BMP antagonism by Noggin is required in presumptive notochord cells for mammalian foregut morphogenesis

Developmental Biology 391 (2014) 111–124

Contents lists available at ScienceDirect

Developmental Biology journal homepage: www.elsevier.com/locate/developmentalbiology

BMP antagonism by Noggin is required in presumptive notochord cells for mammalian foregut morphogenesis Sarah R. Fausett a, Lisa J. Brunet b, John Klingensmith a,n a b

Department of Cell Biology, Duke University Medical Center, Durham, NC, United States Department of Molecular & Cell Biology, University of California Berkeley, Berkeley, CA, United States

art ic l e i nf o

a b s t r a c t

Article history: Received 3 October 2013 Received in revised form 21 January 2014 Accepted 10 February 2014 Available online 12 March 2014

Esophageal atresia with tracheoesophageal fistula (EA/TEF) is a serious human birth defect, in which the esophagus ends before reaching the stomach, and is aberrantly connected with the trachea. Several mouse models of EA/TEF have recently demonstrated that proper dorsal/ventral (D/V) patterning of the primitive anterior foregut endoderm is essential for correct compartmentalization of the trachea and esophagus. Here we elucidate the pathogenic mechanisms underlying the EA/TEF that occurs in mice lacking the BMP antagonist Noggin, which display correct dorsal/ventral patterning. To clarify the mechanism of this malformation, we use spatiotemporal manipulation of Noggin and BMP receptor 1A conditional alleles during foregut development. Surprisingly, we find that the expression of Noggin in the compartmentalizing endoderm is not required to generate distinct tracheal and esophageal tubes. Instead, we show that Noggin and BMP signaling attenuation are required in the early notochord to correctly resolve notochord cells from the dorsal foregut endoderm, which in turn, appears to be a prerequisite for foregut compartmentalization. Collectively, our findings support an emerging model for a mechanism underlying EA/TEF in which impaired notochord resolution from the early endoderm causes the foregut to be hypo-cellular just prior to the critical period of compartmentalization. Our further characterizations suggest that Noggin may regulate a cell rearrangement process that involves reciprocal E-cadherin and Zeb1 expression in the resolving notochord cells. & 2014 Published by Elsevier Inc.

Keywords: BMP signaling Noggin EA/TEF Foregut Notochord Mouse development

Introduction The trachea and esophagus are parallel tubular organs that connect the pharynx with the lungs and stomach, respectively. Though they are ultimately distinct organs, they both arise from a single tube of anterior foregut endoderm, which undergoes a series of complex morphogenetic events over the course of its development (reviewed in Fausett and Klingensmith, 2012). During gastrulation in the mouse, endodermal and mesodermal cells arise from the primitive streak and migrate anteriorly along the ventral aspect of the embryo as its rostro-caudal axis lengthens (Tam et al., 2007). Endodermal cells form a single epithelial cell layer ventrally, while most of the mesodermal cells lie in between the endoderm and the dorsal ectoderm. At the midline, just ventral to the developing neural floorplate, a narrow band of cells lies within the endodermal epithelium and expresses Brachyury (Wilkinson et al., 1990). These cells will separate from the endoderm to form the notochord, an essential signaling center

n

Corresponding author. Tel.: þ 1 919 684 9402. E-mail address: [email protected] (J. Klingensmith).

http://dx.doi.org/10.1016/j.ydbio.2014.02.008 0012-1606/& 2014 Published by Elsevier Inc.

and the defining feature of the Chordate lineage (Jurand, 1974; Cleaver and Krieg, 2001). Remarkably little is known about the cellular basis of notochord separation from the endoderm layer in mammals; however, the descriptive histology by Jurand (1974) provides the current model. During endoderm development, the midline cells appear to evaginate and resolve from the endoderm to become the notochord, a distinct tissue with its own basement membrane. Following notochord resolution, the embryonic endoderm folds ventrally to form the primitive gut tube. Next, lung buds form on the ventral surface of the anterior foregut endoderm (FGE), heralding the commencement of foregut compartmentalization. Finally, in a process that is still poorly understood, the ventral endoderm just rostral to the lung buds gives rise to the trachea while the dorsal endoderm gives rise to the esophagus. The model most consistent with experimental evidence suggests that the lateral sides of the endoderm meet across the lumen to divide the single foregut tube in half lengthwise, creating dorsal and ventral tubes, in a caudal to rostral fashion. This apparent septation process occurs concurrently with lengthening of the entire foregut (see Fig. 1A) (Fausett and Klingensmith, 2012). Incorrect compartmentalization of the trachea and esophagus can result in a defect known as esophageal atresia with tracheoesophageal

112

S.R. Fausett et al. / Developmental Biology 391 (2014) 111–124

Wildtype Septation model of foregut compartmentalization

fp nc

E12 E10

es

BRE-LACZ

nc

id1

msx1 msx2

sox2 nkx2-1 barx1 axin2

fg fg

shh SOX2

nog

fp

tr

fg

fg

NKX2-1

1

0.1

DAPI

Log relative transcript quantitiy

10

Nog-/-

0.01

* 0.001 Fig. 1. Dorsal–ventral patterning of the anterior foregut endoderm is not disrupted in Nog  /  mutants. (A) In the septation model of foregut compartmentalization, the undivided anterior foregut at embryonic day 10 (E10) displays dorsal/ventral patterning (pink/green, respectively). Over the course of two days, the lateral edges of the endoderm meet across the lumen and divide the foregut starting caudally and progressing rostrally. (B, C) Transverse E9.5 wildtype (B) and Nog  /  (C) anterior foregut sections labeled with a BMP-response element reporter, BRE-LacZ (blue) (scale ¼50 μm). (D) Relative transcript quantities of genes involved in D/V patterning of the FGE. (E, F) Immunohistochemistry showing no difference in dorsal SOX2 or ventral NKX2-1 in Nog  /  embryos (F) and wildtype littermates (E) at E10.5 in transverse section (scale ¼25 μm). Abbr: fp, floorplate; nc, notochord; fg, foregut; es, esophagus; tr, trachea.

fistula (EA/TEF), in which the pharynx communicates with the lungs, but not the stomach. EA/TEF occurs in humans at roughly 1 in 3000 live births (Torfs et al., 1995). This is a serious birth defect, because food and water can enter the lungs, while air enters the stomach. Despite the health concern, and the growing number of mouse models of EA/TEF (Fausett and Klingensmith, 2012), much remains to be discovered regarding the normal morphogenesis of the trachea and esophagus and what goes wrong to cause EA/TEF. Gene expression patterns prior to foregut compartmentalization presage the distinct tissue derivatives of the ventral and dorsal foregut. At E10.5 in the mouse, the ventral FGE expresses early respiratory-tract markers including Nkx2-1, while the dorsal FGE expresses Sox2 (Que et al., 2006, 2007). Mutations disrupting dorsal/ventral (D/V) patterning of the foregut cause failure of compartmentalization and yield a phenotype similar to EA/TEF (Fausett and Klingensmith, 2012). When genes and signaling pathways important for ventral specification are disrupted, the entire foregut becomes esophagus-like and fails to compartmentalize (Que et al., 2007; Harris-Johnson et al., 2009; Goss et al., 2009). By contrast, as Sox2 levels are reduced, EA/TEF often occurs, with the single foregut tube resembling a trachea (Que et al., 2007). Noggin null mutants have a very similar defect (Que et al., 2006). NOG, a secreted BMP-antagonist, is expressed in the notochord, floorplate, and dorsal FGE. Conversely, BMP are ligands are expressed primarily in the ventral endoderm and mesoderm (Que et al., 2006). Recently, it was shown that ventral BMP signaling directly represses Sox2 transcription (Domyan et al.,

2011). Thus, dorsal NOG may act to antagonize BMP signaling there, allowing dorsal Sox2 expression and maintaining correct D/V patterning. While it is now clear that incorrect D/V patterning by genetic misregulation can cause defective foregut compartmentalization, other defects may lead to EA/TEF as well. One such case is in a teratogenic model developed in rats. By injecting pregnant rats or mice with Adriamycin, an anthracycline antibiotic, researchers can induce a high incidence of EA/TEF that closely resembles the most common form of human EA/TEF (Thompson et al., 1978; Ioannides et al., 2002). The molecular and cellular bases of the foregut phenotype in the Adriamycin model remain unknown; however, the foregut does not appear to be significantly mis-patterned (Ioannides et al., 2010). Instead, EA/ TEF consistently occurs along with notochords that are abnormally large, disorganized, and sporadically attached to the dorsal foregut (Possoegel et al., 1999). Nog null mice have an identical notochord defect, and roughly 75% have EA/TEF (Que et al., 2006; Li et al., 2007). Many theories have been put forth to explain the coincidence of the notochord and foregut defects in these models, but there is still no conclusive evidence to show that they are linked (Fausett and Klingensmith, 2012). In 2006 and 2007, two groups published the Nog null foregut and notochord phenotypes, and both suggested that the notochord defect might cause EA/TEF (Que et al., 2006; Li et al., 2007). A key insight was the discovery by Li et al. (2007) that not all the cells in the mutant notochord express the notochord marker, Brachyury (T). This led them to propose a model in which the T-negative cells were actually dorsal FGE cells that ended up in the

