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Title Developmental robustness in the Caenorhabditis elegans embryo.

Permalink https://escholarship.org/uc/item/5pt2w94n

Journal Molecular reproduction and development, 82(12)

ISSN 1040-452X

Author Maduro, Morris F

Publication Date 2015-12-01

DOI 10.1002/mrd.22582

Peer reviewed

eScholarship.org Powered by the California Digital Library University of California Molecular Reproduction and Development

Developmental Robustne ss in the C. elegans Embryo

Journal:For Molecular Peer Reproduction Review and Development Manuscript ID: MRD-15-0169.R1

Wiley - Manuscript type: Review Article

Date Submitted by the Author: n/a

Complete List of Authors: Maduro, Morris; University of California, Riverside, Biology

Keywords: Endoderm, C. elegans, Robustness, Gene Regulatory Networks

John Wiley & Sons Page 1 of 42 Molecular Reproduction and Development

1 2 3 4 5 6 7 Developmental Robustness in the C. elegans Embryo 8 9 10 11 12 13 14 15 Morris F. Maduro* 16 17 18 *Department of Biology,For University ofPeer California, Riverside, Review Riverside, CA 92521 19 20 21 22 23 24 University of California, Riverside 25 26 27 Riverside, CA 28 29 30 USA 92521 31 32 33 [email protected] 34 35 36 37 38 39 Running title: ROBUSTNESS OF EMBRYONIC DEVELOPMENT 40 41 42 43 44 45 Abbreviations: bZIP, basic leucine zipper domain; GFP, green fluorescent protein; GRN, gene regulatory 46 47 48 network; MAPK, mitogen-activated protein kinase; smFISH, single-molecule fluorescent in situ 49 50 hybridization 51 52 53 Funding : National Science Foundation Grant IOS#1258054 54 55 56 57 58 59 60 John Wiley & Sons Molecular Reproduction and Development Page 2 of 42

Maduro, p. 2 1 2 3 4 5 6 SUMMARY 7 8 9 10 Developmental robustness is the ability of an embryo to develop normally despite many sources of 11 12 variation, from differences in the environment to stochastic cell-to-cell differences in gene expression. 13 14 The nematode, Caenorhabditis elegans , exhibits an additional layer of robustness: Unlike most other 15 16 17 animals, the embryonic pattern of cell divisions is nearly identical from animal to animal. The endoderm 18 For Peer Review 19 (gut) lineage is a model for the study of developmental robustness: The juvenile gut has a simple 20 21 anatomy, consisting of 20 cells that are derived from a single cell, E; and the gene regulatory network 22 23 (GRN) that controls E specification shares features in common with developmental GRNs in many 24 25 26 systems, including genetic redundancy, parallel pathways and feed forward loops. While early studies 27 28 were more concerned with identifying the genes in the network, recent work has turned to 29 30 understanding how the endoderm produces a robust developmental output in the face of many sources 31 32 33 of variation. Genetic control exists at three levels of endoderm development: Progenitor specification, 34 35 the cell divisions within the developing gut, and maintenance of gut differentiation. Recent findings 36 37 show that specification genes regulate all three of these aspects of gut development, and that mutant 38 39 40 embryos can experience a "partial" specification state in which some, but not all, of the E descendants 41 42 adopt a gut fate. Ongoing studies using newer quantitative and genome-wide methods promise further 43 44 insights into how developmental GRNs buffer variation. 45 46 47 48 49 50 51 INTRODUCTION 52 53 54 Embryos of triploblastic animals are incredible macromolecular machines. Cells descend from 55 56 the zygote through multiple mitotic divisions, precursor cells become specified, and ultimately tissue- 57 58 59 60 John Wiley & Sons Page 3 of 42 Molecular Reproduction and Development

Maduro, p. 3 1 2 3 specific patterns of gene expression will drive morphogenesis and differentiation. Despite widely varying 4 5 6 conditions, embryos of many species are able to form very similar juveniles with minimal phenotype 7 8 variability. This phenomenon can be called developmental robustness and it is synonymous with 9 10 canalization , the notion that only a finite number of developmental trajectories are possible 11 12 13 (Waddington, 1942): Small variations are buffered so that cells become "canalized" toward a stable, 14 15 consistent outcome. 16 17 18 In discussing robustness,For biologists Peer recognize Review three sources of variation: Environmental 19 20 variation, genetic variation and stochastic noise (Felix and Wagner, 2008). Environmental variation 21 22 23 includes differences in nutrition and temperature. Genetic variation includes effects of different alleles 24 25 segregating in a population as a result of mutations or recombination. Stochastic noise is the result of 26 27 cell-to-cell (and embryo-to-embryo) differences in protein levels caused by inherent variability in the 28 29 30 many steps of gene expression, including the availability of transcription factors, DNA accessibility, RNA 31 32 polymerase initiation rates, mRNA degradation, and rate of translation (Chalancon et al., 2012). While 33 34 stochastic variation is useful in certain contexts, such as in a population of cells that are experiencing a 35 36 novel stress, during development the effect of noise must be minimized (Macneil and Walhout, 2011). 37 38 39 The ability of embryos to buffer sources of variation will determine both the consistency of form and 40 41 function from one individual to the next, and the success of the species over time. 42 43 44 While robustness helps development, it actually works against the goals of a developmental 45 46 geneticist. Genes are classically identified through mutations that result in a phenotype – that is, one 47 48 49 must observe a loss of robustness in order to assign a function to a gene. Hence, the first genes 50 51 identified in a developmental process tend to be the most important core regulators at the 'top' of a 52 53 hierarchical network. Once a core set of genes is known, it becomes possible to identify more genes by 54 55 56 other methods, such as by reverse genetics, analysis of expression, protein-DNA interactions, or by the 57 58 59 60 John Wiley & Sons Molecular Reproduction and Development Page 4 of 42

Maduro, p. 4 1 2 3 ability of knockdowns to synergize with weaker alleles of known mutants. Over time, a comprehensive 4 5 6 regulatory network can be assembled that accounts for development of a particular tissue, from its 7 8 embryonic progenitors to differentiated cells. In turn, this allows the effects of upstream perturbations 9 10 to be studied on all downstream target genes. 11 12 13 The features that make C. elegans a good model system also make it useful for the study of 14 15 16 developmental robustness. Many sources of variation can be minimized. Animals can be fed the same 17 18 food source and be grownFor at the same Peer temperature. ReviewFemales are capable of self-fertilization, so that 19 20 self-progeny of homozygous strains will be genetically identical. While mutations can occur during 21 22 23 development or in the germline, these are likely to be rare, and their effects can be minimized by 24 25 regularly obtaining reference strains from frozen storage (Brenner, 1974). Compared with most animals, 26 27 C. elegans embryonic development is robust in an additional way: Cell divisions follow a highly 28 29 30 stereotyped pattern that is nearly identical among wild-type embryos (Sulston et al., 1983). This 31 32 stereotyped cell lineage means that even minor changes to early development can be detected by 33 34 changes in cell division pattern or the tissue types made by specific cells. The combination of 35 36 hermaphrodite genetics and a stereotyped lineage means that the major sources of variation in 37 38 39 development are stochastic noise in gene expression, and any introduced genetic differences. 40 41 42 Several recent reviews cover gene regulatory networks and robustness in general (Macneil and 43 44 Walhout 2011), mechanisms by which expression noise and networks work together (Chalancon et al., 45 46 2012), developmental robustness in other systems (Gursky et al., 2012), and robustness from an 47 48 49 evolutionary perspective in Caenorhabditis (Felix and Wagner, 2008). Rather than duplicate these 50 51 efforts, this review will describe how the C. elegans embryo serves as a model for studies of 52 53 developmental robustness, focusing on the gene network that controls development of the endoderm 54 55 56 57 58 59 60 John Wiley & Sons Page 5 of 42 Molecular Reproduction and Development

Maduro, p. 5 1 2 3 (gut) as this network is the best-understood. I will describe gut development from the perspective of 4 5 6 genetic mechanisms that assure its robustness, and pose questions for future exploration. 7 8 9 10 11 12 C. elegans EMBRYONIC DEVELOPMENT 13 14 15 During C. elegans embryogenesis, a single-celled zygote develops into a 558-cell juvenile over 14 16 17 h at 25 °C (Fig. 1) (Sulston et al., 1983). Embryogenesis takes place entirely within an eggshell made 18 For Peer Review 19 20 primarily of chitin (Olson et al., 2012). During the first half of development, the zygote undergoes 21 22 mitotic divisions to generate hundreds of genetically identical cells, while in the second half, the embryo 23 24 undergoes morphogenesis and elongation to adopt the shape of a worm (Sulston et al., 1983). Wild-type 25 26 27 embryos follow nearly the same pattern of cell divisions from the zygote. This nearly-invariant pattern 28 29 can be represented by a cell lineage diagram as shown in Fig. 1. The early cleavages generate six founder 30 31 cells that will each generate a unique set of tissues among their descendants (Sulston et al., 1983). The 32 33 34 founder cell E generates the 20 cells that form the larval intestine (the endoderm). As another example, 35 36 the sister cell of E, called MS, generates primarily mesodermal cell types, including 31 pharynx cells and 37 38 28 muscle cells (Sulston et al., 1983). 39 40 41 Developing C. elegans embryos demonstrate some variability in cell positions, particularly in the 42 43 44 early embryo, although later development appears to be very similar (Schnabel et al., 1997). Overall 45 46 embryonic development is robust to temperatures between 12 °C and 25°C, but cell divisions take longer 47 48 as temperature is lowered (Schnabel et al., 1997; Prasad et al., 2011; Neves et al., 2015). The related 49 50 species C. briggsae has a similar range for embryo viability (14 C-27 C), and even though C. elegans and 51 ° ° 52 53 C. briggsae are separated by 18-100 million years, they share a nearly identical embryonic cell lineage 54 55 (Sulston et al., 1983; Stein et al., 2003; Cutter, 2008; Zhao et al., 2008). Therefore, embryogenesis is 56 57 58 59 60 John Wiley & Sons Molecular Reproduction and Development Page 6 of 42

