Original Paper Received: July 22, 2011 Returned for revision: August 25, 2011 Accepted after revision: August 31, 2011 Published online: December 6, 2011
Brain Behav Evol DOI: 10.1159/000332804
Development of the Olfactory Pathways in Platypus and Echidna Ken W.S. Ashwell Department of Anatomy, School of Medical Sciences, The University of New South Wales, Sydney, N.S.W., Australia
Key Words Monotreme ⴢ Nasal cavity ⴢ Olfactory bulb ⴢ Piriform cortex ⴢ Brain evolution
Abstract The two groups of living monotremes (platypus and echidnas) have remarkably different olfactory structures in the adult. The layers of the main olfactory bulb of the shortbeaked echidna are extensively folded, whereas those of the platypus are not. Similarly, the surface area of the piriform cortex of the echidna is large and its lamination complex, whereas in the platypus it is small and simple. It has been argued that the modern echidnas are derived from a platypus-like ancestor, in which case the extensive olfactory specializations of the modern echidnas would have developed relatively recently in monotreme evolution. In this study, the development of the constituent structures of the olfactory pathway was studied in sectioned platypus and echidna embryos and post-hatchlings at the Museum für Naturkunde, Berlin, Germany. The aim was to determine whether the olfactory structures follow a similar maturational path in the two monotremes during embryonic and early post-hatching ages or whether they show very different developmental paths from the outset. The findings indicate that anatomical
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differences in the central olfactory system between the short-beaked echidna and the platypus begin to develop immediately before hatching, although details of differences in nasal cavity architecture emerge progressively during late post-hatching life. These findings are most consistent with the proposition that the two modern monotreme lineages have followed independent evolutionary paths from a less olfaction-specialized ancestor. The monotreme olfactory pathway does not appear to be sufficiently structurally mature at birth to allow olfaction-mediated behaviour, because central components of both the main and accessory olfactory system have not differentiated at the time of hatching. Copyright © 2011 S. Karger AG, Basel
Introduction
The modern monotremes are confined to Australia and New Guinea and are represented in the modern world by several species of echidna (long- and shortbeaked; Zaglossus species and Tachyglossus aculeatus, respectively) and the platypus (Ornithorhynchus anatinus) [Musser, 2003]. The short-beaked echidna (T. aculeatus) and the platypus (O. anatinus) both consume invertebrates, but occupy very different habitats. The shortProf. Ken Ashwell Department of Anatomy, School of Medical Sciences The University of New South Wales Sydney, NSW 2052 (Australia) Tel. +61 2 9385 2482, E-Mail k.ashwell @ unsw.edu.au
beaked echidna lives in a wide range of habitats from alpine through woodland to desert and is adapted for hunting arthropods in termite mounds and rotten logs, whereas the platypus is specialized for hunting arthropods and other invertebrates in fresh water using electroand pressure-wave reception [Scheich et al., 1986; Gregory et al., 1987, 1988]. Both the short-beaked echidna and the platypus have unusual cytoarchitecture of the olfactory bulb (OB), in that mitral cell somata are not arranged in a monolayer as seen in eutherians, but are spread through the external plexiform layer [Switzer and Johnson, 1977]. In the echidna, the accessory OB occupies only a small proportion of the total bulb volume [Ashwell, 2006a]; in the platypus, it is not only as large as its main bulb, but is also substantially larger than the accessory OB of echidnas of 6- to 8-fold greater body weight. The olfactory tubercle is small and simple in both monotremes [Ashwell, 2006b], but there are profound differences between the two species in other components of the olfactory system. In the echidna, the anterior olfactory nucleus is negligible in extent and merges at very rostral levels with a large, chemically