Molecular Events Surrounding Embryonic Patterning During LateGastrulation in Danio rerio Open Access
Esterberg, Robert Michael (2008)
Abstract
Abstract Molecular Events Surrounding Embryonic Patterning During Late Gastrulation in Danio rerio By Robert Michael Esterberg Cell signaling events are essential throughout development, playing inductive roles in axis establishment, embryonic patterning, and tissue specification. How a relatively small number of distinct signaling pathways are integrated to reliably pattern an embryo is still largely unknown. As perturbation of even a single signaling pathway often disrupts many aspect of embryogenesis, it can be difficult to examine signaling requirements in a particular spatial-temporal context. Further complicating this matter is mounting evidence that suggests that signaling activity gradients vary dramatically as development proceeds. In this work, we have begun to address the spatial-temporal requirements for signaling activity during events that occur during late gastrulation and early somitogenesis in zebrafish. We focus particular attention on two tissues, chordamesoderm and the pre-placodal region, both of which require local modulation of BMP activity, particularly Bmp4, for proper development. In these tissues, we demonstrate the mechanisms through which appropriate levels of BMP activity are maintained. Chordamesoderm achieves this regulation through the secretion of two genes structurally related to Follistatin, a classical BMP antagonist. Appropriate levels of BMP activity are attained in the pre-placodal region through the regulation of a multifunctional BMP modulator, Crossveinless 2, which is necessary for lowering BMP activity within the pre-placodal region while likely promoting it in adjacent domains. Our results demonstrate the different approaches that tissues utilize to regulate signaling activity levels during development.
Table of Contents
Table of Contents CHAPTER 1: GENERAL INTRODUCTION 1 A. Cell fate specification during gastrulation 3 B. Signaling affecting embryonic patterning during gastrulation 5 1. BMP signaling in axis patterning and cell fate specification 6 2. FGF signaling and dorsoanterior patterning 10 a. FGF inhibition of BMP signaling 11 3. Nodal signaling and mesoderm induction 12 a. Nodal inhibition of BMP signaling 14 4. WNT signaling and AP patterning 16 a. WNT and BMP signaling integration 18 C. Additive effects of signaling integration on cell fate 19 1. Neural induction 19 2. Integrating DV and AP patterning through SMAD1 20 D. Signaling integration in tissue induction and patterning 22 1. Signaling events surrounding the formation of the inner ear 23 a. Positioning the PPR 24 b. Signaling integration in PPR induction 26 c. Specification of otic fate 27 d. Maintenance of otic fate 30 e. Integrating multiple signals over distinct stages of otic development 31 2. Signaling events surrounding the formation of the notochord 32 1. Establishment of the dorsal organizer 33 2. Induction of chordamesoderm 33 3. Notochord elongation and outgrowth 34 4. Chordamesodermal transition into notochord 34 E. Concluding remarks - combining signals to build an embryo 35 CHAPTER 2: TAILBUD-DERIVED BMP4 DRIVES PROLIFERATION AND INHIBITS MATURATION OF ZEBRAFISH
CHORDAMESODERM 38 ABSTRACT 39 INTRODUCTION 40 MATERIALS AND METHODS 42 RESULTS 46 Two BMP antagonists redundantly antagonize BMP activity after DV axis specification 46 Bmp4 activity during late gastrulation is required for chordamesoderm patterning 49 A critical window of Bmp4 activity establishes the proliferative state and size of the CNH and notochord 50 Ventral margin as the source of axially required BMP ligands 53 BMP activity levels influence the temporal state of the notochord 54 The proliferative state of chordamesoderm establishes the timing of notochord maturation 55 A direct role for BMP signaling in notochord development 57 The timing of notochord maturation influences myotome patterning 58 DISCUSSION 59 Axial requirements for BMP activity change as gastrulation proceeds 60 Selective requirement of BMP activity in the chordamesoderm 61 A balance between proliferation of the CNH and differentiation of chordamesoderm 62 ACKNOWLEDGEMENTS 65 CHAPTER 3: DLX3B/4B ARE REQUIRED FOR THE FORMATION OF THE PREPLACODAL REGION AND OTIC PLACODE
THROUGH LOCAL MODULATION OF BMP ACTIVITY 94 ABSTRACT 95 INTRODUCTION 96 MATERIALS AND METHODS 99 RESULTS 102 dlx3b/4b transiently regulate BMP activity 102 dlx3b/4b mediate BMP signaling through cv2 104 dlx3b/4b-mediated expression of cv2 is necessary for PPR marker expression 106 Cv2 antagonism of BMP activity promotes FGF activity 108 Inhibition of BMP activity can rescue the otic phenotype of Dlx3b/4b morphants 110 DISCUSSION 112 Modulation of BMP activity by dlx3b/4b at the neural plate border 112 Integration of BMP and FGF signaling in PPR and placode formation 114 Dlx and Cv2: evolutionary implications for PPR establishment 116 ACKNOWLEDGEMENTS 118 CHAPTER 4: THE ROLE OF FGF RECEPTORS IN OTIC DEVELOPMENT 145 INTRODUCTION 146 MATERIALS AND METHODS 148 RESULTS 150 fgfr expression in otic cells 150 Fgfr1-3 are required for otic development 151 Ligand-receptor