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In utero development of fetal breathing movements in C57BL6 mice
Respiratory Physiology & Neurobiology 271 (2020) 103288
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In utero development of fetal breathing movements in C57BL6 mice Mary M. Niblock Sydney Gilkeyb,c a b c
a,b,⁎
a
b
b,c
T
a
, Alanis Perez , Shahar Broitman , Brigitte Jacoby , Elana Aviv ,
Department of Biology, Dickinson College, Carlisle, PA, United States Neuroscience Program, Dickinson College, Carlisle, PA, United States Biochemistry and Molecular Biology Program, Dickinson College, Carlisle, PA, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: Fetal breathing movements Mice Respiratory development Ultrasound
Fetuses of many species, including humans, breathe during development. This fetal breathing aids in lung development, strengthens respiratory muscles, and is posited to fine-tune the neural circuitry that drives breathing. Previous studies suggested that fetal breathing could begin as early as the fifteenth day of gestation in the mouse, but fetal breathing movements (FBMs) had not been observed in mice in utero. We aimed to determine if and when FBMs commence in mice and if they change over time. We examined unanesthetised pregnant C57BL6 mice with ultrasound beginning on the seventh day of gestation. We first reliably observed episodic FBMs in mice on embryonic day 16. FBMs were sporadic, clustered, or rhythmic, and their frequency increased with age. Ultrasound examination of FBMs in mice has great potential utility in the study of transgenic mouse models to help us understand the prenatal characteristics of breathing related human developmental disorders, including Congenital Central Hypoventilation Syndrome (CCHS) and apnea of prematurity.
1. Introduction Even though fetal breathing movements are not necessary for gas exchange, they are important because they help strengthen the respiratory muscles and prepare the lungs for breathing (See Inanlou et al., 2005, for review). They also are hypothesized to fine-tune the neural circuitry that will allow the animal to breathe regularly and adaptively at birth (See Greer et al., 2006; Feldman et al., 2009; Greer, 2012, for review). Fetal breathing movements have been observed in humans, as well as in numerous experimental animal models, including sheep (Dawes et al., 1970; Merlet et al., 1970; Dawes et al., 1972) and rats (Kobayashi et al., 1985), but have not been observed in mice in utero. Mice are important models for human breathing disorders with neural developmental underpinnings, including Congenital Central Hypoventilation Syndrome (CCHS) and apnea of prematurity. Genetic mouse models are available that are directly related to these disorders but the prenatal onset and subsequent fetal development of breathing have not been studied successfully in mice in utero. Previous studies have documented fetal breathing-like activity in reduced preparations (Viemari et al., 2003; Thoby-Brisson et al., 2005). Some studies have looked for fetal breathing movements (FBMs) in mice using creative approaches such as plethysmography following premature cesarean
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delivery beginning at embryonic day 16 (E16, Viemari et al., 2003) and examination of externalized pups on the day before birth (Kleven and Ronca, 2009). In spite of these efforts, no one has observed FBMs in unanesthetized mice in utero. Lung development in the mouse begins on embryonic day 9 and lung buds are distinct structures by E12.5 (See Herriges and Morrisey, 2014, for review). Diaphragm development commences early on E11 with the migration of muscle progenitors and subsequent projection of the phrenic nerves towards the pleuroperitoneal folds (Sefton et al., 2018). The first phrenic nerve axons reach the primordial diaphragm late on E11 and by E13.5 the phrenic nerve has branched and innervated the developing diaphragm (Sefton et al., 2018). Studies of in vitro preparations have shown that the embryonic parafacial nucleus begins producing rhythmic activity at E14.5 in the mouse (ThobyBrisson et al., 2009). One day later, the presumptive respiratory pattern generator known as the Pre-Bötzinger Complex begins to oscillate in synchrony with the embryonic parafacial neurons, generating the first activity capable of driving fetal respiration in the mouse (Abadie et al., 2000; Thoby-Brisson et al., 2005, 2009). In vivo data from instrumented fetal lambs and ultrasound observations in rats suggest that FBMs mature over time, becoming more frequent and more rhythmic as animals approach birth (Dawes et al., 1970; Kobayashi et al., 1985), which presumably reflects the maturation and fine-tuning of the underlying
Corresponding author at: Department of Biology and Neuroscience Program, Dickinson College, P.O. Box 1773, Carlisle, PA, 17013, United States. E-mail address: [email protected] (M.M. Niblock).
https://doi.org/10.1016/j.resp.2019.103288 Received 9 July 2019; Received in revised form 6 August 2019; Accepted 5 September 2019 Available online 07 September 2019 1569-9048/ © 2019 Elsevier B.V. All rights reserved.
Respiratory Physiology & Neurobiology 271 (2020) 103288
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were videotaped for later review and confirmation of FBM and heart rate counts. Fetal breathing movements were characterized by visible contractions of the diaphragm that displaced the gut bag and the ribcage. Data were collected from embryos in a frontal or side orientation only when body parts could be identified and confirmed to belong to that pup. Following ultrasound measurements and observations, pregnant mice were returned to the colony and checked twice daily for pup delivery. Some pregnant mice were scanned on successive days and in those cases an effort was made to locate the same embryo(s) scanned on the previous day(s). A limited number of pups were scanned on the day of birth in order to compare the ultrasound appearance of in utero breathing movements with the appearance of early postnatal breathing. Neonatal pups were placed lengthwise on top of the transducer on a thin layer of gel and video was captured with the ultrasound system. In total, the data presented here come from 91 different ultrasound sessions. Photos and videos collected using the SonoSite system were projected using a computer monitor and captured using a high resolution digital camera. Photo brightness and contrast were adjusted using Adobe Photoshop (Creative Suite 5). Figures were created using Adobe Illustrator (Creative Suite 5) and movies were created using iMovie.
neural circuitry in addition to progressive diaphragm and lung morphogenesis. We sought to observe FBMs in C57BL6 mice, a common background strain for transgenic models, and confirm the developmental timeline observed in vitro and predicted by the developmental timeline of the underlying neural circuitry and respiratory structures, in addition to looking for any potential evidence of age-related changes in fetal breathing. In our study, we used ultrasound to examine developing pups in awake, pregnant C57BL6 mice between E10 and E18; E19 is the day of birth (Murray et al., 2010). Our goals were (1) to observe if and when FBMs commence in mice and (2) to determine if there are age-related changes in FBMs in mice. In addition, we recorded biometrics and made corresponding qualitative observations of morphological and behavioral development in C57BL6 mice. We found that the onset of episodic fetal breathing movements in C57BL6 mice occurs at E16. We observed statistically nonsignificant trends of increasing numbers of FBMs and increasing frequency of clustered and rhythmic breathing over the three days prior to birth. 2. Methods 2.1. Animals
2.3. Data analysis These experiments were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Dickinson College Institutional Animal Care and Use Committee (Protocol #617). Adult C57BL6 mice were housed in ventilated caging in breeding pairs (one male, one female) or trios (one male, two females) and maintained on a 12:12 light:dark cycle with ad libitum access to food and water. After pairing with males, females were checked each morning for the presence of a vaginal plug. The day a plug was observed was designated embryonic day (E) 0. Females were also visually monitored for pregnancy- related weight gain. The data, observations, photos, and videos reported here were collected from scans of 58 litters, at the following ages: E9/10 (n = 5), E12 (n = 8), E13 (n = 5), E14 (n = 2), E15 (n = 6), E16 (n = 8), E17 (n = 13), and E18 (n = 11). In addition, two P0 pups were scanned for a comparison between the ultrasound appearance of fetal and early postnatal breathing.
