A series of timed laboratory matings were arranged to generate normally developing Carollia embryos of known gestational age. These specimens were then used to create a standardized staging system based on the classic Carnegie system for human development (Streeter, 1942 ; O'Rahilly and Müller, 1987 ). Here, we illustrate this staging system and describe a set of criteria for staging embryos on the basis of the morphology of anatomical features that are easily distinguished either in freshly dissected or fixed specimens under the dissecting microscope. This staging system provides the foundation for further embryological studies in Carollia and other bat species.

A staging series is a fundamental tool for developmental studies in any species (McCrady, 1938 ; Streeter, 1942 ; Nieuwkoop and Faber, 1967 ; Hamburger and Hamilton, 1951 ; Hendrickx, 1971 ; Eyal‐Giladi and Kuchar, 1976 ; Theiler, 1989 ; Mate et al., 1994 ; Kimmel et al., 1995 ; Selwood and Hickford, 1999 ; Iwamatsu, 2004 ). This is in part because, even among genetically homogeneous populations of animals such as inbred strains of mice or clonal strains of fish, there is some variability in the rate of development between individual progeny (Streisinger et al., 1981 ; Theiler, 1989 ; Downs and Davies, 1993 ; Kimmel et al., 1995 ). Such a tool is particularly important for species displaying characteristics such as significant variability in normal gestation length, developmental delays, or where specimens are often harvested from the wild and the day of fertilization is unknown. Staging by morphological criteria relative to a standard series minimizes the effects of developmental variability and delay, obviates the requirement for knowing when conception occurred, and facilitates comparison between independent studies. Moreover, a morphology‐based staging system allows comparison to different bat species and with other mammals.

The short‐tailed fruit bat, Carollia perspicillata (commonly known as Seba's short‐tailed bat or simply Carollia ), would appear to be the bat species of choice for molecular developmental and embryological studies. This bat is a predominantly fruit‐eating microchiropteran, which also takes some insects and floral parts, belonging to the American leaf‐nosed bats (family Phyllostomidae ). This is one of the largest and most successful groups of bats. Carollia occurs from northern Argentina to southern Mexico and is probably the most abundant mammal inhabiting the humid lowland tropics of the New World (McLellan, 1984 ; Fleming, 1988 ; Eisenberg, 1989 ; Rasweiler, unpublished observations). First described in 1758 by Linnaeus in his classic Systema naturae , Carollia ' s behavioral ecology and reproductive physiology have been well described since (Kleiman and Davis, 1979 ; Fleming, 1988 ; Cosson and Pascal, 1994 ; Badwaik and Rasweiler, 2000 ; de Mello and Fernandez, 2000 ; Rasweiler and Badwaik, 1999 ). Carollia adults weigh approximately 19 grams and have a wingspan of approximately 21–25 cm. Carollia in the wild usually breeds twice per year and gives birth to a single offspring that can weigh up to 32% of its mother's postpartum mass (Fleming, 1988 ; Cloutier and Thomas, 1992 ; Rasweiler and Badwaik, 1999 ). Gestation is normally 113 to 120 days (Rasweiler and Badwaik, 1997 ; Rasweiler, unpublished observations) and weaning occurs between 6 and 8 weeks postpartum (Fleming, 1988 ). Juveniles reach adult mass at 10–13 weeks, and both males and females achieve sexual maturity in the wild between 1 and 2 years of age (Fleming, 1988 ). Simple, efficient, and economical methods are now available for maintaining, breeding, and propagating Carollia in the laboratory (Rasweiler and Badwaik, 1996 ). Furthermore, it is routine to safely capture and handle animals on a daily basis, for example to check for breeding activity. In captivity, the females are polyestrous and can be successfully bred at any time of the year. Finally, large numbers of young can be reared and become fertile.

Most bats are nocturnal, and aerial navigation in darkness is guided by means of a phenomenon known as echolocation, another unique feature of bats (Neuweiler, 2000 ). These bats emit vocal sounds through the mouth and/or nose as they fly. The sounds are generally at frequencies above the limit of human hearing and are reflected back to the bat in flight. Neural processing of these reflected echoes enable the bat to avoid obstacles and to locate food in darkness. Echolocation has been found in all bat families investigated thus far, but not all species of bats echolocate (Nowak, 1999 ).

Bats are all members of the order Chiroptera . According to Walker's Mammals of the World (Nowak, 1999 ), the order Chiroptera is divided into 2 superfamilies (Mega‐ and Microchiroptera), 18 families, and 186 genera. The characteristic feature of bats is the presence of wings, making them the only mammals capable of powered flight. Several other groups of mammals such as flying squirrels and flying lemurs do not in fact fly but rather glide. Bats' wings are membranous and supported by the skeletal elements of the limbs and tail. The first digit of the forelimb is short, usually possesses a claw, and is not enclosed within the wing, whereas the other four digits are significantly elongated relative to the thumb, are usually clawless, and support a large portion of the wing membrane. The third (and longest) digit of the forelimb is generally approximately equal in length to the height of the animal from head to foot. Bats display numerous additional adaptations for flight, including light and slender long bones, a robust pectoral girdle and ribcage to support the wings, and a prominent keel extending from the ventral midline of the sternum for the attachment of enlarged pectoral flight muscles.

