An updated classification of hair follicle morphogenesis.

1 |
INTRODUCTION
The mature hair follicle (HF) is structurally complex, belying its small
size. It is predominantly comprised of concentric rings of epithelial cells that
form the hair shaft and inner root sheath (1),
with reserve stem cells in the bulge region (2–7) and their progenitors,
transit-amplifying matrix cells, at the bulbar base. Surrounded by the matrix is a
central cluster of mesenchymal cells, the dermal papilla (DP), which acts as an
instructive signaling niche (8–10) for these transit-amplifying progenitors to
proliferate, migrate upwards and differentiate into the several layers of shaft and
inner root sheath cell lineages during the hair growth phase (10–12).
Adding to the complexity is the presence of other HF resident cell types: sebocytes
that make up the mature sebaceous gland (13–15), and melanocytes
that pigment the hair (16). The specification
of the epithelial cell types of the HF and of the mesenchymal DP, from the embryonic
placode (Pc) and dermal condensate (DC), respectively, and later emergence of other
HF resident cell types, is a tightly controlled process during embryogenesis, both
temporally and spatially (17).
Hair inductive capacity lies within the dermis, which has been demonstrated
in “cut-and-paste” tissue recombination experiments; recombined
hair-forming dermis and non-hairy glabrous epidermis, both derived from murine
embryonic skin before HF morphogenesis, are able to form hair, while dermis from
glabrous regions is unable to induce follicles even in hairy epidermis (18,19).
Remarkably, xenograft experiments recombining feather-forming dermis from chicken
with scale-forming epidermis from lizards or hair forming dermis from mouse with
chicken epidermis results in the production of scale or feather structures,
respectively, suggesting that the induction of epidermal appendages is controlled by
the mesenchyme (19,20). While the “first dermal signal” for
patterned initiation of HF morphogenesis in the epidermis remains elusive (21–23), it is known that widespread Wnt signaling activity in the upper
dermis by embryonic day (E) 12.5 precedes HF formation (24) and is required for HF induction (25). This signal acts on the epidermis to induce Pc
formation at ~E14.0 (17,26), which produce the “first epithelial
signal” that acts on the underlying fibroblasts. One or more such signals,
including FGF20 (27), lead to the formation
of the DC, local condensations of upper dermal fibroblasts, at E14.5 (17). The DC then secretes still unknown
“second dermal signals” to catalyze proliferation of Pc progenitors
and downgrowth of the HF (17). Xenograft
experiments have failed to completely form feathers, scales or HFs suggesting that
species-specific signaling crosstalk between the epithelial and mesenchymal
compartments initiated by the “second dermal signal” is necessary for
proper downgrowth and differentiation (28,29). Complete understanding of
these crucial reciprocal signals in early HF morphogenesis remains elusive, owing to
the rapidity with which epithelial-mesenchymal crosstalk and subsequent
morphological changes occur, although many individual components have been
parsed.
The evaluation of HF morphogenetic stages has historically relied upon
progressive changes in cellular shape and morphology, dynamic aggregation of cells,
emergence of follicle resident cell types, extent of HF downgrowth (17,30), and the
usage of few known molecular markers, IL-1RI (30), TGF-βRII (31), and
Alkaline Phosphatase (AP) (30,32). In a seminal classification guide from 20 years ago,
Paus and colleagues summarized these key characteristics of HF morphogenesis to
provide a well-defined classification system for greater spatiotemporal
clarification of the major HF morphogenesis stages (30). Since its establishment, advanced mouse genetic methods have
enabled numerous functional studies that uncovered the essential roles of major
signaling pathways, such as Wnt, Eda/Edar, Fgf, Bmp, Shh and TGFβ signaling
(21–23,26).
Furthermore, many emerging technologies such as live imaging, multicolor fluorescent
labeling and isolation of distinct cell types from specific stages, as well as high
sensitivity transcriptomics at both the population and single-cell level, have
enabled a more fine-toothed dissection of the cellular and molecular dynamics of HF
morphogenesis. Such advances have permitted the definition of molecular signatures
of Pc and DC, and neighboring cell types (33–35), as well as
identified migration as the main cellular mechanism of Pc and DC formation (36–38). They have also allowed for the discovery of precursors to the Pc
(pre-PC) (24,39–41), multipotent
fibroblasts that give rise to the DC (42) and
the fated precursors of DC (pre-DC) (43), by
their molecular properties and prior to identifiable changes in cell morphologies
and arrangement. Finally, they have enabled identification of suprabasal
SOX9+ precursors to HF stem cells after placode formation (44,45).
In this review, we update the well-established classification guide of HF
morphogenesis stages by incorporating the recently discovered early precursor cell
states and the many new cellular and molecular insights into early HF fate
specification and formation. We then describe the current knowledge of reciprocal
mesenchymal-epithelial interaction to provide a comprehensive overview of the
dynamism of HF morphogenesis, focusing on early cellular, molecular and signaling
events during the first wave of embryonic hair follicle formation.

