ABSTRACT
The initial generation of the pattern of mystacial vibrissae (whiskers) in the mouse is described. The maxillary process is present in 10-day embryos but has a relatively flat surface. Beginning at approximately 11·5 days, the first sign of vibrissal development is the formation of ridges and grooves on the maxillary and lateral nasal processes. The first vibrissal rudiment to form subsequently appears posterior to the most ventral groove on the maxillary process. It is the most ventral whisker of the posterior, vertical row. The next few rudiments appear: (1) dorsal to the first, also in the vertical row; and (2) anterior to the first, on the ventral-most ridge and in the groove beneath it. Formation of vibrissal rudiments continues in a dorsal and anterior progression usually by an apparent partitioning of the ridges into vibrissal units.
The hypothesis that this patterning of mystacial vibrissae might be determined by the pattern of innervation in the early mouse snout was investigated. Nerve trunks and branches are present in the maxillary process well before any sign of vibrissal formation. Because innervation is so widespread there appears to be no immediate temporal correlation between the outgrowth of a nerve branch to a site and the generation of a vibrissa there. Furthermore, at the time just prior to the formation of the first follicle rudiment, there is little or no nerve branching to the presumptive site of that first follicle while branches are found more dorsally where vibrissae will not form until later. Thus, a one-to-one spatial correlation between nerve and follicle sites also appears to be lacking.
The developmental changes in ultrastructure within the neurites of the trunks and branches as well as the apparent rearrangements of the nerve trunks suggest that early innervation of the snout is a labile phenomenon and that the vibrissal pattern begins to be established before the neural pattern is completely developed. The results indicate that vibrissal pattern formation is likely to be a complex process relying on the interplay of the cells and tissues involved, rather than on unidirectional instructions from neurons to other cell types.
INTRODUCTION
The development of the mystacial vibrissae (sinus hairs, whiskers) of the mouse is marked by a high degree of order and pattern in time and space (Danforth, 1925; Gruneberg, 1943; Davidson & Hardy, 1952; Yamakado & Yohro, 1979; Van Exan & Hardy, 1980). The earliest signs of vibrissal formation occur between 11 and 12 days of gestation as two parallel grooves and two ridges appear on the maxillary process of the developing snout. In general, the rudiments of the most posterior (caudal) and ventral vibrissae appear first and succeeding vibrissae form in an anterior (rostral) and dorsal progression. By day 14 most, if not all, rudiments have formed and the final adult pattern of five horizontal rows with one vertical row posterior to them is apparent. This pattern is maintained in most strains of mice studied although there are small variations in the number of follicles in the horizontal rows (Dun, 1958), and supernumerary follicles sometimes occur between rows (Yamakado & Yohro, 1979). The general pattern, however, is quite constant.
The search for the source of the pattern has centred on the fact that vibrissae are highly innervated sensory organs (Vincent, 1913; Winkelman, 1959) which are represented in the somatosensory cortex of the brain by their cortical ‘barrels’ (Woolsey & Van der Loos, 1970). These are discrete groups of neurons, one unit for each vibrissa, which are arranged in a pattern corresponding to the pattern of the vibrissae on the snout. This spatial correspondence between the central nervous system and the periphery suggests that the organization of one might influence the patterning of the other. Several studies have shown that nerve trunks are present in the snout both directly beneath the early vibrissal rudiments and also more anteriorly in the region where rudiments will subsequently form (Tello, 1923; Wessells & Roessner, 1965). Van Exan & Hardy (1980) studied the innervation pattern of 12-day embryonic snouts with light microscopy. They made reconstructions of embryos in which the more caudal vibrissae were already well established and found that the formation of a nerve ‘plexus’ preceded the development of the more rostral follicles. These observations suggest the possibility that, since innervation precedes vibrissal formation, the pattern of that innervation determines the arrangement of vibrissae.
The studies reported here were undertaken to investigate the initial generation of the vibrissal pattern and its relationship, if any, to the pattern of innervation at that time.
MATERIALS AND METHODS
Scanning electron microscopy
Mouse embryos were obtained by crossing Balb/c females with C3H male mice and were staged by day of development with the date of vaginal plug discovery set at day 0. Embryos of 10, 11, 11·5, 12 and 13 days were taken from pregnant females which had been killed by cervical dislocation. The uteri were removed and placed in Hank’s balanced salt solution. The heads of the embryos were removed and immediately put into the primary fixative of 2·5 % glutaraldehyde in 0·07 M-Sorenson’s buffer with 0·06 M-sucrose, pH 7·4. There the heads were cut into right and left halves and trimmed, depending on their age and size, to leave at least the eye and the area beneath it, the medial and lateral nasal processes, and the maxillary process. For almost all but the youngest embryos the right side of the snout was left whole and the left side was cut into two parts in a plane horizontal through the length of the maxillary process. The tissues remained in the primary fix for approximately 1 h before being washed three times with the same Sorenson’s buffer.
Postfixation was in 1 % osmium tetroxide in 0·028 M-Veronal buffer, pH 7·4, for 30 to 60 min, after which the tissues were dehydrated through a series of acetone and dried with CO2 in a Sorvall critical-point drying system. The tissues were then mounted on stubs in various orientations with double-sided tape and sputtered with gold in an argon atmosphere in a Denton vacuum evaporator. The specimens were examined in a Coates and Welter Model 100-6 field emission scanning electron microscope.