Morphological, physiological, and molecular comparisons between diverse species beyond the standard rodent models will be necessary to truly understand mammalian embryonic development. As the second largest order of mammals in terms of the number of recognized species (more than 1,000; Simmons, 2001 ), bats are also one of the most successful with respect to geographic range and biological diversity. Bats are extremely important from agricultural and ecological perspectives for control of pest insects and as pollinators and seed dispersers. Bats are of interest to epidemiologists as known and suspected carriers of a variety of pathogens. Finally, the bat's unique abilities of powered flight and echolocation are fascinating from biomechanical, auditory, and neuroscience perspectives.

Short generation time, large numbers of progeny, modest husbandry requirements, and a long history of genetics have all contributed to several members of Rodentia (predominantly mice and rats) becoming the major models for laboratory study of mammalian biology. Consequently, rodent embryology has been studied in great detail, whereas comparatively little is known about the other 25+ orders of mammals. Although knowledge gained from the study of mouse and rat development is of immense value, and much of this knowledge is likely to be applicable to mammals in general, it should be noted that the rate and fecundity of rodent propagation is accomplished through a set of highly specialized and species‐specific reproductive and developmental adaptations. Thus, the lessons learned using rodents to model mammalian reproduction and development may be misleading due to the very characteristics that have facilitated and promoted rodent models for laboratory research. This suggests that a complete picture of mammalian development cannot be obtained by studying one or two species from within a single order (Eakin and Behringer, 2004 ).

There are approximately 4,800 species of mammals currently living on Earth (Nowak, 1999 ). These mammals are divided into three subclasses: Prototheria (or monotremes), Metatheria (or marsupials), and Eutheria . These three subclasses are further divided into 26 or more orders. Unlike all other animals, mammals nourish their young with milk, possess body hair, and have three middle ear bones. As a class, mammals display enormous diversity in form and function. From the tiny Kitti's hog‐nosed bat ( Craseonycteris thonglongyai ) that weighs between 1.5 and 2 grams to giant 150 ton Blue whales ( Balaenoptera musculus ), mammals walk, run, jump, swim, glide, or fly through nearly every terrestrial, aquatic, or aerial habitat on the planet (Nowak, 1999 ). However, most of our knowledge of development comes from studying a single group of mammals: the rodents (order Rodentia ).

RESULTS

Variation in Gestation Length and Rate of Development Specimens carried by females born, reared, and mated in captivity anchor the staging series presented here. Females in this group have gestation periods of 113–120 days, and this represents the normal (nondelayed) gestation period for this species (Rasweiler and Badwaik, 1997; Rasweiler, unpublished observations). To stage normal development beyond the primitive streak stage, embryos are examined that fall close to the lines of best fit for changes in uterine diameter and embryo size as gestation progresses in captive‐reared and ‐bred females (Supplementary Figure S1, which can be viewed at http://www.interscience.wiley.com/jpages/1058‐8388/suppmat; Badwaik and Rasweiler, 2001). It is important to note that some variability in embryo size and/or developmental state was observed among the specimens collected at all time points. There exists up to 24 hr of variation in the time of ovulation, and up to 2 days of variation in oviduct transit time, relative to the onset of breeding, between individual pregnancies (de Bonilla and Rasweiler, 1974; Olivera et al., 2000; Rasweiler and Badwaik, unpublished observations). This finding presumably accounts for some of the observed variability in normal gestation length (7 days) and in developmental state on any particular day post coitum (dpc). Normal differences in the rate of development between individuals may also contribute to this variability. Additional variability can be introduced by the occurrence of developmental delay at the primitive streak stage in captive‐bred animals. Such delays are more common and sometimes of substantial length in wild‐caught, captive‐bred females. They are much less frequent and tend to be of shorter duration in captive‐reared and ‐bred females. In a very few cases, the delays in captive animals have exceeded 100 days. All of the available evidence indicates that the delays occurring in captive‐bred animals are induced by stress (Rasweiler and Badwaik, 1997, unpublished observations). Delays also occur under natural conditions in the wild. On the West Indian island of Trinidad, most adult female Carollia are reproductively synchronized and carry two pregnancies in succession. The first, initiated late in the rainy season, includes a period of developmental delay estimated to be at least 44–50 days. The timing of this delay is apparently controlled by some unknown seasonal factor. The second, conceived in most parous females at a postpartum estrus during the dry season, usually progresses without significant delay (Rasweiler and Badwaik, 1997; Badwaik and Rasweiler, 2001; Rasweiler et al., unpublished observations).

