PNAS PLUS

Biogeography of a human oral microbiome at the

micron scale
Jessica L. Mark Welcha,b,1, Blair J. Rossettia,b, Christopher W. Riekenb, Floyd E. Dewhirsta,c, and Gary G. Borisya,b,1
a
The Forsyth Institute, Cambridge, MA 02142; bMarine Biological Laboratory, Woods Hole, MA 02543; and cHarvard School of Dental Medicine, Boston,
MA 02115

Contributed by Gary G. Borisy, December 24, 2015 (sent for review November 10, 2015; reviewed by Patricia I. Diaz, Michael A. Fischbach, and
John J. Mekalanos)

The spatial organization of complex natural microbiomes is critical commonly interact. When the microbiota is host-associated, its
to understanding the interactions of the individual taxa that com- physiology and ecology become intimately connected with those of
prise a community. Although the revolution in DNA sequencing has the host at both micron scales and host scale and are capable of
provided an abundance of genomic-level information, the biogeog- critically influencing the promotion of health or the progression
raphy of microbiomes is almost entirely uncharted at the micron scale. toward disease. Thus, it is necessary to know not only who is next
Using spectral imaging fluorescence in situ hybridization as guided by to who but also, who is next to what.
metagenomic sequence analysis, we have discovered a distinctive, Dental plaque is a human microbiome community with study
multigenus consortium in the microbiome of supragingival dental that dates back to the initial observations of Leeuwenhoek over
plaque. The consortium consists of a radially arranged, nine-taxon 300 years ago (6). Modern studies have analyzed taxon–taxon
structure organized around cells of filamentous corynebacteria. The associations through pairwise binding interactions between
consortium ranges in size from a few tens to a few hundreds of members of different oral microbial species. These interactions,
microns in radius and is spatially differentiated. Within the structure, termed “coadhesion” or “coaggregation,” have been described in
individual taxa are localized at the micron scale in ways suggestive of an extensive body of literature (7, 8) and form the basis for an
their functional niche in the consortium. For example, anaerobic taxa influential model describing the structure and development of
tend to be in the interior, whereas facultative or obligate aerobes dental plaque as an ecological succession (9). This model begins
tend to be at the periphery of the consortium. Consumers and with the salivary pellicle coating the teeth and the initial attach-
producers of certain metabolites, such as lactate, tend to be near
ment of Streptococcus spp. and Actinomyces spp. to the pellicle.
each other. Based on our observations and the literature, we propose
These attached microbes then serve as a substrate for the binding
a model for plaque microbiome development and maintenance
of a variety of other colonizers, including Fusobacterium nucleatum,
consistent with known metabolic, adherence, and environmental
which functions as a bridge between the early colonizers and the
considerations. The consortium illustrates how complex structural
late-colonizing pathogens by virtue of its capacity to bind physically
organization can emerge from the micron-scale interactions of its
to both sets of microbes. This model synthesizes in vitro and in vivo
constituent organisms. The understanding that plaque community
organization is an emergent phenomenon offers a perspective that is
observations to make testable predictions about the spatial struc-
general in nature and applicable to other microbiomes.
ture of mature dental plaque, but a direct test of the model by high-
resolution imaging has not previously been undertaken.
The study of microbial communities has been revolutionized by
biofilm | imaging | microscopy | microbial ecology metagenomic and metatranscriptomic approaches, which have

B iogeography—the study of the distribution of organisms across

the globe—seeks to recognize patterns in the spatial distri-
bution of organisms and discover the forces that underlie those
Significance

patterns. Bacteria are micron-sized, and many of the forces and The physiology and ecology of complex microbial communities
factors that underlie their distributional patterns operate at mi- are strongly dependent on the immediate surroundings of
cron scales and are qualitatively different from the large-scale each microbe, including the identity of neighboring microbes;
factors, such as climate, that drive traditional biogeography. To however, information on the micron-scale organization of

MICROBIOLOGY
frame the analysis of microbial distribution patterns at the scale microbiomes is largely lacking. Using sequencing data com-
that microbes themselves experience, we introduce the concept of bined with spectral fluorescence imaging, we have discovered
micron-scale biogeography: the study of the distribution of mi- a multigenus, highly organized microbial consortium in human
crobes relative to micron-scale features of their environment. dental plaque. The spatial structure of the consortium reveals
These features include the host or inanimate surfaces on which the unanticipated interactions and provides a framework for un-
microbes reside as well as local gradients of nutrients and oxygen. derstanding the organization, metabolism, and systems biology
Key components of the micron-scale environment, particularly in of the microbiome and ultimately, its effect on the health of the
biofilms and other densely populated habitats, are the microbes human host. Our synthesis of high-throughput sequencing data
themselves, serving as substrates for attachment of other mi- with spatial and structural information shows the informative
crobes, creating spatial structure, and acting as point sources for value of microbial biogeography at the micron scale.
diffusible metabolites.
Micron-scale biogeography is critical to understanding the Author contributions: J.L.M.W., F.E.D., and G.G.B. designed research; J.L.M.W., B.J.R., C.W.R.,
and G.G.B. performed research; J.L.M.W., B.J.R., and F.E.D. contributed new reagents/
physiology and ecology of the community as well as its systems analytic tools; J.L.M.W., B.J.R., C.W.R., F.E.D., and G.G.B. analyzed data; and J.L.M.W.
biology and its effects on human health and disease. Close and G.G.B. wrote the paper.
proximity or physical contact between two microbes can sub- Reviewers: P.I.D., University of Connecticut Health Center; M.A.F., University of California,
stantially alter their physiology, for example conferring on an San Francisco; and J.J.M., Harvard Medical School.
anaerobe the ability to survive in an aerobic environment (1) or The authors declare no conflict of interest.
dramatically altering the range of metabolites produced com- 1
To whom correspondence may be addressed. Email: [email protected] or
pared with those produced by the same organism in isolation (2–5). [email protected].
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Thus, a mechanistic understanding of the physiology of key players This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
depends on knowing the identity of the neighbors with which they 1073/pnas.1522149113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1522149113 PNAS | Published online January 25, 2016 | E791–E800

