Cell Lineage Patterns Research Articles

CRISPR/Cas9 Directed Reprogramming of iPSC for Accelerated Motor Neuron Differentiation Leads to Dysregulation of Neuronal Fate Patterning and Function.

Neurodegeneration causes a significant disease burden and there are few therapeutic interventions available for reversing or slowing the disease progression. Induced pluripotent stem cells (iPSCs) hold significant potential since they are sourced from adult tissue and have the capacity to be differentiated into numerous cell lineages, including motor neurons. This differentiation process traditionally relies on cell lineage patterning factors to be supplied in the differentiation media. Genetic engineering of iPSC with the introduction of recombinant master regulators of motor neuron (MN) differentiation has the potential to shorten and streamline cell developmental programs. We have established stable iPSC cell lines with transient induction of exogenous LHX3 and ISL1 from the Tet-activator regulatory region and have demonstrated that induction of the transgenes is not sufficient for the development of mature MNs in the absence of neuron patterning factors. Comparative global transcriptome analysis of MN development from native and Lhx-ISL1 modified iPSC cultures demonstrated that the genetic manipulation helped to streamline the neuronal patterning process. However, leaky gene expression of the exogenous MN master regulators in iPSC resulted in the premature activation of genetic pathways characteristic of the mature MN function. Dysregulation of metabolic and regulatory pathways within the developmental process affected the MN electrophysiological responses.

Cell lineage analysis reveals signal tracing and compartment characterisation in Drosophila haltere.

Insect halteres, as specialised hind wings, play an important role during aerial manoeuvres. In Drosophila, halteres and wings are homologous appendages with different morphology. Previous studies have focused on the metamorphosis of halteres, while current knowledge about its cell lineage and regional compartmentalization is still limited. In this study, we performed cell-lineage tracing of canonical landmark signals in halteres and present a simple model for haltere development. Cell lineage tracing in wings was used as a reference. The nub showed wing-like expressions in halteres, whereas hth and pnr exhibited different expressions in adult wings and halteres. The lineage tracing revealed that the pouch region gives rise to end-bulb, and hinge cells contribute to proximal haltere formation. Moreover, we demonstrated that twi-expressing cells participate in the cell population of the distal end-bulb. Haematoxylin and eosin staining indicated that muscle cells were present at the distal end-bulb. These results indicated that adult halteres displayed unique cell lineage patterns and the muscle cells are important components of end-bulbs.

Long-term organoid culture of a small intestinal neuroendocrine tumor.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare and highly heterogeneous neoplasms whose incidence has markedly increased over the last decades. A grading system based on the tumor cells' proliferation index predicts high-risk for G3 NETs. However, low-to-intermediate grade (G1/G2) NETs have an unpredictable clinical course that varies from indolent to highly malignant. Cultures of human cancer cells enable to perform functional perturbation analyses that are instrumental to enhance our understanding of cancer biology. To date, no tractable and reliable long-term culture of G1/G2 NET has been reported to permit disease modeling and pharmacological screens. Here, we report of the first long-term culture of a G2 metastatic small intestinal NET that preserves the main genetic drivers of the tumor and retains expression patterns of the endocrine cell lineage. Replicating the tissue, this long-term culture showed a low proliferation index, and yet it could be propagated continuously without dramatic changes in the karyotype. The model was readily available for pharmacological screens using targeted agents and as expected, showed low tumorigenic capacity in vivo. Overall, this is the first long-term culture of NETs to faithfully recapitulate many aspects of the original neuroendocrine tumor.

Volume segregation programming in a nematode's early embryogenesis.

Nematode species are well-known for their invariant cell lineage pattern during development. Combining knowledge about the fate specification induced by asymmetric division and the anti-correlation between cell cycle length and cell volume in Caenorhabditis elegans, we propose a minimal model to simulate lineage initiation by altering cell volume segregation ratio in each division, and quantify the derived pattern's performance in proliferation speed, fate diversity, and space robustness. The stereotypic pattern in C. elegans embryo is found to be one of the most optimal solutions taking minimum time to achieve the cell number before gastrulation, by programming asymmetric divisions as a strategy.

Pattern of Repetitive Element Transcription Segregate Cell Lineages during the Embryogenesis of Sea Urchin Strongylocentrotus purpuratus.