S.R. Fausett et al. / Developmental Biology 391 (2014) 111–124

notochord at the expense of the dorsal FGE during notochord resolution. They reasoned that a dorsal FGE deficient in cells might be too small to yield a patent esophagus after compartmentalization. Experimental data available to date can be reconciled with either of these models for the occurrence EA/TEF in Nog mutants. It is conceivable that NOG has two separate roles, one in notochord development and one in active compartmentalization of the of the FGE. However, it is also possible that EA/TEF arises because of the notochord defect (Li et al., 2007). In this study we set out further test both hypotheses. We additionally sought to clarify the cellular behaviors that underlie notochord resolution from the foregut endoderm. Our genetic studies pinpoint the requirement for Nog in foregut compartmentalization to be in the notochord at the time of notochord resolution. Additionally, we present quantitative data suggesting that a role of NOG in this domain is to facilitate a cellular rearrangement event involving transcriptional downregulation of E-cadherin (Cdh1) in notochord cells.

Results Dorsal–ventral patterning of the foregut endoderm is not disrupted in Noggin null mutants Nearly all mouse models of EA/TEF display aberrant D/V patterning of the FGE prior to compartmentalization (Fausett and Klingensmith, 2012). Since ventral BMP signaling indirectly promotes tracheal fates (Domyan et al., 2011), we hypothesized that a loss of the BMP antagonist NOG would lead to an inappropriate expansion of BMP signaling, thus perturbing D/V patterning in the early foregut. Results by Li et al. (2007) indicate that BMP signaling occurs at low levels if at all in the early dorsal foregut, and that loss of Noggin causes no major change in BMP signaling levels in this domain. We investigated this issue further with a variety of BMP signaling assays, including the use of a BMP response element (BRE)-driven LacZ reporter that labels cells responding to BMP signaling (Blank et al., 2008). Consistent with the results of Li et al. (2007), we found that the BMP signaling reporter marks a greater proportion of the foregut in Nog null mutants relative to wildtype, stage-matched littermates. However, the foregut is also smaller in these mutants (Fig. 1B, C); therefore, we cannot conclude that BMP signaling is expanded in its dorsal extent. We next examined transcript levels of several targets of BMP signaling (Id1, Msx1, Msx2), as well as other presumptive targets of the D/V patterning network (Sox2, Barx1, Nkx2-1, Axin2) (Miyazono and Miyazawa, 2002; Tribulo et al., 2003; Woo et al., 2011; Domyan et al., 2011; Que et al., 2007; Goss et al., 2009). While levels of Nog transcript were virtually undetectable, we found no significant changes in expression of BMP target genes or foregut patterning genes (Fig. 1D). Finally, we used SOX2 and NKX2-1 antibodies to label the dorsal and ventral domains, respectively. While the cross-sectional circumference of the Nog null FGE varies along the rostro-caudal axis, comparison of wildtype and mutant foregut sections of similar circumference showed no D/V patterning defects at E10.5 (Fig. 1E, F). Together, these data support the conclusion that loss of Nog has a negligible effect on D/V patterning of the foregut prior to compartmentalization. The Noggin mutant foregut endoderm is hypocellular, but not due to increased cell death or decreased proliferation In order to investigate the mechanism(s) behind the occurrence of EA/TEF in Nog null mutants in the absence of a clear patterning defect, we examined the physical attributes of the foregut prior to compartmentalization. Previous work by Li et al. (2007) determined that the foregut endoderm appears smaller in mutants than

113

in wildtype at E9.5. They suggested that the dorsal FGE is reduced relative to the ventral domain, and that it may be insufficient to yield a patent esophagus. While we were able to confirm that the FGE in the trunk and neck regions contains fewer cells at E9.5, we found no significant difference in the relative size of the dorsal vs. ventral domains (Fig. 2A, B). By contrast, the mutant FGE is not significantly hypocellular at E8.5. This suggests that the foregut becomes hypocellular in mutants between E8.5 and E9.5 (Fig. 2C). One possibility is that the reduced foregut size results from either decreased proliferation or increased apoptosis, especially given that BMP signaling is known to promote apoptosis or cell senescence in other contexts (Guha et al., 2002; Buckley et al., 2003). Li et al. (2007) concluded that apoptosis is unlikely to play a significant role in foregut size at E9.0. We extended our analysis earlier to E8.5. We quantified the levels of proliferation and apoptosis in mutant and control foreguts at E8.5 using antibodies against phosphohistone H3 and cleaved caspase-3, respectively. We found that there was no significant difference in apoptosis in Nog null foreguts at E8.5 compared to wildtype, and there was a significant increase rather than decrease in proliferation (Fig. 2D). We therefore conclude that the hypocellularity in Noggin mutants is not caused by either decreased proliferation or increased apoptosis, and that it must be the result of a different mechanism. Noggin expression in the foregut endoderm is not required for proper foregut compartmentalization To isolate the potential roles for Nog in notochord and foregut development, we turned to a genetic approach. Since Nog is expressed in the neural floorplate, notochord, and dorsal FGE just prior to and during compartmentalization (Fig. 3A, B), we tested which of these domains is required for foregut compartmentalization and/or notochord morphogenesis. To do this, we genetically dissected the spatial and temporal requirements for Nog. We first sought to ascertain whether Nog expression in the dorsal foregut is required for correct foregut compartmentalization. To ablate Nog in this domain we employed Cre-Lox technology using Foxg1Cre (Hebert and McConnell, 2000) in conjunction with a conditional, “floxed” allele of Nog (NogFx) (Stafford et al., 2011). Though the recombination domain for this Cre driver is quite large, it does not include the floorplate or the notochord; it preserves Nog expression in these axial domains while ablating it in the endoderm (Fig. 3A, B, and D). To confirm successful ablation of Nog from the endoderm, we performed quantitative PCR (qPCR) on cDNA from isolated foreguts (Fig. 3G). While this genetic manipulation produced several phenotypes found in Nog nulls (exencephaly and small telencephalons, data not shown), it did not produce any discernable notochord phenotype (n ¼0/11) or foregut compartmentalization defects (n ¼0/12) (Fig. 3L). We conclude that the dorsal FGE domain of Nog expression is not necessary for correct morphogenesis of the notochord or compartmentalization of the foregut. BMP antagonism is required in the early notochord for proper notochord and foregut morphogenesis Having ruled out the FGE as a necessary source of NOG, we considered the floorplate and notochord expression domains. Given that NOG is a secreted molecule, it seemed possible that these nearby domains could influence notochord development, foregut compartmentalization, or both. To ablate Nog from these domains, while leaving the dorsal FGE domain intact, we used ShhCre, as Shh itself and the ShhCre driver are expressed in notochord and floorplate but not in the dorsal FGE at relevant stages (Figs. 3A, C, and 4A–C) (Harfe et al., 2004). Quantitative PCR and in situ hybridization confirmed that ShhCre successfully ablates

114

S.R. Fausett et al. / Developmental Biology 391 (2014) 111–124

0.5

WT 25

MT

* 20 15 10

* 5

Ratio of the average # of cells in the dFGE to the vFGE

Average # of cells per crosssection in the E9.5 anterior foregut

30

0

dFGE

vFGE

0.45 0.4

WT MT

0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

MT

Percent of cells in the E8.5 anterior foregut

Average # of cells per crosssection in the E8.5 anterior FGE

7

WT

WT

*

6

MT 5 4 3 2 1 0

dying

dividing

Fig. 2. Neither increased apoptosis nor decreased proliferation can account for the hypo-cellularity of the anterior FGE of Nog  /  mutants at E9.5. (A) The average number of cells per cross-section in E9.5 anterior FGE is significantly lower in Nog  /  mutants compared to wildtype littermates (p o 0.05). (B) The ratio of the number of dorsal to ventral FGE cells (as defined by ShhCre expression) is not significantly lower in Nog  /  mutants compared to wildtype littermates. (C) The average number of cells per crosssection in the E8.5 anterior FGE is not significantly lower in Nog  /  mutants compared to wildtype littermates. (D) There is no significant difference in the level of endodermal apoptosis at E8.5 in Nog  /  mutants compared to wildtype littermates, and the level of proliferation is significantly higher (p o 0.05). Abbr: dFGE, dorsal foregut endoderm; vFGE, ventral foregut endoderm.

