Richard Park

Characterization of Skin from SPINK5 Knockout MiceCaractérisation de la Peau des Souris Knockout SPINK5

Charackterisierung der Haut von Knockout-Mäusen SPINK5

I N T R O D U C T I O N

Inspection of KO skin showed that certain cells near the basal lamina, the border between the upper epidermis and the lower dermis, were becom-

ing spindle-shaped and migratory.

These cells had begun to display signs typical of a process known as the epithelial-mesenchymal transition (EMT).

So the question was raised,

“ Could the presence of LEKTI in highly differentiated cells influence the action of less differentiated cells

much closer to the basal lamina? ”Thus, we next analyzed the skin for markers of EMT.

M O T I V A T I O N

R E S U L T S

T R A N S P L A N T A T I O N

a b c d e f g

During my 8 weeks, I focused on skin transplantation, immunohistochemistry (IHC), and HE staining to characterize the abnormal skin of SPINK5 knockout (KO) mice. This poster summarizes the motivation, the initial questions, and the methods used to accomplish this characterization.

Netherton Syndrome... is a rare, autosomal skin disorder.

Its symptoms include overall inflamma-

tion, accelerated desquamation (skin

peeling), and hair defects. See Figure 1.

SPINK5... is a gene located in a region known

to encode inhibitors and proteases. A

frame-shift mutation in this gene leads to

the decreased production of a protein

known as LEKTI. See Figure 5f-g.

FIGURE 1.Figures 1a-g display the

effects of Netherton Syndrome on human

patients.

LEKTI... contributes

to the regulation of

desquamation. See

Figure 2.

dermis

epidermis

mouse green = LEKTIblue = nuclei

dermis

epidermis

human

100µm100µma b

FIGURE 2.Figures 2a & 2b show the presence of LEKTI in normal, healthy skin

stratum corneum

stratum granulosum

Protein Detection... E-cadherin and vimentin are two markers for EMT. E-cadher-

in aids in cell-to-cell adhesion and is expressed in the epidermis,

and vimentin anchors organelles in the cytosol and is normally ex-

pressed in the dermis. During EMT, e-cadherin is downregulated

and vimentin is upregulated. Consequently, cells in the epidermis

begin losing adhesion to one another as their junctions become

degraded. See Figures 5a-c on right.

The proteins K14 and KI-67 are not exclusive markers of EMT.

However, their presence is used to characterize the actions of cells

near the basal lamina. K14 marks cells lining the basal lamina, and

KI-67 mark cells undergoing cellular division (stages G1 to M). See

Figures 5d-e below.

LEKTI was also stained for. See Figures 5f-g on the bottom

right. I used IHC methods to test for the presence of these five

markers.

FIGURE 3. Figures 3a-c show the healing patters of a WT and a KO. In Figure 3d, 1, 2, 4, & 6 represent the 700bp –/– KO mice, 3, 7, & 8 represent the 600/700bp +/– WT mice, and 5, 9, & 10 represent the 600bp +/+ WT mice.

Dorsal skin from embryonic mice were transplanted onto rectangular flaps of immu-

nodeficient nude mice. DNA samples were prepared from the tails of the mice and then

genotyped using PCR to preview which mice would result in +/+, +/–, and –/–. See Figure

3c. At four weeks after transplantation, the rectangular flaps were imaged. See Figure 3a

& 3b. Unforeseen circumstances led to the use of KO and wild type (WT) samples taken in

2011 to finish the characterization.

a b c d1 2 3 4 5 6 7 8 9 10

Morphological Examination... Samples were examined

using HE staining and light mi-

croscopy to compare. See

Figure 4.FIGURE 4.

It can be clearly seen that the KO mouse skin has a less organized structure. 100µm 100µma b

FIGURES 5d-e (on left)

These figures display fluorescent IHC images of KI-67 and K14.

In both figures, white arrows point to-wards the KI-67 expressing cells (light green). These areas show that cellular proliferation is going unregulated.

Abnormal K14 expression throughout the epidermis in the KO shows a much thicker, disorganized skin when com-pared to the WT.

100µm

100µm

green=KI-67red=K14

basal lamina

basal lam

ina

FIGURES 5a-c (on right)

These dis-play pan-kera-tin, e-cadherin, and nuclei in KO samples.

In Figure 5a, the decrease in epidermal e-cadherin expression near the basal lamina could suggest a downregulation of the protein. Accordingly, an increase in epidermal vimentin expression should be clearly visible in Figure 5b. However, this is not seen but in a few areas (white arrows). Figure 5c displays a negative control for the presence of pan-keratin, vimentin, and e-cadherin. Pan-keratin is often stained to differentiate between the epidermis and the dermis.

FIGURES 5f-g (above). These figures show the pres-ence of LEKTI (brownish red). In 5f, the stark con-trast between the absence of LEKTI (black arrow) and the presence of LEKTI (gray arrow) can be seen at the edge of transplantation.

f g100µm 100µm

100µm 100µm

green = PanKeratinred = e-cadherinblue = nuclei

green = PanKeratinred = vimentinblue = nuclei

a b c100µm

transplantation edge

d

e

basal lamina

basal lamina

WT KO

KO KO KO

KO WT

WT KO KO

KO

WT