HIGHLIGHTED ARTICLE| INVESTIGATION

Rapid DNA Synthesis During Early DrosophilaEmbryogenesis Is Sensitive to Maternal Humpty

Dumpty Protein FunctionShera Lesly,* Jennifer L. Bandura,† and Brian R. Calvi*,1

*Department of Biology, Indiana University, Bloomington, Indiana 47408 and †Biology Department, Lock Haven University ofPennsylvania, Pennsylvania 17745

ORCID ID: 0000-0001-5304-0047 (B.R.C.)

ABSTRACT Problems with DNA replication cause cancer and developmental malformations. It is not fully understood how DNAreplication is coordinated with development and perturbed in disease. We had previously identified the Drosophila gene humpty dumpty (hd),and showed that null alleles cause incomplete DNA replication, tissue undergrowth, and lethality. Animals homozygous for the missense allele,hd272-9, were viable, but adult females had impaired amplification of eggshell protein genes in the ovary, resulting in the maternal effects ofthin eggshells and embryonic lethality. Here, we show that expression of an hd transgene in somatic cells of the ovary rescues amplification andeggshell synthesis but not embryo viability. The germline of these mothers remain mutant for the hd272-9 allele, resulting in reduced maternalHd protein and embryonic arrest during mitosis of the first few S/M nuclear cleavage cycles with chromosome instability and chromosomebridges. Epistasis analysis of hd with the rereplication mutation plutonium indicates that the chromosome bridges of hd embryos are the resultof a failed attempt to segregate incompletely replicated sister chromatids. This study reveals that maternally encoded Humpty dumpty protein isessential for DNA replication and genome integrity during the little-understood embryonic S/M cycles. Moreover, the two hd272-9 maternal-effect phenotypes suggest that ovarian gene amplification and embryonic cleavage are two time periods in development that are particularlysensitive to mild deficits in DNA replication function. This last observation has broader relevance for interpreting why mild mutations in thehuman ortholog of humpty dumpty and other DNA replication genes cause tissue-specific malformations of microcephalic dwarfisms.

KEYWORDS Drosophila; DNA replication; Humpty dumpty; Donson; nuclear cleavage; microcephalic dwarfism

GENOMIC DNA must be fully and accurately replicatedwhen a cell divides. To achieve this, multiple steps of

DNA replication are coordinated with different phases of theeukaryotic cell cycle. Prereplicative complexes (pre-RC) as-semble onto replication origins in G1 phase, and are thenactivated by cell cycle kinases to initiate replication during Sphase (Diffley et al. 1994; Parker et al. 2017; Tocilj et al.2017; Yuan et al. 2017; Zhai et al. 2017). Upon initiation, alarge replisome complex of proteins assembles at replicationforks, which then migrate bidirectionally outward from theorigin to synthesize new DNA strands (Pellegrini and Costa2016). Reassembly of new pre-RCs is then inhibited until

after the next mitosis, ensuring that each genomic region isduplicated only once per cell division (Hills and Diffley2014). Problems with any of these steps of DNA replicationcan cause “replication stress” and DNA damage, which issensed and corrected by cell cycle checkpoints (Hills andDiffley 2014). However, high levels of replication stress andcheckpoint failure are major contributors to genome instabil-ity and cancer (Hills and Diffley 2014; Zeman and Cimprich2014). In addition, hypomorphic mutations in a number ofhuman genes that encode pre-RC and other replication pro-teins are the genetic cause of a class of developmental mal-formations known as microcephalic primordial dwarfisms(MPDs) (Klingseisen and Jackson 2011; Khetarpal et al.2016). While much has been learned, it is still not fully un-derstood how the DNA replication program is coordinatedwith development and perturbed in disease.

An extreme example of developmental modification to theDNA replication program occurs during early Drosophila em-bryogenesis. Embryogenesis begins with division of nuclei in

Copyright © 2017 by the Genetics Society of Americadoi: https://doi.org/10.1534/genetics.117.300318Manuscript received June 28, 2017; accepted for publication September 20, 2017;published Early Online September 22, 2017.Supplemental material is available online at www.genetics.org/lookup/suppl/doi:10.1534/genetics.117.300318/-/DC1.1Corresponding author: Department of Biology, Indiana University, 1001 E. 3rd St.,Bloomington, IN 47405. E-mail: bcalvi@indiana.edu

Genetics, Vol. 207, 935–947 November 2017 935

a common syncytium, and the first nine of these “nuclear cleav-age”divisions consist of only S andMphases (Rabinowitz 1941;Foe and Odell 1993; O’Farrell et al. 2004). The duration ofthese first nine S/M cycles are rapid, �8 min, during whichgenomic DNA is replicated in as little as 3 min by initiatingreplication from numerous, closely spaced origins (�10 kbapart) (Blumenthal et al. 1974; Sasaki et al. 1999). Becausethe majority of zygotic transcription does not begin until laterduring the mid-blastula transition (MBT), the early nuclearcleavage divisions are completely dependent on maternallyencoded proteins (Edgar and O’Farrell 1989). Much remainsunknown, however, about the regulation of the first nine S/Mcycles, and how complete genome duplication exactly onceper cycle is ensured. These early cycles lack a DNA replica-tion or DNA damage checkpoint, although they do have aspindle assembly checkpoint (SAC) that monitors microtubule-kinetochore attachments (Raff and Glover 1988; Girdham andGlover 1991; Fogarty et al. 1997; Sibon et al. 1997, 1999, 2000;Takada et al. 2003, 2007, 2015; Perez-Mongiovi et al. 2005;Crest et al. 2007; Oliveira et al. 2010). While the early, rapidS/M cycles may be an advantage by shortening developmentaltime, they also increase the risk of propagating DNA mutationinto multiple cells that are descended from these early foundernuclei (O’Farrell et al. 2004).

