The topic of embryological development has always been an interest of mine. I wanted to learn how cells knew to go to specific areas, whether they were going to be skin cells or hardened nail cells. Specifically, I wanted to learn about how cells knew how many fingers, toes, or arms to form. This leads me to the topic of embryological limb development and the possible deformities that can result from them. Researchers hypothesized that mutations of the p63 protein, which functions as a transcription factor for gene expression of ectodermal development, play a major factor in the abnormal development of limbs (Kouwenhoven et al., 2010). The most prominent abnormalities of this protein mutation include, ectrodactyly ectodermal dysplasia and cleft lip/palate syndrome (Kouwenhoven et al., 2010).

Ectrodactyly ectodermal dysplasia (EEC), also known as Split Hand/Foot Syndrome (SHFM), is a malformation of the middle area of hands or feet which include disformed or missing digits. Cleft lip/palate, which is closely related to EEC, is the malformation of the upper lip which results in a gap, or hole, in the upper lip leading to the roof of the mouth or to the nose (NORD., 2018). These conditions typically come from the ectoderm, which is the outermost layer that includes the skin, hair, nails. The researchers hypothesized that these abnormalities are the result of a mutation that affects the DNA-binding of the p63 protein.

In order to test this hypothesis, the researchers found that p63 proteins are mostly concentrated in the basale layer of the epidermis, which is the lowermost layer. Thus, they gathered cultures of human primary keratinocytes from different adults and performed high-resolution global binding profiles of p63 using chromatin immunoprecipitation sequencing (ChIP-seq). After analyzing the sequencing, they found that there were 11,369 overlapping peaks of p63 within the cultures, validating that p63 was a reliable protein for use (Figure 1A), (Kouwenhoven et al., 2010). Figure 1A, showed the similarities of the peak height and distance of p63 as a result of the ChIP-seq.

To find the specific binding sites of p63, the researchers performed a Position Weight Matrix (PWM), which generated that 10,702/11,369 of the p63 binding sites directly correlated with p63 (Kouwenhoven et al., 2010). In order to specify the target genes and how they related to p63 diseases, the human malformation disease database (POSSUM) was searched and the researchers found 20 genes that corresponded to the SHFM loci (Kouwenhoven et al., 2010). Of the 20 SHFM genes, p63 binding sites were found near only 12 of them, which is highly beneficial in establishing their relation.

A patient with non-syndromic SHFM was observed to have microdeletions of chromosome 7q21, however, the deletions left genes DLX5 and DLX6 (Kouwenhoven et al., 2010). The researchers believed that the genes DLX5 and DLX6 played a major role in SHFM abnormalities, thus they searched p63 binding sites in chromosomal regions within DLX5 and DLX6 and found 9 p63 binding sites using ChIP-seq (Kouwenhoven et al., 2010). They hypothesized that these must be where the enhancers for the p63 protein are located. The researchers tested the enhancers’ sensitivity to p63 by cloning the enhancers in front of a luciferase receptor, which is used to test gene expression, and found that only one enhancer was extremely responsive, SHFM1-BS1 (Figure 4A), (Kouwenhoven et al., 2010). Figure 4A displays how SHFM1-BS1 was highly activated by the p63 activation, by almost 3 times,  compared to that of the other possible enhancers.

After finding their primary enhancer element, the researchers conducted the same trial on mice without the enhancer and found that there was no activation of the p63 protein on the DLX5 gene. Thus, indicating that without the enhancer, there is no regulation of embryonic limb development by p63. To further conclude their findings, researchers injected a p63 morphlino, a “knock-down”, into zebrafish embryos and found that the fins were either non-existent or extremely small (Figure 5C). The figure also shows how the DLX5 and DLX6 genes were affected by the p63 morpholino by comparing the fin deficiency to that of a control.  They also tested that in the affected embryos, the DLX5 and DLX6 gene expressions were reduced (Kouwenhoven et al., 2010).

In class we’ve repetitively learned that in relation to proteins, structure equals function. When proteins undergo a mutation, or in the case of this research, a missense mutation, an entire nucleotide is changed which results in a different amino acid contributing to the protein. This tiny change is responsible for the complete change in function or inhibition of the protein. When the missense mutation occurs on the p63 protein, it either knocked down the protein or changed its shape, disallowing the enhancer to attach, which results in the downregulation of embryonic limb development. The researchers concluded that this disruption of DLX5/6 gene expression controlled by p63 is the likely cause of SFHM (Kouwenhoven et al., 2010).

 

 

Kouwenhoven E.N., van Heeringen, S.J., Tena, J.J., Oti, M., Dutilh, B.E., et al. (2010). Genome-Wide Profiling of p63 DNA–Binding Sites Identifies an Element that Regulates Gene Expression during Limb Development in the 7q21 SHFM1 Locus. PLoS Genetics 6(8), 1-15.

NORD (National Organization for Rare Disorders). (2018). Ectrodactyly Ectodermal Dysplasia Cleft Lip/Palate – NORD (National Organization for Rare Disorders). [online] Available at: https://rarediseases.org/rare-diseases/ectrodactyly-ectodermal-dysplasia-cleft-lippalate/.