 revealed enormous complexity (10). However, the groundbreaking plaque. This genus was present in only trace amounts in saliva
methods that revealed the complexity have the drawback that the and on six of eight oral surfaces sampled (the tongue, buccal
sample must be homogenized for nucleic acid extraction, thereby mucosa, keratinized gingivae, hard palate, tonsils, and throat)
destroying any spatial structure at the micron scale that might have but made up 8% of the bacterial community in SUBP and more
existed. The absence of detailed spatial information represents a than 12% in SUPP (Fig. 1C) (19, 20). The HOMD recognizes six oral
fundamental gap in knowledge that precludes a full understanding species within the genus Corynebacterium. However, of these six, only
of the assembly and interactions of complex microbial communities. two, Corynebacterium matruchotii and Corynebacterium durum, were
The integration of spatial information with high-throughput present at significant levels in plaque. Although C. matruchotii was
sequencing data by direct visualization of spatial structure opens the dominant species in most individuals, C. durum was domi-
an entirely different window into understanding community nant in some (Fig. 1D). Taking the two species together, the ge-
structure. Fluorescence in situ hybridization (FISH) targeting nus not only had a high mean abundance, but also, it was
rRNA (11, 12) can be used to identify nearly any microbe, but consistently abundant, with a relative abundance of 3% or more
because of technical limitations, it is generally used to differentiate in 90% of the individuals. The abundance and prevalence of Co-
only two or three microbial types simultaneously. The resulting rynebacterium suggest that it plays an important role in the plaque
images reveal distinctive distributions of individual organisms (13– community, whereas its plaque specificity suggests that it occupies a
15) but not the overall structure of the community. However, niche that is dependent on properties of the tooth surface and/or
fluorescence spectral imaging allows the differentiation of many the gingival crevice.
fluorophores and creates an opportunity to take a systems-level Habitat analysis of 12 other abundant plaque genera (Fig. 1E)
view of the spatial structure of the microbiota (16), simultaneously showed large differences in their degree of specificity to plaque
imaging and identifying all members of a complex microbial but only modest differences in their relative abundance in SUPP
consortium. Here, we analyze sequencing data from the Human compared with SUBP. Genera with strong plaque specificity, in
Microbiome Project (HMP) to identify the major bacterial taxa addition to Corynebacterium, included Capnocytophaga, which was
likely to be important in the structure and function of supra- 10-fold more abundant in plaque than at nonplaque sites, as well
gingival plaque, and by imaging the spatial organization of these as Lautropia and Rothia. By contrast, genera such as Strepto-
most abundant taxa, we describe a complex, spatially organized, coccus occupied a broad range of habitats. Despite being the
multigenus consortium. This synthesis of high-throughput se- single most abundant genus in SUPP, Streptococcus was sub-
quencing data with spatial and structural information may serve as stantially more abundant at nonplaque sites than in plaque on
a case study in microbial biogeography at the micron scale. average. This wide-ranging habitat preference likely reflects the
capacity of Streptococcus to be an efficient colonizer of multiple
Results oral surfaces. Additional genera with broad habitat range in the
Identification of Bacterial Taxa Important in Supragingival Plaque. mouth include Haemophilus and Veillonella. Supragingival plaque is
The Human Oral Microbiome Database (HOMD) (17) contains often characterized as being composed primarily of Gram-positive
707 entries at the species level. This enormous diversity poses an aerobes, whereas Gram-negative anaerobes come to dominate
enormous challenge for efforts to sort out the spatial and struc- subgingival plaque, particularly in individuals affected by peri-
tural relationships of the taxa. In an attempt to reduce the com- odontitis (21). However, habitat analysis of genera shows that the
plexity to manageable proportions, we sought guidance from the similarities between the two plaques are more striking than their
16S rRNA gene sequencing data generated by the HMP (18). We differences in the healthy individuals sampled by the HMP. Most of
previously applied an information theory approach to analysis of the abundant genera are enriched in SUPP compared with SUBP
the oral microbiome at the single-nucleotide level, resulting in by a small and relatively constant factor of ∼1.3–1.6. Some genera
high-resolution sequence groups termed oligotypes (19). The oligo- (Actinomyces, Porphyromonas, and Veillonella) are equally abundant.
types were assigned to HOMD species and analyzed for each of A few predominantly anaerobic genera, notably Prevotella and
nine oral habitats defined by the HMP. This analysis showed that Fusobacterium, are more abundant in SUBP but only by a factor of
most species of oral bacteria are habitat specialists and that the ∼2. Thus, the overall similarity of distribution suggests a close
complexity can be reduced simply by considering only the bacteria connection between these two spatially adjacent communities.
resident in the oral habitat of interest. In the following discussion,
we consider plaque to mean specifically the biofilm that forms on Plaque Microbiota Is Organized into Highly Structured, Multigenus
teeth as opposed to other oral substrates, such as gums or tongue, Consortia. Bacteria are micron-sized and live in a chemical and
and we focus on the microbiota resident in plaque above the gum structural environment with micron-scale heterogeneity. There-
line, supragingival plaque. fore, an understanding of the micron-scale spatial organization of
As an initial basis for identifying key taxa in supragingival bacterial communities is necessary for understanding how these
plaque, we assessed the abundance and prevalence of the oligo- communities function. A striking degree of spatial organization can
types grouped by genus. This analysis readily identified a group of become visible, even with simple procedures, when plaque samples
bacterial genera that were both abundant and prevalent (Fig. 1A). are prepared gently to retain their structure. For example, when
Of 57 genera detected in supragingival plaque (SUPP), most had plaque was spread out on a slide, conventional FISH with two
both low abundance and low prevalence. In contrast, 13 genera had probes revealed clumps of Corynebacterium, with long filaments
at least 3% mean abundance and were also highly prevalent, each that were coated at their tips by brightly staining cocci (Fig. 2).
being detected in more than 90% of SUPP samples. Collectively, Associations between cocci and filaments in plaque were docu-
these 13 genera accounted for 85% of the sequencing data from mented over 40 years ago and have been called “corncobs” (22,
SUPP. The same 13 genera also accounted for more than 80% of 23). However, the arrangement in dissected plaque, which we vi-
the subgingival plaque (SUBP) data (Fig. 1B), indicating a close sualized with FISH, is striking in that the filaments are clearly
relationship between the two plaque sites. Because of their abun- continuous from the base of the clump to the tips, but the cocci
dance and prevalence, these taxa are likely to form both the spatial are restricted to the tips or distal ends of the filaments (Fig. 2).
and the metabolic framework of the healthy plaque microbiome. This spatial arrangement suggests a role for Corynebacterium as a
Taxa that are present primarily or exclusively in one site may foundational taxon that structures the environment in a way that
provide clues to the distinctive features of the habitat and the creates a microenvironment favorable to the growth of the cocci.
role that those taxa contribute to the site. Habitat analysis of the Why the cocci are restricted to the distal ends is a key question,
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oral microbiome suggested that one genus, Corynebacterium, in the answer to which requires more complete information about
particular was strikingly specific to supragingival and subgingival the surrounding structure.