Noggin expression in the notochord (Fig. 4B, C), but leaves it intact in the foregut endoderm (Fig. 3G). We found that this cross (ShhCre; NogFx/  ) produced notochord (n ¼ 9/9) and foregut defects (n ¼ 10/17) identical to those seen in the Nog null (Figs. 3K and 4D, E). These data reveal that axial midline expression of Nog is required both for proper notochord morphogenesis and for compartmentalization of the common foregut into esophagus and trachea. To supplement this finding, we employed a Cre-inducible constitutively active Bmpr1a (CAB) transgene targeted to the Rosa26 locus (Rodriguez et al., 2010). Driving this transgene with ShhCre (ShhCre;CAB) allowed us to determine whether the role of NOG from the notochord and floorplate domains is to attenuate BMP signaling only within those domains, or whether a broader domain also requires BMP antagonism by axial NOG (Figs. 3E, F and 4F, G). If the role of NOG from the notochord and floorplate were to attenuate BMP-signaling in the foregut endoderm, for example, we would not expect this cross to produce EA/TEF. However, this experiment also produced notochord (n ¼15/15) and foregut defects (n ¼27/34) very similar to those of the Nog null (Figs. 3I and 4H, I). These data imply a critical role for BMP signaling attenuation in the axial midline. However, because ShhCre is also expressed in the ventral foregut endoderm, it may be that over-activation of BMP signaling in this ventral domain could impact the phenotype. In order to test whether activation of BMP signaling in the FGE could cause EA/TEF, we used Foxg1Cre to induce BMP signaling in the foregut endoderm and mesenchyme. We found that Foxg1Cre;CAB mutants to not display EA/TEF (n ¼5)

(Supplemental Fig. 1). Together, these data reveal that BMP signaling must be attenuated specifically in the floorplate, notochord, or both to ensure proper formation of the notochord and the compartmentalization of the foregut. In order to distinguish Noggin action between its notochord and floorplate domains, we turned to a tamoxifen-inducible Cre driven by Shh, ShhCreER (Harfe et al., 2004). We found that under our experimental conditions, tamoxifen induces Cre-mediated DNA recombination within 24 h of administration, and no longer induces recombination in new Cre expression domains after 48 h (Fig. 5A, C–F). Since Shh expression in the notochord begins at E7.5 in the node, but floorplate expression does not begin until E8.5, we were able to induce recombination specifically in the notochord by administering one dose of tamoxifen 24 h prior, at E6.5 (Fig. 5C). Additionally, by comparing the phenotypes generated with progressively later administration of tamoxifen (i.e. at E6.5, E7.5, E8.5, and E9.5) in ShhCreER;Nogfx/  mutants, we tested whether or not it is possible to uncouple the notochord and foregut defects by inducing recombination after notochord resolution and before foregut compartmentalization. As expected, administering tamoxifen at E9.5, and thus causing recombination at E10.5, produced neither notochord defects (n ¼0/6) nor EA/TEF (n ¼0/6) (Fig. 5B, F). Tamoxifen at E8.5 caused mild notochord defects in about half of the mutant embryos (n ¼10/18) (Fig. 5B and E), while tamoxifen at E7.5 caused mild to severe notochord defects in 100% of mutant embryos (n¼ 16/16). Strikingly, neither administration at E8.5 (n ¼0/18) nor at

S.R. Fausett et al. / Developmental Biology 391 (2014) 111–124

NogLacZ/+

Nog-LacZ

115

Foxg1Cre

ShhCre

fp fp

fp BMP signaling nc

nc

nc fp and nc domains targeted by ShhCre

fg fg

fg

FGE domain targeted by Foxg1Cre

ShhCre;CAB

nc

nc

fg

fg

ShhCre;NogFx/-

Foxg1Cre;NogFx/-

0.5 Log relative nog transcript quantity

Wildtype

0.05

* 0.005

Wildtype

es

ShhCre;CAB

Nog-/-

ShhCre;NogFx/-

Foxg1Cre;NogFx/-

tr es

tr

Fig. 3. Noggin expression from the notochord and/or floorplate is both necessary and sufficient to allow proper foregut compartmentalization. (A) Cartoon showing how ShhCre and Foxg1Cre target the domains of Nog expression (blue). Tissue experiencing the highest levels of BMP signaling (as judged by BRE-LacZ) is represented in orange. (B) Noggin-LacZ expression at E10.5 (scale¼ 50 μm). (C, D) Recombination patterns of Shh Cre (C) and Foxg1Cre (D) reported by R26RLacZ (blue) or R26ReYFP (brown) at E10.5 (scale ¼50 μm). (E, F) BRELacZ-reporter activity in the E9.5 foreguts of Shh Cre;CAB mutants (F) and wildtype littermates (E) (scale ¼50 μm). (G) Quantitative PCR showing reduced Nog expression in the dissected E10.5 foreguts of Foxg1 Cre;NogFx/  mutants, but not Shh Cre;NoxFx/  mutants relative to wildtype littermates; (H) wildtype E12.5 foregut showing a complete esophagus and trachea; (I–K) esophageal atresia (arrowheads) and tracheoesophageal fistula (arrows) in ShhCre;CAB mutants (I), Nog null mutants (J), and ShhCre;NogFx/  mutants (K). (L) Foxg1Cre;NogFx/  mutants do not have EA/TEF (scale ¼ 200 μm). Brown in H–L ¼anti-CDH1. Abbr: fp, floorplate; nc, notochord; fg, foregut; es, esophagus; tr, trachea.

116

S.R. Fausett et al. / Developmental Biology 391 (2014) 111–124

E8.5

E9.5

WT Nog in situ

MT Nog in situ

fp

fp

A ShhCre;NogFx

fp

WT nc

MT nc fp

fp

nc nc fg

nc

nc

fg

nc

fg fg fg

eYFP WT BRE-LacZ

MT BRE-LacZ

H fp ShhCre;CAB

fp

fp

fp

nc

nc

nc nc

fg fg

fg

fg

DAPI Cre

Brachyury

Laminin

eYFP

Fig. 4. Axial domains require BMP antagonism for proper notochord development. (A) Shh recombination at E8.5, reported by R26R (green). (B, C) In situ hybridization showing absence of Nog transcripts (purple) from the notochord at E8.5 in mutants (C) but not wildtype littermates (B). (D, E) Large notochord in which not all cells express Fx/  Brachyury seen in ShhCre;Nog embryos (E) but not wildtype littermates at E9.5 (D). (F,G) BRE reporter activity (blue) showing increased BRE-LacZ expression in the notochords of ShhCre;CAB embryos (G) but not wildtype littermates at E8.5 (F). (H, I) Large notochord in which not all cells express Brachyury seen in ShhCre;CAB embryos (I) but not wildtype littermates (H) at E9.5. In (D, E, H, I) laminin (red) marks the basement membranes and DAPI (white) marks the nuclei. Arrows indicate cells in the notochord that do not express Brachyury (green). Arrowhead indicates example of background fluorescence. Abbr: fp, floorplate; nc, notochord; fg, foregut. Scale ¼ 25 μm.

E7.5 generated EA/TEF (0/16) (Fig. 5B, D). We also note that administration of tamoxifen at E8.5 did not cause EA/TEF in ShhCreER;CAB mutants either (n ¼14). This is further evidence that activation of BMP signaling in the vFGE does not contribute to EA/ TEF. In contrast, we were able to induce EA/TEF (n ¼ 4/16) along with the notochord defects (n ¼16/16) by administering tamoxifen at E6.5 (Fig. 5B, C, G, and H). These data, together with our previous results, suggest a strong connection between BMP antagonism in the notochord during the period of notochord resolution and the subsequent success of foregut compartmentalization. Furthermore, by pooling data from each of our genetic experiments, we determined via statistical analysis that the presence of severe notochord defects (4 300% the size of wildtype notochords) is a strong predictor of the presence of EA/TEF (p ¼0.0047; Fig. 5B). This relationship suggests that larger notochords are more likely to accompany EA/TEF than smaller ones, and is consistent with a model in which large notochords occur at the expense of the adequate endoderm and dispose the embryo towards EA/TEF.

