Dermal papilla cell number specifies hair size, shape and cycling and its reduction causes follicular decline

Abstract

Although the hair shaft is derived from the progeny of keratinocyte stem cells in the follicular epithelium, the growth and differentiation of follicular keratinocytes is guided by a specialized mesenchymal population, the dermal papilla (DP), that is embedded in the hair bulb. Here we show that the number of DP cells in the follicle correlates with the size and shape of the hair produced in the mouse pelage. The same stem cell pool gives rise to hairs of different sizes or types in successive hair cycles, and this shift is accompanied by a corresponding change in DP cell number. Using a mouse model that allows selective ablation of DP cells in vivo, we show that DP cell number dictates the size and shape of the hair. Furthermore, we confirm the hypothesis that the DP plays a crucial role in activating stem cells to initiate the formation of a new hair shaft. When DP cell number falls below a critical threshold, hair follicles with a normal keratinocyte compartment fail to generate new hairs. However, neighbouring follicles with a few more DP cells can re-enter the growth phase, and those that do exploit an intrinsic mechanism to restore both DP cell number and normal hair growth. These results demonstrate that the mesenchymal niche directs stem and progenitor cell behaviour to initiate regeneration and specify hair morphology. Degeneration of the DP population in mice leads to the types of hair thinning and loss observed during human aging, and the results reported here suggest novel approaches to reversing hair loss.

Fig. 1.

Follicles produce different hair types in successive cycles. (A) Hair was dyed at the end of the first hair cycle and follicles were dissected at the end of the second hair cycle. Examples of each type observed are shown: G, guard; A→A, awl to awl; Au→A, auchene to awl; Z→A, zigzag to awl; Z→Au, zigzag to auchene; Z→Z, zigzag-zigzag. Within follicles that made each hair class in the first cycle, the percentage making different hair classes in the second cycle is shown above. (G, n=51; A, n=160; Au, n=128; Z, n=1889 follicles from five mice). (B) The average frequency and s.d. of each hair type in the first (black) and second (white) hair coats is shown (n=21 mice, minimum of 300 hairs scored in each hair coat). G, guard; A, awl; Au, auchene; Z, zigzag. ^*P<7×10^-12, ^**P<8×10^-13.

Fig. 2.

Follicles producing different hair types have different numbers of DP cells. (A,B) Optical sections showing hair bulb from a Sox2GFP/+ mouse (nuclei indicated in red) (A) and overlay of the same section with DP cells that express GFP (green) (B). Scale bar: 25 μm. (C) DP cell numbers were counted in optical sections of isolated intact hair follicles producing each hair type during first (morphogenetic) anagen (P11, black bars), first telogen (P20, grey bars) and second anagen (P30, white bars). Mean and s.d. are shown. Differences between hair types are significant (P<0.001) at all time points. Differences within hair type between the first and second cycle are significant (^*P<0.01; ^**P<0.001; ^***P<0.03). All follicles producing auchene hairs in the second hair cycle produced zigzag hairs in the first cycle. Guard hairs were not analysed during telogen. (Minimum n=20 hairs from two mice per type/time point, except guards, n=6/time point.) (D) Examples of two and three medulla cell thick awls are shown above. There is a significant difference (^*P=0.002) between the number of DP cells in follicles that produce thin (2) versus thick (3) awl hairs in the same hair cycle (mean and s.d., n=5 and n=9, respectively). (E) The average DP cell number and s.d. for the 20% of zigzag follicles with the most DP during the first hair cycle (1Z, n=7), and that for follicles producing an auchene in the second hair cycle (2Au, n=20) are shown. All follicles that produced an auchene in the second cycle produced a zigzag hair in the first cycle. The significant difference (^*P=1.5×10^-8) between these two populations demonstrates that DP cell number has increased as these follicles shift from making zigzag to making auchene hairs.

Fig. 3.

DP cell depletion results in smaller hairs and failure to re-enter the anagen phase of the hair cycle. (A) All cells in DPtet^Off mice start with inactive r26^tTA and tetO-DTA alleles. Corin-cre expresses cre-recombinase in DP cells, which removes the stop sequence from r26^tTA and leads to the production of the tet transactivator protein (tTA). This binds to the tetO sequence and initiates transcription of the DTA transgene. DTA kills the cell. (B) Control (Con) and DPtet^Off are shown. Animals were shaved between the red and white lines after the first hair cycle (P21), and between the yellow and white lines after the second hair cycle (P63). Above the red line the pelage consists of the first, second and, in control, third hair coats. Between the red and yellow lines, the shorter, thinner, darker hairs produced in the second cycle by DPtet^Off contrast with the normal second and third hairs of the control. Between the yellow and white lines, the lack of hair in DPtet^Off reveals the failure to re-enter the anagen phase, while the control has completed the production of a third hair coat. (C) Close-up view of the thin second hair coat that does not completely cover the skin in DPtet^Off (i) and corresponding region of the control (ii). (D) Dissected follicles from a DPtet^Off animal reveal largely normal first hairs. However, many follicles that produced awl or auchene hairs in the first cycle produce zigzag hairs in the second cycle (A→z and Au→z, respectively), and all second cycle hairs are shorter and thinner than normal. Scale bar: 1 mm. (E) Optical sections of follicles harvested at 10 weeks of age that had produced zigzag hairs in control (Con) and DPtet^Off mice. The DP is marked with a broken green line, the secondary germ region with a broken yellow line. Scale bar: 25 μm. (F) The number of cells in the secondary germ region differs significantly between control and DPtet^Off follicles that produced a zigzag hair in both hair cycles (mean and s.d., n=20 follicles from two mice each,^*P=1.0×10^-12). (G) DP cell number per follicle in control (black bars) and DPtet^Off (white bars) after the first and second hair cycles in follicles that made awl, auchene or zigzag hairs in the first hair cycle. DP cell number and hair morphology are only slightly affected after the first cycle, but dramatically reduced after the second cycle (mean and s.d., n=20/type second cycle, n=10/type first cycle). The difference between DP number in control and DPtet^Off after the second cycle is significant for all three hair types shown (P<6×10^-16). (H) Follicle length during second telogen, measured from the skin surface to the base of the DP differs significantly between control and DPtet^Off follicles that produced a zigzag hair in both hair cycles (mean and s.d., n=44 follicles con, n=53 follicles DPtet^Off from 3 mice each, ^* P=6.8×10^-36).

