Chemokine Signaling during Midline Epithelial Seam Disintegration Facilitates Palatal Fusion

Abstract

Disintegration of the midline epithelial seam (MES) is crucial for palatal fusion, and failure results in cleft palate. Palatal fusion and wound repair share many common signaling pathways related to epithelial-mesenchymal cross-talk. We postulate that chemokine CXCL11, its receptor CXCR3, and the cytoprotective enzyme heme oxygenase (HO), which are crucial during wound repair, also play a decisive role in MES disintegration. Fetal growth restriction and craniofacial abnormalities were present in HO-2 knockout (KO) mice without effects on palatal fusion. CXCL11 and CXCR3 were highly expressed in the disintegrating MES in both wild-type and HO-2 KO animals. Multiple apoptotic DNA fragments were present within the disintegrating MES and phagocytized by recruited CXCR3-positive wt and HO-2 KO macrophages. Macrophages located near the MES were HO-1-positive, and more HO-1-positive cells were present in HO-2 KO mice compared to wild-type. This study of embryonic and palatal development provided evidence that supports the hypothesis that the MES itself plays a prominent role in palatal fusion by orchestrating epithelial apoptosis and macrophage recruitment via CXCL11-CXCR3 signaling.

Keywords: apoptosis; chemokine; cleft palate; embryology; heme oxygenase; macrophage.

Figure 1

(A) Isolation of the fetuses and measurement of body length, body surface, head surface. Plugged mouse (e.g., HO-2 KO at E15) was sacrificed by CO₂/O₂ inhalation for 10 min, the uterus and organs were removed. Fetuses were isolated from the uterus. Location of the 6 fetuses in the uterus before they were removed (green arrows). Location of the 5 non-viable/hemorrhagic embryonic implantations (red arrows). (B) A square scale bar was drawn at the ruler in each photograph of 10 × 10 mm (1 cm²) and the total number of pixels within the square was determined (e.g. 13,225 pixels). (C) A line in the length of the body of the fetus was drawn and the number of pixels was recorded (e.g. 158 pixels). The length was calculated (e.g. 158/√13,225 = 13,7 mm). (D) The outline of the total body surface of the fetus was drawn and the number of pixels was recorded (e.g. 11,733 pixels). The total body surface was calculated (e.g. 11,733/13,225 = 0.89 cm²). (E) The outline of the head surface was drawn and the number of pixels was recorded (e.g. 4,761 pixels). The head surface was calculated (e.g. 4761/13,225 = 0.36 cm²).

Figure 2

Palatal fusion observed in both wt and HO-2 KO fetuses at E15. HE stainings demonstrated horizontal orientation of the palatal shelves, midline adhesion and fusion within the same fetus. (A) Wt fetus at E15 (magnification: x100). Palatal shelves in a later stage of the palatal fusion increased in size. The MES changed from a multi-cell-layer into a continuous one-single-cell-layer, to a disintegrating MES, during which islands of epithelium in the midline were observed. (B) HO-2 KO fetus at E15 (magnification: x100). Palatal shelves in a later stage of the palatal fusion increased to some extent in size. Several islands of epithelium in the midline were observed.

Figure 3

Palatal morphology classification: The CXCL11, CXCR3 and HO-1 immunostained sections were categorized in two stages according to their morphological characteristics: fusing palatal shelves, and fusing palatal shelves with adhesion to the nasal septum. Epithelium region classification: For each section epithelial layers were subdivided in 3 regions of interest according to morphological characteristics: epithelium of the palatal shelves from the edge, including the MES, to half of the width of the shelves (in RED), epithelium of the lateral half of the palatal shelves (in BLUE), epithelium of the lateral wall of the nasal cavity, this region is positioned outside the palatal shelves and served as a control region (in YELLOW). Immunoreactivity scoring scale: Semi-quantitative scoring of CXCL11, CXCR3 and HO-1 immunoreactivity in epithelium of the palatal shelves. Each epithelial region was semi-quantitatively scored according to the following scale: HIGH, Immunoreactivity present in the entire epithelial region; MODERATE, Immunoreactivity present only partially in the epithelial region; LOW, Almost no immunoreactivity present in the epithelial region. Right lower panel: Immunoreactivity scored for the 3 epithelial locations in a CXCL11 immunostained section (e.g. wt fetus, E15, section with adhesion of the palatal shelves and adhesion to the nasal septum). RED region was scored as HIGH, BLUE region was scored as MODERATE, YELLOW region was scored as LOW for CXCL11 immunoreactivity.