Characterization of notochord resolution Because our genetic experiments strongly indicated a link between the notochord defects and EA/TEF in Nog conditional mutants, we decided to look closely at notochord resolution and how it is disrupted in Nog null mutants. In their model, Li et al. (2007) suggested that if the notochord cells were unable to successfully resolve from the dorsal foregut endoderm, that part of the

endoderm would be incorporated into the notochord structure. Such a model is consistent with their observations of a reduced foregut endoderm, with only some of the notochord cells in Nog nulls being positive for the notochord marker Brachyury (T). We wanted to build on their observations by testing the hypothesis that the T-negative cells in the notochord expressed the dorsal endoderm marker, SOX2. Indeed, we detected SOX2 protein in cells of the Nog null notochord at E9.5 (Fig. 6A). This result strongly suggests that some of the cells in the mutant notochordal tissue, as defined by the basement membrane surrounding it, were fated to be dorsal endoderm, rather than notochord. These data support a mechanism in which faulty resolution of the notochord from the endoderm results in some presumptive endoderm cells being sequestered along with notochord cells. To further refine our observations, we sought to define distinct stages of wildtype notochord resolution. We stained 6–21 somite embryos using antibodies against E-cadherin and laminin to highlight the endoderm itself and the local tissue boundaries, respectively. Examining sections through the anterior foregut region, we identified at least 6 distinct stages of basement membrane morphology (Fig. 6B). These stages progress in a rostral to caudal fashion along the trunk of the embryo. During Stage 1, the basement membrane appears to interpose between the notochord cells and endoderm cells, and by Stage 2, the basement membrane adopts an angular arrangement around the dorsal and lateral aspects of the notochord cells. Stage 3 is characterized by the rounding of the notochord cells, down-regulation of CDH1, and a basement membrane that begins to surround the ventral aspect of the notochord (also see Fig. 7C). At Stage 4, the basement membrane completely separates the notochord from the dorsal

S.R. Fausett et al. / Developmental Biology 391 (2014) 111–124

ShhCreER; R26RLacZ

117

Notochord size as a percent of wildtype

EA/TEF

Nog-/ShhCre;CAB ShhCreER;NogFx/-

nc

Tamox. E6.5 100%

200%

300%

400%

500%

no EA/TEF

Tamox. E7.5 Tamox. E8.5 likelihood ratio test: p=0.0047

ShhCreER;NogFx/- , havest E12.5 WT MT

ShhCreER;R26RLacZ , harvest E12.5 tam. E6.5

tam. 7.5

tam. 9.5

tam. 8.5

Tamox. E9.5

es

fp

fp

fp

nc nc

nc

nc

Rostral to EA

fp

es

tr

es

es

es

es Level of EA

es

tr

tr tr

tr

tr

tr

Fig. 5. Large notochords in Nog  /  mutants are a good predictor of EA/TEF, and Noggin is required early in the notochord for proper foregut compartmentalization. (A) Strong R26RLacZ reporter activity (blue) in the notochord just 24 h after tamoxifen administration at E7.5 (scale ¼ 20 μm). (B) Chart showing distribution of notochord sizes of individuals in various mutant classes against the presence of EA/TEF. Likelihood ratio test reveals that severe notochord defects are an excellent predictor of EA/TEF (p¼ 0.0047). (C, C0 ) Recombination pattern of ShhCreER at E12.5 in the notochord but not the floorplate or endoderm after a dose of tamoxifen at E6.5. (D, D0 ) Recombination pattern of ShhCreER at E12.5 in the notochord and floorplate but not the endoderm after a dose of tamoxifen at E7.5. (E, E0 ) Recombination pattern of ShhCreER at E12.5 in the notochord, floorplate, and ventral foregut endoderm after a dose of tamoxifen at E8.5. (F, F0 ) Recombination pattern of ShhCreER at E12.5 in the notochord, floorplate, and ventral foregut endoderm after a dose of tamoxifen at E9.5. Blue color reflects R26RLacZ reporter activity (scale¼ 40 μm). (G, H) Transverse sections through the foregut at E12.5 showing esophageal atresia in ShhCreER;NogFx/  embryos that were administered tamoxifen at E6.5 (H), but not wildtype littermates (G) (scale ¼40 μm). Abbr: fp, floorplate; nc, notochord; es, esophagus; tr, trachea.

endoderm, and during Stage 5, the notochord moves away from the endoderm along with the floorplate of the neural tube. As Stage 5 progresses, a trail of basement membrane material can be seen linking the notochord with the endoderm, but by Stage 6 it

has been degraded and the notochord is situated tightly against the floorplate. Lastly, we wanted to confirm the published expression patterns of Nog and various Bmp genes (McMahon et al., 1998;

118

S.R. Fausett et al. / Developmental Biology 391 (2014) 111–124

Stage 1

Nog-/-

Wildtype

Stage 3

Stage 2 fp

fp

fp

fp

fp

Brachyury

nc

nc

nc

nc

nc Stage 4

Stage 5

Stage 6

fp fp nc

SOX2

DAPI

I

a c e

g

i

DAPI

LAMININ

Brachyury in situ, 7s WT

a c

i e

g

LAMININ

Bmp7 in situ, 7s WT

Noggin in situ, 7s WT II

nc

nc

nc

nc

III a

i c

e

CDH-1

BRE LacZ, 7s WT IV

g

i a c

e

g

Ia

Ib

IIa

IIb

IIIa

IIIb

IVa

IVb

Ic

Id

IIc

IId

IIIc

IIId

IVc

IVd

Ie

If

IIe

IIf

IIIe

IIIf

IVe

IVf

Ig

Ih

IIg

IIh

IIIg

IIIh

IVg

IVh

Ii

Ij

IIi

IIj

IIIi

IIIj

IVi

IVj

Fig. 6. Detailed characterization of notochord resolution. (A) Immunohistochemistry on serial sections showing Brachyury-negative and SOX2-positive cells in the notochord of Nog  /  mutants (scale¼ 20 μm). (B) 3D-rendered confocal sections of Stages 1–6 of notochord resolution (scale¼ 20 μm). CI–CIII) In situ hybridization (purple) showing Nog, Brachyury, and Bmp7 expression in selected transverse sections (a–j) from the primitive streak to the anterior foregut in 7-somite wildtype embryos. (CIV) BRE-LacZ reporter activity (blue) in selected transverse sections (a–j) from the primitive streak to the anterior foregut in 7-somite embryos. Abbr: fp, floorplate; nc, notochord; fg, foregut. Section scale bars in panel C are 20 μm. Whole mount scale bars in panel C are 500 μm.

Danesh et al., 2009; Arkell and Beddington, 1997; Solloway and Robertson, 1999). Importantly, genetic reduction of either Bmp4 (Que et al., 2006) or Bmp7 (Li et al., 2007) suppresses the Nog null EA/TEF phenotype. Previous work has shown that both Bmp4 and Bmp7 are expressed between E8.0 and E8.5 in relevant domains. Bmp4 is expressed mainly in the ventral endoderm and mesoderm during notochord resolution (Danesh et al., 2009), with much lower levels more dorsally. However, Bmp7 expression has been reported in the FGE and notochordal plate (Arkell and Beddington, 1997; Solloway and Robertson, 1999; Danesh et al., 2009). We looked carefully at BMP-signaling, Nog expression, Bmp7

expression, and Brachyury expression along the length of 7somite wildtype embryos. Brachyury is expressed in the primitive streak as cells are undergoing EMT to accomplish gastrulation (Herrmann, 1991). It is also expressed in the node, notochordal plate, and notochord more rostrally along the midline (Fig. 6CI). Along the midline and throughout notochord resolution, Brachyury appears to be expressed at a roughly constant level. However, as the axial midline cells migrate rostrally from the node (Tam et al., 2007), there is a striking dynamic quality to Nog expression (Fig. 6CII). Most caudally, at these stages, Nog is expressed in the node. Between the node and the region at which notochord

S.R. Fausett et al. / Developmental Biology 391 (2014) 111–124

resolution is beginning, Nog transcript is nearly undetectable by in situ (Fig. 6CII:e–g). Nog is once again expressed in cells undergoing resolution from the endoderm (Fig. 6CII:h–j). Interestingly, Bmp7's expression pattern is nearly identical to that of Nog

Nog+/-

Nog-/-

119

(Fig. 6CIII). BMP antagonism in the notochord by NOG, and perhaps by other BMP antagonists, is suggested by the low levels of BMP signaling seen in the resolving notochord at later stages of resolution (Fig. 6CIV:j). Together, these data prompted us to hypothesize that Nog and Bmp7 are part of a dynamic regulatory system that governs the changing behaviors of midline cells as they migrate from the node to the trunk region and begin to resolve from the endoderm.