2 |
UPDATED STAGING OF HF MORPHOGENESIS
2.1 -
Early Morphogenesis
To account for the recently discovered precursor cell states and new
molecular events during the earliest phase of HF formation (“molecular
placode” pre-Pc before morphological Pc; fated pre-DC before DC
formation; HFSC precursors before bulge formation), we subdivided the previous
stage 0 from the original
classification (30) into 2 new stages,
stages 1 and 2 (Figure 1).
These are prefaced by a new stage 0 during
which HF induction from the dermis is set up before any patterned molecular or
cellular events. The previously classified advanced Pc/DC and germ stages then
succeed the new precursor stages (Figure
1). All molecular markers related to early morphogenesis described in
this review are featured in a comprehensive Figure 5 that is, for easy
reference, color-coded by cell type and stage, and lists the corresponding cited
publications.
For all stages, we describe the updated classification in the context of
the first wave of primary guard hair formation, for which most new cellular and
molecular insights have been discovered in recent years, likely due to the
ability to study first wave hair formation in isolation. In the experimental
mouse model, the touch sensitive (tylotrich) guard hairs are induced starting at
approximately embryonic day (E)13.5, before the formation of secondary,
non-tylotrich coat hair types (2nd wave: awl, auchene, initiated at
~E15.5; 3rd wave: zigzag, initiated at ~E17.5-E18.5)
that make up the majority of adult hairs (27,46,47). Finally, while we provide the approximate
gestational ages for each HF stage as they first appear, it is important to note
that, due to variability of developmental timing, the early stages of first wave
hair formation co-exist in parallel (e.g. at E15.0) and can be identified and
distinguished by the stage-specific criteria defined below.

Stage 0 –
Before HF morphogenesis begins at ~E13.5, basal epidermal
cells are a uniform layer without any morphological signs or patterned
molecular distinctions of HF formation (Figure
1, Figure 2, stage 0). At
stage 0, widespread Wnt signaling activity in the upper dermis (24) is important for setting up HF
inductivity by supplying the critical, but still unknown “first
dermal signal”: Epidermal ablation of Wntless (Wls), a mediator of
broad epidermal Wnt ligand secretion, at E13.5 and broad dermal ablation of
β-catenin both result in a loss of Wnt signaling activity in the
dermis and subsequent absence of Pre-Pc induction (25), demonstrating the requirement of Wnt
signaling upstream of still unknown target genes that act as inductive
signals towards the epidermis. Knockout of the epidermal transcription
factor ΔNp63 prevents expression of Wnt target genes
in early epidermal progenitors and subsequent HF formation (48), further confirming the important role of
epidermis-derived Wnts and broad dermal Wnt signaling activity in pre-Pc
fate specification and HF induction. Interestingly, recent single-cell RNA
sequencing analyses suggest that upper dermal fibroblasts are transitioning
toward DC fate specification at this early induction stage prior to
morphogenesis (42).
Before pre-Pc specification, neural crest-derived melanoblasts,
precursors of HF-resident melanocytes, are already present in the dermis.
During Stage 0, melanoblasts begin migrating upward into the epidermis
(49). At this stage of skin
development, melanoblasts express Sox10 (50), Mitf (51), Pax3
(52), Dct (53), Kit (54) and Tyrp1 (53).

Stage 1 –
Still unknown signals from Wnt-responsive upper dermal cells act on
the uniform epidermis to induce Pc formation, termed the “first
dermal signal”. At stage 1 around E13.5 – 13.75, the molecular
Pc precursor (pre-Pc) cell fate is focally induced in epidermal progenitors
at sites of future HF morphogenesis, prior to any morphological signs (Figure 1, Figure 2 stage 1). Several markers for the pre-Pc state
(“molecular placode”) have been identified, such as active Wnt
signaling (24,55,56),
Edar (57–59) and Fgf20 (27). Other genes are expressed in molecular placodes, but the
precise timing of their expression with relation to DC fate acquisition in
Stage 2 follicles is unclear. These
include Wnt10b (60), its downstream
target Dkk4 (40,61), and Cxcr4 (62) (Figure 2, Figure 5). The timing of signaling
pathways and other inter- and intracellular molecular machinery that
regulate expression of these genes has yet to be fully dissected. At this
earliest pre-Pc stage, Wnt signaling remains widely active in the upper
dermis.