Another extreme example of developmental modificationto the DNA replication program occurs during Drosophilaoogenesis when genes required for eggshell (chorion)synthesis are selectively amplified in DNA copy number(Spradling 1981). This developmental gene amplification oc-curs through DNA rereplication from amplicon origins at sixloci in somatic follicle cells of the ovary (Claycomb and Orr-Weaver 2005; Calvi 2006; Nordman and Orr-Weaver 2012).Similar to other origins, amplicon origins are bound by thepre-RC and activated by cell cycle kinases, and have beenused as a model system to uncover conserved DNA replica-tion mechanisms (Calvi et al. 1998; Asano and Wharton1999; Royzman et al. 1999; Loebel et al. 2000; Aggarwaland Calvi 2004; Zhang and Tower 2004; Nordman et al.2011; Alexander et al. 2016). Among other methodologicaladvantages, gene amplification can be used for forward ge-netic identification of DNA replication genes. Mild, hypomor-phic mutations in a number of pre-RC and other essentialreplication protein genes are homozygous viable, but defec-tive for gene amplification in the ovary, resulting in a distinctthin eggshell phenotype (Landis et al. 1997; Landis andTower 1999; Whittaker et al. 2000, Yamamoto et al. 2000;Claycomb et al. 2002; Schwed et al. 2002; Calvi 2006).

We used this forward genetic system to identify the genehumpty dumpty (hd) (Snyder et al. 1986; Bandura et al.2005). Animals homozygous for a mild hypomorphic allele,hd272-9, are homozygous viable, but adult females are defec-tive for amplification, resulting in eggs with thin shells thatdehydrate and fail to develop. In contrast, animals homozygousfor null alleles of hd die as pupaewith severely underdevelopedbrains and imaginal discs. Further analysis showed that hdexpression is regulated by E2F1 and peaks at G1/S, and is

essential in all proliferating cells to prevent incomplete DNAreplication and DNA damage (Bandura et al. 2005). Hd proteinsequence is conserved in multicellular eukaryotes, with oneortholog in each genome, suggesting a conserved essentialrole in genome integrity (Bandura et al. 2005). The mouseortholog of Humpty dumpty was named Downstream of Son(Donson orDons) because of its genomic position (Wynn et al.2000). Consistent with evidence in flies, human Donson ex-pression peaks at G1/S, and RNAi screens indicated that it isrequired for genome integrity (Whitfield et al. 2002; Fuchset al. 2010).

It has been reported recently that human Dons associateswith replication forks, and thatDonsmissensemutations are agenetic cause of microcephalic primordial dwarfisms (MPDs)(Evrony et al. 2017; Reynolds et al. 2017). MPDs are charac-terized by severe pre and postnatal undergrowth with micro-cephaly, and display variably expressive other phenotypes(Klingseisen and Jackson 2011; Khetarpal et al. 2016).Hypomorphic mutations in a number of replication proteinsresult in a subtype of MPD known as Meier-Gorlin Syndrome(MGS) (Khetarpal et al. 2016). It remains unclear, however,why different hypomorphic mutations in Dons and other es-sential replication proteins have different clinical presenta-tions and affect some tissues more than others.

In this study we show that maternally encoded Hd proteinis essential to supportDNAsynthesis during the rapid Sphasesof the enigmatic embryonic cleavage cycles, In addition, ourresults uncover that there are two time periods in Drosophiladevelopment that are sensitive to deficits of DNA replicationproteins: ovarian developmental gene amplification andembryonic nuclear cleavage cycles. We discuss the broaderimplications of our findings for interpreting why mild defi-cits in the function of the hd ortholog, Donson, and otherDNA replication proteins result in the tissue-specific pheno-types of microcephalic primordial dwarfisms.

Materials and Methods

Drosophila strains and genetics

All of the hd mutant strains have been described previously(Bandura et al. 2005), and other strains were obtained fromthe Bloomington Drosophila Stock Center (BDSC, Blooming-ton, IN). The plu3/CyO strain was a gift from T. Orr-Weaver(Shamanski and Orr-Weaver 1991). For the epistasis analysisof Figure 6, C323-Gal4; plu3/CyO-GFP;Df(3R)3-4 ru th st/TM6Tb Hu females were created and crossed to P{w+mC UAS-hd};plu3/SM1a; h th st hd272-9 e/TM3 Sb males. Different adultfemale progeny from this cross were either homozygous mu-tant for plu, or hd, both, or neither, and the nuclear cleavagephenotype of their embryos analyzed. Clonal analysis of hdFf inthe ovary was performed by crossing males from the strainw1118; P{ry +t7.2 neor FRT}82B P{w +mC Ubi-GFP(S65T)nls}3R/TM6B Tb to virgin females that were either w; FRT82B hdFf/TM3, or w; FRT82B for control clones, as previously described(Bandura et al. 2005).

936 S. Lesly, J. L. Bandura, and B. R. Calvi

Construction of hd rescue transgenes

The P-element transgene P{w+mC UAS-hd} expresses the hdcDNA under control of the UASt promoter, and was previouslydescribed (Bandura et al. 2005). The P-element transgeneP{ w+mC hd-GFP} expresses an Hd-GFP fusion protein undercontrol of the hd promoter. It contains a genomic subclone ofthe hd locus that is fused in frame on its 39 end to eGFP, in-cluding 575 bp of genomic DNA upstream of the hd transcrip-tion start site, which contains a putative E2F binding site thatresponds to E2F1/Dp overexpression. The details of its con-struction using the Gateway Cloning System (ThermoFisher)are available upon request. An insertion of P{ w+mC hd-GFP}on the third chromosome was used for rescue experiments ofFigure 4.