E792 | www.pnas.org/cgi/doi/10.1073/pnas.1522149113 Mark Welch et al.

 PNAS PLUS
We used two complementary methods designed to preserve
A and visualize the spatial structure of the plaque community:
whole-mount preparations and methacrylate embedding. Whole
mounts permitted the imaging of entire 3D structures, including
long filaments, but at the expense of slight spatial distortion resulting
from compression. Embedding and sectioning preserved micron-
scale spatial relationships more accurately but at the expense of loss
of 3D continuity. Regardless of the preparation method, we de-
tected similar microbial consortia in all samples. For a systems-level
analysis of the spatial organization of these samples, we used
Combinatorial Labeling and Spectral Imaging FISH (CLASI-FISH)
(16) to differentiate up to 15 taxa simultaneously. In our previous
proof of concept of CLASI-FISH, we labeled plaque that was
partially dispersed to single-cell thickness (16), so that spectral sig-
B natures created by binary combinations of fluorophores could be
read unambiguously. For this study, we wished to analyze more
intact 3D structures, in which multiple cells may lie on top of one
another, even in a single optical plane of focus. In such samples,
overlapping cells with different binary signals in the same pixel
could generate ambiguity in taxon identification. To avoid this
ambiguity, we used a simplified labeling strategy, in which a single
fluorophore served as the spectral signature to identify each taxon,
and as many as 10 distinct fluorophores were used simultaneously.
The combination of sequence analysis with imaging allowed an
assessment of spatial organization that was taxonomically both
C wide-reaching and refined. We used FISH probes with broad
coverage, using probes for four phyla, two classes, three families,
and 15 genera (Table S1). More specificity was provided by HMP
sequencing data, which showed that, for most plaque genera, a
small number of species was dominant. Of the 13 most abundant
genera, one genus was represented by only a single major plaque
species (Lautropia mirabilis), and six were represented primarily
by two or three major species or small clusters of species (Table S2).
D Collectively, the probes that we used targeted 96–98% of the cells in
a healthy supragingival plaque microbiome as judged by rRNA tag
sequencing data from the HMP (Table S2). Among these probes, 2
family- and 11 genus-level probes covered 88% of the sequencing
data and are shown in Figs. 2–8 and Figs. S1–S4. When describing
imaging results in the following section, we will use the taxon name
as shorthand for cells in the image that are reactive with the taxon-
E specific probe, but it should be kept in mind that these organisms
are likely to be members of the species shown in Table S2. The
genera Haemophilus and Aggregatibacter are phylogenetically inter-
twined in the family Pasteurellaceae and targeted by probe Pas111,
which we refer to as Haemophilus/Aggregatibacter. The genera
Neisseria, Kingella, and Eikenella are likewise intertwined in the

MICROBIOLOGY
family Neisseriaceae and targeted by probe Nei1030, which we refer
to as Neisseriaceae.
We detected in plaque a complex microbial consortium char-
acterized by the presence of a mass of Corynebacterium filaments
Fig. 1. Metagenomic sequence analysis points to Corynebacterium as a key with Streptococcus at the periphery. We refer to this structure as a
taxon in supragingival plaque. (A) Prevalence abundance plot for supragingival “hedgehog” because of its spiny, radially oriented filaments. We
plaque. (B) Cumulative abundance of genera in both supra- and subgingival identified nine taxa as regular participants in hedgehog structures:
plaque. Genera with greater than 3% abundance in SUPP, mean across 148
Corynebacterium, Streptococcus, Porphyromonas, Haemophilus/
subjects, are indicated by colored dots in A and bar segments in B; B also shows
the abundance of these genera in SUBP. Data are from the HMP (18) V3–V5
Aggregatibacter, Neisseriaceae, Fusobacterium, Leptotrichia, Capno-
region of 16S rRNA, analyzed by oligotyping (19), and grouped by genus. cytophaga, and Actinomyces. Other genera were detected rarely or
(C) Corynebacterium is far more abundant in plaque than in other oral sites. inconsistently in the hedgehog structures. To visualize the regular
Mean abundances of C. matruchotii and C. durum are shown for each oral site constituents of the consortium simultaneously, we constructed a
analyzed by oligotyping (19). BM, buccal mucosa; HP, hard palate; KG, keratinized probe set consisting of 10 probes: the 9 probes targeting these taxa
gingiva; PT, palatine tonsils; SV, saliva; TD, tongue dorsum; TH, throat. (D) Cory- plus the universal probe Eub338 reactive with essentially all bac-
nebacterium is a major component of most plaque samples. Relative abundance teria. Each of these 10 probes consisted of a unique oligonucleo-
of Corynebacterium in the HMP SUPP samples from 148 individuals (19).
tide conjugated to a unique fluorophore (Table S3). To validate
C. matruchotii is usually more abundant, but C. durum dominates some samples.
(E) Habitat analysis identifies genera that are strongly characteristic of SUPP. The
plaque to nonplaque ratio measures the relative abundance of each genus in two
plaque sites compared with seven nonplaque sites sampled by the HMP [calcu- Capnocytophaga as the taxa most preferentially abundant in plaque. The
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lated as (mean SUBP + mean SUPP)/(mean BM + mean KG + mean HP + mean SUPP to SUBP ratio identifies these genera as relatively more abundant in
SV + mean PT + mean TH + mean TD)]. This ratio identifies Corynebacterium and SUPP than in SUBP. Colors in E are the same as those in A and B.