differentiated, 5-layered piriform cortex [Ashwell and Phillips, 2006]. By contrast, the platypus has a distinct anterior olfactory nucleus and 3-layered piriform cortex with no evidence of chemically distinct sub-regions [Ashwell and Phillips, 2006]. The volume of the paleocortex of the echidna is comparable to prosimians of similar body weight and exceeds that for many eutherian olfactory specialists. In other words, the piriform cortex of the echidna shows evidence of regional differentiation, which, in turn, suggests a highly specialized olfactory function for the main olfactory pathway; whereas the platypus shows limited differentiation in the main olfactory pathway, but some development of the accessory olfactory pathway. How and when do these profound anatomical differences in the olfactory pathways between echidna and platypus emerge during development? It has been argued [Pettigrew, 1999; Phillips et al., 2009] that the modern echidnas are derived from a platypus-like ancestor that shifted to a terrestrial niche, lost much of its electroreceptive ability and presumably underwent an enhancement of those senses that would be of greater benefit in a nonaquatic environment (e.g. olfaction). Camens [2010] has argued against this hypothesis, concluding that the poor fossil record does not provide a sufficient basis for this conclusion. Nevertheless, if Phillips et al. [2009] are correct, one might expect to see the olfactory specializations of the short-beaked echidna emerge during late develop2
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ment as a terminal adjustment of a developmental trajectory leading to the type of olfactory structures seen in the modern platypus. In this study, the embryological collections at the Museum für Naturkunde in Berlin were used to determine whether the early development of the components of the main and accessory olfactory pathways [olfactory epithelium and vomeronasal organ (VNO), main and accessory OB, olfactory cortex] is similar or different in platypus and short-beaked echidna and how it compares with olfactory development in therians. Further goals were to determine how the time course of anatomical olfactory development correlates with the known timetable of behavioural development in the two monotremes, and to assess whether olfactory pathways are sufficiently structurally mature at hatching to play a role in vital behaviours such as locating the milk source within the pouch. Materials and Methods Specimens and Processing The findings in this study were based on 33 monotreme specimens (22 platypus and 11 echidna) held in the embryological collections of the Museum für Naturkunde in Berlin. Most of the material is part of the Hill embryological collection, but 7 platypuses and 1 echidna specimen were originally from collections in American museums (AMNH, USNM numbers) and had been sectioned by Professor Ulrich Zeller [Zeller, 1989]. Tables 1 and 2 summarize salient details of the material studied. All of the material had been collected at the close of the 19th century and the beginning of the 20th century. The fixatives used for the material were not recorded, but aldehydes were probably used. The embryonic monotreme material had been embedded in paraffin and sectioned at 8- or 10-m thickness, usually in the transverse plane (except for M37Sag, cut in the sagittal plane), before being stained with haematoxylin, haematoxylin and eosin or Alcian blue and nuclear red. Post-hatching platypus specimens had been embedded in paraffin (M44, M45, MO38, AMNH201969) or celloidin (all others) and sectioned at thicknesses of 10 m (M44, AMNH201969, M45, MO38), 35 m (AMNH202030, MO39, AMNH201311, AMNH201312), 35–50 m (AMNH202002, AMNH202003) or 35–80 m (USNM221112, adult platypus) and stained with azan. Post-hatching echidnas had been embedded in Paraplast or celloidin (adult), sectioned at 15 or 35 m and stained with haematoxylin and eosin, carmine or azan. Analysis of Material Material was photographed either with a Zeiss Axioplan2 fitted with an AxioCam MRc5 camera or with a Leica M420 macroscope fitted with an Apozoom 1:6 lens and Leica DFC490 camera. All images were calibrated by photographing a scale bar at the same magnification. Measurements of structures were made with the aid of ImageJ 1.37v software. All olfactory system structures were identified by reference to published accounts of the anatomy
