Expression Pattern of Axin2 During Chicken Development

Canonical Wnt-signalling is well understood and has been extensively described in many developmental processes. The regulation of this signalling pathway is of outstanding relevance for proper development of the vertebrate and invertebrate embryo. Axin2 provides a negative-feedback-loop in the canonical Wnt-pathway, being a target gene and a negative regulator. Here we provide a detailed analysis of the expression pattern in the development of the chicken embryo. By performing in-situ hybridization on chicken embryos from stage HH 04+ to HH 32 we detected a temporally and spatially restricted dynamic expression of Axin2. In particular, data about the expression of Axin2 mRNA in early embryogenesis, somites, neural tube, limbs, kidney and eyes was obtained.


Introduction
Axin2 (also called Axil or Conductin) is a homologue of Axin protein. It shares approximately 45% of amino acids with Axin [1,2]. The Axin protein houses four highly conserved functional domains throughout the species [3][4][5]. The N-terminal RGS-domain has been found to interact with the tumour suppressor gene adenomatous polyposis coli (APC) [1,6]. Central in the Axin protein, binding sites for β-catenin and for the glycogen-synthase kinase 3 beta (GSK-3β) were described [4]. At the C-terminal region, the DIX-domain is located that resembles the sequence of a DIX-domain in dishevelled protein (Dvl) and promotes its interaction with Axin [7]. At its C-terminus, Axin also interacts with the protein phosphatase 2A (PP2A) [3,8,9]. Being responsible for the degradation of the downstream canonical Wnt-signalling pathway molecule β-catenin, Axin and Axin2 function as negative regulators of the canonical Wnt-signalling pathway [5,[10][11][12]. The Wnt-signalling pathway is one of the best elucidated signalling pathways. First, the canonical Wnt-pathway was described, followed by at least two non-canonical pathways. The pathway of planar cell polarity (PCP) and the Wnt/Ca 2+ -pathway are referred to as non-canonical pathways [13][14][15]. These are described to establish orientation in epithelia (PCP) and to play a role in early embryonic ventral patterning (Wnt/Ca 2+ -pathway) ( [16] for review). Canonical and non-canonical Wnt-signalling are known to be enmeshed with each other, as their members partially contribute to more than one pathway [17,18] and several Wnt ligands were described to activate both canonical and non-canonical pathways [19][20][21][22]. However, in this study, only the canonical pathway is of relevance, constituting a venue for the [11] and exhibited malformations of the skull due to premature fusion of cranial structures [54]. This malformation is an equivalent to the human disease craniosynostosis, that is described to develop on the basis of Axin2 mutations [55]. Another developmental defect associated with Axin2 abnormalities in mice and human is familial tooth agenesis and oligodontia [56,56,57]. Further, Axin is related to hepatocellular cancer [58,59], ovarian cancers [60] and to medulloblastomas [61]. Axin2 mutations play a secondary role in familal adenomatous poliposis coli (FAP), when the causal mutation is not situated in APC and because proper function of APC requires Axin [62,63]. Predisposition to colorectal cancer, when carrying mutations in Axin2 is described [58,63]. Shedding similar functions than Axin, Axin2 was previously tested on its functional redundancy [54]. Axin2 was shown to be able to at least partially compensate for mutated Axin when expressed in the respective cells. Axin however, is expressed in small amounts in all embryonic tissues, while Axin2 expression was described to be restricted and dynamic during mouse development [11,45]. Interestingly, Axin was described to be the limiting factor in Wnt regulation, referring to its low cytoplasmatic levels [64]. Axin2 on the other hand, is highly expressed, suggesting an extensive role for Axin2 regulation in certain tissues. This observation, together with the fact that Axin2 is a target of Wnt-signalling, indicates the importance of Axin2 mediated negative regulation in certain tissues. In this study, we demonstrate the dynamic expression pattern of Axin2 in the development of the chick.