Light and electron microscopy
Sample preparation for transmission microscopy was the same as for SEM through postfixation in osmium. After that, specimens for transmission microscopy were dehydrated in a graded series of ethanol which was then replaced by propylene oxide. The tissues were infiltrated with an Epon 812-propylene oxide mixture, followed by Epon alone; then they were embedded in fresh Epon and were cured for one day at 45 °C and two days at 60 °C.
Sections of 1 μm thickness for light microscopy were cut on an LKB Ultrotome III and stained with a 1:1 mixture of 1 % azure II in water and 1 % methylene blue in 1 % sodium borate (Richardson, Jarett & Finke, 1960). Thin sections for electron microscopy were also cut on the LKB. They were stained with uranyl acetate and lead citrate before being examined in a Hitachi HU-11E.
Half mounts
Some specimens were treated with acetylthiocholine according to the methods of Karnovsky & Roots (1964) and Barald & Berg (1979) to detect cholinesterase activity and thereby aid in viewing nerve cells and their neurites.
RESULTS
Whole mounts
The initial generation of vibrissal rudiments, once started, occurs so rapidly that embryos must be examined in half-day increments of age in order to resolve the formation of the early pattern. The search for the order of development is aided, however, by the wide range of developmental characteristics within the half-day increments. Not even apparently healthy litter mates will have equally developed facial features at the same hour of gestation (Gruneberg, 1943). This report will include the range of sizes and features at each age, although the illustrations will be of specimens selected to show the progression of development. The elements of the pattern, the rows and the individual vibrissae, will be labelled according to Fig. 1, with row 1 being the most ventral row.
A diagrammatic representation of the right side of an embryonic mouse face, ca. 13 days of gestation. The rows and the individual whiskers are labelled in accord with their approximate order of development so that row 1 is the most ventral row and row 5, the most dorsal. In Figs 2 through 10 of this paper, only the right side of the whole snout will be shown.
A diagrammatic representation of the right side of an embryonic mouse face, ca. 13 days of gestation. The rows and the individual whiskers are labelled in accord with their approximate order of development so that row 1 is the most ventral row and row 5, the most dorsal. In Figs 2 through 10 of this paper, only the right side of the whole snout will be shown.
10 days
At 10 days all embryos show the maxillary process diverging from the mandibular process beneath the eye (Fig. 2). The lens of the eye is invaginating and the olfactory pit in various specimens ranges from broad and relatively shallow, to narrower and more groove like. The maxillary and lateral nasal processes are quite short compared to their later outgrowth. The length of the nasolacrimal groove between them, anterior to the eye, ranges from approximately 100 μm to 160 vm (mean = 132 μm, S.D. =21 μm) in the eight specimens studied. No vibrissal rudiments are evident at this stage.
The right half of the facial region of a 10-day mouse embryo. The lens (l) of the eye is in an early stage of invagination. The lateral (ln) and medial (mn) nasal processes surround the olfactory groove. The maxillary process (mx) diverges from the mandibular process (md) beneath the developing eye. The nasolacrimal groove (ng) separates the maxillary and lateral nasal processes. Bar = 100 μm.
The right half of the facial region of a 10-day mouse embryo. The lens (l) of the eye is in an early stage of invagination. The lateral (ln) and medial (mn) nasal processes surround the olfactory groove. The maxillary process (mx) diverges from the mandibular process (md) beneath the developing eye. The nasolacrimal groove (ng) separates the maxillary and lateral nasal processes. Bar = 100 μm.
11 days
The primary change in the 11-day specimens (Fig. 3) is the forward growth of the facial processes. The distance from the anterior edge of the eye to the most anterior point of the maxillary process ranges from approximately 260 μm to 480 μm (mean = 360 μm, S.D. = 70 μm in 20 specimens). This wide range reflects the differences in growth and development in the same-aged embryos reported by Gruneberg (1943). The nasolacrimal groove provides a deeper separation between the maxillary and lateral nasal prominences. No vibrissal rudiments are seen.
An 11-day facial region. The lens (l) is completing its invagination; only a small opening remains in the surface epithelium of the eye. The maxillary (mx) and lateral nasal (ln) processes have both grown out anterior to the eye and the nasolacrimal groove between them has deepened. Bar = 100 μm.
An 11-day facial region. The lens (l) is completing its invagination; only a small opening remains in the surface epithelium of the eye. The maxillary (mx) and lateral nasal (ln) processes have both grown out anterior to the eye and the nasolacrimal groove between them has deepened. Bar = 100 μm.
days
As the facial processes continue their outgrowth, the nasolacrimal groove becomes shallower in its anterior portion. In smaller, less-developed specimens (Fig. 4) the length of the maxillary process anterior to the eye overlaps with the length of 11-day samples, beginning at about 320 μm. In more developed 11·5day snouts (Fig. 5), that measurement of the maxillary process ranges up to 680 μm in length (mean = 470 μm, S.D. = 80 μm in 17 samples).
An early 11·5-day mouse snout. The surface epithelium (e) of the eye is now continuous over the lens. The nasolacrimal groove (ng) is quite deep between the maxillary (mx) and lateral nasal (ln) processes which have continued their outgrowth. Bar= 100 μm.
A more advanced 11·5-day facial region. Note the two depressions (arrows) which appear on the maxillary process and the single slight groove (arrow) on the lateral nasal process. The raised ridges between the grooves are the precursors of horizontal rows 2 through 5 of vibrissae. Bar = 100 μm.
