Interplay between morphogen-directed positional information systems and physiological signaling

Abstract

The development of an organism from an undifferentiated single cell into a spatially complex structure requires spatial patterning of cell fates across tissues. Positional information, proposed by Lewis Wolpert in 1969, has led to the characterization of many components involved in regulating morphogen signaling activity. However, how morphogen gradients are established, maintained and interpreted by cells still is not fully understood. Quantitative and systems-based approaches are increasingly needed to define general biological design rules that govern positional information systems in developing organisms. This short review highlights a selective set of studies that have investigated the roles of physiological signaling in modulating and mediating morphogen-based pattern formation. Similarities between neural transmission and morphogen-based pattern formation mechanisms suggest underlying shared principles of active cell-based communication. Within larger tissues, neural networks provide directed information, via physiological signaling, that supplements positional information through diffusion. Further, mounting evidence demonstrates that physiological signaling plays a role in ensuring robustness of morphogen-based signaling. We conclude by highlighting several outstanding questions regarding the role of physiological signaling in morphogen-based pattern formation. Elucidating how physiological signaling impacts positional information is critical for understanding the close coupling of developmental and cellular processes in the context of development, disease and regeneration.

Introduction.

During the late nineteenth century, Hans Driesch demonstrated that spatially complex organisms develop from undifferentiated single cells as opposed to growing from microscopic, preformed versions of mature organisms (Reinitz, 2012). Two key paradigms have emerged to provide a framework for explaining pattern formation in developing tissues. First, Alan Turing proposed how pattern formation can begin de novo through instability of chemical substances, termed “morphogens”, that are triggered from random disturbances (Turing, 1952). Later, Lewis Wolpert proposed positional information as a mechanism whereby differential gene expression of cells results into spatial patterns of cellular differentiation (Wolpert, 1969). Within the concept of positional information, a coordinate system defines the magnitude and directionality of the positional information sensed by cells. This positional information specifies gene expression and subsequent fates of cells in tissues (Figure 1). Following Wolpert, Gierer and Meinhardt proposed models of morphogen distribution to demonstrate that relatively simple molecular mechanisms can explain the formation of a spatially patterned tissue (Gierer and Meinhardt, 1972). Subsequent experimental work has demonstrated that a core set of morphogens generate and relay positional information to cells to directly induce cellular responses based on the cells’ location (reviewed in (Li and Elowitz, 2019; Tabata and Takei, 2004)).

Figure 1. A classical view of positional information and morphogenesis.

The classical view of positional information stems from the French Flag Model in which source cells secrete morphogens that are transported through a tissue due to diffusion (Wartlick et al., 2009; Wolpert, 1969). A) Morphogen molecules (green) are secreted from source cells where morphogen concentration will be the highest. Morphogens travel to neighboring cells to establish a concentration gradient. Cells will have differential biological responses to the morphogen gradient dependent upon multiple threshold levels. Blue cells are located where morphogen concentrations are above the higher threshold. White cells sense morphogen concentrations that are above the lower threshold. Red cells detect low to no morphogens. B) Cells will respond to morphogens to determine cell fates and cell morphology in a dose-dependent response (Widmann and Dahmann, 2009a, 2009b). C) The ability of progenitor cells to create morphogen gradient-based patterns is dependent upon tissue geometry, size, temporal signaling of morphogens (Sorre et al., 2014; Warmflash et al., 2014) (Figure created based on concepts from Wartlick et al., 2009). D) Governing diffusion equation of the morphogen concentration in accordance with Fick’s second law of diffusion with a non-linear degradation profile (k₁), a source term dependent upon location (k₂), and effective diffusion of the molecule (D_eff). This results in a power-law gradient in which the gradient is position-dependent and reflects the local steepness of the gradient (Wartlick et al., 2009).