Staging The majority of specimens shown below were collected from timed mating of captive bred and reared females for which the day of initial insemination was determined. Between two and six specimens (combined n = 29), which fell near the lines of best fit for changes in uterine diameter and embryo size with time (Badwaik and Rasweiler, 2001) were examined for stages 12, 14, 15, 16, 17, 18, 20, 22, and 24. Table 1 summarizes the measurements and key features for all 29 specimens; the Supplementary Figure S1 illustrates the rate of growth in uterine diameter and embryo mass. The individual pictured (the reference standard) is the most developmentally advanced specimen examined for each time point. Table 1. Summary of Bat Embryo Reference Specimens Stage Key features Age (dpc) Uterus diameter (mm) Crown–rump length (mm) Mass (mg) 12 Forelimb buds form; tail bud forms; caudal neuropore closes; 3 pharyngeal arches; 21–29 somite pairs. 40 5.75 (+/− 0.64) 3.4 (+/− 0.42) 4.3 (+/− 1.7) 6.2 3.7 5.5 5.3–6.2 3.1–3.7 3.1–5.5 14 Retinal pigment; nasal pits; end of somitogenesis; propatagium and plagiopatagium primordia; hindlimb AER; 36–40 somite pairs. 44 6.95 (+/− 0.44) 5.35 (+/− 0.24) 24.6 (+/− 3.6) 6.9 5.5 27.1 6.9–7.0 5.2–5.5 22.0–27.1 15 Hand plate and footplate form; lens vesicle; auditory hillocks; premaxillary centers. 46 8.65 (+/− 1.20) 7.45 (+/− 0.92) 56 (+/− 13) 9.5 8.1 65 7.8–9.5 6.8–8.1 47–65 16 Nose‐leaf primordium; pinna and tragus form; forelimb digital condensations, uropatagium primordium. 50 12.06 (+/− 1.45) 8.66 (+/− 1.05) 110 (+/− 30) 12.5 9.2 124 10.3–14.0 7.3–10.1 60–130 17 Tongue protruding; cervical flexure straightens; hindlimb interdigit tissue receding; eyes begin to close. 54 13.45 (+/− 1.34) 9.15 (+/− 1.34) 114 (+/− 45) 14.4 10.1 146 2.5–14.4 8.2–10.1 82–146 18 Free thumb; head and body smoother, rounder; eyes half‐closed; postaxial flexure at wrist; calcar. 60 16.32 (+/− 0.98) 12.35 (+/− 1.16) 278 (+/− 83) 17.2 13 345 15.0–17.2 10.9–13.9 185–358 20 Distal forelimbs overlap over face; head larger; eyelids cover pigmented retina; claw primordia form. 70 20.0 (+/− 3.54) 16.35 (+/− 1.06) 617 (+/− 156) 22.5 17.1 727 17.5–22.5 15.6–17.1 507–727 22 Prominent, triangular nose‐leaf; eyelids reopening; wing membranes corrugated; claws pigmented, hooked. 80 23.03 (+/− 2.68) 20.02 (+/− 0.26) 1527 (+/− 208) 24 20.3 1457 20.0–25.1 19.9–20.4 1363–1760 24 Fetal period commences; eyes completely open; face and nose‐leaf pigmenting. 90 23.53 (+/− 0.64) 21.13 (+/− 0.06) 2097 (+/− 199) 24 21.2 2327 22.8–24.0 21.1–21.2 1980–2327 Figure 2 Open in figure viewer PowerPoint Stage 12–17. A–T: The first column (A,E,I,M,Q) shows lateral views with dorsal to the left, the second column (B,F,J,N,R) shows ventral views, the third column (C,G,K,O,S) shows dorsal views of the head and trunk, and the fourth column (D,H,L,P,T) shows dorsal views of the trunk and tail. A–D: A Stage 12 specimen collected from a timed mating at 40 days post coitum (dpc) with 26 somite pairs formed. E–H: A Stage 14 (36–40 somites) specimen collected from a timed mating at 44 dpc, with 37 somite pairs formed. I–L: A Stage 15 specimen collected from a timed mating at 46 dpc. M–P: A Stage 16 specimen collected from a wild‐caught female and morphologically stage‐matched to a specimen collected from a timed mating at 50 dpc. Q–T: A Stage 17 specimen collected from a timed mating at 54 dpc. aer, apical ectodermal ridge; ah, auditory hillocks; chp, chiropatagium; cn, caudal neuropore; cvf, cervical flexure; dc, digital condensation; el, eyelid; ela; endolymphatic appendage; fl, forelimb bud; fp, foot plate; ga, glossopharyngeal arch; gt, genital tubercle; h, heart; hp, hand plate; md, mandible; mt, auditory meatus; mx, maxilla; nl, nose‐leaf; nt, neural tube; og, oral groove; ope, optic evagination; otv, otic vesicle; pi, pinna; pig, pigment; plp, plagiopatagium; prp, region of the propatagium primordium; tb, tail bud; tg, tragus; ub, unicorn bump; urp, uropatagium. All specimens are oriented with anterior at the top. Scale bars = 1 mm in D (applies to A–D), in H (applies to E–H), in L (applies to I–L), in P (applies to M–P), in T (applies to Q–T). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] Figure 3 Open in figure viewer PowerPoint Stage 18–24. A–T: The first column (A,E,I,M,Q) shows lateral views with dorsal to the left, the second column (B,F,J,N,R) shows ventral views, the third column (C,G,K,O,S) shows dorsal views of the head and trunk, and the fourth column (D,H,L,P,T) shows dorsal views of the trunk and tail. A–D: A Stage 18 specimen collected from a wild‐caught female and morphologically stage‐matched to a specimen collected from a timed mating at 60 days post coitum (dpc). E–H: A Stage 19 specimen collected from a wild‐caught female and staged by morphological comparison to preceding and subsequent stages. We expect that embryos at this stage of development would be found at approximately 65 dpc from timed mating. I–L: A Stage 20 specimen collected from a timed mating at 70 dpc. M–P: A Stage 22 specimen collected from a timed mating at 80 dpc. Q–R: A Stage 24 specimen collected from a timed mating at 90 dpc. a, autopod; cal, calcar; chp, chiropatagium; cl, claw primordium; el, eyelid; mc, metacarpal; nl, nose‐leaf; pi, pinna; pl, phalange; s, stylopod; tg, tragus; urp, uropatagium; z, zeugopod. All specimens are oriented with anterior at the top. Scale bars = 1 mm in D (applies to A–D), in H (applies to E–H), in L (applies to I–L), in P (applies to M–P), in T (applies to Q–T). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] In two cases, stages 16 and 18, a reference standard specimen, having become fragile after fixation and prolonged storage, was damaged in handling. In these cases, we replaced the damaged specimen with a morphologically stage‐matched specimen collected from the wild. The individuals pictured for stage 10, 11, and 13 were collected from the wild and ordered in this series according to the number of somite pairs present. The individual pictured for Stage 19 was collected from the wild and staged by comparison with the preceding and subsequent stages. The actual time of gestation for these specimens is unknown.