Stage 2 –
The establishment of the pre-Pc then sparks the induction of the DC
cell fate in the closest neighboring fibroblasts at ~E14.0 that
precedes stereotypic aggregation of the mesenchymal DC cluster (Figure 2, stage 2) (43). These DC precursors (pre-DC) are at a
transitional state from fibroblasts towards the DC fate. Pre-DC
specification requires the pre-placodal production of Fgf20, an important
component of the “first epithelial signal” (43) that is also required for DC aggregation and
maintenance at the following stages (36,37). At this stage,
the pre-Pc shows active Wnt signaling and expression of Edar, as well as
Cxcr4 (62), Dkk4 (40,61) and
Wnt10b (60). Whether Shh (24,55), Pcad (34) and Lhx2
(33,34), three bona-fide stage 3 Pc markers, are already expressed at stage 1 or 2 is currently unclear.
In contrast to the other fibroblast-type cells in the mesenchyme,
including fibroblasts that will transition into pre-DC, pre-DC cells are
already post-mitotic, indicating that acquisition of DC fate is concomitant
with the shutdown of the cell cycle machinery (37,42,43). Like the pre-Pc,
there are high levels of Wnt signaling activity in pre-DC (24), when compared to other dermal fibroblasts;
in fact, Wnt activation is necessary for acquisition of DC fate (42) and DC formation (46). Pre-DC cells, ranging from 1 to
~15–20 cells, are randomly dispersed amongst dermal
fibroblasts and located right below pre-Pc cells (separated by the basement
membrane), which by contrast form a contiguous unit (Figure 2). Pre-DC can be discerned by de
novo expression of Foxd1 and Sox2, as well as by upregulation
of Tbx18 and highest expression of pandermal fibroblast marker Twist2 (43), which begins ramping up expression
prior to acquisition of DC fate amongst upper dermal fibroblasts (42) (Figure 2). Foxd1 (35),
Sox2 (8,63), Tbx18 (64), Bmp4 (25,65,66), Bmp7 (25), Hhip
(62), and Fgf10 (41) have been previously identified as highly
expressed signature genes in the aggregated DC (Figure 5).

Stage 3 –
Stage 3 closely resembles Stage
1 from Paus et al (30), and both the Pc and DC are morphologically
identifiable in addition to expression of several signature genes (Figure 1, Figure 3, stage 3). The Pc, starting at ~E14.5, can now
be distinguished from the rest of the epidermis because it appears thicker
and is comprised of larger, tightly packed vertically oriented keratinocytes
that slightly invaginate into the underlying dermis with basement membrane
at the leading edge (Figure 3, stage
3). Most pre-Pc genes remain expressed joining an expanded Pc signature
(Figure 3) (35) (http://hair-gel.net). Intriguingly, Cxcr4 expression wanes
as the Pc matures, and is completely lost by Stage 4 HF morphogenesis, although the gene, itself, appears
dispensable for HF morphogenesis (62). Shh (24,55),Pcad (34) and Lhx2 (33,34), a downstream target of Edar, are
now expressed within these basal matrix progenitors. At this stage,
Sox9+ suprabasal HFSC precursors are specified during
perpendicular asymmetric cell divisions of Shh-expressing basal Pc
progenitors with high Wnt signaling activity (45,67)
(Figure 1, Figure 3, stage 3), and, once suprabasal, have
very low levels of Wnt signaling. Shh and Sox9 expression in basal Pc and
suprabasal HFSC precursors, respectively, is largely non-overlapping (45,67). These HFSC precursors will, through the course of
morphogenesis give rise to the all epithelial cells of the hair follicle,
including the sebaceous gland (44,68) and Merkel cells
(69) (Figure 4). Basal Pc progenitors themselves are
precursors of the progenitors at the leading edge of downgrowing HFs that
become replaced by HFSC progeny by the end of morphogenesis (Figure 4) (44).
The DC can now be recognized as an early cluster of aggregating
cells. At this stage, the DC contains a greater number of cells
(~35+) than pre-DC (Figure 3,
stage 3). It begins to become polarized along the anterior-posterior (A-P)
axis; this polarization becomes more prominent at stage 4, described below. They are also
molecularly distinct from pre-DC: Sox2, Foxd1, Tbx18 expression is further
upregulated and Twist2 expression is lost (43) (Figure 3). Earlier
molecular studies of aggregated DC at E14.5 showed upregulated expression of
genes associated with cell migration, axon guidance, canonical Wnt
signaling, and Notch signaling (35),
as well as additional molecular signature genes that can be queried at the
accompanying web database (http://hair-gel.net) (Figure
3, Figure 5): Cxcr4 (62), Enpp2 (70), Nrp2 (71), Prdm1 (72,73), Sdc1 (74), Prom1 (75), and Trps1 (76). At
this stage, DC weakly express AP, which is readily used as a marker for
mature DP in postnatal skin (30).

