
The anterior head of the swim bladder is becoming smaller relative to the growth
of the abdominal cavity and other organs but remains round. At 12.4 mm SL, the tunica
externa is deeply red staining collagen but remains roughly as thick as the tunica
interna. The tunica interna remains blue-gray staining with loose fibers. By 15.1 mm SL,
the tunica externa (Figure 5E-G, te) becomes thicker and more fibrous, especially
dorsally in the region of the tripus and os suspensorium, which continues diverging in
thickness at 17.1 mm SL. In the adult, the entire tunica externa is thicker than the tunica
interna, and the two layers are markedly different in fiber type, fiber density, and
thickness.
3. DISCUSSION
3.1 Distinct phases of Weberian apparatus development and maturation

This study has identified critical phases of formation and maturation within the
hard and soft tissues of the zebrafish Weberian apparatus, each with unique aspects of
development that are critical for proper future function of the system. Generally, these
main processes can be organized as: 1) Establishment (early larval), 2) Morphogenesis

and Integration (mid larval), 3) Growth and Expansion (late larval), and 4) Refinement
(juvenile).
Early larval development of the zebrafish Weberian apparatus (3.8-5.0 mm NL,
Figure 2) is typified by the establishment of initial developmental structures in the proper
locations. This is exemplified by the formation and expansion of mesenchymal
condensations that will develop into the Weberian ossicles. Both the ear and swim
bladder, while already underway, grow to appropriate sizes to begin
compartmentalization within their structure (pars inferior and anterior head of the swim
bladder) to allow differentiation of the key regions related to Weberian apparatus
function.
Mid-larval development (5.5-7.0 mm SL, Figure 3) is a key time of
morphogenesis and integration within the developing Weberian apparatus. It is within
this time frame that the ear fully segregates into a distinct pars superior and pars
inferior, the swim bladder segregates into anterior and posterior heads, and the ossicles
rapidly develop and obtain shapes relatively close to their adult form. In addition, the
necessary ligaments develop, insert, and increase in fibrosity. Ossification patterns
within the skeletal components are very dynamic in this phase, with significant levels of
both cartilage and bone present.
The key aspect of late larval growth (8.0-9.9 mm SL, Figure 4) is growth,
expansion, and ossification. Overall space surrounding the Weberian ossicles increases
allowing increased freedom of motion of the ossicles, and shape and ossification
changes across the apparatus are close to adult morphology. The sinus impar
increases in size substantially and its walls get thinner, while the ear increases in
volume, likely decreasing resistance to fluid flow within the ear. The swim bladder
anterior head diversifies layers substantially, and integration among the tripus, os
suspensorium, and tunica externa increases rapidly.

Finally, the juvenile (12.4-17.1 mm SL, Figure 5) and adult stages are marked
maturation of the system, with shape and composition of skeletal and ligamentous
elements reaching adult form. In addition, the swim bladder-os suspensorium-
transformator relationship becomes highly integrated via tunica layers and fibrous
connective tissue.
3.2 Results in the context of previous morphological research

Several studies of whole-mount development in the zebrafish have been
published in the last 20 years. Our study adds critical new histological data regarding
ossification patterns and data on soft tissue structures absent from whole-mount
analyses.
Initial development of the otic vesicle begins very early in zebrafish
organogenesis and is well underway in the earliest size examined here (3.8 mm NL,
Figure 2A). Haddon and Lewis (1996) noted the rapid formation but slow maturation of
the inner ear. In this study (whole-mount and histology), the ear was already developing
at the earliest examined size (3.8 mm NL), with the sacculolagenar sac present and
containing the presumptive sagitta. Overall structure and developmental sequence did
not deviate from previous descriptions (Haddon and Lewis 1996, Bever and Fekete
2002) in comparable early stages.
Within the swim bladder, Robertson et al. 2007 and Parichy et al. 2009 have
presented developmental timing of histological and external development of the
zebrafish swim bladder, respectively. Both studies describe early swim bladder
development (inflation) happening between 3.5-4.0 mm NL. The swim bladder was
inflated at the earliest stages we examined (3.8 mm NL), in congruence with their data.
Both studies also show the initial formation of the anterior head of the swim bladder
beginning around 6.0 mm SL, with substantial separation of the two heads by 6.5 mm
SL. This timing coincides with our data on fiber and staining divergence between the