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Table 1. Summary of platypus (O. anatinus) specimens used
No. M43 M37Sag M37 M39 M39x M38 M40 M41 M07 M42 M44 AMNH201969 M45 MO38 AMNH202030 MO39 AMNH202002 AMNH202003 AMNH201311 AMNH201312 USNM221112 Adult
CRLa, mm 6.5 8.5 8.5 8.5 8.5 9 9 9 9 10 16.75 29.3 33.0 44.0 65 70 81.3 87 – – 160 –
Dorsal contour length, mm – – – – – – – – – – 28.0 74.0 56.0 122 193 180 215 240 – – 333 400
Head length mm – – – – – – – – – – 6.0 14.2 – 22.5 – 31.0 – 41.7 43.0 55.5 60 100
Estimated ageb
Stain
H-9 H-6.5 H-6.5 H-6.5 H-6.5 H-6 H-6 H-6 H-6 H-5 PH0–PH2 PH6 PH7 PH11 PH40 PH42 PH45 PH49 PH52 PH110 PH140 Adult
H&E H&E Haem H&E H&E H&E Haem H&E AB&NR H&E Haem Azan H&E Azan Azan Azan Azan Azan Azan Azan Azan H&E/Azan
AB&NR = Alcian blue and nuclear red; H&E = haematoxylin and eosin; H&E/Azan = sections alternately stained; Haem = haematoxylin. a For embryonic ages this is listed as GL (greatest length) in museum records. b For pre-hatching days this is estimated on the basis of 10–11 days between egg laying and hatching, e.g. H-5 = 5 days prior to hatching and PH = days post-hatching, estimated on the basis of CRL, dorsal contour length and head length dimensions with reference to published tables [Manger et al., 1998].
of the olfactory pathways in these two species [Ashwell, 2006a, b; Ashwell and Phillips, 2006], marsupials [Brunjes et al., 1992; Malun and Brunjes, 1996; Chuah et al., 1997; Ashwell et al., 2008] and a reference work on the anatomy of olfactory regions in the rat [Shipley et al., 2004].
Results
Development of the Main and Accessory Olfactory Systems in Incubation Phase Platypus and Echidna Both the platypus and short-beaked echidna have an incubation phase (development within the egg) lasting about 10–11 days [Renfree et al., 2009]. The first third of the incubation phase has the initial formation of the pharyngeal arches as a major feature (stages 12–15 [Werneburg and Sánchez-Villagra, 2011]), the middle third has the elaboration of the arches and their derivatives (stages Olfactory Pathway Development in Platypus and Echidna
16–20), while the final third has the overgrowth of the arches to form a smooth contour of the head and neck (stages 21–24). The olfactory placode develops at around the end of the first phase of incubation, when the crownto-rump length (CRL) is 5.5–6 mm (stage 15). Its appearance in the platypus and echidna is similar (fig. 1a, b); it is approximately 40 m thick and 0.5 mm wide at this stage and faces a 50- to 70-m-thick olfactory vesicle neuroepithelium across a space of approximately 100 m of mesenchymal tissue. Nares become defined at approximately 8.0- to 8.5-mm CRL (stage 17), at which point the nasal cavity has reached a rostro-caudal length of 0.7–0.8 mm and a dorso-ventral height of 0.5 mm, the olfactory epithelium has thickened to 80 m and the VNO primordium has fully invaginated (fig. 1c, d). The VNO opens into the nasal cavity at the junction of the rostral and middle thirds of the nasal cavity and has reached a length of 200 m. Outgrowing axons from both the main olfacBrain Behav Evol
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Table 2. Summary of short-beaked echidna (T. aculeatus) specimens used
No.
CRLa, mm
Estimated ageb
Stain
M155 MOF142c M157 MOF161 M153 M154 MO55 M158 M161 M162 Adult
5.5c 6c 6.5c 6.6 7.3 7.5c 8 12.5 24 25 400
H-10 H-9 H-9 H-9 H-8 H-7.5 H-7 H-2 PH6 PH6 Adult
H&E H&E H&E H&E H&E H&E H&E H&E H&E Carmine Azan
a For embryonic ages this is listed as GL (greatest length) in museum records. b Estimated on the basis of 10–11 days between egg laying and hatching for pre-hatching ages, e.g. H-5 = 5 days prior to hatching. Post-hatching (PH) age for M161 and M162 is estimated on the basis of platypus growth tables and can only be considered approximate, but the specimens must be at least 4–5 days PH, given their size. c Estimated by measuring available sections and/or multiplying section number by thickness.