Stage 4 –
By Stage 4, the Pc has elongated into the hair germ at
~E15.0, which has a more pronounced invagination into the dermis
(Figure 1, Figure 3, stage 4). During Pc downgrowth, the germ
is now prominently polarized as, through PCP-dependent cell rearrangements,
the Sox9+ HFSC precursors for the entire pilosebaceous unit
migrate toward the posterior side of the developing HF with Pcad, Shh, Lhx2
expressing matrix precursors at the leading anterior edge (77). Bipotent HFSC precursors, for both the
future bulge stem cells and sebaceous gland, additionally express Lrig1
(68,78). DC at this stage are positioned on the
anterior cap of the developing HF, which is crucial for maintaining
appropriate spatial organization of HFSC precursors (77). DC at Stage 4 are further clustered than DC
at Stage 3, and are comprised of more
aggregating non-proliferative cells (up to 90+ cells). Though, they are also
molecularly distinct, although many markers are shared (35,43)
(Figure 3, Figure 5). Finally, they express AP more highly,
indicating differentiation toward the mature DP (30).

2.2 -
Late Morphogenesis
Late morphogenetic events of Stage
5, beginning at ~E15.5, through Stage 10 closely resemble
Stage 3 to Stage 8 in the original classification guide (30) (Figure
1). As such, we will reinforce the previously established staging,
but will integrate the previously undescribed Sox9+ HFSC precursors
colonizing the bulge region. (Figure
4).

Stage 5 –
This stage is marked by more pronounced downgrowth of the HF, from
the hair germ stage to the hair peg stage (Figure 1, Figure 4, stage
5). This is associated with an elongated epithelial cell morphology and
concentric orientation around the axis of the future HF. Basal Pc cells
continue to express Shh and Pcad, while the entire developing outer root
sheath (ORS) expresses K5 (79). The
upper portion of the stage 5 HF, where the eventual bulge is formed,
co-expresses Sox9 and Lrig1 (44,78). The DC remains on the leading edge
of the downgrowing hair peg and assumes a more rounded morphology, preceding
its eventual engulfment by the HF matrix and transition into the mature
DP.

Stage 6 –
By Stage 6, the hair peg begins to resemble a mature HF more
closely. The lower portion of the hair peg, in closest contact with the DC,
begins to form a bulb-like shape (Figure
1, Figure 4, stage 6). The
HF lineages are also becoming specified; at this stage the inner root sheath
(IRS) begins to develop, expressing Blimp1 in the Henle layer (72) and Gata3 in its Huxley and cuticle
layers (80,81). Sox9 and Lrig1-expressing HFSC bipotent
precursors remain in the upper ORS (78). The DC is in the process of engulfment; by Stage 6 the DC
is more than 50% engulfed by the surrounding matrix cells at the base of the
hair peg. The morphology of the DC is also becoming more akin to the mature
DP; the DC is elongating and is longer than it is wide. Sox10+
melanoblasts begin migrating into the developing HF from the epidermis
(50,82) while melanoblasts at the epidermis gradually
disappear (49).

Stage 7 –
During Stage 7, the IRS is elongating up through the developing
follicle, and the hair canal begins developing (Figure 1, Figure
4, stage 7). The hair shaft begins to form and expresses nuclear
Lef1 and has active Wnt signaling in its pre-cortex (83). In pigmented mice, melanin granules are also
detectable in the differentiating precortex region, above the DC.
Sox9+ HFSC precursors reside in the location of the future
bulge of the mature HF in the upper ORS. The sebaceous gland lineage is
delineated at this stage; Oil Red O+ Lrig1+ sebocytes
are first visible in the upper part of the hair follicle, at the site of the
future sebaceous gland (pre-SG) (30,78,84). The DC is almost entirely engulfed by the
surrounding matrix.