Materials and Methods Embryos
Fertilized eggs of Gallus gallus domesticus were incubated at 37°C and 80% relative humidity. Eggs were provided by a local breeder (Sörries-Trockels Vermehrungszucht). Staging was performed according to Hamburger and Hamilton [65].
The obtained chicken embryos were isolated, fixed in 4% PFA for at least 24h. For description and analysis of the expression pattern of Axin2 during chicken development, chicken embryos in developmental stages HH 04 to HH 32 were proceeded in in-situ hybridization.

Whole mount in-situ hybridization
Whole mount in-situ hybridization was performed as previously described [66], using cAxin2 riboprobe for detection of Axin2 transcripts in all embryonic tissue.
Generation of a riboprobe for in-situ hybridization. The probe for cAxin2 in-situ hybridization was generated from a pCMS-EGFP plasmid containing a full length Axin2 coding sequence. It was restricted using EcoRV and SmaI to obtain a 835bp fragment binding from bp926 to bp1788 on Axin2 mRNA (NCBI Reference Sequence: NC_006105.4). The purified fragment was blunted and cloned to pJET1.2/blunt Cloning Vector. From here, the fragment was excised using XbaI and XhoI and ligated to pBluescript II KS+ Vector. The obtained plasmid was suitable for generating a riboprobe in in vitro transcription.
Ethic statement. According to German legislation, the use of embryonic vertebrates in an animal experiment needs approval only if the animal is in the last third of its embryonic development. In the case of chicken, this means that experiments done on animals before embryonic day 14 (E14) are not regarded as an animal experiment by the Tierschutzgesetz, and therefore, do not need approval or governmental permission.
The chicken embryos sacrificed for this work were between developmental stages HH+04 (E1) and HH32 (E7.5). All embryos were sacrificed at the end of the study by opening the shell and tearing the allantois and amnion with forceps. Thereafter, the embryos were immersed in 4% PFA/PBS solution for fixation. No permits were required for the described study, which complied with all relevant regulations.
Results and discussion 0.1 Expression pattern of cAxin2 during early chicken embryogenesis After whole mount in-situ hybridization, a dynamic expression pattern of Axin2 was found from stages HH 04 to 32. In early embryogenesis, Axin2 expression was observed in the primitive streak (ps) (Fig 1, A black arrow, B, C, D) and in the Hensen's node (hn) (Fig 1, B red arrow, C red arrow, D, E). Additionally, the head fold (hf) heavily expresses Axin2 from stage HH 07 + onwards (Fig 1, B, C black arrows). During further development, in stage HH 10, Axin2 transcripts were detectable in the Hensen 0 s node (hn), posterior presomitic mesoderm (psm) (Fig 1,  E) and medially in the freshly segmented paraxial mesoderm (dml-dorso-medial lip) (Fig 1, E.1 red arrow). Transversal sections were performed to analyse the expression of Axin2 during early embryogenesis in detail. They present gastrulation and neurulation processes, where the maturation can be observed in a cranial to caudal axis. The green bars in the whole mount specimens indicate the sectioning level. Sections of HH stage 08 (Fig 1, C.1, C.2, C.3) show the caudally regressing primitive streak (ps) with the primitive groove (pg). The primitive folds (pf) of the ectoderm