Zebrafish mutant ninjaos5 (nij) is required for enteric neuron and craniofacial cartilage development and Zebrafish mutant hatchbackos20 (hbk) is required for trunk neural crest development.
THESIS
Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University
By
Tamara Y. Robinson
Graduate Program in Molecular, Cellular and Developmental Biology
The Ohio State University
2010
Master's Examination Committee:
Paul D. Henion “Advisor”, Susan Cole, Mark Seeger, James Jontes
Copyright by
Tamara Y. Robinson
2010
Abstract
The neural crest (NC) is an ectoderm derived embryonic cell population that is
specific to all vertebrate embryos. The NC is induced during gastrulation at the neural
plate border (NPB) and migrates throughout the developing embryo to give rise to a
number of derivatives including neurons and glia of the peripheral nervous system,
pigment cells, and craniofacial cartilage and bone. Although much effort has been put
into understanding neural crest diversification, the genetic regulatory network involved in
this process is still not completely understood. The study of ENU induced zebrafish mutants with defective neural crest development is one approach that has been employed to address this issue.
The zebrafish mutant ninjaos5(nij) is an ENU-induced, recessive, larval lethal
mutation that was identified based on reduced cranial neural crest expression of crestin
during embryogenesis. Reduced crestin expression in nij mutants is evident at hindbrain
levels as well as in more anterior regions. NC precursors of the jaw elements are present
in nij mutants, but neural crest derived elements of the craniofacial skeleton do not differentiate. In addition, we also find that enteric neuron precursors are severely reduced in nij mutants. As a result, very few cells are undergoing overt differentiation, accounting for the absence of enteric neurons. The development of other neural crest derivatives such as sensory neurons and chromatophores, in contrast, is comparatively normal. These results indicate an essential role for nij function in the development of the neural crest
derived craniofacial skeleton and enteric nervous system. nij appears to be required for
ii
terminal differentiation of craniofacial cartilages and for establishment of enteric neurons
precursors.
The zebrafish mutant hatchback (hbk) is an ENU induced recessive embryonic
lethal mutation that was identified based on reduced trunk neural crest expression of
crestin. All zebrafish chromatophore cell types and trunk neuronal derivatives including
enteric neurons, sympathetic neurons, and dorsal root ganglia are missing in hbk mutant embryos. In contrast, derivatives of cranial neural crest cells including the pharyngeal arches that give rise to the head skeleton, cranial satelite glia, and cranial ganglia are present. Assessment of early crest development reveals that the NPB and the NC are induced. However, pre-migratory NC expression of the early NC transcription factors that function in specification of neural crest sub-lineages is perturbed in the trunk. These results suggest that the function of hbk is required for specification of neural crest sub- lineages.
iii
Dedication
This document is dedicated to my family. Thank you for your unconditional love and support. You are there through thick and thin, good or bad, right or wrong. I treasure you
all and promise never to take you for granted. I love you dearly.
iv
Acknowledgment I would like to acknowledge my advisor Paul D. Henion for allowing me to
become a member of his lab and introducing me to the world of science. The exposure I
have received, laboratory experience I have obtained, the growth that has taken place, and
the relationships I have developed would not have been possible if you had not given me
a chance. Secondly I would like to thank you for encouraging, supporting, pushing me to
go after what I want and pursue an alternative career path into medicine. This was a bitter
sweet decision, but it is better to do what’s best for me than to conform to the
expectations of others.
I would also like to thank my committee James Jontes, Susan Cole, Mark Seeger,
as well as Paul D. Henion. I would like to thank you all for your support and kindness. I
have been blessed to be able to learn from such distinguished scientists.
I want to also acknowledge my past and present lab mates, Marsha Lucas, Ph.D.,
Kevin Bosse, Arife Unal, Min An, Ph.D., Myron Ignatious, Ph.D., Brigitte Arduini, PhD.,
and Smitha Malireddy. My experience in this lab would not have been the same without
you. Thanks for all your help, advice, encouragement, stimulating and sometimes random
conversations, and laughs.
I would also like to thank the members of the Beattie and Jontes lab. You too
have been an integral part of my experience here at OSU. Thanks for all of your help and words of encouragement.
v
Vita
June 2001…………………………………..Fairley High School, Memphis, TN
2005...... B.S. Biology, University of Arkansas at Pine
Bluff
2006...... Graduate Teaching Associate, Department
of Biological Sciences, Biology 101, Ohio
State University
2005- to present ...... Graduate Research Associate, Department
of Molecular Cellular Developmental
Biology, The Ohio State University
Fields of Study
Major Field: Molecular, Cellular and Developmental Biology
vi
Table of Contents
Abstract...... ii
Dedication...... iv
Acknowledgment ...... v
Vita...... vi
Fields of Study ...... vi
Table of Contents...... vii
List of Figures...... x
List of Abbreviations ...... xii
CHAPTER1 ...... 1
Introduction...... 1
1.1: Neural Crest Development: Neural Plate Border Induction ...... 1
1.2: Neural Crest Development: Specification of Neural Crest Sub-lineages ...... 6
1.3: Neural Crest Development: Terminal Differentiation of Neural Crest Sub-lineages
...... 7
1.3.1: Terminal Differentiation: Pigment ...... 7
1.3.2: Terminal Differentiation: Craniofacial Skeleton...... 9
1.3.3: Terminal Differentiation: Sympathetic Neurons...... 14
vii
1.3.4: Terminal Differentiation: Enteric Neurons ...... 16
1.3.5: Terminal Differentiation: Dorsal Root Ganglia Sensory Neurons...... 18
1.3.6: Terminal Differentiation: Conclusion ...... 22
Chapter 2...... 23
Characterization of the zebrafish mutant ninjaos5 (nij) ...... 23
2.1 Introduction ...... 23
2.2: Isolation of zebrafish mutant nijos5...... 24
2.3 Zebrafish mutant nijos5 is required for craniofacial cartilage development...... 25
2.4 Zebrafish mutant nij is required for enteric neuron development...... 29
2.5 Neural Plate Border (NPB) and Neural Crest Induction Are Unaffected in nijos5 .. 34
2.6 Enteric neuron and craniofacial abnormalities may be due to an increase in cell
death...... 35
2.7 Mapping of nijos5 ...... 37
2.8 Discussion ...... 38
2.9 Materials and Methods...... 43
2.9.1 Zebrafish Husbandry ...... 43
2.9.2 Whole mount in situ Hybridization and immunohistochemistry...... 44
2.9.3 Alcian Blue Staining...... 44
2.9.4 TUNEL Assay ...... 44
viii
2.9.5 Genetic Mapping ...... 44
Characterization of the zebrafish mutant hatchbackOS20 (hbk) ...... 45
3.1 Introduction ...... 45
3.2 Isolation of zebrafish mutant hbkos20 ...... 46
Section 3.3 Cranial neural crest derivatives are unaffected in hbk embryos...... 49
Section 3.4 Early neural crest development is disrupted in hbk...... 50
Section 3.5 Mapping hbk...... 52
3.6 Discussion ...... 56
3.7 Materials and Methods...... 58
3.7.1 Zebrafish Husbandry ...... 58
3.7.2 Whole mount in situ Hybridization and immunohistochemistry...... 58
3.7.3 Genetic Mapping ...... 58
Chapter 4...... 60
Discussion...... 60
ix
List of Figures
Figure 1.1 Schematic diagram of neurulation and neural crest induction...... 2
Figure 1.2 Pharyngeal arch primordium that forms the craniofacial cartilages in zebrafish.
...... 10
Figure 1.3 Pathways of neural crest migration...... 15
Figure 2.1 Crestin expression...... 24
Figure 2.2 Live Phenotype...... 25
Figure 2. 3 Crest derived craniofacial cartilages...... 26
Figure 2. 4 Craniofacial Cartilage Precursors...... 28
Figure 2. 5 Endodermal, mesodermal, and epidermal components of pharyngeal arches..1
Figure 2. 6 Enteric neurons...... 30
Figure 2. 7 All other crest derived neuronal elements cell types...... 31
Figure 2. 8 Enteric neuron precursors...... 1
Figure 2. 9 sox10 expression...... 33
Figure 2. 10 Neural Plate Border Induction...... 34
Figure 2. 11 Neural Crest Induction...... 35
Figure 2. 12 Increased cell death in ninja...... 37
Figure 3. 1 crestin expression in hbk...... 46
x
Figure 3. 2 hbk live phenotype...... 47
Figure 3. 3 Chromatophore Precursors in hbk...... 48
Figure 3. 4 Neuronal Derivatives in hbk...... 49
Figure 3. 5 Craniofacial cartilage precursors in hbk...... 50
Figure 3. 6 Neural crest induction and cell fate specification in hbk...... 51
Figure 3. 7 Neural plate border induction in hbk...... 52
Figure 3. 8 ctr9 expression in hbk...... 53
Figure 3. 9 Rescue of pigment with BAC containing ctr9...... 54
xi
List of Abbreviations
B-cell leukemia/lymphoma 2 bcl2
Beta β
Bone Morphogenic Proteins BMP
Collagen 2 alpha col2a
Complementary DNA cDNA days post fertilization dpf
Deoxyribonucleic acid DNA dominant negative DN dopachrome tautomerase dct
Dopamine β hydroxylase DBH
Dorsal Root Ganglia DRG eukaryotic translation initiation factor 4, gamma 2a eif4g2a
Ethyl Nitrosourea ENU
Endothelin Edn
Fibroblast Growth Factor FGF
Glial Derived Neurotrophic Factor GDNF
GTP-cyclohydrolase gch hatchback hbk hour post fertilization hpf
xii jellyfish jef lockjaw low
Mammalian achaete-scute homolog 1 mash1
Messenger RNA RNA microphthalmia associated transcription factor mitf monocytic leukemia zinc finger moz morpholino MO
Neurogenin ngn
Neural Plate Border NPB
Neural Crest NC ninja nij receptor tyrosine kinase c-kit c-kit
Retinaldehyde dehydrogenase-1 Raldh2
Retinoic Acid RA
Ribonucleic acid RNA
Sonic Hedgehog shh
Terminal deoxynucleotidyl transferase dUTP nick end labeling TUNEL
Tyrosine hydroxylase TH tyrosinase related protein 1 trp1 xanthine dehydrogenase xdh
xiii
CHAPTER1
Introduction
The neural crest (NC) is a vertebrate specific, ectoderm-derived embryonic cell population that gives rise to a number of derivatives, including craniofacial cartilage and
bone, neurons and glia of the peripheral nervous system, and pigment cells. The neural
crest is induced at the neural plate border, migrates from the dorsal neural tube to various
locations within the embryo, and differentiates into multiple derivatives. The neural crest
must undergo several cellular changes such as induction events, cell fate specification,
epithelial mesenchymal transitions, migration, and terminal differentiation, in order for
diversification of NC sub-lineages from a seemingly homogenous cell population to
occur. Insight gained from the study of the aforementioned developmental events in the
neural crest can serve as a model for other cellular populations that undergo similar
molecular and morphological changes. The study of these developmental processes could
also provide insight into the underlying mechanisms which lead to the manifestation of
neurocristopathies, which are diseases that occur as a direct result of improper neural
crest development.