Repetitive elements (REs) occupy a significant part of eukaryotic genomes and are shown to play diverse roles in genome regulation. During embryogenesis of the sea urchin, a large number of REs are expressed, but the role of these elements in the regulation of biological processes remains unknown. The aim of this study was to identify the RE expression at different stages of embryogenesis. REs occupied 44% of genomic DNA of Strongylocentrotus purpuratus. The most prevalent among these elements were the unknown elements—in total, they contributed 78.5% of REs (35% in total genome occupancy). It was revealed that the transcription pattern of genes and REs changes significantly during gastrulation. Using the de novo transcriptome assembly, we showed that the expression of RE is independent of its copy number in the genome. We also identified copies that are expressed. Only active RE copies were used for mapping and quantification of RE expression in the single-cell RNA sequencing data. REs expression was observed in all cell lineages and they were detected as population markers. Moreover, the primary mesenchyme cell (PMC) line had the greatest diversity of REs among the markers. Our data suggest a role for RE in the organization of developmental domains during the sea urchin embryogenesis at the single-cell resolution level.

Comparative Embryogenesis in Angiosperms: Activation and Patterning of Embryonic Cell Lineages.

Following fertilization in flowering plants (angiosperms), egg and sperm cells unite to form the zygote, which generates an entire new organism through a process called embryogenesis. In this review, we provide a comparative perspective on early zygotic embryogenesis in flowering plants by using the Poaceae maize and rice as monocot grass and crop models as well as Arabidopsis as a eudicot model of the Brassicaceae family. Beginning with the activation of the egg cell, we summarize and discuss the process of maternal-to-zygotic transition in plants, also taking recent work on parthenogenesis and haploid induction into consideration. Aspects like imprinting, which is mainly associated with endosperm development and somatic embryogenesis, are not considered. Controversial findings about the timing of zygotic genome activation as well as maternal versus paternal contribution to zygote and early embryo development are highlighted. The establishment of zygotic polarity, asymmetric division, and apical and basal cell lineages represents another chapter in which we also examine and compare the role of major signaling pathways, cell fate genes, and hormones in early embryogenesis. Except for the model Arabidopsis, little is known about embryopatterning and the establishment of the basic body plan in angiosperms. Using available in situ hybridization, RNA-sequencing, and marker data, we try to compare how and when stem cell niches are established. Finally, evolutionary aspects of plant embryo development are discussed.

Analysis of Gene Expression Using lacZ Reporter Mouse Lines.

Reporter mice transgenically expressing the bacterial (E. coli) lacZ gene encoding β-galactosidase (β-gal, EC 3.2.1.23) are a versatile and extensively used tool to study gene expression and cell lineage patterns. Enzymatic activity of the β-gal reporter can be effectively visualized at cellular resolution either histochemically using 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) or by immunofluorescent detection using a β-gal-specific antibody. Here, we summarize protocols for the localization of β-gal expressing cells in whole embryos or organs as well as in histological tissue sections of lacZ reporter mice and discuss their limitations and common pitfalls.

Live Visualization of ERK Activity in the Mouse Blastocyst Reveals Lineage-Specific Signaling Dynamics.

FGF/ERK signaling is crucial for the patterning and proliferation of cell lineages that comprise the mouse blastocyst. However, ERK signaling dynamics have never been directly visualized in live embryos. To address whether differential signaling is associated with particular cell fates and states, we generated a targeted mouse line expressing an ERK-kinase translocation reporter (KTR) that enables live quantification of ERK activity at single-cell resolution. 3D time-lapse imaging of this biosensor in embryos revealed spatially graded ERK activity in the trophectoderm prior to overt polar versus mural differentiation. Within the inner cell mass (ICM), all cells relayed FGF/ERK signals with varying durations and magnitude. Primitive endoderm cells displayed higher overall levels of ERK activity, while pluripotent epiblast cells exhibited lower basal activity with sporadic pulses. These results constitute a direct visualization of signaling events during mammalian pre-implantation development and reveal the existence of spatial and temporal lineage-specific dynamics.

