Download Crocodilian Forebrain: Evolution and Development

Integrative and Comparative Biology
Integrative and Comparative Biology, volume 55, number 6, pp. 949–961
doi:10.1093/icb/icv003
Society for Integrative and Comparative Biology
SYMPOSIUM
Crocodilian Forebrain: Evolution and Development
Michael B. Pritz1
Molecular Neurosciences Department, Krasnow Institute for Advanced Study, George Mason University, 4400 University
Drive, MS 2A1, Fairfax, VA 22030, USA
From the symposium ‘‘Integrated Biology of the Crocodilia’’ presented at the annual meeting of the Society for
Integrative and Comparative Biology, January 3–7, 2015 at West Palm Beach, Florida.
1
E-mail: [email protected]
Synopsis Organization and development of the forebrain in crocodilians are reviewed. In juvenile Caiman crocodilus, the
following features were examined: identification and classification of dorsal thalamic nuclei and their respective connections with the telencephalon, presence of local circuit neurons in the dorsal thalamic nuclei, telencephalic projections to
the dorsal thalamus, and organization of the thalamic reticular nucleus. These results document many similarities
between crocodilians and other reptiles and birds. While crocodilians, as well as other sauropsids, demonstrate several
features of neural circuitry in common with mammals, certain striking differences in organization of the forebrain are
present. These differences are the result of evolution. To explore a basis for these differences, embryos of Alligator
misissippiensis were examined to address the following. First, very early development of the brain in Alligator is similar
to that of other amniotes. Second, the developmental program for individual vesicles of the brain differs between the
secondary prosencephalon, diencephalon, midbrain, and hindbrain in Alligator. This is likely to be the case for other
amniotes. Third, initial development of the diencephalon in Alligator is similar to that in other amniotes. In Alligator, alar
and basal parts likely follow a different developmental scheme.
Introduction
In the telencephalon of all adult amniotes, a layered
structure of varying complexity is located above the
lateral ventricle in both the transverse and sagittal
plane and is termed the cortex. Similarly, the telencephalon of all amniote brains contains nonlaminated areas usually surrounded by borders of
varying degrees of distinctiveness. These areas are
commonly referred to as nuclei and are located internal to the lateral ventricle in all planes of section.
In the telencephalon of mammals, most of these
latter regions comprise the basal ganglia. However,
in reptiles and birds, located between the cortex and
basal ganglia and internal to the lateral ventricle, lies
a nuclear area seemingly unique to sauropsids and
known as the dorsal ventricle ridge (Ulinski 1983). In
crocodilians, the dorsal ventricular ridge includes:
the dorsolateral area, the intermediolateral area,
and the nucleus of the lateral olfactory tract
(Crosby 1917). Of these, experimental observations
have been limited to the dorsolateral area. A representative transverse section of a crocodilian
telencephalon is shown beside that of a comparable,
transverse section of a mouse’s brain to illustrate
these differences (Fig. 1).
A variety of sophisticated approaches have been
used to unravel the organization of the forebrain
(telencephalon and diencephalon) in amniotes.
Despite considerable information on a variety of amniotes, comparisons between cortical areas in the telencephalon, as well as homologies between the dorsal
ventricular ridge of sauropsids and the telencephalic
regions of mammalian brains remain in dispute
(Bruce 2007; Butler et al. 2011). Regardless of the
ultimate interpretation, examination of crocodilian
brains should prove key to understanding these relationships since crocodilians are the reptiles most closely related to birds (Walker 1972; Whetstone and
Martin 1979; Hedges 1994).
Experimental observations on the forebrain both
of ‘‘adult’’ (juvenile Caiman crocodilus) and developing (embryonic Alligator mississippiensis) crocodilians
are summarized. These findings demonstrate both
differences and similarities in the organization of
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M. B. Pritz
Fig. 1 Cytoarchitecture of telencephalons of Caiman and mice. Transverse Nissl-stained sections through the telencephalon of Caiman
(A) and a mouse (B) illustrate similarities and differences. Note the presence of a nuclear area, the dorsal ventricular ridge (DVR),
located internal to the lateral ventricle (marked by an asterisk, *) in Caiman (A) and the location of the basal ganglia in these two
species.
the forebrain between these reptiles and other amniotes. These data provide an overview of present
knowledge and point to experimental approaches
that should provide information to address some of
the unanswered questions.