1.1: Neural Crest Development: Neural Plate Border Induction
During gastrulation, the embryo is divided into three germ layers: the ectoderm,
mesoderm, and endoderm. The ectoderm gives rise to three derivatives which include: the
non-neural ectoderm (future epidermis), neural ectoderm (gives rise to the neural tube
which eventually forms the future central nervous system) and the cells of the neural
1
plate border (NPB). Within the NPB, three cell populations exist which will give rise to the neural crest, Rohon Beard sensory neurons, and ectodermal placodes.
The NPB is induced at the border between the neural and non-neural ectoderm.
During neurulation the neural ectoderm thickens to form a neural plate.1 The neural plate
folds into a cylindrical structure termed the neural tube. As the neural tube closes, the
cells of the NPB converge at the dorsal midline of the embryo. After converging at the
dorsal midline, neural crest cells delaminate from the neural tube, migrate along distinct
tracks to various locations within the embryo and differentiate into multiple cell types.
Figure 1.1 Schematic diagram of neurulation and neural crest induction. A. Formation of the neural plate. B. Neural plate folds. C. Closure of the neural tube. The neural crest is located at the dorsal part of the neural tube. D. Neural crest cells migrate. Non-neural ectoderm in orange. Neural Plate and neural tube in blue. Neural plate border and neural crest in green. (Courtesy of Marsha E. Lucas) (Adapted from Knecht et. al., 2002)
2
This complex chain of events, ranging from induction of the NPB, induction of the neural crest proper, specification of neural crest sub-lineages, and migration and overt differentiation of NC derivatives, is guided by a carefully orchestrated gene regulatory network. This regulatory network is comprised of multiple signaling pathways and transcription factors that guide the acquisition of specific properties, such as multipotency and migratory capacity, which are required for each stage of neural crest development to proceed.1
As previously stated, several signaling pathways, which include Bone Morphogenic
Protein (BMP), Fibroblast growth factor (FGF), Wnt, and Notch signaling, have all been shown to play major roles in NPB induction. It is believed that a gradient of BMP signaling plays a role in establishing a distinction between the cells that will be specified to become the epidermis, the neural plate, and the NPB. High levels of BMP signaling are required for induction of the epidermis. Low levels of BMP signaling are required for induction of the neural plate. Intermediate levels of BMP signaling are required for induction of the NPB. The intermediate BMP levels necessary for NPB induction, are established by BMP antagonists, chordin, noggin, and follistatin, which are secreted by underlying paraxial mesoderm.2 Although BMP signaling has been shown to play a role in induction of the NPB, it is now evident that BMP signaling alone is not sufficient to induce the NPB. 3, 4
As previously stated, FGF signaling has also been implicated in NPB induction. For example, in Xenopus animal cap assays, FGF2 protein, in combination with BMP antagonists, was capable of inducing the NPB leading to an up regulation of neural crest
3
specific markers.3-5 In addition, Xenopus animal caps that were injected with Fgf8
mRNA alone at the one to two cell stage and grown in isolation resulted in a strong
induction of NPB marker, Zic5, and pre-migratory NC markers, Sox9 and Foxd3.6
Notch signaling has also been implicated in NPB induction. Based on studies in birds
and frogs, Notch signaling is believed to act upstream of BMP signaling to promote NPB
induction. 7 Activation of the Notch signaling pathway or over-expression of Hairy2, a downstream effecter of Notch signaling which is expressed in the cells of the NPB, results in an expansion of Xslug expression in the NC-forming territory in frogs.8
Wnt signaling has also been indicated in NPB induction and thus neural crest induction. In Xenopus, injection of dominant negative (DN) XWnt-8 mRNA into one cell of two cell stage embryos resulted in a significant loss of the expression of Xslug, a marker of pre-migratory NC cells, on the injected side.3 In zebrafish, injection of
translation blocking morpholinos targeted towards wnt8.1 resulted in severe disruption in
the expression of the NPB marker, pax3.9 Also in chick, cells expressing a dominant
negative Wnt-1 construct were injected either adjacent to the open neural plate or into the
closing neural tube. Injection of these DN Wnt-1 expressing cells resulted in a marked
reduction in Slug expression. However, the results detailing the role of Wnt signaling in
mouse are ambiguous. Analysis of mouse mutants with targeted inactivation of Wnts or
downstream components of Wnt signaling reveals severe defects in NC derivatives such
as cranial and trunk ganglia, melanocytes, and craniofacial skeletal elements. However,
the results from these studies have been interpreted to support the idea that Wnts are
involved in specification of NC sub-lineages, rather than NPB induction. 1
4
The activity of the signaling pathways mentioned above have been shown to facilitate
the expression of transcription factors that uniquely define the NPB. These transcription
factors include: Zic1, Msx1, Msx2 Dlx3, Dlx5, Pax3, and Pax7.1 An intermediate level of
BMP activity has been shown to play a role in facilitating the expression of neural plate
border specifiers, Msx1, Pax3, and Zic1. Fgf and Wnt signaling have also been shown to
induce the expression of Msx1, Pax3, and Zic1. For example, injection of a Wnt-8 DNA
expression construct into Xenopus embryos expanded the Pax-3 expression domain in the
neural plate border. Conversely, blocking Wnt signaling by injecting embryos with a
dominant negative XWnt-8 construct eliminated or strongly down-regulated Pax3 and
Msx1 expression. Mis-expression of Zic1 was found to induce ectopic formation of neural
crest cells in the ventral ectoderm. However, this induction was suppressed when Wnt
signaling was blocked by injection of β-catenin morpholinos or dominant negative TCF3
mRNA. Similarly, over-expression of Fgf8 up-regulated the expression of Msx1. In
contrast, injection of Fgf8 morpholinos abolished Msx1 expression and also reduced the expression of Pax3. 10-12
The expression of these NPB transcription factors are indicative of NPB induction
and confer the competency of these cells to form the neural crest proper.1 Studies show
that up-regulation of Msx1 in the NPB is essential for the formation of bona fide NC
cells. Injection of Msx1 morpholinos into Xenopus embryos blocked the activation of
Slug and FoxD3, which are markers of early pre-migratory neural crest cells.
Additionally, morpholino knockdown of Zic1 or Pax3 indicates that these transcription
factors are also required for NPB formation and thus NC induction. Morpholinos targeted
5
towards Zic1 and Pax3 also suppressed the formation of Foxd3 and Slug expressing
neural crest cells 11, 12 Interestingly, in Xenopus, it seems that a balance of Zic1 and Pax3
gene products are required for induction of the neural crest proper. High levels of one of
these transcription factors in the absence of the other favors alternative NPB fates, such
as hatching gland, which is promoted by Pax3, or pre-placodal fate, which is promoted
by Zic1.13, 14
1.2: Neural Crest Development: Specification of Neural Crest Sub-lineages
After the competency of the NPB has been established by the activity of the neural
plate border transcription factors, a new set of transcription factors begins to be expressed. These transcription factors include Snail/Slug, Sox E family genes, Foxd3, and Ap2. The expression of these NC transcription factors define the neural crest domain and initiate the early stages of cell fate specification of NC sub-lineages.1
These early NC transcription factors are expressed by the majority of pre-migratory
neural crest cells. Analysis of zebrafish mutants harboring mutations in these genes has
shown that although these genes are widely expressed in pre-migratory neural crest cells,
they are selectively required for specification and development of specific neural crest
sub-lineages. For example, analysis of the zebrafish foxd3 mutant, sym1, has shown that
disruption of foxd3 function results in a loss of sympathetic neurons, enteric neurons, and
abnormal development of the craniofacial cartilages.15 Similarly, lack of tfap2a function
in zebrafish low mutants has also highlighted a requirement for tfap2a in the development
of the craniofacial cartilages, enteric neurons and sympathetic neurons.16 Lack of sox10
function disrupts the development of pigment and neuronal and glial cell types.17, 18 In
6
addition, loss of sox9 function, in which there are two orthologs in zebrafish, sox9a and sox9b, highlights a requirement for sox9 in craniofacial cartilage development of the head skeleton.19
Gain of function and loss of function studies in several vertebrate model systems have also shown that early NC transcription factors act together to regulate and maintain each other’s expression and ultimately function within a regulatory network to bring about overt specification of NC sub-lineages.1 For example, loss of tfap2a in zebrafish low mutants, results in a reduction of foxd3 expression in hindbrain neural crest cells. In contrast, sox9b is initially expressed normally in pre-migratory NC cells in low mutants, but its expression fails to be maintained.16 Similarly, loss of foxd3 function in sym1
mutants resulted in a reduction in sox10 and snail1b expression in pre-migratory neural
crest cells. However, while the expression of tfap2a in foxd3 mutants was initially
normal, tfap2a expression fails to be maintained at later stages. Likewise, loss of sox9b
results in a reduction of snail1b, foxd3, and sox10 expression in sox9b mutants. Although
it is clear that the early NC transcription factors function in regulating and/or maintaining
each other’s expression, the hierarchial relationships between these genes remain
unresolved.
1.3: Neural Crest Development: Terminal Differentiation of Neural Crest Sub-lineages
1.3.1: Terminal Differentiation: Pigment
Chromatophores are one of the many cells types that are derived from the neural
crest. Mice and chick possess only one chromatophore cell type, melanophores.