tunica layers (Figure 3E,F), especially anterodorsally near the tripus and os
suspensorium.
Within the ossicles, Bird and Mabee (2003) presented a comprehensive table of
whole-mount development and ossification within the developing skeletal elements of
the Weberian apparatus. Our histological data do not conflict with their published timing
of initial cartilaginous development within the ossicles. Slight differences were found in
the timing of development (membranous ossification) of a few specific elements. We
observed scutulum claustri development at 6.0 mm SL, versus 6.3 mm SL, which is not
shocking due to the small bony extension that we identified which would be difficult to
see in whole-mount. In the transformator process of the tripus, our timing was later than
stated by Bird and Mabee (2003), 6.0 mm SL versus 5.1 mm NL (5.6 mm SL in the
swim bladder), and we attribute that difference to our current strict definition of the
transformator process, which is limited to the anteriorly projecting portion of the wing
that inserts in the swim bladder, whereas Bird and Mabee (2003) include portions of
what we classify as the posterior process. We also noted a slightly earlier development
of the anterior process of the tripus (6.0 mm SL versus 6.2 mm SL). Lastly, Bird and
Mabee (2003) stated noted development of the os suspensorium at 5.4 mm NL,
whereas we did not note a definitive os suspensorium at 5.5 mm SL, rather the initial
perichondral ossification at the presumptive bifurcation site of rib 4 and the os
suspensorium, which we assume is what they have classified as the initial os
suspensorium development. Grande and Young (2004) found similar trends in
developmental sequence and timing within the Weberian apparatus as Bird and Mabee
(2003).
3.1 Weberian apparatus function – more than just having all the pieces of the
puzzle?

Research into ontogenetic changes in hearing ability in the zebrafish have
yielded confusing results regarding when (or if) improvements are found. Early
developmental analyses (less than 7 dpf, before sizes examined here) typically found
small but clear increases in either frequency detection or reduced thresholds (Zeddies &
Fay 2005, Lu & DeSmidt 2013, Bhandiwad et al. 2013, Yao et al. 2016), while studies
on more developed individuals (stage 10-47 mm TL, roughly equivalent to our late larval
and juvenile stages) generally found no consistent difference in hearing ability across
the sizes examined (Higgs et al. 2001, 2003). The earlier studies are likely limited to
aspects of particle motion-based hearing, while the later studies are clearly trying to
isolate the timing the addition of sound pressure-based hearing and tying the predicted
shifts in hearing ability with formation of the Weberian apparatus. Our data conclusively
show that the required auditory machinery of the Weberian apparatus (saccule,
transverse canal, sinus impar, ossicles, ligaments, and swim bladder) are all present
and morphologically integrated well before the late-stage hearing studies indicate
functionality. This suggests a clear separation between the morphological integration
within the Weberian apparatus, and the functionality seen in later stages.
There are several aspects of the early integrated Weberian apparatus, in all
regions, that could account for the diminished overall capability of the system.
Compared to adults and juveniles, the shape of the lagena and saccule still undergo
significant changes, which may affect flow of the endolymphatic fluid within the ear. The
size and shape of the sinus impar and transverse canal are also a potential sources of
limitation, as both remain fairly small until later stages, which also may impede flow of
perilymphatic fluid and endolymphatic fluid, respectively. Within the ossicles, significant
changes in shape and ossification continue to occur, which may affect their ability to
properly transmit motion anteriorly. Lastly, the divergence of the tunica layers of the
swim bladder is minimal at mid-larval stages, and the highly integrated relationship
between the transformator process of the tripus, the os suspensorium, and the tunica

externa is just being established. This lack of tight integration could lead to both limited
ability of the swim bladder to physically rock the tripus, and limit the ability of the tripus
to transmit the rocking. Alexander (1962) noted specific fiber differences between the
tunica externa (ichthyocol and elastic fibers) and the tunica interna (smooth muscle and
basic collagen), and these differences are necessary for the proper auditory function of
the anterior head of the swim bladder. The same composition of the tunica externa is
also seen in the interossicular ligament and triple ligament, while the tripus-
parapophysis 4 ligament is composed of elastin, suggesting differing roles among the
ligaments, and full development is necessary for proper function (Alexander 1962).
Taken together, our data suggest that the continued maturation of the Weberian
apparatus, across all regions, is vital for proper function.
4. CONCLUSION
This study provides the first comprehensive histological description of Weberian
apparatus development in zebrafish, including both hard and soft tissues. New data,
such as ligament composition and developmental timing, changes in sinus impar shape,
and ossification patterns across the entire system are critical to our understanding how
the Weberian apparatus forms and functions. Morphological integration was found early,
by 6.5 mm SL, well before increases in hearing ability were detected in functional
studies (>10 mm TL), indicating that morphological integration occurs well before
defined sound pressure hearing is present. Each phase of development within the
Weberian apparatus serves a specific and needed role that collectively will result in a
functional complex structure. Further research is needed to examine the nature of the
functional delay, and how maturation of the Weberian apparatus influences functionality.
5. EXPERIMENTAL PROCEDURES