Noggin null notochords maintain epithelial characteristics F-actin

nc

nc

PRKCZ

DAPI

SOX9

Nog+/-

Nog-/-

nc

nc

DAPI

CDH1 Nog+/-

nc

LAMININ Nog-/-

nc

DAPI

CDH2

SOX9

Previously published work, shows that CDH1 (E-cadherin) is not expressed in the notochord after resolution (Nose and Takeichi, 1986). Since the notochord precursors are initially contiguous with the endodermal epithelium, we suspected that timely loss of CDH1 from the notochord precursors would be important for notochord resolution from the endodermal epithelium. Furthermore, while the dorsal endoderm is a typical epithelium with both apical/basal polarity and a lumen, the notochord does not appear to have a lumen. We suspected that the notochord cells also lack markers of apical polarity. If such a cell rearrangement event were disrupted in Nog null notochords, it could cause the notochord cells to remain adhered to the endoderm cells and help explain the large notochord phenotype. We sought to determine when the down-regulation of CDH1 occurs, whether this down-regulation is disrupted in Nog nulls, and whether other epithelial characteristics are inappropriately maintained in Nog null notochords. To evaluate epithelial characteristics, we observed cortical apical F-actin using phalloidin, and atypical PKCzeta (PRKCZ) by immunofluoresence. While in wildtype notochords, there was no obvious apical domain and very little F-actin or PRKCZ, the apical domain in null mutants was sporadically persistent (n¼ 8). Strong phalloidin and anti-PRKCZ staining was observed in multiple sections along the rostro-caudal axis of the trunk, at what was once the apical domain of these cells (Fig. 7A). Immunohistochemistry against CDH1 and CDH2 revealed that wildtype notochord cells down-regulate CDH1 and up-regulate CDH2 during notochord resolution (Fig. 7B, C). While levels of CDH-2 do not appear to be altered in Nog null mutants as compared to wildtype littermates (Fig. 7B), CDH-1 protein was seen to persist during Stages 3 and 4 of notochord resolution (Figs. 7C and 8A–C). To quantify the persistence of CDH1 protein accurately, wildtype and mutant littermates were processed and embedded together in a single block such that they were sectioned and stained side by side on the same slides. They were then examined by confocal microscopy using the same settings to quantify the integrated density of fluorescence. As a control we quantified DAPI (a nuclear stain) and found no significant difference in fluorescence between wildtype and mutant notochords (Fig. 8A–C). At Stage 4, when the basement membrane normally separates the endoderm from the notochord, CDH1 fluorescence was significantly greater in Nog nulls (Fig. 8A–C). This suggests that CDH1 is not down-regulated appropriately in the forming notochord of Nog mutants. Furthermore, we were able to detect Fig. 7. Epithelial characteristics persist abnormally in Nog  /  notochords after resolution. (A) Phalloidin stain (green, upper) or anti-PRKCZ staining (green, lower) showing persistence of cortical filamentous actin and apical PRKCZ in the notochords of Nog  /  mutants compared to wildtype littermates. SOX9 (pink) marks notochord cells (scale ¼15 μm). (B) Immunohistochemistry showing that CDH2 (red) is up-regulated in Nog  /  notochords and those of their wildtype littermates (scale ¼ 15 μm) DAPI (white) marks nuclei. Anti-laminin (green) marks the basement membrane. (C) Immunohistochemistry showing that CDH1 (red) persists in Nog  /  notochords past the Stage 3 when it is down-regulated in wildtype littermates (scale ¼15 μm). DAPI (white) marks nuclei. Anti-SOX9 (green) marks notochord. Abbr: nc, notochord.

120

S.R. Fausett et al. / Developmental Biology 391 (2014) 111–124

Nog+/-

Nog-/Cdh1 Nog

nc

+/-

Nog-/-

nc nc

nc

ZEB1 DAPI

CDH1

Nog

LAMININ

Nog-/-

+/-

nt

Integrated Density of Fluorescence Normalized to WT Controls

nt 2.5 2

*

WT MT

nc

1.5

nc

1 0.5 0 DAPI

α-CDH1 /

Fig. 8. The ZEB1 Cdh1-regulatory system is altered in Nog mutants during notochord resolution. (A, B) Representative images of anti-CDH1 fluorescence used to quantify CDH1 in wildtype (A) and Noggin null (B) notochords at Stage 4. (C) Quantification of the integrated density of fluorescence of DAPI and CDH1 in Nog null mutants and wildtype littermates that were processed and imaged under identical conditions, showing increased CDH1 in mutant notochords at Stage 4 (p o 0.05). (D, E) In situ hybridization showing persistence of Cdh1 transcript in Nog  /  notochords (E) compared to those of wildtype littermates (D). (F, G) Immunohistochemistry showing that ZEB1 expression is reduced in the neural tube and notochord of Nog null mutants (G) as compared to wildtype littermates (F). Dashed lines show the boundaries of the neural tube and notochord tissues (scale¼ 15 μm). Abbr: nc, notochord.

persistence of Cdh1 transcript in the resolving notochord of Nog mutant embryos relative to stage-matched wildtype littermates (Fig. 8D, E). Taken together, these data demonstrate a persistence of epithelial characteristics in Nog mutant notochords relative to stage-matched wildtype embryos in the foregut region, perhaps indicating that disruption of epithelial characteristics must occur efficiently for successful notochord resolution Bmp signaling may regulate Cdh1 levels during notochord formation via Zeb1 The data presented above support a model in which normal notochord formation involves a partial loss of epithelial integrity that allows the resolution of the notochord cells from the dorsal foregut epithelium. Since such events usually involve the expression of Cdh1 repressors (Yang and Wineberg, 2008), we looked for altered expression of known Cdh1 repressors in the resolving notochord of Nog  /  mutants. In situ hybridization revealed that snai1, snai2, and twist1 are not significantly expressed in the notochord during resolution (Supplemental Fig. 2). The transcriptional repressor Zeb1 is reportedly expressed in the notochord at low levels (Takagi et al., 1998), with notochordal expression also revealed by a LacZ knock-in allele of Zeb1 (Miyoshi et al., 2006). We confirmed this expression via in situ hybridization, and determined that while Zeb1 is expressed at low levels in wildtype notochords during notochord resolution, Zeb1 mRNA is nearly absent in Nog  /  notochords (Supplemental Fig. 3A and B). We

further assayed this expression using an antibody against ZEB1 protein, which also revealed low levels in wildtype notochord but none detectable in Nog mutant notochords (Fig. 8F and G). Work in kidney cell lines has shown that ZEB1 is a transcriptional repressor not only of Chd1 but also of a primary transcript that encodes the microRNAs miR-200a, -200b, and -429 (Eger et al., 2005). These microRNAs, in turn, target Zeb1 transcripts for degradation (Park et al., 2008; Gregory et al., 2008). This negative feedback loop appears to regulate Cdh1 expression and EMT/MET downstream of BMP7 and TGF-beta (Bracken et al., 2008; Samavarchi-Tehrani et al., 2010). We hypothesized that if this system were active during notochord resolution, given the decrease in Zeb1, we would see persistent miR-200a/200b/429 expression in Nog  /  notochords during resolution. Indeed, we detected miR-200 primary transcripts in the endoderm, but not in the notochords of wildtype embryos. In Nog mutants, miR-200 continued to be expressed in the notochords after resolution (Supplemental Fig. 3C and D). Together, these data suggest a model in which the ZEB1 Cdh1-regulatory system, downstream of BMP signaling, participates in the transcriptional regulation of Cdh1 during resolution of the notochord from the dorsal foregut endoderm.

Discussion Several mouse models of EA/TEF have demonstrated that correct dorsal/ventral patterning of the anterior foregut endoderm

S.R. Fausett et al. / Developmental Biology 391 (2014) 111–124

is essential for correct compartmentalization of the trachea and esophagus. Using spatiotemporal genetic dissection of the requirements for the various domains of Nog expression, we find that Noggin is dispensable in the endoderm itself, but is required in the early notochord. We further show that this action of Noggin results in an essential attenuation of BMP signaling in the resolving notochord. Our conclusions lend further support to the view that the EA/TEF defects in Noggin arise as a consequence of too few cells in the endoderm for compartmentalization (Li et al., 2007). Finally, we present mechanistic evidence suggesting that BMP antagonism by notochordal NOG allows a cell rearrangement event involving Cdh1 down-regulation in the notochord cells as they resolve from the foregut endoderm. Loss of ventral BMP signaling or other ventral patterning genes/ pathways results in loss of the ventral domain of the foregut endoderm, and consequently, failure of compartmentalization (Harris-Johnson et al., 2009; Goss et al., 2009; Minoo et al., 1999; Li et al., 2008; Domyan et al., 2011). Conversely, reduced levels of SOX2 from the dorsal domain appear to ventralize the endoderm and induce EA/TEF (Que et al., 2007). Furthermore, these genes and pathways antagonize one another such that SOX2 expression is low in the ventral domain and NKX2-1 is absent from the dorsal domain (Que et al., 2007; Domyan et al., 2011). Given that Nog is expressed dorsally, it was possible that loss of Nog could induce ventralization. Our data, however, support the surprising conclusion that dorsal/ventral patterning in Nog  /  mutants is not disrupted. The result is even more striking considering that in nearly every model of EA/TEF in which D/V patterning has been assessed, the phenotype has been attributed to mis-patterning. In light of this finding, we turned to the alternative hypothesis that the smaller foregut endoderm prevents successful compartmentalization (Li et al., 2007). Li et al. (2007) observed that the Nog  /  dorsal FGE appeared reduced compared to the ventral domain. However, our quantification revealed a smaller foregut endoderm overall. The discrepancy may be dependent on the specific rostro-caudal region observed. Our quantification included the entire region of the foregut thought to give rise to the trachea and esophagus, from the lung buds to the fourth pharyngeal arch at E9.5. Importantly, we found no difference in the size of the foregut endoderm at E8.5, indicating that the deficiency arises between E8.5 and E9.5. Few possibilities exist that could account for such tissue loss. First, rates of cell proliferation or apoptosis could be altered. Second, endoderm cells could be lost to the notochord during resolution. We directly addressed both possibilities. We found no significant change in the amount of apoptosis at E8.5, but rather, significantly more cell proliferation. The increase in proliferation was puzzling, but could have been a response to tissue loss during this time period. To address the second possibility we looked for SOX2expressing cells in the Nog  /  notochord, and found that they do indeed exist, though SOX2 is never seen in wildtype notochords. We therefore conclude that the endoderm is reduced due to impaired notochord resolution. The results of our genetic experiments also support a strong connection between the notochord defect and EA/TEF in Nog  /  mutants. While it was possible that Nog expressed in and around the foregut during foregut compartmentalization was important for that process, we were surprised to find that these spatiotemporal domains of Nog expression were completely dispensable for compartmentalization. Additionally, we discovered using the constitutively active BMPR1A (CAB) in conjunction with ShhCre, Foxg1Cre, and ShhCreER that it is the notochord cells themselves that require BMP antagonism during notochord resolution in order for foregut compartmentalization to occur correctly. Despite our efforts, we were not able to uncouple the notochord and foregut defects by removing NOG or activating BMP signaling after

