FIGURE SUMMARY
Title

Cis-regulatory characterization of sequence conservation surrounding the Hox4 genes

Authors
Punnamoottil, B., Herrmann, C., Pascual Anaya, J., D'Aniello, S., Garcia-Fernàndez, J., Akalin, A., Becker, T.S., and Rinkwitz, S.
Source
Full text @ Dev. Biol.

The tested hoxb3a regulatory element is a tissue-specific enhancer. Lines from four different founders had strong GFP expression in hindbrain r5. Furthermore almost each founder showed another additional expression feature, which are also within the hoxb3a expression domain — founder #9a: pharyngeal endoderm (blue arrow), founder #8a: spinal cord (yellow arrow), founder #1a: migrating neural crest (blue arrow).

Schematic of the genomic region from hoxa5a to hoxa3a with the tested elements in a color code (red: tissue-specific enhancer activity, blue: unspecific, white: no activity) and the distance of the element from the exons in kb. The expression of GFP under the regulatory control of the tested CNEs is shown in representative zebrafish larvae between stage 2 dpf to 4 dpf. The asterisk marks the level of the inner ear and the arrows follow a color code: red, hindbrain; yellow, spinal cord; brown, cranial ganglia; light blue, neuronal projection; pink, pectoral fin. a4-2. Expression of GFP in a dorsal and ventral subpopulation of cells in the most posterior part of the hindbrain and in the anterior spinal cord. a4-2′. Dorsal view of the posterior hindbrain and spinal cord with GFP expression in cells along the ventricular surface. a4-2″. Anti-GFP stained transgenic larva with sections trough the posterior hindbrain and anterior spinal cord (indicated by black lines). a4-3. Wide GFP expression in the medial posterior hindbrain and anterior spinal cord. a4-3′. Dorsal view that shows GFP expression in the vagal ganglion, in the pectoral fins and in fibers innervating the pectoral fins and the somites. a4-3″. Anti-GFP stained transgenic larva with sections showing signal in the hindbrain (red arrow points to the vagal nucleus) and in the spinal cord. The level of sections are indicated by the black lines. Neuronal projections are visible (1) from the vagal nuclei towards the vagal ganglion (brown arrow) and (2) into the pectoral fin and (3) along the somites.

Schematic of the hoxb5a to hoxb3a genomic region. Tested elements are indicated as ovals (red: tissue-specific enhancer activity, blue: unspecific, white: no activity) with its distance from the exons in kb. The regulatory activity of the elements is illustrated by representative larvae at 2 to 4 dpf. The asterisk marks the level of the inner ear. The arrows follow a color code: red, hindbrain; yellow, spinal cord; brown, cranial ganglia; light blue, neuronal projection; white, migrating neural crest. b4-2. GFP expression in the posterior hindbrain, extending with decreasing intensity into the anterior spinal cord. b4-5. Larva with strong and broad GFP expression in the hindbrain r7 and r8 extending posterior into the spinal cord. GFP is also visible in a pectoral fin neuron, in the developing pharyngeal arches and a cranial ganglion. b4-5′. Anti-GFP stained larva with sections at the level of the 2nd somite and the spinal cord. The level of sections are indicated by the black lines. Strong GFP expression is indicated in the dorsal and lateral domains of hindbrain r7 and r8 with projections into the pectoral fins. In the spinal cord the signal is also stronger dorsally with two clusters of motoneurons projecting laterally along the adjacent somites. b4-7. Larva with strong GFP expression in the entire spinal cord. b4-10. Larva with GFP expression in the hindbrain r7 and r8 with a sharp anterior expression boundary at r6. Expression extends posterior into the spinal cord. GFP is also visible in cranial ganglia around the inner ear. b4-10′. Anti-GFP stained larva of the same transgenic line with sections through the hindbrain r6/7 and r7, which shows the distribution of the GFP positive cells in the medial hindbrain and in cells of the posterior lateral line ganglion.