Figure 4

Palatal shelf surface measurement for determining the number of CXCL11, CXCR3 and HO-1 positive immunostained cells cells/mm² within the mesenchyme of the palatal shelves. A square scale bar was drawn in the microscopic picture (magnification: x100) of the section of 1,000 × 1,000 μm (1 mm²) and the total number of pixels within the square was determined (e.g. 1 mm² = 3,442,880 pixels). The contour of the mesenchyme of the shelves was drawn (yellow line). The number of pixels for this area was determined by the ImageJ (1.48 v) software (323,768 pixels). The number of positive immunostained cells within this mesenchymal area of the palatal shelves were counted by direct observation using the Zeiss microscope (e.g. 52 CXCL11 positive cells). The number of cells/mm² was calculated (3,442,880/323,768 × 52 = 553 cells/mm²).

Figure 5

Fetal growth restriction and malformations occur in the absence of HO-2 expression. (A) HO-2 mRNA was not found in HO-2 KO fetuses. HO-2 mRNA was observed in wt fetuses E15 (n = 5), but not in HO-2 KO fetuses E15 (n = 4). Wt fetuses (E15; n = 15), HO-2 KO fetuses (E15; n = 4), and HO-2 KO fetuses (E16; n = 11) were compared for (B) weight, (C) body surface, and (D) length (P = 0.25), (E) head/body surface ratio (P = 0.97). Data presented as mean ± SD. (^*P < 0.05; ^**P < 0.01), (^***P < 0.001). (F) wt fetus at E15 (0.28 g; 12.9 mm). (G) HO-2 KO fetus at E15 (0.13 g; 12.5 mm). (H) HO-2 KO fetus at E16 (0.31 g; 12.4 mm). (I) HO-2 KO fetus at E16 demonstrating severe malformations (0.065 g; 10 mm). (J) HO-2 KO fetus at E16 demonstrating a craniofacial anomaly (0.20 g, 12.2 mm). (K) HO-2 KO fetus at E15 appeared to be the smallest fetus without anomalies (0.10 g, 10.3 mm).

Figure 6

CXCL11 expression in the MES and mesenchyme in both wt and HO-2 KO fetuses. (A) CXCL11 mRNA expression was both present in wt (n = 5) and in HO-2 KO E15 fetuses (n = 4; P = 0.88). Data presented as mean ± SD. CXCL11 overexpression in the MES in wt and HO-2 KO fetuses. Scoring was performed according to Figure 3. (B) Significant higher CXCL11 expression was observed in the MES (in RED; ^***P < 0.001) compared to the other epithelial regions in the fusing palatal shelves (in BLUE) and the nasal cavity (in YELLOW) in the wt group and HO-2 KO group. (C) Significant higher CXCL11 expression was observed in the MES (in RED; ^***P < 0.001) compared to the BLUE region and YELLOW region in the HO-2 KO sections with fusing palatal shelves with adhesion to the nasal septum. (D) No significant difference in the number of CXCL11 positive cells/mm² in the mesenchyme of the fusing palatal shelves was found between the wt and HO-2 KO fetuses) (P = 0.97). Data presented as mean ± SD. (E) No significant difference in the number of CXCL11 positive cells/mm² was found in the mesenchyme between the wt and HO-2 KO group in the sections with fusing palatal shelves with adhesion to the nasal septum(P = 0.97). Data presented as mean ± SD. (F) Representative CXCL11 immunostaining in fusing palatal shelves without adhesion to the nasal septum of a wt fetus (E15) (magnification: x100). The MES (in RED) was highly CXCL11 positive compared to the other epithelial regions; epithelium of the lateral half of the palatal shelves (in BLUE) and epithelium of the lateral wall of the nasal cavity (in YELLOW). (G) Several CXCL11 positive cells in the mesenchyme were observed (e.g. black arrow indicates a CXCL11 positive cell in the mesenchyme) (magnification: x400). This was found in both wt and HO-2 KO fetuses. (H) Moderate CXCL11 expression in the epithelium of the lateral half of the palatal shelve (in BLUE), and in the epithelium of the lateral wall of the nasal cavity (in YELLOW) (magnification: x400).

Figure 7

CXCR3 expression in the MES and mesenchyme in both wt and HO-2 KO fetuses. (A) CXCR3 mRNA expression was found in wt fetuses E15 (n = 5) and in HO-2 KO fetuses E15 (n = 4; P = 0.16). Data presented as mean ± SD. (B) Statistically significant higher CXCR3 expression was observed in the wt group in the MES (in RED; ^*p < 0.05) compared to the YELLOW region. (C) Significant higher CXCR3 expression was observed in the MES (in RED) (^***P < 0.001) compared to the BLUE region and YELLOW region in the HO-2 KO sections with adhesion of the palatal shelves and adhesion to the nasal septum. (D) No significant difference in the number of CXCR3 positive cells/mm² in the mesenchyme of the fusing palatal shelves was found between the wt and HO-2 KO fetuses (P = 0.96). Data presented as mean ± SD. (E) No significant difference in the number of CXCR3 positive cells/mm² in the mesenchyme was found between the wt and HO-2 KO group of the sections with adhesion of the palatal shelves and adhesion to the nasal septum(P = 0.47). Data presented as mean ± SD. (F) Representative CXCR3 immunostaining of fusing palatal shelves without adhesion to the nasal septum of a wt fetus (E15) (magnification: x100). The MES (in RED) had higher CXCR3 expression compared to the other epithelial regions (in BLUE) and (in YELLOW). (G) Some CXCR3 positive cells in the mesenchyme were observed. This was found for the wt sections and HO-2 KO sections (black arrow indicates a CXCR3 positive cell in the mesenchyme) (magnification: x400). (H) Moderate CXCR3 expression in the epithelium of the lateral half of the palatal shelve (in BLUE), and in the epithelium of the lateral wall of the nasal cavity (in YELLOW) (magnification: x400).