and the developing mesoderm underlying the primitive groove (pg) express Axin2 (Fig 1, C.1, C.2, C.3). Further, the transversal section of the head fold (hf) in HH stage 08 (C.4) shows intense expression of Axin2 in medial parts, facing towards the lumen of the anterior neuropore. In HH 09, during the primary neurulation process, cranially to Hensen's node (hn) (Fig 1, D, D.1, D.2), only little Axin2 is expressed in the neural groove (ng) (Fig 1, D.1, D.2) and in the elongating notochord (nc) (Fig 1, D.1 and D.2). At this stage the head folds (hf) at midbrain (mb) level have converged (Fig 1, D.3) and Axin2 expression is increased in the medial neural folds. In Fig 1, E.2, E.3, E.4 and E.5 (HH 10), the segmental plate mesoderm (spm) is formed, as the neural folds (nf) extend distally to form the neural tube (nt). Axin2 is expressed in the neural groove (ng) and in the notochord (nc) (Fig 1, E.4, E.5). At HH stage 10 more cranially, first somites (so) are shaped in the segmental plate mesoderm (Fig 1, E.2, E.3), as the neural folds (nf) fuse to form the neural tube (nt). In sections E.6 to E.8 (Fig 1), the development of the caudally shifted Hensen's node (hn) is depicted. Axin2 expression is restricted to the central Hensen's node (Fig 1, E.6, E.7, E.8) expanding towards the ventral axial mesoderm (am). In picture E.6 (Fig 1), the prechordal mesoderm (pcm) is heavily stained for Axin2.
In stage HH 14 at mid-brain level (Fig 2, B.1), the anterior neuropore has closed and Axin2 expression has shifted to a patch in the ventral mesoderm, flanking the mid-brain (mb) (Fig 2, B.1 black arrow). In HH stage 15, Axin2 expression is detectable in the developing sensory organs, eye and ear, for the first time (Fig 2, C.5). Axin2 mRNA was detected in the otic cup (oc) (Fig 2, C.5) and adjacent hind-brain (hb), as well as in the out-pocketing optic vesicle (ov) (Fig 2, C.5). The optic vesicle (ov) forms laterally from the prosencephalon, where Axin2 is transcribed in the medial wall. Previous studies have investigated the role of Wnt-signalling during gastrulation, neurulation, axis-and head formation. In the early patterning events of the vertebrate body, canonical Wnt-signalling is believed to first act as dorsalizing and later as posteriorizing signal [67,68]. In concordance to that, several Wnt-mutant mice exhibit truncated posterior axis, lost tail formation and disturbed somitogenesis [69,70]. Experiments in chicken and Xenopus resulted in axis duplication and disturbed head formation after Wnt overexpression [71]. Proper formation of the head requires Wnt inhibition in the anterior embryonic tissue [72][73][74]. Ectopic expression of Wnt inhibitors was found to induce notochord formation [75]. The examination of Axin knockouts revealed its function in ventralizing the respective tissue and in inhibiting posterior axis formation [11]. Furthermore, Axin loss of function in Xenopus resulted in disturbed closure of neural folds, head folds and the duplication of the allantois [76]. These findings together with the our new observed expression of Axin2 during chicken embryogenesis support the idea that appropriate regulation Wnt-signalling via Axin2 influences body patterning, axis elongation and head formation. The expression of several Wnts in the chicken primitive streak and Hensen's node reinforce this hypothesis [77].