Maduro, p. 6 1 2 3 robust to slight differences in positions of cells in the early embryo, environment (i.e. temperature) and 4 5 6 to the accumulation of genetic changes over evolutionary time. 7 8 9 Unlike the early embryos of vertebrates, development of C. elegans is mosaic: If an embryonic 10 11 cell is killed with a laser microbeam, or it fails to adopt the correct identity, its normal descendants will 12 13 be missing from the larva (Sulston et al., 1983). Because the embryo does not compensate for missing or 14 15 16 altered cells, changes to the cell lineage persist until the end of development and even adulthood. The 17 18 earlier a change occurs toFor the lineage, Peer the more descendants Review will be affected; hence, mutations 19 20 affecting the earliest cell specification events will affect more descendants and usually cause overall 21 22 23 developmental arrest. Such is the case with strong mutations that affect gut specification, which were 24 25 among the first embryonic developmental mutants to be identified (Bowerman et al., 1992; Lin et al., 26 27 1995). 28 29 30 31 32 33 34 THE ENDODERM GENE REGULATORY NETWORK 35 36 37 Maternal Factors 38 39 40 Early C. elegans development requires many maternally-supplied gene products. Screens for 41 42 maternal-effect lethal mutations affecting gut identified the gene skn-1 (Bowerman et al., 1992). 43 44 Embryos lacking skn-1 activity arrest as a clump of cells lacking the normal descendants of MS, and 45 46 47 specify gut only 20-30% of the time (Bowerman et al., 1992). In such embryos, the MS blastomere, and 48 49 E, if gut is absent, produce epidermal and muscle cells, similar to the C founder cell. Consistent with 50 51 SKN-1 being at the top of a gene network that specifies both MS and E, the SKN-1 protein is a 52 53 54 transcription factor similar to bZIP proteins, and it is found in EMS, the mother of MS and E (Bowerman 55 56 et al., 1993; Blackwell et al., 1994). 57 58 59 60 John Wiley & Sons Page 7 of 42 Molecular Reproduction and Development

Maduro, p. 7 1 2 3 Two other maternal genes contribute directly to specification of gut in addition to skn-1. The 4 5 6 first, pop-1, encodes a protein that functions in specification of many cells throughout development (Lin 7 8 et al., 1998). POP-1 is TCF/LEF transcription factor that works with the co-activator SYS-1/β-catenin to 9 10 contribute to E specification (Shetty et al., 2005; Phillips et al., 2007). The contribution of POP-1/SYS-1 to 11 12 13 E specification results from transduction of a Wnt/MAPK/Src signaling pathway that occurs between 14 15 EMS and its posterior neighbor P 2 (shown as an arrow on the four-cell embryo in Fig. 1) (Rocheleau et 16 17 al., 1997; Thorpe et al., 1997; Bei et al., 2002). Although POP-1 contributes to gut specification in E in 18 For Peer Review 19 parallel with SKN-1, its role in E specification is not apparent unless skn-1 activity is first eliminated: Loss 20 21 22 of skn-1 and pop-1 together results in a higher number of embryos missing gut than loss of skn-1 alone, 23 24 while loss of pop-1 by itself does not affect gut specification (Lin et al., 1995; Maduro et al., 2005b; 25 26 Shetty et al., 2005). The second maternal gene, pal-1, encodes a transcription factor similar to those in 27 28 29 the Caudal family of homeodomain proteins (Hunter and Kenyon, 1996). Like pop-1, the contribution of 30 31 pal-1 is apparent only when gut specification has been partially compromised. Simultaneous loss of skn- 32 33 1, pop-1 and pal-1 results in complete elimination of gut (Maduro et al., 2005b). There are many other 34 35 36 maternal factors that affect gut specification, but these generally affect the activity of SKN-1 or POP-1 in 37 38 E, or cause inappropriate specification of gut in other cells (reviewed in Maduro, 2009). 39 40 41 Zygotic Factors 42 43 44 All three of SKN-1, POP-1 and PAL-1 are present in the early E lineage, though they soon 45 46 disappear after the E cell divides, suggesting that they activate target genes in the embryo that are 47 48 49 responsible for activating gut specification (Lin et al., 1995; Hunter and Kenyon, 1996; Bowerman, 1998). 50 51 Screens for essential zygotic genes required for gut specification led to identification of end-1 and end-3, 52 53 which encode GATA-type transcription factors that are expressed in the early E lineage (Zhu et al., 1997; 54 55 56 57 58 59 60 John Wiley & Sons Molecular Reproduction and Development Page 8 of 42

Maduro, p. 8 1 2 3 Maduro et al., 2005a). Mutation of both genes together abolishes gut specification, resulting in arrested 4 5 6 embryos and hatched inviable larvae completely lacking gut (Owraghi et al., 2010). 7 8 9 The nearly-identical genes med-1 and med-2 were identified by searching the C. elegans genome 10 11 for putative GATA factor genes (Maduro et al., 2001). When med-1 and med-2 are both absent, all med- 12 13 1,2(-) embryos are missing the normal descendants of MS, and 50%-85% of embryos make gut (Maduro 14 15 16 et al., 2001; Goszczynski and McGhee, 2005; Maduro et al., 2007). Hence, like skn-1, the med-1,2 genes 17 18 are required for MS fateFor more than forPeer E fate. Cons istentReview with their role in specification of both MS and 19 20 E, med-1 and med-2 are expressed in EMS, the mother of MS and E, and their gene products appear in 21 22 23 MS and E (Maduro et al., 2001; Robertson et al., 2004; Maduro et al., 2007). 24 25 26 The GATA factor ELT-2 was identified as a regulator of the gut-specific esterase gene ges-1 27 28 (Hawkins and McGhee, 1995). Expression of elt-2 occurs later than the med and end genes, and unlike 29 30 these genes, occurs for the lifespan of the animal. Loss of elt-2 does not prevent specification, but 31 32 33 causes gut differentiation to fail, resulting in arrested larvae with abnormal intestine (Fukushige et al., 34 35 1998). This is consistent with an essential role for ELT-2 in maintenance of intestine fate (McGhee et al., 36 37 2009). A related GATA factor gene, elt-7, was identified by sequence, and found to have similar 38 39 40 expression to elt-2, although elt-7 expression starts earlier (Sommermann et al., 2010). Loss of elt-7 can 41 42 enhance the phenotype of loss of elt-2, though loss of elt-7 alone has no phenotype. A partial 43 44 duplication of elt-2, called elt-4, does not appear to contribute a detectable function to gut development 45 46 (Fukushige et al., 2003). 47 48 49 50 The E Specification Genes Form a Simple Network 51 52 53 The aforementioned regulators have been assembled into a Gene Regulatory Network (GRN) as 54 55 shown in Fig. 2. Most of the proposed regulatory interactions have been confirmed experimentally, 56 57 suggesting that the model reflects the real network topology (Fukushige et al., 1999; Maduro et al., 58 59 60 John Wiley & Sons Page 9 of 42 Molecular Reproduction and Development

Maduro, p. 9 1 2 3 2001; Maduro et al., 2002; Shetty et al., 2005). Many features of the network are apparent that suggest 4 5 6 mechanisms by which endoderm specification is made robust. 7 8 9 Robustness to genetic change: Gene Duplication/Redundancy. The zygotic endodermal regulators are 10 11 present as pairs of duplicated genes: med-1,2 , end-1,3 and elt-2,7 . Each gene pair exhibits overlapping 12 13 function, as mutation of one of the two has a weaker effect than mutation of both genes together, as 14 15 16 shown in Table 1 (Maduro et al., 2005a; Maduro et al., 2007; Sommermann et al., 2010). Therefore, the 17 18 endoderm GRN is robustFor to genetic variationPeer because Review many genes make both overlapping and additive 19 20 contributions to gut specification. Redundancy of this type has been seen in other lineages in the C. 21 22 23 elegans embryo, suggesting it is a general strategy for network robustness in cell specification GRNs 24 25 (Good et al., 2004; Baugh et al., 2005; Fukushige et al., 2006; Broitman-Maduro et al., 2009; Walton et 26 27 al., 2015). 28 29 30 Robustness to noise: Network architecture. The genes in the endoderm network participate in a mostly 31 32 33 linear cascade with a defined hierarchy. Upstream regulators interact with only the most immediate 34 35 downstream tier and the one after that, in what is termed a feed-forward loop (Davidson, 2010) . For 36 37 example, MED-1,2 activates end-3, and both MED-1,2 and END-3 activate end-1 (Broitman-Maduro et 38 39 40 al., 2005; Maduro, 2006; Maduro et al., 2007). The feed-forward loop is widespread in developmental 41 42 GRNs, as it favors stabilization of a regulatory state that drives development forward (Davidson, 2010). A 43 44 second important motif is the positive feedback loop that occurs with the terminal regulator elt-2: Once 45 46 elt-2 is activated, it maintains its own expression (Fukushige et al., 1999). In embryos lacking end-1 and 47 48 49 end-3 (which never specify endoderm), forced expression of elt-2 is sufficient to initiate the endoderm 50 51 differentiation program in most, if not all, embryonic cells (Owraghi et al., 2010). This suggests that END- 52 53 1, END-3 and ELT-7 initiate activation of elt-2, which is then maintained by autoregulation. As end-1, 54 55 56 end-3, elt-7 and elt-2 are all GATA-type transcription factors, it is likely that these regulators act through 57 58 59 60 John Wiley & Sons Molecular Reproduction and Development Page 10 of 42

