Hoxa2 null embryos display a high incidence of cleft secondary palate which has previously been described as secondary to altered tongue development. The experiments described in this thesis demonstrate that expression of Hoxa2 does occur within the developing palate, with the highest levels appearing in the early stages of palatogenesis (E12.5 and E13.5). Increased cell proliferation was observed throughout the palate in the absence of Hoxa2, without a detectable difference in apoptosis or the ability of the shelves to fuse. In addition, the palate shelves of the null embryos failed to elevate above the tongue, suggesting a mechanism by which the increased cell proliferation results in cleft palate.<p> Numerous downstream targets of Hoxa2 were also identified in the palate (Msx1, Bmp4, Barx1, Ptx1, Six2, Lef1 and Tbx1). In all cases, Hoxa2 appears to act as a transcriptional repressor. Increases in palatal Msx1, Bmp4 and Barx1 expression have all been previously described to lead to increases in cell proliferation. Hoxa2, Ptx1, Lef1 and Tbx1 may be involved in a novel pathway that regulates proliferation in the palate. In addition, three novel gene targets were identified in the palate, Six2, Fgf8 and Htra3.<p> Together these data show that there is a direct role for Hoxa2 in regulating palate development, apparently through regulating the expression of downstream genes involved in maintaining normal cell proliferation rates.

Hoxa2 null embryos display a high incidence of cleft secondary palate which has previously been described as secondary to altered tongue development. The experiments described in this thesis demonstrate that expression of Hoxa2 does occur within the developing palate, with the highest levels appearing in the early stages of palatogenesis (E12.5 and E13.5). Increased cell proliferation was observed throughout the palate in the absence of Hoxa2, without a detectable difference in apoptosis or the ability of the shelves to fuse. In addition, the palate shelves of the null embryos failed to elevate above the tongue, suggesting a mechanism by which the increased cell proliferation results in cleft palate.<p> Numerous downstream targets of Hoxa2 were also identified in the palate (Msx1, Bmp4, Barx1, Ptx1, Six2, Lef1 and Tbx1). In all cases, Hoxa2 appears to act as a transcriptional repressor. Increases in palatal Msx1, Bmp4 and Barx1 expression have all been previously described to lead to increases in cell proliferation. Hoxa2, Ptx1, Lef1 and Tbx1 may be involved in a novel pathway that regulates proliferation in the palate. In addition, three novel gene targets were identified in the palate, Six2, Fgf8 and Htra3.<p> Together these data show that there is a direct role for Hoxa2 in regulating palate development, apparently through regulating the expression of downstream genes involved in maintaining normal cell proliferation rates.

Cleft palate is one of the most common congenital malformations in humans which occurs at a frequency of approximately 1:700 live births worldwide. Sine Oculis-related homeobox 2 (Six2) is a member of the vertebrate Six gene family that encode proteins that are transcription factors. Six2 has been reported to be a downstream target of Homeobox a2 (Hoxa2), a gene that plays a direct a role in mouse secondary palate (SP) development. In my thesis, I utilized quantitative real time Polymerase Chain Reaction (qPCR), Western blot analysis and fluorescence immunohistochemisrty (IHC) to characterize the spatial and temporal distribution patterns of Six2 in the developing SP. Additionally, I also employed in vivo cell counting analysis and in vitro cell proliferation assays to investigate the role of Six2 during palate mesenchymal cell proliferation. My study examined the temporal and spatial distribution of Six2 in the developing mouse palatal mesenchyme and epithelia in both wild-type and Hoxa2 null mice. Six2 was expressed throughout the period of embryonic palatogenesis, with the highest levels of Six2 mRNA and protein observed in palatal shelves at E13.5 in both wild-type and Hoxa2 null mice. Six2 protein expression at all stages of SP development (E12.5 to E15.5) increased in the anterior to posterior (A-P) direction with highest expression in the posterior regions of the developing SP. In addition, expression of Six2 protein was higher in the oral half of the palatal mesenchyme compared to the nasal half of the palatal mesenchyme. Interestingly, Six2 protein was expressed in the nasal palatal epithelium but was completely absent from the oral palatal epithelium. Loss of the Hoxa2 gene induced up regulation of Six2 protein and mRNA in the developing palate across all stages of palatogenesis. In the Hoxa2 null mice, there was a significant increase in cell proliferation (Ki-67 positive cells) and the percentage of actively proliferating cells that were co-expressing Six2 protein (Six2/Ki-67 double positive cells) along both the A-P and oral-nasal (O-N) axes of the developing SP. Also, the highest percentage of actively proliferating cells and Six2/Ki-67 double positive cells was observed in the nasal half of the posterior palatal mesenchyme. Furthermore, Six2 siRNA knock down in mouse embryonic palatal mesenchyme (MEPM) cell cultures restored cell proliferation and Cyclin D1 expression in the Hoxa2 null cell cultures to wild-type levels. Collectively, my data reveals a novel spatial and temporal expression profile for Six2 in the developing mouse SP and the potential role it might play during the epithelial-mesenchymal cross talk that drives palatal shelf cell proliferation and out growth.

Our understanding of protein lysine methyltransferases and their substrates remains limited despite their importance as regulators of the proteome. The SMYD (SET and MYND domain) methyltransferase family plays pivotal roles in various cellular processes, including transcriptional regulation and embryonic development. Among them, SMYD2 is associated with oesophageal squamous cell carcinoma, bladder cancer and leukemia as well as with embryonic development. Initially identified as a histone methyltransferase, SMYD2 was later reported to methylate p53, the retinoblastoma protein pRb and the estrogen receptor ERalpha and to regulate their activity. Our proteomic and biochemical analyses demonstrated that SMYD2 also methylates the molecular chaperone HSP90 on K209 and K615. We also showed that HSP90 methylation is regulated by HSP90 co-chaperones, pH, and the demethylase LSD1. Further methyltransferase assays demonstrated that SMYD2 methylates lysine K* in proteins which include the sequence [LFM]-₁-K*-[AFYMSHRK]+₁-[LYK]+₂. This motif allowed us to show that SMYD2 methylates the transcriptional co-repressor SIN3B, the RNA helicase DHX15 and the myogenic transcription factors SIX1 and SIX2. Finally, muscle cell models suggest that SMYD2 methyltransferase activity plays a role in preventing premature myogenic differentiation of proliferating myoblasts by repressing muscle-specific genes. Our work thus shows that SMYD2 methyltransferase activity targets a broad array of substrates in vitro and in situ and is regulated by intricate mechanisms.