Mark Welch et al. PNAS | Published online January 25, 2016 | E793
 cells of two other taxa, Porphyromonas and Haemophilus/Aggre-
gatibacter, both of which are in close contact with Streptococcus
cells. On their way to this corncob region in the periphery, the
Corynebacterium filaments traverse the annulus that is densely
populated with elongated rods of Fusobacterium, Leptotrichia,
and Capnocytophaga, with cells of all four taxa oriented in
roughly the same direction (Fig. 3G).
Completing the overview of the structure, a comparison of the
fluorescent signal from the universal probe (Fig. 3H) to the overlay
of nine specific probes (Fig. 3D) shows that the taxon-specific
probes identify nearly all of the cells in the structure. A second focal
plane near the exterior of the structure (Fig. 3 I–L) shows a view of
the outer shell composed primarily of corncobs. Toward the center
of the image, the edge of the Fusobacterium–Leptotrichia annulus
can be seen in end-on view (Fig. 3K). In summary, the plaque
hedgehog is a radially organized, multigenus consortium with a
framework composed primarily of Corynebacterium, a multitaxon
filament-rich annulus, and a periphery of corncob structures.
Corncobs are defined morphologically as structures in which
coccoid cells, “kernels,” surround a central filament. Our CLASI-
Fig. 2. Corncob structures formed by Corynebacterium and cocci in plaque.
FISH results revealed that the kernels were of different taxo-
Corynebacterium cells (magenta) are visible as long filaments, with cocci nomic types and could be either single or double layer (Fig. 4).
(green) bound to the tips of the filaments. Partially disrupted plaque was Single-layer corncobs had kernels of either Streptococcus or Por-
hybridized with a probe for Corynebacterium and a universal bacterial phyromonas; double-layer corncobs consisted of a combination of
probe. Image was acquired using a Zeiss AxioImager 63× Plan-Apochromat Streptococcus as the inner layer and Haemophilus/Aggregatibacter
1.4 N.A. objective and Apotome structured illumination. (Scale bar: 20 μm.) as the outer layer. The most common corncob had a single layer
of Streptococcus kernels surrounded by a partial or complete layer
of Haemophilus/Aggregatibacter. Porphyromonas kernels could be
the probes for specificity, we applied the 10-probe set to pure colinear with Streptococcus around the same filament, or could
cultures, which we hybridized and imaged under the same condi- form entire corncobs of their own, but in either case were
tions as natural plaque samples. All probes reacted strongly with always organized in a single layer. In contrast, cells of Haemophilus/
the target taxon and insignificantly with the nontarget taxa Aggregatibacter were never observed to form their own corncobs with
(Fig. S1). Corynebacterium filaments. When present, they were always found
This 10-probe set revealed large, organized hedgehog structures adjacent to Streptococcus cells. The Haemophilus/Aggregatibacter–
with a generally consistent composition and spatial arrangement Streptococcus association was evidently specific, because Haemophilus/
(Fig. 3). The fluorescence signal from each of the probes was ac- Aggregatibacter was not found adjacent to cells of Porphyromonas
quired with a spectral, confocal microscope, was differentiated or other taxa in the absence of Streptococcus. Overall, the close
using a linear unmixing algorithm (Materials and Methods), and is spatial proximity of multiple taxa in corncobs suggests the pos-
presented in false color, with combinations of probes shown sibility of significant competitive, exploitative, or mutualistic in-
superimposed as detailed in Fig. 3. Fig. 3 A–D and F–H shows teractions among these taxa.
a single focal plane near the middle of the structure. Co- In a substantial fraction of corncob structures, weak or no
rynebacterium filaments radiate outward from near the center of fluorescence signal was detected from the central filament in the
the image. The coccoid Streptococcus cells are arranged around the region where the kernels were present. Lack of hybridization to
distal tips of the Corynebacterium filaments (Fig. 3A). Also located the central filament was particularly frequent in whole-mount
at the periphery of the structure, in the same region as the Strep- preparations (Fig. 4A). In embedded and sectioned preparations,
tococcus, are cells of Haemophilus/Aggregatibacter and Porphyr- the central filament was more consistently visualized (Fig. 4B).
omonas (Fig. 3B). Capnocytophaga occupies a wide band just inside Higher magnification images of longitudinal and cross-section
the periphery (Fig. 3B). Also occupying this band but forming a views (Fig. 4C) illustrate the visualization or lack thereof of the
more complete ring or annulus between the periphery and the base central filament. However, in all cases in which the central fila-
are Fusobacterium and Leptotrichia (Fig. 3C). Neisseriaceae forms ment was clearly labeled, it hybridized with the Corynebacterium
clusters in and near the periphery (Fig. 3C). Actinomyces, which was probe, even in cases in which cells of other taxa, including Fuso-
represented by only a small number of cells in this particular bacterium, Leptotrichia, and Capnocytophaga, were present in
structure, tended to be located near the base. All taxa are shown abundance immediately adjacent to the corncobs (Fig. S2). This
superimposed in Fig. 3D. observation indicates that, rather than binding promiscuously to
The spatial arrangement of Corynebacterium relative to other any available filamentous substrate, the cocci are engaged in a
taxa in the structure is detailed in Fig. 3 E–G. Long filaments highly specific interaction with Corynebacterium.
that move in and out of the plane of focus can be only partially The filaments or elongated rods inhabiting plaque hedgehogs
captured in a single optical section (∼1-μm thickness). To visu- were striking in both their density and their spatial organization.
alize the continuity of these filaments, we generated a maximum Both Fusobacterium and Leptotrichia showed elongated mor-
intensity projection of three adjacent optical sections (Fig. 3E), phology and were dispersed thinly at the periphery of the hedgehog
which shows single filaments that are continuous for more than but reached very high densities in the region immediately proximal
50 μm and reach from the core to the periphery of the structure. to the periphery (Fig. 3C), a region that we call the filament-rich
Some filaments remain visible after they enter the region that annulus. Capnocytophaga, likewise, reached high densities in the
contains Streptococcus, whereas others apparently disappear annulus but also extended into regions of the consortium that were
when they enter this zone. A detail of the periphery (Fig. 3F) rich in Neisseriaceae (Fig. 3 B and D). In whole-mount prepara-
shows that the corncob structures are composed of a filamentous tions, many cells in the filament-rich annulus overlapped in images
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core (sometimes visualized as Corynebacterium but frequently in which all taxon channels were superimposed (Fig. 3D). This
not stained) surrounded primarily by Streptococcus but also by overlap was likely caused, in part, by compression of the 3D

E794 | www.pnas.org/cgi/doi/10.1073/pnas.1522149113 Mark Welch et al.