Maduro, p. 10 1 2 3 similar cis-regulatory sites in the elt-2 promoter, which is a further source of redundancy built into the 4 5 6 network. 7 8 9 Another type of network topology that gives rise to robustness is distributed robustness (Felix 10 11 and Wagner, 2008). In this paradigm, multiple different factors work in parallel to achieve the same 12 13 goal. Distributed robustness is a property of the signaling pathways that specify the fate of the vulval 14 15 16 precursors in C. elegans (Felix and Wagner, 2008). In the embryonic endoderm network, the maternal 17 18 factors demonstrate thisFor type of robustness, Peer as SKN -1,Review PAL-1 and POP-1 converge on end-1 and end-3 19 20 independently of one another (Maduro et al., 2005b; Shetty et al., 2005; Maduro et al., 2015). The signal 21 22 23 between P 2 and EMS also occurs via maternal, parallel ligands and receptors (Rocheleau et al., 1997; 24 25 Thorpe et al., 1997; Bei et al., 2002). In contrast, the downstream zygotic genes in gut development 26 27 exhibit a linear set of interactions reinforced by feed forward loops, as described above (Maduro, 2009). 28 29 30 Characterization of mutants that affect the endoderm has revealed three levels at which 31 32 33 robustness breaks down: The E lineage cell division pattern, acquisition of gut fate by the E descendants, 34 35 and differentiation of intestine cells. These are described below, in terms of the trait of the endoderm 36 37 that loses robustness, the types of genetic perturbation that cause variability of the trait, and the 38 39 40 mechanism by which robustness is lost. 41 42 43 44 45 46 GUT FUNCTION IS ROBUST TO CHANGES IN THE E LINEAGE PATTERN 47 48 49 As shown in Fig. 1, the E progenitor follows nearly the same pattern of cell divisions in wild-type 50 51 embryos to generate 20 cells by the end of embryogenesis (Sulston et al., 1983). This pattern of divisions 52 53 54 is highly reproducible among C. elegans embryos, and even in other related species, which suggests that 55 56 there has been a strong selection for its maintenance (Sulston et al., 1983; Zhao et al., 2008). But the 57 58 59 60 John Wiley & Sons Page 11 of 42 Molecular Reproduction and Development

Maduro, p. 11 1 2 3 pattern is not completely invariant, as a small number of newly-hatched larvae contain one extra gut 4 5 6 cell, making 21 intestinal cells in total (Sulston and Horvitz, 1977; Sulston et al., 1983; Leung et al., 7 8 1999). Such rare (but detectable) variations in the lineage are seen in other tissues. For example, in the 9 10 post-embryonic development of the vulva, rare phenotype variation occurs under stress or in particular 11 12 13 wild-type genetic backgrounds (Felix and Wagner, 2008). 14 15 16 Two types of genetic perturbation can significantly increase the number of cell divisions within 17 18 the E lineage, resulting inFor extra cells (hyperplasiPeera) of theReview intestine. The first are mutations directly 19 20 affecting cell cycle progression. The clearest examples are gain-of-function mutations in cdc-25.1 that 21 22 23 result in extra cell divisions specifically in the E lineage, resulting in over 35 gut cells by the end of 24 25 embryonic development (Clucas et al., 2002; Kostic and Roy, 2002). Surprisingly, the presence of the 26 27 extra cells affects neither the differentiation of E descendants into gut, nor the function of the adult 28 29 30 intestine. The apparently normal phenotype of adults with intestinal hyperplasia suggests that intestine 31 32 function does not depend strongly on the number of cells in the gut, or that there is some compensatory 33 34 mechanism that suppresses the effects of extra cells. As a result, intestine function is robust to changes 35 36 in the pattern of cell divisions. 37 38 39 40 The second type of perturbation that causes gut hyperplasia is partial loss of gut specification. 41 42 All endoderm specification mutants that cause at least some loss of gut specification also cause some 43 44 embryos to experience hyperplasia. For example, approximately one-third of end-3(-) embryos generate 45 46 over 20 gut nuclei from E (Maduro et al., 2007; Maduro et al., 2015). Hyperplasia likely results from a 47 48 49 loss of cell cycle control due to a transformation to an alternate fate (see below for details). Embryos 50 51 exhibiting gut hyperplasia generally develop to adulthood, however, there is evidence that hyperplasia 52 53 resulting from perturbation of the specification GRN correlates with defects in adult gut function (also 54 55 56 57 58 59 60 John Wiley & Sons Molecular Reproduction and Development Page 12 of 42

Maduro, p. 12 1 2 3 discussed below) (Maduro et al., 2015). 4 5 6 7 8 9 ROBUSTNESS OF GUT SPECIFICATION: WHEN DOES THE E CELL ADOPT A NON-GUT FATE? 10 11 12 Specification is compromised by loss of multiple endoderm genes together 13 14 15 As discussed earlier, the ability of the endoderm GRN to robustly specify a gut fate likely derives 16 17 from the parallel nature of many of the upstream regulatory interactions, and the existence of some 18 For Peer Review 19 20 components as pairs of genes with overlapping function (Fig. 2 and Table 1). The network is robust to 21 22 loss of some components individually, such as med-1 or end-1, as null mutations in these genes do not 23 24 result in a recognizable phenotype (Maduro et al., 2005a; Maduro et al., 2007; Boeck et al., 2011). 25 26 27 Similarly, loss of pop-1 or pal-1 individually does not result in significant loss of endoderm, although 28 29 other lineages become affected and hence embryos undergo developmental arrest (Lin et al., 1995; 30 31 Hunter and Kenyon, 1996; Maduro et al., 2005b). 32 33 34 When multiple components of the endoderm GRN become compromised, embryos become less 35 36 37 likely to specify gut (Table 1). For example, among genetically identical med-1; end-3 double mutant 38 39 embryos from the same mother, some embryos lack gut (and hence undergo developmental arrest as 40 41 late embryos or first-stage larvae), while others make a gut and develop to adulthood (Maduro et al., 42 43 44 2007). A similar effect occurs when skn-1 activity is lost, because expression of med-1, med-2, end-1 and 45 46 end-3 is highly dependent on SKN-1 (Fig. 2) (Bowerman et al., 1992; Maduro et al., 2005b; Maduro et al., 47 48 2007; Raj et al., 2010). In the case of loss of skn-1, the ability to specify gut becomes stochastic because 49 50 expression of end-1 becomes stochastic: Those embryos that fortuitously accumulate enough end-1 51 52 53 mRNA can specify gut and activate elt-2, as described below (Raj et al., 2010). Similarly, loss of 54

55 components of the Wnt/MAPK/Src signal between P 2 and EMS compromises gut specification because 56 57 POP-1 becomes a repressor of gut fate, preventing MED-1,2 from activating end-1,3 (Lin et al., 1998; 58 59 60 John Wiley & Sons Page 13 of 42 Molecular Reproduction and Development

Maduro, p. 13 1 2 3 Shetty et al., 2005; Maduro et al., 2007; Maduro et al., 2015); the partial loss of gut conferred by 4 5 6 mutation of mom-2/Wnt is shown by example in Table 1. As with loss of skn-1, gut specification 7 8 becomes stochastic when Wnt signaling is blocked because of stochastic activation of early E lineage 9 10 markers including end-1 (Robertson et al., 2014). 11 12 13 When none of the E descendants make gut, E adopts an alternate fate, usually that of either C or 14 15 16 MS. Loss of activity of SKN-1, MED-1,2 or END-1,3 results in a transformation of E into a C-like cell, 17 18 resulting in E producing Formuscle and epidermalPeer cells (BowermanReview et al., 1992; Zhu et al., 1997; Maduro et 19 20 al., 2001; Maduro et al., 2005a). In contrast, blockage of components of the overlapping Wnt/MAPK/Src 21 22 23 signal between P 2 and EMS results in a transformation of E to an MS-like precursor, producing pharynx 24 25 and body muscle (Rocheleau et al., 1997; Thorpe et al., 1997; Bei et al., 2002). E can adopt 26 27 characteristics of both MS and C, as occurs when end-1,3 and pop-1 are blocked simultaneously 28 29 30 (Owraghi et al., 2010). Hence, it is possible to view robust E specification as a requirement to activate 31 32 [END-1+END-3] to high enough levels to overcome mechanisms that specify an alternate fate. 33 34 35 E specification regulates the cell cycle 36 37 38 In normal development, the E daughter cells slow their cell cycle to allow their internalization at 39 40 gastrulation, while in gut specification mutants, the E daughter cells often divide early on the ventral 41 42 43 surface of the embryo (Bowerman et al., 1992; Zhu et al., 1997). This suggests that E specification slows 44 45 down the cell cycle. The MS and C lineages both exhibit faster cell divisions than the E lineage, and both 46 47 make more descendants in the embryo: C makes 46 cells, while MS makes 63 (Sulston et al., 1983). 48 49 50 Therefore, perturbations of E specification likely cause an increase in the number of cell divisions 51 52 because of the loss of cell cycle slowing and a transformation to the MS or C fates (Bowerman et al., 53 54 1992; Zhu et al., 1997; Maduro et al., 2001; Maduro et al., 2005a; Boeck et al., 2011; Robertson et al., 55 56 57 2014). As all the upstream factors ultimately regulate end-1 and end-3, it can be inferred that END- 58 59 60 John Wiley & Sons Molecular Reproduction and Development Page 14 of 42