Because morphogens play crucial roles during the specification of cell fates, they contribute to many aspects of development. A core set of key morphogens regulate development. Examples include members of the Hedgehog (Hh) family that are involved in Drosophila appendage formation (Mullor et al., 1997; Strigini and Cohen, 1997) and chick neural tube development (Briscoe et al., 2001). As a second example, Wingless (Wg)/Int-1 (Wnt) proteins contribute to Drosophila appendage development and human degenerative diseases (Neumann and Cohen, 1997; Nusse, 2005; Zecca et al., 1996). Bone morphogenetic proteins (BMPs), such as Drosophila Decapentaplegic (Dpp), are utilized in dorsal-ventral patterning of the Drosophila embryo and pattern formation and growth control of limb primordia (Estella et al., 2008; Francois et al., 1994; Irish and Gelbart, 1987; Lecuit et al., 1996; Matsuda et al., 2016; Nellen et al., 1996). BMPs also regulate the formation of early germ layers of mammals (Winnier et al., 1995).

Combined experimental and computational efforts have aided in piecing together key aspects of morphogen signaling, including their identification and interactions, how they convey positional information, and their ability to induce pattern formation through gradients. For example, experimental studies have demonstrated that one morphogen can control expression of another during morphogenetic processes, such as Hh-induced Dpp activity in developing Drosophila (Chang et al., 2001; Ingham and Fietz, 1995), and Hh participating in crosstalk with Wnt in cancer (Song et al., 2015). On the other hand, computational modeling has proven critical for explaining increasingly complex datasets and counter-intuitive results from genetic perturbations to morphogenetic patterning systems (Cohen et al., 2014). Additional computational efforts have uncovered the role of the nervous system in facilitating regeneration in Planaria (Pietak et al., 2019). This among many other studies support a close analogy between embryonic patterning and brain-like signal processing (Durant et al., 2019; Fukumoto et al., 2005; Levin, 2006). However, in many cases the underlying kinetics and dynamics in gradient formation and maintenance remain poorly understood.

Morphogen-based signaling during development requires active cellular and physiological processes, as has been noted through analysis of the importance of lipid metabolism in morphogen transport (Panáková et al., 2005). Here, we denote the term physiological signaling to represent cell regulatory mechanisms at the ionic and molecular level that control key cellular functions such as trafficking through exo- and endocytosis, metabolic processes of cellular growth and division, or the regulation of cell mechanics. Increasingly, evidence demonstrates the central role of physiological signaling events in mediating morphogen activity and emerging parallels between neurotransmission and morphogen transport across non-neural tissues (Fukumoto et al., 2005; Levin, 2006; Pezzulo and Levin, 2015). In particular, we highlight the functional roles of secondary messengers, such as calcium (Ca²⁺), in mediating morphogen secretion, transport, downstream information processing, and providing robustness of positional information. In the concluding section, we pose open questions for the field regarding how physiological signaling cooperates with morphogen signaling to regulate positional information and morphogenesis.

Cellular mechanics directly influence pattern formation and morphogen gradients.

Early studies of morphogenesis posited the notion that chemical-based morphogen gradients are the primary signals to pattern cell differentiation. However, there is emerging evidence demonstrating that mechanical events can also be a primary trigger in pattern formation. For example, Shyer et al. discovered that mechanical forces drive cellular self-organization and structural rearrangements of a feather follicle’s shape and gene expression in chicken embryos (Shyer et al., 2017). In particular, they demonstrated that mechanical activation of β-catenin initiated downstream follicle gene expression of bmp2, a key component of the TGF-β signaling pathway. This study thus serves as a striking example of the general importance of mechanical forces in regulating growth and morphogenesis (LeGoff and Lecuit, 2016).

