Jeremiah J. Zartman

Coordination of Patterning and Growth by the Morphogen DPP

The elegance of animal body plans derives from an intimate connection between function and form, which during organ formation is linked to patterning and growth. Yet, how patterning and growth are coordinated still remains largely a mystery. To study this question the Drosophila wing imaginal disc, an epithelial primordial organ that later forms the adult wing, has proven to be an invaluable and versatile model. Wing disc development is organized around a coordinate system provided by morphogens such as the TGF-β homolog Decapentaplegic (DPP). The function of DPP has been studied at multiple levels: ranging from the kinetics of gradient formation to the establishment and maintenance of target gene domains as well as DPPs role in growth control. Here, we focus on recent publications that both enrich our view of DPP signaling but also highlight outstanding questions of how DPP coordinates patterning and growth during development.

A Combinatorial Code for Pattern Formation in Drosophila Oogenesis

Two-dimensional patterning of the follicular epithelium in Drosophila oogenesis is required for the formation of three-dimensional eggshell structures. Our analysis of a large number of published gene expression patterns in the follicle cells suggests that they follow a simple combinatorial code based on six spatial building blocks and the operations of union, difference, intersection, and addition. The building blocks are related to the distribution of inductive signals, provided by the highly conserved epidermal growth factor receptor and bone morphogenetic protein signaling pathways. We demonstrate the validity of the code by testing it against a set of patterns obtained in a large-scale transcriptional profiling experiment. Using the proposed code, we distinguish 36 distinct patterns for 81 genes expressed in the follicular epithelium and characterize their joint dynamics over four stages of oogenesis. The proposed combinatorial framework allows systematic analysis of the diversity and dynamics of two-dimensional transcriptional patterns and guides future studies of gene regulation.

Flow-visualization during macrovoid pore formation in dry-cast cellulose acetate membranes

Video-microscopy flow-visualization (VMFV) is adapted to study the development of macrovoid (MV) pores in the dry-casting of cellulose acetate (CA)/acetone/water solutions. Particle tracer velocities provide the first direct evidence for the presence of solutocapillary-driven convection that can enhance mass-transfer to a MV. Three phases of MV development are observed: fast initial growth, slow growth, and collapse. During the latter, MVs were observed on occasion to initiate far from the demixing front. These studies have led to a significantly modified hypothesis for MV development. Extremely rapid initial MV growth is thought to occur owing to coalescence of dispersed phase microdroplets. To ensure net mass-transfer to a growing MV, it is postulated that a homogeneous supersaturated solution layer must exist between the demixed fluid layer and the homogeneous stable solution layer. Fast growth also involves convective mass-transfer to the MV whose surface is initially entirely immersed in this homogeneous supersaturated solution layer. Slow growth involves net transport that results from both convective mass-transfer to the MV across the portion of its surface in contact with the homogeneous supersaturated solution layer, and convective mass-transfer from the portion of its surface that extends into the homogeneous stable solution layer. Active collapse is thought to occur owing to skin formation at the MV surface. Passive collapse occurs when the convective mass-transfer from the MV in the homogeneous stable solution layer exceeds that entering the MV in the homogeneous supersaturated solution layer.

A high-throughput template for optimizing Drosophila organ culture with response-surface methods

The Drosophila wing imaginal disc is a key model organ for molecular developmental genetics. Wing disc studies are generally restricted to end-point analyses of fixed tissues. Recently several studies have relied on limited data from discs cultured in uncharacterized conditions. Systematic efforts towards developing Drosophila organ culture techniques are becoming crucial for further progress. Here, we have designed a multi-tiered, high-throughput pipeline that employs design-of-experiment methods to design a culture medium for wing discs. The resulting formula sustains high levels of proliferation for more than 12 hours. This approach results in a statistical model of proliferation as a function of extrinsic growth supplements and identifies synergies that improve insulin-stimulated growth. A more dynamic view of organogenesis emerges from the optimized culture system that highlights important facets of growth: spatiotemporal clustering of cell divisions and cell junction rearrangements. The same approach could be used to improve culture conditions for other organ systems.

Feedback control of the EGFR signaling gradient: superposition of domain-splitting events in Drosophila oogenesis.

The morphogenesis of structures with repeated functional units, such as body segments and appendages, depends on multi-domain patterns of cell signaling and gene expression. We demonstrate that during Drosophila oogenesis, the two-domain expression pattern of Broad, a transcription factor essential for the formation of the two respiratory eggshell appendages, is established by a single gradient of EGFR activation that induces both Broad and Pointed, which mediates repression of Broad. Two negative-feedback loops provided by the intracellular inhibitors of EGFR signaling, Kekkon-1 and Sprouty, control the number and position of Broad-expressing cells and in this way influence eggshell morphology. Later in oogenesis, the gradient of EGFR activation is split into two smaller domains in a process that depends on Argos, a secreted antagonist of EGFR signaling. In contrast to the previously proposed model of eggshell patterning, we show that the two-domain pattern of EGFR signaling is not essential for specifying the number of appendages. Thus, the processes that define the two-domain patterns of Broad and EGFR activation are distinct; their actions are separated in time and have different effects on eggshell morphology.

