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Hox genes are key players in defining positional information along the main body axis of vertebrate embryos. In Xenopus laevis, Hoxc6 was the first homeobox gene isolated. It encodes two isoforms. We analyzed in detail their spatial and temporal expression pattern during early development. One major expression domain of both isoforms is the spinal cord portion of the neural tube. Within the spinal cord and its populations of primary neurons, Hox genes have been found to play a crucial role for defining positional information. Here we report that a loss-of-function of either one of the Hoxc6 products does not affect neural induction, the expression of general neural markers is not modified. However, Hoxc6 does widely affect the formation of primary neurons within the developing neural tissue. Manipulations of Hoxc6 expression severly changes the expression of the neuronal markers N-tubulin and Islet-1. Formation of primary neurons and formation of cranial nerves are affected. Hence, Hoxc6 functions are not restricted to the expected role in anterior-posterior pattern formation, but they also regulate N-tubulin, thereby having an effect on the initial formation of primary neurons in Xenopus laevis embryos.
Figure 1. Temporal and spatial expression pattern of the Hoxc6 isoforms. A: Structure of Hoxc6 isoforms in Xenopus laevis. Hoxc6 isoforms derive from 2 different promoters. The distal promoter (PR I) leads to a 2.2-Kb precursor transcript, coding for the short form (SF) protein of 152 aa. The proximal promoter encodes a 1.8-Kb transcript leading to the long form (LF) protein of 234 aa. Black boxes indicate the open reading frame, grey boxes are the homeodomain. Red bars indicate the binding sites of the morpholino-oligonucleotides. The blue and green arrows show the primer sites that served for the RT-PCR and to generate the probes for in situ hybridization. B: Temporal expression pattern of Hoxc6 isoforms. RT-PCR was carried out using embryos at the indicated stages from 1 to 27. H4 is used as a loading control; -RT is shown for stage 12 RNA. C-K: SF expression patterns from beginning of gastrulation until tadpole stages. Stages are indicated by the numbers. L-T: LF pattern from beginning of gastrulation until tadpole stages. The arrowheads in K and T indicate the location of the pronephric anlage. The squared brackets indicate the different distances between the anteriormost border of expression of each isoform and the otic vesicle area.
Figure 2. Tissue localisation of Hoxc6 isoforms in cross-sections. A: SF expression in the somitic tissue and neural tube. B: Expression of LF in somitic tissue and neural tube; in addition, LF is expressed in the pronephric anlage (arrowheads). C, D: Close-ups of the neural tubes shown in A and B. Expression of the Hoxc6 isoforms is detected in all cells of the neural tube with exception of the floorplate. E: MyoD expression in the somites. F: Xlim-1 expression in the neural tube and in the pronephric anlage (arrowheads). G: FoxF1 expression in the lateral plate mesodem. H: Position of the shown sections within stage-27 embryos, and localization of different tissues in the sections. Neural tube (blue); somitemesoderm (red); notochord (brown); endoderm (yellow); lateral plate mesoderm (dark orange); the pronephric anlage (light orange). I: Cross-section at the level of the branchial arches (br) as indicated in the inlay. Expression of SF is found in the neural tube (ne), even though it was not visible in whole mount in situ hybridization. J: Expression of LF in the neural tube at the same level.
Figure 3. The effect of Hoxc6 knockdowns on neural markers. A: Specificity of MO nucleotides was tested in vitro using a coupled transcription-translation assay. In vitro synthesis of LF was inhibited by MO3, but not by the ctMO (black arrowheads). In vitro synthesis of the SF was reduced by MO2, but not by ctMO (open arrowheads). Translation of an LF construct lacking the MO3 binding site (LF insensitive) is neither inhibited by MO2 nor by MO2 combined with MO3 (arrows). This demonstrates that the SF-specific MO2 does not affect synthesis of the LF, even though its target sequence is present in the LF mRNA. B-F: The neural marker Sox2 was analyzed by in situ hybridization in uninjected (uninj.) embryos and after injection of the control morpholino (ctMO), the MO2, the MO3, or the combination of MO2 and MO3, as indicated above the photographs. G-K: Expression of the neural marker N-CAM after the same set of injections. L-P: Expression of the neural crest marker Xslug after the same set of injections.
Figure 4. The effect of Hoxc6 knockdowns on formation of primary neurons and cranial nerves. Markers specific for primary neurons (N-tubulin, Xisl-1) and for cranial nerves (Xisl-1) were analyzed at neurula and tadpole stages by in situ hybridization upon Hoxc6 depletion. Shown are uninjected (uninj.) embryos, and embryos injected with ctMO, MO2, MO3, or a combination of MO3 with an MO-insensitive mRNA for LF, as indicated above the photographs. A-E: Analysis of the neuronal marker N-tubulin. Arrowheads indicate ectopic N-tubulin expression. F-J: Expression of Xisl-1 at a neurula stage for the same set of injections. No ectopic expression was found. K-O: Expression of Xisl-1 in the head of a tadpole. Numbers in K indicate the different branchial arches and the corresponding cranial nerves.
Figure 5. Effect of overexpression of the LF and the SF on neural and neuronal markers. A-C: Expresssion of the neural marker N-CAM in uninjected embryos (uninj.), and in embryos injected with mRNA encoding either the LF or the SF as indicated above the photographs. D-F: Expression of the neuronal marker N-tubulin after the same set of injections. Arrowheads indicate ectopic activation of N-tubulin expression. G-I: Expression of the neuronal determination factor X-ngnr-1 after the same set of injections. J-L: Expression of the neuronal factor CRMP-4 after the same set of injections. M-O: Expression of the neuronal marker Xisl-1 after the same set of injections.
Figure 6. The early temporal and spatial pattern of N-tubulin and its activation by Hoxc6 isoforms. A: A time course of N-tubulin expression analyzed by RT-PCR. Numbers indicate the stages. H4 is used as loading control. B: Activation of N-tubulin in animal caps by Noggin (nog), SF, and LF at early neurulation. As additional controls uninjected animal caps (ni) and whole embryos (we) are shown. H4 is used as loading control. C-F: Spatial expression pattern of N-tubulin at gastrula and early neurula stages as indicated by the numbers. G: Ectopic activation of N-tubulin during gastrulation after half-sided injection of LF mRNA.
Figure 7. Effects of LF and SF mRNA on embryos either injected with MO2 or with MO3. A-E: Expression of N-tubulin is shown in embryos after injection with MO2 alone, and in combination with different doses of either LF or SF mRNA, as indicated above the photographs. The numbers describe the amount of mRNA in pg per embryo. Arrowheads indicate ectopic activation of N-tubulin expression. F-J: Expression of N-tubulin in embryos after injection with MO3 alone and in combination with different doses of either LF or SF mRNA. Arrowheads in G indicate ectopic activation of N-tubulin expression. Expression labeled with arrowheads in I and J may be ectopic or may result from the defect of gastrulation.