Maduro, p. 14 1 2 3 1+END-3 together regulate cell cycle genes in the E lineage, although this has not yet been 4 5 6 demonstrated. A connection between specification and the cell cycle is known for the WEE-1.1 kinase, 7 8 whose product regulates cell cycle progression in other systems (Kellogg, 2003). The wee-1.1 gene is 9 10 expressed in the early E lineage, its promoter has binding sites for MED-1,2, and early E lineage 11 12 13 expression of a wee-1.1 reporter is reduced by perturbation of Wnt/MAPK components (Wilson et al., 14 15 1999; Broitman-Maduro et al., 2005; Robertson et al., 2014). Paradoxically, alteration of some cell cycle 16 17 components can affect E specification. The β-TrCP (beta-transducin repeat containing protein) 18 For Peer Review 19 orthologue LIN-23 regulates CDC-25.1 abundance and also affects gut specification (Hebeisen and Roy, 20 21 22 2008; Segref et al., 2010). Loss of cdc-25.1 by RNAi can also result in an absence of gut, though this is 23 24 likely due to an effect on cell cycles prior to the birth of E (Clucas et al., 2002). 25 26 27 Gut specification is not an "all-or-none" phenomenon 28 29 30 By definition, when gut specification fails to occur in an embryo, intestine tissue does not get 31 32 33 made. Differentiated intestine cells produce birefringent gut granules that can be easily visualized under 34 35 polarized light, facilitating identification of embryos that have gut or lack gut (Fig. 3A) (Bowerman et al., 36 37 1992). The earliest screens to identify specification genes recovered embryonic lethal mutations, in 38 39 40 which embryos contained differentiated tissue, but which were unable to hatch (Priess and Thomson, 41 42 1987). When skn-1 was first identified, arrested skn-1 embryos were described as either having 43 44 intestinal cells or not based on the presence of a patch of gut granules in terminally-arrested embryos 45 46 (Bowerman et al., 1992). This binary characterization persisted through subsequent studies examining 47 48 49 phenotypes in the Wnt/MAPK/Src pathways, as these phenotypes also result in failure of 50 51 morphogenesis (Rocheleau et al., 1997; Thorpe et al., 1997; Bei et al., 2002). 52 53 54 A molecular model for binary gut fate choice was advanced when analysis of mRNAs in the 55 56 57 endoderm GRN revealed that the number of end-1 transcripts, quantified by single-molecule fluorescent 58 59 60 John Wiley & Sons Page 15 of 42 Molecular Reproduction and Development

Maduro, p. 15 1 2 3 in situ hybridization (smFISH), could predict whether or not gut specification had occurred in a skn-1 4 5 6 mutant embryo (Raj et al., 2010). Although smFISH requires fixation of embryos, end-1 and elt-2 mRNAs 7 8 could be detected simultaneously during a time window when both are present in the early E lineage. It 9 10 was found that while wild-type embryos achieve a certain level of end-1 transcripts, skn-1 mutant 11 12 13 embryos have highly variable amounts of end-1 mRNA, from almost none to amounts similar to wild- 14 15 type (Raj et al., 2010). Hence, the apparent binary specification of gut in skn-1 mutant embryos could be 16 17 attributed to highly stochastic end-1 mRNA (and hence of END-1 protein) coupled to a threshold 18 For Peer Review 19 requirement for END-1 for elt-2 activation and commitment to a gut fate (Raj et al., 2010). As skn-1 20 21 22 mutants were previously classified as having gut or not, it was thought that this binary choice occurs in 23 24 the E cell, as diagrammed in Fig. 4A. 25 26 27 Specification is not appropriately described as being all or none, however. It has recently been 28 29 30 shown that under particular conditions, some (but not all) of the E descendants can adopt a gut fate, a 31 32 phenotype that can be called "partial specification" or "hypomorphic specification" (Maduro et al., 33 34 2015). The isogenic embryos shown in Fig. 3A are mutant for end-1 and end-3, and carry a chromosomal 35 36 end-1 transgene in which the MED-1,2 binding sites have been mutated (Maduro et al., 2015). In this 37 38 39 strain, most terminal embryos lack gut granules, many have a normal-looking patch of gut (black 40 41 arrowheads), and some have tiny patches of gut (white arrowheads). These different-sized regions 42 43 correspond to varying numbers of gut cells as analyzed by gut-specific transgene reporters. Most 44 45 46 perturbations that cause loss of gut specification part of the time (i.e. between 0% and 100) result in 47 48 production of at least some embryos that have fewer than 20 gut nuclei (hypoplasia), in addition to 49 50 embryos with greater than 20 gut nuclei (hyperplasia) (Maduro et al., 2007; Robertson et al., 2014; 51 52 53 Maduro et al., 2015). The stronger the defect in gut specification, measured as the proportion of 54 55 embryos made that have no gut, the more likely that hypoplasia will be seen over hyperplasia among 56 57 embryos making gut (Maduro et al., 2007; Maduro et al., 2015). Paradoxically, both types of embryos 58 59 60 John Wiley & Sons Molecular Reproduction and Development Page 16 of 42

Maduro, p. 16 1 2 3 co-occur with increased cell divisions within the E lineage. In the case of gut hyperplasia, most of these E 4 5 6 descendants adopt a gut fate, while in the case of hypoplasia, fewer descendants of E have adopted a 7 8 gut fate. Figs. 3B-3J show examples of embryos in a partial gut specification strain in which all, some, or 9 10 none of the E descendants have adopted a gut fate. 11 12 13 A model for partial specification of E 14 15 16 17 Partial gut specification can be explained if a binary fate choice can also occur stochastically in 18 For Peer Review 19 the descendants of E (Fig. 4B). When [END-1+END-3] become compromised, activation of elt-2 becomes 20 21 delayed and stochastic, so commitment to gut fate occurs in some E lineage descendants, but not 22 23 necessarily all. Although a more comprehensive analysis of the dynamics of elt-2 expression in such 24 25 26 embryos has yet to be reported, variability in onset of elt-2::GFP expression was observed in partial gut 27 28 specification strains as predicted by this model (Maduro et al., 2015), and computer modeling suggests 29 30 that stochastic, later specification can easily generate the type of partial guts observed in late-stage 31 32 33 embryos (Farhad Ghamsari and MM, unpublished). 34 35 36 37 38 39 ROBUSTNESS OF DIFFERENTIATION: IF GUT IS MADE, DOES IT FUNCTION NORMALLY? 40 41 42 Intestine differentiation requires elt-2 but not elt-7 43 44 45 The main regulator of intestine fate is ELT-2 (McGhee et al., 2009). Both elt-2 and a related 46 47 48 gene, elt-7, are expressed from the time of specification through adulthood (Fukushige et al., 1998; 49 50 Sommermann et al., 2010). Loss of elt-2 is compatible with gut specification, however intestine 51 52 differentiation is not properly executed, and animals arrest as L1 larvae with various intestine defects 53 54 55 (Fukushige et al., 1998). As described above, loss of elt-7 by itself results in no detectable phenotype, 56 57 however a double mutant of elt-2 and elt-7 results in a stronger phenotype than loss of elt-2 alone, 58 59 60 John Wiley & Sons Page 17 of 42 Molecular Reproduction and Development

Maduro, p. 17 1 2 3 suggesting that ELT-7 plays a reinforcing role (likely in parallel with END-1 and END-3) in elt-2 activation 4 5 6 (Sommermann et al., 2010). Given the multiple inputs that activate end-1 and end-3, it is surprising that 7 8 the maintenance of intestinal fate is not at all robust to the loss of a single gene ( elt-2). One explanation 9 10 may be that it may be difficult to recruit new pathways into maintenance of intestine fate. There are 11 12 13 thousands of genes that appear to be intestine-enriched and regulated by ELT-2, and co-option of a new 14 15 regulator into endoderm differentiation would require that thousands of target genes acquire 16 17 regulatory input from such a new factor (Maduro, 2006; Pauli et al., 2006; McGhee et al., 2007). 18 For Peer Review 19 20 Another interpretation of the requirement for elt-2 in gut fate is that robustness in the 21 22 23 endoderm has evolved only in the earliest steps of the network to assure a highly invariant level of elt-2 24 25 expression downstream. Quantification of mRNA showed that elt-2 appears to have a relatively constant 26 27 number of transcripts per cell in the early embryo (Raj et al., 2010). Although variation in elt-2 28 29 30 expression has not been examined in adults, expression of a single-copy reporter for hsp-16.2 in the 31 32 intestine is surprisingly similar from animal to animal (75%-88% correlation) (Mendenhall et al., 2015), 33 34 suggesting that expression variation is generally low in the adult gut. Hence, C. elegans may not have 35 36 evolved mechanisms to accommodate variation in gut gene expression, relying instead on upstream 37 38 39 components to assure consistency of expression of elt-2. 40 41 42 43 44 45 Partial E specification compromises adult gut function 46 47 48 As mentioned above, consistent output of embryonic development is synonymous with the 49 50 existence of only a finite number of stable end states (Waddington, 1942). A recent work in Drosophila 51 52 53 provides cellular-level support for the idea that even when a major upstream regulator is lost, later 54 55 embryonic cells themselves are driven to stable differentiated states. When the key maternal regulator 56 57 bcd was blocked in early embryos, cell fates nonetheless adopted fates present in wild-type embryos, 58 59 60 John Wiley & Sons Molecular Reproduction and Development Page 18 of 42