Macrovoid pore formation in dry-cast cellulose acetate membranes: buoyancy studies

Experiments were conducted onboard a NASA KC-135 aircraft in order to assess the validity of two hypotheses proposed for the growth of macrovoid (MV) pores formed during the dry-casting of cellulose acetate (CA)/acetone/water casting solutions. The KC-135 aircraft provides the capability for greatly reducing the effective gravitational body forces that influence the buoyancy force on MVs. Buoyancy should have no effect on MV growth as proposed in the purely diffusive growth hypothesis but should influence MV growth via the solutocapillary convection hypothesis since the latter involves a balance between Marangoni, viscous drag, and buoyancy forces. CA membranes were cast in low-gravity (low-g) (KC-135) and normal-gravity (1-g) (ground-based control) from CA/acetone/water solutions as a function of the solvent/non-solvent (S/NS) ratio. Morphological analysis indicated that MV growth was enhanced in low-g only for the case in which the S/NS ratio = 2.0; no effect was observed for higher values of the S/NS ratio. These studies provide support for the solutocapillary convection hypothesis; however, the present data do not unambiguously demonstrate the occurrence of solutocapillary convection. Further research is required to provide such proof.

Pattern formation by a moving morphogen source.

During Drosophila melanogaster oogenesis, the follicular epithelium that envelops the germline cyst gives rise to an elaborate eggshell, which houses the future embryo and mediates its interaction with the environment. A prominent feature of the eggshell is a pair of dorsal appendages, which are needed for embryo respiration. Morphogenesis of this structure depends on broad, a zinc-finger transcription factor, regulated by the EGFR pathway. While much has been learned about the mechanisms of broad regulation by EGFR, current understanding of processes that shape the spatial pattern of broad expression is incomplete. We propose that this pattern is defined by two different phases of EGFR activation: an early, posterior-to-anterior gradient of EGFR signaling sets the posterior boundary of broad expression, while the anterior boundary is set by a later phase of EGFR signaling, distributed in a dorsoventral gradient. This model can explain the wild-type pattern of broad in D. melanogaster, predicts how this pattern responds to genetic perturbations, and provides insight into the mechanisms driving diversification of eggshell patterning. The proposed model of the broad expression pattern can be used as a starting point for the quantitative analysis of a large number of gene expression patterns in Drosophila oogenesis.

Bistability coordinates activation of the EGFR and DPP pathways in Drosophila vein differentiation

Cell differentiation in developing tissues is controlled by a small set of signaling pathways, which must coordinate the timing and levels of activation to ensure robust and precise outcomes. Highly coordinated activation of signaling pathways can result from cross‐regulatory interactions in multi‐pathway networks. Here we explore the dynamics and function of pathway coordination between the EGFR and DPP pathways during Drosophila wing‐vein differentiation. We show that simultaneous activation of both the EGFR and DPP pathways must be maintained for vein cell differentiation and that above‐threshold ectopic activation of either pathway is sufficient to drive vein cell differentiation outside the proveins. The joint activation of the EGFR and DPP signaling systems is ensured by a positive feedback loop, in which the two pathways stimulate each other at the level of ligand production.

Cad74A is regulated by BR and is required for robust dorsal appendage formation in Drosophila oogenesis

Drosophila egg development is an established model for studying epithelial patterning and morphogenesis, but the connection between signaling pathways and egg morphology is still incompletely understood. We have identified a non-classical cadherin, Cad74A, as a putative adhesion gene that bridges epithelial patterning and morphogenesis in the follicle cells. Starting in mid-oogenesis, Cad74A is expressed in the follicle cells that contact the oocyte, including the border cells and most of the columnar follicle cells. However, Cad74A is repressed in two dorsolateral patches of follicle cells, which participate in the formation of tubular respiratory appendages. We show genetically that Cad74A is downstream of the EGFR and BMP signaling pathways and is repressed by the Zn-finger transcription factor Broad. The correlation of Cad74A repression in the cells that bend out of the plane of the follicular epithelium is preserved across Drosophila species and mutant backgrounds exhibiting a range of eggshell phenotypes. Complete removal of Cad74A from the follicle cells causes defects in dorsal appendage formation. Ectopic expression of Cad74A in the roof cells results in shortened, flattened appendages due to the hindered migration of the roof cells. Based on these results, we propose that Cad74A is part of the adhesive machinery that enables robust dorsal appendage formation, and as such provides a link between the patterning of the follicle cells and eggshell morphogenesis.

Sizing it up: the mechanical feedback hypothesis of organ growth regulation.

The question of how the physical dimensions of animal organs are specified has long fascinated both experimentalists and computational scientists working in the field of developmental biology. Research over the last few decades has identified many of the genes and signaling pathways involved in organizing the emergent multi-scale features of growth and homeostasis. However, an integrated model of organ growth regulation is still unrealized due to the numerous feedback control loops found within and between intercellular signaling pathways as well as a lack of understanding of the exact role of mechanotransduction. Here, we review several computational and experimental studies that have investigated the mechanical feedback hypothesis of organ growth control, which postulates that mechanical forces are important for regulating the termination of growth and hence the final physical dimensions of organs. In particular, we highlight selected computational studies that have focused on the regulation of growth of the Drosophila wing imaginal disc. In many ways, these computational and theoretical approaches continue to guide experimental inquiry. We demonstrate using several examples how future progress in dissecting the crosstalk between the genetic and biophysical mechanisms controlling organ growth might depend on the close coupling between computational and experimental approaches, as well as comparison of growth control mechanisms in other systems.