tory epithelium and VNO have reached the region of the OB at this point in time, but the OB primordium is still at a very primitive stage, consisting of only a 100-mthick neuroepithelium and an unlaminated 80-m-thick mantle layer (fig. 1c, d). The latter contains only a few post-mitotic neurons and does not give rise to a lateral olfactory tract. During the last third of incubation (fig. 2), the olfactory system remains undifferentiated in structure in both monotremes, although some of the distinctive anatomical features of the echidna olfactory system begin to emerge close to hatching. The nasal cavity, olfactory epithelium and VNO (fig. 2a, d–f) are essentially similar in structure during this period to the mid-incubation phase, but the terminal nerve and terminal ganglion become more prominent than at earlier ages (fig. 2b, g). The internal surface of the nasal cavity continues to be relatively smooth in the platypus, whereas folding of the nasal epithelium becomes more prominent in the echidna close to hatching (fig. 2d). The sub-mucosa of the olfactory epithelium is free of glandular elements, but is well vascularized. Although the OB has grown since mid-incubation, it remains poorly differentiated (fig. 2b, g). In both 4
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species, the OB is comprised of a 100-m-thick olfactory vesicle neuroepithelium surrounded by a very poorly differentiated mantle zone with a diffuse band of post-mitotic neurons. There is no defined olfactory nerve fibre layer and very few of the macroneurons that will provide output from the OB (i.e. mitral and tufted neurons) have been produced. Although vomeronasal and main olfactory nerves are clearly distinguishable, it is not possible to discriminate separate main and accessory OBs in either species. Consistent with the poor differentiation of the OB, there are neither lateral nor dorsal lateral olfactory tracts visible within the OB or along the path to the more central olfactory structures. The deep fissure that will separate the OB from the rest of the telencephalon in the mature echidna brain has emerged even before hatching (fig. 2h), whereas the platypus OB primordium is in smooth continuity with the rest of the telencephalon. The central parts of the olfactory system are similarly undifferentiated in late incubation. In both species there are only a few post-mitotic cells in the region of the future piriform cortex (fig. 2c) and no differentiation of the cortex into the 3 or 5 layers that characterize this region in the adult platypus and echidna, respectively. Furthermore, none of the tracts that might convey olfactory information to more caudal levels of the neuraxis (e.g. medial forebrain bundle) are present and the pallial (olfactory) amygdala is not visible. Post-Hatching Development of the Olfactory System in Platypus and Echidna Post-hatching development in the platypus occurs in a vegetation-lined nest in the maternal burrow and involves a lactational period (i.e. hatching to weaning) of about 135–145 days [Holland and Jackson, 2002]. Posthatching life in the short-beaked echidna involves an initial pouch phase of about 55 days within a lactational phase of 139–210 days, the duration depending on the region within Australia [Morrow et al., 2009]. Only a platypus specimen was available for the immediate post-hatching period (M44; 16.75 mm CRL). In contrast to the immediate pre-hatching echidna, the nasal cavity continues to be relatively smooth (fig. 3a, b). Both the main olfactory and vomeronasal epithelium are similar in thickness and morphology to at pre-hatching ages, and sub-mucosal (Bowman’s) glands have not yet developed. The OB is also quite undifferentiated in appearance, with a large olfactory ventricle germinal neuroepithelium, no granule cells generated and a poorly defined zone of presumptive output neurons. No differentiation into main or accessory OBs is evident and the more cauAshwell
a
c
b
d
Fig. 1. Development of the olfactory path-
way in the early-to-middle incubation phase of monotremes embryos. a, b Transverse sections through the rostral head of platypus and echidna embryos, respectively. c, d Transverse and sagittal sections, respectively, through the rostral head of mid-incubation platypus embryos. The olfactory placode develops at 5–6-mm CRL and the olfactory (olf epith) and VNO epithelium form and give rise to the main olfactory nerve (on) and vomeronasal nerve (vn) axons during the middle third of incubation. * = Limited population of postmitotic neurons in the developing OB. The midline is to the left in a–c and caudal is to the left in d. LV = Lateral ventricle; OV = olfactory vesicle.
dal components of the olfactory pathways (not shown) appear to be as structurally immature as in late incubation. No lateral or dorsal lateral olfactory tracts are visible. During the first week of post-hatching life (fig. 3d–f), folding of the lateral and dorsal wall of the echidna nasal cavity (fig. 3d) continues to be more advanced than that seen in the platypus, but the morphology of main olfactory epithelium (fig. 3e) and VNO epithelium (fig. 3f) are similar in both. The first signs of sub-mucosal glands emerge in both species in the first post-hatching week
(fig. 3e), although their ducts and acini are not yet canalized and the glands are unlikely to be functional. Infolding of the lateral and dorsal nasal wall in the platypus begins to emerge towards the end of the first post-hatching week (fig. 3g) and differentiation of central components of the olfactory system allows identification of key elements in the main and accessory olfactory pathways. A distinct accessory OB is identifiable at the dorso-lateral margin of the primitive bulb (fig. 3h) at the end of the first post-hatching week and the first suggestions of lamination are visible within it. Key events at the end of the
Olfactory Pathway Development in Platypus and Echidna
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a
d
g
Fig. 2. Development of the olfactory pathway in the late incubation phase of monotreme embryos. a–c In the platypus, the OB remains poorly differentiated and pallial parts of the olfactory system are little more than neuroepithelium surrounded by scattered post-mitotic cells. In the immediately pre-hatching echidna, folding of the olfactory epithelium first appears (d), processes of olfactory receptor neurons form a brush border to the olfactory epithelium (e: arrows), but the VNO remains small and immature
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c
b
e
f
h
(f). The OB is also very immature at this stage (g), with only a few neurons settled outside the neuroepithelium, but a distinct fissure (*) has developed between the OB primordium and the rest of the telencephalon (h). Hi = Hippocampal primordium; lge = lateral ganglionic eminence; ne = neuroepithelium; Pir = piriform cortex primordium; ri = rhinal incisure; termg = terminal ganglion; termn = terminal nerve. Other abbreviations are as in figure 1. The midline is on the left in a, c–h and in the centre of b.
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a
d
e
g
h
Fig. 3. Olfactory pathway components in monotremes during the 1st week of post-hatching life. a–c A platypus immediately after hatching. d–f An echidna at about the middle of the 1st post-natal week. g–i Two platypuses at about the end of the 1st post-natal week. e The 1st week is the period when sub-mucosal glandular
elements begin to emerge in the nasal cavity (arrow) and differentiation of the central olfactory nuclei and pathways accelerates.
Olfactory Pathway Development in Platypus and Echidna
c
b
f
i
i The arrow marks the boundary between the piriform cortex and cortical amygdala. The midline is on the left and dorsal at the top in all photomicrographs. AOB = Accessory OB; CxA = cortical amygdala; ec = external capsule; lo = lateral olfactory tract; 5oph = ophthalmic division of trigeminal nerve; Pu = putamen. Other abbreviations are as for figures 1 and 2.