Expression pattern of cAxin2 in stages HH 17 to 32
At HH stage 17, the chicken limb buds (lb) are distinguishable, expressing Axin2 mRNA from their onset (

Axin2 expression during somitogenesis
In transversal sections of in-situ hybridized chicken embryos, Axin2 expression was found during somitic differentiation (Fig 5). Green bars in the whole mount specimens (A, B, C, D, E, F, G) indicate the levels, where the sections have been performed. In the segmented paraxial mesoderm, Axin2 is expressed in the epithelial somites and in the differentiating dermomyotome. At HH stage 15 transcripts are mainly detectable in the medial and medio-dorsal wall of the  C.1 black arrow) and in a patch adjacent to the ventral neural tube (nt) (Fig 5, C.1 red arrow). In stage HH 19, when the dermomyotome is fully established, transcripts are visible in the ventrally facing margin of the dml, neighbouring the sclerotome (Fig 5, HH 19: D.1 black arrow). In HH stage 20, at limb level Axin2 expression is detectable also in the ventro-lateral lip (vll) (Fig 5, E.1 wing level). In further development, this expression gets restricted to the dorsal half of the dermomyotome (dm), the epaxial myotome and appears more faintly (Fig 5, E.2 interlimb level). In mice Axin2 expression was found to oscillate in the segmental plate mesoderm and to occupy a central role for the segmentation of the presomitic mesoderm [50,78]. We were able to detect Axin2 expression in the posterior psm in chicken from stage HH 09 to HH 16 (Figs 1  and 3). In mice, the expression of Wnt-genes alternates with the expression of FGFs in the PSM [78], indicating a similar mechanism in chicken. Interestingly Axin2 mutant mice still undergo segmentation with slight to average deviation [55,78]. Additionally, Axin2 transcripts were found during the maturation of the somites. In this process, a network of many different Wnt-molecules and other signals is described to play a role. The patterning of the somites is controlled by dorsalizing Wnt1 and Wnt3a from the dorsal neural tube [79][80][81], such as Wnt6 from the overlying ectoderm [82]. Wnt11 was described to maintain the epithelial status of the dml, while Wnt6 from the ectoderm maintains the epithelial ventro-lateral lip (VLL) [83]. Additionally, it was found that Wnt1 and Wnt3a are required for the formation of the dml [81]. Axin2 expression in the dml and its progenitors (Fig 5) indicate a potential role in the proper development of the dml and the deriving dermis. This hypothesis is supported when regarding the expression of Axin2 in the dermal derived feather buds (Fig 4, H.1, I.1).

Expression pattern in the developing neural tube
Regarding the development of the neural tube, Axin2 is expressed from neurulation to the differentiated mature neural tube (nt) (Figs 2 and 5). In Fig 5, the maturation of the neural tube (nt) is depicted. First, Axin2 mRNA was detected in a sprinkled distribution all over the neural tube (nt) (Fig 5, HH 15 Axin2 transcripts were also found in secondary neurulation in the tail bud (Fig 2, C.2, C.3, C.4). After secondary neurulation, the differentiating secondary neural tube (snt) heavily expresses Axin2 (Fig 5, H.1, H.2, H.3). First, this expression is well distributed over the entire neuroepithelium (Fig 5, HH 24: H.1). During maturation, transcripts were observed in HH 27, (Fig 5, H.2) mainly in the dorsal half of the secondary neural tube (snt) as in the overlying subectodermal mesenchyme and ectoderm. By HH 28 the Axin2 is missing in the ventral two thirds of the secondary neural tube (snt), but is expressed intensively in the dorsal third, such as in the ectoderm and subectodermal mesenchyme (Fig 5, H.3).
During the development and maturation of the neural tube, the establishment of a dorsoventral axis through ventralizing Shh activity versus dorsalizing Wnt-signals has been described [84,85]. The main Wnt-genes expressed in the dorsal neural tube and roof plate are Wnt1 and Wnt3a [84,86]. These promote neural proliferation [84,87]. Therefore, after activation of dorsal Wnt-signalling in the chick, dorso-ventral patterning of the neural tube was perturbed and mitogenic activity of neural progenitors was increased [88]. Wnt1 and Wnt3a inhibition in mice, besides incomplete closure of the neural folds, displayed phenotypic alterations throughout the neural tube including partially absent basal-, roof-and floor plates [89]. In addition, Wnts have been identified to play a role in ventrally specified neural progenitors [86,90]. The countless signalling molecules interacting with the Wnt-signalling pathway during neural tube maturation imply that Axin2 expression and its negative-feedback-loop in canonical Wnt-signalling impact this neural development and the basic molecular functions will be of special interest in future research.