 PNAS PLUS
A B C D

E F G H

I J K L

Fig. 3. A hedgehog structure in plaque showing spatial organization of the plaque microbiome. Plaque was hybridized with a set of 10 probes each labeled
with a different fluorophore. Each panel shows the superposition of several of these individual fluorophore channels. A–D and F–H show a single focal plane
near the center of the structure, with two to three fluorophore channels shown in each of A–C and all nine specific probes superimposed in D. (E) Maximum
intensity projection of three planes, representing a total of ∼2 μm of thickness, to visualize the continuity of Corynebacterium filaments from the center
toward the edge of the structure. F is a detailed view of corncob structures. G is a detailed view of mixed filaments. H shows the fluorophore channel
corresponding to the universal bacterial probe, showing that the specific probes (D) identify most of the cells that hybridize to the universal probe. I–L show a

MICROBIOLOGY
second focal plane near the periphery of the structure. Fluorophore channels shown correspond to the following genera in the figure: (A, E, and I) Co-
rynebacterium and Streptococcus; (B and J) Capnocytophaga, Porphyromonas, and Haemophilus/Aggregatibacter; (C and K) Fusobacterium, Leptotrichia, and
Neisseriaceae; (D and L) all nine specific probes; (F) Corynebacterium, Streptococcus, Porphyromonas, and Haemophilus/Aggregatibacter; (G) Co-
rynebacterium, Fusobacterium, Leptotrichia, and Capnocytophaga; and (H) Bacteria. The plaque sample was fixed in 2% (wt/vol) paraformaldehyde, stored in
50% (vol/vol) ethanol, and spread onto the slide in 50% (vol/vol) ethanol in preparation for FISH.

structure in whole-mount preparations, so that the cells were more within the base of the hedgehog or adjacent to hedgehogs, as
densely packed than would occur in uncompressed material. In shown in Figs. 6 and 7. The presence of Actinomyces near the base
plaque embedded in methacrylate and sectioned, the compression of a hedgehog is suggestive of the possibility that Corynebacterium
was eliminated, and the images showed cells that were tightly finds its attachment site in plaque not directly on the tooth but on
packed but clearly resolved and distinct from one another (Fig. 5). a preexisting biofilm containing Actinomyces.
Notably, these images showed that bacteria do not form large Application of a nested probe set allowed identification of the
single-taxon clusters within hedgehogs. Instead, cells of at least framework Corynebacterium taxon to the species level. As shown
four different taxa were intermingled at micron scales. These in Fig. 7 and Fig. S3, only three genera, Corynebacterium, Actino-
images show that the local environment of a cell in hedgehog myces, and Rothia, comprised virtually all of the Actinobacteria
consortia includes cells of several other taxa, even when we define present in plaque, and the species C. matruchotii comprised nearly
local to mean within a radius of as little as 5–10 μm. all of the Corynebacterium in the hedgehog structure.
By contrast, the localization of Actinomyces relative to Co- Hedgehog structures showed near-universal prevalence among
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rynebacterium was characterized more by patchy clusters than by individuals, but the fraction of plaque consisting of hedgehogs
intermingling. Actinomyces cells were generally detected in clumps was highly variable from sample to sample, even within a single

Mark Welch et al. PNAS | Published online January 25, 2016 | E795
 A this spatial organization indicate that micron-scale organization
reflects a finely tuned interaction among the cells comprising
oral microbial communities.
Discussion
Organization of Hedgehog Structures. The spatial organization of
hedgehog consortia provides a framework for understanding the
community structure and metabolism of the plaque microbiome.
A modest number of abundant taxa makes up the clear majority
of the cells in the structure, and these taxa are arranged in an
organized spatial framework, within which each microbe oc-
cupies a characteristic position. Based on the literature, we in-
terpret the metabolic, adhesive, and environmental drivers of
plaque spatial structure as diagrammed in Fig. 9.
B The radial organization of hedgehogs, built on a framework of
Corynebacterium, suggests that Corynebacterium is the founda-
tion taxon of the consortium: it structures the environment,
thereby creating habitat for other organisms and nucleating a
plaque-characteristic consortium. Consistent with this view, our
habitat analysis showed that Corynebacterium was the genus most
characteristic of plaque. The physical environment of plaque is
distinctive from all other oral habitats because of the tooth
surface itself: the tooth represents a solid surface permanently
exposed in the mouth, whereas all other oral surfaces are cov-
ered in epithelial cell layers that frequently shed. Our model
suggests that Corynebacterium proliferates in plaque and struc-
tures the plaque environment because it has adopted a strategy
of filamentous growth outward from the tooth, anchored in a
C base cemented to that permanent, exposed surface. By embed-
ding itself in a biofilm matrix attached to the tooth, Co-
rynebacterium could anchor the entire structure and create a
protected reservoir from which it can regrow after its removal by
abrasion or oral hygiene procedures, thus accounting for how

Fig. 4. Complex corncob structures in SUPP. (A and B) Clusters of corncobs at

the perimeter of hedgehog structures. (A) Whole mount of plaque hybridized
with probes for Corynebacterium, Fusobacterium, Streptococcus, Porphyr-
omonas, and Haemophilus/Aggregatibacter. (B) Methacrylate-embedded section
hybridized with probes for Corynebacterium, Streptococcus, Porphyromonas,
and Haemophilus/Aggregatibacter. (C) Gallery of representative images showing
types of corncobs frequently observed. (Scale bar: C, 5 μm.)

individual. We detected hedgehogs in every individual who was

sampled on multiple occasions and in ∼80% of individuals sampled
only once. The most exposed surface sampled, the tooth surface on
the buccal side, yielded hedgehogs; so did plaque from the
gingival margin. Some samples contained multiple hedgehog
structures adjacent to one another (Fig. S4). Other samples lacked
hedgehogs but contained other consortia. For example, clusters of
Lautropia formed the center of a structure that also contained
Streptococcus, Haemophilus/Aggregatibacter, and Veillonella and was
reminiscent of a cauliflower (Fig. 8). Most samples contained a
mixture of hedgehogs and other consortia. Because of this extensive
variability and the time-intensive nature of spectral imaging analysis,
higher-throughput imaging methods will be required to conduct a Fig. 5. Filaments and rods of several genera intermingle at micron scales in
an annulus of the hedgehog structure. The two images shown are from
comprehensive analysis of spatial, temporal, and individual variation
methacrylate-embedded, sectioned plaque from two different donors. Both
in the abundance of hedgehogs and other consortia in plaque. samples were hybridized with probes for Corynebacterium, Fusobacterium,
In summary, we have discovered distinctive, multigenus con- Leptotrichia, Streptococcus, Porphyromonas, Haemophilus/Aggregatibacter,
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sortia in dental plaque, with each taxon localized in a precise and and Neisseriaceae; the probe set in Upper also included a probe for Capno-
well-defined spatial zone. The precision and reproducibility of cytophaga.