5.1 Husbandry and Specimen Collection. Adult zebrafish (Danio rerio, AB line) were
obtained from the Zebrafish International Resource Center (Eugene, Oregon). Adult
zebrafish were maintained at 28.5 +/- 0.5° on a 12:12 light cycle in a Z-Hab Mini
zebrafish housing rack (Pentair Aquatic Eco-Systems, Apopka, FL), and fed live
hatched brine shrimp (Brine Shrimp Direct, Ogden, UT), and/or commercial zebrafish
pellet (Pentair Aquatic Eco-Systems) twice daily. Larval zebrafish were maintained in
incubators at 28.5°C (±0.5°C), and fed live paramecia (Carolina Biological, Burlington,
NC) on days 5-15 post-fertilization, then fed newly hatched San Francisco strain brine
shrimp (Brine Shrimp Direct) starting on day 8 in increasing amounts as the larvae were
slowly weaned off of paramecia. For collection, fish were anesthetized using buffered
0.04% MS-222, then fixed in chilled 10% buffered formalin for 24h at 4°C. All
procedures followed approved UNI IACUC protocol 2015-05-1.
5.2 Histology. Sizes examined were 3.8 mm NL (Notochord Length), 4.0 mm NL, 4.5
mm NL, 5.0 mm NL, 5.5 mm SL (Standard Length), 6.0 mm SL, 6.5 mm SL, 7.0 mm SL,
8.0 mm SL, 9.0 mm SL, 9.9 mm SL, 12.4 mm SL, 15.1 mm SL, 17.1 mm SL, and adult
(30.5-33.7 mm TL, Total Length). Four specimens (two cross-section, one each for
horizontal and sagittal) for each length (except 17.1 mm SL, two cross-section only)
were paraffin embedded and sectioned at 8 µm. Specimens were processed as per Bird
and Webb (2014) with modifications. Whole fish were embedded except for adults,
where heads and anterior vertebral region were dissected away from posterior
vertebrae. Larger specimens were decalcified in Cal‐Ex (CS511‐1D, Fisher Scientific,
Pittsburgh, PA USA) while shaking (6-7 mm SL for 2 h, 8-9.9 mm SL for 3.5 h, >12.5
mm SL and adult heads for 8 h). Specimens were washed in PBS for 2 h while shaking,
then dehydrated through an increasing concentration of ethanol to xylene, then vacuum-
infiltrated twice with paraffin (Paraplast Plus, 12‐646‐111, Fisher Scientific, Pittsburgh,
PA USA). Tissue was embedded using a Leica Histoembedder, and sectioned using a

Micron HM315 microtome. Sections were stained using a modified Hall-Brunt
Quadruple stain (Hall 1986; Webb et al., 2014), allowing for visualization of ossification
sites within cartilage models.
5.3 Whole-mount clearing and staining. Two additional specimens for each size were
enzyme cleared and double-stained for cartilage and bone following Bird and Mabee
(2003) and references therein (Dingerkus & Uhler 1977, Potthoff 1984). Whole-mounts
were used to help orient, reconstruct, and analyze the histological data.
5.4 Imaging. Histological images were collected using a Zeiss Axio ScopeA.1
microscope with a ProgRes CF Scan camera and CapturePro v2.8.8 software
(Jenoptik). Cleared and stained images were collected on a Dell OptiPlex 7060 using a
Leica EZ4W using Leica Airlab. Cleared and stained images were stacked and
projections produced using Helicon Focus 7 Pro (Helicon Soft Ltd.). Composite images
and figures were assembled in Adobe Photoshop CC 2018. No alterations were made
to individual images other than overall brightness when images were merged in
composite assembly.
ACKNOWLEDGEMENTS
Research was supported by a UNI Provost-Pre-Tenure Research Grant, a UNI
Graduate College Faculty Summer Research Fellowship, and Departmental Research
Funds to NCB; Summer Undergraduate Research Funds to JRA and SSR; A Dr. Robert
and Brenda Good Research Fellowship to JRA. We thank Dr. Julie Kang for use of
histological and imaging equipment. We also thank Daniel W. Bird and Jill D. Maroo for
helpful discussions and comments on previous versions of this manuscript.
Figure Legends