Stage 8 –
The growing HF has elongated past the boundary of the lower dermis,
and the hair canal is morphologically visible (Figure 1, Figure 4, stage
8). The IRS has reached the level of the hair canal and starts to express
trichohyalin, detected by AE15 antibodies (85), also expressed in the medulla of the hair shaft (80)(81). The maturing hair shaft can be recognized by AE13-stained
hair keratins and marking of the cortex by Foxn1 (86). Other markers including K71
(K6irs1/Krt2–6g) (87), Cutl1
(88), and Hoxc13 (89) are expressed by the IRS at later stages, but
it is unclear at which stage they first appear. The companion layer, which
expresses K6 (81), can be found
between the IRS and the K5+ ORS. Sebocytes form the sebaceous gland (SG),
just above the bulge on the posterior side of the HF. The DP is now fully
engulfed by matrix cells, and appears morphologically thinner than in Stage 7 DC. Melanoblasts are separated
into two populations at two distinct locations: Sox10−
precursors to melanocyte stem cells localize at the bulge region and
Sox10+ melanocyte precursors localize next to the DP in the
hair matrix compartment (49,50,82).

Stage 9 –
By Stage 9 of HF development, the tip of the hair shaft leaves the
IRS and enters the hair canal (Figure
1, Figure 4, stage 9). The DP is
even more fully narrowed.

Stage 10 –
The HF has reached maximal length by Stage 10; it extends to the
subcutaneous level (Figure 1, Figure 4, stage 10). The hair shaft
emerges through the epidermis.

3 -
ESSENTIAL SIGNALING PATHWAYS FOR HAIR FOLLICLE MORPHOGENESIS
3.1 -
Initial HF induction
Broad dermal Wnt signaling activity is the upstream initiating event for
HF morphogenesis. Subsequent Wnt signaling activation, alternating in epidermal
Pc and dermal DC, leads to downstream signaling events to control HF formation.
Several additional pathways, such as Eda, Fgf, Bmp and Shh signaling, have been
identified over the past two decades to be essential in either compartment and
their stage-specific roles will be described in the following.
3.1.1 -
Sequential Wnt activity in dermis and epidermis
The importance of Wnt/β-catenin signaling in HF formation has
been well demonstrated over the years (21–23,26,90–92). Mutant
mice with eliminated expression of the transcription factor Lef1, a
β-catenin binding partner, lack HF as well as other skin appendages
such as teeth and mammary gland (93).
Conversely, transgenic epidermal overexpression of Lef1 leads to abnormal HF
clustering and ectopic HF formation in hairless epithelium (94), while mice expressing stabilized
β-catenin in the epidermis exhibited de novo HF morphogenesis (95). Wnt10b expression (60) and Wnt signaling is localized in the nascent
stage 1 and 2 pre-Pc (83), and it is essential for Pc induction as epidermal
β-catenin ablation (24,55) prevented Pc formation. Likewise,
forced broad epidermal misexpression of Wnt inhibitors Dkk1, normally found
in the dermal fibroblasts surrounding early HFs, and Dkk2, a surrogate for
pre-Pc marker Dkk4, both blocked Pc formation (40,56). In
fact, the balance between Wnt signaling activators and inhibitors is thought
to limit the number of HFs by setting up a pre-pattern through lateral
inhibition that follows the reaction-diffusion model (40,96),
famously proposed by Alan Turing nearly 70 years ago (97,98). In
this model, short-range activation signals are counteracted by long-range
inhibition signals, consistent with a wider diffusion range of smaller Dkks
compared to larger, hydrophobic Wnts (96,99,100), thereby limiting the HF induction field.
Besides driving Pc initiation, localized epidermal Wnt activity is also
required for Pc formation during the transition from pre-Pc (stages 1 and 2) to Pc (stage 3) by
coordinating cell migration (36):
live imaging and tracking cell divisions during early Pc morphogenesis
demonstrated that Wnt and Eda signaling mediate Pc formation through
directed migration and cytoskeletal rearrangements, rather than cell
proliferation.
Preceding focal Wnt signaling and Pc formation, broad uniform Wnt
signaling activity in the upper dermis (24) is essential for pre-Pc induction at stage 0: dermal specific
β-catenin ablation blocked localized Pc Wnt
signaling and subsequent Pc formation (25). While broad dermal Wnt signaling is an absolute requirement
for Pc fate specification, the Wnt target gene(s) that serve as key Pc
inductive signal(s), i.e. the first dermal signal(s) is/are still unknown.
Dermal Wnt activity itself requires production of epidermal Wnt ligands, as
blocking Wnt ligand secretion in Wntless (Wls) mutants results in a failure
of dermal Wnt signaling (25).
Epidermal Wnt ligand expression is controlled by the transcription factor
ΔNp63 (48), a key regulator of
epidermal fate specification (101,102).
Finally, after HF induction and following localized Wnt signaling in
the pre-Pc and Pc, intensified Wnt activity was also found in the early
clustered DC of stage 3 HFs, which is
required for HF progression (24,46). Also at stage 3, localized high Wnt signaling in basal Pc
progenitors together with active Shh signaling (45) and a suprabasal Wntlow signaling
environment (67) are essential for
suprabasal Sox9+ HFSC fate acquisition before HF downgrowth in the following
stages. For this process, asymmetric cell division in the basal layer of
stage 3 Pcs, perpendicular to the
basal-suprabasal plane, is required for the emergence of suprabasal
SOX9+ HFSC precursors (45).