Expression pattern of cAxin2 during limb development
Limb development in chicken starts from an out-bulged ridge of the somatic lateral plate mesoderm by stage HH 15. At HH stage 17 the wing bud heavily express Axin2 predominantly in the dorsal mesenchyme (Fig 6, A.1 black arrow). The hind-limb bud at the same stage is slightly further developed and transcripts of Axin2 are present in the thickened ectoderm, which gives rise to the apical ectodermal ridge (aer) (Fig 6, A.2), as well as at proximo-ventral margin of the lateral plate mesoderm (Fig 6, A.2 black arrow). In stages HH 18 to HH 20 Axin2 is expressed in the dorsal mesenchyme of the rapidly outgrowing limb buds (Fig 6, B Several members of the Wnt family are expressed in the developing limb ([37] for review). The outgrowth of the limb bud is mediated by the apical ectodermal ridge (aer) [91]. Wnt genes are described to initiate the formation of the limb bud (Wnt2b) from the lateral plate mesoderm as well as the aer (Wnt3a) [92]. The aer in chicken expresses Wnt3a that, by initiating fibroblast growth factor (FGF) expression, mediates the rapid cell proliferation in the mesenchymal progress zone (PZ) underlying the aer [93]. Non-canonical Wnt7a is expressed in the dorsal ectoderm of the chicken limb, being responsible for dorsalization [94,95]. Its expression site overlaps an additional expression site for Wnt3a in the ectoderm during early limb growth [96]. As Wnt7a target genes are expressed in the mesenchyme underlying the dorsal ectoderm, it was suggested that their signalling ranges as far as the target gene expression [97]. We postulate a similar distance of signalling for the canonical Wnt3a from early dorsal limb ectoderm as a source for early Axin2 expression in the dorsal limb mesenchyme. Mutations of Wnt3a and Wnt7a and FGFs in chicken embryo induced the expression of a gene responsible for a form of polydactyly in human, the Townes-Brock-Syndrome [98]. Later in the limb development, canonical Wnt-signalling is described to promote cell proliferation and the differentiation of connective tissue [99]. Axin2 expression in accordance to our results was reported in the perichondrium of mice [99]. By describing the expression of Axin2 in the chicken developing limb, we want to reveal its presumable function in regulating Wnt-signals that are involved in outgrowth, proliferation and differentiation.

Expression patten of Axin2 during chicken nephrogenesis
The kidney development in birds and mammals takes place in three generations of nephric precursors [100]. In this study, an Axin2 expression in mesonephric development is described (Fig 7). In stage HH 19 the mesonephric duct (md) at leg level faintly expresses Axin2 (Fig 7, A.1 black arrow). By HH 20 at interlimb level, the staining expands to the overlying coelomic epithelium (coe) (Fig 7, B.1 black arrow).
The role of Wnt in the developing kidney has been extensively studied in the past. Wnt4 and Wnt9b were described to be expressed in the nephric duct and coeloemic epithelium [101][102][103]. The initiation of tubulogenesis of the developing kidney requires canonical Wnt4 and Wnt9b signals from the nephric duct [101,102]. Later in development both Wnt-ligands were described to act through the PCP and the Ca2+-dependent pathway as well [104][105][106][107]. As the Wnt-ligands partially activate different intracellular responses in the course of kidney development, the research faces a challenging aim in understanding this network. In Xenopus a model mediating the switch from canonical to non-canonical Wnt-signalling during nephrogenesis was proposed [108,109]. However, canonical Wnt-signalling is known to mediate not only nephron induction, but also its orientation, cell proliferation, specification and differentiation [107,[110][111][112][113]. Alterations in canonical and non-canonical Wnt-signalling are known to cause polycystic kidney diseases [114,115]. Taken together, we suggest that Axin2 might impact kidney development by regulating Wnt-signalling as indicated, through its expression in the nephric duct and coelom epithelium. The Axin2 expression in the coelomic epithelium could possibly hint a role for Axin2 in the development of the derived Mullerian-duct that develops to form the female genitals. As male gonads develop from the nephric or Wolffian-duct, Axin2 might be involved in this developmental process as well.