Organization of the forebrain in
C. crocodilus
Dorsal thalamus: identification of nuclear
groups and neural circuitry
While a number of characters have been used to
define the dorsal thalamus, the most common attribute, and the one most frequently used in mammalian
studies, is the connections with the telencephalon
(Jones 2007). Accordingly, a similar approach was
employed in crocodilians using the following strategies. One series of experiments made large injections
of a retrograde tracer into various regions of the
telencephalon with the object of injecting all parts
of this area. The goal was to identify all thalamic
nuclei that projected to the telencephalon. These observations were supplemented by injections of anterograde tracers into specific dorsal thalamic areas.
Additional experiments examined the neural circuitry
of a variety of sensory systems from the periphery
centrally to the telencephalon. These latter experiments focused on specific circuits and specific neuronal aggregates.
Using these approaches, 11 dorsal thalamic nuclei
that project to the telencephalon in Caiman have
been identified (Pritz 2014). The neural circuitry of
some of these nuclei is known; for others, many details remain incomplete. Nevertheless, these data suggest that dorsal thalamic nuclei in crocodilians can
be grouped into several categories based on the following features: the telencephalic target, the fiberbundle connecting these forebrain structures, and
whether thalamic projections to the telencephalon
are ipsilateral or bilateral. Using this scheme, six categories of dorsal thalamic nuclei have been recognized based on the available data (Pritz 2014).
These groups include the following subdivisions:
(1) nuclei that project to the cortex bilaterally and
utilize the medial forebrain bundle (dorsolateralis anterior), (2) nuclei that project ipsilaterally to the
cortex (diagonalis), (3) nuclei that project to the ipsilateral primordial general cortex (dorsal geniculate),
(4) nuclei that project both to the ipsilateral cortex
and to the dorsal ventricular ridge (dorsomedialis
anterior), (5) nuclei that project to the ipsilateral
dorsal ventricular ridge and utilize the lateral forebrain bundle (rotundus, reuniens pars centralis,
reuniens pars diffusa, medialis complex posterior,
posterocentralis, and the area ventrolateralis), and
(6) nuclei that project to the ipsilateral basal ganglia
via the lateral forebrain bundle (medialis complex
anterior). Of these nuclei mentioned above, only
the medialis complex anterior likely has reciprocal
connections with the telencephalon (Pritz unpublished observations) Reciprocal connections of other
thalamic nuclei that project to the telencephalon in
crocodilians have yet to be demonstrated. These observations are summarized in Table 1.
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Table 1 Summary of thalamo–telencephalic connections in Caiman
Thalamic nucleus
Telen. Target
Ipsi-
Contra-
FB
Reciprocity
Dorsolateralis anterior
General cx
Yes
Yes
MFB
No
Dorsomedialis anterior
General cx & DVR
Yes
No
?
?
Dorsal geniculate
10 General cx
Yes
No
?
?
Diagonalis
General cx
Yes
No
?
?
Rotundus
DVR
Yes
No
LFB
No
Reuniens pars centralis
DVR
Yes
No
LFB
No
Reuniens pars diffusa
DVR
Yes
No
LFB
No
Medialis complex posterior
DVR
Yes
No
LFB
No
Area ventrolateralis
DVR
Yes
No
LFB
?
Posterocentralis
DVR
Yes
No
LFB
?
Medialis complex anterior
Basal ganglia
Yes
No
LFB
Probably
Abbreviations: contra-, contralateral; cx, cortex; DVR, dorsal ventricular ridge; FB, forebrain; ipsi, ipsilateral; LFB, lateral forebrain bundle; MFB,
medial forebrain bundle; Telen., telencephalon; ?, unknown; 10, primordial.