A more advanced 11·5-day facial region. Note the two depressions (arrows) which appear on the maxillary process and the single slight groove (arrow) on the lateral nasal process. The raised ridges between the grooves are the precursors of horizontal rows 2 through 5 of vibrissae. Bar = 100 μm.
Most importantly, in these larger, more-developed samples there is an indication of two new grooves appearing on the surface of the maxillary process, more or less parallel to the length of the nasolacrimal groove. With the two depressions appear two ridges (the precursors of vibrissal rows 2 and 3), one between the two new grooves and one between the more dorsal of the depressions and the nasolacrimal groove. In addition, in some specimens another groove and two ridges (rows 4 and 5) appear on the surface of the lateral nasal process.
12 days
12-day embryos exhibit a very broad range of facial and vibrissal development (Figs 6–10). In size, the length of the maxillary process anterior to the eye varies from approximately 650 μm to 1200 μm (mean = 910 μm, S.D. = 150 μm) in the 33 samples studied. The nasolacrimal groove becomes much shallower to form the groove between rows 3 and 4.
An early 12-day snout. At the posterior end of the most ventral groove is a slight hillock, the first sign of the first vibrissal rudiment (α) to form. The nasolacrimal groove (ng) has become much shallower than it was at 11 days. Bar = 100 μm.
The earliest, shortest samples show that the first vibrissa appears as a hillock at the posterior end of the ventral-most groove on the maxillary process (Fig. 6). This is the rudiment of the most ventral whisker in the vertical row, vibrissa a. A second hillock, vibrissa β, appears dorsal to the first (Fig. 7), at the posterior end of the more dorsal groove of the maxillary process. The next rudiments appear at or about the same time anterior to the first two (Figs 7, 8), one on the ventral ridge (horizontal row 2) and one in the groove beneath the ridge (presumptive row 1).
A slightly later stage of a 12-day mouse embryo. The first vibrissa (α) of the vertical row, is more apparent, and the second (β) has begun to form. Likewise, the initial signs of vibrissa 2a appear on its ridge. Bar = 100 μm.
A later stage of facial development at 12 days. Vibrissa la is forming in the groove beneath 2a. There are hints of hillock development at 3a and in the vertical row at positions γ and Δ. The remnant of the nasolacrimal groove (ng) separating rows 3 and 4 appears to be similar in depth to the more recently formed grooves on the maxillary process. Bar = 100 μm.
A later stage of facial development at 12 days. Vibrissa la is forming in the groove beneath 2a. There are hints of hillock development at 3a and in the vertical row at positions γ and Δ. The remnant of the nasolacrimal groove (ng) separating rows 3 and 4 appears to be similar in depth to the more recently formed grooves on the maxillary process. Bar = 100 μm.
More advanced 12-day specimens show vibrissal formation proceeding dorsally and rostrally as two more hillocks appear in the vertical row posterior to the ridges, new vibrissae form as the ridges themselves are blocked off into new hillocks, and more rudiments form in the ventral-most groove (Figs 9,10).
Another 12-day specimen. All four rudiments in the vertical row are present as are more rudiments in the horizontal rows, including a hint of vibrissa 4a above the nasolacrimal groove (ng). Bar = 100 μm.
A late 12-day embryo. At least 12, and possibly 14, rudiments have formed and development is proceeding in ventral-(V)-to-dorsal (D) and posterior-(P)-to-anterior (A) directions. Bar = 100 μ m.
Late 12-day and 13-day embryos continue to form new whiskers in the established pattern from ventral to dorsal and posterior to anterior. The whisker pad becomes elevated above the level of the surrounding head tissue and the epithelium of the earliest rudiments begins its downward growth into the mesenchyme to form whisker follicles. These later stages have been thoroughly described by Hardy (1951), Davidson & Hardy (1952), and Yamakado & Yohro (1979).
Half mounts and sectioned snouts
Large nerve trunks are present in the maxillary process very early as it is growing out, before any signs of vibrissal formation. Fig. 11 shows an 11·5-day maxillary process which has been favourably bisected frontally for scanning electron microscopy. Lengths of nerve trunk course through the mesenchyme after leaving the trigeminal ganglion which is the source of sensory innervation to the vibrissae (Vincent, 1913).
The bottom half of an 11·5-day maxillary process and adjacent tissue which has been bisected frontally, i.e., in a horizontal plane with the anterior (A), nasal portion of the process at the bottom and the posterior (P) portion toward the top. The posterior boundary of the process proper is approximately at the level of the P. Vibrissae will form on the outer surface to the right. At the top lies the trigeminal ganglion (tg). Nerves (n) are seen leaving the ganglion in a large trunk and smaller sections of nerve tracts (arrows) are present in much of the length of the process. Bar = 100 μ m.
The bottom half of an 11·5-day maxillary process and adjacent tissue which has been bisected frontally, i.e., in a horizontal plane with the anterior (A), nasal portion of the process at the bottom and the posterior (P) portion toward the top. The posterior boundary of the process proper is approximately at the level of the P. Vibrissae will form on the outer surface to the right. At the top lies the trigeminal ganglion (tg). Nerves (n) are seen leaving the ganglion in a large trunk and smaller sections of nerve tracts (arrows) are present in much of the length of the process. Bar = 100 μ m.
Thiocholine staining of snouts, bisected as above, permits the visualization of nerves on and beneath the surface of the cut so that their branching patterns are more easily viewed. Although it is not known how deeply the stain penetrates in these half mounts, it is clear that vibrissal rudiments in 13-day snouts (Fig. 12) are associated with well-defined nerve branches coming to them and that other nerve branches course more rostrally where vibrissal rudiments are not yet apparent.