Maduro, p. 18 1 2 3 and not novel fates, as revealed by patterns of downstream regulator gene expression (Staller et al., 4 5 6 2015). There is evidence that this is also true in the C. elegans endoderm. When E specification is 7 8 partially compromised, three markers – expression of reporters for elt-2 and the differentiation marker 9 10 pept-2, and gut granules – appear in those E descendants that make gut (Maduro et al., 2015). This 11 12 13 suggests that cells that adopt a gut fate in such embryos are comparable to wild-type gut cells. 14 15 Furthermore, when embryos do not activate both end-1 and end-3, the E cell undergoes a complete 16 17 transformation into the C lineage, as shown by following the early cell divisions of the transformed E cell 18 For Peer Review 19 (which become similar to those of C), and by appearance of markers of C fates (PAL-1-dependent muscle 20 21 22 and epidermis) in descendants of E (Zhu et al., 1997; Maduro et al., 2005a; Owraghi et al., 2010). 23 24 However, recent findings suggest that, just as specification is not an "all-or-none" phenomenon, 25 26 activation of differentiation may not be self-correcting for earlier defects in specification, even when an 27 28 29 apparently functional embryonic intestine is made. 30 31 32 To examine intestine function downstream of partially compromised gut specification, 33 34 endoderm specification must be compromised in a way that leaves other lineages unaffected, so that 35 36 gut function in surviving animals can be evaluated in a complete larva or adult. However, most 37 38 39 perturbations in E specification either affect other lineages (resulting in embryonic or larval arrest), 40 41 cause complete loss of gut, or cause only weak effects (Bowerman et al., 1992; Lin et al., 1995; 42 43 Rocheleau et al., 1997; Thorpe et al., 1997; Rocheleau et al., 1999; Maduro et al., 2001; Maduro et al., 44 45 46 2005a; Maduro et al., 2005b). An engineered strain that meets this requirement is one in which end-1 47 48 and end-3 activity derives solely from single-copy end-1 and end-3 transgenes with mutated MED-1,2 49 50 binding sites (Maduro et al., 2015). This strain exhibits gut specification in 75% of embryos, and most 51 52 53 embryos are able to survive to adulthood with at least a wild-type number of gut cells. However, these 54 55 animals exhibit a developmental delay and fat accumulation that resembles the phenotypes of adults 56 57 subjected to caloric restriction during larval development (Palgunow et al., 2012; Maduro et al., 2015). 58 59 60 John Wiley & Sons Page 19 of 42 Molecular Reproduction and Development

Maduro, p. 19 1 2 3 Similar phenotypes are observed among surviving adults of a med-1; end-3 double mutant strain, which 4 5 6 exhibits gut specification in 42% of embryos (Maduro et al., 2007; Maduro et al., 2015). These results 7 8 suggest that proper adult gut function is not robust to perturbations of the early endoderm lineage, 9 10 even though an apparently normal gut has been made. In turn, it can be inferred that proper gut 11 12 13 function is assured by the robustness of gut specification. 14 15 16 There are several mechanisms that could explain why partial specification might result in 17 18 reduced quality of differentiation.For First, Peer many of the adultsReview that survive partial specification 19 20 demonstrate intestinal hyperplasia, suggesting that extra gut cells may be the cause of poor-quality 21 22 23 differentiation (Maduro et al., 2015). However, the average amount of hyperplasia, ~5 extra intestinal 24 25 nuclei per adult, is much milder than what is seen in cdc-25.1 gain-of-function mutants (~25 extra 26 27 nuclei), and major defects in gut function were not seen in the latter (Kostic and Roy, 2002; Maduro et 28 29 30 al., 2015). Second, there is a delay in elt-2 activation in partial gut specification strains, suggesting that 31 32 late activation of differentiation misses an optimal window for activation of other unknown factors by 33 34 end-1 and end-3 (Boeck et al., 2011; Maduro et al., 2015). Finally, it may be that weak initiation does not 35 36 allow elt-2 to achieve the same level of postembryonic expression as in wild-type. In other systems, 37 38 39 positive autoregulation can be vulnerable to weaker initial input (Maeda and Sano, 2006), suggesting 40 41 that robust, high-level initiation of elt-2 is essential for proper differentiation and intestine function. 42 43 Loss of stable [END-1+END-3] might therefore propagate into weak and unstable activation of elt-2 that 44 45 46 could persist for many cell generations. 47 48 49 50 51 52 A framework that connects cell cycle, specification and differentiation 53 54 55 A model is presented in Fig. 5 that relates the levels of END-1+END-3 with the development of 56 57 the gut. At maximal END activity, robust specification occurs and E produces 20 descendants and a 58 59 60 John Wiley & Sons Molecular Reproduction and Development Page 20 of 42

Maduro, p. 20 1 2 3 normal intestine. When all END activity is abolished, the E cell fails to generate intestinal cells and all 4 5 6 animals arrest development at the end of embryogenesis or immediately after hatching. At intermediate 7 8 levels, the E lineage, E specification and intestine function all become compromised due to reduced and 9 10 stochastic activation of END target genes. In most of these embryos, the number of E descendants 11 12 13 increases, due to a failure of the ENDs to slow down the cell cycle. If all of these E descendants adopt an 14 15 intestinal fate, the result is a functional gut with (possible) concomitant defects in adult intestine 16 17 function. If only some of these adopt a gut fate, the result is hypoplasia of the gut, and an inability to 18 For Peer Review 19 survive embryonic development. Cell cycle regulator defects within the E lineage can induce hyperplasia 20 21 22 but these generally do not influence specification. 23 24 25 26 27 28 Evolutionary Changes in the Endoderm Gene Regulatory Network 29 30 31 The embryonic cell lineage, which includes the cell division patterns in the intestine, is nearly 32 33 34 identical between C. elegans and the related species C. briggsae (Zhao et al., 2008). However, there is 35 36 evidence that the underlying gene regulatory network is subject to more rapid evolution compared with 37 38 the differentiation network. For example, the med GATA factor genes appear to be rapidly-evolving, 39 40 with many more med genes in C. remanei and C. briggsae (Coroian et al., 2005). Perhaps more surprising 41 42 43 is the observation that knockdown of skn-1 and pop-1 has different effects on endoderm specification in 44 45 C. briggsae as compared with C. elegans (Lin et al., 2009). These differences reveal that regulatory 46 47 interactions in the endoderm GRN can change over evolutionary time without any effect on the 48 49 50 specification of E and the development of the gut. Changes in underlying GRNs among related species, in 51 52 the absence of phenotype change, are generally called developmental system drift (True and Haag, 53 54 2001). In this paradigm, the ability of multiple pathways to drive the same processes allows them to 55 56 57 acquire different relative importance over evolutionary time. When examined closely, underlying 58 59 60 John Wiley & Sons Page 21 of 42 Molecular Reproduction and Development

Maduro, p. 21 1 2 3 regulatory mechanisms can be quite different, although they robustly cause the same developmental 4 5 6 outcome, and usually involve at least some regulators in common. Examples of developmental system 7 8 drift in C. elegans and its close relatives include changes in the core sex determination pathway, 9 10 particularly in the modifications that lead to hermaphroditism (Ellis and Lin, 2014), and in the 11 12 13 development of the vulva (Felix and Barkoulas, 2012). Given enough evolutionary distance, larger 14 15 changes become apparent: In the endoderm network, while elt-2 is conserved, the med-1,2 and end-1,3 16 17 genes appear to be absent from genomes outside of the Elegans supergroup of Caenorhabditis species, 18 For Peer Review 19 suggesting that early gut specification outside this group does not involve GATA factors (Christian Turner 20 21 22 and MM, unpublished observations) (Kiontke et al., 2011). Presumably, different zygotic regulators exist 23 24 in these other species that perform the function of the MEDs and ENDs. Identifying these may reveal 25 26 cryptic mechanisms that may be partially conserved in C. briggsae and could account for the differences 27 28 29 in skn-1 and pop-1 knockdown with C. elegans . The existence of multiple pathways in gut specification, 30 31 therefore, may be a way to achieve robustness of development while providing the means by which 32 33 developmental system drift can occur over evolutionary time. 34 35 36 37 38 39 40 FUTURE PROSPECTS 41 42 43 Although the proposed structure of the C. elegans endoderm Gene Regulatory Network has 44 45 remained largely unchanged in over a decade, there is still much to be learned. For example, newer 46 47 methods allow quantitative measurements to be made of gene expression at the level of individual 48 49 50 mRNAs (Raj et al., 2010) and protein (Raj et al., 2010; Tanenbaum et al., 2014; Mendenhall et al., 2015). 51 52 Embryonic cell lineages can be followed semi-automatically using fluorescent reporters (Bao et al., 53 54 2006). It is also possible to define the transcriptome of individual embryonic cells (Hashimshony et al., 55 56 57 2012). High-resolution analysis of the effect of specific cis -regulatory sites could be made by introducing 58 59 60 John Wiley & Sons Molecular Reproduction and Development Page 22 of 42

Maduro, p. 22 1 2 3 promoter mutations into genes in their native chromosomal context (Waaijers and Boxem, 2014). These 4 5 6 newer approaches could be used in combinations to probe gene expression variation under normal 7 8 conditions and as a result of specific network perturbations; thus, it should be possible to determine the 9 10 roles of all the known factors in controlling variation at the level of gene products, and in overall 11 12 13 phenotype. 14 15 16 There remain unanswered questions about the architecture of the endoderm gene regulatory 17 18 network and how embryosFor assure robustness Peer of intest Reviewinal development. For example, are there more 19 20 regulators in the endoderm specification network that act to reinforce cell fate and differentiation? Do 21 22 23 different perturbations of the endoderm GRN all cause the same type of partial gut phenotypes? What 24 25 happens to non-gut derivatives of E in partial specification strains? What subtle phenotypic effects result 26 27 from the presence of extra gut cells? Is expression noise in the endoderm specification network buffered 28 29 30 by microRNAs as occurs in other developmental contexts (Burke et al., 2015; Schmiedel et al., 2015)? 31 32 How do differences in genetic background (e.g. from wild isolates of C. elegans ) or in environment (e.g. 33 34 temperature) influence the proportion of embryos that make gut in partial specification mutants 35 36 (Francesconi and Lehner, 2014; Vu et al., 2015)? What is the role of more general gene expression 37 38 39 factors in buffering gene expression noise (Burga et al., 2011)? How is gene expression noise different in 40 41 other species as a result of developmental system drift (True and Haag, 2001)? And finally, how do the E 42 43 specification genes control the robust pattern of cell divisions in the E lineage? 44 45 46