Certain of the nuclei that comprise one of these
categories (see group 5, above) have been studied in
greater detail. This analysis revealed additional features shared by three of these nuclei: reuniens pars
centralis, rotundus, and medialis complex posterior.
These similar features include: neural circuitry and
topographic projection to the telencephalon. Each of
these nuclei utilizes the lateral forebrain bundle and
projects to the ipsilateral dorsal ventricular ridge.
Because these fibers pass through the ventrolateral
area, axons from each of these thalamic nuclei are
likely to synapse on intervening neurons of the basal
ganglia although definitive proof would require
ultrastructural confirmation. The topography of termination in the telencephalon, as well as the position
of efferent axons in the lateral forebrain bundle, reflects the location of each respective nucleus in the
dorsal thalamus (Fig. 2). Nucleus reuniens pars centralis, a caudo-medial nucleus, which, in Caiman is
fused at the midline, projects to a caudo-medial portion of the dorsal ventricular ridge. Axons from this
nucleus travel in a medial portion of the lateral forebrain bundle (Pritz 1974b, 1995). Nucleus rotundus,
which is located anterior and lateral to the nucleus
reuniens pars centralis, has efferents located in a
lateral part of the lateral forebrain bundle that end
in an antero-lateral portion of the dorsal ventricular
ridge (Pritz 1975, 1995). The medialis complex posterior, which is located between nuclei reuniens pars
centralis and rotundus, has fibers that travel in a
central portion of the lateral forebrain bundle to
terminate in a part of the dorsal ventricular ridge
that lies between the projection zones of nucleus
reuniens pars centralis and nucleus rotundus
(Pritz and Stritzel 1994c; Pritz 1995). Summaries
illustrating these fiber paths are available elsewhere
(Fig. 9, Pritz [1975] for audition and vision and Fig.
16, Pritz and Stritzel [1994c] for somatosensation
from the body surface). These neural circuits and
their termination in the dorsal ventricular ridge
are not unique to crocodilians but have been
described in other reptiles and in birds
(Nieuwenhuys et al. 1998; Butler and Hodos 2005;
Bruce 2007). However, unlike reptiles and birds, similar circuits in mammals end in the cortex (Jones
2007) rather than in the dorsal ventricular ridge of
nonmammalian amniotes, which is organized as a
nucleus.
Furthermore, each of these three nuclei shares additional similarities in their neural circuitry, beginning at the periphery (Table 2). In this simplified
scheme, receptors for audition, vision, and somatosensation from the body surface are connected to
‘‘bipolar’’ cells, which, in turn, synapse on cells
with ‘‘long axons’’. Regardless of the modality,
these ‘‘long-axon cells’’ project to the contralateral
midbrain (Burns and Goodman 1967; Braford
1973; Repérant 1975; Pritz and Stritzel 1989;
Derobert et al. 1999). From this ‘‘third’’-order element, neurons terminate bilaterally in a dorsal thalamic target with the densest projection located
ipsilaterally (Braford 1972; Pritz 1974a; Pritz and
Stritzel 1990a). A similar grouping of neural circuits
is also present in other reptiles as well as in birds
(Nieuwenhuys et al. 1998; Butler and Hodos 2005;
Bruce 2007). Other features shared by this dorsal
thalamic category in Caiman are certain patterns of
histochemical staining in the dorsal ventricular ridge
(Pritz and Northcutt 1977).
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Local circuits neurons in the dorsal thalamus
Fig. 2 Thalamo–telencephalic topography in Caiman. Horizontal
sections projected onto a single-dimension of the diencephalon
(A) and dorsal ventricular ridge (B) are shown. Note that the
topography of similarly coded thalamic areas is preserved in the
dorsal ventricular ridge. Because these areas are projected onto a
single dimension in the dorsal ventricular ridge, areas of seeming
overlap (open areas in B) are actually separate. Abbreviations:
Dla, nucleus dorsolateralis anterior, MCp, medialis complex
posterior; OT, optic tract; Rc, nucleus reuniens pars centralis; Rd,
nucleus reuniens pars diffusa; Rt, nucleus rotundus; TRT, tectoreuniens tract; c, caudal; m, medial; IIIv, third ventricle. This figure
was re-drawn from Fig. 3 in Pritz (1995).
