«Joshua Frederick Coulcher UCL Submitted for the Degree of Doctor of Philosophy September 2011 Declaration I, Joshua Frederick Coulcher, confirm that ...»
Fig.6.4. Alignment of the bZIP domain of At cnc with other CNC and bZIP family members confirms that At cnc is a homologue of cnc. The basic region, the DNA binding domain, of the bZIP domain is between positions -25 to -3 and consists of numerous basic residues. The Leucine zipper consists of heptad repeats, every seventh amino acid is a Leucine or hydrophobic residue. The leucine zipper positions are indicated with asterisks present at positions 1, 8, 15, 22, 29 and 36. CNC family members are Tc cnc from Tribolium, cnc from Drosophila, NFE2-RF1 (Nrf1) from Homo sapiens and Skn-1 from C.
elegans. bZIP family members include AP-1 (also known as JUN) from Homo sapiens, Jun related antigen from Drosophila melanogaster, FOS from H. sapiens, Kayak (synonymous with Fos related antigen) from Drosophila, and cAMP response element binding protein (CREB) from Drosophila. At cnc is clearly more similar to other CNC family members than other bZIP family members. CNC family members have 6 heptad repeats with some substitutions of alternative amino acids in some of the leucine zipper postions. Charged residues present in some postions in the leucine zipper region can prevent homodimerization. Skn-1 has lost its Leucine zipper domain but retains the basic region which is highly similar to other CNC family members. Amino acid residues that are shared by 50% or more taxa are highlighted.
domain and the Leucine zipper that is clearly more similar to the CNC family members than the remaining bZIP family of transcription factors.
At cnc transcripts were detected by RT-PCR at all studied stages of embryogenesis from very early blastoderm stages through to late stages prior to hatching of spiderling larval instars.
Control in situ hybridizations with the genes At en, At Dll, At Dfd-1
In order to optimize the in situ hybridization protocol and study the developing embryo of Achaearanea, in situ hybridizations were performed with the homologues of the genes engrailed (At en), Distal-less (At Dll), and to one of the spider Dfd homologues (At Dfd-1). Sequences for these genes were downloaded from Genbank (Benson et al., 2005). The expression of these genes is shown in fig.6.5., fig.6.6. and fig.6.7. At en is expressed in the posterior of each developing segment in the embryo and the posterior of the developing appendages (see fig.6.5.A-D). Additional segmental domains of At en expression are added to the posterior of the embryo as the growth zone progressively adds segments to the germ band extending embryo. At Dll is expressed in the developing appendages (see fig.6.5. E-H). The appendages in order from anterior to posterior are the chelicerae (Ch), pedipalps (Pp), and the first to the fourth leg appendage segments (L1-L4). The first leg segment, which is homologous to the mandibular segment, is indicated with an arrow.
As the anterior domain of cnc in Tribolium and Drosophila is expressed in the developing labrum, the development of the spider labrum was visualized using At Dll as a marker (fig.6.6A-D), and compared to the development of the Tribolium labrum which was visualized with Tc cnc and Tc Dll expression (see fig.6.6F,G). The spider labrum (or At Dll expression in the developing labrum) develops relatively late compared to Tribolium. The labrum develops as a pair of labral buds in both species which later fuse to form the labrum. At Dll is also expressed in a domain around the developing stomodeum (arrowhead in fig.6.5C) which is reminiscent of the Tc cnc expression domain around the developing stomodeum in Tribolium (arrowhead in fig.6.6F). Conversely, there is no stomodeal domain of Tc Dll expression in Tribolium (fig.6.6G).
The expression of At Dfd-1 is shown in fig. 5.6. At Dfd-1 is expressed in the developing L1 to L4 segment limb buds (fig.6.7C,D). Expression is localized in the tips of these appendages (marked with an arrow in fig.6.7E). Expression of the Tribolium homologue of Dfd is shown in fig.6.7F. Comparison of the anterior expression domains homologizes the first leg segment to the mandibular segment. The anterior domains of Dfd in Tribolium and Achaearanea is depicted schematically in fig.6.7G. Expression of At Dfd-1 was visualized by in situ hybridization using tyramide signal amplification. This Fig.6.5. Expression of homologues of segment polarity gene engrailed and the PD domain gene Dll in Achaearanea embryos. Gene expression was detected by in situ hybridization. The L1 segment is indicated with an arrow. Anterior is to the left. (A-D) Expression of segment polarity gene At en. At en is expressed in the posterior of each segment. (A) lateral view of early germ band extending stage. (B) Later germ band extending stage after the limb buds have formed, lateral view. (C) Ventral view of embryo at a similar stage to B. (D) Late stage embryo, lateral view. (E-H) Expression of At Dll. At Dll is expressed in the distal tips of every appendage. (E) Germ band extending embryo after the limb buds have formed, lateral view. (F) Lateral view of a slightly later stage than E. (G) Ventral view of an embryo at the same stage as F. (H) Later stage germ band retracting embryo.
Fig.6.6. Comparison of the labrum of Achaearanea to Tribolium marked by the expression of Dll. Gene expression was detected by in situ hybridization. All views are ventral with anterior to the left unless otherwise indicated. (A-E) Expression of At Dll in developing spider embryos. (F-G) Tribolium embryos.
The developing labrum is marked with an arrow. Stomodeal expression domains is marked with arrowhead. (A) Embryo just after the limb buds have formed. There is no expression of At Dll in the labrum. (B) Slightly later Germ band extending embryo than A. The stomodeum is visible, but there is no At Dll expression. (C) Germ band extending embryo, the stomodeum is surrounded by At Dll expression.