The lower half of a frontally bisected 13-day maxillary process stained with acetylthiocholine to demonstrate a portion of the innervation pattern. Developing vibrissae (v) appear as small, rounded contours on the epithelial surface and some of the nerve branches (n) terminate near them. The trigeminal ganglion (tg) appears at the upper right of the photo. Bar = 100 μ m.
The lower half of a frontally bisected 13-day maxillary process stained with acetylthiocholine to demonstrate a portion of the innervation pattern. Developing vibrissae (v) appear as small, rounded contours on the epithelial surface and some of the nerve branches (n) terminate near them. The trigeminal ganglion (tg) appears at the upper right of the photo. Bar = 100 μ m.
The information provided by examining bisected snouts is, however, severely limited. Only those portions of nerves in or near the plane of the cut can be seen. Furthermore, it is impossible to distinguish between small nerve fibres or terminals and the surrounding mesenchymal cells.
To gain more information about the distribution of neurites relative to the positions of developing vibrissae it was necessary to section the snouts in various planes for light and transmission electron microscopy. In general, 1 μm-thick plastic sections were taken first and examined for areas of special interest requiring higher resolution. When such areas were seen, sections 50 nm to 70 nm thick were taken of the adjacent tissue and examined in the TEM. In this way very small neural elements could be identified, their ultrastructure observed, and their connections followed to larger nerve bundles, thus providing maps of the innervation pattern at the time of initial vibrissal formation.
Frontal sections of 11-day snouts, parallel to the length of the maxillary process, show that nerve trunks are present in the process approximately one day before any sign of the first vibrissal rudiment. There is limited branching of the trunks toward the periphery (Fig. 13). Light micrographs show that mesenchymal cells of the maxillary process are very densely packed at 11 days, especially in the areas adjacent to the nerve trunks. Away from the nerve tracts, toward the epithelium, the mesenchyme is slightly less densely packed but, compared to the later stages, there is little extracellular matrix. A network of capillaries lies in the mesenchyme between the nerves and the epithelium. At this stage the capillaries appear to remain at least 6 or 7 cell diameters away from the epithelium.
A light micrograph of an 11-day maxillary process sectioned frontally with the anterior (A) end of the process to the right and the posterior (P) end to the left. Large nerve trunks (n) with little branching are present in the cell-dense mesenchyme (m). A network of capillaries (c) filled with red blood cells lies about six or seven cell diameters from the epithelial surface (e). Bar = 50 μm.
A light micrograph of an 11-day maxillary process sectioned frontally with the anterior (A) end of the process to the right and the posterior (P) end to the left. Large nerve trunks (n) with little branching are present in the cell-dense mesenchyme (m). A network of capillaries (c) filled with red blood cells lies about six or seven cell diameters from the epithelial surface (e). Bar = 50 μm.
Ultrastructurally, neurites in the large nerve trunks appear somewhat ‘immature’ (Fig. 14) compared to those seen at later stages. Some of the individual neurites of a bundle contain the organelles typical of axons: numerous neurotubules, neurofilaments, and mitochondria in a ribosome-free cytoplasm. These organelles are often loosely arrayed, however, in neurites whose profiles may measure up to 4 μm or 5 μm in diameter, larger than the 1 μm-wide profiles which, as we shall see, are typical of axons at later stages. Other neurites in a bundle contain few, if any, of these organelles. These neurites appear to be more like growth cones with much fine filamentous material, dense-core granules, and membranous structures, some vesicular and some reminiscent of smooth endoplasmic reticulum, in a homogeneous, ribosome-free cytoplasm. Finally, some neurites have an intermediate appearance with both groups of organelles present. Occasionally small filopodia with few organelles are seen extending from the larger profiles. In general, the appearance of the large nerve trunks at 11 days suggests that most of the neurites have just recently grown out or are in the process of growing out even as the maxillary process itself is enlarging.
A transmission EM view of a region of nerve tract (11 days) similar to those seen in Fig. 13. The nerve is composed of individual neurites of varying size and contents. Some neurites (ax) have regions containing the organelles typical of mature axons, i.e., neurotubules and neurofilaments. Other neurites look more like growth cones (gc) with dense-core granules and membranous structures in a rather homogeneous background cytoplasm. Many neurites are intermediate (i) in appearance with some features of both types. Note how closely the mesenchymal cells (m) at the bottom are apposed to the neurons. Bar = 1 μm.
A transmission EM view of a region of nerve tract (11 days) similar to those seen in Fig. 13. The nerve is composed of individual neurites of varying size and contents. Some neurites (ax) have regions containing the organelles typical of mature axons, i.e., neurotubules and neurofilaments. Other neurites look more like growth cones (gc) with dense-core granules and membranous structures in a rather homogeneous background cytoplasm. Many neurites are intermediate (i) in appearance with some features of both types. Note how closely the mesenchymal cells (m) at the bottom are apposed to the neurons. Bar = 1 μm.
By 12 days of development ridges are present on the maxillary process and vibrissae have begun to form (Figs 6–10). Frontal sections parallel to the ridges (the same orientation as in the 11-day sections described above) show the epithelial hillocks and their underlying dermal condensations. In Fig. 15 the vibrissa marked β on the right is a member of the vertical row (Fig. 1). The plane of section passes through one side of the structure. Vibrissa a is the first to develop in its horizontal row, and, to its left, b is just beginning to form. Within the more advanced condensations as in β, the mesenchymal cells are closely apposed. Outside of the vibrissal dermal condensations the mesenchymal cells are much less densely packed than they were at 11 days. The growth of the maxillary process mentioned above appears to be at least partly attributable to the increase in extracellular matrix within the process.