3.1.2 -
Eda signaling
Upon binding of TNF family member ligand Ectodysplasin (Eda) to its
receptor Edar, downstream NFκB activation triggers transcriptional
regulation essential for placode development (103,104).
Mutations in Eda (tabby) and Edar
(downless) in both humans and mouse models fail to form
skin appendages such as HFs and teeth (105,106). During HF
initiation, Eda is uniformly expressed throughout the epidermis, while Edar
expression is confined to the stage 1
and 2 pre-Pc and later to the stage 3 Pc (39). Eda signaling is downstream of Wnt
signaling; abolishing Wnt activity in the epidermis eliminates Edar
expression and NFκB activation, while Wnt activity persists even
after genetic abrogation of Eda signaling (24). Although Edar is one the earliest markers for pre-Pc,
Eda/Edar signaling appears to be dispensable for pre-Pc induction of first
wave HF. In the absence of Eda pre-Pcs remain stuck at the stage 1–2 stage, and is therefore required for further development to
stage 3 Pc (24). Second and third wave HFs did form, but
third wave HFs lost the characteristic zigzag shape, suggesting that Eda
signaling also plays a role in establishing the molecular mechanism for hair
shaft bending (107). In addition,
this also indicates that molecular controls of HF morphogenesis can have
intrinsic differences between first, second and third wave HFs.

3.1.3 -
Fgf20 signaling
After pre-Pc fate initiation at stage
1, a “first epithelial signal” leads to
specification and formation of the underlying DC. So far, Fgf20 has been the
only identified epithelial signal that is directly required for DC
formation; upon gene ablation of this Pc-derived factor, formation of
aggregated DC and subsequent HF morphogenesis was abolished in all first and
most second wave HFs (27). The same
group very recently demonstrated that formation of the clustered DC is
achieved through Fgf20-dependent cell migration and aggregation, and not
proliferation (37). The intercellular
machinery driving migration to form the condensed DC remains unknown, but
recent profiling of DC suggested that actin remodeling and, intriguingly,
axonal guidance genes may play a role (35). Preceding its role in DC cluster formation (stage 3), very recent work demonstrated that Fgf20
is already required for DC fate specification from fibroblasts before
aggregation takes place (43). DC
precursors, or pre-DC, are unclustered cells at stage 2 underneath pre-Pcs that transition from a
fibroblast fate to acquire the DC gene expression program. In the absence of
Fgf20, pre-DC fail to become specified.
Besides failure of DC specification and formation, Pc morphology was
also severely altered in Fgf20 mutants, with Pcs forming stripe-like pattern
instead of rounded shapes (27). Pc
expansion beyond normal size may be due to impaired lateral inhibition from
the KO pre-Pcs and the absent DCs that normally produce Dkk4 and Bmp2, and
Bmp4, respectively. While Fgf20 is a downstream target of Wnt and Eda
signaling, Edar expression levels were also severely reduced in Fgf20
knockout Pcs (27). Both cases raise
the question of whether Fgf20 acts cell-autonomously on the pre-Pc, or
whether perturbed placode development could be a secondary consequence of
altered signaling inputs from the pre-DC (stage 2) or DC (stage 3).
Taken together, as the first known epithelial signal from the pre-Pc (stage 1) towards the dermis, Fgf20 is
responsible for promoting the transition of fibroblasts to the DC fate
(43). Then Fgf20 instructs pre-DC
cells to migrate and aggregate to form the clustered DC (37).