Comparable information on specific circuits in
other categories of thalamo–telencephalic projections
in crocodilians is fragmentary. These gaps in knowledge point to areas where further morphological information (see Table 1) is needed. These additional
data will likely reveal similarities yet to be described
and/or differences in the organization of pathways in
the forebrain.
In the dorsal thalamus of amniotes, two types of
neurons are present: local circuit neurons (also
called interneurons) and relay cells. Axons of local
circuit neurons remain within their region of origin
whereas axons of relay (projection) cells terminate
outside of this area (Jones 2007).
With the exception of the dorsal geniculate nucleus (Pritz and Stritzel 1994b), these previously
identified dorsal thalamic nuclei in crocodilians
lack local circuit neurons and contain only projection cells. These observations were based on two
types of experiments. One approach used massive
injections of a tracer into various telencephalic targets and subsequent counting of retrogradely labeled
neurons in specific dorsal thalamic nuclei. Only
rarely was an unlabeled neuron observed in the following dorsal thalamic nuclei: rotundus (Pritz and
Stritzel 1986), reuniens pars centralis (Pritz and
Stritzel 1986), and dorsolateralis anterior (Pritz
and Stritzel 1987). The other technique independently confirmed these latter findings utilizing a different approach: immunocytochemistry. Based on
the observations that local circuit neurons are immunoreactive to antibodies to gamma amino butyric
acid (GABA) or to glutamic acid decarboxylase
(GAD), neurons in these dorsal thalamic nuclei
were examined using this methodology. With the
exception of the dorsal geniculate nucleus, the following dorsal thalamic nuclei were unlabeled: dorsolateralis anterior, dorsomedialis anterior, reuniens
pars centralis, reuniens pars diffusa, rotundus, diagonalis, posterocentralis, and medialis complex posterior (Pritz and Stritzel 1988; 1994a). Lack of GABA/
GAD immunoreactive neurons in certain dorsal thalamic nuclei is not unique to crocodilians but has
been described in turtles (Belekhova et al. 1991)
and chameleons (Bennis et al. 1991) and pigeons
(Domenici et al. 1988; Granda and Crossland
1989). However, some GABA immunoreactive cells
have been noted surrounding the borders of certain
dorsal thalamic nuclei in turtles (Belekhova et al.
1991) and a few such immunoreactive neurons
have been observed in certain dorsal thalamic
nuclei in pigeons (Veenman and Reiner 1994).
Similar to crocodilians (Pritz and Stritzel 1994b),
GABA immunoreactive neurons are present in the
dorsal geniculate of turtles (Belekhova et al. 1991;
Rio et al. 1992; Kenigfest et al. 1995) and chameleons
(Bennis et al. 1991) and in its avian homolog in
pigeons (Domenici et al. 1988; Granda and
Crossland 1989; Veenman and Reiner 1994; Miceli
et al. 2008). In mammals, the percentage of local
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Table 2 Generalized pattern of synaptic elements of sensory systems that synapse in the midbrain of Caiman crocodilusa
Sensory modality
Synaptic element
Audition
Vision
Body surface somatosensation
Receptors
Hair cells
Rods & cones
Somatosensory receptors
‘Bipolar’ cells
Spiral ganglion
Retinal bipolar cells
Dorsal root ganglion
Long axon cells
Cochlear nuclei
Retinal ganglion cells
Dorsal column nucleus
Midbrain
Torus semicircularis cn
Optic tectum
Intercollicular area
Thalamus
N. reuniens pc
N. rotundus
Medialis complex posterior
Telencephalon
DVR
DVR
DVR
a
Modified from Pritz and Stritzel (1994c).
Abbreviations: cn, central nucleus; DVR, dorsal ventricular ridge; N., nucleus; pc, pars centralis.
circuit neurons in individual dorsal thalamic nuclei
varies. In some small-brained species, GABA immunoreactive neurons are either absent or sparsely present in some dorsal thalamic nuclei while being
present in other dorsal thalamic nuclei in the same
species. On the other hand, large-brained mammals
have local circuit neurons present throughout dorsal
thalamic nuclei in varying percentages (Jones 2007).