Light micrographs of sections of 12-day maxillary process taken approximately 50 μm apart. The anterior (A) of the snout is to the left and the posterior (P ) is to the right. The vibrissa marked β in each figure is the same structure cut at different levels. It is a member of the posterior, vertical row of follicles. Vibrissa a in Fig. 15 is the first to form in its horizontal row, and the beginning of vibrissa b is suggested by the slight thickening of the epithelium seen in this section. In both figures numerous small sections of nerve trunks are scattered through the mesenchyme (arrows); some are far anterior to the youngest developing vibrissa. Fig. 16 shows a longitudinal section through a major nerve (n) with a small branch coming from it (br). Other nerve trunks (nt) lie deep in the mesenchyme. Bars = 100 μm.
Light micrographs of sections of 12-day maxillary process taken approximately 50 μm apart. The anterior (A) of the snout is to the left and the posterior (P ) is to the right. The vibrissa marked β in each figure is the same structure cut at different levels. It is a member of the posterior, vertical row of follicles. Vibrissa a in Fig. 15 is the first to form in its horizontal row, and the beginning of vibrissa b is suggested by the slight thickening of the epithelium seen in this section. In both figures numerous small sections of nerve trunks are scattered through the mesenchyme (arrows); some are far anterior to the youngest developing vibrissa. Fig. 16 shows a longitudinal section through a major nerve (n) with a small branch coming from it (br). Other nerve trunks (nt) lie deep in the mesenchyme. Bars = 100 μm.
The nerves of the 12-day snout are more highly branched than at 11 days. Fig. 16 shows several main nerve trunks near the bottom of the picture and portions of nerve tracts nearer the periphery. On the right a large nerve trunk splays out beneath the dermal condensation of vibrissa β (the same vibrissa as seen in Fig. 15 but sectioned more centrally). Transmission EM of the same nerve bundle in a section approximately 5 μm away from that in Fig. 16 shows that beneath the condensation the neurites have a typical growth-cone appearance (Fig. 17). At the end of that portion of nerve trunk away from the condensation several neurites resemble axons with arrays of neurotubules and neurofilaments (Fig. 18), but most of the neurites at this level have an intermediate appearance similar to that described for the neurites of 11-day nerve trunks.
Light micrographs of sections of 12-day maxillary process taken approximately 50 μm apart. The anterior (A) of the snout is to the left and the posterior (P ) is to the right. The vibrissa marked β in each figure is the same structure cut at different levels. It is a member of the posterior, vertical row of follicles. Vibrissa a in Fig. 15 is the first to form in its horizontal row, and the beginning of vibrissa b is suggested by the slight thickening of the epithelium seen in this section. In both figures numerous small sections of nerve trunks are scattered through the mesenchyme (arrows); some are far anterior to the youngest developing vibrissa. Fig. 16 shows a longitudinal section through a major nerve (n) with a small branch coming from it (br). Other nerve trunks (nt) lie deep in the mesenchyme. Bars = 100 μm.
Light micrographs of sections of 12-day maxillary process taken approximately 50 μm apart. The anterior (A) of the snout is to the left and the posterior (P ) is to the right. The vibrissa marked β in each figure is the same structure cut at different levels. It is a member of the posterior, vertical row of follicles. Vibrissa a in Fig. 15 is the first to form in its horizontal row, and the beginning of vibrissa b is suggested by the slight thickening of the epithelium seen in this section. In both figures numerous small sections of nerve trunks are scattered through the mesenchyme (arrows); some are far anterior to the youngest developing vibrissa. Fig. 16 shows a longitudinal section through a major nerve (n) with a small branch coming from it (br). Other nerve trunks (nt) lie deep in the mesenchyme. Bars = 100 μm.
The distal end of the nerve tract directly beneath the dermal condensation (de) of vibrissa β in Fig. 16 (arrow 1). Most of the neurite profiles are enlarged and contain the organelles such as dense-core granules (deg) and smooth endoplasmic reticulum (ser) which are typical of growth cones. Bar = 1 μm.
The distal end of the nerve tract directly beneath the dermal condensation (de) of vibrissa β in Fig. 16 (arrow 1). Most of the neurite profiles are enlarged and contain the organelles such as dense-core granules (deg) and smooth endoplasmic reticulum (ser) which are typical of growth cones. Bar = 1 μm.
The proximal end of the portion of nerve tract beneath vibrissa β, i.e., the end away from the dermal condensation (arrow 2 in Fig. 16). At this level of the nerve tract, the population of neurites is mixed in appearance: some resemble growth cones (gc), some resemble axons (ax) with arrays of neurotubules (ntu), and some are intermediate (/), containing the organelles of both types. The general appearance of the nerve is similar to that seen in nerve trunks at 11 days (Fig. 14). Bar = 5 μm.
The proximal end of the portion of nerve tract beneath vibrissa β, i.e., the end away from the dermal condensation (arrow 2 in Fig. 16). At this level of the nerve tract, the population of neurites is mixed in appearance: some resemble growth cones (gc), some resemble axons (ax) with arrays of neurotubules (ntu), and some are intermediate (/), containing the organelles of both types. The general appearance of the nerve is similar to that seen in nerve trunks at 11 days (Fig. 14). Bar = 5 μm.
