Expression of the Wnt Receptor Frizzled‐4 in the Human Enteric Nervous System of Infants

Abstract

The Wnt signalling pathway plays a crucial role in the development of the nervous system. This signalling cascade is initiated upon binding of the secreted Wnt ligand to a member of the family of frizzled receptors. In the present study, we analysed the presence of frizzled-4 in the enteric nervous system of human infants. Frizzled-4 could be identified by immunohistochemistry in a subpopulation of enteric neuronal and glial cells in the small and large intestine. Detection of frizzled-4 in the tunica muscularis by RT-PCR confirmed this receptor's expression on the mRNA level. Interestingly, we observed distinct cell populations that co-expressed frizzled-4 with the intermediate filament protein nestin and the neurotrophin receptor p75NTR, which have been reported to be expressed in neural progenitor cells. Flow cytometry analysis revealed that 60% of p75NTR positive cells of the tunica muscularis were positive for frizzled-4. Additionally, in pathological samples of Hirschsprung's disease, the expression of this Wnt receptor correlated with the number of myenteric ganglion cells and decreased from normoganglionic to aganglionic areas of large intestine. The expression pattern of frizzled-4 indicates that this Wnt receptor could be involved in postnatal development and/or function of the enteric nervous system.

Wnt Receptor Frizzled-4 as a Marker for Isolation of Enteric Neural Progenitors in Human Children

“…The present study confirmed our previous findings [25] showing the expression of frizzled-4 and p75 NTR in myenteric ganglia in cryosections of the human colon (Figure 1A). We found considerable expression of both markers within the ganglia (also with a large overlap), but no immune-reactivity in surrounding muscle layers.…”

“…In order to obtain purified spheroids containing exclusively frizzled-4 positive cells, we stained cell suspensions from human Tunica muscularis with a PE-conjugated anti-frizzled-4 antibody and subsequent used fluorescence-activated cell sorting (FACS). Similar to our previous results by Nothelfer et al [25], we found that frizzled-4 positive cells make up a numerically stable (15.0 ± 7.6% of counts in parent population in five independent patients, Figure 2) and very distinct cell pool, clearly distinguishable from the rest of the Tunica muscularis cell population (Figure 2). As previously shown, these cells are furthermore a subpopulation of the P75 NTRpositive cell population [25], which is commonly used for the isolation of enteric neural progenitors [27,28].…”

“…Extensive ligand-receptor-interaction studies also indicate that frizzled-4 and Wnt3a bind with high affinity to initiate the canonical Wnt pathway [26]. Additionally, we found that frizzled-4 is expressed by some mature neurons and glial cells but also by a subpopulation of cells expressing the potential stem cell marker P75 NTR [25] and also exhibits a considerable co-expression with nestin in human enteric ganglia [25]. Thus, we hypothesize that frizzled-4 is expressed by putative neural progenitors of the human ENS and might be a more precise marker than the commonly used P75 NTR stainings.…”

“…Moreover, our previous immunohistological study shows the co-expression of the Wnt-receptor frizzled-4 (Fzd4) with the putative stem cell markers nestin and the neurotrophin-receptor P75 NTR in the human ENS. Flow-cytometric analysis verified that frizzled-4 positive cells represent a subpopulation of the P75 NTRpositive cell pool [25].…”

Recent developments in cell-based ENS regeneration – a short review

“…Although a concentration of proliferating neural cells in vitro can be achieved by cell sorting, the combination of markers that will isolate the highest and purest amount of enteric neural progenitors is still unknown. We recently found Fizzled-4, a Wnt receptor, to define a subpopulation of p75-sorted cells [33]. Preliminary data demonstrate that only p75+/Fzd4+, but not p75+/Fzd4− cells proliferate in vitro .…”