RESEARCH REPORT

Single-cell murine genetic fate mapping reveals bipotentialhepatoblasts and novel multi-organ endoderm progenitorsGabriel K. El Sebae, Joseph M. Malatos, Mary-Kate E. Cone, Siyeon Rhee, Jesse R. Angelo, Jesse Mager andKimberly D. Tremblay*

ABSTRACTThe definitive endoderm (DE) is the embryonic germ layer that formsthe gut tube and associated organs, including thymus, lungs, liverand pancreas. To understand how individual DE cells furnish gutorgans, genetic fate mapping was performed using the Rosa26lacZ

Cre-reporter paired with a tamoxifen-inducible DE-specificCre-expressing transgene. We established a low tamoxifen dosethat infrequently induced heritable lacZ expression in a single cellof individual E8.5 mouse embryos and identified clonal celldescendants at E16.5. As expected, only a fraction of the E16.5embryos contained lacZ-positive clonal descendants and a subset ofthese contained descendants in multiple organs, revealing novelontogeny. Furthermore, immunohistochemical analysis was used toidentify lacZ-positive hepatocytes and biliary epithelial cells, whichare the cholangiocyte precursors, in each clonally populated liver.Together, these data not only uncover novel and suspected lineagerelationships between DE-derived organs, but also illustrate thebipotential nature of individual hepatoblasts by demonstrating thatsingle hepatoblasts contribute to both the hepatocyte and thecholangiocyte lineage in vivo.

KEY WORDS: Lineage tracing, Liver, Definitive endoderm,Hepatoblast, Mouse

INTRODUCTIONThe definitive endoderm (DE), the embryonic germ layer thatproduces the gut tube as well as the parenchyma of the liver,pancreas, lung, thymus and thyroid, arises by embryonic day (E) 7.5as an epithelial sheet on the ventral surface of the developing mousethat is broadly patterned by overlapping transcription factors. Asdevelopment proceeds, the transcription factor network is furtherrefined and by E9.5 distinct and non-overlapping transcriptionalboundaries are apparent throughout the gut tube, representingadjacent organ progenitor populations (Sherwood et al., 2009).Murine fate mapping has demonstrated that hepatoblasts, theembryonic hepatic progenitors, arise from discrete non-contiguouspopulations of E8.25 DE (Tremblay and Zaret, 2005). Hepatoblastscan be distinguished from the remainder of the endoderm bythe expression of liver-specific genes such as α-fetoprotein andalbumin, starting at the 7-8 somite stage (7-8S; ∼E8.5) (Deutschet al., 2001; Gualdi et al., 1996; Serls et al., 2005). By E9.0 thehepatoblasts are contained in a recognizable liver bud from which

they delaminate and invade adjacent mesenchyme, initiating aperiod of rapid hepatic outgrowth and proliferation (Bort et al.,2006; Sosa-Pineda et al., 2000).

The liver comprises two functional cell types: hepatocytes andcholangiocytes. Hepatocytes interact with blood and produce bile,which is then shuttled through a biliary network composed ofcholangiocytes. Defects in either of these cell types can haveenormous clinical consequences and can result in liver fibrosis,cirrhosis, biliary atresia, cholangiocarcinoma, hepatitis and Alagillesyndrome (Friedman, 2003; Rowe, 2017). Furthermore, the liversustains injury and repairs itself through an unparalleled regenerativeplasticity. A complete understanding of such capacity must includea developmental understanding of the hepatoblast population.

Although it is clear that hepatoblasts are the embryonicprogenitors of adult cholangiocytes and hepatocytes, and thatindividual progenitors can initiate both differentiated cell typesin vitro (Suzuki et al., 2002), lineage tracing of individualhepatoblasts in vivo has not yet been performed and thebipotential nature of individual hepatoblasts has been indirectlyassumed despite some contradictory evidence (Koga, 1971; Shiojiriet al., 2001). To investigate the potency of individual DE cells, weused a genetic labeling strategy to perform retrospective fatemapping of single E8.5 DE cells and analyzed descendants at E16.5,when the gut tube has formed and differentiated tissues and celltypes can be discerned. By analyzing the ontogeny of single E8.5DE progenitors, we uncovered shared organ progenitors, linking acell in the E8.5 endoderm to multiple endoderm-derived organs.Furthermore, we used this in vivo system to demonstrate thatindividual hepatoblasts contribute to both the hepatocyte and biliaryepithelial cell (BEC) lineages of the E16.5 liver. Together, thesedata provide important insights into endoderm organogenesis thatcould link pathologies to defects during normal developmentalprocesses and could guide in vitro differentiation strategies aimed atthe production of endoderm-derived cell types.