3.1.4 -
Bmp signaling
While Wnt, Eda and Fgf signaling promote HF morphogenesis, BMP
signaling acts in an inhibitory fashion, likely to fine-tune and reinforce
the lateral inhibition already set up by Wnt/Dkk diffusion gradients for
proper spacing of HFs in the reaction-diffusion model (97). During HF morphogenesis, the ligands Bmp2
and Bmp4 are enriched in Pc and DC (25), respectively, and thought to inhibit a HF fate in
neighboring epidermis where the receptor Bmpr1a is expressed (65). Conversely, the BMP inhibitor
Noggin is enriched in the DC and thought to activate and promote Pc
formation as short-range Bmp inhibitory signal: Overexpression of Noggin
results in formation of excessive Pcs (108), while secondary HF fail to form in Noggin null mice (109,110).

3.2 -
Hair follicle downgrowth
After initial HF induction, Pc and DC are formed after sequential first
dermal and epithelial signals, respectively. The DC then produces the secondary
dermal signal, which triggers proliferation of Pc progenitors for HF downgrowth.
Continued signal crosstalk between the epidermal and dermal compartments is
thought to be crucial for HF formation. To date, none of the second dermal
signals have been identified yet, but epithelial Shh, Pdgfa, and Tgfβ
signals have been shown to act on the dermal compartment during subsequent
signal interplay for HF downgrowth (111–114). Shh
signaling was also shown to be key for regulating specification of
Sox9+ suprabasal future bulge SCs (45), as well as for maintaining the proper cellular
movement of developing Pc cells (77).
3.2.1 -
Shh signaling
Shh is expressed in the pre-Pc and Pc at all stages, while the
receptor Patched (Ptch) can be found in both epidermal and dermal
compartments (113). Shh null mice
display an arrest of HF development at the stage 4 germ, despite normal induction of both Pc and DC (111,112). Dermal specific ablation of Shh pathway component Smo
abolished dermal Shh signaling and was shown to be important for Noggin
expression in the DC and for maintaining the DC (115). Conversely, Pcs in Noggin null skins failed
to express Shh mRNA and protein (110), suggesting that Shh signaling in DCs and Noggin-mediated BMP
signaling inhibition in Pcs establish a positive feedback loop in developing
HFs.
In addition to its role in maintaining the DC, two recent studies
have shown that Shh is also indispensable for the development of the HF
epithelial fate and formation. Ouspenskaia and colleagues demonstrated that
Shh signaling is essential for the expansion of suprabasal Sox9+
HFSC precursors (45). In Shh-null
mice, there was a significant decrease in both the number of
Sox9+ suprabasal cells and levels of Sox9 expression.
Moreover, high Wnt activity in basal Pc progenitors, the precursors of
future matrix cells, is required for SHH expression, which then triggers
symmetric divisions of overlying Sox9+ suprabasal cells. Besides
establishing HFSC, Shh signaling is also essential for placode invagination
and counter-rotational placode cell movements that set up polarization
during the stage 3 to stage 4 transition of early HF morphogenesis
(Figure 4) (77). Sox9+ suprabasal cells, which
first appear around the Shh-expressing basal cells, migrate to the posterior
position, while Shh-expressing cells move anteriorly. This cellular
rearrangement is also DC dependent, as DC laser ablation resulted in
aberrant Sox9 expression in anterior placode cells (77).

3.2.2 -
PDGFA signaling
During HF morphogenesis, the ligand Pdgfa is broadly expressed in
the epidermis while the receptor, Pdgfra, is uniformly expressed in the
dermis (113). Pdgfa ablation
revealed a requirement of Pdgfa signaling for HF downgrowth; despite normal
HF induction, the mice had a sparse hair coat due to retarded HF development
(113). However, whether this a
specific HF development defect has been called into question with a more
recent study: Rezza and colleagues reported unperturbed HF induction and
development following dermal specific Pdgfra ablation (116). As arrested HF phenotypes were only found
in Pdgfa mutants with a severe systemic phenotype (113), and given that Pdgfa signaling is essential
for many developmental aspects (117), the retarded HF development could stem from secondary effects
of abrograted systemic Pdgfa signaling.