Data were entered into an Excel spreadsheet. The average and standard error of the mean were calculated for FBMs at each age. For the biometric data, an online linear regression and correlation coefficient calculator were used (www.alcula.com/calculators/statistics/ linear-regression/). 3. Results 3.1. Biometrics and qualitative observations As we looked for FBMs, we collected age-appropriate biometric data, including heart rate when the beating heart was visible, crown rump length, and biparietal diameter when the orientation of the head was clear (Table 1, Fig. 1). We also recorded our observations of agerelated changes in pup behavior and the relative echogenicity of prominent anatomical features. These data and observations are presented first to provide a progressive picture of the changes we saw leading up to and subsequent to the onset of fetal breathing. Crown rump length and biparietal diameter were used to estimate and confirm gestational ages along with dated observations of seminal plugs. The beating heart could be readily detected by E10 (Movie 1) and the ventricular septum and atrial and ventricular valves could be seen clearly at this age (Fig. 2). Heart rate did not have a strong correlation with age (r = 0.33) and was highly variable within age, and within litter, in our limited sample (n = 21 total), but did tend to increase with age, as indicated by the slope of the line of best fit (Fig. 1; slope = 4.08). CRL and BPD both increased with age as expected (Fig. 1). CRL had a higher correlation coefficient (0.90) than BPD (0.60) so we found it more useful for estimating gestational age in cases for which no plug was observed. Bones first became visible via ultrasound on E13 (Fig. 2F and G) and became more echogenic, and therefore more distinct, as they calcified with increasing age (Fig. 3). Embryonic day 13 was the earliest age at which we could reliably distinguish the head from the trunk. Overall pup movement was evident as early as E13, increased with age, and included rolling, squirming, head movements, mouth and throat movements, and limb movements (Movie 2). The developing ribs, lungs, and diaphragm could be seen as early as E14 and by E18 these breathing related structures were easily identified (Fig. 4).
2.2. Ultrasound Ultrasound (SonoSite MicroMaxx portable unit with the L25e 136 MHz transducer) was used to confirm pregnancy around the estimated seventh day of pregnancy. Unanesthetized pregnant mice were held by the scruff in a supine position, rotated slightly to the side, with the tail held firmly by the experimenter’s ring and pinky finger. The ultrasound probe was coated with a thin layer of ultrasound gel. The presence of gestational sacs was considered evidence of pregnancy. Measurements (cm) of gestational sac diameter (GSD), biparietal diameter (BPD), and crown-rump length (CRL) were used to estimate and confirm gestational age, along with the day the vaginal plug was observed, if a plug was found (Fig. 1). Once pregnancy was confirmed by ultrasound, pregnant mice were returned to their housing until the ninth or tenth day of gestation. At that time, depilatory cream (Nair) was applied with a cotton swab to unanesthetised pregnant mice held by the scruff in a supine position for one minute to remove belly fur for better image resolution. Depilatory residue was removed with a micellar cleansing cloth. Following depilation, pregnant mice were scanned as described above to locate, collect biometric data for, and observe their pups. If a breathing pup was found, fetal breathing episodes and movements were manually recorded as they were observed at 10 min intervals by at least two trained observers for an additional 20–80 min, depending on the case. Observations and count of FBMs were made in the same pup(s) during each ultrasound session. Movies of selected recording sessions
3.2. Fetal breathing movement observations The earliest diaphragmatic activity we observed occurred on E15, 4 2
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Fig. 1. Scatter plots of heart rate, crown rump length (CRL), and biparietal diameter (BPD) in embryonic C57BL6 mice. (A) Heart rate between E12 and E18. (B) Crown rump length between E12 and E18. (C) Biparietal diameter between E14 and E18. The line of best fit is included for each scatter plot. (D, top) Schematic diagram illustrating how we defined and collected CRL (blue doubleheaded arrow) BPD (green doubleheaded arrow) measurements. (D, bottom) Photo of an E15 embryo with corresponding BPD (line A) and CRL (line B) measurements. The data points for this pup are designated on the scatter plots by the blue and green stars labeled D in (B) and (C).
displacement and compression of the gut bag and abdominal cavity (Fig. 5, Movie 3). We observed that FBMs occurred in mouse pups that were still (not otherwise moving). In one case scanned at 10-minute intervals for 90 min within 6 h of birth, pups were observed to be
days before birth, in 2 of 6 E15 animals we scanned. Episodes of FBMs were reliably observed beginning on E16, 3 days before birth, in 8 of 8 animals scanned on E16. FBMs were distinguished from other movements based on the visible expansion of the ribs coupled with the rapid Table 1 C57BL6 Embryonic Mouse Biometrics by Age.
Average Heart Rate+/- SEM (bpm) Average Crown Rump Length+/- SEM (cm) Average Biparietal Diameter+/- SEM (cm)
E12
E13
E14
E15
E16
ad
E18
132.5 +/− 16.7 0.81 +/−0.06
152 +/−10.0 0.95 +/−0.13
132 +/− 6.0 1.37 +/−0.08
137.3 +/− 9.2 1.49 +/− 0.23
155.1 +/− 8.9 1.50 +/− 0.09
138 +/−18.1 1.75 +/− 0.18
171 +/−9.0 1.77 +/− 0.13
nd
nd
0.51 +/−0.010
0.55 +/− 0.018
0.60 +/− 0.017
0.59 +/− 0.009
0.64 +/− 0.012
3
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Fig. 2. Early features of embryonic C57BL6 mice visualized with ultrasound. (A–C) The beating heart could be readily detected by E10 and the ventricular septum and heart valves could be seen clearly at E10 as shown in this coronal view. The measured gestational sac diameter is shown as the line A between the crosses (A–E). (D–G) Pups became more distinct as gestation progressed as seen in a sagittal view of an E12 pup (D and E) and a frontal view of an E13 pup (F and G). The measured crown rump length of the E13 pup can be seen as the line A between the crosses (F and G). The bones are just becoming distinct at E13. Dashed white lines in (G) indicate the pup’s trunk (left) and skull (right). Solid white outlines mark the developing orbits (top and bottom, black fill) and nasal cavities (middle, no fill).
occurring in clustered episodes defined as two or more regularly spaced FBMs preceded or followed by an apneic period. We did not find a significant difference among the mean numbers of clustered breathing episodes observed at the three ages examined (Fig. 7, mean +/−SEM: E16 = 0.33 +/−0.33; E17 0.5 +/−0.27; E18 0.61 +/−0.22; ANOVA, F = .38; P = .688076), but their incidence did tend to increase with age (frequency at E16 = 0.17, at E17 = 0.38, at E18 = 0.50) (frequency = number of times clusters were present (present = 1, not present = 0)/total number of observations). In two E18 cases and one E17 case, lengthy strings of FBMs that could be classified as shallow and highly rhythmic (roughly 1 breath per 1–2 s) were observed (Movie 4). These rhythmic fetal breathing episodes appeared somewhat similar to the breathing we observed with ultrasound in postnatal day (P)0 pups (Movie 5), but the breaths during late gestation were less frequent and the rib expansion and gut compression were both more evident and appeared more pronounced.
squirming and rolling but no FBMs were observed. Fetal breathing movements in our mice could be classified as sporadic (separated by more time than one expected interbreath interval based on our rhythm as we counted), clustered (clusters of 2–15 regular breaths separated from the preceding or succeeding sporadic breaths by more than one expected interbreath interval), or, occasionally, rhythmic (regular and lasting 15 s or more with roughly 1 FBM per second; Fig. 6). Each type was observed at each age, although there was a trend for the frequency of clustered FBMs and rhythmic FBMs to increase with age (see below). We also observed that individual pups varied their type of breathing over the course of a recording session. For example, the sporadic breathing illustrated in Fig. 6 for an E16 pup was followed by clustered breathing 10 min later. On E18, one day before birth, there were significantly more FBMs per minute than on E16 (4.0 +/−0.58 at E18 (n = 6) vs 2.3 +/−1.3 at E16 (n = 3), p < 0.05), however the differences between E16 and E17 (2.5 +/−0.65, n = 4) and between E18 and E17 were not statistically significant (Fig. 7). As previously noted, we often observed FBMs 4
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Fig. 3. Features of embryonic C57BL6 mouse pups visualized and measured using ultrasound. (A) Three pups photographed at E14. (B) CRL measurements for pups A–C. (C) A pup from the same litter photographed 2 days later at E16, showing CRL and BPD measurements. (D) Anatomical features that can be seen in the E16 pup shown in (C) include the hind paws, abdominal cavity, stomach, liver, lung, heart, right forepaw, skull, right orbit, and snout. (E) Illustration of the orientation of the E16 pup shown in (C) and (D). Note that the head is tilted to the pup’s right and the pup’s right forelimb is resting next to his head.