7
However, zebrafish possess three chromatophore cell types including black
melanophores, yellow xanthophores, and iridescent iridiphores. Of the three zebrafish chromatophore cell types, melanophore development has been well defined and shown to be conserved across species. A great amount of information has been obtained from
studies done in mouse and zebrafish mutants. More than 800 coat color mouse mutants
corresponding to 127 genetic loci have been identified. 20 Likewise, in zebrafish, almost
one hundred pigmentation mutants have been identified from genetic screens. 21-23
In many species it has been shown that microphthalimia associated transcription
factor (mitf) functions as the master regulator of melanophore development.24 mitf is one
of the earliest genes to be expressed in melanophore precursors and has been shown to be
necessary and sufficient for melanophore development.25 Previous work has placed mitf upstream of several genes that are necessary for melanophore terminal differentiation and melanin synthesis which include in most cases dopachrome tautomerase (dct), tyrosinase, tyrosinase-related protein1 (trp1), receptor tyrosine kinase c-kit (ckit), and B- cell leukemia/lymphoma 2 (bcl2).26
Many transcriptional regulators of mitf expression have also been identified.
sox10 has been shown to directly regulate melanophore cell fate via the mift promoter.27
mitf is also positively regulated by Wnt signaling.28 Other positive regulators of mift
expression include: CREB and pax329-36
Unlike melanophore development, the other zebrafish chromatophore derivatives have not been as extensively studied. However, some of the mechanisms involved in xanthophore and iridiphore development have been elucidated. For example, the kit
8
ortholog, fms, is required for migration of xanthophores precursors. 37, 38 xanthine
dehydrogenase (xdh) is expressed in xanthoblasts and is required for the synthesis of
yellow pteridine pigments.37, 39, 40 The enzyme GTP-cyclohydrolase (Gch) is expressed in both melanophores and xanthophores. Gch is required for the conversion of intermediates
of both melanin and pteridine. 37, 41-43 The endothelin receptor (ednrb) is also expressed in
neural crest derived chromatophores. Homozygous Ednrb-/- mutant mice are almost
completely devoid of melanocytes.20 In zebrafish, ednrb1 is initially expressed by all chromatopohores but later becomes localized in iridiblasts and iridiphores. Zebrafish ednrb1 mutants display defects in subsets of adult melanophores and iridiphores, but these mutants lack an embryonic phenotype. 37
1.3.2: Terminal Differentiation: Craniofacial Skeleton
Craniofacial cartilage and bone that make up the head skeleton are also
derivatives of the neural crest. During development, cranial neural crest cells delaminate from the neural tube and migrate in separate streams to form pharyngeal arches. These craniofacial cartilage precursors arise from the hindbrain and emigrate in three distinct streams to form the mandibular (stream 1), hyoid (stream 2), and five branchial arches
(stream 3). In zebrafish, as well as other species, each of these pharyngeal arches give rise to distinct craniofacial cartilage elements that lay down the framework for the head skeleton. (Fig 1.6) In later stages of development of the craniofacial skeleton these cartilage elements are subsequently replaced with bone.
9
Figure 1.2 Pharyngeal arch primordium that forms the craniofacial cartilages in zebrafish. A.) Lateral view of 28hpf zebrafish embryo. Diagram depicts the dorsal (D1, D2) and ventral (V1, V2) crest derived skeletal precursors of the first two arches and ventral crest derived skeletal precursors of the branchial arches. Purple oval (arrow) represent the neurocranium skeletal precursors. B.) The cylindrical organization of arch primordia. Mesoderm (brown) is surrounded by CNC (red, white and blue) which form dorsal and ventral condensations. Joint precursors (white) express bapx1 in the mandibular arch. These groups of arch mesenchyme are surrounded by endodermal (yellow) and ectodermal (green) epithelia. C.) Drawing of larval cartilages of the mandibular (red), hyoid (blue) and branchial (green) arches, as well as the neurocranium (purple). D.) drawing of flat-mounted, dissected cartilages of the mandibular and hyoid. Abbreviations: ch, ceratohyal; hs, hyosymplectic; ih, interhyal; mc, meckel's cartilage; pq, palatoquadrate; tr, trabeculae. (Knight R. D. et. al.)
The skeletal derivatives of the cranial neural crest are patterned through a combination of intrinsic differences within crest cells and extrinsic signals from adjacent tissues, such as pharyngeal endoderm and ectoderm.44 Differences in Hox gene
expression have been shown to play significant roles in conferring positional identity of
each of the neural crest streams that give rise to the pharyngeal arches. For instance,
cranial neural crest cells in stream 2, which give rise to the hyoid arch, arise from the
10
hox2 expressing region of the hindbrain and expresses Hox group 2 genes. However,
cranial neural crest cells of stream 1, which form the mandibular arch, arise from the hox
negative region of the anterior hindbrain and midbrain and do not possess hox2 gene
expression.44 Loss of Hox2 gene expression in the hyoid arch results in a transformation
of hyoid skeletal elements into mandibular skeletal elements.45, 46 In contrast, over
expression of hox genes in stream 1 results in the formation of ectopic hyoid skeletal
elements.18, 47
Recent evidence has shown that, despite the origin of each cranial neural crest
stream, regulation of their hox2 gene expression identity is independent of the hox2 gene expression in the hindbrain.44 For example, in zebrafish moz mutants, which contain a
mutation in the histone acetyltransferase (monocytic leukemia zinc finger), hoxa2 and
hoxb2 expression are disrupted in the hyoid cranial neural crest but hox2 gene expression
is unaltered in the hindbrain.48 In these mutants, specific loss of cranial neural crest hox2
gene expression resulted in the hyoid arch cartilages being partially transformed into
mandibular arch elements.49 Likewise, zebrafish lockjaw (low) mutants, which possess a
mutation in the transcription factor tfap2a, exhibit segment specific changes in hox2 gene
expression and fate changes in the pharyngeal cartilages, but hox2 gene expression in the
hindbrain is unaffected.16
Each pharyngeal arch is arranged cylindrically with each arch containing a
mesodermal core that is surrounded by cranial neural crest cells. These cells are, in turn,
surrounded by endodermal and ectodermal epithelia.44 (Fig. 1.6) Several studies have
shown that not only are there intrinsic signals within neural crest cells that are required
11
for patterning of cranial neural crest cells and development of crest-derived skeletal
elements, but extrinsic signals from adjacent tissues are also necessary. For example, studies in amphibians and avians highlight a requirement for endoderm in the formation of pharyngeal cartilages.50 Genetic studies in zebrafish have also confirmed a role for
endoderm in patterning and development of the head skeleton. For example, in mutants
that lack pharyngeal endoderm, such as the sox32 mutants, Casanova (cas), and tbx1
(vgo) mutants, pharyngeal cartilages fail to develop properly. In cas mutants, pharyngeal
cartilages are completely absence.51 In vgo mutants, pharyngeal arches are variably lost and the cartilage elements that are present are reduced or fused.52 As studies have shown,
removal of surface ectoderm also results in the failure of craniofacial cartilages to
development suggesting that interactions between the cranial neural crest and surface
ectoderm are also essential for craniofacial cartilage development.44
Several signaling molecules from both the endoderm and the ectoderm have been
shown to play roles in craniofacial cartilage development. Some examples include:
Fibroblast Growth Factors (fgf3 and fgf8) 53-55, Endothelin (Edn) signaling 56, Bone
Morphogenetic Proteins (BMP) 57, and Sonic Hedgehog (Shh) 58. Studies have shown
that, fgf3, is expressed specifically in the endoderm of the pharyngeal arches. Removal of
Fgf3 function specifically from pharyngeal endoderm revealed that fgf3 is required for
the formation of the posterior branchial cartilages. 51 Subsequent studies also suggest that
Fgf3 and Fgf8 work together to pattern the pharyngeal arches. Loss of both fgf3 and fgf8 function by morpholino knockdown results in a loss of all craniofacial cartilages.53, 54
12
Studies have also shown that removal of Fgf8 specifically from the mandibular ectoderm
results in a loss of all but the most ventral skeletal elements.55
Similarly, several studies show that loss of Edn signaling also results in abnormal
development of the craniofacial cartilages and highlight a role for Edn signaling in
dorsal/ventral patterning of the pharyngeal arches.44 In the zebrafish edn1 mutant, sucker,
a variable phenotype is observed. Some sucker mutants lack the lower jaw pharyngeal
elements; while others appear to exhibit partial duplications of dorsal skeletal elements in the ventral arches.59 Likewise, mice with mutations in the Edn receptor, EdnrA, exhibit a proximal-distal transformation that results in ectopic dorsal structures in place of ventral structures.60
During the latter stages of craniofacial cartilage development, chondrocytes are
surrounded by a collagen matrix. The major collagen that encodes for this matrix is
collagen 2 alpha (col2a). As development continues, chondrocytes undergo directional
proliferation, hypertrophy, and then apoptosis. Before chondrocytes undergo apoptosis,
the extracellular matrix mineralizes and replaces the cartilage tissue with bone forming
cell replacements.61
dlx2a and sox9a are believed to be major players involved in overt differentiation
of craniofacial cartilage and thus facilitating the expression of col2a. Studies have shown
that dlx2a plays a role in specifying a distinct population of cells to become pharyngeal arch precursors and is necessary for the survival of these cells. Knockdown of dlx2a also
resulted in a loss of sox9a expression in the mandibular arch and a reduction in sox9a
13
expression in the more posterior arches suggesting that dlx2a plays a role in modulating
the expression of sox9a.62
sox9a has been shown to be required for overt differentiation of the craniofacial cartilages. Zebrafish sox9a mutants, jellyfish (jeftw37), have severely reduced crest-derived cartilage elements. Alcian blue staining, which binds the proteoglycan components of the chondrocyte extracellular matrix, demonstrated that most of the cartilage elements of the pharyngeal arches were missing from homozygous jeftw37 mutants. In these mutants,
sox9a has been shown to be required for col2a1 expression. In homozygous jeftw37 mutants, col2a1 is severely reduced and appears in only small regions of the pharyngeal arches. 61 Additionally, sox9a has been shown to directly activate the transcription of
col2a1 by binding to a chondrocyte-specific enhancer present in its first intron.63-66
1.3.3: Terminal Differentiation: Sympathetic Neurons
The autonomic nervous system regulates body function homeostasis in
vertebrates. Its function is essential for adaptation to internal and external environmental
changes. Sympathetic neurons of the autonomic nervous system arise from the neural
crest and play a major role in regulating vascular tone, exocrine gland and gut function.67
After leaving the neural tube, NC cells that will give rise to sympathetic ganglia,
migrate along a ventral route between the somites and through the rostral half of the each
somite to their final destination, which is adjacent to the dorsal aorta.(Fig. 1.7) 68 Signals from the aorta, as well as somatic mesoderm, ventral neural tube, and notochord all play an integral role in terminal differentiation of sympathetic neurons.69-74 For example,
14
development of the autonomic nervous system heavily depends on the presence of BMP
signaling. A number of BMPs including, BMP-2, BMP-4 and BMP-7 are expressed by
the aorta during the developmental stages that crest-derived cells are coalescing into
sympathetic ganglia.70 Inhibition of BMP signaling in chick embryos by treating them
with the BMP antagonist, noggin, prevents neuronal differentiation of sympathetic
ganglia.74
Figure 1.3 Pathways of neural crest migration. Cells that migrate just beneath the ectoderm will form pigment cells. Cells that migrate along the medial pathway adjacent to the somites form DRG and sympathetic ganglia. Cells that form neurons and glial cells of the enteric ganglia are formed from neural crest cells that migrate along the length of the body. (Molecular Biology of the Cell. Fourth Edition.)