Biophysical phenotypes and determinants of anterior vs. posterior primitive streak cells derived from human pluripotent stem cells

Formation of the primitive streak (PS) marks one of the most important developmental milestones in embryonic development. However, our understanding of cellular mechanism(s) underlying cell fate diversification along the anterior-posterior axis of the PS remains incomplete. Furthermore, differences in biophysical phenotypes between anterior and posterior PS cells, which could affect their functions and regulate their fate decisions, remain uncharacterized. Herein, anterior and posterior PS cells were derived using human pluripotent stem cell (hPSC)-based in vitro culture systems. We observed that anterior and posterior PS cells displayed significantly different biophysical phenotypes, including cell morphology, migration, and traction force generation, which was further regulated by different levels of Activin A- and BMP4-mediated developmental signaling. Our data further suggested that intracellular cytoskeletal contraction could mediate anterior and posterior PS differentiation and phenotypic bifurcation through its effect on Activin A- and BMP4-mediated intracellular signaling events. Together, our data provide new information about biophysical phenotypes of anterior and posterior PS cells and reveal an important role of intracellular cytoskeletal contractility in regulating anterior and posterior PS differentiation of hPSCs. Statement of SignificanceFormation of the primitive streak (PS) marks one of the most important developmental milestones in embryonic development. However, molecular and cellular mechanism(s) underlying functional diversification of embryonic cells along the anterior-posterior axis of the PS remains incompletely understood. This work describes the first study to characterize the biophysical properties of anterior and posterior PS cells derived from human pluripotent stem cells (hPSCs). Importantly, our data showing the important role of cytoskeleton contraction in controlling anterior vs. posterior PS cell phenotypic switch (through its effect on intracellular Smad signaling activities downstream of Activin A and BMP4) should shed new light on biomechanical regulations of the development and anterior-posterior patterning of the PS. Our work will contribute significantly to uncovering new biophysical principles and cellular mechanisms driving cell lineage diversification and patterning during the PS formation.

Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches

BackgroundCanine MuStem cells have demonstrated regenerative efficacy in a dog model of muscular dystrophy, and the recent characterization of human counterparts (hMuStem) has highlighted the therapeutic potential of this muscle-derived stem cell population. To date, these cells have only been generated in research-grade conditions. However, evaluation of the clinical efficacy of any such therapy will require the production of hMuStem cells in compliance with good manufacturing practices (GMPs). Because the current use of fetal bovine serum (FBS) to isolate and expand hMuStem cells raises several ethical, safety, and supply concerns, we assessed the use of two alternative xeno-free blood derivatives: human serum (HS) and a human platelet lysate (hPL).MethodshMuStem cells were isolated and expanded in vitro in either HS-supplemented or hPL-supplemented media and the proliferation rate, clonogenicity, myogenic commitment potential, and oligopotency compared with that observed in FBS-supplemented medium. Flow cytometry and high-throughput 3′-digital gene expression RNA sequencing were used to characterize the phenotype and global gene expression pattern of hMuStem cells cultured with HS or hPL.ResultsHS-supplemented and hPL-supplemented media both supported the isolation and long-term proliferation of hMuStem cells. Compared with FBS-based medium, both supplements enhanced clonogenicity and allowed for a reduction in growth factor supplementation. Neither supplement altered the cell lineage pattern of hMuStem cells. In vitro differentiation assays revealed a decrease in myogenic commitment and in the fusion ability of hMuStem cells when cultured with hPL. In return, this reduction of myogenic potential in hPL-supplemented cultures was rapidly reversed by substitution of hPL with HS or fibrinogen-depleted hPL. Moreover, culture of hMuStem cells in hPL hydrogel and fibrinogen-depleted hPL demonstrated that myogenic differentiation potential is maintained in heparin-free hPL derivatives.ConclusionsOur findings indicate that HS and hPL are efficient and viable alternatives to FBS for the preparation of hMuStem cell batches in compliance with GMPs.

LIN-42, the Caenorhabditis elegans PERIOD homolog, negatively regulates microRNA transcription.

During C. elegans development, microRNAs (miRNAs) function as molecular switches that define temporal gene expression and cell lineage patterns in a dosage-dependent manner. It is critical, therefore, that the expression of miRNAs be tightly regulated so that target mRNA expression is properly controlled. The molecular mechanisms that function to optimize or control miRNA levels during development are unknown. Here we find that mutations in lin-42, the C. elegans homolog of the circadian-related period gene, suppress multiple dosage-dependent miRNA phenotypes including those involved in developmental timing and neuronal cell fate determination. Analysis of mature miRNA levels in lin-42 mutants indicates that lin-42 functions to attenuate miRNA expression. Through the analysis of transcriptional reporters, we show that the upstream cis-acting regulatory regions of several miRNA genes are sufficient to promote highly dynamic transcription that is coupled to the molting cycles of post-embryonic development. Immunoprecipitation of LIN-42 complexes indicates that LIN-42 binds the putative cis-regulatory regions of both non-coding and protein-coding genes and likely plays a role in regulating their transcription. Consistent with this hypothesis, analysis of miRNA transcriptional reporters in lin-42 mutants indicates that lin-42 regulates miRNA transcription. Surprisingly, strong loss-of-function mutations in lin-42 do not abolish the oscillatory expression patterns of lin-4 and let-7 transcription but lead to increased expression of these genes. We propose that lin-42 functions to negatively regulate the transcriptional output of multiple miRNAs and mRNAs and therefore coordinates the expression levels of genes that dictate temporal cell fate with other regulatory programs that promote rhythmic gene expression.

