Foa L et al. (2005), Homer expression in the Xenopus tadpole nervous...

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J Comp Neurol 2005 Jun 20;4871:42-53. doi: 10.1002/cne.20496.

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Homer expression in the Xenopus tadpole nervous system.

Foa L , Jensen K , Rajan I , Bronson K , Gasperini R , Worley PF , Tu JC , Cline HT .

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Homer proteins are integral components of the postsynaptic density and are thought to function in synaptogenesis and plasticity. In addition, overexpression of Homer in the developing Xenopus retinotectal system results in axonal pathfinding errors. Here we report that Xenopus contains the homer1 gene, expressed as the isoform, xhomer1b, which is highly homologous to the mammalian homer1b. The mammalian homer1 gene is expressed as three isoforms, the truncated or short form homer1a and the long forms homer1b and -1c. For Xenopus, we cloned three very similar variants of homer1b, identified as Xenopus xhomer1b.1, xhomer1b.2, and xhomer1b.3, which display up to 98% homology with each other and 90% similarity to mammalian homer1b. Furthermore, we demonstrate that Xenopus also contains a truncated form of the Homer1 protein, which could be induced by kainic acid injection and is likely homologous to the mammalian Homer1a. xHomer1b expression was unaffected by neuronal activity levels but was developmentally regulated. Within the brain, the spatial and temporal distributions of both Homer isoforms were similar in the neuropil and cell body regions. Homer1 was detected in motor axons. Differential distribution of the two isoforms was apparent: Homer1b immunoreactivity was prominent at junctions between soma and the ventricular surface; in the retina, the Mueller radial glia were immunoreactive for Homer1, but not Homer1b, suggesting the retinal glia contain only the Homer1a isoform. Homer1b expression in muscle was prominent throughout development and was aligned with the actin striations in skeletal muscle. The high level of conservation of the xhomer1 gene and the protein expression in the developing nervous system suggest that Homer1 expression may be important for normal neuronal circuit development.