Embryo and ovary preparation andimmunofluorescent analysis

Ovaries were dissected, fixed, and labeled as previously de-scribed (Bandura et al. 2005). Embryos were collected byallowing females to lay eggs on grape plates supplementedwith fresh yeast paste for 1 hr. For nuclear counts in Figure 1,however, embryos were collected for 30 min, and fixed im-mediately or transferred to a humid chamber to develop at25� for 30 or 60 min as indicated before fixing. Embryoswere fixed by first dechorionating with a 1:1 mixture ofhousehold bleach: 0.2% NaCl/0.02% Triton-X 100, andthen slow fixed using a modification of previous protocols(Rothwell and Sullivan 2000). Briefly, dechorionated em-bryos were brushed into a glass vial, and then 1 ml heptanewas added followed by 1 ml of 3.7% formaldehyde in PEMbuffer. After shaking the vial for 15 sec, the embryos wereallowed to fix at room temperature for 15 min. The bottomlayer was removed and 1 ml of methanol was added. Aftervortexing for 10 min, the embryos that were permeablizedand sank were collected. Embryos were stored at 220�. Be-fore antibody labeling, embryos were rehydrated through aPBTA/Methanol series as previously described (Rothwelland Sullivan 2000). Embryos that were labeled with anti-bTubulin were fixed in 500 ml heptane and 500 ml of roomtemperature 97% methanol with 15 mM EGTA for 10 minwith constant shaking. The embryos were allowed to settle forup to 1 min and the fix removed. Embryos were washed 33with methanol-EGTA solution, then fresh methanol-EGTAwas added and the embryos stored at 220� for a minimumof 24 hr. Before fixation embryos were rehydrated using pro-tocols appropriate for the fixation as described (Rothwell andSullivan 2000).

The following primary antibodies were used for labelingfixed embryos or ovaries at the indicated concentrations:Rabbit anti-full length Hd antibody (1:1,000) (Banduraet al. 2005), mouse anti-bTubulin 1:500 [DevelopmentalStudies Hybridoma Bank, (DSHB)], mouse anti-Cyclin B(1:100) (DHSB), rabbit polyclonal anti-GFP (1:50) (Invitro-gen), rabbit anti-Cnn antibody was used at (1:200) (Eismanet al. 2009). Fluorescent secondary antibodies were anti-mouse

or anti-rabbit Alexa 488 and Alexa 568 (Invitrogen), bothused at 1:500–1:750. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) or TOTO-3 (MolecularProbes) as described (Calvi and Spradling 2001). Embryos

Figure 1 Expression of hd in ovarian follicle cells rescues eggshell synthesis butnot embryo survival. (A and B) Eggs laid by wild type (A) and hd272-9 mutant (B)mothers. Bar, 50 mm. (C) Somatic rescue (SR) of hd mutant mothers. Strategyfor rescue of gene amplification defects in hd272-9/Df(3R)3-4 by driving expressionof UAS-hd with c323GAL4 in somatic follicle cells (pink), but not germline nursecells and oocytes. (D) Egg laid by hd SR female with fully rescued eggshellsynthesis. Bar, 50 mm. (E) Eggs from hd SR mothers fail to hatch. Numbersindicate percent of wild type or hd SR embryos that hatched, n = 200 embryos.(F and G) Cleavage stage embryos from wild type (F) and hd SR (G) femaleslabeled with DAPI, with higher magnifications shown in insets. Arrowhead ininset of (G) points to a chromosome bridge. Bar in (F and G) are 100 and 25 mmin the insets. (H) Quantification of the number of nuclei in wild type and hd SRembryos during three time windows after egg deposition (AED). Females wereallowed to lay eggs on plates for 30 min, after which the population of embryoswas either fixed immediately (0–30 min) or allowed to age for 30 min (30–60 min) or 60 min (60–90 min) before fixing and DAPI labeling. Nuclei werecounted in.30 embryos, and the mean and SD of nuclear number per embryoare indicated. The error bars for hd SR embryos are not apparent because mostembryos had two nuclei and the SD is very small.

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and ovaries were mounted in Vectashield (VECTOR labora-tories). Slides were analyzed using Leica SP5 laser scanningconfocal microscope with LAS AF software, and a LeicaDMRA2 epifluorescence microscope with Openlab software.Bright field images of the eggshell were taken with a NikonSMZ1500 microscope and DMX1200 Nikon digital camerawith Act-1 imaging software.

Protein preparation and Western blotting

Protein was prepared from 0 to 1 hr. dechorionated embryosby grinding in a microcentrifuge tube followed by boiling inLaemmli loading buffer (Adolphs et al. 1977; Thomer et al.2004). Protein was transferred to membrane by electroblot-ting and Hd protein detected with rabbit anti- full-length Hdantibody at 1:10,000 as previously described (Bandura et al.2005). Mouse anti-bActin (1:250) (Sigma A1978) or mouseanti-aTubulin (1:1000) (Eisman et al. 2009) were used asloading controls.

Data and reagent availability

More information about reagents used in this study can befound in the Supplemental Material, Reagent Table. All re-agents from this study are freely available upon request.

Results

Expression of a humpty dumpty cDNA in ovariansomatic follicle cells rescues eggshell synthesis but notembryo viability

We previously showed that flies homozygous for the humptydumpty (hd) hypomorphic allele, hd272-9 (G463D), are viable,but adult females have a severe defect in developmental geneamplification, and produce eggs with severely thin shells thatdehydrate and collapse after they are laid (Figure 1, A and Band Table 1) (Bandura et al. 2005). As part of our initialidentification of hd, we had used c323GAL4 to drive expres-sion of a hd cDNA (UAS-hd) specifically in somatic folliclecells of the ovary, which completely rescued eggshell geneamplification and eggshell synthesis in these hd272-9 mutantfemales (Bandura et al. 2005). Despite rescue of eggshell syn-thesis, the embryos from these rescued females failed to hatch

into larvae. Crossing these rescued females to wild-type malesresulted in embryos heterozygous for the recessive hd272-9, butthese embryos again failed to hatch, indicating that this failurein development is the result of amaternal effect. To ensure thatthis maternal-effect, embryonic lethality is not caused by ho-mozygosity for an undefined mutation on the hd272-9 chromo-some, we repeated this experiment by expressing UAS-hd infollicle cells of females who were trans-heterozygous forhd272-9 and a deletion allele of hd, Df(3R)3-4 (Figure 1C andTable 1) (Bandura et al. 2005). These rescued females laid eggswhose shells and overall morphology were indistinguishablefrom wild type, yet their embryos again failed to develop andhatch into larvae (Figure 1, D and E and Table 1). Given thatthe GAL4/UAS system that we used is active in somatic folliclecells but not the germline, we hypothesized that the embryoniclethality was the result of a deficit of hd that is supplied to theembryo through thematernal germline (Figure 1C). Because ofthis special genotype, we will hereafter refer to the c323GAL4/UAS-hd; hd272-9/Df(3R)3-4 females as hd Somatic Rescue (SR)mothers, and their offspring as hd SR embryos (Figure 1D).