4. Discussion
movements became visible the following day, and by E16 we also saw mouth and throat movements, including yawning and swallowing. The ages at which these behaviors were observed is clearly tied to the relative ease of seeing these movements as bones calcified but provides a reliable base timeline for their development as visualized by ultrasound. Fetal mouse motor behaviors in the C57BL6 strain, with the exception of fetal breathing, have been well described previously beginning at E16 (Kleven and Ronca, 2009). Our ultrasound observations complement and extend those observations and corroborate the conclusion that C57BL6 fetal mouse behavior is an excellent model for fetal human motor development. Our observations of FBMs suggest that ultrasound examination of pregnant mice will be a useful model for studying breathing development in the available genetic mouse models of human breathing related disorders. Even with our relatively inexpensive ultrasound equipment, we were able to visualize all of the relevant breathing related structures (diaphragm, ribs, and lungs) and the commencement of fetal breathing, as well visualize other anatomical features and activity, including the beating heart, that allowed us to collect relevant behavioral and biometric data. Furthermore, the types of episodic breathing – sporadic, clustered, and rhythmic – and the relative amounts that we observed over the last three days of embryonic mouse development are consistent with the types and amounts of fetal breathing observed in sheep, rats, and humans at equivalent developmental stages (Greer, 2012, for review). Mouse development occurs on a continuum over just 19.3 days (462.4 +/−1.0 h) in the C57BL6 strain (Murray et al., 2010) and it is
To our knowledge this is the first report of fetal breathing movements observed in uterovia ultrasound in unanesthetized mice. We reliably observed FBMs beginning at E16 in our C57BL6 mice, one day after the onset of rhythmic activity observed in in vitro studies of both the timing of the development of the respiratory circuitry and fetal breathing-like movements in reduced preparations (Viemari et al., 2003; Thoby-Brisson et al., 2005). This apparent delay is likely due to the additional time required for all of the necessary components to mature enough to work in concert to produce observable breathing in the whole animal. The earliest diaphragmatic activity we observed was at E15, in just two cases of six confirmed E15 litters. This activity looked very much like hiccups; the development of hiccups precedes the development of regular breathing in humans (Pillai and James, 1990). It has been hypothesized that hiccups exist in higher vertebrates because the vertebrate respiratory pattern generator is built upon the motor units that produce gill ventilation in lower vertebrates (Straus et al., 2003). The occurrence of hiccups prior to fetal breathing movements may reflect this phylogenetic relationship. It also is possible that due to the episodic nature of FBMs and the fact that animals increase the amount of time they spend breathing in utero as they approach birth (Dawes et al., 1970; Kobayashi et al., 1985; Greer, 2012 for review) that we missed FBMs in our E15 animals due to unfortunate timing. The development of several other motor behaviors preceded regular fetal breathing by three days. Overall pup movement was evident as early as E13, when we first observed rolling. Head movements and limb 5
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Fig. 4. Structures relevant to breathing that were visible via ultrasound in the chest cavity in the C57BL6 embryonic mouse pup. (A) The diaphragm, lung, heart, and ribs (as well as the skull) can be seen in this ultrasound photo of an E18 mouse. (B) The schematic diagram on the right shows the cross-sectional anatomy of the E18 mouse at the sagittal plane illustrated on the left and approximates the ultrasound plane in (A). V = heart ventricle, A = heart atrium.
to increase over the last several days of gestation, although this trend was not statistically significant in our study. A previous study that we used for guidance while conducting our study documented the fetal development of the CD-1 mouse strain using ultrasound (Brown et al., 2006) and demonstrated the usefulness of biometric data in staging mouse pregnancy. Our biometric data and observations of morphological development in the C57BL6 mouse showed similar age-related patterns to those that they observed, with a few exceptions. For example, we first observed the fetal heart beating on E10 and they were able to detect fetal heart activity on E10.5, an equivalent time because they designated the day a seminal plug was observed as E0.5. Likewise, they first observed the spine on E12.5 and we saw it a bit later, on E13. We observed rib ossification at E14, about one day earlier than they did (E15.5). Unlike Brown et al. (2006), we
important to note that we are comparing a relatively small number of animals based on discrete, whole day age groups, so the trends we see may be important and physiologically relevant even though they are not statistically significant. In other words, within a given age group there are bound to be younger, less developed and older, more developed animals. Although numbers of FBMs did not differ significantly between E17 pups and either E16 or E18 pups, the numbers of FBMs did differ significantly between the E16 and E18 animals. A lack of a statistically significant difference between E16 and E17 or E17 and E18 does not preclude a physiologically significant difference across the age range of E16-E18. The duration of single FBMs and tidal volume equivalent were not measured so we do not know if those parameters changed. As previously reported in sheep and rats (Dawes et al., 1970; Kobayashi et al., 1985), FBM frequency and rhythmicity in mice tended 6
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Fig. 5. Time lapse sequence of images captured at 1 s intervals during 5 s of a single recorded fetal breathing movement in an E18 C57BL6 mouse pup. (A) In these images the mouse is oriented facing the viewer, with the trunk on the left and head on the right. The unfilled blue ovals represent the boundaries of the abdominal cavity in each photo. Double-sided blue arrows indicate the distance between the fourth/fifth pair of visible ribs on each side. The breathing movement begins between 1 and 2 s. As the ribs expand and rise, the abdominal cavity is compressed along the anterior-posterior axis. (B) Illustration of relative changes in the size of the abdominal cavity (filled blue ovals) and distance between the ribs (blue double-sided arrows) during a single FBM. (B,Top) The oval from each panel in (A) has been filled and aligned tangentially to the X-axis and Y-axis on the underlying grid (arbitrary units) for comparison of relative abdominal cavity size across the 5 s time frame. (B, Bottom) The doublesided arrows from each panel have been aligned in the center of the underlying grid (arbitrary units) for comparison of the relative distance between the ribs during the breath. Note that prior to (0 s.) and just after (5 s.) the FBM, the ribs at rest are not clearly visible in the plane of the ultrasound, hence there are no double-sided arrows for these time points.
collection began once a particular pup was located, which typically took less than a minute, but occasionally took several minutes because we sought pups in the best orientation for observing the ribs and abdominal cavity. Future studies might benefit from a longer data collection period, as well as more frequent scans. Also, we scanned pregnant mice only during the day between 10 a.m. and 4 p.m., never at night when the mother and her pups might have been more active. In our study, FBMs were only observed in otherwise still pups. Fetuses that were rolling or squirming were never observed to also be breathing, in spite of the fact that we could see their ribs, lungs, and diaphragm moving in and out of the ultrasound plane. Studies in sheep and rats have shown that FBMs occur primarily during REM sleep (Dawes et al., 1970; Pagliardini et al., 2012). We were unable to determine sleep state in our fetal mice, but our observations of their overall activity, or lack there of, suggest that they were asleep when we observed fetal breathing. In one litter observed just hours before birth
found CRL a more useful measure than BPD for confirming gestational age. Notably, fetal breathing was not observed in their study, possibly because their mice were anesthetized with isoflurane. As noted in their study, it can be a challenge to determine which mouse body parts belong to an individual mouse pup because of crowding in the mother’s abdomen. We performed a few ultrasound scans in the CD-1 strain (data not shown) and note that it was easier to distinguish individual mice and their body parts in the relatively smaller litters in the C57BL6 strain compared with the larger litters in the CD-1 strain. Also of note, we did observe FBMs in unanesthetized CD-1 mice 4 days before their birth. The amount of time over which we collected data is a limitation of our approach. Several minutes (typically 3–7 min in our study) is a long time to hold a mouse still and collect ultrasound data but it is not a long time period for observation. In order to see if FBMs changed over the latter part of gestation, we counted FBMs observed over one minute and collected at 10 min intervals over 20–80 additional minutes. Data 7
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and Platt, 1979 [human]), and hypercapnia (Boddy et al., 1974 [sheep]; van Weering and Wladimiroff, 1982 [human]). Carbon dioxide in particular appears to have a crucial role in the control of fetal breathing (Darnall, 2010, for review). Our lab is especially interested in the prenatal development of the central chemoreflex and its underlying neural circuitry, so the effect of hypercapnia on FBMs in mice is an area we are currently studying using our ultrasound approach. Our preliminary data suggest that even as early as E16, the first day we reliably observe FBMs, mice quickly alter their fetal breathing in response to exposure of the mother to carbon dioxide. In summary, we have demonstrated that observation and quantification of FBMs via ultrasound in the C57BL6 mouse strain has great potential utility as a model for future studies of both the normal and abnormal development of fetal breathing and the factors that impact that development. Due to the availability of transgenic mouse models, these studies could help us better understand developmental disorders of breathing in humans.
Fig. 6. Types of fetal breathing movements observed in embryonic C57BL6 mice. We observed episodic sporadic, clustered, and rhythmic breathing in our mice. Each schematic drawing represents one minute of recording time in an individual animal. The sporadic sample is from an E16 pup, the clustered sample is from an E18 pup, and the rhythmic sample is from an E18 pup, but each type of breathing was observed at each age and individual pups varied their type of breathing over the length of the recording session. The counts for each were: (Top) three sporadic FBMs, (Middle) seven total FBMs with one episode of four clustered FBMs and one episode of two clustered FBMs, followed by one sporadic FBM, and (Bottom) nine slow, rhythmic FBMs.
Funding sources This work was supported by the Dickinson College Biology Department and Neuroscience Program through departmental studentfaculty research funds. This research did not receive any additional money from funding agencies in the public, commercial, or not-forprofit sectors. Acknowledgements The authors wish to thank Dr. Scott Boback, Ms. Madison Parks, Ms. Jessica Bell, Ms. Justine Hayward, and Ms. Alice Kuklina for their technical help in the early stages of this study. We also thank Ms. Katie Landis for animal care and Dr. Charles Zwemer for technical help and critical reading of the manuscript. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.resp.2019.103288. References Abadie, V., Champagnat, J., Fortin, G., 2000. Branchiomotor activities in mouse embryo. Neuroreport. 11 (January 1), 141–145. Boddy, K., Dawes, G.S., Fisher, R., Pinter, S., Robinson, J.S., 1974. Foetal respiratory movements, electrocortical and cardiovascular responses to hypoxaemia and hypercapnia in sheep. J. Physiol. 243 (December 3), 599–618. Brown, S.D., Zurakowski, D., Rodriguez, D.P., Dunning, P.S., Hurley, R.J., Taylor, G.A., 2006. Ultrasound diagnosis of mouse pregnancy and gestational staging. Comp. Med. 56 (August 4), 262–271. Darnall, R.A., 2010. The role of CO(2) and central chemoreception in the control of breathing in the fetus and the neonate. Respir. Physiol. Neurobiol. 173 (October 3), 201–212. https://doi.org/10.1016/j.resp.2010.04.009. Dawes, G.S., Fox, H.E., Leduc, B.M., Liggins, G.C., Richards, R.T., 1970. Respiratory movements and paradoxical sleep in the foetal lamb. J. Physiol. 210 (September 1), 47P–48P. Dawes, G.S., Fox, H.E., Leduc, B.M., Liggins, G.C., Richards, R.T., 1972. Respiratory movements and rapid eye movement sleep in the foetal lamb. J. Physiol. 220 (January 1), 119–143. Feldman, J.L., Kam, K., Janczewski, W.A., 2009. Practice makes perfect, even for breathing. Nat. Neurosci. 12 (August 8), 961–963. https://doi.org/10.1038/nn0809961. Greer, J.J., Funk, G.D., Ballanyi, K., 2006. Preparing for the first breath: prenatal maturation of respiratory neural control. J. Physiol. 570 (February Pt 3), 437–444. Greer, J.J., 2012. Control of breathing activity in the fetus and newborn. Compr. Physiol. 2 (July 3), 1873–1888. https://doi.org/10.1002/cphy.c110006.Greer, 2012. Herriges, M., Morrisey, E.E., 2014. Lung development: orchestrating the generation and regeneration of a complex organ. Development. 141 (February 3), 502–513. https:// doi.org/10.1242/dev.098186. Inanlou, M.R., Baguma-Nibasheka, M., Kablar, B., 2005. The role of fetal breathing-like movements in lung organogenesis. Histol. Histopathol. 20 (October 4), 1261–1266. https://doi.org/10.14670/HH-20.1261. Kleven, G.A., Ronca, A.E., 2009. Prenatal behavior of the C57BL/6J mouse: a promising model for human fetal movement during early to mid-gestation. Dev. Psychobiol. 51
Fig. 7. Average numbers of FBMs and episodes of clustered FBMs between E16 and E18 in C57BL6 mice. Numbers of FBMs and episodes of clustered FBMs observed per minute at 10 min intervals over 20–30 min were averaged by age. The average number of FBMs per minute differed significantly between E16 and E18 (indicated by the asterisk). The number of episodes of clustered FBMs did not differ among the age groups.
we did not observe any FBMs. It has been found previously that FBMs cease in preparation for parturition (see Greer, 2012 for review), so this feature in the mouse might add to its usefulness as a model for human disorders of breathing development. In addition to sleep state and the onset of labor, other factors have been observed to impact the incidence or frequency of fetal breathing in animal models as well as in humans. These factors, which may act independently or interact, include maternal glucose levels (Natale et al., 1978 [humans]; Richardson et al., 1982 [sheep]), body temperature (Walker, 1988 [sheep]), hypoxia (Boddy et al., 1974 [sheep]; Manning 8
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Sefton, E.M., Gallardo, M., Kardon, G., 2018. Developmental origin and morphogenesis of the diaphragm, an essential mammalian muscle. Dev. Biol. 440 (August 2), 64–73. https://doi.org/10.1016/j.ydbio.2018.04.010. Straus, C., Vasilakos, K., Wilson, R.J., Oshima, T., Zelter, M., Derenne, J.P., Similowski, T., Whitelaw, W.A., 2003. A phylogenetic hypothesis for the origin of hiccough. Bioessays 25 (February 2), 182–188. Thoby-Brisson, M., Trinh, J.B., Champagnat, J., Fortin, G., 2005. Emergence of the preBötzinger respiratory rhythm generator in the mouse embryo. J. Neurosci. 25 (April 17), 4307–4318. Thoby-Brisson, M., Karlén, M., Wu, N., Charnay, P., Champagnat, J., Fortin, G., 2009. Genetic identification of an embryonic parafacial oscillator coupling to the preBötzinger complex. Nat. Neurosci. 12 (August 8), 1028–1035. https://doi.org/10. 1038/nn.2354. van Weering, H.K., Wladimiroff, J.W., 1982. The effect of maternal hypercapnia and hyperoxia on breathing movements in the normal and growth-retarded fetus. Acta Obstet. Gynecol. Scand. 61 (1), 69–74. Viemari, J.C., Burnet, H., Bévengut, M., Hilaire, G., 2003. Perinatal maturation of the mouse respiratory rhythm-generator: in vivo and in vitro studies. Eur. J. Neurosci. 17 (March 6), 1233–1244. Walker, D.W., 1988. Effects of increased core temperature on breathing movements and electrocortical activity in fetal sheep. J. Dev. Physiol. 10 (December 6), 513–523.