RESULTS AND DISCUSSIONEstablishing a protocol to produce a single geneticallylabeled cell per embryoDespite in vitro evidence supporting the bi-potency of individualhepatoblasts, in vivo experiments are conspicuously absent.Furthermore, the existence of at least two spatially distinct liverbud populations with differential requirements for BMP and FGFsuggest distinct hepatoblast subpopulations (Palaria et al., 2018;Wang et al., 2015). To probe this putative heterogeneity and toassess the potential of individual hepatoblasts we used an in vivofate mapping strategy that pairs a tissue-specific TMX-inducibleCre-transgene and a lacZ reporter.

The Foxa2mcm allele, which is produced by insertion of a TMX-inducible Cre-recombinase into the 3′ UTR of the Foxa2 locus,recapitulates endogenous Foxa2 expression in the DE, notochordReceived 10 June 2018; Accepted 6 September 2018

Department of Veterinary and Animal Sciences, University of Massachusetts,Amherst, MA 01003, USA

*Author for correspondence (kdtrembl@umass.edu)

J.M., 0000-0002-4123-0633; K.D.T., 0000-0002-3059-9910

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and floorplate of the neural tube (Park et al., 2008). When present inembryos also harboring the ubiquitously expressed Rosa26lacz

Cre-reporter (Soriano, 1999), TMX administration activateslacZ expression specifically in Foxa2-expressing cells. lacZrecombination produces a chromogenically identifiable andgenetically heritable cellular mark. Because the level of Cre-mediated recombination can be altered by dose, we hypothesizedthat we could identify a TMX concentration that reliably inducesrecombination in a single cell (Hayashi and McMahon, 2002; Parket al., 2008).To assess the TMX dose response, Rosa26lacZ/lacZ females were

mated with Foxa2mcm/mcm males, producing embryos heterozygousfor both alleles. Pregnant dams received a single dose of TMX onE7.75 and embryos were X-gal stained on E8.5. A 50 mg kg−1

TMX dose, half the typical maximal dose, produced numerouslabeled cells in all embryos (Fig. S1A). Decreasing the dose to6 mg kg−1 resulted in notably fewer labeled cells [3±3 (mean±s.d.);Fig. S1B] while only 50% (n=16/32) of embryos underwentrecombination. At 4 mg kg−1 TMX, 48% (n=10/21) of embryosdemonstrated one or two labeled cells (±1; Fig. S1C). At 3 mg kg−1

TMX, 13.9% (n=11/79) of embryos were labeled and only a singlelacZ-positive cell was present in each of those embryos (Fig. S1D).A final reduction to 2 mg kg−1 TMX did not produce any labeledembryos (n=0/30; Fig. S1E) nor did control animals receiving onlypeanut oil (n=0/49; data not shown). Statistical analysis of these dataindicate that, at 6 mg kg−1 and below, there is a linear relationshipbetween dose and the number of labeled cells (Fig. S1F; ANOVA***P=2.23e−9). Furthermore, although the ventral neural tube islabeled at high TMX doses (Fig. S1G), section analysis of a subsetof embryos provided with low TMX doses (n=4 at 4 mg kg−1 andn=9 at 3 mg kg−1) reveals that most labeled cells are confined to theDE layer, whereas a minority (1/15) are found in the notochord(Fig. S1H). These experiments demonstrate that, at 3 mg kg−1

TMX, if recombination occurs it is confined to a single cell perembryo, indicating the utility of this system for lineage tracing.When establishing this retrospective lineage analysis, it was

essential to confirm that Cre-mediated recombination did not occuroutside of the expected parameters (Liu et al., 2010; Vooijs et al.,2001). We considered the possibility that recombination could beinduced beyond E8.5 by lingering TMX. However the half-life ofTMX in mouse is less than 12 h (Robinson et al., 1991), and thus in12 h a single 3 mg kg−1 dose would decay to below 2 mg kg−1, adose at which we have seen no recombination.