This hint of a proximodistal sequence of neurite ultrastructure is reinforced by further study of nerve trunks at various distances from the epithelium. In general most of the individual neurites of the main nerve trunks deep in the mesenchyme are axon like with neurotubule and neurofilament arrays in neuritic cell profiles of 0·5 μm to 1 μm in diameter (Fig. 19). This gives the trunks an appearance of ‘maturity’ compared to those at 11 days. The population is mixed, however, and neurites with an intermediate appearance (described above) and growth cones are also present at this level. The closer the nerves get to the periphery, the greater the proportion of their neurites which look like growth cones. In all cases these remain at least four or five mesenchymal cell diameters away from the epithelium. No nerve branches or endings have been observed to contact either the basal lamina or the epithelium.
A portion of the major nerve trunk (nt) seen deep in the mesenchyme of Fig. 16. At this level of the nerve most of its neurites are axon-like (ax) with smaller profiles and neurotubules and neurofilaments. However, there are some larger profiles which resemble growth cones (gc). This suggests that later neurites continue to grow out along the established nerve tract. Bar = 1 μm.
A portion of the major nerve trunk (nt) seen deep in the mesenchyme of Fig. 16. At this level of the nerve most of its neurites are axon-like (ax) with smaller profiles and neurotubules and neurofilaments. However, there are some larger profiles which resemble growth cones (gc). This suggests that later neurites continue to grow out along the established nerve tract. Bar = 1 μm.
Figs 15 and 16 show a number of nerve bundles across the width of their sections. These are typical in that virtually any single 1 μm-thick, frontal section will have nerve branches both in the vicinity of obvious dermal condensations and also further nasally where there is, as yet, no sign of vibrissal development. In an attempt to represent the distribution of nerves in one portion of serially sectioned 12-day maxillary process, drawings were made on clear plastic of approximately every second 1 μm-thick section (electron microscopy was used for nerve identification when necessary). The drawings were mounted with air space between them to approximate the depth of the intervening sections. The three-dimensional representations thus obtained (Fig. 20) show that neural elements are distributed through the length of the maxillary process including the rostral portion some distance beyond the last vibrissal site. If one were to divide the area between the site of the youngest recognizable rudiment and the site of the most nasal nerve endings into units approximately the same size as the already developing vibrissae, one could say that nerves are present at least two or three units in advance of the next site to develop. In truth, however, the great forward growth of the maxillary process during early vibrissal formation makes such a division into units meaningless in terms of predicting future follicle sites. The best one can say is that nerves are present at sites in the mesenchyme well in advance of the time that dermal condensations would be forming near those sites.
Three reconstructions from different angles of the same portion of 12-day maxillary process in which a few vibrissae have begun to form. In each view solid lines indicate nerves on the surface toward the viewer. Nerve trunks and branches are distributed throughout the tissue, including in the anterior region (A) well beyond the last vibrissal site. Posterior = P.
Three reconstructions from different angles of the same portion of 12-day maxillary process in which a few vibrissae have begun to form. In each view solid lines indicate nerves on the surface toward the viewer. Nerve trunks and branches are distributed throughout the tissue, including in the anterior region (A) well beyond the last vibrissal site. Posterior = P.
To investigate the possibility that the nerve pattern may play a role at an earlier stage, before the first vibrissa begins to form, sections were taken of 11·5-day embryonic snout in a plane perpendicular to the length of the ridges on the maxillary process (Figs 21–24). The sectioning was done in a posterior-to-anterior direction through the trigeminal ganglion beneath the eye and continuing well into the maxillary and nasal processes. In this manner one can trace the smaller branches of the superior maxillary branch of the trigeminal ganglion and can see the position of those branches relative to the ridges and to the sites of initial vibrissal formation.
These illustrate the distribution of nerve trunks and branches relative to the ridges and presumptive sites of vibrissal formation in an 11·5-day maxillary process. Sections were taken perpendicularly to the length of the process (they are, consequently, perpendicular to the plane of the previous figures) beginning at the trigeminal ganglion and continuing anteriorly (see diagram inset, Fig. 21, for locations of these sections). The ventral edge (V) is to the left, and the dorsal (D) is to the right.
Fig. 21. The first sign of branching (arrows) from the major trunks (n) occurs in the dorsal region of the process at the level where the dorsal groove (dg) and ridge (dr) are just beginning to appear. This region will eventually become horizontal row 3 of follicles but not until after vibrissae begin to develop more ventrally (V) where, as yet, no sign of branching occurs. Bar = 100 μm.
Fig. 22. A higher power view of the region in the box in Fig. 21. The smaller nerve branches (arrows) are more easily seen. Bar = 10 μm.
Fig. 23. No branching of the ventral nerve trunks (in box) occurs until this level of section where the ventral groove (vg) is already well established. The site where the first vibrissa would have formed is in previous sections, at the posterior end of the ventral groove where no ventral branching was seen. Branching continues more dorsally (arrows) under presumptive row 3. Bar = 100 μm.
Fig. 24. A higher power view of the region in the box in Fig. 23. A number of small nerve branches (arrows) are seen in the area under the ventral groove. Bar = 10 μm.