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 PNAS PLUS
hydrogen peroxide can be inhibitory to susceptible microbes but
not to other participants in corncobs: hydrogen peroxide is de-
toxified by catalase-producing members of the commensal
microbiota, including Corynebacterium and Aggregatibacter, and
lactate is used preferentially as a growth substrate by Aggregati-
bacter (28). Capnocytophaga requires CO2 for growth, and its
abundance just inside the corncob shell suggests that it is making
use of a CO 2 -rich environment generated by Streptococcus.
Fusobacterium and Leptotrichia are generally considered anaer-
obes, but strains of both have recently been found to grow
efficiently as microaerophiles as well (1, 29–31), and their pro-
liferation in the annulus proximal to the corncob shell suggests a
low-oxygen environment there. In summary, the precise localiza-
tion of taxa within this complex structure is consistent with the
modulation of the chemical environment that we would expect to
be carried out by Streptococcus but likely includes other, as yet
unknown metabolic interactions among neighboring cells. In con-
trast, the localization of Porphyromonas in the outer shell of the
hedgehog structure, in a presumably aerobic environment, is un-
expected. Both of the widely studied species of Porphyromonas,
Porphyromonas gingivalis and Porphyromonas endodontalis, are
strict anaerobes. However, those two species are rare in the plaque
Fig. 6. Localization of Actinomyces within hedgehogs, in patches within of healthy individuals, whereas two additional species, Porphyr-
the base region of hedgehogs, and adjacent to them. omonas catoniae and Porphyromonas pasteri (previously Porphyr-
omonas sp. HOT 279), are abundant (19). It is likely that the
Porphyromonas spp. that we detect in hedgehogs are represen-
this organism maintains a high relative abundance and a prom-
tatives of these less well-known taxa and are at least
inent position specifically in the microbiota associated with the
moderately aero-tolerant.
tooth surface, i.e., dental plaque. Within each of the strata in the hedgehog, cells of each taxon
Other taxa involved in the hedgehog are stratified into distinct
are arranged not as single-taxon microcolonies but in close
zones within the structure, so that different sets of taxa are
proximity to cells of other genera. Corncob structures, for ex-
characteristic of the perimeter, the filament-rich annulus, and
ample, are characterized by direct physical contact between the
the base. A consequence of this stratification is to reduce the
number of taxa likely to be the major drivers of community me-
tabolism in each of the zones. Of the nine taxa that we show to be
abundant participants in hedgehogs, the three that are most likely to
be key participants in the aerobic metabolism at the perimeter are A
Streptococcus, Haemophilus/Aggregatibacter, and Porphyromonas. In
addition, Neisseriaceae may contribute, although it is less regular
in its presence at the perimeter. Key participants in metabolism of
the filament-rich annulus are likely to be the three taxa that
proliferate most specifically and abundantly there: Fusobacterium,
Leptotrichia, and Capnocytophaga. The base is likely dominated
by the metabolism of Corynebacterium itself, modulated by other
taxa, such as Actinomyces, when they are present. Imaging the
spatial organization of the structure focuses attention on small

MICROBIOLOGY
sets of taxa that are highly likely to interact with one another in
the healthy human mouth, and can serve to guide studies of
metabolic interaction in the most promising direction.
The selection and spatial stratification of taxa in the hedgehog
are likely driven by environmental and biochemical gradients. B C D
Plaque is bathed in a flow of saliva that presents an oxidizing
environment, but microbes at the surface of a biofilm can rapidly
consume the O2, resulting in a hypoxic environment within 10–20 μm
of the surface (24). Streptococcus, a facultative aerobe, can thrive
in an oxygen-rich environment, consistent with its location at
the periphery of the consortium. In agreement with our lo-
calization of the Streptococcus-containing corncobs to the pe-
riphery of the hedgehog, earlier electron micrographs of the
plaque colonizing teeth and enamel crowns clearly show the lo-
calization of corncob structures at the periphery of plaque, on
the side away from the enamel surface (23, 25), although these Fig. 7. Nested probing for species-level identification of Corynebacterium.
Methacrylate-embedded, sectioned plaque was hybridized with a nested
earlier studies were unable to assign a taxonomic identity to the
probe set targeting cells at the taxonomic levels of phylum, genus, and
participants in the corncob structures. The location of Strepto- species. (A) Low-magnification image shows the three major oral genera of
coccus at the periphery, in turn, drives biochemical gradients. phylum Actinobacteria: Corynebacterium, Actinomyces, and Rothia. High-
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Streptococci consume sugars and oxygen and generate lactate, magnification views show (B) all Actinobacteria, (C) the three genera, and
acetate, CO2, and hydrogen peroxide (26, 27). Both lactate and (D) C. matruchotii.

Mark Welch et al. PNAS | Published online January 25, 2016 | E797
 Relation to Previous Models of Plaque Structure and Development.
The prevalence of hedgehog structures alters our understanding
of the dynamics of colonization of oral surfaces and the succes-
sional development of plaque. Clean enamel, glass, or hydroxy-
apatite surfaces in the mouth are initially colonized by a mixed
community in which Streptococcus and Actinomyces are prominent
(32–34). Previous models of development and succession in
plaque, after initial colonization, assign a central role to Fuso-
bacterium spp. in physically linking early and late colonizers (9, 35)
or creating the conditions necessary for colonization of plaque by
pathogens (1, 36, 37). Whether these models were meant to de-
scribe interactions in supragingival as opposed to subgingival
plaque is not entirely clear; the work on initial colonization gen-
erally used substrates mounted supragingivally in the mouth,
whereas the pathogens in the climax community were subgingival
anaerobes. The genus Corynebacterium is conspicuously absent
from the early microbiota colonizing enamel and from these
models but is one of the more abundant plaque taxa detected in
cultivation-independent analyses based on sequencing of rRNA
genes (18, 20, 38). These cultivation-independent analyses repre-
sent neither the very earliest stages in colonization nor the highly
mature and complex subgingival biofilm associated with perio-
dontitis, but instead represent ordinary daily plaque accumulation
sampled from healthy subjects. The results that we present here,
using HMP sequencing data and samples of ordinary plaque from
healthy volunteers, show Corynebacterium as the taxon that pro-
Fig. 8. A cauliflower structure in plaque composed of Lautropia, Strepto-
coccus, Haemophilus/Aggregatibacter, and Veillonella. Scattered cells of Pre-
vides a physical link to each of the other taxa in the hedgehog
votella, Rothia, and Capnocytophaga are also visible. structure. Our results do not suggest a central role for Fusobacterium