Stages 1–9: Fertilization to Open Neural Plate (Not Shown) The Carollia equivalents of Carnegie Stage 1 (fertilization), Stage 2 (two‐cell to morula), Stage 3 (free blastocyst), Stage 4 (attaching blastocyst), and Stage 5 (implantation) previously have been described thoroughly (de Bonilla and Rasweiler, 1974; Badwaik et al., 1997; Oliveira et al., 2000; Rasweiler et al., 2002). Useful descriptions of the Carollia equivalents of Carnegie Stage 6 (primitive streak), Stage 7 (notochordal process), Stage 8 (primitive pit), and Stage 9 (open neural plate) will require histological and/or ultrastructural analyses beyond the scope of this study. During these early stages, the morphology of Carollia embryos is planar in geometry, like that of human and most other mammals, but unlike the rodent egg cylinder (Streeter, 1942; Butler and Juurlink, 1987; O'Rahilly and Müller, 1987; Theiler, 1989; Downs and Davies, 1993; Behringer et al., 2000).

Stage 10: Neural Fold Fusion Stage 10 embryos appear as an hourglass‐shaped multilayered region (embryo proper) surrounded by the optically clearer extraembryonic membranes (Fig. 1A–C). The long axis of the opaque region defines the anterior–posterior (A‐P) axis of the embryo. The most prominent features at this stage are the forming neural tube and somites (Fig. 1A). Somite formation begins during Stage 9. The first pair of somites form at the junction of the head and trunk, and as development proceeds, additional somite pairs form progressively from anterior to posterior. Stage 10 is defined by the presence of 4–12 pairs of somites. Figure 1 Open in figure viewer PowerPoint Stage 10–13. A–C show two specimens at Stage 10 (4–12 somites). A: Dorsal view of a four‐somite embryo collected from a wild‐caught female. The extraembryonic membranes have been removed to expose the dorsal surface of the embryo. B,C: Lateral view with dorsal to the left (B) and dorsal view (C) of an eight‐somite embryo collected from a wild‐caught female. D,E: Stage 11 (13–20 somites); lateral view with dorsal to the left (D) and dorsal view (E) of an 18‐somite embryo collected from a wild‐caught female. F,G: Stage 12 (21–29 somites); lateral view with dorsal to the left (F) and dorsal view (G) of a 26‐somite embryo collected from a wild‐caught female. H,I: Stage 13 (30–35 somites); lateral view with dorsal to the left (H) and dorsal view (I) of a 31‐somite embryo collected from a wild‐caught female. aer, apical ectodermal ridge; al, allantois; cn, caudal neuropore; crf, cranial flexure; ela; endolymphatic appendage; ga, glossopharyngeal arch; fl, forelimb bud; h, heart; ha, hyoid arch; hl, hindlimb bud; lp, lens placode; ma, mandibular arch; nt, neural tube; opc, optic cup; ope, optic evagination; otv, otic vesicle; rn, rostral neuropore; so, 1st somite; tb, tail bud; v, ventricle. All specimens are oriented with anterior at the top. Scale bars = 1 mm in A, in C (applies to B,C), in E (applies to D,E), in G (applies to F,G), in I (applies to H,I). The open neural plate folds medially and fuses dorsally to form the neural tube. Early in Stage 10 the neural tube is fused at the level of the trunk. By the end of this stage, fusion extends anterior to the hindbrain region and posterior to around the level of the newest‐formed somite pair. As the lateral edges of the anterior neural plate elevate relative to the midline, and more somite pairs form in the posterior trunk, the previously planar embryo bends toward ventral in the midbrain region (cranial flexure) and in the region posterior to the newest somite pair. This bending of the embryo toward the ventral side reaches maximum curvature during late Stage 11 or early Stage 12 (Fig. 1D–G), after which the embryo progressively, but never completely, straightens. By the middle of this stage, a pair of slight depressions, called the optic sulcii, are seen in the forebrain region of the neural plate. The optic sulcii will form the rudiments of the eyes. The otic placodes, or rudiments of the ears, are visible in the ectoderm overlying the hindbrain. The heart tube and allantois are apparent at the anterior and posterior ventral midline, respectively, by the end this stage (Fig. 1B).