Central cell-derived peptides regulate early embryo patterning in flowering plants.

Plant embryogenesis initiates with the establishment of an apical-basal axis; however, the molecular mechanisms accompanying this early event remain unclear. Here, we show that a small cysteine-rich peptide family is required for formation of the zygotic basal cell lineage and proembryo patterning in Arabidopsis. EMBRYO SURROUNDING FACTOR 1 (ESF1) peptides accumulate before fertilization in central cell gametes and thereafter in embryo-surrounding endosperm cells. Biochemical and structural analyses revealed cleavage of ESF1 propeptides to form biologically active mature peptides. Further, these peptides act in a non-cell-autonomous manner and synergistically with the receptor-like kinase SHORT SUSPENSOR to promote suspensor elongation through the YODA mitogen-activated protein kinase pathway. Our findings demonstrate that the second female gamete and its sexually derived endosperm regulate early embryonic patterning in flowering plants.

Culture of Isolated Floor Plate Tissue and Production of Conditioned Medium to Assess Functional Properties of Floor Plate-released Signals

During development, progenitors and post-mitotic neurons receive signals from adjacent territories that regulate their fate. The floor-plate is a group of glial cells lining the ependymal canal at ventral position. The floor-plate expresses key morphogens contributing to the patterning of cell lineages in the spinal cord. At later developmental stages, the floor-plate regulates the navigation of axons in the spinal cord, acting as a barrier to prevent the crossing of ipsilateral axons and controlling midline crossing by commissural axons1. These functions are achieved through the secretion of various guidance cues. Some of these cues act as attractants and repellents for the growing axons while others regulate guidance receptors and downstream signaling to modulate the sensitivity of the axons to the local guidance cues2,3. Here we describe a method that allows investigating the properties of floor-plate derived signals in a variety of developmental contexts, based on the production of Floor-Plate conditioned medium (FPcm)4-6. We then exemplify the use of this FPcm in the context of axon guidance. First, the spinal cord is isolated from mouse embryo at E12.5 and the floor-plate is dissected out and cultivated in a plasma-thrombin matrix (Figure 1). Second two days later, commissural tissue are dissected out from E12.5 embryos, triturated and exposed to the FPcm. Third, the tissue are processed for Western blot analysis of commissural markers.

Culture of Isolated Floor Plate Tissue and Production of Conditioned Medium to Assess Functional Properties of Floor Plate-released Signals

During development, progenitors and post-mitotic neurons receive signals from adjacent territories that regulate their fate. The floor-plate is a group of glial cells lining the ependymal canal at ventral position. The floor-plate expresses key morphogens contributing to the patterning of cell lineages in the spinal cord. At later developmental stages, the floor-plate regulates the navigation of axons in the spinal cord, acting as a barrier to prevent the crossing of ipsilateral axons and controlling midline crossing by commissural axons(1). These functions are achieved through the secretion of various guidance cues. Some of these cues act as attractants and repellents for the growing axons while others regulate guidance receptors and downstream signaling to modulate the sensitivity of the axons to the local guidance cues(2,3). Here we describe a method that allows investigating the properties of floor-plate derived signals in a variety of developmental contexts, based on the production of Floor-Plate conditioned medium (FP(cm))(4-6). We then exemplify the use of this FP(cm) in the context of axon guidance. First, the spinal cord is isolated from mouse embryo at E12.5 and the floor-plate is dissected out and cultivated in a plasma-thrombin matrix (Figure 1). Second two days later, commissural tissue are dissected out from E12.5 embryos, triturated and exposed to the FP(cm). Third, the tissue are processed for Western blot analysis of commissural markers.