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a
b
c
h
d
e
f
g
i
4
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first week are the dispersal of migrated neurons by an increase in neuropil to separate the layers of the piriform cortex (fig. 3i) and provide definition to the boundaries of the pallial (cortical) amygdala, and the first sign of a robust lateral olfactory tract (fig. 3i). The period of development after the end of the first post-hatching week can only be followed in the platypus (fig. 4a–h). During the period from 2 to 6 post-hatching weeks, differentiation within the main and accessory OBs proceeds rapidly, so that by the middle of the second post-hatching month all the laminae of the mature and accessory OBs can be identified and glomeruli surround the circumference (fig. 4b). The anatomy of central olfactory nuclei and cortex is essentially mature by the middle of the 2nd month of post-hatching life (fig. 4c). The most significant event in the olfactory system after post-hatching day 40 is the elaboration of the nasal turbinates. Bony ridges in the platypus nasal cavity during the second month of post-hatching life are on the dorsal and lateral wall (fig. 4d). Olfactory epithelium covers the dorsal septum and dorsal ridge, whereas the lateral turbinate ridge appears to be covered only by the respiratory epithelium. The lateral turbinate expands by a tree-like branching process during months 2–6 of post-hatching life (2ndorder branches visible at about post-hatching day 50, head length 43 mm; 4th-order branches emerging at about post-hatching day 140, head length 60 mm), where-
Fig. 4. Development of the olfactory system and nasal cavity from the end of the first post-hatching week to adulthood. a, b Lamination of the main and accessory OB of the platypus develops during the first 6 weeks of post-hatching life. c Structure of the central olfactory cortex and nuclei appears mature by the middle of the 2nd month of post-hatching life. Elaboration of nasal turbinates proceeds progressively from the 2nd post-hatching week (d), to adulthood (h). h, i Arrows indicate the ventral boundaries of the olfactory epithelium (olf epith) in the adult platypus and echidna, respectively. Note the gyrified OB and extensive sheet of olfactory nerve fibres (on) in the adult echidna. Medial is to the left and dorsal at the top of all photomicrographs. EPl = External plexiform layer of main OB; Gl = glomerular layer of main OB; Gla = glomerular layer of accessory OB; GrO = granule cell layer of main OB; GrOa = granule cell layer of accessory OB; HL = head length; IPl = internal plexiform layer of main OB; IPla = internal plexiform layer of accessory OB; 5max = maxillary division of the trigeminal nerve; Mi = mitral cell layer of main OB; Mia = mitral cell layer of accessory OB; ON = olfactory nerve fibre layer of main OB; ONa = olfactory nerve fibre layer of accessory OB; Pir1, 2 and 3 = layers of piriform cortex; rf = rhinal fissure; Tu = olfactory tubercle; VEn = ventral endopiriform nucleus. Other abbreviations are as for figures 1, 2 and 3.
Olfactory Pathway Development in Platypus and Echidna
as the dorsal turbinate retains the simple structure of a vertical ridge. Elaboration of the lateral turbinate must continue progressively during the subsequent juvenile period, because by adulthood (fig. 4h), as many as 8 orders of branching have been attained. Note the contrast in the structure of nasal turbinates in the adult platypus and echidna. In the adult platypus, the olfactory epithelium is laid over a single vertical sheet and the dorsal nasal septum, whereas the nasal cavity of the adult echidna (fig. 4i) has the olfactory epithelium laid over a complex series of vertically oriented plates that extend beneath the rostral forebrain.