???displayArticle.pubmedLink??? 15861458
???displayArticle.link??? J Comp Neurol
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Species referenced: Xenopus laevis
Genes referenced: actl6a homer1
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	Fig. 1. Xenopus homer1b isoforms are highly homologous to rat and human isoforms. The open reading frames of the three Xenopus homer1b variants are aligned (Clustal analysis, Megalign) with the corresponding homer1b sequence from rat and human. Exact matching sequence is in gray and boxed. Examples of the conserved substitutions can be seen at amino acid positions 62, 97, and 136.
	Fig. 2. Xenopus homer1b has been highly conserved through evolution. By Clustal analysis (Megalign), the pylogenetic tree demonstrates the close relationship of Xenopus homer1b with its mammalian counterparts. Numbers indicate percentage similarity.
	Fig. 3. Different Homer antibodies were used to detect specific Homer isoforms. A: By Western blotting, the pan Homer antibody detected both Homer1b (band at approximately 45 kD) and Homer1a (at approximately 28 kD) in whole Xenopus larvae (stage 47/48 shown). The 28-kD band routinely appeared as a doublet. At the same stage, the specific Homer1b antibody detected only xHomer1b at approximately 45 kD. B: Expression of Homer1a and Homer1b in the developing Xenopus brain. The pan Homer1 antibody was used to illustrate the expression of xHomer1a in the developing Xenopus brain, which remained constant during the later stages of larval brain development. The same Western blot was reincubated with the Homer1b antibody, demonstrating that expression levels of Homer1b increased with age and appeared to plateau at metamorphosis.
	Fig. 4. Spatiotemporal pattern of Homer1b expression in the developing Xenopus brain. A: Homer1b was first detected at stage 34 in the brain neuropil (arrow). B: At stage 44, Homer1b-IR was evident in both the cell body regions (CB) and the neuropil (NP), but Homer1b-IR was absent from the proliferative zone (PZ). C: At stage 48, Homer1b-IR was more intense in the neuropil regions compared with the cell body regions throughout the brain. D: Specific Homer1b-IR was abolished with the omission of the primary antibody, stage 48 brain. E: A transverse section at stage 55 shows a mediallateral gradient of Homer1b expression across the tectum. In addition, large immunoreactive cell bodies were seen along the medial boundaries of the neuropil (arrows). The boxed areas in E are shown at higher magnification in F and G. F: Perisomatic puncta suggest immunopositive axon terminals (arrow) or possibly cell junctions (arrowhead). G: Homer1b-IR puncta line the ventricular border (arrow). In A–D, anterior is upward. In E–G, dorsal is upward. Scale bars 50 m in D (applies to A–D); 50 m in E; 10 m in G (applies to F,G).
	Fig. 5. Spatiotemporal pattern of Homer1a and -1b expression in the developing Xenopus brain. A: At stage 40, Homer1-IR was clearly evident within cell body (CB) and neuropil (NP) regions throughout the brain, including the olfactory bulb (OB) and tectum. B: At stage 44, Homer1-IR appeared high in cell body regions compared with the neuropil and remained absent from the proliferative zone (PZ). The boxed region in B is shown at higher magnification in D. C: At stage 48, predominant Homer1 expression has shifted from somata to the neuropil regions and axon tracts. In the olfactory bulb, expression remains high in the cell body region (arrowhead). D: Intense Homer1-IR at stage 44 is evident within the cell body region (arrowheads). Homer1-IR axon tracts were also evident (arrows). In all panels anterior is upward. Scale bars 50 m in B (applies to A,B); 50 m in C; 20 m in D.
	Fig. 6. Homer1 expression in the developing retina. A: No Homer1-IR was detected at stage 34 in the retina. The only evidence of lamination in the retina at this stage was the border of the presumptive lens (arrow). B: By stage 39–40, Homer1-IR was detected throughout the retina and was particularly strong in the retinal ganglion cells (arrowhead), the Mueller glia (small arrow) and the plexiform layers (large arrows). Homer1-IR was not present in the retinal proliferative zone (asterisk). C: Homer1-IR intensity increased at stage 44. Immunoreactive axons were seen in the optic nerve (arrow). D: At stage 48, Homer1-IR appeared most intense in the inner plexiform layer (arrow). E: At stage 55, Homer1-IR was detected in the cytoplasm of cell bodies in the bipolar layer (arrow). F: At metamorphosis, Homer1-IR was still evident and was strongest in the plexiform layers. Inset: Homer1-IR axons within the optic nerve at metamorphosis (arrow). In all panels, nasal retina is upward. G: A control section, incubated without primary antibody. Nonspecific staining was not detected in the retinal ganglion cells, bipolar cells or in the inner and outer plexiform layers. Scale bar 50 m.
	Fig. 7. Homer1 and 1b-IR in the retina at stage 45. A: Within the retina, Homer1-IR appears strongest within the inner plexiform layer (IPL). The Mueller glia (arrowhead) were immunoreactive for Homer1, but not Homer1b (compare with B). No Homer1 was evident in the undifferentiated cells of the proliferative zone (PZ). ON, optic nerve. B: Homer1b-IR was found concentrated in puncta in the inner plexiform layer and in the cytoplasm of the retinal ganglion cell (RGC) and inner nuclear (INL) layers (arrows). In both panels, nasal retina is upward. Scale bar 50 m in A (applies to A,B).
	Fig. 8. Homer1-IR in the Xenopus spinal cord. A: Homer1-IR was first detected in the Xenopus embryo at stage 26 in the midline (bracketed region labelled M) and developing myocytes (arrows). B: The boxed region in A is shown at higher magnification to illustrate the early detection Homer1-IR in axons at stage 26 (arrows). C: At stage 34, Homer1-IR puncta were found in the neuropil (NP) and in the muscle (boxed region, enlarged in D). D: Homer1-IR localized to the muscle striations of skeletal muscle and is concentrated at the myotome junctions (arrow). E: By stage 40, cytoplasmic localization of Homer1-IR was evident in motor neuron cell bodies and axons (boxed region enlarged in F), and discrete Homer1-IR puncta were evident in the neuropil. F: Homer1-IR extends throughout the cytoplasm of the developing motor neurons, including the axons (arrow). G: At stage 45, Homer1b-IR appeared strongest in the neuropil. H: Discrete Homer1b-IR puncta were evident around cell bodies (arrowhead) and along the ventricular border (arrow). In all panels, anterior is upward. Scale bars 50 m in G (applies to A,C,E,G); 10 m in H (applies to B,D,F,H).
	Fig. 9. Xenopus Homer1a is induced by neuronal activity. A: Kainic acid injection into the ventricle was used to increase neuronal activity. The pan-Homer1 antibody showed that Homer1a expression (band at approximately 28 kD) increased with kainic acid concentration and stimulation time. B: Homer1b expression was not altered by the kainic acid stimulation, as shown by the pan-homer1 antibody (A) and the Homer1b antibody (B).
	Fig. 10. Kainic acid induced Homer1a expression in neuronal and glial cell bodies. A: Pan Homer1-IR at stage 48 is normally concentrated in the neuropil (NP) compared with the cell body regions (CB; see also Figs. 4 and 5 with the Homer1b and Homer1a antibodies, respectively). B: Kainic acid stimulation increases Homer1-IR in the cell body region (arrows). In both panels, anterior is upward. Based on the Western blot shown in Figure 9, this is likely an increase in Homer1a expression. Scale bar 50 m in B (applies to A,B).