Stage 11: Rostral Neuropore Closure The embryo is increasingly curved toward ventral. The posterior end of the embryo spirals past the head, usually but not invariably to the right side (Fig. 1D,E). Stage 11 is defined by the presence of 13–20 pairs of somites. This stage is marked by the closure of the anterior end of the neural tube, or rostral neuropore (Fig. 1E), when there are 15–16 pairs of somites formed. Cranial flexure reaches approximately 90 degrees by the middle of this stage (Figs. 1D, 6A) and the ventricles of the brain (Fig. 1E) are readily apparent. The first two pharyngeal arches (mandibular and hyoid) become distinct to either side of the hindbrain region and begin to extend distally (Figs. 1D, 6A). The heart tube is larger, segmented into two distinct chambers, and looped rightward (Fig. 1E). The optic sulcii, seen as evaginations from the lateral forebrain after neural tube closure (Figs. 1D,E, 6A), become more prominent, and by the end of this stage, have begun to form optic vesicles. The otic placodes invaginate and take on a saclike morphology (Figs. 1D, 6A), but remain open until the early part of the next stage. The allantois expands, becoming spherical in morphology and highly vascularized (Fig. 1D,E). By the end of the stage, the allantois extends ventrally from the posterior trunk region and makes contact with the chorion (Rasweiler and Badwaik, 1997). Figure 6 Open in figure viewer PowerPoint Craniofacial development. A: Lateral view of right side of Stage 11 head with dorsal to top and anterior to the right. B: Lateral view of right side of Stage 12 head with dorsal to top and anterior to the right. C: Lateral view of right side of Stage 13 head with dorsal to top and anterior to the right. D: Lateral view of right side of Stage 14 head with dorsal to top and anterior to the right. E: Face‐on view of Stage 15 head with anterior to top. F: Face‐on view of Stage 16E head with anterior to top. G: Face‐on view of Stage 16L head with anterior to top. H: Face‐on view of Stage 17E head with anterior to top. I: Face‐on view of Stage 17L head with anterior to top. J: Face‐on view of Stage 18 head with anterior to top. K: Face‐on view of Stage 19 head with anterior to top. L: Face‐on view of Stage 20 head with anterior to top. Views are not to scale. ah, auditory hillocks; cc, chin cleft; crf, cranial flexure; cw, chin wart; el, eyelid; ela; endolymphatic appendage; ga, glossopharyngeal arch; ha, hyoid arch; lj, lower jaw; lp, lens placode; lv, lens vesicle; ma, mandibular arch; md, mandible; mx, maxilla; mo, mouth; nl, nose‐leaf; np, nasal pit; og, oral groove; opc, optic cup; ope, optic evagination; otv, otic vesicle; pi, pinna; pig, pigment; pmx, premaxillary center; tg, tragus; to, tongue; ub, unicorn bump; uj, upper jaw; vb, vibrissa. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Stage 12: Forelimb Bud The corkscrew‐like curl of the main body axis reaches maximum extent by early Stage 12 (Figs. 1F,G, 2A–D). This stage is defined by the presence of 21–29 pairs of somites and marked by the first appearance of forelimb buds adjacent to the 7th through 12th somite when 21–22 pairs of somites are present (Figs. 1F,G, 2B,C). The allantois is fused with the chorion early in this stage, initiating the chorioallantoic placenta (Rasweiler and Badwaik, 1997; Rasweiler et al., 2000; Badwaik and Rasweiler, 2001). The optic evaginations enlarge and approach the overlying ectoderm (Figs. 1G, 6B), but the retina and lens are not yet seen. The otic vesicles close early in Stage 12 and take on a spherical morphology (Figs. 1F, 2A, 6B). The posterior end of the neural tube, or caudal neuropore, closes by the time 25 somite pairs are formed, and a tail bud is present (Figs. 1F, 2C). The first and second pharyngeal arches are larger and extend distally (Figs. 1F, 2A, 6B). A third pair of pharyngeal arches (glossopharyngeal) appears immediately posterior to them in the first half of this stage (Figs. 2A, 6B). By the end of this stage, the distal end of this outgrowth is partly concealed under the second (hyoid) arch (Fig. 1F). A regular heartbeat, with blood circulating through the pharyngeal region and around the neural tube, is seen in freshly dissected specimens by the time there are 26 somite pairs formed.

Stage 13: Hindlimb Bud Overtly similar in overall appearance to those of the previous stage, Stage 13 embryos are defined by the presence of 30–35 pairs of somites (Fig. 1H,I). This stage is marked by the first appearance of hindlimb buds adjacent to the 22nd through 28th somite pairs when there are 30–31 pairs of somites (Fig. 1H,I). The first two pharyngeal arch pairs are larger and show distinct proximal and distal parts: A cleft, called the oral groove, is visible on the anterior margin of the first arch, dividing the distal mandible (or lower jaw) component from the proximal maxilla (or upper jaw and palate) component by the time there are 30 pairs of somites formed (Figs. 1H, 6C). The second, or hyoid, arch is dumbbell shaped when viewed laterally (Figs. 1H, 6C). The third arch extends under the hyoid and by the end of this stage only the most proximal portion is visible in lateral view (Figs. 1H, 6C). From the second half of the stage, an apical ectodermal ridge (AER) is seen at the distal edge of the forelimb bud (Fig. 1H). The optic evaginations form optic cups, and lens placodes are seen in the overlying ectoderm by the end of this stage (Figs. 1H, 6C). The otic vesicle loses its spherical shape as the endolymphatic appendage initiates dorsally (Figs. 1H, 6C).