interactions in otic development 152 Fgfr2 mediates Fgf3 signaling in pituitary and epibranchial development 153 DISCUSSION 154 Making sense of multiple FGF signals 155 Conservation of FGF Ligand-receptor interaction among vertebrates 155 Role of maternally loaded fgfrs in otic induction 156 Direct and indirect roles of FGF signaling in otic induction 157 FUTURE DIRECTIONS 158 CHAPTER 5: CHARACTERIZATION OF TBX2A/B REVEALS A NOVEL PATHWAY IN OTIC INDUCTION 175 INTRODUCTION 176 MATERIALS AND METHODS 178 RESULTS 180 Identification of tbx2a/b as potential targets of Foxi1 180 tbx2a/b expression 181 Tbx2a/b are required for proper otic development 182 tbx2a/b lie upstream of pax8 183 tbx2a/b act in a pathway parallel to dlx3b/4b 183 The requirement of Tbx2a/b in otic gene regulation 184 Tbx2a/b specify anterior and lateral domains of the otic vesicle 185 Tbx2a/b regulation of fgf24 is critical in specification of some hindbrain neurons 185 DISCUSSION 187 Three parallel pathways in otic development 187 Mammary placode induction as a model for Tbx2a/b function 188 The otic placode is a signaling center involved in hindbrain neurogenesis 190 FUTURE DIRECTIONS 191 Elucidation of pathways mediating early phases of otic induction 191 The requirements of the otic signaling center in hindbrain neurogenesis 193 CHAPTER 6: GENERAL CONCLUSIONS 214 REFERENCES 232 Figures and Tables CHAPTER 2: TAILBUD-DERIVED BMP4 DRIVES PROLIFERATION AND INHIBITS MATURATION OF ZEBRAFISH
CHORDAMESODERM 38 Figure 2.1: Expression of follistatin-like genes 1 and 2 67
Figure 2.2: Expansion of the tailbud in Fstl1/2 morphant embryos 69
Figure 2.3: Phenotype of translation-blocking fstl1/2 morpholinos 71
Figure 2.4: Bmp4 is required in the establishment of chordamesoderm during late gastrulation 73
Figure 2.5: BMP attenuation in tBR embryos heat-shocked during late gastrulation 75
Figure 2.6: Sizes of the notochord and chordoneural hinge (CNH) are influenced by Bmp4 activity during late gastrulation 77
Figure 2.7: BMP activity establishes proliferation in axial mesoderm 79
Figure 2.8: The ventral margin as the source of Bmp4 81
Figure 2.9: The temporal state of the notochord changes with the alteration of BMP activity 83
Figure 2.10: Disruption of the temporal state of the notochord can be observed through notochord cell morphology 85
Figure 2.11: BrdU incorporation of embryos treated with the cell cycle inhibitors hydroxyurea and aphidicolin 87
Figure 2.12: A requirement for Bmp4 on the dorsal side of the gastrula 89
Figure 2.13: Bmp2/7 do not affect notochord morphology 91
Figure 2.14: Myotome patterning as a proxy of notochord maturity 93
CHAPTER 3: DLX3B/4B ARE REQUIRED FOR THE FORMATION OF THE PREPLACODAL REGION AND OTIC
PLACODE THROUGH LOCAL MODULATION OF BMP ACTIVITY 94 Figure 3.1: BMP activity is transiently increased during early somitogenesis in Dlx3b/4b morphants 120 Figure 3.2: Levels of bmp4 and fgfr1/2/3/4 transcript are a function of dlx3b/4b activity 122 Figure 3.3: Depletion of Chd reveals anti-BMP function of Dlx3b/4b 124 Figure 3.4: chd expression is reduced in Dlx3b/4b morphants, but can be rescued when cv2 is ectopically
expressed 126 Figure 3.5: cv2 expression is lost from the PPR, otic placode, and pharyngeal arches in Dlx3b/4b morphants 128 Figure 3.6: dlx3b overexpression dorsalizes the zebrafish embryo 130 Figure 3.7: dlx3b/4b and cv2 are required for PPR marker expression 132 Figure 3.8: Ectopic dlx3b or cv2 expression can induce ectopic FGF activity 134 Figure 3.9: fgfr expression is lost from the otic placode and hindbrain of Dlx3b/4b morphants 136 Figure 3.10: FGF activity is not compromised in Dlx3b/4b morphant embryos prior to the onset of somitogenesis 138 Figure 3.11 The reduction of FGF activity in Dlx3b/4b morphant embryos is transient, and begins to return to control levels by mid-somitogenesis 140 Figure 3.12: pax2a expression in the otic placode requires Cv2 142 Figure 3.13: Manipulation of PPR-inducing signals can rescue the otic phenotype of Dlx3b/4b morphants 144 CHAPTER 4: THE ROLE OF FGF RECEPTORS IN OTIC DEVELOPMENT 145 Figure 4.1: fgfr expression during otic development 162 Figure 4.2: The requirements of fgfrs for pax8 expression 164 Figure 4.3: Otic vesicle formation in Fgfr morphants 166 Figure 4.4: Interactions between Fgf8 and Fgfr1-3 168 Figure 4.5: Interactions between Fgf3 and Fgfr1-3 170 Figure 4.6: Fgfr2 mediates Fgf3 signaling in epibranchial placode development 172 Figure 4.7: Fgfr2 mediates Fgf3 signaling in pituitary development 174 CHAPTER 5: CHARACTERIZATION OF TBX2A/B REVEALS A NOVEL PATHWAY IN OTIC INDUCTION 175 Table 5.1: Microarray analysis of gene expression in Foxi1 morphants 197 Figure 5.1: Differential regulation of tbx2a/b by Foxi1 199 Figure 5.2: tbx2a/b expression 201 Figure 5.3: Tbx2a/b are required for proper otic formation 203 Figure 5.4: Tbx2a/b lie upstream of pax8 205 Figure 5.5: tbx2a/b act in a pathway parallel to dlx3b/4b 207 Figure 5.6: The role of Tbx2a/b in otic gene expression 209 Figure 5.7: Tbx2a/b specify the anterior and lateral domains of the otic vesicle 211 Figure 5.8: Tbx2a/b and Fgf24 are required for hindbrain neurogenesis 213
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