Telencephalic projections to the dorsal thalamus
Another characteristic feature of organization of the
forebrain in mammals is reciprocal connections between thalamic nuclei and their respective areas of
cortical projection (Jones 2007). To date, this feature
does not appear to be present in nuclei that project
to the anterior dorsal ventricular ridge in crocodilians (Pritz 2014). Limited observations suggest that
this may be a feature of the medialis complex anterior (Pritz unpublished observations). As data are
incomplete, other dorsal thalamic nuclei may possess
this property. Rather than having reciprocal connections with the non-cortical telencephalon directly,
telencephalic efferents arise from the basal ganglia
in crocodilians (Brauth and Kitt 1980; Brauth
1988). Similar neural circuits are shared by other
reptiles and birds (Hoogland 1977; Voneida and
Sligar 1979; Russchen and Yonker 1988; Reiner
et al. 1998). In turtles (Hall et al. 1977; Ulinski
1986; Kenigfest et al. 1998), reciprocal connections
between the dorsal geniculate nucleus and the cortex
have been documented. A similar feature also has
been noted in birds (Adamo 1967; Karten et al.
1973; Miceli et al. 1987). Whether this circuit is present in crocodilians remains to be seen.
Organization of the thalamic reticular nucleus
One nucleus that is integral to the organization and
function of the forebrain in mammals is the thalamic
reticular nucleus. Several features characterize this
nucleus in mammals. First, the thalamic reticular nucleus projects to all dorsal thalamic nuclei. Second,
its neurons are located within the internal capsule,
the fiber bundle interconnecting the dorsal thalamus
and cerebral cortex. Third, the thalamic reticular nucleus is composed of a homogeneous group of inhibitory neurons that utilize GABA and GAD. These
same neurons also contain the calcium-binding protein, parvalbumin. Fourth, dendrites of the thalamic
reticular nucleus are oriented perpendicular to the
fibers of the internal capsule (Jones 2007).
In crocodilians, a thalamic reticular nucleus was
determined based on injections of a retrograde tracer
into two caudal dorsal thalamic nuclei: rotundus and
the medialis complex posterior (Pritz and Stritzel
1990b). Subsequent to these injections, retrogradely
labeled cells were located within the fibers of the
dorsal peduncle of the lateral forebrain bundle
(Pritz and Stritzel 1990b), a fiber tract connecting
the dorsal thalamus with the telencephalon. In
Caiman, the thalamic reticular nucleus contains at
least two groups of neurons based on immunocytochemical properties. One cell-type projects to the
dorsal thalamic nuclei, is immunoreactive for parvalbumin (Pritz and Stritzel 1991, 1993), and has dendrites that are oriented parallel to the fibers of the
lateral forebrain bundle (Pritz and Stritzel 1991). The
other neuronal group contains neurons immunoreactive to GAD and has its processes oriented perpendicular to the axons of the lateral forebrain bundle
(Pritz and Stritzel 1990b). In turtles (Kenigfest et al.
2005) and lizards (Diaz et al. 1994), similar dorsal
thalamic projections of a thalamic reticular nucleus
have been described. In turtles, some neurons in this
nucleus that are immunoreactive both to GAD and
to parvalbumin project to the dorsal thalamus
(Kenigfest et al. 2005). In pigeons, a thalamic reticular nucleus that projects to the dorsal thalamus has
been identified experimentally (Benowitz and Karten
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1976). Its neurons are immunoreactive to GABA
(Domenici et al. 1988; Granda and Crossland 1989;
Veenman and Reiner 1994).
Neural development in A. mississippiensis
Despite sharing certain features of similar neural circuitry, the forebrain in adult sauropsids, including
crocodilians, appears quite different from that of
adult mammals. These differences must have occurred through evolution and development.