Stage 14: Pigmented Retina Defined by the presence of 36–40 pairs of somites, the first appearance of faint scattered pigment in the retina of the eye marks the start of this stage, and during its course, retinal pigmentation is progressively more uniform (Figs. 2E, 6D). The curve of the main body axis is interrupted by a sharp angle at the head/trunk junction called the cervical flexure (Fig. 2E–H). The axis is also straighter, and by the end of this stage, the head and tail are no longer adjacent to each other. The first pharyngeal arch is further divided into maxillary and mandibular components by the deepening oral groove (Figs. 2E,H, 6D). A pair of nasal pits are clearly evident just anterior to the distal end of the maxilla by the time there are 36 somite pairs (Figs. 2E, 6D). The hindlimb bud is approximately as wide as it is long, whereas the forelimb bud is distinctly longer than it is wide (Fig. 4A,B). When there are 37 somite pairs, a prominent bulge appears proximally on the anterior edge of the forelimb bud (Figs. 2E, 4A). It is from this region that the primordium of the propatagium, or the portion of the wing membrane that will eventually stretch between the shoulder and wrist, emerges. A second bulge appears at the junction of the posterior edge of the forelimb bud and flank (Fig. 2H). Again based on position and subsequent morphogenesis, this structure is the primordium of the plagiopatagium, or the portion of the wing membrane that will eventually stretch from the end of the fifth digit of the forelimb to the ankle (Fig. 4K,L). When there are 38 somite pairs, the heart appears to be enclosed by the body wall but is still protruding ventrally (Fig. 2E). The rudiment of the genital tubercle first becomes evident as a midline thickening of the ventral body wall at the level of the hindlimb buds, between the umbilicus and tail. AERs are present at the distal edges of the hindlimb buds (Figs. 2H, 4B). The otic vesicle is longer along the A–P axis, and the endolymphatic appendage extends, resulting in a trefoil shape (Fig. 6D). All late Stage 14 and early Stage 15 specimens that we have examined to date (n = 26) displayed no more than 40 pairs of somites. By these criteria, we infer that somitogenesis is complete by the end of Stage 14, and 40 is the maximum number of somite pairs in Carollia. Subsequent stages are defined primarily by the morphology of limb and craniofacial structures and in the latest stages by the extent of body pigmentation. Figure 4 Open in figure viewer PowerPoint Limb morphology. A: Forelimb at Stage 14. B: Hindlimb at Stage 14. C: Forelimb at Stage 15E. D: Hindlimb at Stage 15E. E: Forelimb at Stage 15. F: Hindlimb at Stage 15. G: Forelimb at Stage 16. H: Hindlimb at Stage 16. I: Forelimb at Stage 16L. J: Hindlimb at Stage 16L. K: Forelimb at Stage 17. L: Hindlimb at Stage 17. M: Forelimb at Stage 18. N: Hindlimb at Stage 18. O: Forelimb at Stage 19. P: Hindlimb at Stage 19. Q: Forelimb at Stage 20. R: Hindlimb at Stage 20. a, autopod; aer, apical ectodermal ridge; ca, calcar; chp, chiropatagium; cl, claw primordium; dc, digit condensation; fp, foot plate; hp, hand plate; id, interdigit; mc, metacarpal; pl, phalange; plp, plagiopatagium; prp, region of the propatagium primordium; s, stylopod; tm, thumb; urp, uropatagium; z, zeugopod. All panels show the dorsal surface of the right limb with anterior toward top and the proximal at left, views are not to scale. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Stage 15: Hand/Foot Plate Until Stage 15, the width of the trunk, discounting the limbs, is relatively uniform when viewed dorsally or ventrally, but from this stage on, the trunk is significantly broader at the shoulders compared with the hips (Fig. 2L). The main body axis is straighter in the trunk region, and the anterior and posterior ends of the embryo are well separated (Fig. 2I,J). The beginning of this stage is marked by the formation of the forelimb hand plate (Figs. 2I, 4C,E). The distal segment of the forelimb bud (or forelimb autopod) expands along the A–P axis, while flattening dorsoventrally as it continues to extend distally to form a paddle‐like structure called the hand plate. In more advanced specimens, the hand plate is asymmetric, with the posterior portion expanded to a greater extent than the anterior. The hand plate mesenchyme has begun to organize into condensations that will give rise to the skeleton of the five digits, separated by four interdigit regions of uncondensed mesenchyme by the end of the stage (Fig. 5C). By late Stage 15, the distal segment of the hindlimb bud (or hindlimb autopod) displays a foot plate similar to the early hand plate in appearance (Figs. 2I, 4D,F). Unlike the hand plate, the foot plate remains symmetrical along the A–P axis throughout its subsequent development (Fig. 4H,J,L). The plagiopatagium primordia extend distally along the posterior edge of the forelimb to approximately the level of the wrist and caudally along the flank to the base of the hindlimb (Fig. 2L). In the eye, a lens vesicle is apparent (Figs 2I, 6E). The external opening of the auditory canal (or meatus) is seen in the groove between first and second pharyngeal arches (Fig. 2I). The proximal pharyngeal arch tissue surrounding the meatus organizes into auditory hillocks (Figs. 2I, 6E). The distal mandibular components of the first pharyngeal arch pair meet at the midline to form the lower jaw. The maxillary components of the first pharyngeal arch pair remain separate and premaxillary centers are first seen as distinct bulges between the distal maxilla and the midline (Fig. 6E). Figure 5 Open in figure viewer PowerPoint Limb skeleton and pattern. A–F: Dorsal views with proximal to the left of right forelimbs and hindlimbs stained with Alcian blue for cartilage and cleared. A: Stage 14 forelimb. B: Stage 14 hindlimb. C: Stage 15 forelimb. D: Stage 15 hindlimb. E: Stage 17 forelimb. F: Stage 17 hindlimb. Forelimb and hindlimb views are to scale within each stage but not between stages. G: Posterior view of a freshly dissected Stage 19 specimen; dorsal is to the bottom. H: Posterior view of a freshly dissected Stage 20 specimen; dorsal is to the bottom. Note the symmetry of the hindlimb digits but asymmetry of the hindlimb vasculature (arrowheads in G,H).