These illustrate the distribution of nerve trunks and branches relative to the ridges and presumptive sites of vibrissal formation in an 11·5-day maxillary process. Sections were taken perpendicularly to the length of the process (they are, consequently, perpendicular to the plane of the previous figures) beginning at the trigeminal ganglion and continuing anteriorly (see diagram inset, Fig. 21, for locations of these sections). The ventral edge (V) is to the left, and the dorsal (D) is to the right.
Fig. 21. The first sign of branching (arrows) from the major trunks (n) occurs in the dorsal region of the process at the level where the dorsal groove (dg) and ridge (dr) are just beginning to appear. This region will eventually become horizontal row 3 of follicles but not until after vibrissae begin to develop more ventrally (V) where, as yet, no sign of branching occurs. Bar = 100 μm.
Fig. 22. A higher power view of the region in the box in Fig. 21. The smaller nerve branches (arrows) are more easily seen. Bar = 10 μm.
Fig. 23. No branching of the ventral nerve trunks (in box) occurs until this level of section where the ventral groove (vg) is already well established. The site where the first vibrissa would have formed is in previous sections, at the posterior end of the ventral groove where no ventral branching was seen. Branching continues more dorsally (arrows) under presumptive row 3. Bar = 100 μm.
Fig. 24. A higher power view of the region in the box in Fig. 23. A number of small nerve branches (arrows) are seen in the area under the ventral groove. Bar = 10 μm.
Serial 1 μm-thick sections at 11·5 days reveal that the maxillary branch is composed of approximately 20 to 25 discrete nerve trunks interspersed with mesenchymal cells and small capillaries as it leaves the trigeminal ganglion. The trunks remain together as a loosely associated group until they enter the region of the maxillary process proper, i.e., in sections beyond the eye where the nasolacrimal groove separates the maxillary and nasal processes. Then the trunks begin to splay out in a dorsoventral direction across the base of the process. Continued sectioning passes through the posterior end of the dorsal groove on the maxillary process as the trunks continue to spread dorsoventrally. Within the next 10 to 20 sections (Figs 21–22) the more dorsal nerve trunks send several branches into the mesenchyme beneath the dorsal ridge (the area between the dorsal groove and the nasolacrimal groove, presumptive row 3 of vibrissae). Approximately 40 sections further on, the ventral groove begins to appear. It is within the area of these sections, just before the groove, that the first vibrissa would have begun to form if development had continued for another half day. But within these sections there is no sign of the more ventral nerve trunks branching toward the epithelium. A small branch does appear approximately 20 μm more anteriorly, but it is not until another 40 or so sections are taken that the ventral nerve trunks begin to branch (Figs 23,24) as profusely as those under presumptive row 3. Those more dorsal nerve bundles have continued their branching throughout these sections into regions where vibrissae would not be developing until after they form in the ventral region. Thus, at 11·5 days the site of the first vibrissal rudiment is not as highly innervated as areas that would develop vibrissae later. It is also important to note that at this stage the many nerve trunks are splayed out dorsoventrally through the mesenchyme with no clear segmentation or other patterning corresponding to the positions of the ridges and grooves above. Therefore, at the time just prior to the development of the earliest vibrissa, there appears no obvious spatial nor temporal one-to-one correlation between the innervation pattern and the positions of the ridges, the grooves, or the first whisker.
DISCUSSION
The variation in the rate of facial development in mouse embryos, even among litter-mates, facilitates constructing what we interpret to be a precise sequence of vibrissal rudiment formation. This study, in establishing the locations of the first three or four rudiments, demonstrates that the common method of labelling the sinus hairs from dorsal to ventral (Danforth, 1925; Davidson & Hardy, 1952; Van der Loos & Woolsey, 1973, are examples) is developmentally inaccurate and confusing in the description of early pattern formation. Fig. 1 presents an alternative nomenclature which reflects the ventral-to-dorsal developmental pattern.
The early appearance of the ridges and the linearity of the horizontal rows suggest that ridge formation might be an essential precondition for whisker formation. In considering this possibility, one must remember that there are two distinct facets of vibrissal development: (1) the generation of the overall whisker pattern, and (2) the generation of a single vibrissa. It seems quite likely that the ridges play a role in the first case, establishing the overall pattern, since the positions of all five horizontal rows and the vertical row are closely correlated with the positions of the four original ridges and the grooves. Indeed, the linearity of horizontal rows 2 through 5 arises directly from the partitioning of the ridges. Ridge formation is a feature also found in mammary gland and tooth development and may be a common mechanism for linear alignment of organs as discussed by Van Exan & Hardy (1980).
In the second case, however, this study shows that it is not necessary for a particular site to be on a ridge for that site to develop into a vibrissa. The vertical row does not arise directly from a ridge nor do the first few vibrissae to form in row 1. Therefore, while it may be true that ridge formation provides a general plan for vibrissal pattern, it probably does not provide a specific and essential morphogenetic signal to position a vibrissa in a specific spot.
Another conspicuous feature of facial development concurrent with early vibrissal formation is the great amount of growth and remodelling of the maxillary and nasal processes. It seems likely that the forward growth of the two processes provides new tissue to participate in vibrissal formation anterior to rudiments which have already begun to form. To the authors’ knowledge, no studies on mice have yet been published, but Minkoff (1980) has shown that in the chick embryo there are higher labelling indices at the tip of the maxillary process and at the boundaries of the maxillary and nasal processes than in the centres of the processes. If the same differential could be shown in the mouse snout, it would help to explain the posterior-to-anterior gradient of development.
