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First published online August 4, 2003
doi: 10.1242/10.1242/dev.00631

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Role of Islet1 in the patterning of murine dentition

Thimios A. Mitsiadis1,2,*, Irene Angeli1,*, Chela James1, Urban Lendahl3 and Paul T. Sharpe1,{dagger}

1 Department of Craniofacial Development, GKT Dental Institute, Kings College London, Floor 28 Guy's Tower, Guy's Hospital, London SE1 9RT, UK
2 Laboratoire de Biologie Moléculaire et Cellulaire, UMR 5665 CNRS/ENS Lyon, Ecole Normale Supérieure de Lyon, 46 allée d'Italie, 69364 Lyon Cedex 07, France
3 Department of Cell and Molecular Biology, Karolinska Nobel Institutet, SE-171 77 Stockholm, Sweden



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  		Fig. 1. Expression of the Isl1 gene and protein (ISL1) in the developing mandibular and maxillary processes of E9-E10.5 mouse embryos. Whole-mount in situ hybridisation using a digoxigenin-labelled probe (A-D) and whole-mount immunohistochemistry (E). (A,B) Frontal views of E9 and E9.5 mouse embryos showing strong Isl1 mRNA expression (violet colour) in the distal parts of the oral epithelium and in a part of the nasal epithelium. (C) Lateral view of an E10 embryo showing Isl1 expression in distal parts of the oral epithelium. (D) Higher magnification of C showing Isl1 expression in oral epithelium and the trigeminal ganglion. (E) Lateral view of the head of an E10.5 mouse embryo demonstrating the distribution of the ISL1 protein in the distal parts of the oral epithelium and in the trigeminal ganglion. e, eye; fb, forebrain; fn, frontonasal process; md, mandibular process; mx, maxillary process; oc, oral cavity; t, tail; tg, trigeminal ganglion.

 


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  		Fig. 2. Patterns of the Isl1 gene and protein expression in developing teeth of E10-E13.5 mouse embryos. Digoxigenin in situ hybridisation (A,B,F-H) and immunohistochemistry (C-E). (A) Sagittal section through the head of an E10 mouse embryo shows Isl1 expression in the oral epithelium where incisors will develop (placodes of incisors). (B) Section of an E10 mouse embryo demonstrating Isl1 expression in the epithelium of the upper incisor and the absence of Isl1 transcipts in epithelium of the lower molar. (C) Sagittal section of an E11.5 mouse embryo demonstrating ISL1 protein distribution in the distal part of the oral epithelium and the epithelium of the incisors. (D) Section of an E11.5 mouse embryo showing ISL1 protein expression in the oral epithelium of the maxilla and the trigeminal ganglion. Note the absence of ISL1 in the epithelium of the lower molar (red dotted line). (E) Frontal section. Higher magnification of an E11.5 incisor. Note the nuclear localisation of the protein. (F) Frontal section. Higher magnification of an E11.5 molar. Isl1 transcipts are observed in a restricted part of the epithelium (lingual side) of the developing molar. (G) Frontal section through the head of an E13.5 mouse embryo showing Isl1 expression in the oral epithelium and the epithelium of a developing upper incisor. The gene is absent in the aboral epithelium. (H) Frontal section through the head of an E13.5 mouse embryo demonstrating the absence of Isl1 in the epithelium of a developing upper molar. ab, aboral epithelium; de, dental epithelium; ey, eye; i, incisor territory; inc, incisor; Lab, labial side; Lin, lingual side; m, molar territory; md, mandibular process; mes, mesenchyme; mol, molar; mx, maxillary process; frontonasal process; oc, oral cavity; oe, oral epithelium; tg, trigeminal ganglion.

 


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  		Fig. 3. Patterns of ISL1 protein distribution in developing mouse incisors. Frontal (A,C) and longitudinal (B,D-I) sections. (A) ISL1 expression in the epithelium of an E14 upper incisor. (B) ISL1 distribution in the epithelium of an E16.5 lower incisor. The line represents the level of the frontal section in C. (C) ISL1 protein is restricted in the epithelium of the E16.5 incisor. (D,E) Higher magnifications showing in detail the expression pattern of ISL1 in the E16.5 incisor. Note the strong nuclear staining in pre-ameloblasts and inner dental epithelial cells. ISL1 is absent in dental papilla mesenchyme. (F) High magnification showing in detail the distribution of ISL1 in epithelial cells of the posterior part of a P1 incisor. Note the heavy nuclear labelling in pre-ameloblasts, stratum intermedium and outer dental epithelium. (G) High magnification showing the expression of ISL1 in epithelial cells of the anterior part of a P1 incisor. Note the strong nuclear staining in functional ameloblasts and the faint staining in cells of the outer dental epithelium. a, ameloblasts; ab, alveolar bone; d, dentin; de, dental epithelium; df, dental follicle; e, enamel; ide, inner dental epithelium; La, labial side; Li, lingual side; o, odontoblasts; ode, outer dental epithelium; p, dental papilla; pa, pre-ameloblasts; pd, predentine; si, stratum intermedium; sr, stellate reticulum.

 


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  		Fig. 4. ISL1 protein distribution in developing mouse molars. Frontal (A) and longitudinal (B-D) sections. (A,B) ISL1 protein is absent in dental epithelial cells of E15.5 (A) and P1 (B) molars. Red dotted lines in A indicate the cup shape of the enamel organ of a developing lower molar. (C) High magnification showing the absence of ISL1 in functional ameloblasts of a P1 molar. (D) High magnification demonstrating nuclear ISL1 staining in ameloblasts of the enamel-free cusp region of a P4 molar. a, ameloblasts; ab, alveolar bone; d, dentin; df, dental follicle; e, enamel; eo, enamel organ; o, odontoblasts; oe, oral epithelium; p, dental papilla; si, stratum intermedium.