Evolutionary explanations require further data on
features described above with subsequent comparisons with other amniotes. These include characters
such as fiber connections and cellular properties including the molecular signature both of areas of the
brain and of individual neurons. On the other hand,
studies focusing on development should determine
which processes have occurred to produce these different adult forebrains.
To address some of these latter questions, experiments were undertaken to address three basic questions. First, are the very early development of both
the forebrain and other individual regions of the
brain in Alligator similar to that of other amniotes?
Second, is the later development of each of these
primary brain vesicles of the brain similar after
each of these areas has undergone segmental divisions but before each respective area has undergone
internal cytoarchitectonic differentiation? Third, specifically focusing on the diencephalon, is its initial
development similar among amniotes? Without answering these basic questions, further studies to investigate later development would be difficult to
interpret accurately.
Early development of the brain in Alligator
and other amniotes
Early in development, the brains of all amniotes, including those of crocodilians, follow a similar plan.
The brain begins as a single, hollow vesicle
(Nieuwenhuys et al. 1998), which subsequently
undergoes a series of subdivisions. Initially, these
transformations occur in the transverse plane as
this single vesicle is divided into three, with the formation of: forebrain or prosencephalon, midbrain or
mesencephalon, and hindbrain or rhombencephalon
(Vaage 1969; Vieira et al. 2010). Subsequently, two
events occur. One separates the forebrain transversely
into an anterior, secondary prosencephalon, and a
posterior, diencephalon (Vaage 1969; Puelles et al.
1987). The other divides the entire neural tube longitudinally into a dorsal, alar, and a ventral, basal
plate. At this time, a section perpendicular to the
M. B. Pritz
long axis of this longitudinal plane contains four
parts: roof plate, alar plate, basal plate, and floor
plate (Puelles 1995). While these four areas are present in a section perpendicular to this longitudinal
axis, the area of a given component varies, depending on which part of the brain’s vesicle is examined
(Fig. 3). Crocodilians undergo these general developmental transformations in a manner similar to that
of other amniotes (Pritz 2008).
At this four-vesicle stage in amniotes, the diencephalon becomes further subdivided transversely
into segments known as prosomeres. Each prosomere
ultimately forms the following divisions observed
in the brains of adults: ventral thalamus (prethalamus), dorsal thalamus (thalamus, including the
epithalamus), and pretectum (Puelles and Rubenstein
2003). Each diencephalic prosomere contains both an
alar and a basal component (Puelles and Rubenstein
2003). At this time in development in Alligator, prior
to stage 14.5, each prosomere is cytoarchitectonically
undifferentiated (see Pritz [2008] for Alligator and
for review of other amniotes).
While the prosomeres of the diencephalon remain
morphologically homogenous at this time in development in Alligator (before stage 14.5), other major
vesicles of the brain are not. The secondary prosencephalon shows distinctly different patterns of cortical layering at stage 11 depending on the location in
the cerebral hemisphere (Fig. 4). On the other hand,
at stage 13, the alar midbrain contains a layered
structure destined to become the optic tectum,
whereas an internal structure, the torus semicircularis, exhibits a nuclear organization (Fig. 5). At
this time in the development of Alligator embryos,
at stage 11, the hindbrain has already begun differentiation (Pritz 1999). While formation of the hindbrain in Alligator is similar to that observed in other
species (Pritz 1999), its pattern of development differs from that of the secondary prosencephalon, diencephalon, and midbrain. Early development of the
hindbrain follows a ‘‘two segment’’ rule in which
odd-numbered rhombomeres share particular cellular and molecular characteristics alternate with evennumbered segments that display different molecular
and cellular properties (Lumsden 2004).