The early development of the onychopod cladoceran Bythotrephes longimanus (Crustacea, Branchiopoda).

IntroductionWithin arthropods, several crustacean groups are unique in their early development due to their stereotyped cell division patterns and cell lineages. However, it is still unclear whether these cell division patterns are homologous between the various crustacean groups and whether they could indicate the ground pattern of Tetraconata (Crustacea and Hexapoda). In this study we describe the early development of the raptorial water flea Bythotrephes longimanus as a representative of the Cladocera within branchiopods.ResultsIn B. longimanus the early cell lineage and the cell division pattern are stereotyped up to the fifth cell division cycle. As a morphological marker a nurse cell remnant (ncr) identifies the cell lineage of the smallest and division delayed blastomere up to the 16-cell stage. This marker might be indicative of the germ line. By combining histology, confocal laser scanning microscopy, and 4D microscopy, we reconstruct the early cell lineage and cell division pattern and follow transient formations of cell morphological structures in their temporal and spatial behavior up to gastrulation.ConclusionsCorrespondences to the early cleavage pattern of other Cladocera suggest that the described pattern can be assumed to be ancestral for either the entire Cladocera or for the majority of the Cladocera comprising Anomopoda, Ctenopoda and Onychopoda. The comparison to the cell division patterns of other crustacean groups such as Malacostraca, Ostracoda, and Copepoda reveals similarities that allow for a discussion of a common pattern for the crustacean groups and a ground pattern for the Tetraconata.

Intracholecystic papillary-tubular neoplasm of the gallbladder

The pathologic description for neoplastic polyps of the gallbladder (GB) was controversial. Although current WHO suggest adenoma and papillary neoplasm for precursor lesions, the distinction is not clear. In recent study for 123 GB cases, we found tumoral preinvasive neoplasms (≥ 1 cm) were analogous to their pancreatic and biliary counterparts. They show variable cellular lineages, a spectrum of dysplasia, and a mixture of papillary or tubular growth patterns, often with significant overlap, warranting their classification under one unified parallel category, intracholecystic papillary-tubular neoplasm (ICPN). We found that ICPN have similarity with intraductal papillary-mucinous neoplasm of pancreas (IPMN) and intraductal papillary neoplasm of bile duct (IPNB). ICPN of GB showed similar cell lineage and mucin core protein expression pattern with IPMN and IPNB. It is suspected that ICPN might be a GB counterpart with IPMN and IPNB. We think these polypoid precursor lesions of pancreatobiliary tract and GB may correspond to tumoral intraepithelial neoplasia, whereas the pancreatic intraepithelial neoplasia and biliary intraepithelial neoplasia correspond to the flat intraepithelial neoplasia. We also found there is a geographic difference of ICPN incidence between East and West. ICPN was more common in East whereas conventional flat dysplasia was more frequently found in West.

Wnt/β-catenin signalling regulates Sox17 expression and is essential for organizer and endoderm formation in the mouse

Several signalling cascades are implicated in the formation and patterning of the three principal germ layers, but their precise temporal-spatial mode of action in progenitor populations remains undefined. We have used conditional gene deletion of mouse β-catenin in Sox17-positive embryonic and extra-embryonic endoderm as well as vascular endothelial progenitors to address the function of canonical Wnt signalling in cell lineage formation and patterning. Conditional mutants fail to form anterior brain structures and exhibit posterior body axis truncations, whereas initial blood vessel formation appears normal. Tetraploid rescue experiments reveal that lack of β-catenin in the anterior visceral endoderm results in defects in head organizer formation. Sox17 lineage tracing in the definitive endoderm (DE) shows a cell-autonomous requirement for β-catenin in midgut and hindgut formation. Surprisingly, wild-type posterior visceral endoderm (PVE) in midgut- and hindgut-deficient tetraploid chimera rescues the posterior body axis truncation, indicating that the PVE is important for tail organizer formation. Upon loss of β-catenin in the visceral endoderm and DE lineages, but not in the vascular endothelial lineage, Sox17 expression is not maintained, suggesting downstream regulation by canonical Wnt signalling. Strikingly, Tcf4/β-catenin transactivation complexes accumulated on Sox17 cis-regulatory elements specifically upon endoderm induction in an embryonic stem cell differentiation system. Together, these results indicate that the Wnt/β-catenin signalling pathway regulates Sox17 expression for visceral endoderm pattering and DE formation and provide the first functional evidence that the PVE is necessary for gastrula organizer gene induction and posterior axis development.