Additional support for the emerging role of mechanical events in morphogenesis was observed in a murine model that exhibited knockdown of the pericellular matrix (PCM) molecule Perlecan (Xu et al., 2016). Perlecan is a highly conserved proteoglycan that is responsible for cross-linking extracellular matrix (ECM) components and cell-surface molecules (Iozzo, 1994). Xu et al. demonstrated that perlecan knockdown significantly decreased cell and ECM stiffness through microscopy techniques that measured stiffness of live cells and surrounding tissue (Xu et al., 2016). More specifically, reduced Perlecan resulted in reduced stiffness of murine chondrocytes and the interstitial matrix during cartilage development in embryonic and postnatal mice. Considering the work done by Shyer et al., the reduced cell and tissue stiffness could potentially influence pattern formation through altered cell mechanics. Further, heparan sulfate side chains of Perlecan have demonstrated the capability of sequestering and presenting growth factors to their respective receptors, which is crucial for establishing morphogen gradients in developmental processes (Gubbiotti et al., 2017). Thus, mechanical forces, cellular mechanics, and tissue mechanics are key components of morphogenesis and can influence pattern formation. The underlying mechanisms in which mechanics provide positional information to cells require further elucidation.

Morphogen sources are dynamic and controlled by physiological signaling.

The conceptual French Flag model, an iconic description of positional information proposed by Wolpert, specifies that morphogens are secreted from a cluster of cells and form a graded distribution throughout the tissue to specify multiple cell types dependent on the concentration sensed by cells (Wolpert, 1969) (Figure 1A). Within this model, the secretion of morphogens from source cells is the initial step in formation of positional information (Figure 1B). This initial framework has since expanded from a static viewpoint to include dynamic changes to the size, geometry, location, mechanics, and temporal signaling of morphogen secreting cells (Figure 1C).

An example of a dynamic morphogen source occurs during the morphogenesis of dorsal appendages of the Drosophila melanogaster eggshell (Zartman et al., 2011). The morphological boundaries of these structures depend on the spatial patterning transcription factor Broad (BR), which is regulated by the epidermal growth factor receptor (EGFR) signaling pathway through a feedback regulatory network. The patterning of BR is established when Gurken, an EGFR ligand, is secreted from the underlying oocyte forming a posterior-to-anterior gradient. Later, a dorsoventral gradient forms after translocation of the oocyte nucleus to the dorsal anterior cortex (González-Reyes et al., 1995; Roth et al., 1995; Zhao et al., 2012). This suggests that morphogen sources are spatiotemporally dynamic and do not always adhere to the framework of passive diffusion-based transport from a stationary source (Figure 1D). This computational-based analysis of how subsequent rounds of EGFR activation refines spatial patterns was later experimentally confirmed (Fregoso Lomas et al., 2013). Given the potential for spatiotemporally dynamic morphogen sources, secretion mechanisms of morphogens may provide insight into how this is possible.

Source cells secrete morphogens through exocytosis, an active transport mechanism of molecules to the cell membrane through vesicles. Fusion of vesicles to the cell membrane releases the morphogen. Therefore, morphogen secretion is influenced by physiological regulation of exocytosis. For example, inwardly rectifying potassium (Irk) channels regulate the release of Dpp and cytosolic Ca²⁺ in the developing Drosophila wing imaginal disc (Dahal et al., 2017) (Figure 2). Irk channels facilitate flow of potassium (K⁺) into the cell and have a fundamental role in restoring the membrane potential to its resting potential. Inhibition of Irk channels in Dpp producing cells, reduced Dpp secretion independent of Dpp expression (Dahal et al., 2017). Furthermore, Dahal et al. demonstrated that Irk channels regulate vesicle release by changing membrane potential while also altering intracellular Ca²⁺ dynamics. A potential explanation for similar outcomes in Dpp and Ca²⁺ after Irk channel inhibition is that intracellular Ca²⁺ dynamics regulate Dpp release, although whether or not Ca²⁺ has a causative effect requires further study. Several studies have shown that Ca²⁺ is crucial to regulation of epithelial physiology during development (Balaji et al., 2017; Brodskiy et al., 2019; Brodskiy and Zartman, 2018; Dahal et al., 2017; Li et al., 2018; Slusarski and Pelegri, 2007). However, whether Ca²⁺ directly regulates Dpp secretion is currently unknown. Given that misregulation of Ca²⁺ is frequently associated with developmental genetic disorders resulting in neoplasms (Hörtenhuber et al., 2017), it is necessary to further characterize the roles of Ca²⁺ and other second messengers in morphogen transport and morphogenesis to provide insight into therapies to treat these genetic disorders.