Assessing the potency of individual E8.5 DE cells at E16.5With our single-cell labeling strategy defined, pregnant dams wereadministered 3 mg kg−1 TM on E7.75 and embryos collected atE16.5. The entire gut tube and visceral organs, including kidney andheart, were dissected from each embryo and then subject to X-galstaining to identify any labeled descendants in all gut derivatives.Because the vertebral areas were discarded, we would not expect todetect the few embryos harboring labeled notochord descendants,which are confined to the intervertebral disks at E16.5 (McCannet al., 2012). Importantly, all viscera containing lacZ-expressingclonal descendants were confined to DE-derived organs andwere never found in attached mesoderm-derived tissues, furthervalidating the specificity of the experimental system. Finally, thelow frequency of recombination at E16.5 (11.8%; 57/472) ensures alow risk of independently labeling two cells in the same embryo(1.4%=0.118×0.118).The liver is one of the first foregut organs specified (at 7-8S,

E8.5), with the ventral and dorsal pancreas being specified shortly

thereafter (Deutsch et al., 2001; Gannon and Wright, 1999; Gualdiet al., 1996). Thus, in accordancewith our labeling strategy, the liverwas the only foregut organ that produced descendants that weremainly confined to a single organ (n=22/25 or 88% of embryos withlabeled livers were restricted to the liver). Of the embryos withclonal descendants in other foregut organs, 9/20 (45%) wereconfined to a single organ and seven of those were restricted to thepancreas (Table 1, Fig. S2).

The presence of clonal cell descendants in multiple organs ofindividual embryos reveals the multipotentcy of the originallylabeled E8.5 DE cell. Interestingly, of those embryos with clonaldescendants in multiple organs, several patterns were apparent.These patterns highlight known or suspected ontogeny and alsorevealed novel progenitor relationships. For example, we previouslyused prospective DiI fate-mapping to demonstrate that at E8.25 asubset of hepatic progenitors contribute to the ventral pancreasbud (Angelo et al., 2012; Tremblay and Zaret, 2005). Indeed, ofthe limited number of embryos with multi-organ contribution thathad descendants in liver, all three contributed to the pancreas(Table 1, Fig. 1C). Similarly, although only a single embryoproduced labeled descendants in both the pancreas and in thegallbladder (Table 1), it too supports the previously described

Table 1. Distribution of E16.5 extrahepatic clones

Embryo # TR* TH LNG ST L GB PH PTR PT DUO INT

41.2 W‡ W122.1 W R W122.2 R44.1 W W39.1 R109.1 R R R118.1 W W W W71.1§ R W W41.1§ W R46.1§ W R120.2 W W120.3 W W79.1 W W61.1 W W W121.5 R R121.4 W W121.2 W81.2 W121.1 R R R81.1 R W108.1 W W42.1 R48.1 W48.2 W64.1 W64.2 W75.1 W108.2 W109.2 W109.3 W110.1 W112.1 R118.2 W120.1 W121.3 R

All organs were collected and examined in each embryo. *TR, trachea; TH,thymus; LNG, lung; ST, stomach; L, liver; GB, gall bladder; PH, pancreas head;PTR, pancreas trunk; PT, pancreas tail; DUO, duodenum; INT, intestine.‡W, widespread clonal distribution; R, restricted clonal distribution. A blankspace means that no clonal progeny were detected.§Embryos also included in Table 2.

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common ventral pancreas/gall bladder progenitor (Spence et al.,2009; Uemura et al., 2015).Unlike other endoderm-derived organs, the pancreas is produced

by the fusion of discrete and non-contiguous dorsal and ventralpancreas buds, which contribute by E11.5 to the pancreas tail andhead, respectively (Spooner et al., 1970; Wessels and Cohen, 1967).Therefore, it is not surprising that, of the embryos with labeleddescendants in the pancreas and other organs, the contribution to thepancreas reflects a dorsal or ventral origin. For example, of the threeembryos with clonal descendants that spanned the pancreas andintestine (n=3; Fig. 1D, Fig. S1), all pancreatic descendants wereconfined to the trunk and tail of the pancreas, consistent with adorsal pancreas origin. In other embryos, clonal descendants were

observed in the ventral pancreas-derived pancreatic head, as well asin the duodenum and stomach (n=2; Fig. 1E).