47 48 49 50 CONCLUSIONS AND PERSPECTIVES 51 52 53 The C. elegans endoderm GRN is a hierarchical network of regulators that exhibits a broad array 54 55 of inputs in its earlier steps that gives way to a more linear topology that is reinforced by feed-forward 56 57 loops, and in the case of the gut fate determinant elt-2, positive autoregulation. Through mutational 58 59 60 John Wiley & Sons Page 23 of 42 Molecular Reproduction and Development

Maduro, p. 23 1 2 3 analysis, it is apparent that the structure of the network determines how all of its components 4 5 6 contribute to robustness at the level of the cell cycle pattern of the E lineage, the acquisition of gut fate 7 8 by the E descendants, and expression of differentiation genes. A surprising result has been that adult 9 10 intestinal defects can result from "hypomorphic" specification, challenging the notion that cell fates 11 12 13 always canalize toward stable "normal" fates (Waddington, 1942). This result has important implications 14 15 for the study of development in any metazoan system, and the programming of stem cells, and in 16 17 specialized instances of transdifferentiation (Ladewig et al., 2013). Cells that acquire a (new) 18 For Peer Review 19 differentiated state, especially in an in vitro system, may overtly acquire the properties of a particular 20 21 22 tissue type, for example, by expression of a tissue-specific transcription factor. However, the tissue may 23 24 not exhibit completely normal function. It is therefore important to confirm a correct differentiated 25 26 state by additional means, such as by phenotype, or through genome-wide profiling of the 27 28 29 transcriptome or proteome. This may be especially important for induced pluripotent stem cells, which 30 31 can vary in the quality of their differentiation depending on their lineage of origin (Lee et al., 2014). 32 33 34 Looking forward, newer methods will allow a more complete understanding of how gene 35 36 expression noise is buffered in developmental GRNs, and determine the generality of the observation 37 38 39 that early effects on specification may manifest as subtle (but physiologically detectable) changes in the 40 41 function of the differentiated tissue. The C. elegans endoderm will therefore continue to be a useful 42 43 model for the study of robustness in developmental gene regulatory networks. 44 45 46

47 48 49 50 ACKNOWLEDGMENTS 51 52 53 I thank Gina Broitman-Maduro, Hailey Choi, Farhad Ghamsari and Christian Turner for contributing 54 55 unpublished results; two anonymous reviewers for their helpful comments; and Alex Mendenhall for 56 57 useful insights. This work was supported by NSF grant IOS#1258054 to M.M. 58 59 60 John Wiley & Sons Molecular Reproduction and Development Page 24 of 42

Maduro, p. 24 1 2 3 4 5 6 Figure Legends 7 8 9 10 Fig. 1. Embryonic origin of the C. elegans intestine. Top: Diagrams of embryos at various stages of 11 12 development. Approximate times are for development at 25 °C. Anterior is to the left and dorsal is up. 13 14 Nuclei of cells in the endoderm lineage are shown as green circles when the E lineage comprises 1, 8 and 15 16 17 20 cells (1E, 8E and 20E, respectively). After 430 minutes, embryonic divisions within the gut are 18 For Peer Review 19 complete. The embryo hatches at approximately 12 h after fertilization. Bottom: Abbreviated cell 20 21 lineage showing origin of the founder cells AB, MS, E, C, D and P 4, after Sulston et al., (1983). A vertical 22 23 line is a cell and a horizontal line is a cell division. The founder cells undergo further divisions. The 24 25 26 pattern that generates the 20 cells of the intestine is shown in green. A diagram of a larva shows the 27 28 intestine along with other components of the digestive tract and the blastomere of origin. ABa is the 29 30 anterior daughter of AB and ABp is the posterior daughter. The egg and larva are approximately 50 µm 31 32 33 and 200 µm long, respectively. Diagrams were adapted from WormAtlas ( http://wormatlas.org/ ) and 34 35 other sources (Sulston et al., 1983; Maduro and Rothman, 2002). 36 37 38 Fig. 2. Simplified diagram of the Endoderm Gene Regulatory Network (GRN) in C. elegans . Green font 39 40 indicates factors that are contributed maternally, while factors in black are zygotic. Most interactions 41 42 43 are known to be direct: SKN-1 to med-1,2 (Maduro et al., 2001); MED-1,2 to end-1 and end-3 (Broitman- 44 45 Maduro et al., 2005; Maduro et al., 2015); POP-1 on end-1 and end-3 (Robertson et al., 2014; Shetty et 46 47 al., 2005); and PAL-1 on end-1 and end-3 (Maduro et al., 2015). Other interactions are implied 48 49 50 genetically, including END-3 on end-1 (Maduro et al., 2007) and SKN-1 on end-1 and end-3 (Maduro et 51 52 al., 2005b). There is also some evidence for a maternal contribution of MED-1,2 (Maduro et al., 2007). 53 54 Left to right: The wild-type network; network missing END-1 but which results in normal intestine 55 56 57 (Maduro et al., 2005a; Boeck et al., 2011); loss of robust specification in the absence of MED-1,2 input 58 59 60 John Wiley & Sons Page 25 of 42 Molecular Reproduction and Development

Maduro, p. 25 1 2 3 into the ENDs (Maduro et al., 2015); loss of most gut when SKN-1 is absent, which also removes MED- 4 5 6 1,2 (Bowerman et al., 1992; Maduro et al., 2001); and a malformed intestine when ELT-2 is absent 7 8 (Fukushige et al., 1998). 9 10 11 Fig. 3. Evidence for partial specification of gut when the endoderm GRN is perturbed at the level of end- 12 13 1,3 . (A) Polarized light showing gut granules (white spots) in a collection of isogenic end-1,3(-) embryos 14 15 16 partially rescued by an end-1 transgene in which the MED binding sites have been mutated (Maduro et 17 18 al., 2015). Most embryosFor lack gut granules, Peer some ha veReview a near-normal amount (yellow arrowheads), and 19 20 others have very small amounts (white arrowheads). (B-J) Examples of embryos in which all, some or 21 22 23 none of the E descendants have adopted a gut fate. (B, C, D) Differential Interference Contrast (DIC) 24 25 microscopy images of embryos at 8E-12E stage. The cells forming the embryonic intestine are outlined in 26 27 white dashed lines in B and C. (E, F, G) Stacks of images showing a nuclear-localized end-3::mCherry 28 29 30 transcriptional reporter in the descendants of the E cell. Nuclei adopting a non-gut identity are outlined 31 32 by a dashed cyan line. The reporter was made previously (Murray et al., 2008). Because all upstream 33 34 regulators of end-3 are present, the reporter is activated in the early E lineage even when no gut is 35 36 made. (H, I, J) Expression of a nuclear-localized elt-2::GFP reporter shows nuclei of cells that have 37 38 39 adopted a gut fate. In (H), all E descendants express elt-2::GFP; in (I), only some descendants do; and in 40 41 (J), none of the E descendants express elt-2::GFP. In (E-J) the eggshell is outlined in yellow. A C. elegans 42 43 embryo is approximately 50 µm long. In (B-J), anterior is to the left. 44 45 46 Fig. 4. Models for how gut specification can fail. (A) Binary (all-or-none) model in which gut specification 47 48 49 either occurs or it does not. (B) A schematic diagram of how "partial" specification leads to fewer gut 50 51 nuclei due to a later commitment to gut fate by some E descendants and not others. For simplicity, the 52 53 same pattern of cell divisions is maintained across the various examples. 54 55 56 57 58 59 60 John Wiley & Sons Molecular Reproduction and Development Page 26 of 42

Maduro, p. 26 1 2 3 Fig. 5. Relationship of the activity of END-1+END-3 with gut specification and organogenesis of the 4 5 6 intestine. At high levels, specification and development of the intestine are robust. At intermediate 7 8 levels, development becomes stochastic, and animals can experience the absence of gut (aplasia), 9 10 changes in gut cell number (hypo- or hyperplasia), or no change (normoplasia). Animals with 11 12 13 compromised END-1+END-3 can survive hyperplasia, although they exhibit defects in metabolism. 14 15 Modified from a prior work (Maduro et al., 2015). 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Page 27 of 42 Molecular Reproduction and Development

Maduro, p. 27 1 2 3 REFERENCES 4 5 6

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Maduro, p. 31 1 2 3 Lin KT, Broitman-Maduro G, Hung WW, Cervantes S, Maduro MF. 2009. Knockdown of SKN-1 and the 4 5 6 Wnt effector TCF/POP-1 reveals differences in endomesoderm specification in C. briggsae as 7 8 compared with C. elegans. Developmental Biology 325(1):296-306. 9 10 Lin R, Hill RJ, Priess JR. 1998. POP-1 and anterior-posterior fate decisions in C. elegans embryos. Cell 11 12 13 92(2):229-239. 14 15 Lin R, Thompson S, Priess JR. 1995. pop-1 encodes an HMG box protein required for the specification of 16 17 a mesoderm precursor in early C. elegans embryos. Cell 83(4):599-609. 18 For Peer Review 19 Macneil LT, Walhout AJ. 2011. Gene regulatory networks and the role of robustness and stochasticity in 20 21 22 the control of gene expression. Genome Research 21(5):645-657. 23 24 Maduro M, Hill RJ, Heid PJ, Newman-Smith ED, Zhu J, Priess J, Rothman J. 2005a. Genetic redundancy in 25 26 endoderm specification within the genus Caenorhabditis. Developmental biology 284:509-522. 27 28 29 Maduro MF. 2006. Endomesoderm specification in Caenorhabditis elegans and other nematodes. 30 31 Bioessays 28(10):1010-1022. 32 33 Maduro MF. 2009. Structure and evolution of the C. elegans embryonic endomesoderm network. 34 35 36 Biochimica et Biophysica Acta 1789(4):250-260. 37 38 Maduro MF, Broitman-Maduro G, Choi H, Carranza F, Chia-Yi Wu A, Rifkin SA. 2015. MED GATA factors 39 40 promote robust development of the C. elegans endoderm. Developmental Biology 404:66-79. 41 42 Maduro MF, Broitman-Maduro G, Mengarelli I, Rothman JH. 2007. Maternal deployment of the 43 44 45 embryonic SKN-1-->MED-1,2 cell specification pathway in C. elegans. Developmental Biology 46 47 301(2):590-601. 48 49 Maduro MF, Kasmir JJ, Zhu J, Rothman JH. 2005b. The Wnt effector POP-1 and the PAL-1/Caudal 50 51 52 homeoprotein collaborate with SKN-1 to activate C. elegans endoderm development. 53 54 Developmental Biology 285:510-523. 55 56 57 58 59 60 John Wiley & Sons Molecular Reproduction and Development Page 32 of 42