Stage 16: Nose‐Leaf Primordium During the course of this stage, the cervical flexure straightens and the head rises relative to the rest of the body (Figs. 2M, 6F). Stage 16 is marked by the appearance of the nose‐leaf primordium on the midline of the face between the eyes and above the nasal pits (Figs. 2N, 6F). This structure will continue to grow throughout the remainder of development, forming the defining characteristic of the phyllostomid (or leaf‐nosed) family of bats. The nose‐leaf plays a role in the projection of echolocation calls, which are emitted through the nose in this family (Hartley and Suthers, 1987; Nowak, 1999; Neuweiler, 2000). In addition to the beginning of nose‐leaf development, the rest of the facial region undergoes dramatic morphogenesis in this stage (Fig. 6F,G). The lower jaw extends and a slight midline cleft appears (Fig. 6G). The morphological distinctions between the distal maxillary processes and premaxillary centers disappear as the upper jaw unifies across the midline, and the first indication of vibrissae, or whisker follicles, are visible as slight bumps on lateral surface of the upper jaw (Fig. 6F,G). As the upper jaw unifies and the lower jaw extends, the lower facial region takes on the appearance of a snout or muzzle (Fig. 2M). The auditory hillocks are larger and elaborate distinct pinnae and tragii (Figs. 2N,O, 6F,G). The mammalian outer ear is composed of the auditory meatus and the surrounding external structures called pinnae, which function to enhance the directional properties of sound (Neuweiler, 2000). Some mammals, including most bats, also possess a projection that extends over the meatus called the tragus. The optic fissures close, and eyelid primordia are seen as raised swellings of tissue surrounding each eye (Fig. 6F). The foot plate mesenchyme is organized into five digit condensations separated by four uncondensed interdigit regions, and by the end of this stage, the distal ends of the digit condensations extend beyond the interdigit regions, giving the foot plate a slightly “scalloped” appearance (Fig. 4J). The plagiopatagium extends distally along the anterior edge of the hindlimb to the level of the foot plate, while on the forelimb it extends to the tip of the fifth digit (Figs. 2O,P, 4I,J). The posterior‐most portion of the wing membrane, or uropatagium, begins to enclose the tail (Figs. 2P, 4J).

Stage 17: Tongue Out The head continues to rise, and the snout is noticeably longer (Fig. 2Q). The mouth opens and the developing tongue extends until the tip protrudes (Fig. 6I). Vibrissae follicles are prominent features on either side of the upper jaw (Figs. 2Q, 6H). A series of bumps, or chin warts, are seen on the lower jaw (Fig. 6H). Chin warts of variable size and number are fairly common among different species of bats: these have no known function to the best of our knowledge. The tip of the nose‐leaf extends from the end of the snout, and a ridge appears along its midline, ending between the nasal pits (Fig. 6H,I). The developing eyelids begin to cover the surface of the eye, giving it a characteristic almond shape (Figs. 2R, 6H,I). The pinnae are larger and curl inward at the distal tips, obscuring the meatus and partially covering the tragus (Figs. 2R, 6H,I). The cranial vault expands and a transitory button‐like epithelial structure we call the unicorn bump appears on the midline of the head‐crown just posterior to the forebrain–midbrain junction by the middle of this stage (Figs. 2Q, 6I). The limbs are longer, with pronounced flexures at the elbow and knee joints, and the wrist displays a slight flexure (Figs. 2Q,R, 4K,L). The tissue between all of the digits of the hindlimb and between the first and second digit of the forelimb begins to recede, perhaps by apoptosis as has been determined in other vertebrates (Fig. 4K,L; Zou and Niswander, 1996; Laufer et al., 1997; Salas‐Vidal et al., 2001). The tissue between the remaining forelimb digits persists to form the chiropatagium, or the wing membrane webbing between the fingers (Figs. 2R, 4K). The uropatagium encloses most of the tail and extends along the posterior edges of the hindlimbs to the level of the ankle (Figs. 2R, 4L).