These observations suggest that differentiation of
the secondary prosencephalon, diencephalon, midbrain, and hindbrain follows a different time-course
and, most likely, a different, developmental plan for
each of these vesicles (Pritz 2010b). Furthermore, in
the diencephalon of Alligator when individual prosomeres remain undifferentiated, basal portions
differ from alar parts in the expression of Pax6
(Pritz and Ruan 2009) and in the orientation of
Crocodilian forebrain
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Fig. 3 Early development of the brain in vertebrates. Schematic, lateral views of an idealized vertebrate brain are shown at: one-vesicle,
three-vesicle, and four-vesicle stages. A section perpendicular to the long axis of the brain is shown at the four-vesicle stage for the:
secondary prosencephalon, diencephalon, midbrain, and hindbrain. Abbreviations: ap, alar plate; bp, basal plate; fp, floor plate; rp, roof
plate; 28, secondary.
fiber tracts (Pritz 2010a). In Alligator, at least, this
suggests that alar and basal components in the diencephalon may also follow a different developmental
program at this time when prosomeres are homogeneous in appearance and have yet to undergo internal cytoarchitectonic differentiation. If a similar
pattern also is present in other vesicles of the
brain, one potential consequence is that evolution
could act in a seemingly independent fashion on
each of the alar and basal parts of the secondary
prosencephalon, diencephalon, midbrain, and hindbrain. Such features are unlikely to be unique to
crocodilians but common to other amniotes.
Thus, the responses to the three developmental
questions posed previously are as follows. First,
very early development of the brain in crocodilians
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M. B. Pritz
Fig. 4 Laminar organization of the cerebral cortex in an Alligator embryo. Differences in cortical lamination are illustrated in an
horizontal section of the left hemisphere of a stage-11 Alligator embryo stained with peanut agglutinin at low (A) and higher (B–D)
magnification to show variation between anterior (B), lateral (C), and medial (D) cortical areas. Abbreviations: m, medial; r, rostral,
s, stage.
is similar to that observed in other amniotes. Second,
development of individual regions of the brain in
Alligator embryos differs. This is likely to be the
case for other amniotes. Third, initial development
of the diencephalon in Alligator is similar to that in
other amniotes, although alar and basal parts in
Alligator are likely to follow a different developmental scheme.
Later development of the brain
What about subsequent development of the brain
after individual segments have become internally differentiated? Although many details are incomplete,
several generalized features are likely common to a
process that transforms an embryonic brain into its
adult morphology (Fig. 6). Initially, an uncommitted
cell will become either a neuron or a glial cell. While
glia are clearly important, the following discussion
focuses on neurons. This uncommitted cell undergoes mitoses and proliferation before exiting the
cell cycle and acquiring a phenotype as either a
neuron or a glial cell. Neurons migrate in a variety
of ways, radially, tangentially, and over long distances, before ultimately becoming either a relay
(projection) or local circuit neuron. These two
types of neurons align themselves in only one of
two ways: either in layers or as a nucleus surrounded
by borders. These processes are influenced by expression of transcription factors and signaling molecules
as well as by local environmental factors. Although
variation among vertebrates occurs, in general, these
events take place before birth.
Early in vertebrate development, a continuous
layer of cells between the ventricular border and
the pial surface of the neural tube stretches from
the hindbrain rostrally to the secondary prosencephalon (Senn 1970). In certain regions of the brain, this
laminated pattern persists, while in others, neuronal
aggregates lose their layered appearance and become
organized as nuclei. In the diencephalon of amniotes,
a nuclear pattern is seen. However, this is not the
morphology observed in other vertebrates. For example, in amphibians and fish a ‘‘primitive’’ layering
pattern persists into adulthood rather than being organized as nuclei (Nieuwenhuys et al. 1998; Butler
and Hodos 2005).
In the telencephalon, a layered morphology dominates in mammals. In this class, the cortex occupies
a much greater extent of tissue than does the comparatively thin rim of layered neurons seen in reptiles
and birds (Nieuwenhuys et al. 1998; Butler and
Hodos 2005). Other features of the forebrain’s organization in amniotes display similarities as well as
differences. Some of these characters have been
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Crocodilian forebrain
locus of termination in mice as opposed to chicks
(Bielle et al. 2011) and turtles (Bielle et al. 2011; Tosa
et al. 2015). In chicks and turtles, these studies have
focused mainly on the course of axons ending in the
dorsal ventricular ridge (Bielle et al. 2011; Tosa et al.