central Corynebacterium filament and the surrounding Strepto-

coccus (23). We detected Porphyromonas cells sometimes forming
separate corncobs, but also present in mixed corncobs immediately
adjacent to Streptococcus. This arrangement raises the question
of whether the Porphyromonas cells are in direct competition
with Streptococcus, for example competing for attachment sites
on the Corynebacterium filament that allow the attached cell to
be bathed in the surrounding nutrient-rich saliva. Alternatively, it
is conceivable that Streptococcus and Porphyromonas may facilitate
each other’s attachment in the corncob and thereby gain some
cross-feeding metabolic advantage. The positioning of Haemo-
philus/Aggregatibacter in corncobs, by contrast, was not directly
adjacent to the central filament in the absence of Streptococcus.
This arrangement could arise simply from the binding properties
of the cells, if they find attachment sites only on Streptococcus, or
alternatively could reflect a metabolic need for close proximity to
Streptococcus. Whether each taxon–taxon interaction in corncobs
is primarily mutualistic, commensal, or parasitic is a subject for
future research, as is the degree to which the relationships might
change depending on local conditions, and the degree to which
active competition shapes the composition and spatial organiza-
tion of the assemblage. Similar questions arise for the cells in the
filament-dense annulus and the hedgehog base.
From the point of view of microenvironments inhabited by the
Fig. 9. Summary hypothesis for interpretation of hedgehog structures.
component cells of the hedgehog, one taxon stands out as being part Corynebacterium filaments bind to an existing biofilm containing Strepto-
of multiple communities: a single filament of Corynebacterium may coccus and Actinomyces. At the distal tips of the Corynebacterium filaments,
experience several distinctive microenvironments along its length. corncob structures form in which the filaments are surrounded by cocci,
The proximal part of the filament inhabits the hedgehog base, which including Streptococcus and Porphyromonas, in direct contact with the Co-
is largely dominated by Corynebacterium but may contain Actino- rynebacterium filament as well as Haemophilus/Aggregatibacter in contact
myces and is also populated by a scattering of other cells. Farther with Streptococcus. Clusters of Neisseriaceae also occupy the periphery of the
along its length, the Corynebacterium filament traverses the filament- hedgehog. The Streptococcus cells create a microenvironment rich in CO2, lac-
tate, and acetate, containing peroxide, and low in oxygen. Elongated filaments
rich annulus, where its neighbors consist of Fusobacterium, Lepto-
of Fusobacterium and Leptotrichia proliferate in this low-oxygen, high-CO2
trichia, and Capnocytophaga. At its distal end, the Corynebacterium environment in an annulus just proximal to the corncob-containing peripheral
filament is encased in a corncob shell of Streptococcus and Por- shell of the hedgehog. The CO2-requiring Capnocytophaga also proliferates
phyromonas, with frequently abundant Haemophilus/Aggregatibacter abundantly in and around this annulus. The base of the hedgehog is
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and Neisseriaceae. These different environments may alter the local dominated by Corynebacterium filaments and thinly populated by addi-
physiology of Corynebacterium, even within a single filament. tional rods, filaments, and/or cocci.

E798 | www.pnas.org/cgi/doi/10.1073/pnas.1522149113 Mark Welch et al.

 PNAS PLUS
in physically connecting members of the consortium. Although support the greatest amount of life. In his words, “the advantages of
it may contribute to consortium organization, Fusobacterium is diversification of structure, with the accompanying differences of
only one of four filamentous taxa in hedgehogs, and it is habit and constitution, determine that the inhabitants, which thus
neither the most abundant nor the most spatially extensive. jostle each other most closely, shall, as a general rule, belong to
Bioinformatic analysis suggests that the genus Fusobacterium what we call different genera and orders” (41). Although our im-
as a whole is substantially more abundant in subgingival than in aging studies to date have mostly been restricted to genus-level
supragingival plaque (20). Our FISH probe detects the entire analysis, we suggest that the members of each genus are not in-
Fusobacterium genus, but within the genus, only F. nucleatum has terchangeable with one another. Future, more taxonomically refined
high abundance in plaque. Furthermore, several distinct subspecies analyses will reveal which representatives of each genus participate
of F. nucleatum are recognized in the HOMD, and in HMP se- in hedgehog structures. The likely identity of these representatives
quencing data, these subspecies showed distinctive habitat prefer- can be inferred from sequencing data and is generally small in
ences: some for subgingival and some for supragingival plaque (19). number, as noted above for genus Corynebacterium with only two
We conclude that the Fusobacterium in hedgehogs is likely one of major oral species.
the supragingival-abundant subspecies of F. nucleatum.
As a slowly growing taxon that is not prominent or even fre- Potential Significance of Plaque Spatial Structure for Health and Disease
quently represented among initial colonizers of enamel, how does and for Modeling Using Synthetic Communities. The sources of both
Corynebacterium and the hedgehog structures that it apparently spatial and temporal heterogeneity in the plaque community and
organizes come to be so abundant in plaque? It seems likely that their significance are a rich field for additional investigation.
Corynebacterium finds attachment sites on the preexisting biofilm The existence of heterogeneity is clearly visible in the sequencing
consisting of Streptococcus and Actinomyces, which are among the data, and its functional significance is suggested by morphological
early colonizers (33) and can be found near the base of hedgehog studies showing, for example, that primarily filamentous and pri-
structures. EM examination of the microbial community colo- marily coccoid communities coexist side by side in supragingival
nizing removable enamel chips worn inside the mouth showed plaque, and that the filamentous communities are more likely to
scattered filamentous cells oriented perpendicularly to the pri- form calculus (40). Would plaque rich in hedgehog structures,
marily coccus-covered surface at 24 hours and a mixed com- compared with other alternate consortia, make the preservation of
munity of abundant filamentous organisms by 48 hours (39), health or transition to disease more or less likely? Critical topics
suggesting that colonization with Corynebacterium may take for future study include whether there are features of host biology,
place around the 24-hour stage in plaque development. In the behavior, or immune system that encourage the development of
subgingival crevice or within the dental calculus that precipitates the hedgehog consortium over other microbial assemblages in
around these filamentous aggregates (40), the attachment sites plaque and what, if any, role hedgehog structures play in the
of Corynebacterium would be protected, and when distal por- maintenance of oral health or progression toward disease. The
tions of the structure are lost, by abrasion or oral hygiene ability to create realistic in vitro models of the plaque biofilm is
procedures similar to our sampling methods, the filaments critical for advancing the testing of antimicrobial or microbe-
could rapidly regrow from these reservoirs. modulating compounds as well as exploring biochemical and
metabolic interactions among taxa. Plaque as currently recon-
Understanding Microbiomes: Genes Vs. Organisms. The imaging ap- stituted in flow cells or other in vitro systems bears little re-
proach cuts through the overwhelming complexity of detail in mi- semblance to the structures that we see in plaque harvested from
crobial communities and allows common patterns to shine through. the mouth. Improving these reconstituted models, with success
With deep sequencing, it has become clear that many taxa in the judged by their ability to recapitulate the spatial structure of
oral microbiota are shared across individuals but are abundant in natural plaque, will open up a vast area of mechanistic inquiry.
some samples and almost vanishingly rare in others (19, 38). These In summary, the biogeography of a microbial community on
differences in abundance may result from real differences be- the micron scale reveals a spatial organization critical to un-
tween individuals, fluctuations within a single individual over derstanding the physiology, ecology, and functional significance of
time, or a combination of the two. Whatever its cause, the lack the community. Whether the community in question is host-
of a consistently abundant microbial “core” has led to the idea associated or environmental, these insights are generalizable to the
that perhaps it is not organisms but genes and functions that are study of any microbiome.
conserved within the microbiome, distributed across a variety of