While ridge formation and growth of the snout may have roles in establishing the general vibrissal pattern, neither could explain the precise temporal and spatial ordering of the vibrissae in their vertical and horizontal rows. Certainly a likely patterning agent would seem to be the distribution of nerves within the snout. The results of Van Exan & Hardy (1980) support this idea. Those investigators examined follicle formation at stages we believe correspond to our late 12-day or early 13-day embryos, at times when many vibrissae had already begun to form and few sites remained to begin development. Their reported correlation between innervation pattern and vibrissal location appears to be similar to the patterns seen in the 13-day bisected snouts stained with an acetylthiocholine procedure to identify nerves. Indeed, nerve branches do seem to be intimately associated with the positions of vibrissae at this stage. Furthermore, with so few vibrissae left to form in each row it would be possible to visualize their future sites and see their associated neural elements as Van Exan & Hardy have done.
The results reported here, however, show that at earlier times, as the arrangement of whiskers is just beginning to be established, innervation is already widespread within the mesenchyme. This profusion of nerve trunks, of neurites and growth cones within the maxillary process complicates any attempt to show a one-to-one correlation between a nerve ending (termed a ‘plexus’ by Van Exan & Hardy) and the later development of a vibrissa. In the first place, there appears to be no immediate temporal correlation between many nerve branches at a certain site and the beginning of a dermal condensation at that place. Secondly, any precise spatial correlation would be difficult, if not impossible, to show because of the interplay of two factors: (1) the close proximity of the early rudiments (see Fig. 15), and (2) the prodigious growth of the maxillary process as the rudiments are forming (from approximately 650 μm to 1200 μm long in the 12-day samples studied). With so little space between the rudiments (each measures ca. 100 μm in diameter) one would have to postulate a very precisely located set of growth cones corresponding to a specific site of a dermal condensation with no growth cones between them. In fact, the nerve tracts in this study do not appear so localized. Neurites do appear between the developing condensations. Furthermore, even if the growth cones were localized in the early 12day snout, one could not be confident that the sites above them would be future centres of dermal condensation because of the unknown effects of growth within the process on the spatial relationships between the elements of the process.
It appears, then, that at the time the follicular pattern is beginning to be established, the overall pattern of innervation is too complex to account for a simple one-to-one correlation. Furthermore, any influence the nerves may have on vibrissal formation would have to be mediated through some action on the mesenchyme. Nerve endings are never closer than approximately five mesenchymal cell diameters from the epithelium. As the dermal cells condense, the growth cones remain on the outside, forming shallow, cup-like figures beneath the condensations at the latest stages studied. Therefore, whatever influence neurons may have on vibrissal development at this time must not be exerted directly on the epithelium.
The results of this study suggest that early innervation of the snout may be a labile phenomenon. Most interesting is the large number of nerve trunks splayed out in the mesenchyme of the 11·5-day maxillary process as ridges are forming. Van Exan & Hardy (1980) reported that five major branches of the maxillary branch underlie the five ridges across the entire whisker pad in later embryos. Three of these large branches would be expected under the area of the three ridges derived from the maxillary process. The many trunks seen at 11·5 days in perpendicular sections must be rearranged at a later time to form three major branches. If this is so, the positioning of the ridges precedes the positioning of the nerve trunks beneath them.
In addition, the nerve tracts themselves are in a process of development during the time studied. Neurites in the trunks at 12 days look more ‘mature’ than those at 11 days because most of the 12-day neurites have a more axon-like appearance. But even at 12 days growth cones are present in the main trunks and become progressively more prevalent as the nerve tracts are traced toward the periphery. The ultrastructure of the neurons is similar to that found during the outgrowth of the interosseus nerve of the chick wing by Al-Ghaith & Lewis (1982). These authors suggested that ‘pioneer’ growth cones were the first to advance through the wing mesenchyme and that later growth cones followed their tracts. Whether or not the most distal growth cones in the maxillary process are true ‘pioneers’ in the same sense as those first suggested by Harrison (1910), found by Bate (1976) in invertebrates, and studied by others (Keshishian, 1980, and Edwards, Chen & Berns, 1981, are examples), it appears that vibrissae can begin to form well before the time that the innervation of the maxillary process is completely developed.
One further comment deserves mention. The high degree of spatial correspondence between the arrangements of the vibrissae on the snout and of their barrels in the sensory cortex was mentioned earlier as being suggestive of the possibility that the central nervous system may direct the patterning of vibrissae. But the correspondence might equally suggest an influence acting in the opposite direction. Indeed, while the follicular pattern is established at 12 days of embryonic life, the barrel pattern is not morphologically apparent until about 5 days after birth (Rice & Van der Loos, 1977), about the same time that barrel development can no longer be altered by lesions of the vibrissae (Van der Loos & Woolsey, 1973; Weller & Johnson, 1975). Weller & Johnson concluded that ‘the periphery can at least be strongly suspected of exerting an organizational influence on the cortex’. Similarly, after a study of supernumerary whiskers and barrels in mice, Van der Loos & Dorfl (1978) concluded that the vibrissal pattern is responsible for the patterning of the cortical barrels.
This study makes no attempt to investigate the hypothesis of peripheral-to-central patterning. Rather, by focusing on early innervation and the lack of clear spatial or temporal correlations between it and initial vibrissal formation, the study suggests that patterning is not unidirectional but is a complex process requiring mutual interactions between the cells and tissues involved.
ACKNOWLEDGEMENTS
The authors wish to thank Heather Hindman for her patience and careful work in drawing the reconstructions. This project was supported by NIH grant No. HD 04708.