Embryos from hd SR mothers arrest during the first fewnuclear cleavage cycles

To explore why embryos from hd SR mothers failed to hatch,we examined embryonic development using fluorescence mi-croscopy. A population of embryos from wild-type and hd SRfemales distributed from 0 to 1 hr of development were fixedand labeled with the fluorescent DNA dye DAPI. While wild-type embryos had up to several 100 nuclei, all hd embryoshad, at most, a few nuclei, which often had long chromosomebridges between them (up to 30 mm) (Figure 1, F and G).These embryos were fertilized because arrested nuclei werein the interior of the embryo, indicating that maternal pro-nuclear migration had occurred on microtubules that are nu-cleated by sperm-derived centrioles (Figure 1G, see alsocentriole labeling in Figure 2). To quantify the arrest of hdSR embryos, we allowed wild-type and hd SR females to layeggs on plates, and then aged the embryos for differentamounts of time at 25� before fixing and DAPI labeling. Nu-clear number increased exponentially during the rapid cleav-age cycles of wild-type embryos, whereas hd SR embryos had

Table 1 Summary of hd genotypes and phenotypes

Maternal Effects

Genotype Viabilitya Eggshell Synthesisb Embryonic Cleavagec

hdFf/hdFf — N.A. N.A.hd272-9/hd272-9 + — —

hd272-9/Df(3R)3-4 + — —

c323Gal4/UAS-hd; hd272-9/Df(3R)3-4 + + —

hd-GFP/hd-GFP; hd272-9/Df(3R)3-4 + + +/2d

hd-GFP/hd-GFP; hdFf/Df(3R)3-4 +e n.d. n.d.a +, viable to adulthood; 2, not viable to adulthood.b +, adult females have normal gene amplification and produce eggs with normal shells; 2, defective amplification and thin eggshells.c +, embryos from mothers of indicated genotype have normal cleavage divisions; 2, arrest during first few cleavage divisions.d +/2, hd-GFP expression in the maternal germline rescued the early embryonic cleavage arrest, but these embryos arrested during later cleavage division.e Rescue of viability was �100% relative to sibling controls.

938 S. Lesly, J. L. Bandura, and B. R. Calvi

an average of only two nuclei up to 90 min of development,with almost all nuclei connected by chromatin bridges (Fig-ure 1H). Because expression of the zygotic genome does notbegin until the MBT, these early nuclear cleavage cycles arecompletely dependent on the maternal supply of RNA andprotein. These data suggested, therefore, that embryos fromhd SR mothers arrest during the first few nuclear cleavagecycles with incomplete chromosome segregation because of adeficit in the maternal supply of Hd.

hd SR embryos have severe chromosome segregationand spindle abnormalities

To define the nuclear division defect in hd SR embryos, welabeled chromosomes, spindles, and centrosomes with DAPI,anti-b-Tubulin, and anti-Centrosomin (Cnn) respectively(Chu and Klymkowsky 1989; Eisman et al. 2009). Comparedto wild type, spindle morphology and chromosome segrega-tion in hd SRembryos were severely aberrant (Figure 2, A–F).Spindles were variable in size and shape, with some appear-ing barrel-shaped while others were extremely narrow andlong (Figure 2, B and C). In some cases, two spindles sur-rounded two closely adjacent chromatin masses of unequalsize, and were connected by a chromatin bridge, indicative ofincomplete and unequal chromosome segregation during theprevious mitotic division (Figure 2B). Spindles often hadmore than two centrosomes, suggesting that centrosomescontinued to duplicate despite nuclear division arrest, consis-tent with previous evidence that centrosome duplication canbe uncoupled from mitotic divisions during embryonic cleav-age stages (Raff and Glover 1988). Some of the spindles withextra centrosomes were bipolar, with evidence of complete orpartial “centrosome clustering,” while other spindles withextra centrosomes were multipolar (Figure 2, C–E) (Kwonet al. 2008; Ganem et al. 2009). Many of the embryos had

multiple centrosomes that were not associated with spindlesor chromatin, further suggesting that centrosome duplicationcontinued in the absence of nuclear division (Figure 2F).Together, these results suggest that a deficit of maternallysupplied Hd results in severe chromosome instability andembryonic arrest during the first few nuclear cleavagedivisions.

Maternal Hd protein is abundant during earlyembryonic cleavage cycles but severely reduced inembryos from hd SR mothers

The results suggested that maternally encoded Hd protein isrequired to support early cleavage cycles of the embryo. Toexamine maternal Hd protein abundance during these earlycycles, we immunolabeledwild type embryoswith an affinity-purified polyclonal Hd antibody during the first hour ofembryogenesis before zygotic gene expression begins(Bandura et al. 2005). Beginningwith the first division cycles,Hd protein was concentrated in the interphase (S phase)nucleus, with lower levels in the cytoplasm, and more evenlydistributed during M phase after nuclear envelope break-down (Figure 3, A–A”). In contrast, Hd protein was undetect-able by immunofluoresence in embryos from hd272-9 SRmothers (Figure 3, B–B”). We further quantified the level ofHd protein in 0–1 hr wild-type and hd SR embryos by West-ern blotting, which confirmed that maternally encoded Hdprotein is abundant during wild-type early embryonic cleav-age stages, and reduced in embryos from hd272-9 SR mothers(Figure 3C). The ability to detect low levels of Hd protein byWestern blotting but not immunofluoresence is because hdSR embryos arrest during M phase when Hd protein is dis-persed throughout the embryoplasm, which is a limitation fordetection by immunofluorescence but not Western blotting.The lower level of Hd protein in hd SR embryos is consistent

Figure 2 Embryos from hd SR femaleshave chromosome segregation errors andgenome instability. (A–F) Confocal imagesof nuclear divisions in embryos from wild-type (A) and hd SR (B–F) mothers labeledwith DAPI (DNA, blue), b-Tubulin (red), andcentrosome protein Centrosomin (Cnngreen). (A) Wild-type metaphase nucleuswith a bipolar spindle and a single centro-some per pole. (B) hd SR embryo with achromatin bridge that connects two nucleiarrested in metaphase, and of unequalDNA content, presumably a result of un-equal and incomplete chromosome se-gregation in the previous cycle. (C) Anexample of an hd extended spindle withmultiple centrosomes at the poles andstretched chromosomes. (D) An hd multi-polar spindle. (E) Two spindles with incom-plete clustering of excess centrosomes atthe poles. (F) An example of free centro-somes that continue to duplicate uncoupledfrom the arrested nuclear division cycle. Bar,10 mm.