(D) Germ band extending embryo, the labral buds are beginning to fuse. (E) Late stage embryo during dorsal closure. The labrum is completely fused. Anterior is to the top. (F) Expression of Tc cnc in germ band extending Tribolium embryo. cnc is expressed in an anterior domain in the labrum and a posterior domain in the mandibular segment. Expression is also present around the developing stomodeum in a manner similar to that of At Dll. (G) Expression of Tc Dll and Tc prd. The stomodeal domain of Dll present in Achaearanea is missing in Tribolium (arrowhead).
Fig.6.7. Comparison of the anterior expression boundary of Dfd homologues in Achaearanea and Tribolium homologises the mandibular segment to the first leg segment. Gene expression was detected by in situ hybridization. (A) Expression of At Dll in a late germ band extending embryo. The appendages are clearly marked with At Dll expression. There is At Dll expression in the endite on the pedipalp (star). (B) Expression of Tc Dll and Tc prd in Tribolium embryo. Tc Dll is expressed in the tip of every appendage except the mandible. Tc Dll is also expressed in the labrum. Tc prd is expressed in the endites of the mandible, maxilla and labial appendages. (C-E) Expression of At Dfd-1 in Achaearanea.
Expression of At Dfd-1 is present in the L1 to L4 segments. (C) Early germ band extending embryo as the limb buds are forming. Arrow marks the anterior boundary of At Dfd -1 expression. (D) later stage germ band embryo. Arrow marks anterior boundary of At Dfd-1 expression. (E) At Dfd-1 expression is present at the tips of appendages (arrow). (F) Expression of Tc Dfd in Tribolium. The arrow marks the anterior boundary of Tc Dfd expression in the mandibular segment. Tc Dfd is expressed in the mandibular and maxillary segments. (G) Comparison of the expression domains of Dfd between chelicerates and insects represented in the figure by Achaearanea and Tribolium respectively. Figure is adapted from Hughes and Kaufman (2002a). The segmental abbreviations are as follows: ocular (Oc), chelicerae (Ch), pedipalps (Ped) and four Leg segments (L1-L4) opisthosomal segments (Op), antennal (ant), intercalary (Int), mandibular (Mn), maxillary (Mx), labial (Lb), thoracic (T), abdominal (A) alternative in situ hybridization protocol was tested to try to provide additional sensitivity for detecting transcripts that are expressed at a low level.
Expression of At cnc transcripts visualized by in situ hybridization In order to visualize expression of At cnc, numerous in situ hybridizations were performed. No structure specific signal was detected. Instead, ubiquitous or background staining was visible in all performed At cnc in situ hybridization experiments.
Different in situ hybridization parameters were used in different experiments in order to optimize the signal of At cnc expression. The following parameters were changed in different in situ hybridization experiments: i) Length of probe (extending the At cnc specific antisense probe sequence from 366bp to 1.2kb). ii) Concentration of probe was tested over a thousand-fold range (0.01µl-10 l labelled RNA probe/100µl hybridization solution). iii) Incubation time of hybridization of the probe to the embryos from overnight to one week. iv) Incubation time of the anti-hapten antibody from overnight to one week.
In addition, the hybridization temperature was tested over a range of 55 oC to 65oC. The concentration of formamide present in the hybridization solution was also increased to 75% (v/v) to reduce the stringency of RNA binding. The number and duration of washes post-hybridization and post-antibody incubation was substantially increased from several washes over two hours to several washes over more than six hours. The only notable effect of many of these alterations was the overall reduction of background staining visible in sense probe in situ hybridizations and to lengthen the amount of time required for antisense in situ hybridization background/ubiquitous staining to surface.
The staining with 1.2kb of labelled antisense and sense At cnc RNA probe is shown in fig. 6.8. in situ hybridization performed with antisense probe (fig.6.8A-C) has ubiquitous staining throughout the embryo. in situ hybridization performed with sense probe has staining in the yolk of the embryo, but the developing embryo is free of background staining (fig.6.8.D-F). In an attempt to control for non-specific binding, in situ hybridizations were performed both with sense probe and without any probe.
Fig.6.8. in situ hybridations with At cnc antisense and sense probes in Achaearanea embryos. Gene expression was detected by in situ hybridization. Anterior is to the left, ventral is top. (A-C) in situ hybridisation with At cnc anti-sense probe in (A) early germ band extending stage. (B) germ band retracting stage. (C) Late stage embryo. (D-F) in situ hybridisation with At cnc sense probe in (D) early germ band extending stage. (E) germ band retracting stage (F) Late stage embryo. (G-I) Alternative in situ hybridisation experiment that produced assymetric pseudo-expression patterns. (G) Ventral view.
(H) close up of pedipalp and first leg appendage. (I) Dissected leg. Proximal is top.
Spider embryos that did not have any probe added produced no background staining at all (data not shown).
In embryos subjected to one week of hybridization and one week of antibody incubation, asymmetric spots of staining were detected. There was a relative increase in staining at the tips of appendages and the base of appendages (see fig.6.8G-I). A dissected leg appendage is shown in fig.6.8I. As the staining was asymmetric and nonreproducible between different embryos, this staining pattern was interpreted to be an artefact of that particular in situ protocol used.
6.3 Discussion General overview of results The homologue of cnc was discovered in Achaearanea. The short sequence obtained by degenerate PCR was extended by RACE. Alignment of the conserved regions of the protein sequence including the bZIP domain confirms the identity of the cloned gene as a CNC family member, most likely the orthologue of cnc present in single copies in other arthropod species. The identity of Tc cnc was confirmed by aligning the sequence of the bZIP domain with members of the CNC family and bZIP family of transcription factors. It is clear from the sequence comparison that the sequence obtained is nearly identical to other cnc orthologues in other arthropods and CNC family members in C. elegans, H. sapiens and is distinct from other bZIP family transcription factors such as kay, Jra and CREB in D. melanogaster and AP-1, FOS in H.