2015). This has provided an explanation as to why
thalamic efferents in mammals form the internal
capsule to end in the cortex while in sauropsids,
these fibers enter the lateral forebrain bundle to terminate in the dorsal ventricular ridge. In crocodilians, and most likely in other reptiles and in birds,
although not the major fiber tract, several additional
paths to the telencephalon have been identified (Pritz
2014). How these ‘‘other’’ paths relate to these data
remains to be determined but may explain the evolution of thalamo–telencephalic connections in addition to possibly sculpting the cytoarchitecture of the
forebrain in sauropsids.
Formation of the cortex in amniotes
Fig. 5 Organization of the alar midbrain in an Alligator embryo.
A sagittal section of a stage-13 Alligator brain stained for cresyl
violet is shown. Low-power magnification of an enclosed area
of the optic tectum (A) is illustrated at higher magnification
(B). At this early developmental stage, the optic tectum is
layered. On the other hand, the torus semicircularis (*) is
organized as a nucleus (A). Abbreviations: c, caudal; d, dorsal;
s, stage.
discussed previously. While the programs to explain
these observations are far from being understood, the
following two examples suggest developmental
approaches directed at explaining differences in the
morphology of adults between mammals and
sauropsids.
Formation of thalamo–telencephalic fiber tracts in
amniotes
In mammals, the paths of thalamo-cortical axons are
influenced by ‘‘corridor’’ cells (Bielle et al. 2011;
Molnár et al. 2012) as well as by several factors expressed in the surrounding forebrain (Braisted et al.
2000, 2009; Lopez-Bendito et al. 2006; Uziel et al.
2006; Molnár et al. 2012; Garel and Lopez-Bendito
2014). Identification of these factors and their expression at different times during development suggests an explanation for the trajectory, course, and
In mammals, the neocortex develops in an inside-out
fashion (Angevine and Sidman 1961), contains a
subventricular zone, and possesses a prominent and
well-organized cortical plate, which forms in an inside–outside manner (Molnár et al. 2006). On the
other hand, the comparable region in sauropsids,
the dorsal pallium, develops in an outside–inside
fashion (Tsai et al. 1981; Goffinet et al. 1986), has
a rudimentary subventricular zone (if present at all)
(Goffinet 1983; Martinez-Cerdeno et al. 2006;
Cheung et al. 2007), and has a cortical plate that is
rudimentary in turtles although well-developed in
lizards (Goffinet 1983). A variety of explanations
have been advanced to account for some of these
differences in cortical complexity and layering. One
possibility is the migratory behavior of local circuit
neurons originating in the medial ganglionic eminence to enter the cortical plate. In mice, the cortical
plate is permissive to the migration of local circuit
neurons through this structure but not in chicks or
turtles (Tanaka et al. 2011).
The above examples of developmental processes
that sculpt the forebrain provide approaches to understand not only development of the forebrain in
amniotes but also its evolution. In regards to the
diencephalon of crocodilians, the time-period when
homogeneous prosomeres become internally subdivided represents the developmental epoch when
these regions begin to acquire their respective adult
morphologies. How these changes occur will require further information not only on a variety of
morphological features but also on developmental
958
M. B. Pritz
Fig. 6 Development of cortical and nuclear structures of the brain. The influence of local environmental factors and molecular events
over time is shown in this schematic beginning with an undifferentiated cell and ending with transformation into the cortex or into a
nucleus. Although the time course for this scheme of development will vary among vertebrates, for the most part, these events will
occur before birth. Abbreviation: LCN, local circuit neuron.
processes to explain how forebrains of amniotes
evolved.
Acknowledgments
C. Brown helped with Figs. 3 and 6. J. Murphy assisted in the preparation of Figs. 4 and 5. Dr R.M.
Elsey and the Louisiana Department of Wildlife and
Fisheries provided Alligator eggs.
Funding
Partial funding for participation in this symposium
was provided for by the Society for Integrative and
Comparative Biology. Some of the data described
in this report were supported by the National
Institutes of Health [NS 20120] and by a biomedical
research grant from Indiana University School of
Medicine.
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