MICROBIOLOGY
organisms whose identities are irrelevant. The discovery of Materials and Methods
hedgehog consortia argues against this idea, at least for some Sample Collection, Fixation, and Storage. We collected supragingival plaque
microbiomes. The consistency of the composition and structure from 22 healthy volunteers, each of whom had given informed consent. For
of the hedgehog across many individuals suggests that organisms 10 volunteers, oral health was confirmed by clinical examination; the rest
are highly relevant to understanding the roles, organization, and were self-reported as both orally and systemically healthy. Volunteers
refrained from oral hygiene for 12–48 h before sample collection. Plaque was
dynamics of the members of the consortium. We suggest that it
collected using toothpicks to scrape visible plaque from the gingival margin
is neither necessary nor desirable to disregard taxonomy and
or tooth surface, or using floss to collect plaque from throughout the
reduce the microbiome to a collection of genes or metabolites mouth. Samples were prepared in three ways. (i) Plaque was applied directly
unmoored from their source organism. Instead, we suggest the to slides and air-dried, and the samples were fixed directly on the slide,
converse, that an understanding of the ecology and physiology washed, dehydrated through an ethanol series, and subjected to FISH.
of the organisms in the consortium will provide an organizing (ii) Plaque was fixed in paraformaldehyde, stored in 50% (vol/vol) ethanol,
principle for understanding and interpreting metagenomic and spread onto slides in 50% (vol/vol) ethanol, and air-dried immediately be-
metatranscriptomic data. fore FISH. (iii) Plaque was fixed in paraformaldehyde followed by immediate
The consortium that we have described is composed of organ- embedding in methacrylate resin, which was later sectioned, and the sec-
isms of not only different species but nine different genera clas- tions were applied to slides and then subjected to FISH directly on the slide.
Samples were handled gently with a minimum of agitation so as to preserve
sified into eight families in five phyla. Thus, it is a consortium not
as much spatial structure as possible. Fixation was carried out in 2% (wt/vol)
of close relatives but of highly disparate organisms. The intimate paraformaldehyde in PBS or 10 mM Tris (pH 7.5) for at least 1.5 h on ice. For
association of widely diverged taxa may be a general property of samples not fixed directly on the slides, sample was allowed to sediment by
spatially organized microbial consortia. Darwin (41) was the first gravity, and the supernatant was removed by pipetting. Samples were
to recognize that the “struggle for existence” was most severe be-
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washed in 1× PBS or 10 mM Tris (pH 7.5) for 15 min and again sedimented by
tween closely allied forms and that “divergence of character” would gravity, and the supernatant was removed. Samples were resuspended in

Mark Welch et al. PNAS | Published online January 25, 2016 | E799
 PBS or 10 mM Tris, mixed with an equal volume of 100% ethanol, and stored incubated at 46 °C for 2–4 h in a chamber humidified with 20% (vol/vol) form-
at −20 °C, or they were immediately dehydrated through an ethanol series, amide. Slides were then washed in wash buffer (215 mM NaCl, 20 mM Tris, pH
washed with acetone, and embedded in Technovit 8100 methacrylate resin 7.5, 5 mM EDTA) at 48 °C for 15 min, dipped in cold water, air-dried, mounted in
(EMSdiasum.com). Embedded blocks were stored at room temperature and ProLong Gold Antifade Solution (ThermoFisher), covered with a #1.5 coverslip,
sectioned dry using a triangular tungsten carbide knife (Delaware Diamond and allowed to cure in the dark overnight. Slides were imaged using a Zeiss LSM
Knives, Inc.). 780 Confocal Microscope with a 40× 1.4 N.A. Plan-Apochromat objective. Each
field of view was imaged using sequential excitation with the 633-, 594-, 561-,
Sequencing and Sequence Analysis. Sequence analysis was carried out on a 514-, 488-, and 405-nm laser lines. Linear unmixing was performed using the
previously published oligotyping analysis (19) using publicly available data
Zeiss ZEN software using reference spectra acquired from cultured cells hybrid-
from the HMP (18); details of sample collection, sequencing, and human
ized as above with the Eub338 probe labeled with the appropriate fluorophore.
subjects research approvals are contained in the original publications.
Unmixed images were assembled and false-colored using Fiji (43).
Spectral Imaging FISH, Image Acquisition, and Analysis. Fluorophore-labeled
ACKNOWLEDGMENTS. We thank Alex Valm for probe design; Katherine
oligonucleotides were purchased from ThermoFisher.com or www.Biomers.net.
Lemon and Matthew Ramsey for helpful discussions; and Jake Casper, Louie
FISH was carried out using standard protocols (42) on sections or whole-mount Kerr, Carissa McKinney, Janina Schuhmann, Braden Tierney, Steven Wilbert,
samples mounted onto UltraStick Slides (Thermo Scientific). Hybridization Liping Xun, and the members of the MBL 2014 Physiology Summer Course.
solution [900 mM NaCl, 20 mM Tris, pH 7.5, 0.01% SDS, 20% (vol/vol) formamide, This research was supported by NIH National Institute of Dental and
each probe at a final concentration of 2 nM] was applied to samples and Craniofacial Research Grant DE022586 (to G.G.B.).

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