Figure 1.
Schematic diagram of the cuttlefish CNS.
The molluscan ganglia origin of each principle lobe of the central nervous system (CNS) is color coded. On the left the head is shown from a dorsal view, with the right supraesophageal mass (SPM) removed at the mid-line (dashed line). The CNS is represented in lateral view on the right. The grey neuropil structure is approximate and not meant to represent the actual internal structure of the organ. a: anterior subesophageal mass; br.g: branchial ganglia; m: middle subesophageal mass; ol: optic lobe; st: statocyst; p: posterior subesophageal mass; sg: stellate ganglia; spm: supraesophageal mass; wb: white bodies.
Table 1.
Details of recovered HD gene sequences from the European cuttlefish Sepia officinalis.
Figure 2.
Temporal gene expression profiles of homeodomain genes.
A) Reverse transcription polymerase chain reaction (RT-PCR) analysis of ANTP-class genes (left) and PRD- and LIM-class genes (right) amplified for 30 PRC cycles. The ribosomal protein control (Sof-RPS16) shows a saturated profile after 20 cycles, whereas all developmental transcription factors show differential peaks of expression throughout embryogenesis. B) Real time quantitative reverse transcription polymerase chain reaction (qPCR) analysis of ANTP-class genes (left) and PRD- and LIM-class genes (right). Ct data for each gene was normalized to the internal control (Sof-RPS16), and represented relative to its lowest levels of gene expression present in the assay. The ANTP-class genes show oscillating expression profiles throughout embryogenesis, whereas the PRD- and LIM-class genes show very similar expression profiles with an expression peak between stages E25–26.
Figure 3.
Spatial gene expression profiles of PRD-class homeodomain genes.
The first panel of all figures is a schematic diagram as in figure 1, summarizing gene expression domains reconstructed from analysis of serial sections of embryos from stages E24–28. The age of embryo shown is found in the upper right corner of each image. A) Sof-Arx. From left to right: Medial section through the head illustrating expression within the supraesophageal mass; Gene expression within the stellate ganglion; Medial section through the head illustrating retained expression within the supraesophageal mass. B) Sof-Vsx. From left to right: Medial section through the head illustrating expression within cells of the optic lobes, the supraesophageal mass, and the middle subesophageal mass; section through the buccal mass illustrating signal associated with the mesenchyme of the developing beak; medio-lateral section through the head demonstrating gene expression in cells scattered throughout the optic lobes, and within the supraesophageal mass. Abbreviations - asm: anterior subesophageal mass; bm: buccal mass; msm: middle subaosophageal mass; psm: posterior subesophageal mass; st: statocyst; sg: stellate ganglion; spm: supraesophageal mass.
Figure 4.
Spatial gene expression profiles of ANTP-class homeodomain genes.
Figure layout as in Figure 3. A) Sof-Gbx. Clockwise from upper left: Medial section through the head showing signal within the buccal mass, oesophagus, and posterior and middle subesophageal masses; Higher magnification from the same embryos showing gene signal within the posterior subesophageal mass and statocyst; Media-lateral section through the mantle cavity highlighting expression in the gills and posterior subesophageal mass; Expression is detected in the arm epithelium; Medial section of the subesophageal mass, signal is evident in the ventral-most regions; Staining of the stellate ganglion; Medial section through the mantle cavity highlighting gene expression in the intestinal apparatus. B) Sof-Hox3. Clockwise from upper left panel; Medial section through the head showing extensive staining within the posterior and middle subesophageal masses, highlighting a domain of concentrated gene expression posteriorly. Signal is also present at the level of the brachial ganglia (arrows); Section showing medial staining within the funnel; Section shows staining in the beak epithelium in the buccal mass and a small population of 2–4 cells (arrow) in the anterior-most portion of the supraesophageal mass; Buccal mass staining is no longer present by stage E26, whereas the supraesophageal mass cells retain expression (arrows); Section through the base of the arms in a stage E25 embryo shows staining within the arm epithelium and the nerve cord surrounding the neuropil of the arms (np); Similar staining within the arms is not present in earlier stage E24 embryos, while expression in the brachial ganglia primordial is present; Abdominal section showing staining in the stellate ganglia; Lower magnification image showing staining in gills, stellate ganglia, subesophageal mass, and ventral-most portion of the funnel tube. Abbreviations - br.g: brachial ganglia; g: gills; hp: hepatopancreas; int: intestine; iym: inner yolk mass; m: mantle; np: neuropil; oe: oesophagus; all others as in Figure 3.
Figure 5.
Spatial gene expression profiles of Sof-Lhx3/4.
Figure layout as in Figure 4. Clockwise from upper left: Medial section through the head detecting moderate expression levels within all lobes of the CNS and the posterior mesenchyme of the buccal mass; A more lateral section of the same embryo, highlighting the strong expression domain within the middle and anterior subesophageal masses, and expression within the nerve cords of the arms; Lateral section showing expression within the optic lobes and the presumptive olfactory pit epithelium (*); Expression within the stellate ganglion is absent in the ventral-anterior most portion; Medial section at the level of the buccal mass showing expression within the buccal ganglion and neural cord of the arms; Section at the level of the optic lobes demonstrating expression throughout. Expression is also evident in the nerve cords of the arms but absent in the dorsal-lateral portion of the supraesophageal mass; Medial section through the supraesophageal mass showing expression domain in the dorsal-anterior most portion; Medial section through the subesophageal mass illustrating expression domains within the anterior-most portion of the anterior and middle masses, as well as the posterior-most portion of the posterior mass and underlying statocyst. Abbreviations: bg: buccal ganglia; nc: nerve cord; all others as in Figures 3 and 4.
Figure 6.
Schematic representation of HD gene expression domains in the cuttlefish CNS.
Expression territories are extrapolated from analysis of serially sectioned embryos from stages E24–28 (this paper) and previously published data* °. Dorsal view of the head is shown on the left, and lateral view on the right as in Figure 1. Territories with potentially overlapping expression domains are multi-coloured. A) ANTP-class Hox genes Sof-Hox3 (dark green), Sof-Gbx (orange), and Sof-Mnx* (pink). B) PRD-class genes Sof-Arx (red), Sof-Vsx (dark blue), Sof-Phox2* (light green), and Sof-otx° (yellow). C) LIM-class gene Sof-Lhx3/4 (light blue). Note that ANTP-class genes are largely restricted to the subesophageal masses, whereas PRD-class genes are more expansive within the supraesophageal mass. Both Sof-Hox3 and Sof-Lhx3/4 show extensive expression throughout the nervous system, and five of the eight genes represented here are found within the stellate ganglion. Abbreviations are as in Figure 1; * data extrapolated from Nomaksteinsky et al. [26] for stage E29 embryos; ° data extrapolated from Buresi et al. [25] for embryos at stages E25–26.