Maduro, p. 32 1 2 3 Maduro MF, Lin R, Rothman JH. 2002. Dynamics of a developmental switch: recursive intracellular and 4 5 6 intranuclear redistribution of Caenorhabditis elegans POP-1 parallels Wnt-inhibited 7 8 transcriptional repression. Developmental Biology 248(1):128-142. 9 10 Maduro MF, Meneghini MD, Bowerman B, Broitman-Maduro G, Rothman JH. 2001. Restriction of 11 12 13 mesendoderm to a single blastomere by the combined action of SKN-1 and a GSK-3beta 14 15 homolog is mediated by MED-1 and -2 in C. elegans. Mol Cell 7(3):475-485. 16 17 Maduro MF, Rothman JH. 2002. Making worm guts: the gene regulatory network of the Caenorhabditis 18 For Peer Review 19 elegans endoderm. Developmental Biology 246(1):68-85. 20 21 22 Maeda YT, Sano M. 2006. Regulatory dynamics of synthetic gene networks with positive feedback. 23 24 Journal of Molecular Biology 359(4):1107-1124. 25 26 McGhee JD, Fukushige T, Krause MW, Minnema SE, Goszczynski B, Gaudet J, Kohara Y, Bossinger O, Zhao 27 28 29 Y, Khattra J, Hirst M, Jones SJ, Marra MA, Ruzanov P, Warner A, Zapf R, Moerman DG, Kalb JM. 30 31 2009. ELT-2 is the predominant transcription factor controlling differentiation and function of 32 33 the C. elegans intestine, from embryo to adult. Developmental Biology 327(2):551-565. 34 35 36 McGhee JD, Sleumer MC, Bilenky M, Wong K, McKay SJ, Goszczynski B, Tian H, Krich ND, Khattra J, Holt 37 38 RA, Baillie DL, Kohara Y, Marra MA, Jones SJ, Moerman DG, Robertson AG. 2007. The ELT-2 39 40 GATA-factor and the global regulation of transcription in the C. elegans intestine. 41 42 Developmental Biology 302(2):627-645. 43 44 45 Mendenhall AR, Tedesco PM, Sands B, Johnson TE, Brent R. 2015. Single Cell Quantification of Reporter 46 47 Gene Expression in Live Adult Caenorhabditis elegans Reveals Reproducible Cell-Specific 48 49 Expression Patterns and Underlying Biological Variation. PloS One 10(5):e0124289. 50 51 52 Murray JI, Bao Z, Boyle TJ, Boeck ME, Mericle BL, Nicholas TJ, Zhao Z, Sandel MJ, Waterston RH. 2008. 53 54 Automated analysis of embryonic gene expression with cellular resolution in C. elegans. Nature 55 56 Methods 5(8):703-709. 57 58 59 60 John Wiley & Sons Page 33 of 42 Molecular Reproduction and Development

Maduro, p. 33 1 2 3 Neves A, Busso C, Gonczy P. 2015. Cellular hallmarks reveal restricted aerobic metabolism at thermal 4 5 6 limits. eLife 2015;4;e04810. 7 8 Olson SK, Greenan G, Desai A, Muller-Reichert T, Oegema K. 2012. Hierarchical assembly of the eggshell 9 10 and permeability barrier in C. elegans. The Journal of Cell Biology 198(4):731-748. 11 12 13 Owraghi M, Broitman-Maduro G, Luu T, Roberson H, Maduro MF. 2010. Roles of the Wnt effector POP- 14 15 1/TCF in the C. elegans endomesoderm specification gene network. Developmental Biology 16 17 340(2):209-221. 18 For Peer Review 19 Palgunow D, Klapper M, Doring F. 2012. Dietary restriction during development enlarges intestinal and 20 21 22 hypodermal lipid droplets in Caenorhabditis elegans. PloS One 7(11):e46198. 23 24 Pauli F, Liu Y, Kim YA, Chen PJ, Kim SK. 2006. Chromosomal clustering and GATA transcriptional 25 26 regulation of intestine-expressed genes in C. elegans. Development 133(2):287-295. 27 28 29 Phillips BT, Kidd AR, 3rd, King R, Hardin J, Kimble J. 2007. Reciprocal asymmetry of SYS-1/beta-catenin 30 31 and POP-1/TCF controls asymmetric divisions in Caenorhabditis elegans. Proceedings of the 32 33 National Academy of Sciences of the United States of America 104(9):3231-3236. 34 35 36 Prasad A, Croydon-Sugarman MJ, Murray RL, Cutter AD. 2011. Temperature-dependent fecundity 37 38 associates with latitude in Caenorhabditis briggsae. Evolution; International Journal of Organic 39 40 Evolution 65(1):52-63. 41 42 Priess JR, Thomson JN. 1987. Cellular interactions in early C. elegans embryos. Cell 48(2):241-250. 43 44 45 Raj A, Rifkin SA, Andersen E, van Oudenaarden A. 2010. Variability in gene expression underlies 46 47 incomplete penetrance. Nature 463(7283):913-918. 48 49 Robertson SM, Medina J, Lin R. 2014. Uncoupling different characteristics of the C. elegans E lineage 50 51 52 from differentiation of intestinal markers. PloS One 9(9):e106309. 53 54 Robertson SM, Shetty P, Lin R. 2004. Identification of lineage-specific zygotic transcripts in early 55 56 Caenorhabditis elegans embryos. Developmental Biology 276(2):493-507. 57 58 59 60 John Wiley & Sons Molecular Reproduction and Development Page 34 of 42

Maduro, p. 34 1 2 3 Rocheleau CE, Downs WD, Lin R, Wittmann C, Bei Y, Cha YH, Ali M, Priess JR, Mello CC. 1997. Wnt 4 5 6 signaling and an APC-related gene specify endoderm in early C. elegans embryos. Cell 90(4):707- 7 8 716. 9 10 Rocheleau CE, Yasuda J, Shin TH, Lin R, Sawa H, Okano H, Priess JR, Davis RJ, Mello CC. 1999. WRM-1 11 12 13 activates the LIT-1 protein kinase to transduce anterior/posterior polarity signals in C. elegans. 14 15 Cell 97(6):717-726. 16 17 Schmiedel JM, Klemm SL, Zheng Y, Sahay A, Bluthgen N, Marks DS, van Oudenaarden A. 2015. Gene 18 For Peer Review 19 expression. MicroRNA control of protein expression noise. Science 348(6230):128-132. 20 21 22 Schnabel R, Hutter H, Moerman D, Schnabel H. 1997. Assessing normal embryogenesis in Caenorhabditis 23 24 elegans using a 4D microscope: variability of development and regional specification. 25 26 Developmental Biology 184(2):234-265. 27 28 29 Segref A, Cabello J, Clucas C, Schnabel R, Johnstone IL. 2010. Fate specification and tissue-specific cell 30 31 cycle control of the Caenorhabditis elegans intestine. Molecular Biology of the Cell 21(5):725- 32 33 738. 34 35 36 Shetty P, Lo MC, Robertson SM, Lin R. 2005. C. elegans TCF protein, POP-1, converts from repressor to 37 38 activator as a result of Wnt-induced lowering of nuclear levels. Developmental Biology 39 40 285(2):584-592. 41 42 Sommermann EM, Strohmaier KR, Maduro MF, Rothman JH. 2010. Endoderm development in 43 44 45 Caenorhabditis elegans: the synergistic action of ELT-2 and -7 mediates the specification-- 46 47 >differentiation transition. Developmental Biology 347(1):154-166. 48 49 Staller MV, Fowlkes CC, Bragdon MD, Wunderlich Z, Estrada J, DePace AH. 2015. A gene expression atlas 50 51 52 of a bicoid-depleted Drosophila embryo reveals early canalization of cell fate. Development 53 54 142(3):587-596. 55 56 57 58 59 60 John Wiley & Sons Page 35 of 42 Molecular Reproduction and Development