15
The activity of BMP signaling leads to the expression of a number of different transcriptional regulators that are involved in sympathetic neuron differentiation. These transcriptional regulators include Mammalian achaete-scute homolog1 (also known as
Mash1, Cash1 in chick, and zash1a in zebrafish), Hand2 (also known as dHand), the paired homeodomain transcription factors, Phox2a and Phox2b, and the zincfinger proteins, Gata2 and Gata3.75-80 Mash1 has been implicated as a key factor that is necessary for the expression of pan neuronal markers, such as HuC, in sympathetic ganglion.76 The elimination of Mash1 significantly impairs sympathetic ganglion
development. 75, 81 In homozygous Mash1 mutant mice, the brain and spinal cord appear
normal but development of sympathetic ganglia is severely disrupted.81 Similarly, loss of
Phox2b function in mice results in a complete loss of the autonomic nervous system.77
Both Phox2 transcription factors, phox2a and phox2b, are positive regulators of
Dopamine β-hydroxylase (DBH) and Tyrosine hydroxylase (TH) expression.82-84 TH and
DBH are genes that control essential steps that lead to the synthesis of noradrenalin and are terminal differentiation markers of sympathetic neurons.67 Several other transcription
factors, such as Creb, AP-2a, Gata2/3, and Hand2 (dhand), have also been implicated in
controlling the expression of Dbh and Th in sympathetic neuron precursors.79, 80, 82, 85-88, 88,
89
1.3.4: Terminal Differentiation: Enteric Neurons
The neural crest also gives rise to the neurons and glia of the enteric nervous
system. The enteric nervous system (ENS) plays an integral role in mediating basic
16
functions of the gut such as peristalsis, secretion, regulating blood flow within the gut
wall, and water electrolyte transport across the mucosal epithelium.90, 91 The ENS has
often been termed the “the second brain” because some regions of the gastrointestinal
tract can function independently of the CNS.92
The majority of ENS precursors originate from vagal neural crest cells emerging
from the neural tube at the levels of somites 1-7. A smaller subset of ENS precursors
arises from sacral neural crest cells. After delamination from the neural tube, ENS
precursors migrate throughout the developing embryo to colonize the entire length of the
gut. ENS migration occurs in two phases. In the first phase, vagal neural crest cells
migrate ventrally to the anterior part of the gut. Afterwards, these cells migrate caudally
along the entire length of the gut. In the second phase, sacral neural crest cells migrate
ventrally to the hindgut. Afterwards, sacral ENS precursors migrate rostrally giving rise
to only a small portion of total ENS precursors.93 After colonizing the gut, neural crest
cells undergo an extensive amount of proliferation and differentiate into the glial and
neuronal cell types that make up the ENS.90
Glial cell line-derived neurotrophic factor (GDNF) is thought to be a major player
in ENS development. GDNF is a secreted protein that signals through the Ret tyrosine
kinase receptor. GDNF is expressed in the gut mesenchyme, while Ret is expressed
exclusively in neural crest derived enteric neurons.94, 95 The absence of enteric neurons
from GDNF -/- mice and Ret-/- mice suggests that GDNF/Ret signaling is critical for enteric neuron development.96 GDNF/Ret signaling is involved in multiple aspects of
enteric neuron development. Studies have shown that GDNF acts as a chemoattractant of
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neural crest cells97, is required for neural crest cell survival 98, and promotes
differentiation and proliferation of ENS precursors.99-102
Several transcription factors have also been identified and found to play critical
roles in ENS development. For instance, enteric neurons are absent in mice lacking
Phox2b and Sox10.77, 103, 104 Zebrafish sox10 mutants also exhibit a great reduction in the number of enteric neurons aligning the gut.18Morpholino knockdown of phox2b in
zebrafish also results in a marked reduction in the number of enteric neurons colonizing
the gut.93 Also, mice lacking Pax3 function are missing enteric neurons from the large
and small intestines.105 Likewise, disruption of Mash1 function in mice causes an absence
of enteric neurons in the esophagus.81
Each of the aforementioned transcription factors plays a role in facilitating the
expression of Ret. For example, the absence of Ret expression in Phox2b-/- mice suggests
that Phox2b plays a role in regulating the expression of Ret.77 Sox10 has been shown to
regulate the expression of Ret either directly via interactions with Pax3 106 or indirectly
by inducing Phox2b expression.107 Mash1a can also regulate the expression of Ret by interacting with Phox2a.75
1.3.5: Terminal Differentiation: Dorsal Root Ganglia Sensory Neurons
Dorsal Root Ganglia (DRG) are responsible for transmitting all of the somatosensory information from the body. This information includes sensory modalities such as touch, nociception (perception of pain and irritation), proprioception (position and movement of limbs and body), and temperature. DRG send out processes that
18
innervate the skin, muscles, and joints of the limbs and trunk. They also send out central
projections that extend dorsally, entering the spinal cord through the dorsal root. The
branches of these central processes terminate either in the spinal cord or ascend to the
brain stem. 108
Neural crest precursors of DRG must migrate along the same path as neural crest
cells that make up the sympathetic neurons of the autonomic nervous system. The cells of
the autonomic sympathetic ganglia are located along the dorsal aorta. However, DRG
precursors must stop and adopt a sensory neuron cell fate which is located adjacent to the
spinal cord. (Figure 1.7) 108
There are two classes of sensory neurons which are morphologically and
functionally distinguishable. There are large neurons that contain large myelinated fibers
that convey fine touch and proprioceptic information to the brain stem. There are also
small neurons that contain small unmyelinated fibers that convey pain information. 108
Large and small neurons are also characterized by the neurotrophin receptor that each cell type expresses. TrkA is expressed in small diameter neurons. TrkC, however, is expressed in large diameter neurons. 109, 110 Both TrkA and TrkC act as receptors for the
neurotrophins, NGF and NT-3, respectively. 108
Based on the early of TrkC expression, it has been suggested that these receptors
and their neurotrophins play roles in specification of neuronal subtypes and axonal
outgrowth. 111, 112 Genetic analyses provided further confirmation of the role that these
receptors play in sensory neuron development. For example, targeted inactivation of
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TrkC receptors and NT3 resulted in the loss large proprioceptors.113, 114 Also, selective inactivation of TrkA receptors or NGF resulted in a loss of smaller nociceptors.114, 115
While neurotrophins do contribute to the series of events that lead to specification of sensory neurons, there are implications that other factors are also involved in specifying sensory neuron fate. In mammalian and avian species, DRG growth occurs in two phases. First a subpopulation of large neurons is born. Second, a group of both large and small neurons are generated. 116, 117 These two phases of DRG development appear to be regulated by the Neurogenin basic helix loop helix transcription factors. Targeted knockout of Ngn1 resulted in the loss of most of the later small diameter neurons and a reduction in the larger diameter neurons. Knockout of Ngn2 resulted in a delayed onset of the appearance of TrkA and TrkC expressing cells, but by later stages DRG appear normal. However, knockout of both Ngn1 and Ngn2 results in a complete absence of all
DRG. 118-120 In zebrafish, the single neurogenin gene, ngn1, has also been shown to be required for sensory neuron specification. In zebrafish embryos, in which ngn1 was knocked down by morpholinos, DRG do not form.121 In contrast, over expression of neurogenins in zebrafish and Xenopus embryos resulted in the formation ectopic sensory neurons.122, 123 Also, retroviral-mediated mis-expression of ngn in chick pre-migratory neural crest cells was shown to bias cells towards sensory neuron cell fate. 124
Although Neurogenins do play a major role in sensory neuron specification, several studies show that Neurogenin alone is not sufficient to specify neural crest cells as sensory neurons.125, 126 Brn3a, a POU domain transcription factor, has also been implicated in the development and survival of sensory neurons.127 Ngn2 expression
20
precedes Brn3a expression in culture and it is believed that Ngn2 may play a role in
regulating its expression.128 Inactivation of Brn3a results in a loss of both small and large
diameter neurons.129, 130
Runx transcription factors are also implicated in sensory neuron specification.
The mammalian homologue of Runx3 is specifically expressed in large diameter neurons
and loss of Runx3 function disrupts the development of proprioceptors. 131, 132 Runx1 is
expressed in small diameter sensory neurons. However, its function in DRG has not been
tested because knockout mice die early from blocked blood development. 133 There are,
however, binding sites for Brn3a found in regulatory sequences upstream of both Runx3
and Runx1, suggesting that Runx gene expression could be regulated by Brn3a further
supporting a role for Runx transcription factors in sensory neuron development. 131, 134, 135
Several signaling molecules are also believed to play a role in specification of
sensory neurons. In mice, tissue specific inactivation of β-catenin in neural crest cells
results in the specific loss of DRG. In contrast, constitutive activation of β-catenin
promotes the differentiation of sensory neurons and Ngn2 expression. 136, 137 Studies in zebrafish and chick, however, suggest that WNTs may inhibit neuronal differentiation.138,
139
Notch signaling is also believed to play a role in determining if neural crest cells will adopt a sensory neuron fate. Notch1 is expressed in proliferating cells of the DRG, while the Notch ligand, Delta, is expressed in differentiating neurons. Activation of
Notch signaling in vivo and vitro prevents sensory neuron differentiation and promotes
21 glial differentiation. Targeted inactivation of Numb, a negative regulator of Notch signaling, results in a specific loss of DRG neurons. 140-142
In zebrafish, sonic hedgehog (Shh) signaling has also been implicated in specification of DRG. In zebrafish carrying mutations in the Shh signaling pathway and those treated with the Shh inhibitor, cyclopamine, DRG do not form. Similarly, in Shh mutants (smo or gli2), ngn1 expressing sensory neurons are not present. Additionally, transplantation experiments demonstrate that Shh signaling must be intact in DRG precursors in order for them to differentiate into sensory neurons. 143
1.3.6: Terminal Differentiation: Conclusion
Because a great number of derivatives originate from the NC, the NC provides an excellent platform for studying specification and differentiation of various cell types from a homogeneous cell population. While many of the genes that are involved in the overall process of specification and differentiation of NC sub-lineages have been identified, several aspects of NC development remain unresolved.
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Chapter 2
Characterization of the zebrafish mutant ninjaos5 (nij)
2.1 Introduction
The neural crest (NC), often referred to as the fourth germ layer144, is a vertebrate
specific ectoderm-derived embryonic cell population that gives rise to a number of
derivatives, including craniofacial cartilage and bone, neurons and glia of the peripheral
nervous system, and pigment cells. It is induced during gastrulation at the neural plate
border. After being induced at the neural plate border, neural crest cells delaminate from
the dorsal neural tube, migrate throughout the developing embryo, and differentiate into
multiple cell types.
Much effort has been put into understanding the genetic network that is
responsible for diversification of the neural crest into the various derivatives that originate from this cell population. However, despite the great amount of work put into trying to understand this process, the genetic network responsible for the progression of neural crest diversification is still not completely understood. In an effort to understand this process, this study has utilized forward genetic mutagenesis screens to identify novel
genes that are involved in neural crest diversification by specifically isolating zebrafish
mutants that exhibit abnormal neural crest development. The zebrafish mutant, ninjaos5
(nij), is a mutant that has been isolated from a forward genetic screen. nij displays a very
specific phenotype which includes disruption of neural crest derived enteric neurons and
craniofacial cartilage development.
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2.2: Isolation of zebrafish mutant nijos5
To identify genes that play a role in neural crest diversification ENU induced forward genetic mutagenesis screens were employed in an effort to identify zebrafish mutants with defective neural crest development. nijos5 is a recessive larval lethal mutation that was identified based on a reduction in cranial neural crest expression of crestin, a pan neural crest marker (Fig. 2.1). A reduction in crestin expression is seen in the hindbrain as well as more anterior regions of the brain. nij embryos live to about 6dpf.
At 34hpf, the appearance of the head in nij embryos appears to be smaller in size than wt embryos (Fig. 2.2). Also, the presence of melanophores is reduced in number as compared to wt (Fig. 2.2). However, at 5dpf the number of pigment cells recovers and is relatively normal (Fig 2.2). Although pigment recovers, the differences in head size between wt and nij become more evident as nij embryos increases in age. At 5dpf, the head of nij embryos is significantly reduced in size as compared to wt (Fig. 2.2).
Figure 2.1 Crestin expression. Whole mount in situ hybridization of crestin in wt (A) and nij (B) shows a reduction in crestin expressing cranial neural crest cells.
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Figure 2.2 Live Phenotype. nij mutant embryos at 34hpf and 5dpf. nij (B) can be distinguished from its wt (A) siblings at 34hpf by its slightly smaller head size and appearance of fewer pigment cells. At 5dpf pigment is relatively normal but a significant reduction in the size of the head persists in nij (D).
2.3 Zebrafish mutant nijos5 is required for craniofacial cartilage development
To determine the function of nij several experiments were performed to determine if nij is required for the development of any of the various derivatives of the neural crest.
Alcian blue staining, which specifically stains cartilage, reveals that nij plays an integral role in craniofacial cartilage development. As opposed to wt embryos, all crest derived craniofacial cartilage elements of the head skeleton are completely absent in nij embryos
(Fig. 2.3). Although all of the crest-derived craniofacial cartilage elements are absent, the neurocranium, which is not crest derived, is present in nij embryos suggesting that nij is required specifically for the development of crest derived craniofacial cartilage elements
(Fig. 2.3).
25
Figure 2. 3 Crest derived craniofacial cartilages. Alcian Blue staining reveals that as compared to wt embryos (A,B), nij mutant embryos (C,D) lack crest derived craniofacial cartilage elements. The neurocranium, which is not NC derived, is present in ninja embryos. Abbreviations: M, mandibular; H, hyoid; B, branchial.
To determine if nij is required for specification of pharyngeal arch precursors of
the craniofacial cartilages, the expression of dlx2a was assessed. dlx2a is a pharyngeal
arch marker that plays a role in specifying a distinct population of neural crest cells to
become pharyngeal arch precursors and is necessary for their survival. Although the
pharyngeal arches appear to be slightly disorganized, dlx2a expression in the pharyngeal
arches of nij embryos is comparable to wt (Fig. 2.4). The expression of dlx2a in the
pharyngeal arches of nij embryos suggests that craniofacial cartilage precursors are being
26
specified. Based on these results it can be concluded that nij is not required for
specification of pharyngeal arch precursors.
sox9a is a transcription factor that is also expressed in the pharyngeal arches. It is
downstream of dlx2a, required for terminal differentiation of craniofacial cartilages, and
is required for the expression of collagen 2 alpha (col2a), which is a terminal
differentiation marker of mature chondrocytes. To determine if nij is required for
terminal differentiation of cartilage precursors into mature chondrocytes, the expression
of sox9a and col2a was assessed. At 48hpf sox9a expression appears to be normal (Fig.
2.4). However, at 72hpf, sox9a expression is disrupted in nij embryos. sox9a expression
in the mandibular and hyoid arches is slightly reduced. However, sox9a expression in the
branchial arches is nearly absent (Fig. 2.4). The expression of col2a1 is also perturbed in nij embryos. col2a1 expression is severely reduced in the mandibular and hyoid arches and is completely absent in the branchial arches (Fig. 2.4). These results suggest that nij
is required for terminal differentiation of the crest derived craniofacial cartilages.
27
Figure 2. 4 Craniofacial Cartilage Precursors. Whole mount in situ hybridization of dlx2 at 24hpf (A, B), sox9a at 48hpf (C, D), sox9a at 72hpf (E, F), and col2a at 72hpf (G, H). Pharyngeal arch precursors are specified. Branchial arch elements fail to maintain the expression of sox9a at 72hpf. col2a is severely reduced in the first two arches and is completely absent in the branchial arches. Abbreviations: M, mandibular; H, hyoid; B, branchial.
To determine if the perturbed development of the craniofacial cartilages in nij
embryos could be partially due to an absence of pharyngeal endoderm, mesoderm, or ectoderm, the expression of genes that are expressed in and are required for the development of these components of the pharyngeal arches was assessed. Based on the expression of tbx1 in pharyngeal endoderm and edn1 in pharyngeal mesoderm and ectoderm, all three of these components appear to be normal in nij embryos (Fig. 2.5).
Therefore, the function of nij is required specifically for the development of pharyngeal neural crest cells and does not affect adjacent tissues of the pharyngeal arches.
28
Figure 2. 5 Endodermal, mesodermal, and epidermal components of pharyngeal arches. Whole mount in situ hybridization of edn1 (A, B), marker of pharyngeal endoderm, mesoderm, and ectoderm, and tbx1 (C, D), marker of pharyngeal endoderm, show that all other pharyngeal arch components are unaffected in ninja mutant embryos. Abbreviations: M, mandibular; H, hyoid; B, branchial.
2.4 Zebrafish mutant nij is required for enteric neuron development
To determine if the function of nij is required for the development of crest derived neuronal derivatives, whole-mount immunostaining of the pan neuronal marker, anti-Hu, was performed and TH expression in sympathetic neurons was assessed. Hu antibody labeling and TH expression has revealed that while all other crest-derived neuronal cell types are normal (Fig. 2.7), enteric neurons are nearly absent in nij embryos
(Fig 2.6).
29
Figure 2. 6 Enteric neurons. Hu antibody labeling reveals that enteric neurons are severely reduced in nij mutant embryos (B) as compared to wt siblings (A).
30
Figure 2. 7 All other NC derived neuronal cell types are normal. Hu antibody labeling shows that dorsal root ganglia are present in nij. (A, B) foxd3 expression shows that cranial satellite glia are present. (C, D) TH expression shows that sympathetic neurons are present.
To investigate whether nij is required for specification of enteric neuron
precursors, the expression of sox10 and phox2b was assessed. Both sox10 and phox2b are expressed in enteric neuron precursors and loss of the expression of these genes results in a severe reduction or complete absence of enteric neurons precursors. In nij, there is a
severe reduction in the number of sox10 expressing cells aligning the length of the gut
(Fig. 2.8). A reduction in sox10 expression is first detected at 36hpf and does not recover
at later time points (Fig. 2.9). There is also a severe reduction in the number of phox2b
expressing cells that align the gut of nij embryos (Fig. 2.8). To determine if neural crest
cells are present along the length of the gut, the expression of zash1a, another marker of
enteric neuron precursors, was assessed. Normal expression of zash1a would suggest that
there are neural crest cells present along the gut, but these cells fail to express the correct
genes, sox10 and phox2b, that are required for enteric neuron development to proceed.
zash1a expression in nij, however, was not normal and was also markedly reduced 31
suggesting that there is a severe reduction in the number of enteric neurons being specified (Fig. 2.8). Taken together, these results suggest that nij is required for
specification of the correct number of enteric neurons needed to populate the entire
length of the gut.
Figure 2. 8 Enteric neuron precursors. The expression of phox2b (A,B), sox10 (C, D), and zash1a (E, F) show that enteric neuron precursors are severely reduced in ninja embryos.
32
Figure 2. 9 sox10 expression. In situ hybridization of sox10 at 3s (A), 6s (B), 10s (C), 15s (D), 21hpf (E), 25hpf (F), 36hpf (G, H), 48hpf (I, J), 54hpf (K, L). A reduction in sox10 expression is first observed in nij at 36hpf and persists through 54hpf.
33
2.5 Neural Plate Border (NPB) and Neural Crest Induction Are Unaffected in nijos5
To determine if the absence of crest derived enteric neurons and craniofacial
cartilages could be attributed to the NPB not being induced properly, the expression of
genes that are indicative of NPB induction and facilitate downstream events that lead to
the induction of the neural crest proper was assessed. pax3 and msxb are both expressed
in the NPB. No differences in the expression of pax3 and msxb were observed between
wt and nij embryos indicating that the NPB has been induced (Fig. 2.10).
Figure 2. 10 Neural Plate Border Induction. No differences in the expression of NPB markers, pax3 and msxb, were observed suggesting that that NPB induction occurs normally in nij.
To determine if nij is required for induction of the neural crest from the NPB and
initial cell fate specification events that occur during early neural crest development, the
expression of early neural crest transcription factors, foxd3, snail1b, sox10, and sox9b, was assessed. These genes are all expressed by the majority of neural crest cells and the
34
expression of all of these genes is indicative of induction of the neural crest proper. No
differences in the expression of these genes were observed between wt and nij embryos
indicating that early neural crest development is normal in nij embryos (Fig. 2.11). Taken
together these results suggest that the nij is not required for NPB induction, NC
induction, or the initial stages of cell fate specification.