Figure 1. Cleared and stained whole-mount zebrafish from mid-larval to mid-juvenile
stages. Spatial relationship between ear, vertebrae, and swim bladder can be seen, as
well as overall changes in ossification patterns. A,B = 6.5 mm SL. C,D = 9.0 mm SL.
E,F = 15.1 mm SL. cla = claustrum, int = intercalarium, os = os suspensorium, oto =
otolith, sb = swim bladder, sca = scaphium, tri = tripus. Anterior is to the left in all
images. Scale bars: A,B = 250µm, C,D = 500µm, E,F = 1 mm.
Figure 2. Weberian apparatus development at early larval stage (4.5 mm NL). A = Inner
ear at the level of the developing pars inferior, B = Vertebra 1, C = Vertebra 2, D =
Vertebra 3, E = Vertebra 4. c = centrum, int = intercalarium, mac = sensory macula, na
= neural arch, pop = parapophysis, sc = spinal cord, sca = scaphium, tri = tripus, *
denotes it is the presumed element beginning to form as a mesenchymal condensation.

All views in cross section. All scale bars = 100 µm.
Figure 3. Weberian apparatus development at mid-larval stage (6.5 mm SL). A-E, cross
sectional views of the inner ear (A), vertebra 1 (B), vertebra 2 (C), vertebra 3 (D), and
vertebra 4 (E). F-H, horizontal views of the developing interaction between the tripus, os
suspensorium, and tunica externa (F), the scaphium and intercalarium with the
interossicular ligament (G), and the tripus-parapophysis 4 ligament (H). art = articular
process, asi = atrium of the sinus impar, b = body of tripus, c = centrum, con = concha
scaphium, cor = corpus claustri, fp = fibrocartilage pad, iol = interossicular ligament, l =
lagena, lig = ligament, lm = lagenar macula, lo = lagenar otolithic membrane, lp = lateral
process, man = manubrium, na = neural arch, nc = neural complex, os = os
suspensorium, pop = parapophysis, rib = pleural rib, s = saccule, sc = spinal cord, sd =
syndesmosis, scu = scutulum claustri, si = sinus impar, sm = saccular macula, so =
saccular otolithic membrane, sp = saccus paravertebralis, tc = transverse canal, te =

tunica externa, ti = tunica interna, tl = triple ligament, tri = tripus, w = wing of tripus. All
scale bars = 100 µm except G and H (50 µm).
Figure 4. Weberian apparatus development at late larval stage (9.0 mm SL). A-E, cross
sectional views of the inner ear (A), vertebra 1 (B), vertebra 2 (C), vertebra 3 (D), and
vertebra 4 (E). F-H, horizontal views of the developing interaction between the tripus, os
suspensorium, and tunica externa (F), the scaphium and intercalarium with the
interossicular ligament (G), and the tripus-parapophysis 4 ligament (H). art = articular
process, asi = atrium of the sinus impar, b = body of tripus, c = centrum, con = concha
scaphium, cor = corpus claustri, fp = fibrocartilage pad, iol = interossicular ligament, l =
lagena, lig = ligament, lm = lagenar macula, lo = lagenar otolithic membrane, man =
manubrium, na = neural arch, nc = neural complex, os = os suspensorium, pop =
parapophysis, rib = pleural rib, s = saccule, sc = spinal cord, scu = scutulum claustri, si
= sinus impar, sm = saccular macula, so = saccular otolithic membrane, sp = saccus
paravertebralis, tc = transverse canal, te = tunica externa, ti = tunica interna, tra =
transformator process of the tripus, tri = tripus, w = wing of tripus. All scale bars = 100
µm except G and H (50 µm).
Figure 5. Weberian apparatus development at juvenile stage (15.1 mm SL). A-E and G,
cross sectional views of the inner ear (A), vertebra 1 (B), vertebra 2 (C), vertebra 3 (D),
and vertebra 4 (E), and the os suspensorium (G). F and H-L, horizontal views of the
developing interaction between the tripus, os suspensorium, and tunica externa (F),
ossification of the tripus (H), tripus-parapophysis 4 ligament (I), the scaphium and
intercalarium with the interossicular ligament (J), the elongated manubrium (K), and the
insertion of the interossicular ligament on the manubrium (L). art = articular process, asi
= atrium of the sinus impar, b = body of tripus, c = centrum, con = concha scaphium, cor
= corpus claustri, fp = fibrocartilage pad, iol = interossicular ligament, l = lagena, lig =