 


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  		Fig. 5. Effects of SHH, BMP4 and FGF8 signalling molecules on Isl1 gene expression in oral epithelium of E9-E10.5 mouse mandibular explants cultured in vitro. (A,B) Oral views of E9 mouse mandibles implanted with SHH- (A, violet), BSA- (A, black), FGF8- (B, green) and BMP4 (B, red)-soaked beads showing ectopic Isl1 induction in oral epithelium only by BMP4. (C-F) Oral views of E9.5 (C,D), E10 (E) and E10.5 (F) mandibles cultured together with BMP4-beads. Isl1 expression is strongly induced in oral epithelium where molars will develop. b, bead; f, median area of the mandible; i, incisor territory; m, molar territory; t, tongue.

 


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  		Fig. 6. Effects on Isl1 and Bmp4 expression of electroporation of Bmp4 and Isl1 expression constructs into the epithelium of E10 mandibular explants. (A) E10 mandibular explant prior to electroporation. (B) Schematic representation of the position of electrodes (red arrows) and glass needle containing the construct (green colour). (C) GFP expression of the Bmp4-pIRES2-EGFP construct (green colour, orange arrowhead). (D) Superimposition of A and C showing the localised epithelial expression of the Bmp4-pIRES2-EGFP construct (green colour, orange arrowhead). (E) Digoxigenin whole-mount in situ hybridisation showing Isl1 ectopic epithelial expression (yellow arrowhead) after electroporation with the Bmp4-pIRES2-EGFP construct. (F) Whole-mount in situ hybridisation showing Bmp4 expression (violet arrowhead) after electroporation with the Isl1 expression construct. (G) Isl1 expression in a cultured E10 mandible after electroporation with the control pIRES2-EGFP expression construct. (H) Downregulation of Isl1 expression in the distal epithelium of an E10 mandible after electroporation with the Noggin-pIRES2-EGFP expression construct in the distal part, right side of the mandible. i, incisor territory; m, mesenchyme; f, median part of the mandible; md, mandibular explant; ml, molar territory; oe, oral epithelium; +ve, positive-voltage electrode/cathode; -ve, negative-voltage electrode/anode; BMP4, Bmp4-pIRES2-EGFP construct; ISL1, Isl1 expression construct; Noggin, Noggin-pIRES2-EGFP construct; Control, pIRES2-EGFP construct.

 


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  		Fig. 7. Electroporation of Isl1 morpholino expression construct into the epithelium of an E10 mandibular explant and effects on Msx1 and Bmp4 expression in the mesenchyme. (A,E,I) E10 mandibular explants prior to electroporation. (B) GFP expression following electroporation of Sox9B morpholino and pIRES2-EGFP (green colour, yellow arrow). (C) Superimposition of A and B showing the localised epithelial area of electroporated DNA. (D) Digoxigenin whole-mount in situ hybridisation, showing expression of Bmp4 in epithelium (yellow arrow) following Sox9B electroporation. (F) Fluorescence image of explant following electroporation of Isl1 morpholinos (yellow arrow). (G) Superimposition of E and F showing localised epithelial area of electroporated morpholinos. (H) Digoxigenin whole-mount in situ hybridisation showing expression of Bmp4 following electroporation of Islet1 morpholinos (yellow arrow). (J) Fluorescence image of explant following electroporation of Isl1 morpholinos (yellow arrow). (K) Superimposition of I and J showing localised epithelial area of electroporated morpholinos. (L) Digoxigenin whole-mount in situ hybridisation showing expression of Msx1 following electroporation of Isl1 morpholinos (yellow arrow). md, mandibular explant.

 


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  		Fig. 8. Electroporation of Isl1 expression construct into proximal (presumptive molar) epithelium of E10 mandibles. (A,E) Cultured E10 mandibles prior to electroporation. (B,F) Expression of GFP following electroporation of the Isl1 expression construct in proximal epithelium (yellow arrows). (C,G) Superimposition of A,B and E,F to show the location of GFP to identify the region of ectopic Isl1 expression. (D) Downregulation of Barx1 expression in proximal epithelium following Isl1 expression (yellow arrow). (H) Msx1 whole-mount in situ hybridisation of a mandible electroporated with Isl1 showing no change in expression. md, mandibular explant.

 


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  		Fig. 9. Digoxigenin whole-mount in situ hybridisation showing expression of Isl1 following epithelial/mesenchymal recombinations. (A) Distal epithelium recombined with proximal mesenchyme at E10.5. (B) Proximal epithelium recombined with distal mesenchyme at E10.5. (C) Distal epithelium recombined with proximal mesenchyme at E12.5. (D) Proximal epithelium recombined with distal mesenchyme at E12.5.

 


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  		Fig. 10. A model illustrating the molecular events directing murine dentition patterning. A regulatory loop exists between ISL1 and BMP4 in oral epithelium, where Isl1 stimulates Bmp4 expression and Bmp4 activates Isl1 expression. Thereafter, Bmp4 activates Msx1 expression in mesenchyme, whereas it acts as an antagonist for Fgf8 expression from oral epithelium and downregulates Barx1 expression in mesenchyme. Red and blue colours represent the incisor and molar territories respectively of the oral epithelium, green and yellow colours represent the incisor and molar territories respectively of the mesenchyme.

 

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© The Company of Biologists Ltd 2003