Discussion
The Embryological Collections of the Museum für Naturkunde in Berlin The combined embryological collections at the Museum für Naturkunde in Berlin are a unique source of information that is unobtainable in the modern world. The bulk of the material was collected during J.P. Hill’s sojourn as a demonstrator of biology at the University of Sydney at the beginning of the 20th century. Since monotremes do not readily breed in captivity, the amassing of embryological material like the Hill collection requires the capture and death of many adult female monotremes for the acquisition of even a few young in the uterine phase of development, as well as the removal of eggs or pouch young from burrows during the incubation and post-hatching phases. This sort of interference with wild populations of platypus and echidna is unacceptable to the modern Australian public and the ethical research committees that represent the public interest, so the collection of such material is unlikely to be repeated until monotremes can be reliably bred in captivity. At present, this is only an occasional event at wildlife parks and zoos [Temple-Smith and Grant, 2001; Holland and Jackson, 2002; Hawkins and Battaglia, 2009] and is unlikely to provide a reliable source of tissue for neuroscientific research. The material was sectioned and stained using technical best practice current at the time of collection, but naturally there are limitations as to what type of data can be extracted from the material. The relatively thick sections do not permit detailed cytological examination of sensory epithelium or the morphology of neurons in central olfactory nuclei and there are no remaining specimens or sections available in suitable preservation to permit the use of modern histological or immunohistochemical Brain Behav Evol
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techniques. Nevertheless, the material does provide some important clues about key developmental events in the monotreme olfactory system. Major morphological features such as the post-mitotic settling of neuronal populations, the outgrowth of olfactory axons and formation of tracts, the development of lamination and the folding of nasal turbinates can be reliably assessed from the available material and correlated with developmental events. Establishing the Age of Monotreme Embryos and Pouch Young Determining the precise age of embryonic and also nest or pouch young (i.e. post-hatching) monotremes can be difficult, but there are some key facts that have been established with reasonable certainty. The uterine phase of development in both the monotremes is of uncertain duration, but is probably between 15 and 21 days for the platypus [Holland and Jackson, 2002; Hawkins and Battaglia, 2009] and 20–24 days for the short-beaked echidna [Morrow et al., 2009], and brings the embryo from zygote to neurulation. Fortunately, the incubation phase, during which much of the embryonic development of the olfactory system occurs, is fairly reliably clocked at 10–11 days [Renfree et al., 2009] for both species and the changes in the embryo that occur during that period are also well documented [Hughes and Hall, 1998; Wenneburg and Sánchez-Villagra, 2011]. The precise correlation of the 12 incubation phase Standard Event System stages [Wenneburg and Sánchez-Villagra, 2011] with calendar days is uncertain, but given the profound structural changes during this period, it is a reasonable assumption that the incubation phase monotreme embryo passes through these stages at the pace of about 1 per day. The known egg size also sets a dimensional limit to the new hatchling of about 14 mm, even though the process of hatching itself has never actually been observed under rigorous scientific conditions. After hatching, somatic growth is rapid in both the platypus [Holland and Jackson, 2002] and short-beaked echidna [Rismiller and McKelvey, 2003], thanks to a prodigious consumption of milk. Post-hatching growth of the platypus can be timed by matching dimensions with published data on linear growth and change in weight [Manger et al., 1998], although in the case of the platypus, these data are largely reliant on the observations of a single naturalist. Nevertheless, if we consider the observed changes in the olfactory system in the context of major phases of development (i.e. 3 sub-phases of incubation, immediate and later post-hatching life), it is possible to draw useful conclusions about the tempo of development. 10
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Evolutionary Considerations The two lineages of monotremes have very different olfactory pathways in the adult [Ashwell, 2006a, b; Ashwell and Phillips, 2006]. The nuclear and pallial components of the main olfactory system of the platypus are relatively small and simple in structure and the major central component of the accessory pathway (i.e. accessory OB) is comparable in size to the main OB. The small size of the OB has been a feature of the ornithorhynchids since the middle Miocene, as shown by the endocranial morphology of the fossil platypus, Obdurodon dicksoni [Macrini et al., 2006]. On the other hand, the shortbeaked echidna has components of the main pathway (main olfactory epithelium, gyrified main OB, extensive piriform cortex) that are large and structurally complex, suggesting a major behavioural role, whereas the accessory olfactory system components are relatively small. Some authors [Pettigrew, 1999; Phillips et al., 2009] have argued that the echidnas are relatively recent offshoots (19–48 million years ago) of the platypus