Together, these data provide new insights into pancreatic, gastricand intestinal development, and point to the existence of a commondorsal pancreas/intestine progenitor as well as a common stomach/ventral pancreas/duodenum progenitor. This idea is also supportedby the existence of multiple, yet transcriptionally distinct, endodermdomains in the posterior foregut endoderm at E9.5 (Sherwood et al.,2009), and serves to highlight the dynamic nature of the developingposterior foregut at E8.5 (McCracken and Wells, 2017).Furthermore, when the pancreas data are examined in total, theylend credence to the hypothesis that the dorsal and ventral pancreaseach contribute to the pancreatic trunk (Table 1), a process that has

Fig. 1. Lineage tracing reveals individual multi-organ progenitors in the E8.5 DE. Three mg kg−1 TMX was maternally administered at E7.75 to Foxa2mcm/+;R26R−/+ embryos and X-gal staining of dissected viscera performed at E16.5. (A,B) Illustrated and annotated caudal (A) and cranial (B) views of thedissected viscera are provided for reference. (C) A caudal view of embryo 46.1 reveals lacZ-expressing descendants in the right lateral (RL) and right medial(RM; not seen in this view) liver lobes, as well as in the pancreatic trunk (PTR; n=3 embryos with this pattern). (D) A caudal view of the viscera dissectedfrom embryo 121.1 reveals blue lacZ-expressing cells in the pancreatic tail (PT) and intestine (INT; n=2 with this pattern). (E) A caudal view of the visceraof embryo 118.1 reveals lacZ-positive cell descendants in the stomach (ST) as well as in the PTR and pancreatic head (PH). The left/caudal view of thisviscera in the inset reveals additional lacZ-positive descendants in the ST and duodenum (DUO; n=3 with this pattern). (F) A cranial view of embryo 122.1demonstrates lacZ-expressing clonal descendants in the left lung lobe (LLL), left thymus (TH) and in the trachea (TR). (G) A fate map of the E8.5 DE,produced by combining previously published fate mapping with the novel progenitor relationships reported herein. The fate map is drawn on a linearized8S embryo. The midline placed notochord, paired somites, and anterior and posterior intestinal portal are drawn on the embryo as landmarks. The shapesindicate organ progenitor domains and the color represents individual organs as identified on the simplified E16.5 gut tube. Progenitor domains that overlaprepresents endoderm that will contribute to each of those organs. CLL, cardiac lung lobe; GB, gall bladder; HRT, heart; LL, left lateral liver lobe; LM, left medial liverlobe; RC, right caudate liver lobe; RK, right kidney; SLL, superior lung lobe. Scale bars: 1 mm.

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been experimentally verified in Xenopus (Jarikji et al., 2009). Giventhe extensive efforts that have gone into recapitulating pancreaticdevelopment from pluripotent mammalian tissues in vitro, the noveldevelopmental relationships uncovered herein are significant andmay suggest new strategies for generating pancreatic tissues. Onereason these relationships have not been uncovered is because muchof the retrospective fate mapping performed in the pancreas hasrelied on genetic techniques that are restricted to already specifiedpancreatic cells (Gu et al., 2002; Kawaguchi et al., 2002; Kopinkeet al., 2011; Larsen et al., 2017; Offield et al., 1996; Zhou et al.,2007). Similarly, in the case of prospective progenitor fate mapping,the experiments are terminated by E9.5, which is too early to easilydistinguish gastric and intestinal fates (Angelo et al., 2012; Mikiet al., 2012).Finally, four embryos contained clones in distinct but

overlapping combinations of trachea, thymus and lung: threeorgans that originate from the anterior ventral foregut endoderm(Fig. 1F, Table 1). These include independent embryos withdescendants in each of four categories: trachea and lung; thymusand lung; trachea, thymus and lung; thymus alone. Despite theunique organ grouping in each of these embryos, the presence ofthe same organ descendants in at least two embryos supports theexistence of a common E8.5 lung/thymus/trachea endodermprogenitor. These conclusions are further supported by thepresence of thymus and lung descendants, when observed in thesame embryo, on the same side of the embryo (left or right),consistent with the expectation for multi-lobed organs that originatefrom distinct left and right lateral progenitors. Although theexistence of a common DE progenitor pool for trachea and lunghas been assumed, a common trachea/thymus/lung E8.5 progenitoris less well established but not without precedent (Perl et al., 2002).In this study, individual embryos with clonal descendants in more

than one organ reveal the multi-organ fate of an individual E8.5 DEcell. The endoderm emerges during gastrulation in an orderlyfashion, with the foregut emerging first, followed by the midgut andthen the hindgut (Tam et al., 2007). This broad regionalization,although initially overlapping, becomes sharper as developmentproceeds (Tam et al., 2007). Furthermore, it is clear that coherentgroups of endoderm contribute to contiguous domains of celldescendants within the gut tube and associated organs (Angeloet al., 2012; Franklin et al., 2008; Miki et al., 2012; Tremblay andZaret, 2005). Armed with this information, the novel progenitorrelationships described herein and published fate maps, weproduced a fate map of the E8.5 DE (Fig. 1G). Each coloredshape on the E8.5 map represents an organ progenitor domain andthe color a particular organ, as identified on the E16.5 gut tubecartoon. The overlapping progenitor domains on the E8.5 maprepresent the multi-organ progenitors identified herein. The positionof the liver, gallbladder and pancreas progenitor domains are basedon published results (Angelo et al., 2012; Franklin et al., 2008; Mikiet al., 2012; Tam et al., 2007; Tremblay and Zaret, 2005; Uemuraet al., 2015). The remainder of the organ domains are positionedbased on their ultimate arrangement in the embryo relative to theexperimentally identified organ domains. In addition to providing abetter understanding of normal organ ontogeny, the developmentalrelationships described herein may provide novel inductionstrategies for the parenchymal tissues of the thymus, lung,stomach, pancreas, duodenum and intestine.

Assessing hepatoblast potencyTo begin a more in-depth analysis of the E16.5 livers obtained, weexamined the distribution of clonal descendants between the five

readily identifiable liver lobes (Table 2, Fig. 2A). Early prospectivefate mapping and recent retrospective clonal analysis suggests a left/right asymmetry in descendant contribution to the lobes (Angeloand Tremblay, 2013; Weiss et al., 2016). In agreement with thesestudies, we find that clonal descendants mainly contribute tocontiguous lobes (Fig. 2B, Table 2, Fig. S3). Given the largenumber of embryos with descendants in the liver in our study(n=25), we calculated that up to three may result fromtwo independent recombination events. We believe that twoindependent recombination events could have produced the twoembryos with unique and discontinuous hepatic lobe contribution(embryos 49.1 and 53.1; Table 2, Fig. S3).

The hepatoblast-to-hepatocyte transition is completed byE15.5. ByE16.5 hepatocytes and the cholangiocyte precursor, biliary epithelialcells (BECs), can be identified both molecularly and histologically(Yang et al., 2017). Hepatocytes express hepatocyte nuclear factor 4α(HNF4α) and reside throughout the liver parenchyma, whereasSOX9-expressing BECs are confined to the periportal epithelium(Antoniou et al., 2009; Parviz et al., 2003; Shiojiri, 1997). To analyzethe differentiation state of the clonal descendants, each lacZ-expressing liver was sectioned and adjacent or near adjacentsections subjected to immunohistochemistry for HNF4α or SOX9.In each of the 22 livers analyzed, lacZ colocalized with both HNF4αand SOX9 (Fig. 2C,D; Table 2, Fig. S4), demonstrating for the firsttime that individual hepatoblasts differentiate into both hepatocytesand BECs in vivo by E16.5 (Fig. 2E; χ2, ***P=9.11−4). These dataoffer unequivocal results, as 100%of livers analyzed showed that bothpopulations arise from a single cell.

We have shown that carefully timed single-cell retrospective fatemapping is not only useful for confirming suspected ontogeny but

Table 2. Distribution of E16.5 hepatic clones

Embryo # LL* LM RM RL RC

39.1 W‡

41.1§ W49.2 R71.1§ R119.1 R34.2¶ W W52.1 W W56.1 W R36.2 W W W55.2 W W R56.2 W W R78.1 R W R49.1 W R55.1 W R58.1 W R52.4 W74.2 W34.1¶ W53.2 R W R74.1 W W36.1 W R R46.1§ W R R52.3 W W R R32.1¶ W R53.1 W R

Each liverwas collected intact andall lobesexamined. *LL, left lateral lobe; LM, leftmedial lobe; RM, right medial lobe; RL, right lateral lobe; RC, right caudate lobe.‡W, widespread clonal distribution; R, restricted clonal distribution. A blankspace means that there were no clonal descendants in that lobe.§Embryos with clonal descendants also in pancreas, as detailed and includedin Table 1.¶Immunohistochemistry not performed.

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can also be used to uncover novel progenitor relationships. Suchstudies also reveal how cell potential is gradually refined andprovides insight into how individual cells physically contributeto mature organs. Furthermore as single-cell RNA-sequencingbecomes more prevalent, its use as a predictive tool to inferdevelopmental relationships will be strengthened and validated bysingle-cell lineage-tracing methods such as those presented herein.

MATERIALS AND METHODSMiceHomozygous Foxa2mcm/mcm males (C57BL/6J and 129S1 background; age3-12 months) were crossed to homozygous Rosa26lacZ/lacZ reporter females(C57BL/6NJ; age 4-12 weeks) to generate heterozygous, Foxa2mcm/+;Rosa26lacZ/+ embryos (Park et al., 2008; Soriano, 1999). The morning thecopulation plug was identified was defined as E0.5. All animal experimentswere performed in accordance with guidelines issued by the InstitutionalAnimal Care and Use Committee at the University of MassachusettsAmherst (IACUC #2015-45).

Tamoxifen administrationTamoxifen (TMX; Sigma, #T5648) was administered on E7.75 (between16:30 and 17:30) via oral gavage. To prepare the TMX solution, 100 μl ofethanol was aspirated with 5 mg of TMX and heated in a water bath until fullydissolved. The dose (2-50 mg kg−1) was calculated using the body weight atthe time of administration and achieved by adding the appropriate volume ofpeanut oil (human consumption grade). A constant volume (100 µl) wasadministered to all animals, including the controls receiving only peanut oil.

X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)stainingEmbryos were collected at either E8.5 or E16.5 in ice-cold PBS. E8.5embryos were dissected intact, whereas E16.5 embryos were dissected to

collect viscera only. X-gal staining of E8.5 embryos was performed aspreviously described (Tremblay et al., 2001). Although most were stored inethanol, those presented herein were stored in glycerol for imaging. X-galstaining at E16.5 was performed similarly, except modifications to theprotocol were made to reduce background, increase substrate penetrationand ensure that the staining minimally interfered with subsequentimmunohistochemistry. The 0.2% gluteraldehyde fixation step was limitedto 10 min on ice. Although this modification diminished the intensity of theX-Gal stain, particularly on the innermost region of the livers, it allowed forreliable immunohistochemistry after X-gal staining. The X-gal buffer wasadjusted to pH 8.0 and X-gal staining performed overnight on a nutator atroom temperature, rather than at 37°C, which served to reduce the backgroundthat was otherwise prevalent in the liver (Nagy et al., 2003).

ImmunohistochemistrySamples were dehydrated in ethanol, cleared in xylenes, embedded inparaffin wax and sectioned at 7 μm. E8.5 embryos were sectioned onto asingle slide, whereas only the X-gal positive regions of the E16.5 tissueswere serially sectioned and distributed onto three slides. After dewaxing inxylene and rehydration, microwave antigen retrieval was performed inboiling 10 mM Tris buffer (pH 10) for 8 min. Slides were cooled to roomtemperature before incubating in block (0.5%milk powder in PBT) for 2 h atroom temperature, and then incubated in primary antibody (diluted in block)at 4°C overnight. Slides were incubated with secondary antibodies diluted inblock for 1 h at room temperature. ABC peroxidase staining (ThermoFisher,#32020) was followed by development with metal-enhanced DAB(ThermoFisher #34065). Finally, slides were mounted in Cytoseal-60(ThermoScientific, #8310-16) for permanent storage.

The primary antibodies and dilutions used for immunohistochemistrywere anti-HNF4α (SantaCruz, #sc6556, 1:200) and anti-SOX9 (Millipore,#ab5535, 1:1000). The secondary antibodies and dilutions used werebiotinylated goat anti-rabbit (Vector Labs, #ba-1000, 1:500) andbiotinylated rabbit anti-goat (Vector Labs, #ba-5000, 1:500).

Fig. 2. Individual hepatoblasts contribute hepatocytes and BECs to each E16.5 liver. Embryos were maternally administered 3 mg kg−1 TMX at E7.75and then dissected and X-gal stained at E16.5. (A) An annotated illustration of a frontal view of dissected viscera at E16.5. (B) A frontal image of theviscera of embryo 58.1 reveals widespread lacZ expression in the left medial liver lobe (LM) and more restricted expression on the left edge of the right medialliver lobe (RM). (C,D) Immunohistochemistry, performed on sections from the embryo in B, reveals the colocalization of lacZ-expressing clonal descendants(blue) with the hepatocyte marker HNF4α (C, pink arrowheads) and the biliary epithelial cell marker SOX9 (D, pink arrowheads). The black arrowheads indicatelacZ-expressing descendants that are negative for the indicated immunohistochemistry marker. (E) Schematic illustrating that each clonally labeled E8.5hepatoblast contributes to the hepatocyte and biliary epithelial cell lineage by E16.5 (n=22/22; χ2, ***P=9.11−4). Scale bars: 1 mm in B; 50 µm in C,D.INT, intestine; LL, left lateral liver lobe; RC, right caudate liver lobe; RL, right lateral liver lobe.

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ImagingWhole-mount images were captured on a Nikon SMZ1500 dissectingmicroscope fitted with either a QImaging MicroPublisher 5.0 RTV or a Spot29.2 color camera. Bright-field images were taken with a 3DHistechPanoramic MIDI slide scanner under a 20× objective. Images were croppedin Photoshop and arranged using InDesign.

StatisticsAll embryos receiving TMX were collected and the number with recombinedcells were reported herein. A one-way analysis of variance (ANOVA) wasperformed on the number of labeled cells observed in each E8.5 embryocollected at each TMX dose (***P=2.23−9), suggesting that the number oflabeled cells observed is dependent on TMX dose. The frequency of labelingat E16.5 (11.8%) was slightly, but not significantly, lower than at E8.5(13.9%; F-test, P=0.0523). lacZ-expressing cells within E16.5 liversconsistently co-expressed both hepatocyte and biliary transcription factors(n=22/22) so hepatoblasts must be at least bipotential (χ2, ***P=9.11−4) toproduce those cells. Statistics were performed in R v3.3.3 (Team, 2017).

AcknowledgementsWe thank Dr Mary Weiss for her help in reducing background in our X-gal stainedviscera, and members of the Tremblay and Mager labs for their discussion andadvice during this project.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: K.D.T.; Methodology: G.K.E.S., J.M.M., M.-K.E.C., S.R.,J.R.A., K.D.T.; Validation: G.K.E.S., K.D.T.; Formal analysis: G.K.E.S., K.D.T.;Investigation: G.K.E.S., J.M.M., M.-K.E.C., S.R., K.D.T.; Resources: J.M., K.D.T.;Writing - original draft: G.K.E.S., K.D.T.; Writing - review & editing: G.K.E.S., J.M.,K.D.T.; Visualization: G.K.E.S., J.R.A., K.D.T.; Supervision: K.D.T.; Projectadministration: K.D.T.; Funding acquisition: K.D.T.

FundingThis research is funded in part by the National Institutes of Health (R01DK087753 toK.D.T.). Deposited in PMC for release after 12 months.

Supplementary informationSupplementary information available online athttp://dev.biologists.org/lookup/doi/10.1242/dev.168658.supplemental

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