Use of somatic mutations to quantify random contributions to mouse development

BackgroundThe C. elegans cell fate map, in which the lineage of its approximately 1000 cells is visibly charted beginning from the zygote, represents a developmental biology milestone. Nematode development is invariant from one specimen to the next, whereas in mammals, aspects of development are probabilistic, and development exhibits variation between even genetically identical individuals. Consequently, a single defined cell fate map applicable to all individuals cannot exist.ResultsTo determine the extent to which patterns of cell lineage are conserved between different mice, we have employed the recently developed method of “phylogenetic fate mapping” to compare cell fate maps in siblings. In this approach, somatic mutations arising in individual cells are used to retrospectively deduce lineage relationships through phylogenetic and—as newly investigated here—related analytical approaches based on genetic distance. We have cataloged genomic mutations at an average of 110 mutation-prone polyguanine (polyG) tracts for about 100 cells clonally isolated from various corresponding tissues of each of two littermates of a hypermutable mouse strain.ConclusionsWe find that during mouse development, muscle and fat arise from a mixed progenitor cell pool in the germ layer, but, contrastingly, vascular endothelium in brain derives from a smaller source of progenitor cells. Additionally, formation of tissue primordia is marked by establishment of left and right lateral compartments, with restricted cell migration between divisions. We quantitatively demonstrate that development represents a combination of stochastic and deterministic events, offering insight into how chance influences normal development and may give rise to birth defects.

Single-cell level analysis of megakaryocyte growth and development

Several fundamental questions regarding cell growth and development can be answered by recording and analyzing the history of cells and their progeny. Herein, long-term and large-field live cell imaging was used to study the process of megakaryopoiesis at the single cell level (n=9300) from human CD34+ cord blood (CB) in the presence of thrombopoietin (TPO) or the cytokine cocktail BS1 with or without nicotinamide (NIC). Comparative analyses revealed that the cocktail BS1 increased the mitotic and proplatelet rate of diploid and polyploid cells, respectively. Conversely, only NIC treatment increased the endomitotic rate of megakaryocytes (MKs) leading to the formation of CB-MKs with ploidy level frequently observed with BM-MKs. However, NIC failed to enhance platelet production. Rather, a 7- and 31-fold reduction in proplatelet formation was observed in tetraploid and octaploid CB-MKs, respectively, and ex vivo platelet production output was reduced by half due to a reduction in MK output in NIC cultures. Unexpectedly, a significant fraction of di- and polyploid CB-MKs were seen to undergo complete proplatelet regression. Though rare (<0.6%), proplatelet reversal led to the formation of regular round cells that could at times resume normal development. The cell tracking data was then used to investigate the impact of “developmental fate” and ploidy on cell cycling time, and to identify potential developmental patterns. These analyses revealed that cell fate and ploidy level have major impacts on the cell cycling time of the cells, and that four recurrent cell lineage patterns could be identified for CD34+ cells undergoing MK differentiation.

Patterns of cell lineage, movement, and migration from germ layer specification to gastrulation in the amphipod crustacean Parhyale hawaiensis

The acquisition of specific cell fates throughout embryonic development is one of the core problems in developmental and evolutionary biology. In the amphipod Parhyale hawaiensis all three germ layers and the germ line are determined by the eight-cell stage. Despite this early fate determination, multiple cell types can be replaced following ablation of their founder cells, showing that this embryo also has significant regulative properties. Here we present a cellular-level resolution lineage analysis for P. hawaiensis embryos between fertilization and gastrulation, including analysis of cleavage patterns, division times, and clonal behaviors. We compare these cellular behaviors in wild type embryos with those in embryos where specific founder cells have been ablated, or where zygotic transcription has been inhibited. We observe that when germ line, endoderm or mesoderm founder cells are ablated, the remaining cells do not alter their cleavage or migration behaviors before the onset of gastrulation. In the absence of zygotic transcription, ingression movements proceed normally, but epibolic movements are disrupted. This indicates that the embryo's regulative response to germ layer founder loss, in the form of altered cell behavior, is realized in the ~32h between gastrulation and early germ band elongation, and is likely to require zygotic reprogramming rather than alternative deployment of maternally supplied determinants. Combining these data with the observations of previous studies, we propose a framework to elucidate the molecular mechanisms that regulate the determinative and regulative properties of the P. hawaiensis embryo.