Maduro, p. 35 1 2 3 Stein LD, Bao Z, Blasiar D, Blumenthal T, Brent MR, Chen N, Chinwalla A, Clarke L, Clee C, Coghlan A, 4 5 6 Coulson A, D'Eustachio P, Fitch DH, Fulton LA, Fulton RE, Griffiths-Jones S, Harris TW, Hillier LW, 7 8 Kamath R, Kuwabara PE, Mardis ER, Marra MA, Miner TL, Minx P, Mullikin JC, Plumb RW, Rogers 9 10 J, Schein JE, Sohrmann M, Spieth J, Stajich JE, Wei C, Willey D, Wilson RK, Durbin R, Waterston 11 12 13 RH. 2003. The Genome Sequence of Caenorhabditis briggsae: A Platform for Comparative 14 15 Genomics. PLoS Biology 1(2):E45. 16 17 Sulston JE, Horvitz HR. 1977. Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. 18 For Peer Review 19 Developmental Biology 56(1):110-156. 20 21 22 Sulston JE, Schierenberg E, White JG, Thomson JN. 1983. The embryonic cell lineage of the nematode 23 24 Caenorhabditis elegans. Developmental Biology 100(1):64-119. 25 26 Tanenbaum ME, Gilbert LA, Qi LS, Weissman JS, Vale RD. 2014. A protein-tagging system for signal 27 28 29 amplification in gene expression and fluorescence imaging. Cell 159(3):635-646. 30 31 Thorpe CJ, Schlesinger A, Carter JC, Bowerman B. 1997. Wnt signaling polarizes an early C. elegans 32 33 blastomere to distinguish endoderm from mesoderm. Cell 90(4):695-705. 34 35 36 True JR, Haag ES. 2001. Developmental system drift and flexibility in evolutionary trajectories. Evolution 37 38 & Development 3(2):109-119. 39 40 Vu V, Verster AJ, Schertzberg M, Chuluunbaatar T, Spensley M, Pajkic D, Hart GT, Moffat J, Fraser AG. 41 42 2015. Natural Variation in Gene Expression Modulates the Severity of Mutant Phenotypes. Cell 43 44 45 162(2):391-402. 46 47 Waaijers S, Boxem M. 2014. Engineering the Caenorhabditis elegans genome with CRISPR/Cas9. 48 49 Methods 68(3):381-388. 50 51 52 Waddington CH. 1942. Canalization of development and the inheritance of acquired characters. Nature 53 54 150:563-565. 55 56 57 58 59 60 John Wiley & Sons Molecular Reproduction and Development Page 36 of 42

Maduro, p. 36 1 2 3 Walton T, Preston E, Nair G, Zacharias AL, Raj A, Murray JI. 2015. The Bicoid class homeodomain factors 4 5 6 ceh-36/OTX and unc-30/PITX cooperate in C. elegans embryonic progenitor cells to regulate 7 8 robust development. PLoS Genetics 11(3):e1005003. 9 10 Wilson MA, Hoch RV, Ashcroft NR, Kosinski ME, Golden A. 1999. A Caenorhabditis elegans wee1 11 12 13 homolog is expressed in a temporally and spatially restricted pattern during embryonic 14 15 development. Biochimica et Biophysica Acta 1445(1):99-109. 16 17 Zhao Z, Boyle TJ, Bao Z, Murray JI, Mericle B, Waterston RH. 2008. Comparative analysis of embryonic 18 For Peer Review 19 cell lineage between Caenorhabditis briggsae and Caenorhabditis elegans. Developmental 20 21 22 Biology 314(1):93-99. 23 24 Zhu J, Hill RJ, Heid PJ, Fukuyama M, Sugimoto A, Priess JR, Rothman JH. 1997. end-1 encodes an 25 26 apparent GATA factor that specifies the endoderm precursor in Caenorhabditis elegans 27 28 29 embryos. Genes & Development 11(21):2883-2896. 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Page 37 of 42 Molecular Reproduction and Development

1 2 3 Table 1. Examples of loss of gene functions that affect robustness of gut specification 4 5 6 7 Genotype % of embryos making gut 1 reference(s) 8 9 10 11 Weak/no effect 12 pop-1 100% (Lin et al. 1995) 13 14 pal-1 100% (Maduro et al. 2005b) 15 16 pop-1; pal-1 100% (Maduro et al. 2005b) 17 med-1 100% (Maduro et al. 2007) 18 For Peer Review 19 med-2 100% (Maduro et al. 2007) 20 21 end-1 100% (Maduro et al. 2005a) 22 23 elt-7 100% (Sommermann et al. 2010) 24 end-3 95% (Maduro et al. 2005a) 25 26 27 28 Intermediate effect 29 30 med-1; med-2 50-85% (Goszczynski and McGhee 2005; Maduro et al. 2015) 31 med-1; end-3 42% (Maduro et al. 2007) 32 33 34 35 Strong effect 36 skn-1 20-30% (Bowerman et al. 1992) 37 38 end-3; pop-1 14% (Maduro et al. 2005a) 39 40 mom-2 12% (Rocheleau et al. 1997; Thorpe et al. 1997) 41 42 med-1; med-2; pal-1 7% (Maduro et al. 2005b) 43 med-1; med-2; pop-1 6% (Maduro et al. 2005b) 44 45 end-3; mom-2 0% (Maduro et al. 2005a) 46 47 skn-1; mom-2 0% (Thorpe et al. 1997) 48 49 skn-1; pop-1; pal-1 0% (Maduro et al. 2005b) 50 end-1 end-3 0% (Owraghi et al. 2010) 51 52 53 1 54 Generally scored by presence of birefringent gut granules (e.g. (Bowerman et al. 1992)). 55 56 57 58 59 60 John Wiley & Sons Molecular Reproduction and Development Page 38 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Embryonic origin of the C. elegans intestine. Top: Diagrams of embryos at various stages of development. 38 Approximate times are for development at 25°C. Anterior is to the left and dorsal is up. Nuclei of cells in the 39 endoderm lineage are shown as green circles when the E lineage comprises 1, 8 and 20 cells (1E, 8E and 40 20E, respectively). After 430 minutes, embryonic divisions within the gut are complete. The embryo hatches 41 at approximately 12 h after fertilization. Bottom: Abbre viated cell lineage showing origin of the founder cells 42 AB, MS, E, C, D and P4, after Sulston et al. (1983). A vertical line is a cell and a horizontal line is a cell 43 division. The founder cells undergo further divisions. The pattern that generates the 20 cells of the intestine 44 is shown in green. A diagram of a larva shows the intestine along with other components of the digestive tract and the blastomere of origin. ABa is the anterior daughter of AB and ABp is the posterior daughter. The 45 egg and larva are approximately 50 µm and 200 µm long, respectively. Diagrams were adapted from 46 WormAtlas (http://wormatlas.org/) and other sources (Maduro and Rothman 2002; Sulston et al. 1983). 47 159x144mm (300 x 300 DPI) 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Page 39 of 42 Molecular Reproduction and Development

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 Simplified diagram of the Endoderm Gene Regulatory Network (GRN) in C. elegans. Green font indicates 26 factors that are contributed maternally, while factors in black are zygotic. Most interactions are known to be direct: SKN-1 to med-1,2 (Maduro et al. 2001); MED-1,2 to end-1 and end-3 (Broitman-Maduro et al. 2005; 27 Maduro et al. 2015); POP-1 on end-1 and end-3 (Robertson et al. 2014; Shetty et al. 2005); and PAL-1 on 28 end-1 and end-3 (Maduro et al. 2015). Other interactions are implied genetically, including END-3 on end-1 29 (Maduro et al. 2007) and SKN-1 on end-1 and end-3 (Maduro et al. 2005b). There is also some evidence for 30 a maternal contribution of MED-1,2 (Maduro et al. 2007). Left to right: The wild-type network; network 31 missing END-1 but which results in normal intestine (Boeck et al. 2011; Maduro et al. 2005a); loss of robust 32 specification in the absence of MED-1,2 input into the ENDs (Maduro et al. 2015); loss of most gut when 33 SKN-1 is absent, which also removes MED-1,2 (Bowerman et al. 1992; Maduro et al. 2001); and a 34 malformed intestine when ELT-2 is absent (Fukushige et al. 1998). 99x51mm (300 x 300 DPI) 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Molecular Reproduction and Development Page 40 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Evidence for partial specification of gut when the endoderm GRN is perturbed at the level of end-1,3 . (A) Polarized light showing gut granules (white spots) in a collection of isogenic end-1,3(-) embryos partially 48 rescued by an end1 transgene in which the MED binding sites have been mutated (Maduro et al. 2015). 49 Most embryos lack gut granules, some have a nearnormal amount (yellow arrowheads), and others have 50 very small amounts (white arrowheads). (BJ) Examples of embryos in which all, some or none of the E 51 descendants have adopted a gut fate. (B, C, D) Differential Interference Contrast (DIC) microscopy images 52 of embryos at 8E12E stage. The cells forming the embryonic intestine are outlined in white dashed lines in 53 B and C. (E, F, G) Stacks of images showing a nuclearlocalized end-3::mCherry transcriptional reporter in 54 the descendants of the E cell. Nuclei adopting a nongut identity are outlined by a dashed cyan line. The 55 reporter was made previously (Murray et al. 2008). Because all upstream regulators of end-3 are present, the reporter is activated in the early E lineage even when no gut is made. (H, I, J) Expression of a nuclear 56 localized elt-2::GFP reporter shows nuclei of cells that have adopted a gut fate. In (H), all E descendants 57 express elt-2::GFP; in (I), only some descendants do; and in (J), none of the E descendants express elt- 58 59 60 John Wiley & Sons Page 41 of 42 Molecular Reproduction and Development

1 2 3 2::GFP. In (EJ) the eggshell is outlined in yellow. A C. elegans embryo is approximately 50 µm long. In (B 4 J), anterior is to the left. 5 138x191mm (300 x 300 DPI) 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Molecular Reproduction and Development Page 42 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 Models for how gut specification can fail. (A) Binary (all-or-none) model in which gut specification either occurs or it does not. (B) A schematic diagram of how "partial" specification leads to fewer gut nuclei due to 25 a later commitment to gut fate by some E descendants and not others. For simplicity, the same pattern of 26 cell divisions is maintained across the various examples. 27 91x43mm (300 x 300 DPI) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Page 43 of 42 Molecular Reproduction and Development

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 Relationship of the activity of END-1+END-3 with gut specification and organogenesis of the intestine. At 32 high levels, specification and development of the intestine are robust. At intermediate levels, development 33 becomes stochastic, and animals can experience the absence of gut (aplasia), changes in gut cell number 34 (hypo- or hyperplasia), or no change (normoplasia). Animals with compromised END-1+END-3 can survive 35 hyperplasia, although they exhibit defects in metabolism. Modified from a prior work (Maduro et al. 2015). 36 103x75mm (300 x 300 DPI) 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons