Molecular & Genetic Mechanisms of Tooth Development

Cell fate determinants

Cell fate depends on origin, temporal-spatial history, and is determined by genes/environment (autonomous/intrinsic and non-autonomous/extrinsic factors)

Neural crest alone (before day 12)

When cultured alone, forms cartilage and neural crest tissue but NOT teeth or bone

Mandibular epithelium alone (before day 12)

When cultured alone, forms none of teeth, bone, cartilage, or neural crest -- cannot act alone

Neural crest + mandibular (1st arch) epithelium

Recombination that produces teeth, bone, cartilage, AND neural crest -- shows epithelium is instructive

Neural crest + limb (or 2nd arch) epithelium

Produces bone, cartilage, and neural crest but NOT teeth -- shows only first arch epithelium is odontogenic

Odontogenic epithelium + skin mesenchyme (after day 12)

Forms skin, not teeth -- after day 12 epithelium alone can't direct fate

Skin epithelium + odontogenic ectomesenchyme (after day 12)

Forms a tooth -- after day 12 the ectomesenchyme directs tooth development

Key conclusion of recombination experiments

Before E12, epithelium initiates odontogenesis; after E12, epithelium loses odontogenic potential and ectomesenchyme takes over directing tooth development

Three stages of tooth morphogenesis

Initiation (dental lamina) -> Morphogenesis (bud, cap) -> Differentiation (bell)

Hox genes

Family of homeobox-containing genes first discovered in Drosophila (antennapedia); encode homeodomain transcription factors that control segment identity and specialization

Antennapedia gene

Drosophila Hox gene; mutation causes a leg to grow in place of an antenna, showing Hox genes control segment/appendage identity

Homeodomain

~60 amino acid region of a ~400 amino acid transcription factor protein; binds specific DNA sequences to turn genes on/off, can regulate hundreds of genes

Homeobox

The DNA sequence within a Hox/homeobox gene that, when translated, produces the homeodomain protein region

Synteny

The conserved co-linear order of Hox genes across species during evolution

Co-linearity of Hox genes

The spatial order of Hox genes on the chromosome matches the anterior-to-posterior order of their expression in the embryo

HOX gene clusters in mammals

HOXA, HOXB, HOXC, HOXD -- mammalian equivalent of Drosophila Hox complex, arranged in chromosomal clusters

Msx

Homeobox gene family; originally identified in muscle development in Drosophila; in mammals, expressed in incisor-forming ectomesenchyme

Dlx

Homeobox gene family; originally identified in limb development in Drosophila; combines with Msx and Barx to pattern tooth type

Barx

Homeobox gene family; originally identified in retina development in Drosophila; expressed in molar-forming ectomesenchyme

Genes required to form the dental lamina

Msx1, Msx2, Dlx2, and Barx1 expressed in the overlap region of ectomesenchyme

Incisor identity gene code

Msx-1 and Msx-2 expression in ectomesenchyme

Canine identity gene code

Msx-1, Msx-2, and Dlx-2 expression (combination of anterior and posterior signals)

Molar identity gene code

Barx-1 and Dlx-2 expression

Odontogenic homeobox code model

Concept that combinatorial (overlapping) expression of homeobox genes (Barx1, Dlx1/2, Msx1, Msx2, Alx3) in ectomesenchyme before tooth germ initiation determines tooth type and pattern

FGF8

Growth factor from epithelium that induces/promotes tooth germ formation at future tooth positions

BMP4 (epithelial role)

Growth factor from epithelium that inhibits/restricts where tooth germs can form, helping mark tooth position

Pax9

Transcription factor expressed in ectomesenchyme; activated by FGF8, inhibited by BMP4; marks position of each tooth; ectomesenchyme depends on ligands for Pax9 early but becomes independent by bud stage

SHH (Sonic Hedgehog)

Secreted growth factor from dental epithelium; SHH-soaked beads applied to oral epithelium are sufficient to cause invaginations resembling tooth buds; involved throughout development with multiple roles including a concentration gradient later on

Reciprocal signaling stages

Initiation stage = epithelial signaling; Bud stage = mesenchymal signaling; Cap stage = enamel knot signaling

Epithelial signaling (initiation stage) genes

SHH, BMP4, FGF8 acting on dental mesenchyme; activate Msx1, Pax9, Ptc, Gli1, ActbA

BMP4 expression shift

BMP4 is expressed in dental lamina epithelium initially, then switches to the ectomesenchyme during the bud stage -- this switch indicates the shift in developmental control from epithelium to mesenchyme

Enamel knot

Specific inner enamel epithelium (IEE) signaling center at the cap stage; non-dividing epithelial cells at cusp tips that express p21, organize crown/cusp morphogenesis

Ectomesenchymal BMP4 (cap stage)

Required to induce formation of the enamel knot; enamel knot cells then express BMP4 and FGF4

p21

Cell cycle regulator expressed in enamel knot cytoplasm; causes primary and secondary enamel knot cells to stop dividing (non-dividing signaling center)

Primary enamel knot

First enamel knot to form at the cap stage; induces formation of secondary enamel knots and determines cusp pattern

Secondary enamel knots

Form later (bell stage), induced by the primary enamel knot; position determines multi-cusped tooth pattern; regulate dental papilla cells and laterally regulate IEE cells

Single-cusped vs multi-cusped teeth

Determined by whether only a primary enamel knot forms (single cusp) or primary knot induces secondary knots (multi-cusp)

Enamel knot signaling factors

BMP, FGF, SHH -- knot cells signal laterally to tell neighboring IEE cells not to become knot cells and to keep dividing

Cervical loop

Region of the enamel organ at the bell/late bell stage that becomes the root

Dental lamina

Initial thickening of oral epithelium that marks where teeth will form in each jaw

Dental placode

Localized thickening of epithelium marking the start of tooth initiation, associated with SHH expression

Odontogenic ectomesenchyme

Neural crest-derived mesenchyme beneath the epithelium that becomes competent to direct tooth development

Order of tooth structure formation (late stages)

Bell stage -> late bell stage -> root formation and eruption; produces enamel, dentin, pulp, root, and surrounding jaw bone

MSX gene function/product

Msh-like gene; transcription factor; located inside the nucleus

DLX gene function/product

Distaless homologue; transcription factor; located inside the nucleus

BARX gene function/product

BarH1 homologue; transcription factor; located inside the nucleus

BMP4 function/product

Bone morphogenetic protein; growth factor; located outside the cell

SHH function/product

Sonic hedgehog; growth factor; located outside the cell

FGF function/product

Fibroblast growth factor; growth factor; located outside the cell

Number of genes identified in tooth development

More than 90 genes encoding transcription factors and growth factors (signaling molecules)