Figure 2. 11 Neural Crest Induction. No differences in the expression the of early neural crest markers, foxd3 (A), sox10 (B), sox9b (C), and snail1b (D) were observed suggesting that the neural crest has been induced properly in nij embryos.
2.6 Enteric neuron and craniofacial abnormalities may be due to an increase in cell death.
The reduced expression of sox10, phox2b, and zash1a suggests that there is a great reduction in the number of enteric neuron precursors aligning the length of the gut.
To determine if the function of nij is required for the survival of enteric neuron precursors, TUNEL assays were performed to assess the number of apoptotic cells aligning the gut. At 24hpf and 36hpf there is an increase in the number of apoptotic cells that are located in the vicinity of where sox10 and phox2b expressing enteric neuron precursors are located in nij mutants (Fig. 2.12). These results suggest that the reduction
35
in the number of enteric neuron precursors being specified could be due to increased cell
death.
Likewise, despite maintenance of sox9a expression in the mandibular and hyoid
pharyngeal arches, these craniofacial cartilage elements fail to terminally differentiate.
Failure of these pharyngeal arch elements to terminally differentiate could also be due to
an increase in cell death. To determine if cell death contributed to the loss of these pharyngeal arch elements, TUNEL assays were performed. At 60hpf, there is an increase in the number of apoptotic cells in the mandibular and hyoid pharyngeal arches. These results suggest that failure of the mandibular and hyoid arches to terminally differentiate into mature chondrocytes could be due to increased cell death in nij (Figure 2.12). Taken
together, these results suggest that nij is required for the survival of enteric neuron
precursors and mandibular and hyoid pharyngeal arch precursors.
36
Figure 2. 12 Increased cell death in nij. TUNEL assays were done at 24hpf (A, B), 36 hpf (C, D), and 60hpf (E, F). Results show increased cell death in enteric neurons precursors and pharyngeal arches.
2.7 Mapping of nijos5
In an effort to map nij to a chromosome, a WIK mapping line was generated by
crossing nij AB* carriers to a WIK zebrafish line. After generating a mapping line,
simple sequence length polymorphisms (SSLP) were used to try to identify markers that
were linked to the nijos5 mutation. This involved isolation of genomic DNA from both wt
and nij embryos and screening of SSLPs from all chromosomes in an effort find markers
that were linked to the nijos5 mutation. For many of the chromosomes all known SSLP
37
were screened. However, no markers were identified that segregated with the nijos5 mutation. As a result, the nij mutation was not localized to a linkage group.
2.8 Discussion
Overall neural crest development requires the activity of various genes and signaling molecules that work together at different time points in development to facilitate overt differentiation of neural crest sub-lineages. In nij mutant embryos, the initial stages of crest development are unaffected. These results show that NPB induction,
NC induction, and initial cell fate specification events occur normally. However, although the initial requirements for crest development are normal, later crest development is disrupted in nij mutants.
The results presented suggest that nij is required for terminal differentiation of the craniofacial cartilages that make up the head skeleton. The pharyngeal arches of nij embryos initially express the correct genes, dlx2 and sox9a, that are required for chondrogenesis. dlx2 expression at 28hpf and sox9a expression at 48hpf show that pharyngeal arch precursors are specified. However, at later time points, it appears that nij is playing two different roles that are required for terminal differentiation of the craniofacial cartilages. In the absence of nij function, the branchial arches fail to maintain the expression of the genes that are necessary for overt differentiation of the branchial cartilages. As described above, sox9a is initially expressed in the branchial arches at
48hpf. However, at 72hpf, sox9a branchial arch expression is lost. As a result, branchial
38
arch craniofacial cartilage elements fail to differentiate into mature chondrocytes. These
results suggest that nij is required for the maintenance of branchial arch sox9a expression.
In contrast to the branchial arches, the mandibular and hyoid arch elements do
maintain the expression of sox9a. However, despite the maintenance of sox9a expression, the mandibular and hyoid pharyngeal arch elements fail to undergo terminal differentiation into mature chondrocytes. Failure of these pharyngeal arch precursors to terminally differentiate is demonstrated by a severe reduction in mandibular and hyoid arch col2a1 expression in nij. This is further supported by alcian blue staining which reveals a complete absence of all crest derived craniofacial cartilage elements.
To determine why the mandibular and hyoid sox9a expressing cells fail to terminally differentiate and express col2a1, TUNEL assays were performed to determine if these cells are dying. At 60hpf, the results of these assays show that it is evident that there is an increased number of TUNEL positive cells in the mandibular and hyoid arch elements. These results suggest that the function of nij is required for the survival of
mandibular and hyoid pharyngeal arch precursors.
The expression of edn1 and tbx1 was assessed to determine if abnormalities in the
pharyngeal endoderm, mesoderm, or ectoderm could contribute to the failure of the
craniofacial cartilages to develop. The expression of both edn1 and tbx1 expression was
normal suggesting that the function of nij is not necessary for development of adjacent
pharyngeal arch tissues.
All of these results taken together, highlight two distinct roles that nij is playing in
craniofacial cartilage development. nij is required for cell survival of the mandibular and
39
hyoid arches, but is also be required for the maintenance of sox9a expression in the branchial arches. Experiments can be done to confirm these results and the role nij is playing in craniofacial cartilage development. For instance, p53 morpholino injections
could be performed to determine if inhibiting cell death is able to rescue the development
of the mandibular and hyoid arch elements. Also, TUNEL and sox9a co-labeling could be
done to determine if the TUNEL positive cells are indeed neural crest cells. In addition,
mRNA injections of sox9a could be performed to determine if nij and sox9a are acting in
a linear pathway to bring about overt differentiation of the pharyngeal cartilages. Rescue
of the branchial cartilages by sox9a mRNA injections would suggest that nij is upstream
of sox9a and is required for sox9a expression.
nij mutants also lack enteric neurons that make up the enteric nervous system. The
results presented suggest that nij is required for specification of the proper number of
enteric neuron precursors that are necessary to colonize the entire length of the gut.
Decreased sox10 expression, as well as phox2b expression, reveals that there is a severe
reduction in the number of enteric neurons precursors populating the gut of nij embryos.
This result was confirmed by a similar reduction in the expression of zash1a. Because zash1a expression is not dependant on sox10 expression, the reduction in zash1a expression suggests that the reduction in sox10 and phox2b expressing cells seen in nij embryos is not due to a failure of these cells to express the correct genes. Rather it is possible that these cell types are not present. To determine if the function of nij is
required for the survival of enteric neuron precursors, TUNEL assays were performed.
40
The results from these assays suggest that the absence of sox10 and phox2b expressing enteric neuron precursors could be a result of an increase in apoptotic cells in the gut.
Although there is an increase in TUNEL positive cells in the gut, it needs to be confirmed that these cells are neural crest cells. Confirmation of these results is necessary because there is a possibility that a decrease in proliferation could account for the reduction of enteric neuron precursors seen in nij embryos. Once neural crest cells enter the anterior portion of the gut during ENS development, they undergo an extensive amount of proliferation to attain the correct number of cells needed to colonize the entire length of the gut. To rule out the possibility that the reduction in enteric neuron precursors seen in nij embryos is a result of decreased proliferation, proliferation assays, such as histone H3 antibody labeling, can be performed. Also, because increased cell death is seen as early as 24hpf in nij embryos, double labeling by performing crestin in situ hybridization and TUNEL labeling could be done to determine if the dying cells are neural crest cells.
One of the primary tasks left to be done is to map nij to a chromosome. Mapping nij to a chromosome could be very beneficial. As mentioned above, nij can be confidently distinguished from its wt siblings from 48hpf onwards because nij embryos exhibit a significant reduction in head size. However, at earlier stages nij mutants can not be confidently distinguished from its wt siblings by the head phenotype. Number counts of embryos with abnormalities in gene expression determine if a result is true. If a result is true, it is expected that 1/4 of the total number of embryos for each experiment would exhibit a particular phenotype. After mapping nij to a chromosome, all of the above data
41
could be confirmed by genotyping which would provide further evidence that the
phenotypes seen in this mutant are true.
The ultimate goal, however, is to identify the disrupted gene that is causing the phenotypes seen in this mutant. There are a few possibilities as to the type of role that nij
may be playing. For example, because it was shown that nij embryos exhibit an increase
in apoptotic cells in the pharyngeal arches and ENS precursors, nij could be a gene that is
involved in inhibiting programmed cell death or regulating the expression of a gene that
is involved in inhibiting apoptosis.
nij could also be a gene that is genetically interacting with components of
Endothelin signaling. Literature has shown that Endothelin signaling is involved in both
craniofacial cartilage and enteric neuron development. The complementary expression of
Endothelin-1(Edn1) in the mesodermal core of the pharyngeal arches and the endothelin
receptor (EdnrA) in neural crest cells facilitates endothelin signaling.145-147 Loss of Edn1
or EdnrA in mice results in a severe loss of branchial arch derived craniofacial cartilage
elements.145, 148 Inactivation of EdnrA with a specific antagonist results in a phenotype
similar to mice lacking Edn1 signaling.149 In zebrafish, loss of edn1 results in a range of
defects involving the anterior pharyngeal arches that resulted in the loss of cartilage
elements and changes in shape and size of the craniofacial cartilages.59
Likewise, Edn-3 signaling via its receptor EdnrB, which is also expressed in
neural crest cells, is also believed to play a role in enteric neuron development. It is
believed to promote proliferation and reduce the differential role of GDNF maintaining
ENS precursors in a mitotic and migratory pool. Reduction of Edn-3 function leads to
42 unrestrained neuronal differentiation which is driven by GDNF. This premature differentiation is believed to withdraw cells from the migratory and proliferative pool of
ENS precursors leaving too few cells to colonize the entire gut. 100, 150
nij could also be genetically interacting with components of the Retinoic acid
(RA) signaling pathway. Retinoic acid is a derivative of vitamin A. Retinaldehyde dehydrogenase-2 (Raldh2) is the major enzyme that is responsible for the synthesis of retinoic acids. Retinoic acids act as a ligand for RA receptors and retinoid X recpetors.
