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Biol. Bull. 201: 252-254. (October 2001)
© 2001 Marine Biological Laboratory

The Stellate Ganglion of the Squid Loligo pealeii as a Model for Neuronal Development: Expression of a POU Class VI Homeodomain Gene, Rpf-1

J. Peter H. Burbach, Anita J. C. G. M. Hellemons1, Marco Hoekman1, Philip Grant2 and Harish C. Pant2

Laboratory of Neurochemistry, NINDS, NIH, Bethesda, MD 20892.

A major challenge in developmental neurobiology is to understand how neuronal systems are specified, for example, how transmitter phenotype and connectivity are established during development. Molecular cascades of transcription factors and growth factors direct neuronal specification (1). How they operate in terminal differentiation and adult networks is poorly understood. To complement our research to characterize molecular cascades in complex neuronal systems such as midbrain dopaminergic and hypothalamic systems in the mouse (2,3,4), we have turned to a non-mammalian neuronal system that has a functionally and morphologically more homogeneous structure. That structure is the stellate ganglion of the squid Loligo pealeii. It is a compacted cluster of neurons that innervates the muscles of the mantle through the giant nerve fiber system and controls the jet-propelled escape response of the squid (5). The aim of this study was to identify homeodomain genes expressed in the stellate ganglion and to correlate their expression with development of the ganglion.

Working from the concept that transcription factors involved in terminal neuronal differentiation are still operating in the adult system, as demonstrated in mammalian brain (2,3,4), we cloned homeodomain transcripts from the dissected stellate ganglion of the squid Loligo pealeii using RT-PCR with degenerated primers designed to conserved motifs in paired-like homeodomain genes (2,3). Two homeodomain transcripts were identified from 40 cloned PCR fragments (Fig. 1). One fragment (1 out of 40 clones) predicted a homeodomain protein that was highly homologous to a POU class VI homeodomain gene product recently identified in man (6): retina-derived POU factor-1 (RPF-1). The other fragment (6 clones out of 40) was highly similar to the paired-like homeodomain genes phox2a and phox2b, also termed arix/pmx (7). Both types of homeodomain genes have been implicated in the specification of neuronal systems of the mouse. Phox2 genes are required for normal development of central and peripheral components of the autonomous nervous system, while rpf-1 has been implicated in the development of amacrine and retinal ganglion cells (6,7). Other clones represented non-homeodomain-containing genes, including abundant transcripts like alpha-tubulin, actin, and collagen.



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  		Figure 1. Partial amino acid sequences of homeodomain proteins predicted from cloned PCR fragments obtained from the stellate ganglion of the squid Loligo pealeii. The degenerate PCR primers were those used in mammalian brain (2,3): upstream, 5'-GMRSCGMSAVMGSACMMBCTTYAC-3'; downstream, 5'-TGGTTYMRVAAYCGYHGMGCMARRTG-3'. Sequences without primer-coded sequence are shown and compared with the mammalian homologs retina-derived POU factor-1 (RPF-1) of man (A), and Phox2a of mouse (B). In the comparison, identical amino acids are shown and substitutions by physicochemically similar amino acids indicated by +. The predicted RPF-1 protein sequence of Loligo pealeii shows a 31/38 identity and a 35/38 similarity to the human protein. Phox 2 shares a 34/38 identity and 37/38 similarity to mouse Phox2a.

 
We chose to determine the embryonic expression of the rpf-1 gene further by a whole mount in situ hybridization protocol using DIG-labeled cRNA (8), since initial experiments indicated specific labeling for rpf-1, but no signals for phox2. Comparison of anti-sense and sense probes showed specific expression of the squid rpf-1 gene in dorsal structures in the mantle in stage 27 embryos of the squid. Comparison to histologically stained sections of squid embryos (9) indicated that the labeled structures are part of the stellate ganglion. No other neural or non-neuronal structures were labeled at this developmental stage. In earlier stages (22 to 25), results suggested expression in the developing eye. These results suggest that this rpf-1 gene is expressed in the developing and adult stellate ganglion of the squid.

Genes like rpf-1 and others may have a role in developmental events in the stellate ganglion, such as establishment of connectivity and giant axon formation, as well as participating in regulation and maintenance of the adult giant fiber system. If interference with its expression, for example by introduction of morpholinos (10), can be achieved, the role of the rpf-1 gene and other homeodomain genes can be established and can serve as a starting point to delineate molecular cascades in developing neurons. Part of this research was performed at the Marine Biological Laboratory, Woods Hole, Massachusetts, and supported by an MBL Fellowship sponsored by the Baxter Postdoctoral Fellowship Fund, MBL Associates Fund, James A. and Faith Miller Memorial Fund, and the H. B. Steinbach Fellowship Fund.

Footnotes

1 Rudolf Magnus Institute of Neurosciences, Utrecht, The Netherlands Back

2 Department of Medical Pharmacology, Rudolf Magnus Institute of Neurosciences, University Medical Center Utrecht, Utrecht University, 3584CG Utrecht, The Netherlands. Back

Literature Cited

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  2. Smidt, M. P., H. S. van Schaick, C. Lanctot, J. J. Tremblay, J. J. Cox, A. A. van der Kleij, G. Wolterink, J. Drouin, and J. P. H. Burbach. 1997. Proc. Natl. Acad. Sci. USA,94:13,305–13,310.[Abstract/Free Full Text]
  3. Smidt, M. P., C. H. Asbreuk, J. J. Cox, H. Chen, R. L. Johnson, and J. P. H. Burbach. 2000. Nat. Neurosci., 3:337–341.[ISI][Medline]
  4. Burbach, J. P. H., S. M. Luckman, D. Murphy, and H. Gainer. 2001. Physiol. Rev.,81:1197–1267.[Abstract/Free Full Text]
  5. Martin, R. 1965. Z. Zellforsch. Mikrosk. Anat., 67:77–85.[ISI][Medline]
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  7. Stanke, M., D. Junghans, M. Geissen, C. Goridis, U. Ernsberger, and H. Rohrer. 1999. Development, 126:4087–4094.[Abstract]
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  9. Grant, P., D. Tseng, R. M. Gould, H. Gainer, and H. C. Pant. 1995. J. Comp. Neurol., 356:311–326.[ISI][Medline]
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