(January 1), 84–94. https://doi.org/10.1002/dev.20348. Kobayashi, K., Lemke, R.P., Greer, J.J., 1985. Ultrasound measurements of fetal breathing movements in the rat. J. Appl. Physiol. 91 (July 1), 316–320 2001. Manning, F.A., Platt, L.D., 1979. Maternal hypoxemia and fetal breathing movements. Obstet. Gynecol. 53 (June 6), 758–760. Merlet, C., Hoerter, J., Devilleneuve, C., Tchobroutsky, C., 1970. [Demonstration of respiratory movements in lamb fetus in utero during the last month of gestation]. C R Acad Sci Hebd Seances Acad Sci D. 270 (May 20), 2462–2464. Murray, S.A., Morgan, J.L., Kane, C., Sharma, Y., Heffner, C.S., Lake, J., Donahue, L.R., 2010. Mouse gestation length is genetically determined. PLoS One 5 (August 8), e12418. https://doi.org/10.1371/journal.pone.0012418. Natale, R., Patrick, J., Richardson, B., 1978. Effects of human maternal venous plasma glucose concentrations on fetal breathing movements. Am. J. Obstet. Gynecol. 132 (September 1), 36–41. Pagliardini, S., Greer, J.J., Funk, G.D., Dickson, C.T., 2012. State-dependent modulation of breathing in urethane-anesthetized rats. J. Neurosci. 32 (August 33), 11259–11270. https://doi.org/10.1523/JNEUROSCI.0948-12.2012. Pillai, M., James, D., 1990. Hiccups and breathing in human fetuses. Arch. Dis. Child. 65 (October 10 Spec No), 1072–1075. Richardson, B., Hohimer, A.R., Mueggler, P., Bissonnette, J., 1982. Effects of glucose concentration on fetal breathing movements and electrocortical activity in fetal lambs. Am. J. Obstet. Gynecol. 142 (March 6 Pt 1), 678–683.
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Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
In utero development of fetal breathing movements in C57BL6 mice Mary M. Niblock Sydney Gilkeyb,c a b c
a,b,⁎
a
b
b,c
T
a
, Alanis Perez , Shahar Broitman , Brigitte Jacoby , Elana Aviv ,
Department of Biology, Dickinson College, Carlisle, PA, United States Neuroscience Program, Dickinson College, Carlisle, PA, United States Biochemistry and Molecular Biology Program, Dickinson College, Carlisle, PA, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: Fetal breathing movements Mice Respiratory development Ultrasound
Fetuses of many species, including humans, breathe during development. This fetal breathing aids in lung development, strengthens respiratory muscles, and is posited to fine-tune the neural circuitry that drives breathing. Previous studies suggested that fetal breathing could begin as early as the fifteenth day of gestation in the mouse, but fetal breathing movements (FBMs) had not been observed in mice in utero. We aimed to determine if and when FBMs commence in mice and if they change over time. We examined unanesthetised pregnant C57BL6 mice with ultrasound beginning on the seventh day of gestation. We first reliably observed episodic FBMs in mice on embryonic day 16. FBMs were sporadic, clustered, or rhythmic, and their frequency increased with age. Ultrasound examination of FBMs in mice has great potential utility in the study of transgenic mouse models to help us understand the prenatal characteristics of breathing related human developmental disorders, including Congenital Central Hypoventilation Syndrome (CCHS) and apnea of prematurity.
1. Introduction Even though fetal breathing movements are not necessary for gas exchange, they are important because they help strengthen the respiratory muscles and prepare the lungs for breathing (See Inanlou et al., 2005, for review). They also are hypothesized to fine-tune the neural circuitry that will allow the animal to breathe regularly and adaptively at birth (See Greer et al., 2006; Feldman et al., 2009; Greer, 2012, for review). Fetal breathing movements have been observed in humans, as well as in numerous experimental animal models, including sheep (Dawes et al., 1970; Merlet et al., 1970; Dawes et al., 1972) and rats (Kobayashi et al., 1985), but have not been observed in mice in utero. Mice are important models for human breathing disorders with neural developmental underpinnings, including Congenital Central Hypoventilation Syndrome (CCHS) and apnea of prematurity. Genetic mouse models are available that are directly related to these disorders but the prenatal onset and subsequent fetal development of breathing have not been studied successfully in mice in utero. Previous studies have documented fetal breathing-like activity in reduced preparations (Viemari et al., 2003; Thoby-Brisson et al., 2005). Some studies have looked for fetal breathing movements (FBMs) in mice using creative approaches such as plethysmography following premature cesarean
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delivery beginning at embryonic day 16 (E16, Viemari et al., 2003) and examination of externalized pups on the day before birth (Kleven and Ronca, 2009). In spite of these efforts, no one has observed FBMs in unanesthetized mice in utero. Lung development in the mouse begins on embryonic day 9 and lung buds are distinct structures by E12.5 (See Herriges and Morrisey, 2014, for review). Diaphragm development commences early on E11 with the migration of muscle progenitors and subsequent projection of the phrenic nerves towards the pleuroperitoneal folds (Sefton et al., 2018). The first phrenic nerve axons reach the primordial diaphragm late on E11 and by E13.5 the phrenic nerve has branched and innervated the developing diaphragm (Sefton et al., 2018). Studies of in vitro preparations have shown that the embryonic parafacial nucleus begins producing rhythmic activity at E14.5 in the mouse (ThobyBrisson et al., 2009). One day later, the presumptive respiratory pattern generator known as the Pre-Bötzinger Complex begins to oscillate in synchrony with the embryonic parafacial neurons, generating the first activity capable of driving fetal respiration in the mouse (Abadie et al., 2000; Thoby-Brisson et al., 2005, 2009). In vivo data from instrumented fetal lambs and ultrasound observations in rats suggest that FBMs mature over time, becoming more frequent and more rhythmic as animals approach birth (Dawes et al., 1970; Kobayashi et al., 1985), which presumably reflects the maturation and fine-tuning of the underlying
Corresponding author at: Department of Biology and Neuroscience Program, Dickinson College, P.O. Box 1773, Carlisle, PA, 17013, United States. E-mail address: [email protected] (M.M. Niblock).
https://doi.org/10.1016/j.resp.2019.103288 Received 9 July 2019; Received in revised form 6 August 2019; Accepted 5 September 2019 Available online 07 September 2019 1569-9048/ © 2019 Elsevier B.V. All rights reserved.
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were videotaped for later review and confirmation of FBM and heart rate counts. Fetal breathing movements were characterized by visible contractions of the diaphragm that displaced the gut bag and the ribcage. Data were collected from embryos in a frontal or side orientation only when body parts could be identified and confirmed to belong to that pup. Following ultrasound measurements and observations, pregnant mice were returned to the colony and checked twice daily for pup delivery. Some pregnant mice were scanned on successive days and in those cases an effort was made to locate the same embryo(s) scanned on the previous day(s). A limited number of pups were scanned on the day of birth in order to compare the ultrasound appearance of in utero breathing movements with the appearance of early postnatal breathing. Neonatal pups were placed lengthwise on top of the transducer on a thin layer of gel and video was captured with the ultrasound system. In total, the data presented here come from 91 different ultrasound sessions. Photos and videos collected using the SonoSite system were projected using a computer monitor and captured using a high resolution digital camera. Photo brightness and contrast were adjusted using Adobe Photoshop (Creative Suite 5). Figures were created using Adobe Illustrator (Creative Suite 5) and movies were created using iMovie.
neural circuitry in addition to progressive diaphragm and lung morphogenesis. We sought to observe FBMs in C57BL6 mice, a common background strain for transgenic models, and confirm the developmental timeline observed in vitro and predicted by the developmental timeline of the underlying neural circuitry and respiratory structures, in addition to looking for any potential evidence of age-related changes in fetal breathing. In our study, we used ultrasound to examine developing pups in awake, pregnant C57BL6 mice between E10 and E18; E19 is the day of birth (Murray et al., 2010). Our goals were (1) to observe if and when FBMs commence in mice and (2) to determine if there are age-related changes in FBMs in mice. In addition, we recorded biometrics and made corresponding qualitative observations of morphological and behavioral development in C57BL6 mice. We found that the onset of episodic fetal breathing movements in C57BL6 mice occurs at E16. We observed statistically nonsignificant trends of increasing numbers of FBMs and increasing frequency of clustered and rhythmic breathing over the three days prior to birth. 2. Methods 2.1. Animals
2.3. Data analysis These experiments were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Dickinson College Institutional Animal Care and Use Committee (Protocol #617). Adult C57BL6 mice were housed in ventilated caging in breeding pairs (one male, one female) or trios (one male, two females) and maintained on a 12:12 light:dark cycle with ad libitum access to food and water. After pairing with males, females were checked each morning for the presence of a vaginal plug. The day a plug was observed was designated embryonic day (E) 0. Females were also visually monitored for pregnancy- related weight gain. The data, observations, photos, and videos reported here were collected from scans of 58 litters, at the following ages: E9/10 (n = 5), E12 (n = 8), E13 (n = 5), E14 (n = 2), E15 (n = 6), E16 (n = 8), E17 (n = 13), and E18 (n = 11). In addition, two P0 pups were scanned for a comparison between the ultrasound appearance of fetal and early postnatal breathing.
Data were entered into an Excel spreadsheet. The average and standard error of the mean were calculated for FBMs at each age. For the biometric data, an online linear regression and correlation coefficient calculator were used (www.alcula.com/calculators/statistics/ linear-regression/). 3. Results 3.1. Biometrics and qualitative observations As we looked for FBMs, we collected age-appropriate biometric data, including heart rate when the beating heart was visible, crown rump length, and biparietal diameter when the orientation of the head was clear (Table 1, Fig. 1). We also recorded our observations of agerelated changes in pup behavior and the relative echogenicity of prominent anatomical features. These data and observations are presented first to provide a progressive picture of the changes we saw leading up to and subsequent to the onset of fetal breathing. Crown rump length and biparietal diameter were used to estimate and confirm gestational ages along with dated observations of seminal plugs. The beating heart could be readily detected by E10 (Movie 1) and the ventricular septum and atrial and ventricular valves could be seen clearly at this age (Fig. 2). Heart rate did not have a strong correlation with age (r = 0.33) and was highly variable within age, and within litter, in our limited sample (n = 21 total), but did tend to increase with age, as indicated by the slope of the line of best fit (Fig. 1; slope = 4.08). CRL and BPD both increased with age as expected (Fig. 1). CRL had a higher correlation coefficient (0.90) than BPD (0.60) so we found it more useful for estimating gestational age in cases for which no plug was observed. Bones first became visible via ultrasound on E13 (Fig. 2F and G) and became more echogenic, and therefore more distinct, as they calcified with increasing age (Fig. 3). Embryonic day 13 was the earliest age at which we could reliably distinguish the head from the trunk. Overall pup movement was evident as early as E13, increased with age, and included rolling, squirming, head movements, mouth and throat movements, and limb movements (Movie 2). The developing ribs, lungs, and diaphragm could be seen as early as E14 and by E18 these breathing related structures were easily identified (Fig. 4).
2.2. Ultrasound Ultrasound (SonoSite MicroMaxx portable unit with the L25e 136 MHz transducer) was used to confirm pregnancy around the estimated seventh day of pregnancy. Unanesthetized pregnant mice were held by the scruff in a supine position, rotated slightly to the side, with the tail held firmly by the experimenter’s ring and pinky finger. The ultrasound probe was coated with a thin layer of ultrasound gel. The presence of gestational sacs was considered evidence of pregnancy. Measurements (cm) of gestational sac diameter (GSD), biparietal diameter (BPD), and crown-rump length (CRL) were used to estimate and confirm gestational age, along with the day the vaginal plug was observed, if a plug was found (Fig. 1). Once pregnancy was confirmed by ultrasound, pregnant mice were returned to their housing until the ninth or tenth day of gestation. At that time, depilatory cream (Nair) was applied with a cotton swab to unanesthetised pregnant mice held by the scruff in a supine position for one minute to remove belly fur for better image resolution. Depilatory residue was removed with a micellar cleansing cloth. Following depilation, pregnant mice were scanned as described above to locate, collect biometric data for, and observe their pups. If a breathing pup was found, fetal breathing episodes and movements were manually recorded as they were observed at 10 min intervals by at least two trained observers for an additional 20–80 min, depending on the case. Observations and count of FBMs were made in the same pup(s) during each ultrasound session. Movies of selected recording sessions
3.2. Fetal breathing movement observations The earliest diaphragmatic activity we observed occurred on E15, 4 2
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Fig. 1. Scatter plots of heart rate, crown rump length (CRL), and biparietal diameter (BPD) in embryonic C57BL6 mice. (A) Heart rate between E12 and E18. (B) Crown rump length between E12 and E18. (C) Biparietal diameter between E14 and E18. The line of best fit is included for each scatter plot. (D, top) Schematic diagram illustrating how we defined and collected CRL (blue doubleheaded arrow) BPD (green doubleheaded arrow) measurements. (D, bottom) Photo of an E15 embryo with corresponding BPD (line A) and CRL (line B) measurements. The data points for this pup are designated on the scatter plots by the blue and green stars labeled D in (B) and (C).
displacement and compression of the gut bag and abdominal cavity (Fig. 5, Movie 3). We observed that FBMs occurred in mouse pups that were still (not otherwise moving). In one case scanned at 10-minute intervals for 90 min within 6 h of birth, pups were observed to be
days before birth, in 2 of 6 E15 animals we scanned. Episodes of FBMs were reliably observed beginning on E16, 3 days before birth, in 8 of 8 animals scanned on E16. FBMs were distinguished from other movements based on the visible expansion of the ribs coupled with the rapid Table 1 C57BL6 Embryonic Mouse Biometrics by Age.
Average Heart Rate+/- SEM (bpm) Average Crown Rump Length+/- SEM (cm) Average Biparietal Diameter+/- SEM (cm)
E12
E13
E14
E15
E16
ad
E18
132.5 +/− 16.7 0.81 +/−0.06
152 +/−10.0 0.95 +/−0.13
132 +/− 6.0 1.37 +/−0.08
137.3 +/− 9.2 1.49 +/− 0.23
155.1 +/− 8.9 1.50 +/− 0.09
138 +/−18.1 1.75 +/− 0.18
171 +/−9.0 1.77 +/− 0.13
nd
nd
0.51 +/−0.010
0.55 +/− 0.018
0.60 +/− 0.017
0.59 +/− 0.009
0.64 +/− 0.012
3
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Fig. 2. Early features of embryonic C57BL6 mice visualized with ultrasound. (A–C) The beating heart could be readily detected by E10 and the ventricular septum and heart valves could be seen clearly at E10 as shown in this coronal view. The measured gestational sac diameter is shown as the line A between the crosses (A–E). (D–G) Pups became more distinct as gestation progressed as seen in a sagittal view of an E12 pup (D and E) and a frontal view of an E13 pup (F and G). The measured crown rump length of the E13 pup can be seen as the line A between the crosses (F and G). The bones are just becoming distinct at E13. Dashed white lines in (G) indicate the pup’s trunk (left) and skull (right). Solid white outlines mark the developing orbits (top and bottom, black fill) and nasal cavities (middle, no fill).
occurring in clustered episodes defined as two or more regularly spaced FBMs preceded or followed by an apneic period. We did not find a significant difference among the mean numbers of clustered breathing episodes observed at the three ages examined (Fig. 7, mean +/−SEM: E16 = 0.33 +/−0.33; E17 0.5 +/−0.27; E18 0.61 +/−0.22; ANOVA, F = .38; P = .688076), but their incidence did tend to increase with age (frequency at E16 = 0.17, at E17 = 0.38, at E18 = 0.50) (frequency = number of times clusters were present (present = 1, not present = 0)/total number of observations). In two E18 cases and one E17 case, lengthy strings of FBMs that could be classified as shallow and highly rhythmic (roughly 1 breath per 1–2 s) were observed (Movie 4). These rhythmic fetal breathing episodes appeared somewhat similar to the breathing we observed with ultrasound in postnatal day (P)0 pups (Movie 5), but the breaths during late gestation were less frequent and the rib expansion and gut compression were both more evident and appeared more pronounced.
squirming and rolling but no FBMs were observed. Fetal breathing movements in our mice could be classified as sporadic (separated by more time than one expected interbreath interval based on our rhythm as we counted), clustered (clusters of 2–15 regular breaths separated from the preceding or succeeding sporadic breaths by more than one expected interbreath interval), or, occasionally, rhythmic (regular and lasting 15 s or more with roughly 1 FBM per second; Fig. 6). Each type was observed at each age, although there was a trend for the frequency of clustered FBMs and rhythmic FBMs to increase with age (see below). We also observed that individual pups varied their type of breathing over the course of a recording session. For example, the sporadic breathing illustrated in Fig. 6 for an E16 pup was followed by clustered breathing 10 min later. On E18, one day before birth, there were significantly more FBMs per minute than on E16 (4.0 +/−0.58 at E18 (n = 6) vs 2.3 +/−1.3 at E16 (n = 3), p < 0.05), however the differences between E16 and E17 (2.5 +/−0.65, n = 4) and between E18 and E17 were not statistically significant (Fig. 7). As previously noted, we often observed FBMs 4
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Fig. 3. Features of embryonic C57BL6 mouse pups visualized and measured using ultrasound. (A) Three pups photographed at E14. (B) CRL measurements for pups A–C. (C) A pup from the same litter photographed 2 days later at E16, showing CRL and BPD measurements. (D) Anatomical features that can be seen in the E16 pup shown in (C) include the hind paws, abdominal cavity, stomach, liver, lung, heart, right forepaw, skull, right orbit, and snout. (E) Illustration of the orientation of the E16 pup shown in (C) and (D). Note that the head is tilted to the pup’s right and the pup’s right forelimb is resting next to his head.
4. Discussion
movements became visible the following day, and by E16 we also saw mouth and throat movements, including yawning and swallowing. The ages at which these behaviors were observed is clearly tied to the relative ease of seeing these movements as bones calcified but provides a reliable base timeline for their development as visualized by ultrasound. Fetal mouse motor behaviors in the C57BL6 strain, with the exception of fetal breathing, have been well described previously beginning at E16 (Kleven and Ronca, 2009). Our ultrasound observations complement and extend those observations and corroborate the conclusion that C57BL6 fetal mouse behavior is an excellent model for fetal human motor development. Our observations of FBMs suggest that ultrasound examination of pregnant mice will be a useful model for studying breathing development in the available genetic mouse models of human breathing related disorders. Even with our relatively inexpensive ultrasound equipment, we were able to visualize all of the relevant breathing related structures (diaphragm, ribs, and lungs) and the commencement of fetal breathing, as well visualize other anatomical features and activity, including the beating heart, that allowed us to collect relevant behavioral and biometric data. Furthermore, the types of episodic breathing – sporadic, clustered, and rhythmic – and the relative amounts that we observed over the last three days of embryonic mouse development are consistent with the types and amounts of fetal breathing observed in sheep, rats, and humans at equivalent developmental stages (Greer, 2012, for review). Mouse development occurs on a continuum over just 19.3 days (462.4 +/−1.0 h) in the C57BL6 strain (Murray et al., 2010) and it is
To our knowledge this is the first report of fetal breathing movements observed in uterovia ultrasound in unanesthetized mice. We reliably observed FBMs beginning at E16 in our C57BL6 mice, one day after the onset of rhythmic activity observed in in vitro studies of both the timing of the development of the respiratory circuitry and fetal breathing-like movements in reduced preparations (Viemari et al., 2003; Thoby-Brisson et al., 2005). This apparent delay is likely due to the additional time required for all of the necessary components to mature enough to work in concert to produce observable breathing in the whole animal. The earliest diaphragmatic activity we observed was at E15, in just two cases of six confirmed E15 litters. This activity looked very much like hiccups; the development of hiccups precedes the development of regular breathing in humans (Pillai and James, 1990). It has been hypothesized that hiccups exist in higher vertebrates because the vertebrate respiratory pattern generator is built upon the motor units that produce gill ventilation in lower vertebrates (Straus et al., 2003). The occurrence of hiccups prior to fetal breathing movements may reflect this phylogenetic relationship. It also is possible that due to the episodic nature of FBMs and the fact that animals increase the amount of time they spend breathing in utero as they approach birth (Dawes et al., 1970; Kobayashi et al., 1985; Greer, 2012 for review) that we missed FBMs in our E15 animals due to unfortunate timing. The development of several other motor behaviors preceded regular fetal breathing by three days. Overall pup movement was evident as early as E13, when we first observed rolling. Head movements and limb 5
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Fig. 4. Structures relevant to breathing that were visible via ultrasound in the chest cavity in the C57BL6 embryonic mouse pup. (A) The diaphragm, lung, heart, and ribs (as well as the skull) can be seen in this ultrasound photo of an E18 mouse. (B) The schematic diagram on the right shows the cross-sectional anatomy of the E18 mouse at the sagittal plane illustrated on the left and approximates the ultrasound plane in (A). V = heart ventricle, A = heart atrium.
to increase over the last several days of gestation, although this trend was not statistically significant in our study. A previous study that we used for guidance while conducting our study documented the fetal development of the CD-1 mouse strain using ultrasound (Brown et al., 2006) and demonstrated the usefulness of biometric data in staging mouse pregnancy. Our biometric data and observations of morphological development in the C57BL6 mouse showed similar age-related patterns to those that they observed, with a few exceptions. For example, we first observed the fetal heart beating on E10 and they were able to detect fetal heart activity on E10.5, an equivalent time because they designated the day a seminal plug was observed as E0.5. Likewise, they first observed the spine on E12.5 and we saw it a bit later, on E13. We observed rib ossification at E14, about one day earlier than they did (E15.5). Unlike Brown et al. (2006), we
important to note that we are comparing a relatively small number of animals based on discrete, whole day age groups, so the trends we see may be important and physiologically relevant even though they are not statistically significant. In other words, within a given age group there are bound to be younger, less developed and older, more developed animals. Although numbers of FBMs did not differ significantly between E17 pups and either E16 or E18 pups, the numbers of FBMs did differ significantly between the E16 and E18 animals. A lack of a statistically significant difference between E16 and E17 or E17 and E18 does not preclude a physiologically significant difference across the age range of E16-E18. The duration of single FBMs and tidal volume equivalent were not measured so we do not know if those parameters changed. As previously reported in sheep and rats (Dawes et al., 1970; Kobayashi et al., 1985), FBM frequency and rhythmicity in mice tended 6
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Fig. 5. Time lapse sequence of images captured at 1 s intervals during 5 s of a single recorded fetal breathing movement in an E18 C57BL6 mouse pup. (A) In these images the mouse is oriented facing the viewer, with the trunk on the left and head on the right. The unfilled blue ovals represent the boundaries of the abdominal cavity in each photo. Double-sided blue arrows indicate the distance between the fourth/fifth pair of visible ribs on each side. The breathing movement begins between 1 and 2 s. As the ribs expand and rise, the abdominal cavity is compressed along the anterior-posterior axis. (B) Illustration of relative changes in the size of the abdominal cavity (filled blue ovals) and distance between the ribs (blue double-sided arrows) during a single FBM. (B,Top) The oval from each panel in (A) has been filled and aligned tangentially to the X-axis and Y-axis on the underlying grid (arbitrary units) for comparison of relative abdominal cavity size across the 5 s time frame. (B, Bottom) The doublesided arrows from each panel have been aligned in the center of the underlying grid (arbitrary units) for comparison of the relative distance between the ribs during the breath. Note that prior to (0 s.) and just after (5 s.) the FBM, the ribs at rest are not clearly visible in the plane of the ultrasound, hence there are no double-sided arrows for these time points.
collection began once a particular pup was located, which typically took less than a minute, but occasionally took several minutes because we sought pups in the best orientation for observing the ribs and abdominal cavity. Future studies might benefit from a longer data collection period, as well as more frequent scans. Also, we scanned pregnant mice only during the day between 10 a.m. and 4 p.m., never at night when the mother and her pups might have been more active. In our study, FBMs were only observed in otherwise still pups. Fetuses that were rolling or squirming were never observed to also be breathing, in spite of the fact that we could see their ribs, lungs, and diaphragm moving in and out of the ultrasound plane. Studies in sheep and rats have shown that FBMs occur primarily during REM sleep (Dawes et al., 1970; Pagliardini et al., 2012). We were unable to determine sleep state in our fetal mice, but our observations of their overall activity, or lack there of, suggest that they were asleep when we observed fetal breathing. In one litter observed just hours before birth
found CRL a more useful measure than BPD for confirming gestational age. Notably, fetal breathing was not observed in their study, possibly because their mice were anesthetized with isoflurane. As noted in their study, it can be a challenge to determine which mouse body parts belong to an individual mouse pup because of crowding in the mother’s abdomen. We performed a few ultrasound scans in the CD-1 strain (data not shown) and note that it was easier to distinguish individual mice and their body parts in the relatively smaller litters in the C57BL6 strain compared with the larger litters in the CD-1 strain. Also of note, we did observe FBMs in unanesthetized CD-1 mice 4 days before their birth. The amount of time over which we collected data is a limitation of our approach. Several minutes (typically 3–7 min in our study) is a long time to hold a mouse still and collect ultrasound data but it is not a long time period for observation. In order to see if FBMs changed over the latter part of gestation, we counted FBMs observed over one minute and collected at 10 min intervals over 20–80 additional minutes. Data 7
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and Platt, 1979 [human]), and hypercapnia (Boddy et al., 1974 [sheep]; van Weering and Wladimiroff, 1982 [human]). Carbon dioxide in particular appears to have a crucial role in the control of fetal breathing (Darnall, 2010, for review). Our lab is especially interested in the prenatal development of the central chemoreflex and its underlying neural circuitry, so the effect of hypercapnia on FBMs in mice is an area we are currently studying using our ultrasound approach. Our preliminary data suggest that even as early as E16, the first day we reliably observe FBMs, mice quickly alter their fetal breathing in response to exposure of the mother to carbon dioxide. In summary, we have demonstrated that observation and quantification of FBMs via ultrasound in the C57BL6 mouse strain has great potential utility as a model for future studies of both the normal and abnormal development of fetal breathing and the factors that impact that development. Due to the availability of transgenic mouse models, these studies could help us better understand developmental disorders of breathing in humans.
Fig. 6. Types of fetal breathing movements observed in embryonic C57BL6 mice. We observed episodic sporadic, clustered, and rhythmic breathing in our mice. Each schematic drawing represents one minute of recording time in an individual animal. The sporadic sample is from an E16 pup, the clustered sample is from an E18 pup, and the rhythmic sample is from an E18 pup, but each type of breathing was observed at each age and individual pups varied their type of breathing over the length of the recording session. The counts for each were: (Top) three sporadic FBMs, (Middle) seven total FBMs with one episode of four clustered FBMs and one episode of two clustered FBMs, followed by one sporadic FBM, and (Bottom) nine slow, rhythmic FBMs.
Funding sources This work was supported by the Dickinson College Biology Department and Neuroscience Program through departmental studentfaculty research funds. This research did not receive any additional money from funding agencies in the public, commercial, or not-forprofit sectors. Acknowledgements The authors wish to thank Dr. Scott Boback, Ms. Madison Parks, Ms. Jessica Bell, Ms. Justine Hayward, and Ms. Alice Kuklina for their technical help in the early stages of this study. We also thank Ms. Katie Landis for animal care and Dr. Charles Zwemer for technical help and critical reading of the manuscript. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.resp.2019.103288. References Abadie, V., Champagnat, J., Fortin, G., 2000. Branchiomotor activities in mouse embryo. Neuroreport. 11 (January 1), 141–145. Boddy, K., Dawes, G.S., Fisher, R., Pinter, S., Robinson, J.S., 1974. Foetal respiratory movements, electrocortical and cardiovascular responses to hypoxaemia and hypercapnia in sheep. J. Physiol. 243 (December 3), 599–618. Brown, S.D., Zurakowski, D., Rodriguez, D.P., Dunning, P.S., Hurley, R.J., Taylor, G.A., 2006. Ultrasound diagnosis of mouse pregnancy and gestational staging. Comp. Med. 56 (August 4), 262–271. Darnall, R.A., 2010. The role of CO(2) and central chemoreception in the control of breathing in the fetus and the neonate. Respir. Physiol. Neurobiol. 173 (October 3), 201–212. https://doi.org/10.1016/j.resp.2010.04.009. Dawes, G.S., Fox, H.E., Leduc, B.M., Liggins, G.C., Richards, R.T., 1970. Respiratory movements and paradoxical sleep in the foetal lamb. J. Physiol. 210 (September 1), 47P–48P. Dawes, G.S., Fox, H.E., Leduc, B.M., Liggins, G.C., Richards, R.T., 1972. Respiratory movements and rapid eye movement sleep in the foetal lamb. J. Physiol. 220 (January 1), 119–143. Feldman, J.L., Kam, K., Janczewski, W.A., 2009. Practice makes perfect, even for breathing. Nat. Neurosci. 12 (August 8), 961–963. https://doi.org/10.1038/nn0809961. Greer, J.J., Funk, G.D., Ballanyi, K., 2006. Preparing for the first breath: prenatal maturation of respiratory neural control. J. Physiol. 570 (February Pt 3), 437–444. Greer, J.J., 2012. Control of breathing activity in the fetus and newborn. Compr. Physiol. 2 (July 3), 1873–1888. https://doi.org/10.1002/cphy.c110006.Greer, 2012. Herriges, M., Morrisey, E.E., 2014. Lung development: orchestrating the generation and regeneration of a complex organ. Development. 141 (February 3), 502–513. https:// doi.org/10.1242/dev.098186. Inanlou, M.R., Baguma-Nibasheka, M., Kablar, B., 2005. The role of fetal breathing-like movements in lung organogenesis. Histol. Histopathol. 20 (October 4), 1261–1266. https://doi.org/10.14670/HH-20.1261. Kleven, G.A., Ronca, A.E., 2009. Prenatal behavior of the C57BL/6J mouse: a promising model for human fetal movement during early to mid-gestation. Dev. Psychobiol. 51
Fig. 7. Average numbers of FBMs and episodes of clustered FBMs between E16 and E18 in C57BL6 mice. Numbers of FBMs and episodes of clustered FBMs observed per minute at 10 min intervals over 20–30 min were averaged by age. The average number of FBMs per minute differed significantly between E16 and E18 (indicated by the asterisk). The number of episodes of clustered FBMs did not differ among the age groups.
we did not observe any FBMs. It has been found previously that FBMs cease in preparation for parturition (see Greer, 2012 for review), so this feature in the mouse might add to its usefulness as a model for human disorders of breathing development. In addition to sleep state and the onset of labor, other factors have been observed to impact the incidence or frequency of fetal breathing in animal models as well as in humans. These factors, which may act independently or interact, include maternal glucose levels (Natale et al., 1978 [humans]; Richardson et al., 1982 [sheep]), body temperature (Walker, 1988 [sheep]), hypoxia (Boddy et al., 1974 [sheep]; Manning 8
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