Figure 2. Irk channels regulate Dpp secretion and cytosolic Ca²⁺.

In the Drosophila wing disc, inwardly rectifying potassium channels (Irk) regulate both the secretion of bone morphogenic protein Dpp and cytosolic Ca²⁺ levels. Expression of Dpp along the dorsal-ventral (D-V) axis in the wing disc is indicated in red. Dpp expression is not present along the anterior-posterior (A-P) axis of the wing disc. Inhibition of Irk channels using dominant negative Irk2 mutants (Irk2^DN) decreased the duration and amplitude of Ca²⁺ oscillations in Dpp-producing cells (yellow square indicates region of interest) (Dahal et al., 2017). Further, loss of Irk2 channel function increased the baseline concentration level of Dpp-GFP while simultaneously decreasing discrete Dpp release events. Thus, Irk2 inhibition in wing discs simultaneously alters intracellular Ca²⁺ activity and Dpp release (Figure created based on concepts from Dahal et al., 2017).

Parallels between neurotransmission and morphogen-based transport systems.

After morphogens are secreted from a group of cells, they are transported across tissues to establish a gradient (Müller et al., 2013; Vincent and Dubois, 2002). How morphogens disperse and form gradients is still under debate despite progress in understanding the molecular mechanisms of morphogen transport through imaging studies and biophysical measurements (Akiyama and Gibson, 2015). Several morphogen transport models have been proposed in the literature. These range from passive mechanisms, such as free or hindered diffusion (Figure 1D), to cell-based dispersal by transcytosis or cytonemes (Akiyama and Gibson, 2015; Restrepo et al., 2014). Multiple transport mechanisms may be involved, and this likely varies across developmental contexts.

The simplest mechanism of morphogen transport is passive diffusion where molecules disperse by random motion. However, this model does not fully capture the complexity of morphogen transport dynamics due to evidence demonstrating that a single source of morphogen is not always sufficient to establish a gradient (Akiyama and Gibson, 2015; Zhou et al., 2012). For example, during Drosophila wing disc development, DWnt6 is expressed in an identical pattern to Wg (Janson et al., 2001), while both BMP ligands Gbb and Dpp are necessary to establish proper morphogen gradients (Khalsa et al., 1998; Raftery and Umulis, 2012). In facilitated diffusion, morphogens are largely immobile until they bind to a positive diffusion regulator that enhances motility. Shuttling is a special case of facilitated diffusion in which molecular shuttles, not morphogens are generated from a localized source. Morphogens near the local source get attached to the shuttle and the morphogen-shuttle complex is transported across tissues. Subsequently, the morphogen-shuttle complex is degraded, resulting in the morphogen being immobilized to stationary negative diffusion regulators and formation of a gradient (Müller et al., 2013; Zinski et al., 2017).

Beyond extracellular-diffusion-based morphogen transport, several studies suggest that morphogens also can be transported through cell-based mechanisms via transcytosis or along cellular extensions known as cytonemes (Bischoff et al., 2013; Hsiung et al., 2005; Ramírez-Weber and Kornberg, 1999). In the case of transcytosis, signaling molecules are taken up by cells through endocytosis and subsequently released through exocytosis into the extracellular space. Endocytosis is the process in which cargo molecules, including morphogens, are absorbed and distributed into a series of endosomes with distinct physical and biochemical properties (Bökel and Brand, 2014; Gonzalez-Gaitan and Jülicher, 2014; Kruse et al., 2004). An endosome’s dynamics, such as fission, fusion, and maturation, influences its ability to sort and concentrate cargo molecules (Christoforidis et al., 1999; Foret et al., 2012; Gautreau et al., 2014; Gonzalez-Gaitan and Jülicher, 2014; Horiuchi et al., 1997; Simonsen et al., 1998). For the case of morphogens, endosome dynamics are thus able to establish concentration gradients and signaling activities across tissues.