Once activated, these receptors bind as heterodimers to DNA motifs and regulate the transcriptional activity of target genes.151 Previous studies have shown that mice that lacking Raldh2 are missing all pharyngeal arch elements, with the exception of the first arch.151 Rara/g-null mice also display malformations in a large variety of cartilage elements derived from cranial neural crest.152 Mice lacking Raldh2 are also devoid of Ret expressing enteric neuron precursors from the stomach and gut wall.151
2.9 Materials and Methods
2.9.1 Zebrafish Husbandry
Zebrafish mutant lines were reared under standard conditions at 28º C at the Ohio
State University zebrafish facility. The nij mutation was maintained on an AB* background. Zebrafish embryos were staged according to morphological criteria in
Kimmel et al. For in situ hybridization and antibody labeling, embryos were incubated in
PTU (1-phenyl-2-thiourea) to prevent pigment formation.
43
2.9.2 Whole mount in situ Hybridization and immunohistochemistry
In situ hybridizations were performed as previously described in Thisse et. al., 1993.
Whole mount immunohistochemistry was performed as previously described in An et. al,
2002.
2.9.3 Alcian Blue Staining
To detect cartilage, alcian blue staining was performed as previously described in
Kimmel et al., 1998.
2.9.4 TUNEL Assay
TUNEL was performed on embryos fixed with 4% PFA, dehydrated in methanol, and rehydrated in TBS. TdT/Digoxigenin-dUTP (Roche) labeling was done for 1hr. Embryos were then labeled with anti-Digoxigenin (Roche) secondary antibody. NBT/BCIP was used for color developing.
2.9.5 Genetic Mapping
The nijos5 was maintained in an AB* background. Heterozygous carriers were then crossed to a WIK mapping line (Nechiporuk et al.). SSLPs were screened to find
polymorphic markers that were linked to the nij mutation.
44
CHAPTER 3
Characterization of the zebrafish mutant hatchbackOS20 (hbk)
3.1 Introduction
The neural crest is specialized embryonic cell population that is unique to
vertebrate embryos. The neural crest is induced at the neural plate border (NPB) during
gastrulation. After induction at the NPB this seemingly homogenous embryonic cell
population delaminates from the dorsal neural tube and migrates along specific paths
throughout the developing embryo. After migration, these cells differentiate into a
number of different cell types, including neurons and glia of the peripheral nervous
system, craniofacial cartilage and bone, and pigments cells.
Although an extensive amount of work has been done, the genetic mechanisms
that are involved in neural crest diversification are not fully understood. Attempts to decipher the genetic network involved in neural crest diversification, has prompted the use of chemical mutagenesis screens. In this study, mutagenesis screens have been employed to isolate zebrafish mutants in which neural crest development has been specifically disrupted. hbkos20 is a zebrafish mutant that has been isolated from one of
these mutagenesis screens. The hbk mutation specifically disrupts trunk NC development
resulting in the loss of chromatophores and several neuronal cell types. hbk also disrupts the development of derivatives of the lateral plate mesoderm which gives rise to hematopoietic cell types and the vasculature. However, the lateral plate mesoderm phenotype will not be discussed in detail.
45
3.2 Isolation of zebrafish mutant hbkos20
To further understand the genetic mechanisms involved in neural crest diversification, ENU induced forward genetic mutagenesis screens were employed to identify zebrafish mutants with mutations in genes that specifically disrupt neural crest diversification. hbkos20 (hbk) was isolated from one of these screens. hbk is a recessive embryonic lethal mutation that was identified based on a severe reduction in crestin expression in the trunk. hbk lives about 3dpf (Figure 3.1). hbk can be distinguished from its wt siblings by the absence of all chromatophore cell types. (Fig.3.2) hbk also exhibits severe heart edema and lacks circulating blood cells (Fig.3.2).
Figure 3. 1 crestin expression in hbk. The expression of crestin at 12s (A, B) and 18s (C, B) highlights a reduction of crestin expression in the trunk of hbk embryos. (Marsha Lucas)
46
Figure 3. 2 hbk live phenotype. Live image of wt (A) and hbk (B) embryos. As depicted hbk lacks all pigment cell types, exhibits severe heart edema, and lacks circulating blood cell. (Marsha Lucas)
Section 3.3 hbk disrupts chromatophore, sympathetic neuron, enteric neuron, and dorsal
root ganglion development.
The hbk mutation disrupts the development of a number of neural crest
derivatives. In zebrafish, there are three chromatophore cell types. They are black
melanophores, yellow xanthophores, and iridescent iridiphores. In hbk, all three pigment cell types are absent (Fig. 3.2). To determine if chromatophore precursors are being specified in hbk embryos the expression of mitf, dct, and xdh was assessed. The
expression of mitf, a marker of specified melanophore precursors, and dct, an enzyme
involved in the melanin synthesis pathway that is also indicative of melanophore terminal
differentiation, was assessed and found to be completely absent in hbk (Fig.3.3).
Likewise, the expression of xdh, a marker of specified xanthophore precursors, was also
completely absent in hbk. Taken together these result suggests that hbk is required for
specification of melanophore and xanthophore precursors (Fig 3.3).
47
Figure 3. 3 Chromatophore Precursors in hbk. In situ hybridization of mitf (E, F), dct (G, H), and xdh (I, J) show that chromatophore precursors are absent in hbk. (Marsha Lucas)
In addition to chromatophores, several neuronal cell types are disrupted in hbk embryos. As evidenced by the expression of tyrosine hydroxylase (TH), a late differentiation marker of sympathetic neurons, hbk lacks sympathetic neurons (Fig. 3.4).
Whole-mount antibody labeling of the pan neuronal marker, Hu, shows that enteric neurons and dorsal root ganglia (DRG) are absent in hbk (Fig. 3.4). These results suggest that hbk is required for the development of a subset of neural crest derived neuronal derivatives.
48
Figure 3. 4 Neuronal Derivatives in hbk. TH expression shows an absence of sympathetic neurons in hbk (A, B). Hu antibody staining reveals an absence of enteric neurons and DRG in hbk (C, D). Hu antibody staining reveals that cranial ganglia are present in hbk (E.F). foxd3 expression reveals that cranial satellite glia are unaffected by the hbk mutation (G, H). (Marsha Lucas)
Section 3.3 Cranial neural crest derivatives are unaffected in hbk embryos.
It seems that hbk is differentially required for the development of cranial and
trunk neural crest derivatives. hbk does not live long enough to allow for the development of the crest-derived craniofacial cartilages that make up the head skeleton. However, the
initial migration and specification events that are required for craniofacial cartilage
development can be assessed. The expression of dlx2, a specification marker of the
pharyngeal arches, suggests that craniofacial cartilage precursors are specified and
migration into the pharyngeal pouches is not perturbed (Fig. 3.5).
49
Figure 3. 5 Craniofacial cartilage precursors in hbk. dlx2 expression at 28h (A-D), 36h (E,F), and 48h (G-I) reveals that pharyngeal arches are present in hbk. (Marsha Lucas)
While it seems that all trunk neuronal derivatives are disrupted, cranial neuronal
derivatives are not affected in hbk mutant embryos. Whole-mount Hu antibody labeling and foxd3 expression shows that cranial ganglion and cranial satellite glia are present in
hbk embyos (Fig. 3.4).
Section 3.4 Early neural crest development is disrupted in hbk.
To determine if hbk is required for neural crest induction and/or the initial stages
of cell fate specification, the expression of the early neural crest transcription factors was
assessed in wt and hbk embryos. Based on the expression of foxd3, one of the earliest
markers to be expressed in neural crest cells, neural crest development is perturbed very
early in development. foxd3 expression at 1s is severely reduced in the trunk (Fig 3.6). At
3s, the expression of snail1b is also reduced in the trunk (Fig. 3.6). However, tfap2a and
sox10 expression are unaffected at 3s (Fig. 3.6). Normal expression of tfap2a and sox10
suggests that neural crest cells are induced in the trunk, but these cells merely fail to 50
express foxd3 and snail1b. At 6s, foxd3 and snail1b expression remains reduced, and a reduction of tfap2a and sox10 expression becomes evident (Fig. 3.6). These results
suggest that hbk is not required for induction of the neural crest, but is required for the
expression or maintenance of the expression of the NC transcription factors that are
required for the initial stages of specification of neural crest sub-lineages.
Figure 3. 6 Neural crest induction and cell fate specification in hbk. foxd3 expression at 1s (A, B) and 6s (I, J) is severely reduced in the trunk. Snail1b expression at 3s (E, F) and 6s (M, N) is also severely reduced in the trunk. The expression of tfap2a and sox10 at 3s is initially unaffected in hbk (C,D, G, H,). However, the expression of tfap2a and sox10 is disrupted at 12s (K, L, O, P). (Marsha Lucas)
To determine if the lack of expression or failure to maintain the expression of the
early neural crest transcription factors could be attributed to abnormal induction of the
NPB, the expression of NPB markers, msxb and pax3 was assessed. The expression of both of these genes was normal (Fig. 3.7). These results suggest that hbk is acting downstream of NPB induction and is required for specification of trunk neural crest sub- lineages.
51
Figure 3. 7 Neural plate border induction in hbk. The expression of msxb and pax3 suggest that NPB induction occurs normally in hbk. (Marsha Lucas)
Section 3.5 Mapping hbk
In an order to map hbk to a chromosome, hbk AB* carriers were crossed to a WIK mapping line. After generating a hbk WIK mapping line, SSLPs, simple sequence length polymorphisms, were used to map hbk. Initial mapping results localized hbk to chromosome 7 using markers z14401, z42919b, and the EST, fb61f04. Markers between z42919b and fb61f04 were designed using sequenced BAC ends. Beg8-1, the closest marker to hbk, was designed from the BAC end CHORI1073_ 444G8PIBR. This marker is located within exon 12 of the ctr9 gene.
In a different laboratory study, the Takada lab performed morpholino knockdown
of ctr9 in zebrafish. The results from this study show that loss of ctr9 results in a
phenotype that is very similar to hbk. Knockdown of ctr9 resulted in the loss of pigment, cardiac edema, and a reduction in crestin expression in the trunk. 153 Because of these
results, ctr9 was investigated as a candidate gene for hbk. The cDNA of ctr9 was sequenced in hbk embryos, but no mutation was found. An RNA riboprobe of the 5’ end of the ctr9 gene was also made and the expression of ctr9 was found to be severely reduced in hbk (Fig. 3.8). 52
Figure 3. 8 ctr9 expression in hbk. In situ hybridization of ctr9 shows a severe reduction in the expression of ctr9 at 3s and 6s. (Marsha Lucas)
ctr9 was initially ruled out by a former member of the Henion lab as a candidate gene for several reasons. First, no mutation was found in the coding region of the ctr9 gene. Secondly, there were a considerable number of recombinants associated with the marker, Beg8-1, which is located within the ctr9 gene. Genotyping of haploid panels with wt and hbk embryos that were polymorphic for Beg8-1 revealed 6 recombinants out of
288 haploids. Thirdly, morpholino knockdown of ctr9 done in our lab yielded some phenotype differences that suggested that the hbk mutation was not ctr9.
ctr9 was not completely ruled out. Although there was no mutation in the coding region, there could be a mutation in an upstream regulatory sequence. However, because of the high number of recombinants found within the ctr9 gene, it was concluded that it
53
was very unlikely that hbk was ctr9. It was suggested that the phenotypes seen in hbk
could be in part due to the absence of ctr9 expression in hbk. It was also concluded that
hbk and ctr9 genetically interact and that hbk may be in a nearby gene that is regulating
ctr9 expression.