Figure 8

CXCR3 positive macrophages were located near the MES. (A) Representative immunofluorescent histochemical double staining for F4/80 (red) with CXCR3 (green) in HO-2 KO section (E16). Nuclear staining with DAPI (Blue). The mesenchyme demonstrates the presence of a F4/80 positive HO-2 KO macrophages, which also express CXCR3 (white arrows) (magnification: x200). One CXCR3 F4/80 positive HO-2 KO macrophage was located near the disintegrating MES (white arrow within the white square). (B) Magnification of a F4/80 positive HO-2 KO macrophage located near the MES (area between the dotted white lines) (magnification: x400). (C) Magnification of a F4/80 positive HO-2 KO macrophage located in the mesenchym of the palate shelf (magnification: x400). (D) Magnification of a F4/80 positive HO-2 KO macrophage located outside the palatal shelf (magnification: x400).

Figure 9

Macrophages located near the MES phagocytose apoptotic cell fragments. (A) Representative FragEL™ DNA fragmentation assay (brown) in combination with F4/80 (dark blue) macrophage staining. Fusing palatal shelves in a HO-2 KO fetus (E16) (magnification: x100). Multiple macrophages were observed in the mesenchyme of the palatal shelves. (B) Magnification of the disintegrating MES (magnification: x400, black square). Multiple apoptotic DNA fragments are observed within the MES (Red arrows). The only apoptotic DNA fragments in the palatum outside the MES are in macrophages that had taken up epithelial cells seen. Two macrophages were located near the MES (green arrows), one macrophage was in close contact with the MES (red circle). (C) Magnification of the macrophage in close contact with the MES (black square). In this macrophage (red circle), two apoptotic cell fragments within its cell body are present (purple arrows). Apoptotic DNA fragments near the macrophage were observed (red arrows). These findings were representative for both wt and HO-2 KO sections.

Figure 10

More HO-1-positive cells are found in palatal shelves from HO-2 KO fetuses. (A) HO-1 mRNA expression was similar in wt fetuses E15 (n = 5) and in HO-2 KO fetuses E15 (n = 4; P = 0.35). Data presented as mean ± SD. (B) Representative fluorescent immunohistochemical double staining for F4/80 and HO-1 of fusing palatal shelves with adhesion to the nasal septum of a wt fetus (E15) (magnification: x400). A part of the mesenchyme around the MES, showing a F4/80 positive macrophage (red) expressing HO-1 (green) located (white arrow). Nuclear staining with DAPI (Blue). (C) Representative fluorescent immunohistochemical double staining for F4/80 with HO-1 of fusing palatal shelves with adhesion to the nasal septum of a HO-2 KO fetus (E16) (magnification: x400). A F4/80 positive HO-2 KO macrophage (red) expressing HO-1 (green) located near the MES (white arrow). Nuclear staining with DAPI (Blue). (D) Representative HO-1 immunostaining of palatal shelves of a wt fetus (E15) (magnification: x400). This part of the mesenchyme demonstrates the presence of one HO-1 positive cell (black arrow). (E) Representative HO-1 immunostaining of fusing palatal shelves of a HO-2 KO fetus (E16) (magnification: x400). This part of the mesenchyme demonstrates the presence of seven HO-1 positive cells (black arrows). (F) Significant higher numbers of HO-1 positive cells/mm² were observed in the HO-2 KO fetuses compared to the wt fetuses in the fusing palatal shelves (P = 0.02). Data presented as mean ± SD. (G) Numbers of HO-1 positive cells/mm² in the mesenchyme of wt and HO-2 KO fetuses in the fusing palatal shelves with adhesion to the nasal septum(P = 0.60). Data presented as mean ± SD.

Figure 11

MES mediated chemokine signaling facilitates MES disintegration and palatal fusion. Conceptual model: Autocrine and paracrine MES signaling facilitates palatal fusion. CXCL11/CXCR3 autocrine signaling controls migration and/or apoptosis of epithelial cells during disintegration of the MES. CXCL11-CXCR3 paracrine signaling recruits macrophages to clean up the MESs. HO-2 KO macrophages are still able to phagocytize apoptotic DNA fragments from the MES due to induction of HO-1.