Fig. 4.

A fixed period of DP damage alters hair morphology. (A) Schematic of the DPtet^On system. All cells in DPtet^On mice start with inactive r26^rtTA and tetO-DTA alleles. Corin-cre expresses cre-recombinase in DP cells, which removes the stop sequence from r26^rtTA and leads to the production of the reverse tet transactivator protein (rtTA) and EGFP. rtTA is inactive until bound by doxycycline. In the presence of doxycycline it binds to the tetO sequence and initiates transcription of the DTA transgene. DTA kills the cell. (B-K) Dissected follicles from DPtet^On mice treated with doxycycline during the anagen phase (P30-56, B-D), second telogen phase (P42-63, E,G-J) or control (F,K). The hair cycle in which the hair was generated is indicated (1, 2, 3). For animals treated in telogen and control, the first two hairs were dyed red at 9 weeks, prior to formation of the third hair (black). (B,C) Treatment during anagen results in either a shift to production of a smaller hair type (B, awl to zigzag) or production of a smaller hair of the same class (C, zigzag to smaller zigzag). (D) Example of a chimeric hair produced with treatment during anagen. The distal half of the second hair has the morphology of an auchene, whereas the proximal region has the morphology of a zigzag. In follicles that produced awl hairs in the second hair cycle, DP deletion during the second telogen phase causes production of smaller hairs or hair types during third anagen. (E) This follicle produced a normal awl in the second cycle and a smaller awl after DP cell ablation. A small segment is magnified for comparison of thickness and structure. (F) A control awl follicle produces a slightly larger hair in the third cycle. Scale bar: 1 mm. (G,H) A follicle that produced a normal auchene hair in the first cycle (1), an awl hair in the second (2) and made an auchene (G) or reduced zigzag hair (H) in the third cycle after DP cell depletion (3). (I) Follicles that produced zigzag hairs in the first cycle and auchenes in the second produce zigzag hairs after DP cell deletion during second telogen. (J) In contrast to follicles producing larger hair types, the follicles that produced zigzag hairs in the second hair cycle and re-entered the third cycle produce a zigzag hair that is only slightly smaller than its second cycle counterpart. (K) Control follicle that produced zigzag hairs of increasing size in all three cycles.

Fig. 5.

DP number is restored in follicles that enter the anagen phase. (A-C) Follicles from DPtet^On mice treated with doxycyline during the second growth phase have reduced second cycle hairs (2) compared with first cycle hairs (1). Many follicles fail to generate a third hair (A), whereas follicles that generate a slightly larger second hair go on to make a more normal third hair (B). (C) A follicle that produced a reduced awl hair in the second cycle (2) made a normal-sized zigzag in the third cycle (3) and progressed to a normal auchene in the fourth (4). Scale bar: 1 mm. (D) Average DP cell number for follicles that made zigzag hairs in the first and second cycles are shown at 10 weeks (B, before) and 20 weeks (A, after) for five treated mice and one control. For each mouse, the time of doxycycline treatment (Anagen; Tel, telogen; No, None) and average number of DP cells per follicle among the entire zizgzag population at 10 weeks (10wk DP#) is shown below. The data have been separated into the follicles that cycled (green) and those that did not (grey). The fraction that had cycled at 20 weeks is listed below the graph for each mouse (20-100%). Black bars represent the average DP cell number scored at 20 weeks for the fraction that did (green shaded area, ‘Cycle’) or did not (grey shaded area, ‘Arrest’) produce a third hair. The white bar (B) shows the average DP number after doxycycline treatment, before the skin re-enters anagen, inferred for the fraction of the sample that will (Cycle, green) or will not (Arrest, grey) make a third hair (see text). The inferred threshold number of DP cells required to re-enter the anagen phase for each mouse is shown below (Threshold). Mean and s.d., ^*P<3×10^-6, ^**P<0.001 (n=25, 37, 40, 38 and 20 follicles at 10 weeks, 37, 27, 23, 22 and 20 follicles at 20 weeks, for mice 1-5, respectively). (E) Schematic representation of this experiment in which follicles with the fewest DP cells (green) arrest in the telogen phase, while follicles with a few more DP cells cycle and restore DP cell number, hair size and cycling activity.