Stage 18: Free Thumb The overall appearance of the embryo is smoother and rounder than previous stages as the cervical flexure disappears and the cranial vault expands (Fig. 3A–D). The eyelids cover more of the eye and the pinnae remain curled inward, obscuring the tragii (Fig. 6J). The snout is longer, and the tongue no longer protrudes (Fig. 6J). Isolated pigmented melanocytes are first seen on the dorsal head and trunk by the end of Stage 18. The tissue between the first and second digit of the forelimb is absent, and the thumb (first digit) is free from the rest of the hand plate (Figs. 3A,B, 4M). The rest of the forelimb digits are significantly longer than the thumb, and the distal extremities approach each other over the face (Fig. 3A,B). The chiropatagium is thinner between well‐defined metacarpals and phalanges, and a clear postaxial flexure at the wrist joint is evident (Figs. 3B, 4M). The tissue between all of the hindlimb digits disappears, leaving five free digits on each foot (Figs. 3B, 4N). Calcars, or cartilaginous projections from the ankle joint that function in spreading the uropatagium, are first seen extending medially from the ankle into the margin of the uropatagium (Figs. 3B, 4N). By the end of the stage, the tail is completely enclosed within the uropatagium (Figs. 3B, 4N).

Stage 19: Peek‐A‐Boo The overall appearance of the embryo is overtly similar to the previous stage, but the trunk is straighter and the head appears larger relative to rest the body (Fig. 3E–H). By the middle of this stage, the head has approximately the same profile area as the trunk, excepting the limbs (Fig. 3E). The distal extremities of the forelimbs overlap, obscuring the face completely and giving this stage its name (Fig. 3F). The first digit of the forelimb (thumb) and all of the hindlimb digits develop knob‐like claw primordia at their distal tips (Figs. 3E, 4O,P, 5G). The hindlimb autopod displays no apparent A–P patterning in terms of digit morphology; however, the developing blood vessels display A–P asymmetry in branching pattern (Figs. 4P, 5F,G). The ankle displays a clear preaxial flexure and the calcar is more prominent (Figs. 3F, 4P). The developing eyelids cover more of the eyes, and nearly all the pigmented retina is obscured (Fig. 6K). The pinnae are larger and extend away from the head (Figs. 3F, 6K). The tips remain curled inward but the tragii are visible. The tragii are rod‐like and blunt distally (Fig. 6K). Isolated melanocytes are seen on the lateral head, flanks, and over the shoulders.

Stage 20: Eyelids Closed The overall appearance of the embryo is overtly similar to the previous stage, but the trunk appears longer and the head appears comparatively smaller relative to rest the body (Fig. 3I–L). By the beginning of Stage 20, the eyelids are closed (Figs. 2I,J, 6L). The limbs are longer, and greater flexure of the wrist and ankle joints is evident (Figs. 2I, 4Q,R). For the first time the zeugopod and autopod of the forelimb are noticeably longer than the stylopod (Fig. 4Q). The forelimb will continue to increase in length at a greater rate distally compared with proximally for the remainder of development, until it reaches the adult stylopod : zeugopod : autopod proportion of approximately 1 : 2 : 4. The claw primordia are more pointed at the tips (Fig. 4Q,R), and their dorsal surfaces appear iridescent in freshly dissected specimens, indicating that keratinization has commenced (Fig. 5H). The pinnae are larger and only slightly curled in at the tip (Fig. 6L). The tragii are conical and slightly longer (Fig. 6L). The skin is more uniformly pigmented dorsally, with larger numbers of small melanocytes found interspersed between more scattered large ones. Pigmentation covers the entire dorsal trunk and extends rostrally over the dorsal head to approximately the level of the ears (Fig. 6L).

Stage 21: Not Shown By inference to the preceding and later stages and timings, we expect the equivalent of Carnegie Stage 21 to be found between 73 and 77 dpc from females reared and bred in the laboratory colony. We did not collect any reference standard specimens in this gestation range, however.

Stage 22: Triangular Nose‐Leaf The overall appearance of the embryo (Fig. 3M–P) is overtly similar to that of Stage 20. During Stage 22, the eyelids, which were completely closed at the end of Stage 20, are more than halfway open (Fig. 3N). The nose‐leaf is larger, extends above the snout, and is triangular in shape (Fig. 3N). The stylopod of the forelimb is approximately three quarters the length of the zeugopod, which is, in turn, approximately three quarters the length of the autopod (Fig. 3M). The thumb and toe claws are hooked toward ventral, pigmented, and come to sharp points (Fig. 3M,P). The wing membranes have thinned such that they are translucent in freshly dissected specimens and the surface appears corrugated (Fig. 3M).

Stage 23: Not Shown By inference to the preceding and later stages, we expect the equivalent of Carnegie Stage 23 to be found between 83 and 87 dpc from females reared and bred in the laboratory colony. We did not collect any reference standard specimens in this gestation range.

Stage 24: Fetal Period Commences The overall appearance of the early fetus resembles that of the newborn except that it is less than half as large, is less heavily pigmented, and lacks body hair (Fig. 3Q–T). The stylopod of the forelimb is approximately three quarters the length of the zeugopod, which is, in turn, approximately two thirds the length of the autopod (Fig. 3Q,R). The eyes are completely open, and pigmented melanocytes are found on the face and nose‐leaf (Fig. 3R).