Schematic of the hoxc5a to hoxc3a genomic region. Tested CNEs are illustrated as explained in [Fig. 4] and [Fig. 5]. Representative GFP expressing larvae are shown at stages 2 to 4 dpf. The asterisk marks the level of the inner ear. The arrows follow a color code: red, hindbrain; yellow, spinal cord; dark blue, pharyngeal arches; pink, pectoral fins. c4-2. Larva with GFP expression in the hindbrain r7 and r8 extending weaker into the anterior spinal cord. c4-2′. Dorsal view of the same larva as in A with posterior hindbrain and pectoral fin expression. c4-3. Larva with GFP expression in the posterior hindbrain and in the spinal cord. c4-3′. Another line with higher magnification shows single GFP labeled cells in hindbrain r7 and further expression in the pharyngeal arches. c4-4. Larva with expression in the hindbrain r7 and r8, in the spinal cord and also in all pharyngeal arches. c4-4′. A dorsal view of a larva with focus on GFP expression in the pectoral fins.

Schematic of the hoxd9a to hoxd3a genomic region with the tested elements illustrated as described in [Fig. 4] and [Fig. 5]. The arrow color code: red, hindbrain; yellow, spinal cord; grey, somites; light blue, neuronal projection; green, pharyngeal arches. Representative larvae with GFP expression under regulatory control of CNEs that surround hoxd4a are shown at 2 to 4 dpf. The asterisk marks the level of the inner ear. d4-1. Larva showing GFP expression in the posterior spinal cord and in the somites. d4-3. GFP expression in two bilateral columns in hindbrain r2 to r6, adjacent to the inner ear. d4-4. Larva with GFP expression in spinal cord motor neurons and their projections towards the somites. Some neurons in the hindbrain r4–6 are also GFP positive. d4-6. Larva with a strong expression boundary at cerebellum/midhindbrain boundary. Strong GFP expression in the hindbrain and in the dorsal spinal cord. d4-8. Element d4-8 is located in the intron of the gene. A larva with expression in the dorsal and ventral spinal cord with GFP expressing neurons projecting towards the somites. d4-9a. Larva shows GFP expression in the hindbrain with a week anterior boundary at r6/r7. The GFP distribution continues into the spinal cord and GFP expression is also visible in the pharyngeal arches.

A. Three conserved sequences, identified through VISTA alignments of the zebrafish Hox4 loci (bf1, bf2, bf4). B. Alignments of the amphioxus intronic element with the four paralogous zebrafish intronic elements (upper part) and the frog intronic elements (lower part). The width of the line is proportional to the length of the local alignment, the color indicates the level of similarity. C. The intronic element of the AmphiHox4 gene, bf4, was tested in zebrafish transgenesis. Shown is are larvae with GFP expression in the pharyngeal arches (blue arrow) and the pectoral fins (pink arrows) as well as hindbrain (red arrow) and spinal cord (yellow arrow). D. For comparison the expression features that are regulated through the tested zebrafish intron elements are shown. The arrows use the following designated color code: red, hindbrain; yellow, spinal cord; blue, pharyngeal arches and pink, pectoral fins.

Endogenous expression of the hoxc4a gene in zebrafish embryos and larvae. The otic placode is marked by an asterisk. The level of sections are indicated by the red lines. A. Expression analysis of hoxc4a at 1 dpf reveals specific distribution of transcripts in the hindbrain r7, r8 and in the spinal cord. Sections revealed further transcripts in the epibranchial placodes (black arrow), in the paraxial mesoderm underlying the epibranchial placodes and in lateral line neuromasts (blue arrow). B. At 2 dpf strong hoxc4a expression is visible in the hindbrain and in the pharyngeal arches 6 and 7. B′. A section on second somite level shows expression in the developing pectoral fins. C. Hoxc4a expression at 3 dpf. C′. Transcripts are strongly distributed throughout the hindbrain and in addition in the vagal ganglion (black arrow). C″. A section on third somite level shows strong expression in the hindbrain and in the pectoral fins (blue arrow).

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Reprinted from Developmental Biology, 340(2), Punnamoottil, B., Herrmann, C., Pascual Anaya, J., D'Aniello, S., Garcia-Fernàndez, J., Akalin, A., Becker, T.S., and Rinkwitz, S., Cis-regulatory characterization of sequence conservation surrounding the Hox4 genes, 269-282, Copyright (2010) with permission from Elsevier. Full text @ Dev. Biol.