3.2.3 -
TGFβ signaling
Besides Shh signaling, Tgfβ signaling has also been
implicated as crucial for HF downgrowth. The ligand TGFβ2 is secreted
by the dermis and acts on the receptor expressed by the epithelium (31,118,119). Studies in
Tgfβ2 null mice revealed a requirement of Tgfβ signaling for
HF downgrowth as HF development was arrested early (114). A second study of Tgfb receptor ablation
demonstrated fewer and growth-retarded HFs (120). Interestingly, a recent study placed Tgfβ signaling
downstream of Eda signaling in a NF-κB/Lhx2/Tgfβ signaling
axis (33). Both mice with suppressed
NF-κB activity and knockouts of NF-κB target Lhx2 have
impaired TGFβ signalling, demonstrating that Eda signaling is not
only important for Pc maintenance during stage
1 and 2 of HF formation, but
is also essential for HF downgrowth by activating TGFβ signaling.
In summary, after the first dermal signals kick-start the initiation
of HF morphogenesis, activation of Wnt and Eda signaling promotes pre-Pc
formation and Pc stabilization. The nascent pre-Pc in turn provides the
first epithelial signals, such as Fgf20, for inducing DC fate in unclustered
precursors, and then aggregation of the maturing DC. At the same time,
inhibitory signals including Dkk4 and Bmp4 from the Pc and DC, respectively,
suppress HF formation in the interfollicular area to maintain even spacing
between established HF. Second dermal signals from the DC then promote the
proliferation of Pc progenitors for HF downgrowth. Continued signal
interplay through Shh and Tgfb signaling further promotes HF development
towards the formation of the mature HF.

4 –
CONCLUDING REMARKS
The HF morphogenesis staging guide described by Paus and colleagues in 1999
has been widely used for 20 years; it has served as an important standard of HF
development for classifying and comparing HF morphogenetic defects across numerous
HF morphogenesis studies. In light of the recent discoveries of specific early
precursor cell states in both the epithelial and mesenchymal HF compartments, as
well as of the myriad novel molecular insights driving the classification of new
stages, we propose this update here to the classical staging guide. To account for
Pc induction at a “molecular placode” pre-Pc stage, for DC cell fate
acquisition and pre-DC specification, and for the emergence of precursors to HFSC,
we subdivided the previously defined stage 0
into 2 new stages that are prefaced by a new
stage 0 with no specific patterned
molecular or cellular events, while at the same time preserving previous established
advanced stages that succeed the new precursor stages.
Continued and concerted efforts over the past 20 years to parse out
essential signals and controls of HF initiation, in both the epidermal and dermal
compartments, have provided many important insights that we summarized here.
Nevertheless, many details regarding the initiating events of the “first
dermal signal” and relevant crosstalk between the Pc and DC in the
“first epithelial signal” and “second dermal signal”
remain elusive and the full spatiotemporal account of all HF morphogenetic signaling
is complete. The near simultaneous activity of many known signaling pathways, in
both the epidermis and dermis, and interplay of positive and negative regulation
between them further complicates the story. A more granular perspective of early HF
morphogenetic events, in combination with integrated in vivo,
in vitro, and in silico approaches may aid in
deconvoluting the roles of many of these signaling pathways to paint a more complete
picture.
Technological advances have permitted the identification of stage-specific
marker genes in embryonic epidermis and dermis for identification and isolation of
relevant cell types, as well generation of more specific genetic drivers for
in vivo study. Critically, single-cell RNA-sequencing has
allowed for a more dynamic understanding of developmentally associated
transcriptional dynamics, through the use of computational modeling, as well as
identification of cellular heterogeneity. Since pseudotemporal ordering of cell
fates in the dermis allowed for revealing a putative differentiation trajectory from
upper dermal fibroblasts to pre-DC to Stage 3
and 4 DC (42,43), it is conceivable that
cognate analyses of epidermal differentiation will add in the future increased
temporal resolution and, in combination with dermal differentiation analyses, will
shed light on critical signaling crosstalk. Through observations of in
vivo phenomena in genetic abrogation studies, substantial insights into
the staging of early HF morphogenesis have been made, to drive understanding of
signaling necessity. And in combination with the modeling ability of in
silico approaches, we predict that we will continue to move steadily
toward gaining a clearer picture of the identity of crucial dermal and epithelial
signals.