ligament, lm = lagenar macula, lo = lagenar otolithic membrane, man = manubrium, na
= neural arch, nc = neural complex, os = os suspensorium, pop = parapophysis, rib =
pleural rib, s = saccule, sc = spinal cord, scu = scutulum claustri, sd = syndesmosis, si =
sinus impar, sm = saccular macula, sp = saccus paravertebralis, tc = transverse canal,
te = tunica externa, ti = tunica interna, tra = transformator process of the tripus, tri =
tripus, w = wing of tripus. All scale bars = 200 µm except H-K (100 µm) and L (50 µm).
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ccepted Artic XXXXXXXXXXXXXXXXX
A Transient Window Of Resilience During Early Development Minimizes
Teratogenic Effects of Heat In Zebrafish Embryos
Triveni Menon1 and Sreelaja Nair1, 2
1
Department of Biological Sciences, Tata Institute of Fundamental Research, Homi
Bhabha Road, Colaba, Mumbai, 400005, India
2
Corresponding author: Sreelaja Nair, email: s.nair@tifr.res.in
Running Title: Minimizing Heat Teratogenicity In Zebrafish Embryos And Gynogenic

Diploid Production
Main Points:
1. Zebrafish embryos at the end of pronuclear fusion and before initiation of

zygotic mitosis are resistant to teratogenic effects of heat.
2. The teratogenic heat resilient window exists transiently during the maternally
controlled phase of development.
3. Heat shock during the teratogenic heat resilient window enables generation of
morphologically normal zebrafish tetraploids.
4. Diploidization of haploids by transient heat shocks during the teratogenic heat
resilient windows aids in effective generation of gynogenic diploids.
Funded By:
1) Wellcome Trust Department of Biotechnology India Alliance (Intermediate
Fellowship to Sreelaja Nair). Grant Number 13X301
2) Tata Institute of Fundamental Research (Department of Atomic Energy
Government of India). Grant Number 12P0127
ticles for future issues, temporarily published online

019; Accepted: Jul 03, 2019
copyright. All rights reserved.
XXXXXXXXXXXXXXXXX
Accepted Article
1
2

Diploid Production
Main Points:

Funded By:

copyright. All rights reserved. This article is protected by copyright. All rights reserved.
XXXXXXXXXXXXXXXXX
Accepted Article
1
2

Diploid Production
Main Points:

Funded By:

XXXXXXXXXXXXXXXXX
Accepted Article
1
2

Diploid Production
Main Points:

Funded By:

XXXXXXXXXXXXXXXXX
Accepted Article
1
2

Diploid Production
Main Points:

Funded By:


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Developmental Dynamics

PATTERNS &amp; PHENOTYPES

Histological Development and Integration of the Zebrafish Weberian Apparatus

Nathan C. Bird*, Selena S. Richardson, and Jeremy R. Abels

© 2020 Wiley Periodicals, Inc.

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Conclusion: This study provides the first comprehensive histological description of

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1.1 Zebrafish Weberian apparatus development

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2.1 Early Larval Stages (3.8-5.0 mm NL; Figure 2)

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2.2 Mid-Larval Stages (5.5-7.0 mm SL, Figure 3)

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and transformator processes. Perichondral ossification remains limited to the tapering

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interossicular ligament and the triple ligament.

2.3 Late Larval Stages (8.0-9.9 mm SL)

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Intercalarium (Figure 4C,G). At 8.0 mm SL, the articular process of the

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of the os suspensorium, continues laterally from the parapophysis, and is bounded

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2.4. Juvenile-Adult Stages (12.3-17.1 mm SL, Adult (30.1-33.7 mm TL)

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staining hyaline cartilage, with no sign of endochondral ossification. However,

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3.1 Distinct phases of Weberian apparatus development and maturation

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3.2 Results in the context of previous morphological research

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denotes it is the presumed element beginning to form as a mesenchymal condensation.

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NY: Springer. p 269-318.

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Advances in Experimental Medicine and Biology 877. p 321-340.

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PATTERNS & PHENOTYPES