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with our previous evidence that the hd272-9 missense allele(G463D) results in lower levels of Hd protein in imaginaldiscs (Figure 3C) (Bandura et al. 2005). The maternal supplyof Hd protein in early wild type embryos is also consistentwith ModENCODE data that maternal hd mRNA is highlyabundant in 0–2 hr embryos before the onset of zygotic tran-scription (Roy et al. 2010; Marygold et al. 2016; Gramateset al. 2017). Together, these data indicate that embryos fromhd272-9 SR mothers have reduced amounts of maternallyencoded Hd protein that are insufficient to support early nu-clear cleavage cycles.

Expression of humpty dumpty in the maternal germlineand soma partially rescues the embryoniccleavage defects

To further address whether a deficiency of maternal hd isresponsible for the early arrest phenotype in syncytialDrosoph-ila embryos, we created a rescue transgene that expresses Hdprotein in the soma and germline. This transgene contains agenomic subclone of the hd locus that is fused in frame on its 39end to GFP (hd-GFP), and includes 575 bp of genomic DNAupstream of the hd transcription start site, including a putativeE2F binding site that responds to E2F1/Dp (Bandura et al.2005). Unlike UAS-hd, hd-GFP is expressed in both germlineand somatic cells of the ovary. We used this hd-GFP transgeneto rescue hd272-9/Df(3R)3-4, hereafter hd272-9 Germline andSomatic Rescue (GSR) mothers. Embryos from these hd272-9

GSRmothers had high levels ofmaternally encodedHd-GFP inthe nucleus during early cleavage cycles (Figure 4, A–A”). Earlycleavage divisions in these hd272-9GSR embryos appeared nor-mal, with no evidence of the chromosome bridges that wereseen in hd SR embryos, indicating that maternal hd-GFP res-cues the early nuclear cleavage arrest (Figure 4, B and C).During later nuclear division cycles, however, aberrant spin-dles and chromatin bridges began to accumulate as nucleimigrated to the cortex, and all hd272-9 GSR embryos arrestedbefore cellular blastoderm (Figure 4, B–D). Thus, the expres-sion of Hd-GFP in the maternal germline partially rescues em-bryonic nuclear cleavagedivisions. The progressive accumulationof chromosome bridges and aberrant spindles during latercleavage cycles may result from depletion of insufficient

Hd-GFP. Consistent with this, Western blotting indicatedthat Hd-GFP protein was expressed to lower levels than en-dogenous Hd protein, perhaps explaining why rescue wasnot complete, although fusion to GFP may also compromiseHd function (Figure 4E). In contrast, hd-GFP completely res-cued imaginal disc cell proliferation and adult viability of hdnull larvae (hdFf/Df(3R)3-4) (Table 1). The ability of the hd-GFP transgene to fully rescue zygotic hd null animals but notembryos from hd hypomorph mothers is consistent with theinterpretation that cleavage cycles are sensitive to small def-icits of hd function. The penetrant bridging in the partiallyrescued embryos indicates that a deficit of maternal Hd re-sults in a failure of chromosome segregation and a nuclearcleavage cycle arrest.

hd mutant cells arrest during G2 and early M phase ofcanonical mitotic cell cycles

Theresults indicated that adeficit in thematernal supplyofHdresults in chromosome instability and mitotic division arrestduring thefirst fewembryonicnuclearS/Mcleavagedivisions.The penetrant chromosome bridging in these embryos mayreflect a requirement for hd for proper sister chromatid seg-regation during M phase. Alternatively, since early nuclearcleavage cycles lack DNA replication or damage checkpoints,the chromosome bridges could result from an attempt tosegregate sister chromatids that were incompletely repli-cated during the previous S phase.

To begin to evaluate whether the chromosome bridgephenotype reflects a function of Hd during S or M phase,we examined the impact of hd mutations during a canonicalmitotic cell cycle that has DNA replication and damage check-points. We chose proliferating somatic follicle cells of theovary because they have a well-characterized cell cycle andform an epithelial sheet that is amenable to imaging. Thesefollicle cells mitotically proliferate up to stage 6 of oogenesis,after which they switch to G/S endocycles (Mahowald et al.1979; Klusza and Deng 2011).We used a heat inducible FLP/FRT system to make homozygous mutant clones of the nullallele hdFf, and then examined follicle cell cycle behavior byantibody labeling for Cyclin B (Knoblich and Lehner 1993; Xuand Rubin 1993). During canonical mitotic cell cycles, Cyclin

Figure 3 Hd protein is abundant duringembryonic nuclear cleavage cycles and reducedin embryos from hd SR females. Confocal im-ages of embryos fromwild type (A–A”) and hdSR (B and B’) mothers labeled with the DNAdye TOTO-3 (red) (A and B), anti-Hd (green) (A’and B’), and merge (A” and B”). Bar, 10 mm.(C) Western blot of cleavage stage (0–1 hr)embryo extracts from hd SR and wild-typemothers incubated with anti-Hd antibody andanti-b-actin antibody as loading control.

940 S. Lesly, J. L. Bandura, and B. R. Calvi

B protein begins to appear in the cytoplasm during late Sphase, increases in the cytoplasm during G2 phase, becomesboth nuclear and cytoplasmic during the G2 to M phase tran-sition, and is then rapidly degraded at metaphase/anaphase(Knoblich and Lehner 1993; Gavet and Pines 2010). Labelingof wild-type follicle cells with anti-Cyclin B antibody reflectedthis known cell cycle behavior of Cyclin B protein during themitotic proliferation of follicle cells (Figure 5A). As expected,anti-Cyclin B did not label follicle cells after stage 6 whenthey are in the endocycle, and the expression of Cyclin B isrepressed (Figure 5A) (Sun and Deng 2005). In contrast,clones of homozygous hdFf cells had high levels of Cyclin Bin the cytoplasm only, and this labeling persisted past stage 6when neighboring wild type follicle cells had switched toendocycles (Figure 5B). This observation is consistent withour previous clonal analysis in wing discs, which showed thathdmutant cells can divide a limited number of times as stableHd protein is slowly depleted, but then eventually arrest with

incomplete DNA replication and DNA damage (Bandura et al.2005). The accumulation of hdmutant follicle cells with highlevels of Cyclin B in the cytoplasm suggest that these cells arearresting at the G2/M DNA damage checkpoint, perhaps be-cause of incomplete DNA replication in the previous S phase.Unlike hd mutant cleavage stage embryos, we saw no evi-dence of chromosome bridging in hd mutant follicle cells.This difference between hd follicle cells and embryos is notbecause of different alleles or levels of Hd protein because wesaw no evidence of bridging or arrest in the follicle cells ofhd272-9 SR mothers, despite a highly penetrant bridging phe-notype in their embryos. These data are consistent with theidea that the hd chromosome bridge phenotype during cleav-age cycles is caused by a sensitivity to replication defectscombined with an absence of a G2/M checkpoint, which re-sults in a failed attempt to segregate incompletely replicatedsister chromatids.

Maternal Hd is required for DNA rereplication inembryos from plutonium mutant mothers

We wanted to test directly whether maternal Hd protein isrequired for DNA replication during early cleavage cycles. Todo this would require separating the potential function of Hdduring S phase vs. M phase, which is challenging during therapid S/M cycles. We therefore took a genetic approach byconducting an epistasis experiment with the gene plutonium(plu) (Shamanski and Orr-Weaver 1991). The Plu protein is amember of the trimeric giant nucleus complex, which is com-posed of Plu, Giant nucleus (Gnu), and Pan Gu (Png) (Leeet al. 2003). This maternally encoded complex is required fornormal Cyclin B levels and mitosis during embryonic nuclearcleavage cycles (Lee et al. 2001). Embryos frommothers whoare homozygous mutant for the null allele plu3 undergo mul-tiple rounds of S phase without mitosis, and eventually arrestwith one or a few giant polyploid nuclei (Axton et al. 1994;Lee et al. 2003).We reasoned that if Hd is required formitosisand not S phase, embryos from hd; plu double mutant moth-ers should have the plu mutant phenotype: one to severalgiant nuclei. Conversely, if hd is required for DNA replication,embryos form hd; plu double mutant mothers should havethe hd phenotype: several small nuclei.

We created female siblings that haddifferent combinationsof genotypes at the plu and hd loci, and that were somaticallyrescued for hd eggshell defects using c323GAL4; UAS-hd. Asexpected, females heterozygous for the recessive plu3 allelegave rise to embryos in which S/M cleavage cycles occurrednormally, whereas females that were homozygous mutant forplu3 gave rise to embryos with the giant nucleus phenotype,which was not genetically modified when these motherswere heterozygous for the hd deletion Df(3R)3-4 (Figure 6,A–B’). Females that were heterozygous for the recessive plu3

allele (plu3/+) and trans-heterozygous mutant for hd (hd272-9/Df(3R)3-4) gave rise to embryos with the hd early cleavagearrest and chromosome bridge phenotypes (Figure 6, C andC’). Importantly, the plu3; hd272-9/Df(3R)3-4 double mutantfemales had embryos that arrested with the hd single mutant

Figure 4 Expression of hd-GFP in the maternal germline partially rescuesembryonic nuclear cleavage cycles. (A–A”) Germline and somatic rescue(GSR) of hdmothers. Confocal images of nuclei of an early embryo from ahd-GFP; hd272-9/Df(3R)3-9 mother that expresses hd-GFP under control ofthe hd promoter in both germline and somatic cells of the ovary. Shownare DNA (A) Hd-GFP (A’) and merge (A”). Bar, 10 mm. (B–D) Maternallysupplied Hd-GFP partially rescues the hd cleavage defect. Confocal im-ages of embryonic mitoses from wild-type (B) and hd GRM mothersduring early (C) and later (D) nuclear cleavage cycles. Nuclei in hd GSRembryos initially divide normally, partially migrate to cortex, but theneventually arrest with a highly expressive chromosome bridge phenotypeduring cycles 8–9. Bar for (B–D) are 10 mm. (E) Western Blot of 0–1 hrembryo extracts from mothers of genotypes indicated at the top of eachlane. Blots were incubated with anti-Hd, which recognizes both endog-enous Hd and Hd-GFP. Incubation with a-Tubulin was used as a loadingcontrol.

Humpty Dumpty Cleavage 941

phenotype: several small nuclei (Figure 6, D and D’). This lastresult indicates that hd is epistatic to plu, and that hd is requiredfor the multiple rounds of DNA rereplication that occur in plumutant embryos in the absence of mitosis. Thus, in embryosfrom hd single mutant mothers, the long chromosome bridgeslikely result from insufficient Hd function during S phase andsubsequent stretching of incompletely replicated sister chroma-tids by the mitotic spindle. Altogether, the data suggest thatmaternal Hd is essential for DNA replication and genome in-tegrity during early Drosophila development.

Discussion

We previously showed that animals homozygous for thehypomorphic genotype hd272-9/Df(3R)3-4 are viable, butadult females have severe defects in developmental gene am-plification, produce eggs with thin shells, and are sterile. Inthis study, we found that rescue of this amplification defect isnot sufficient for female fertility, and that mothers with ahd272-9/Df(3R)3-4 mutant germline provide insufficient Hdfunction to their embryos, resulting in arrest during the firstfew nuclear cleavage cycles with long chromosome bridgesand genome instability. Although our previous findings wereconsistent with a role of Hd in DNA replication, it remainedformally possible that Hd had a different essential function ingenome maintenance (Bandura et al. 2005). Our current ge-netic and cell biological evidence show that maternallyencoded Hd protein is essential for DNA replication duringrapid nuclear cleavage cycles of early embryogenesis. To-

gether with our previous analysis of the hd272-9 allele, thesefindings suggest that embryonic nuclear cleavage cycles andovarian developmental gene amplification represent twotime periods in Drosophila development that are sensitiveto deficits in DNA replication protein function. These obser-vations have important broader relevance for understandinghow hypomorphic mutations in the human ortholog of hdand other essential replication proteins cause tissue-specificphenotypes in different types of microcephalic primordialdwarfisms (MPDs).

How DNA replication is coordinated with mitosis duringearly, rapid S/Mcycles is not completely understood.Our dataindicate thatmaternalHd is essential forDNAsynthesis duringthese early cycles. Embryos from hd hypomorphic mothersarrested in the first or second mitosis of nuclear cleavagedivision, often with pairs of condensed chromatin masses ofunequal size and connected by chromatin bridges. In con-trast, depletion of humpty dumpty in cells with canonicalmitotic cell cycles and checkpoints resulted in a cell cyclearrest during G2 and early M phase, consistent with a repli-cation checkpoint arrest. Together with our finding that hd isepistatic to the rereplication phenotype of plumutants, thesedata suggest that the long chromosome bridges in hdmutantembryos are likely the result of spindle forces pulling on in-completely replicated and attached sister chromatids. Thisinterpretation is consistent with the known absence of aDNA replication checkpoint during these early S/M cycles(Raff and Glover 1988). Although early cycles lack a DNAreplication checkpoint, they do have a spindle assemblycheckpoint (SAC), which likely contributed to the preponder-ance of M phase arrest that we observed (Oliveira et al.2010). The hd bridging phenotype is similar to that after in-jection of the DNA polymerase alpha inhibitor aphidicolininto early embryos and that of maternal effect mutations inseveral DNA replication and repair genes (Raff and Glover1988; Girdham and Glover 1991; Apger et al. 2010; Gosnelland Christensen 2011; Sakurai et al. 2011). Among thisgroup of mutants, the hd arrest is the earliest, during the firstor second cleavage cycles, suggesting that these early cyclesare extremely sensitive to levels of Hd function. This earlyarrest is not a reflection of a unique requirement for Hd pro-tein during the first few cleavage cycles because partial res-cue with hd-GFP resulted in a similar chromosome bridgingand arrest during later S/M cleavage cycles. Thus, sufficientmaternal Hd function is essential to ensure DNA replicationand genome integrity during early embryogenesis.

The hd embryos had supernumerary centrosomes that nu-cleated multipolar spindles, while other extra centrosomesclustered together to form a bipolar spindle. These supernu-merary centrosomes of multipolar and bipolar spindles likelycontribute to chromosome instability (CIN), and the unequalDNA content of arrested nuclei, similar to that documented incancer cells with amplified centrosomes (Kwon et al. 2008;Ganem et al. 2009; Godinho and Pellman 2014). It was pre-viously reported that overexpression of the human orthologof hd, Donson (Dons), results in its association with

Figure 5 Depletion of hd during canonical mitotic cell cycles results in aG2/M arrest. (A) An ovariole from a control hsp70-FLPase; FRT hdFf/FRThd+ GFP+ female that was not heat induced. Labeling of this ovariole withanti-GFP (green) and anti-cyclin B (red) reveals oscillating levels of Cyclin Bin follicle cells during mitotic division cycles up until stage 6 (S6), but notin endocycling follicle cells of stage 7 (S7) and later egg chambers. Thelarge polyploid nurse cells in the interior of the egg chamber that switchinto endocycles before stage 1 do not express mitotic cyclins during thesestages. Inset: A higher magnification of a follicle cell with Cyclin B incytoplasm and nucleus. (B) An ovariole from an hsp70-FLPase; FRT hdFf/FRT hd+ GFP+ female, in which an hdFf/hdFf null mutant clone (arrow,GFP2) was heat induced 2 days earlier. After initial cell divisions, slowdepletion of Hd protein in the hdFf null cells results in their arrest withhigh levels of Cyclin B in the cytoplasm but not nucleus, suggestive of aG2/M arrest, which persists after stage 6. Inset: A higher magnification ofa hd mutant follicle cell arrested with Cyclin B in the cytoplasm but notnucleus. Bar, 25 mm for main panels, 6 mm for insets.

942 S. Lesly, J. L. Bandura, and B. R. Calvi

centrosomes in human cells, but we did not observe Hd lo-calization to centrosomes using either Hd antibodies orHd-GFP fusions (Fuchs et al. 2010). Therefore, our interpre-tation is that the extra centrosome phenotype is an indirectconsequence of nuclear cycle arrest with continued centro-some duplication, consistent with the known independenceof centrosome and nuclear division cycles in early embryos(Raff and Glover 1988; Archambault and Pinson 2010).

Consistent with our findings, while this manuscript was inpreparation, Reynolds and colleagues reported that humanDons protein associates with replication forks and is requiredfor replication fork progression and the replication stress check-point in human cells (Reynolds et al. 2017). It remains unclear,however, whether Hd associates with replication forks in Dro-sophila (Bandura et al. 2005). Further work is needed in flies

and human cells to define the precise molecular function(s) ofHd/Dons proteins in DNA replication and checkpoint signaling.

Our analysis of different hd genotypes has revealed in-sights into how DNA replication programs are modifiedduring development. We previously showed that animalshomozygous for null alleles of hd (e.g., hdFf) die as pupaewith DNA damage and severe undergrowth of brains andimaginal discs (Table 1) (Bandura et al. 2005). Our evidencefor abundant Hd protein in the early embryo suggests thathdFf homozygous mutant embryos and larvae likely surviveon maternally supplied Hd, with severe defects in the mitoticproliferation of imaginal discs manifesting as pupal lethalitywhen it is time for these tissues to metamorphose into adultstructures. In contrast, animals homozygous for the hypo-morphic allele hd272-9 are viable, but adult females have

Figure 6 hd is epistatic to plu and requiredfor DNA replication. (A–D’) Embryos frommothers that were either single or doublemutant for hd and plu were labeled withDAPI. Lower (A–D) and higher (A’–D’) mag-nifications of nuclear phenotypes areshown. (A and A’) An embryo with normalnuclear cleavage divisions from a motherheterozygous for the recessive plu3 muta-tion and wild type for hd. (B and B’) Anembryo with two large polyploid nucleifrom a mother that was mutant for plubut heterozygous for the recessive hd de-letion Df(3R)3-4. (C and C’) An embryo witharrest at the second cleavage division andchromosome bridges from a hd SR motherwho was also heterozygous for the reces-sive plu mutation. (D and D’) hd is epistaticto plu. An embryo from a hd SR motherthat was also homozygous mutant for pluwith a cleavage arrest/chromosome bridgephenotype similar to that of embryos fromhd SR single mutant mothers. Bar, (A–D)100 mm, (A’–D’) 20 mm.

Humpty Dumpty Cleavage 943

severely reduced gene amplification resulting in thin egg-shells (Figure 7) (Snyder et al. 1986; Bandura et al. 2005).The hd272-9 amplification defect is similar to that caused byhypomorphic mutations in other essential DNA replicationgenes (i.e., Orc1, Orc2, Cdt1, Dbf4, Mcm6, and mus101), in-dicating that thin eggshells are a sensitized phenotype forsmall reductions in DNA replication protein function (Calviand Spradling 1999; Calvi 2006). Through rescuing the am-plification defect of hd272-9 in somatic follicle cells of theovary, we have uncovered that early S/M cleavage cycles ofthe embryo are also sensitive to reductions in maternal hdfunction. Thus, early embryonic cleavage cycles and develop-mental gene amplification are two time periods in Drosophiladevelopment that are highly sensitive to small deficits inreplication protein function (Figure 7). Both of these timesin development require high levels of DNA replication. Dur-ing S/M cycles, the genome is duplicated in �3 min byinitiating replication from numerous, closely spaced origins(Figure 7) (Blumenthal et al. 1974; Sasaki et al. 1999).During ovarian gene amplification, repeated reinitiationfrom specific origins is required to rapidly increase eggshell

protein gene copy number during a several hour time win-dow (Figure 7) (Calvi 2006). The genetic evidence suggeststhat these two rapid DNA replication programs require highlevels of DNA replication protein function. Moreover, earlyS/M cycles and developmental gene amplification havecompromised checkpoints and lack backup dormant originsthat normally respond to replication stress during canonicalcell cycles. Thus, differences in DNA replication demandand replication stress response among cell types likely ex-plain why hypomorphic alleles of essential DNA replicationgenes cause specific developmental phenotypes.

Two recent reports indicate that hypomorphic mutations inhuman Dons are a genetic cause of MPD (Evrony et al. 2017;Reynolds et al. 2017). The clinical presentation of childrenwithsome Dons mutations overlaps that of hypomorphic mutationsin a number of other replication proteins (Orc1, Orc4, Orc6,Cdt1, Cdc6, MCM5, Cdc45, and Geminin) that cause a subtypeofmicrocephalic dwarfismknown asMGS, characterized by preand postnatal undergrowth, microcephaly, small ears (micro-tia), and underdeveloped or absent knee caps (knee patellasyndrome), as well as other variably expressive phenotypes(Bicknell et al. 2011a,b; Balasov et al. 2015; Burrage et al.2015; Fenwick et al. 2016; Khetarpal et al. 2016; Vetro et al.2017). Thus, it appears that mild mutations in hd gene familymembers result in phenotypes that are similar to hypomorphicalleles of these other essential replication protein genes in bothflies (thin eggshell) and humans (microcephalic dwarfism).

While some alleles of Dons resembleMGSwith relativelymildeffects on growth, other alleles resemble other classes of MPDswith more severe pleiotropic phenotypes of impaired skeletal,brain, and lung development, which, in some cases, result inperinatal lethality (Khetarpal et al. 2016; Evrony et al. 2017;Reynolds et al. 2017). It remains unclear, however, why differentmutations in Dons, and other replication genes, have differentpleiotropic clinical presentations, and affect some tissues morethan others. Our finding that specific time periods of Drosophiladevelopment are more sensitive to hypomorphic alleles of hdraises the possibility that the range of tissue-specific clinical pre-sentationsofdifferentDonsallelesmay reflect adifferenceamongdeveloping tissues in the speed of DNA synthesis, availability ofdormant origins, or effectiveness of DNA replication checkpoints.A further examination of Hd–Dons function in specific tissues offly and human should shed light on the developmental and mo-lecular mechanisms of microcephalic primordial dwarfisms.

Acknowledgments

We would like to thank R. Eisman and T. Kaufman foradvice and Cnn antibody, and T. Orr-Weaver for plu flies.We also thank the Bloomington Drosophila Stock Center(BDSC) and Flybase for flies and critical information,J. Powers of the Indiana University (IU) Light MicroscopyImaging Center (LMIC) for help and advice, and M.-Y.Cho for Hd-GFP construction. This work was supportedby funding from National Institutes of Health (NIH)1R01GM113107 to B.R.C.

Figure 7 Developmental gene amplification and embryonic cleavage cy-cles are sensitive to reductions in Hd function. A model of Hd function ingermline and somatic cells of the Drosophila ovary. Hd is required insomatic follicle cells (pink) for DNA rereplication to amplify the copynumber of genes that are required for eggshell synthesis. Ovarian germ-line nurse cells provide maternal Hd to the oocyte and future embryo tosupport rapid DNA synthesis from closely spaced origins during nuclearcleavage cycles. Females homozygous or hemizygous for the hypomor-phic allele hd272-9 are viable but manifest severe maternal effects on bothovarian gene amplification and DNA synthesis during embryonic nuclearcleavage, suggesting that these two time periods in Drosophila develop-ment are especially sensitive to reductions in the function of maternallyencoded DNA replication proteins. We propose that these two time pe-riods are sensitive because of a high replication demand and a reducedreplication stress response. Please see the Discussion for a full descriptionof this model.

944 S. Lesly, J. L. Bandura, and B. R. Calvi

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Communicating editor: G. Bosco

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