121

notochord resolution; we succeeded in inducing EA/TEF only by removing NOG or activating BMP signaling in the notochord prior to resolution. Furthermore, we observed that the most severe defects (largest notochords) seemed to correlate with the presence of EA/TEF, and statistically showed that a large notochord is an excellent predictor of EA/TEF. Noggin is known to be expressed in the early notochord (McMahon et al., 1998), but detailed analysis has not been reported. Bmp4 and Bmp7 are also known to be expressed during notochord resolution. However, while Bmp7 expression has been reported in the notochordal plate, Bmp4 expression has only been reported in the endoderm and mesoderm (Danesh et al., 2009; Arkell and Beddington, 1997; Solloway and Robertson, 1999). Therefore, we looked carefully at BMP signaling and the expression of Nog and Bmp7 during migration of the midline cells from the primitive streak and resolution of those cells from the endoderm. Two striking phenomena were observed. First, Nog expression is dynamic in the midline cells, and correlates with active notochord morphogenesis. Second, Bmp7 is expressed in a nearly identical fashion as Nog in and around the notochord. These expression patterns suggest that BMP signaling and BMP antagonism act in balance to dynamically regulate the morphogenesis of the notochord at this stage. Importantly, reducing the gene dosages of Bmp4 or Bmp7 can rescue the Nog null notochord defects as well as EA/TEF (Que et al., 2006; Li et al., 2007). It would be interesting to test whether removing Bmp7 after notochord resolution can still rescue EA/TEF in Noggin null mutants using an inducible Cre and a floxed allele of Bmp7. Since the process of resolution must involve the notochord cells detaching from their endoderm neighbors, and since BMP7 is known to promote epithelial characteristics in several contexts, we hypothesized that NOG was allowing the notochord cells to lose some of their epithelial characteristics in order to resolve from the endoderm. Indeed, we found that the epithelial markers apical F-actin, PRKCZ, and CDH1 are absent from the notochord cells after resolution. However, these markers persist in Nog  /  notochords at least until E9.5. Furthermore, careful quantification of antiCDH1 fluorescence confirmed significantly higher levels of CDH1 in Stage 4 Nog  /  notochords compared to wildtype. Together, these data support a model in which BMP antagonism in the notochord by NOG is important for reducing epithelial characteristics that prevent proper notochord resolution. In the future, it would be very interesting to develop mouse lines that would allow genetic reduction of epithelial characteristics in the notochords of Nog  /  mutants to rescue these phenotypes. Conversely, genetically enhancing epithelial characteristics in wildtype notochords may induce both large notochords and EA/TEF. We note that CDH1 was also reported to be expressed in the notochord of embryos in the Adriamycin model for EA/TEF (Hajduk et al., 2012). It remains unclear whether there is a molecular connection between the effects of Adriamycin in embryonic organogenesis and the increased BMP signaling resulting from loss of Noggin. It seems likely that the two molecular perturbations converge on some common molecular effectors of notochord development. In order to discover factors that may be downstream of BMP antagonism and upstream of Cdh1, we chose to look at the expression of known Cdh1 repressors. While we found that snai1, snai2, and twist1 are not expressed at the right time or place, Zeb1 is both expressed in wildtype notochords and abnormally reduced in Nog  / notochords. ZEB1 is a transcriptional repressor of Cdh1 as well as of the miR-200a/200b/429 transcript (Bracken et al., 2008). The mir-200a/200b/429 transcript, in turn, generates mature microRNAs that target Zeb1 transcripts for degradation. Thus, ZEB1 and the miR-200-family microRNAs negatively regulate one another, and expression of one over the other acts as a switch that is capable of turning Cdh1 expression on or off (Bracken et al., 2008).

122

S.R. Fausett et al. / Developmental Biology 391 (2014) 111–124

Furthermore, this paradigm is known to be downstream of BMP7 in iPSCs and a mouse model of renal injury, such that BMP7 drives Cdh1 expression and other epithelial characteristics (SamavarchiTehrani et al., 2010; Ziesberg et al., 2003). Our expression data strongly reflect the known roles of these genes. Cdh1 and the miR200a/200b/429 transcripts are expressed highly in the endodermal epithelium, and are absent from the notochord cells during and after resolution as other epithelial characteristics are lost. Conversely, Zeb1 is not highly expressed in the endoderm, but turns on in the notochord during resolution. In Nog  / mutants this arrangement is disrupted and both Cdh1 and the mir-200a/200b/429 transcript are expressed abnormally in the notochord cells while Zeb1 is absent. In light of these results, we hypothesized that Zeb1 null mutants might have also notochord defects. We examined histological sections of E10.5 Zeb1 null embryos provided by Dr. Yujiro Higashi and Dr. Przemko Tylzanowski (Takagi et al., 1998). In the limited number of samples examined (n¼5) only one embryo had an abnormally large notochord (data not shown). The relative lack of notochord defects we observed in Zeb1 null mice could be due to either functional redundancies, or to incomplete penetrance. In any case, it would be very interesting to look more closely at the notochord in Zeb1 mutants and in Zeb1/Zeb2 double knockouts, as well as to develop new genetic tools that target the miR-200 family. Of note, Zeb1 expression is also altered in ventral neural tube in Nog  / mutants. It is unclear whether this results in any functional or physical defects, and it would be interesting to test whether levels of Cdh1 and Cdh2 are altered in the floorplate of Nog nulls. In conclusion, this study has revealed that EA/TEF can arise by a mechanism other than that of failed D/V patterning of the foregut endoderm. We have presented evidence that supports a model, previously put forth by Li et al. (2007), in which faulty notochord resolution causes foregut hypocellularity prior to compartmentalization, and thus causes EA/TEF. We have established that notochord resolution from the foregut endoderm involves gene expression changes that reflect a loss of epithelial characteristics in presumptive notochord cells, perhaps driven by the ZEB/miR200 regulatory system downstream of BMP7. The development of new genetic tools should allow further exploration into the mechanistic details of notochord resolution and its impact on compartmentalization of anterior foregut.

recognizing the 50 and 30 regions of the targeting construct flanking loxP sites (Supplemental Table 1). To induce cre-mediated recombination using the ShhCreER allele, pregnant dams were administered tamoxifen (Sigmas) dissolved in corn oil by oral gavage at doses between 0.08 and 0.1 mg/g body weight. Immunofluorescent and immunohistochemical staining With the exception of the ZEB1 antibody, which was applied to unfixed tissue, embryos were fixed 30 min to 1 h at room temperature in 4% paraformaldehyde. For sectioning, embryos were processed through a sucrose gradient and embedded in 1:3 15% Sucrose:OCT (Tissue-Teks). Tissue was sectioned at 6–10 μm and then stained using standard protocols. Primary antibodies used: goat anti-SOX2 (Neuromics), mouse anti-NKX2-1 (ThermoScientific), rabbit anti-GFP (Abcam), mouse anti-phosphohistone H3 (Cell Signaling Technologies), rabbit anticleaved caspase 3 (Cell Signaling Technologies), rat anti-CDH1 (Invitrogen), rabbit anti-laminin (Lightner et al., 1989), goat antiBrachyury (Santa Cruz), rabbit anti-PRKCZ (Santa Cruz), anti-ZEB1 (E-20:sc-10572, Santa Cruz), rat anti-CDH2 (MNCD2, Developmental Studies Hybridoma Bank), goat anti-ZEB1(E-20) (sc-10572, Santa Cruz). Filamentous actin was visualized using Alexa Fluors-488 conjugated phalloidin at 1:200. Nuclei were visualized using DAPI. For colorimetric visualization of antibodies, an HRP-conjugated secondary was used and subsequently detected with a solution of 3,30 -diaminobenzidine and nickel ammonium sulfate. RNA isolation and quantitative PCR Foreguts (between the 4th pharyngeal pouch and lung buds) were dissected from E10.5 Noggin nulls and wildtype littermates, and stored at  80 1C. Two to five foreguts were pooled for each biological replicate. RNA was extracted using a standard TRIzols protocol. Reverse transcription was carried out using the iScript cDNA synthesis kit from BioRad. Quantitative PCR was performed using SYBRGreen (Applied Biosystems) and an Applied Biosystems StepOnePlus Real Time RT-PCR system. Primers are listed in Supplemental Table 1. H&E and Xgal staining

Methods Generation of mutant embryos and genotyping Except for lines used with Foxg1Cre, which were backcrossed 5– 6 generations to 129/SvEv, all mice were bred on undefined outbred backgrounds. All genotyping was performed on dissected tissues (tails, limbs, or extraembryonic membranes) according to published protocols (except NogDel, see below) and wildtype littermates were used as controls (Supplemental Table 1). Previously published alleles used in this study: BRE-Hspa1alacz reporter strain (BRE-LacZ) (Blank et al., 2008), Gt(ROSA) 26Sortm1Sor (R26R-LacZ) (Soriano, 1999), Gt(ROSA)26Sortm1(EYFP)Cos (R26R-eYFP) (Srinivas et al., 2001), Shhtm1(EGFP/cre)Cjt (ShhCre) (Harfe et al., 2004), Shhtm2(cre/ERT2)Cjt(ShhCreER) (Harfe et al., 2004), Foxg1tm1(cre)Skm (Foxg1Cre) (Hebert and McConnell, 2000), Nogtm1.1Rmh(NogFx) (Stafford et al., 2011), NogLacZ (McMahon et al., 1998), Rosa26CAG-loxpstoploxp-caBmprIa (CAB) (Rodriguez et al., 2010), Zeb1tm2Yhi (Takagi et al., 1998). The null allele of Noggin used in this study (NogDel) was generated in our colony by recombining the NogFx allele in the germline using Alpltm1(cre)Nagy (Lomeli et al., 2000). Excision of the single exon of the Nog gene was confirmed using primers

H&E staining and Xgal staining were conducted using standard protocols. Cell counting assays and statistics Serial sections (6 μm) of E8.5 or 9.5 foreguts were stained with anti-cleaved caspase 3, anti-phosphohistone H3, and DAPI. Stained slides were imaged and images were analyzed using ImageJ. Subsets of labeled cells in the foregut are reported as a percent of the total cells counted in the foregut between the 4th pharyngeal pouch and division of the lung bud at E9.5 or between the 2nd pharyngeal pouch and the anterior intestinal portal at E8.5. Values are reported as mean 7s.e.m and were compared using a Student's T-test. Differences were considered significant at pr 0.05. In situ hybridization and cloning of Zeb1 and miR-200 in situ probes A 947 bp fragment of exon 6 from the Zeb1 mRNA was isolated from E8.5 ICR (Harlan) cDNA. Similarly, a 3 kb fragment spanning most of the miR-200b/mir-200a/mir-429 unprocessed transcript was isolated from E8.5 ICR (Harlan) cDNA. These fragments were

S.R. Fausett et al. / Developmental Biology 391 (2014) 111–124

each cloned into a pGEMs-T easy vector from Promega. All other constructs used to generate riboprobes were previously published: Bmp7 (Ozkaynak et al., 1991), Brachyury (Herrmann, 1991), Noggin (McMahon et al., 1998), Cdh1 (Xu et al., 2002). To detect transcripts in situ, embryos were fixed overnight at 4 1C in 4% PFA then dehydrated to 100% methanol for storage. Hybridization was performed as previously published (Belo et al., 1997), except that the hybridization buffer contained 5% dextran sulfate. After whole-mount in situ hybridization, embryos were embedded for frozen sectioning. Notochord comparative measurements and statistics Serial sections of various mutant classes at E12.5 were used for notochord measurement. The cross-sectional area of the notochord, in each section from the rostral limit of the esophagus to the branching of the main bronchi, was measured using ImageJ. The average cross-sectional area for each embryo was compared to that of a wildtype littermate that had been processed identically, and reported as a percent of wildtype. A likelihood ratio test was performed using JMP8s. Microscopy and CDH1 quantification with statistics All microscopy and imaging was carried out either on a Zeiss 710 inverted confocal, a Zeiss AxioPlan 2, or a Leica MZ12 stereoscope. For quantification of anti-CDH1 fluorescence, mutant embryos were frozen embedded alongside a wildtype littermate so that they were sectioned, stained, and imaged together. Slides were stained with anti-CDH1, anti-laminin (to highlight tissue boundaries), and DAPI. Sections of paired littermates' Stage 4 notochords were imaged on the confocal microscope and images were analyzed using ImageJ. The fluorescence was quantified within the notochord, using laminin to mark the boundaries, and using the “integrated density” measurement in ImageJ. Pairs of embryos were re-scaled to the control embryo for comparison using the formula xnew ¼ (x  xmin/xmin  xmax) where xmin and xmax are taken from the control embryo. Normalized values were reported as mean 7sem and compared using Student's T-test. p r0.05 was considered significant. Quantification of DAPI staining using the same method is presented for comparison.

Acknowledgments We are grateful to members of the Klingensmith and Capel Labs for discussion and technical assistance, particularly Kathy Carmody. Dr. Harold Erikson provided the anti-laminin antibody. We also acknowledge the Duke University Light Microscopy Core. The BMP reporter mouse strain was kindly provided by Dr. Lief Oxenberg. The CAB mouse and the NogFx strains were generously provided by Dr. Fan Wang and Dr. Richard Harland, respectively. Dr. Przemko Tylzanowski and Dr. Yujiro Higashi provided Zeb1 null embryo samples. This work was supported by grants from the National Institutes of Health to Dr. John Klingensmith.

Appendix A. Supplementary materials Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ydbio.2014.02.008. References Arkell, R., Beddington, R.S., 1997. BMP-7 influences pattern and growth of the developing hindbrain of mouse embryos. Development 124 (1), 1–12.

123

Belo, J.A., Bouwmeester, T., Leyns, L., Kertesz, N., Gallo, M., Follettie, M., De Robertis, E.M., 1997. Cerberus-like is a secreted factor with neutralizing activity expressed in the anterior primitive endoderm of the mouse gastrula. Mech. Dev. 68, 45–57. Blank, U., Seto, M.L., Adams, D.C., Wojchowski, D.M., Karolak, M.J., Oxburgh, L., 2008. An in vivo reporter of BMP signaling in organogenesis reveals targets in the developing kidney. BMC Dev. Biol. 8, 86, http://dx.doi.org/10.1186/1471-213X-8-86. Bracken, C.P., Gregory, P.A., Kolesnikoff, N., Bert, A.G., Wang, J., Shannon, M.F., Goodall, G.J., 2008. A double-negative feedback loop between ZEB1-SIP1 and the microRNA-200 family regulates epithelial–mesenchymal transition. Cancer Res. 68, 7846. Buckley, S., Shi, W., Dirscoll, B., Ferrario, A., Anderson, K., Warburton, D., 2003. BMP4 signaling induces senescence and modulates the oncogenic phenotype of A549 lung adenocarcinoma cells. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L81–L86, http://dx.doi.org/10.1152/ajplung.00160.2003. Cleaver, O., Krieg, P.A., 2001. Notochord patterning of the endoderm. Dev. Biol. 234 (1), 1–12. Danesh, S.M., Villasenor, A., Chong, D., Soukup, C., Cleaver, O., 2009. BMP and BMP receptor expression during murine organogenesis. Gene Expr. Patterns 9 (5), 255–265. Domyan, E.T., Ferretti, E., Throckmorton, K., Mishina, Y., Nicolis, S.K., Sun, X., 2011. Signaling through BMP receptors promotes respiratory identity in the foregut via repression of Sox2. Development 138, 971–981. Eger, A., Aigner, K., Sonderegger, S., Dampier, B., Oheler, S., Schreiber, M., Berx, G., Cano, A., Beug, H., Foisner, R., 2005. Delta-EF1 is a transcriptional repressor of E-cadherin and regulates epithelial plasticity in breast cancer cells. Oncogene 24, 2375–2385. Fausett, S.R., Klingensmith, J., 2012. Compartmentalization of the foregut tube: developmental origins of the trachea and esophagus. WIREs Dev. Biol. 1, 184–202, http://dx.doi.org/10.1002/wdev.12. Goss, A.M., Tian, Y., Tsukiyama, T., Cohen, E.D., Zhou, D., Lu, M.M., Yamaguchi, T.P., Morrisey, E.E., 2009. Wnt2/2b and beta-catenin signaling are necessary and sufficient to specify lung progenitors in the foregut. Dev. Cell. 17, 290–298. Gregory, P.A., Bert, A.G., Paterson, E.L., Barry, S.C., Tsykin, A., Farshid, G., Vadas, M.A., Khew-Goodal, Y., Goodall, G.J., 2008. The miR-200 family and miR-205 regulate epithelial to mesechymal transition by targeting ZEB1 and SIP1. Nat. Cell Biol. 10, 593–601. Guha, U., Gornes, W.A., Kobayashi, T., Pestell, R.G., Kessler, J.A., 2002. In vivo evidence that BMP signaling is necessary for apoptosis in the mouse limb. Dev. Biol. 249, 108–120. Hajduk, P., May, A., Puri, P., Murphy, P., 2012. The effect of adriamycin exposure on the notochord of mouse embryos. Birth Defects Res. B Dev. Reprod. Toxicol. 95 (2), 175–183. Harfe, B.D., Scherz, P.J., Nissim, S., Tian, H., McMahon, A.P., Tabin, C.J., 2004. Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities. Cell 118 (4), 517–528. Harris-Johnson, K.S., Domyan, E.T., Vezina, C.M., Sun, X., 2009. Beta-catenin promotes respiratory progenitor identity in mouse foregut. Proc. Natl. Acad. Sci. USA 106, 16287–16292. Hebert, J.M., McConnell, S.K., 2000. Targeting of cre to the foxg1 (BF-1) locus mediates loxp recombination in the telencephalon and other developing head structures. Dev. Biol. 222, 296–306. Herrmann, B.G., 1991. Expression pattern of the Brachyury gene in whole-mount TWis/Wis mutant embryos. Development 113, 913–917. Ioannides, A.S., Chaudhry, B., Henderson, D.J., Spitz, L., Copp, A.J., 2002. Dorsoventral patterning in oesophageal atresia with tracheo-oesophageal fistula: evidence from a new mouse model. J. Pediatr. Surg. 37, 185–191. Ioannides, A.S., Massa, V., Ferraro, E., Cecconi, F., Spitz, L., Henderson, D.J., Copp, A.J., 2010. Foregut separation and tracheo-oesophageal malformations: the role of tracheal outgrowth, dorso-ventral patterning, and programmed cell death. Dev. Biol. 337, 351–362. Jurand, A., 1974. Some aspects of the development of the notochord in mouse embryos. J. Embryol. Exp. Morphol. 32, 1–33. Li, Y., Gordon, J., Manley, N.R., Litingtung, Y., Chiang, C., 2008. BMP4 is required for tracheal formation: a novel mouse model for tracheal agenesis. Dev. Biol. 322, 145–155. Li, Y., Litingtung, Y., Dijke, P.T., Chiang, C., 2007. Aberrant BMP signaling and notochord delamination in the pathogenesis of esophageal atresia. Dev. Dyn. 236, 746–754. Lightner, V.A., Gumbowski, F., Bigner, D.D., Erickson, H.P., 1989. Tenascin/hexabrachion in human skin: biochemical identifcation and localization by light and electron microscopy. J. Cell Biol. 108, 2483–2493. Lomeli, H., Ramos-Mejia, V., Gertsenstein, M., Lobe, C.G., Nagy, A., 2000. Targeted insertion of Cre recombinase into the TNAP gene: excision in primordial germ cells. Genesis 26 (2), 116–117. McMahon, J.A., Takada, S., Zimmerman, L.B., Fan, C.M., Harland, R.M., McMahon, A. P., 1998. Noggin-mediated antagonism of BMP signaling is required for growth and patterning of the neural tube and somite. Genes Dev. 12 (10), 1438–1452. Minoo, P., Su, G., Drum, H., Bringas, P., Kimura, S., 1999. Defects in tracheosophageal and lung morphogenesis in Nkx2.1(  /  ) mouse embryos. Dev. Biol. 209, 60–71. Miyoshi, T., Maruhashi, M., Van De Putte, T., Kondoh, H., Huylebroeck, D., Higashi, Y., 2006. Complementary expression pattern of Zfhx1 genes Sip1 and deltaEF1 in the mouse embryo and their genetic interaction revealed by compound mutants. Dev. Dyn. 235 (7), 1941–1952. Miyazono, K., Miyazawa, K., 2002. id: a target of BMP signaling. Sci. Signal. 151, pe40.

124

S.R. Fausett et al. / Developmental Biology 391 (2014) 111–124

Nose, A., Takeichi, M., 1986. A novel cadherin cell adhesion molecule: its expression patterns associated with implantation and organogenesis of mouse embryos. J. Cell Biol. 103 (6), 2649–2658. Ozkaynak, E., Schnegelsberg, P.N., Oppermann, H., 1991. Murine osteogenic protein (OP1): high levels of mRNA in kidney. Biochem. Biophys. Res. Commun. 179, 116–123. Park, S.M., Gaur, A.B., Lengyel, E., Peter, M.E., 2008. The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev. 22, 894–907. Possoegel, A.K., Diez-Pardo, J.A., Morales, C., Tovar, J.A., 1999. Notochord involvement in experimental esophageal atresia. Pediatr. Surg. Int. 15, 201–205. Que, J., Choi, M., Ziel, J.W., Klingensmith, J., Hogan, B.L.M., 2006. Morphogenesis of the trachea and esophagus: current players and new roles for Noggin and BMPs. Differentiation 74, 422–437. Que, J., Okubo, T., Goldenring, J.R., Nam, K., Kurotani, R., Morrisey, E.E., Taranova, O., Pevny, L.H., Hogan, B.L.M., 2007. Multiple dose-dependent roles for Sox2 in the patterning and differentiation of the anterior foregut endoderm. Development 134, 2521–2531. Rodriguez, P., Da Silva, S., Oxburgh, L., Wang, F., Hogan, B.L.M., Que, J., 2010. BMP signaling in the development of the mouse esophagus and forestomach. Development 137, 4171–4176. Samavarchi-Tehrani, P., Golipour, A., David, L., Sung, H., Beyer, T.A., Datti, A., Woltjen, K., Nagy, A., Wrana, J.L., 2010. Functional genomics reveals a BMPdriven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Cell Stem Cell 7 (1), 64–77. Solloway, M.J., Robertson, E.J., 1999. Early embryonic lethality in Bmp5;Bmp7 double mutant mice suggests functional redundancy within the 60A subgroup. Development 126 (8), 1753–1768. Soriano, P., 1999. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21 (1), 70–71. Srinivas, S., Watanabe, T., Lin, C.S., William, C.M., Tanabe, Y., Jessell, T.M., Costantini, F., 2001. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1 (1), 4.

Stafford, D.A., Brunet, L., Khokha, M.K., Economides, A.N., Harland, R.M., 2011. Cooperative activity of Noggin and gremlin 1 in axial skeleton development. Development 138 (5), 1005–1014. Takagi, T., Moribe, H., Kondoh, H., Higashi, Y., 1998. DeltaEF1, a zinc finger and homeodomain transcription factor, is required for skeleton patterning in multiple lineages. Development 125 (1), 21–31. Tam, P.P.L., Khoo, P.L., Lewis, S.L., Bildsoe, H., Wong, N., Tsang, T.E., Gad, J.M., Robb, L., 2007. Sequential allocation and global pattern of movement of the definitive endoderm in the mouse embryo during gastrulation. Development 134, 251–260. Thompson, D.J., Molello, J.A., Strebing, R.J., Dyke, I.L., 1978. Teratogenicity of adriamycin and daunomycin in the rat and rabbit. Teratology 17, 151–158. Torfs, C.P., Curry, C.J.R., Bateson, T.F., 1995. Population-based study of tracheoesophageal fistula and esophageal atresia. Teratology 52, 220–232. Tribulo, C., Aybar, M.J., Nguyen, V.H., Mullins, M.C., Mayor, R., 2003. Regulation of msx genes by a bmp gradient is essential for neural crest specification. Development 130, 6441–6452. Wilkinson, D.G., Bhatt, S., Herrmann, B.G., 1990. Expression pattern of the T gene and its role in mesoderm formation. Nature 343, 657–659. Woo, J., Miletich, I., Kim, B.M., Sharpe, P.T., Shivdasani, R.A., 2011. Barx1-mediated inhibition of Wnt signaling in the mouse thoracic foregut controls tracheoesophageal septation and epithelial differentiation. PLoS One 6 (7), e22493, http://dx.doi.org/10.1371/journal.pone.0022493. Xu, L., Overbeek, P.A., Reneker, L.W., 2002. Systematic analysis of E-, N-, and P-cadherin expression in mouse eye development. Exp. Eye Res. 74, 753–760. Yang, J., Wineberg, R.A., 2008. Epithelial–mesenchymal transition: at the crossroads of development and tumor metastasis. Dev. Cell 14 (6), 818–829. Ziesberg, M., Hanai, J., Sugimoto, H., Mammoto, T., Charytan, D., Strutz, F., Kalluri, R., 2003. BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat. Med. 9 (7), 964–968.