Based on these conclusions, further investigation was done to determine if ctr9
was indeed hbk. A BAC containing the ctr9 and eif4g2a was injected into hbk embryos to see if it could rescue the hbk phenotype (Fig. 3.9). Injection of this BAC successfully rescued pigment in hbk embryos. Further genotyping of haploid panels with Beg8-1 was also performed. From the genotyping results, the number of recombinants increased from
6/288 to 11/666. From these results, we concluded that ctr9 genetically interacts with
hbk, but the high number of recombinants confirmed that hbk may not be ctr9.
Figure 3. 9 Rescue of pigment with BAC containing ctr9. (A.) Un-injected wt embyos. (B.) Un-injected hbk embryos. (C-F) ctr9 BAC injected hbk embryos.
As a result, further fine mapping was done. New markers were created from
sequenced BAC ends. Only two of the markers that were screened were found to be
polymorphic. They were named TBEP-2 and TBEP-12. Screening haploid panels with
54
TBEP-12 yielded 53 recombinants out 383 haploids. The recombinant number associated
with TBEP-12 suggested that this marker was very far from the hbk mutation. Screening
haploid panels with TBEP-2 yielded 7 recombinants out of 383 haploids. Blasting the
sequence of TBEP-2 to the Ensemble generated map of chromosome 7 revealed that this
marker was very close to the ctr9 gene. However, the number of recombinants suggested
that more fine mapping was necessary. After screening a 35 of markers generated from
sequence BAC ends, no other markers were found to be polymorphic. In addition, every
known marker on the MGH map of chromosome 7 was screened. 6 polymorphic markers
were found, but recombinant numbers revealed that none of these markers were close to
the hbk mutation.
Because all efforts to fine map hbk lead to the region where the ctr9 gene was located, further investigation of ctr9 as a candidate gene for hbk was done. To investigate whether there were any alternative transcripts of ctr9 present in hbk. mRNA from both hbk and wt embryos was isolated. Because the ctr9 gene was so large, primers were designed to amplify the ctr9 cDNA in five overlapping segments. Then the RT-PCR product from each segment was analyzed by gel electrophoresis. No alternative splice forms were found. In addition, each segment was also sent for sequencing. Re- sequencing results were the same as the initial sequencing results.
Although much effort was put into trying identify the mutated gene responsible
for the phenotypes exhibited in this mutant, the identity of hbk is still ambiguous. There
is, however, a possibility to ctr9 could be hbk. As stated before, there could be a mutation
in an upstream regulatory sequence. In addition, the BAC rescue results, suggests that
55
ctr9 is either downstream of hbk or that ctr9 could be hbk. The presence of eif4g2a on the
same BAC as ctr9 makes this result ambiguous.
3.6 Discussion
The neural crest can be divided into two regions: the cranial crest and trunk crest.
Both regions produce similar cell types, with the exception of craniofacial cartilage and
bones which are derived strictly from cranial neural crest cells. hbk is differentially required for the developmental regulation of the two neural crest populations.
The results presented suggest that hbk is specifically required for the development of trunk neural crest derivatives. hbk is lacking all pigment cells types. The expression of
mitf and dct shows that melanophore precursors fail to be specified. xdh expression
highlights a similar absence of xanthophore precursors. In addition to the absence of pigment cells, enteric neurons, sympathetic neurons, and dorsal root ganglia all fail to
development in hbk embryos.
However, while all trunk neural crest derivatives fail to develop, cranial neural
crest derivatives appear normal. hbk does not live long enough to assess whether the
craniofacial cartilages terminally differentiate, but dlx2 expression suggests that craniofacial cartilage precursors are specified and migration into pharyngeal pouches is not perturbed. Hu antibody labeling also shows that cranial ganglia are present. foxd3
expression shows that cranial satellite glia are also normal.
The expression of NPB genes, pax8 and msxb, suggests that NPB induction
occurs normally. Normal expression of the early crest transcription factors, tfap2a and
56
sox10, suggests that the neural crest is also induced. However, abnormal foxd3 and
snail1b expression, in addition to the failure of NC cells to maintain the expression of
tfap2a and sox10 expression, suggests that the initial stages of cell fate specification are
perturbed. Therefore hbk is acting downstream of NPB induction and is necessary
specification of trunk neural crest sub-lineages.
Much effort has been put into finding the identity of hbk. Based on a recent report from the Takada lab, morpholino knockdown of ctr9 showed a phenotype that was very similar to hbk. As a result, ctr9 was investigated as a candidate gene for hbk. The expression of ctr9 was analyzed and it was shown that the expression of ctr9 was down regulated in hbk. In addition injection of a BAC containing ctr9 and eif4g2a, rescued pigment in hbk. These were positive results that support the idea that hbk may be ctr9.
However, the cDNA of ctr9 from hbk embryos was sequenced but no mutation was found. In addition, attempts to identify alternative splice forms yielded no alternatives transcripts in the hbk cDNA. Screening haploid panels with Beg8-1, a marker that is located within exon 12 of the ctr9 gene, and TBEP-2, a marker that is located very close to the ctr9 gene, yielded very high recombinant numbers that do not support the idea that hbk is ctr9.
However, there is a possibility that the recombinants found when screening
haploid panels could be false positive results. In addition, the absence of a mutation in the
coding region does not completely rule out ctr9 either, because a mutation could exist in
an upstream regulatory region. Other experiments could be done to support the idea that
hbk could be ctr9. Morpholino knockdown of ctr9 could be done and the expression of
57
early crest transcription factors could be assessed. The expression of genes, such as mitf
and xdh, that are expressed in the chromatophore precursors could also be assessed. In
addition, the assessment of both cranial and trunk neuronal neural crest derivatives could
be assessed by Hu antibody labeling and foxd3 expression (cranial glia). If knockdown of
ctr9 expression phenocopies hbk, these results would further support the idea that hbk is
ctr9.
3.7 Materials and Methods
3.7.1 Zebrafish Husbandry
Zebrafish mutant lines were reared under standard conditions at 28º C at the Ohio State
University zebrafish facility. The hbk mutation was maintained on an AB* background.
Zebrafish embryos were staged according to morphological criteria in Kimmel et al. For in situ hybridization and antibody labeling, embryos were incubated in PTU (1-phenyl-2- thiourea) to prevent pigment formation.
3.7.2 Whole mount in situ Hybridization and immunohistochemistry
In situ hybridizations were performed as previously described in Thisse et. al., 1993.
Whole mount immunohistochemistry was performed as previously described in An et. al,
2002.
3.7.3 Genetic Mapping
58
The hbkos20 mutation was maintained in an AB* background. Hetorzygous carriers were
crossed to a WIK mapping line (Nechiporuk et al.). SSLPs were screened to find
polymorphic markers that were linked to the hbk mutation. Beg8-1 primer sequences: 5’-
GCGATGGCACGTGATAAGGGAAAT-3’and 5’-
GGTGATGTCATGAGTCCACAATTAGGC-3’. TBEP-2 primer sequences: 5’-
GGCAACAGCGGAGTTAAAAG-3’ and 5’-CGATGCACCAATTACGTCAC-3’.
TBEP-12 primer sequences: 5’-CAAACTGGAATCTCTTCAGACG-3’ and 5’-
CCAAATTTAGGCTTGTTAGCATT-3’. BAC containing ctr9 (CU467652 Zebrafish
DNA sequence from clone DKEY-256O15) was obtained from ImaGenes GmbH.
59
Chapter 4
Discussion
In this work two zebrafish mutants have been analyzed. In nij, NPB induction and
neural crest induction are normal. However despite normal early crest development, nij is
lacking enteric neurons and all crest derived craniofacial cartilage elements. The results
presented have shown that nij is playing multiple roles in the development of these two
sub-lineages. nij is necessary for the survival of enteric neuron precursors, the survival of
mandibular and hyoid arch precursors, and the maintenance of sox9a expression in the branchial arches.
The zebrafish mutant hbk exhibits a very different phenotype. In hbk, NPB and
neural crest induction are normal. In contrast, specification of neural crest sub-lineages is disrupted very early in hbk embryos. hbk, however, does not disrupt cell fate specification of all neural crest sub-lineages. hbk specifically disrupts the development of trunk neural crest derivatives.
Knowledge gained from studying these two mutants will provide valuable insight
that will contribute to the pool of information already known about neural crest
diversification. As a whole, these two mutants highlight the dynamics of neural crest development. We can see from these two mutants that several events must occur in order for overt differentiation of neural crest sub-lineages to proceed. These events happen in a sequential manner. If one step is disrupted, it can affect the downstream cascade of events that are supposed to occur. For instance, nij seems to be acting later in development at the
60
level of terminal differentiation. Disruption in a later event results in a nearly complete
absence of neural crest derivatives, enteric neurons and craniofacial cartilages. hbk is
acting earlier in neural crest development at the level of cell fate specification. Although
hbk disrupts neural crest development at a different time point the result is the same,
which is a loss of neural crest derivatives (specifically trunk NC derivatives).
These two mutants also contrast the differences in the regulation of cranial and
trunk neural crest development. Here we have two mutants that are differentially required
for the development of the two neural crest populations. hbk is required specifically for
trunk neural crest development; whereas nij is required for the development of specific
cranial and trunk neural crest derivatives.
nij also highlights a link between enteric neurons and craniofacial cartilage development. This has already been observed in mice. Now it is being observed in another species.
Although the identity of both of these genes is not known, more effort will be put
into solidifying the identity of the mutagenized genes that are causing the phenotypes
seen in these mutants. Overall, the use of mutagenesis screens will have proved to be a
valuable tool in identifying novel genes that play specific roles in neural crest
diversification.
61
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