cAxin2 expression in developing chicken eye
The chicken eye initially develops, as the prosencephalon out-pockets and the optic vesicle (ov) invaginates to the head mesenchyme. Axin2 in this process is expressed in the proximal layer of the bi-layered optic vesicle (ov) (Fig 8, HH 15: A.1, black arrow). By stage HH 16 the lens vesicle (lv) has formed from the ectoderm (Fig 8, B.1). Axin2 transcripts are still detectable mainly in the proximal layer of the optic cup (oc) (Fig 8, B.1, HH 17: C.1 black arrow). An additional expression in the subectodermal mesenchyme overlying the optic cup (oc) and surrounding the lens vesicle (lv) is established at stage HH 18 (Fig 8, D.1 black arrow). While the lens vesicle (lv) expresses little Axin2 in the inner lens epithelium (Fig 8, HH 19: E.1 and HH 20: F.1, F.2. F.3 red arrows), transcripts in the optic cup (oc) are found in both proximal and distal layer at the epithelial margins facing towards the vesicular space (Fig 8: HH 19: E.1 black arrow, HH 20: F.4 black arrow). In the following observed stages, the proximal layer of the optic cup (oc) has formed the retinal pigmented epithelium (rpe), whereas the distal layer differentiates into the retina [116]. Axin2 expression was found only in the lens (Fig 8, HH 24: H.3 red arrow), ectoderm and subectodermal mesenchyme covering the eye (Fig 8, HH 24: H.1 and H.2 black arrows). Regarding the formation of the optic nerve (on) and optic chiasm (och), Axin2 expression is observable in the approaching and fusing neuroepithelial layers (Fig 8, HH  Anteriorly expressed inhibitors of canonical Wnt signals are required for the initiation of the eye as described in zebrafish [117,118]. Later, Wnt2b is expressed in the proliferative lens epithelium [119], retinal pigmented epithelium (rpe) and periphery of the optic cup [120][121][122]. Further, Wnt3 and Wnt11 were found to be expressed in the outer layer of the chicken optic cup [122]. Wnt2b was described to be responsible for maintaining the proliferative state of neural progenitors in the retina in chick [123]. Previous studies have reported a depigmentation of the retinal pigmented epithelium (rpe) after disruption if Wnt2b signalling in the chicken eye [120]. Our observeded Axin2 expression in the lens overlaps with regions of increased cell proliferation, which express Wnt-ligands as well [122,124,125]. The chicken developing cornea and corneal stroma cells express Wnt3a and Wnt9b [126]. Interestingly, a subgroup of the disease familial adenomatous poliposis coli (FAP), which is caused by a truncation in APC or Axin2, the Gardner syndrome, includes a congenital hypertrophy of the rpe [127]. Additionally, some cases of tetra amelia, which is the result of homozygous Wnt3 mutations, exhibit optic malformations [128].

Conclusion
In the present study, we describe the expression pattern of avian Axin2 during embryonic development. We found a dynamic, temporally and spatially restricted expression pattern in many developing structures and tissues. In the early development of the chick, Axin2 was Expression Pattern of Axin2 During Chicken Development expressed in the primitive streak and underlying mesoderm, in the neural folds and in the head fold. It was additionally expressed during secondary neurulation in the tailbud mesenchyme. Here, the pre-somitic mesoderm as well transcribes Axin2. We were able to detect such expression in the posterior psm and during the maturation of the somites in its medial epithelium and in the dml. By this developmental stage, transcripts were also detectable in the brain and differentiating neural tube. In the developing limb a dynamic expression was found. Furthermore, we detected Axin2 mRNA in the nephric duct and coelomic epithelium. Regarding the head of the chicken embryo, Axin2 was expressed in branchial arches and sensory anlagen. Later in development, expression in feather buds, interdigital spaces, external ear and scleral ossicles on the eye was observed.
The expression of Axin2 in mice was previously found in the primitive streak, head folds, neural tube, branchial arches I and II (maxillary and mandibular arch), psm and dml, tailbud, limbs, kidney and brain [45,78].
These findings are mainly consistent to the expression we found in the chick. Additionally, we were able to show Axin2 expression in the developing eye and in the otic vesicle. With this study we want to point out the often neglected impact of Axin2 in many Wnt-dependant developmental processes. While Wnt-ligands are extensively studied, investigating their regulation through Axin2 in the respective tissues might help understanding the interactions of different signalling factors.