line, and that the common ancestor for all modern monotremes was an aquatic mammal somewhat like the modern platypus. This contention is based on several lines of molecular and general morphological evidence, but can only be convincingly refuted by the discovery of definite tachyglossid fossils from the Cretaceous or very early Tertiary. If the common ancestor for modern monotremes were platypus-like (i.e. a strongly trigeminally specialized, electroreceptive aquatic mammal with a relatively poorly developed main olfactory pathway), then some profound neuroanatomical changes would have been required to arrive at the modern tachyglossid central nervous system. These include a reduction in electroreceptive and trigeminal capacity, extensive gyrification of the cortex with a caudal shift of isocortical functional areas, wide-ranging elaboration of the main olfactory pathway and diminution of the accessory olfactory pathway. With respect to the olfactory system, these changes would have been possible, but several features of olfactory structure in the adult and developing monotremes are more consistent with the common ancestor for modern monotremes being somewhat less specialized than the platypus. Some observations in this report suggest relatively early developmental divergence in the structure of the olfactory pathways and nasal cavity in platypus and echidna. The pronounced folding of the rostral olfactory telencephalon seen in adult echidnas appears even before hatching, at a time when the platypus olfactory telencephalon is quite smooth, and the elaboration of the dorsal and lateral walls of the nasal cavity in the two species follows quite Ashwell
different pathways from the time of hatching. A more parsimonious explanation of these observations would be that the common ancestor of both modern monotreme lineages was relatively generalized with respect to both trigeminal electroreception and olfaction and the rather different structure of the respective modern adults has emerged from divergent sensory evolution in both olfaction and trigeminal somato-sensation. What Is the Functional Capacity of the Olfactory System in Newly Hatched Monotremes? The special challenge facing the newly hatched monotreme is that it must locate the milk source repeatedly, because monotreme mammary glands do not have nipples for semi-permanent attachment (as marsupials do) and the young monotreme must find the areola each time it needs to feed. This might be achieved with the aid of either olfaction or trigeminal somato-sensation, so the question naturally arises as to whether the olfactory system of the newly hatched monotreme is capable of assisting in the repeated location of the milk source. At the time of hatching, the structure of the olfactory system in both platypus and echidnas is extremely immature. Although the main and accessory pathway olfactory epithelia are in place at hatching and olfactory nerve bundles can be seen traversing the distance to the olfactory telencephalic primordium, there are no Bowman’s glands beneath the epithelium. More importantly, the central components of both pathways have not progressed significantly beyond the neural tube stage. A few postmitotic cells (presumably macroneurons generated early, like the mitral and tufted cells) have accumulated in the mantle region of the bulb primordium around the time of hatching, but there is no formation of the lamination that underlies the functional anatomy of the bulb (i.e. formation of glomeruli and plexiform layers), and microneurons, like the granule cells, have yet to be generated. Furthermore, there is no sign of the lateral olfactory tract pathway to more caudal levels of the neuraxis (i.e. piri-
References
Olfactory Pathway Development in Platypus and Echidna
form and entorhinal cortex, anterior olfactory region, olfactory tubercle and medial and cortical amygdala) that is essential to mediate behavioural responses to olfactory stimuli. Taken together, these observations indicate that the monotreme olfactory system is far from structurally mature at birth and is unlikely to engage in olfactionmediated behaviour. On the other hand, the terminal nerve and ganglion are present well before birth. As in other mammals, the terminal nerve projection is probably providing a migration pathway for entry of LHRH+ and FMRFamide+ axons and neurons into the forebrain as has been found for young therian mammals [Schwanzel-Fukuda et al., 1988; Cummings and Brunjes, 1995; Malz and Kuhn, 2002]. As far as is known, this pathway is solely for the transfer of neuroendocrine cells into the hypothalamus and is not substantial enough to mediate olfaction-related behaviours [Ashwell et al., 2008].
Concluding Remarks
The available material has limitations, but it is possible to determine some key facts about the development of the monotreme olfactory system. Although the olfactory epithelium and olfactory nerve develop before birth, central structures in the main and accessory systems do not differentiate until after hatching. The very different olfactory system anatomy in the platypus and echidna has its origin in the immediate pre-hatching period.
Acknowledgements This work was supported by a grant from the Alexander von Humboldt Foundation. I would like to thank Dr Peter Giere of the Museum für Naturkunde, Berlin, for access to the collections and for all his kind help during the work. I am also very grateful to Professor Ulrich Zeller from this museum for kindly providing access to his collection of sectioned platypus and echidna heads.
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