Significant questions remain regarding which is the dominant mode of morphogen transport in a given developmental context. In the case of the Drosophila wing disc, Dpp can move through regions that are mutant for the type I transforming growth factor beta receptor thickveins (tkv) and downstream transcriptional repressor (Brinker) (Schwank et al., 2011). This is significant given that support for transcytosis stems from experiments utilizing the Dpp receptor Tkv (Entchev et al., 2000). Further evidence used to argue against transcytosis in this system resulted from fluorescence recovery after photobleaching (FRAP) experimental data of Dpp transport differing from predicted transcytosis models (Zhou et al., 2012). Due to the apparent conflicts between experimental studies, much work remains to clarify the roles of the physiological processes governing morphogen transport.

An example of morphogen signaling being affected by exo- and endocytosis is seen in Drosophila air sac primordia (ASP), which depends on Dpp signaling (Huang et al., 2019). Cytoneme-based signaling utilizes many of the same components found in neural synapses. Huang and colleagues showed that in the Drosophila ASP (“receiving cells”), specialized filopodia-like cytonemes endocytose Dpp from the adjacent Drosophila wing disc cells (“sending cells”) (Figure 3A). Dpp signaling was compromised when Synaptotagmin-1, Synaptobrevin, the glutamate receptor, or voltage-gated Ca²⁺ channels were inhibited in the secreting disc cells, resulting in reduction in the size of the ASP. This result parallels neurotransmission as Synaptobrevin is an intrinsic membrane protein that regulates neurotransmitter release through Ca²⁺-dependent exocytosis (Quetglas et al., 2002; Südhof et al., 1989). The receiving cells of the ASP require Synaptotagmin-4 and the glutamate receptor GluRII. This is noteworthy given that Ca²⁺ is a crucial regulator of endocytosis and neurotransmitter regulation (Wucherpfennig et al., 2003; Yao et al., 2017, 2009). Huang et al. further demonstrated that Ca²⁺ transients observed in cytonemes correlate with Dpp uptake. This suggests that signal uptake and transport within cytonemes depends on local Ca²⁺ concentrations (Huang et al., 2019). This study, thus, underscores the role of physiological signals, like Ca²⁺, in morphogen mediated transport and internalization that have not yet been fully explored.

Figure 3. Morphogen transport mechanisms.

A) Extended filopodia called cytonemes are present in the Drosophila air sac primordium (ASP). Cytonemes projecting from the ASP take up Dpp from the adjacent wing imaginal disc. Correlated transients of Ca²⁺ concentrations are observed in cytonemes. Cytoneme mediated transport requires Synaptotagmin-4 (Syt4), which helps in vesicle fusion and receptor internalization, and glutamate receptor GluRII in the ASP. Wing disc cells secreting Dpp require Synaptobrevin, Synaptotagmin-1, the glutamate transporter and voltage-gated calcium channels (Huang et al., 2019). Blue circles represent morphogens, red circles represent pMad molecules in cells, and orange circles represent dpERK molecules in cells. Figure is adapted with permission from Huang et al., 2019. B) In Planaria, morphogens are transported on microtubule arrays along the axons from the nervous system to the wound edge during regeneration. Vector transport is identified as a fundamental requirement for scale-free self-assembly of morphogens during Planaria homeostasis and regeneration. Figure is adapted with permission from Pietak et al., 2019.

Further evidence for second messenger signaling and morphogen transport lies in the interaction between Ca²⁺ and endocytic and exocytic regulators. For instance, Yamanaka et al. reported that knockdown of Ca²⁺ signaling dependent exocytic components in the prothoracic gland in Drosophila brain, resulted in the accumulation of unreleased steroid hormone ecdysone (Yamanaka et al., 2015). Whether the same exocytic machinery controls secretion of key morphogens is currently unknown. Reverse genetic RNAi screening could be employed to map out the components regulating morphogen secretion and transport through exocytosis. Additionally, synthesis of new alleles with mutations in Ca²⁺ binding domains of key morphogen regulatory proteins will further enable confirmation of the role of physiological signals in regulating morphogen secretion.

A key player in the endocytic process is the regulatory guanosine triphosphatase (GTP) protein Rab5 (Wucherpfennig et al., 2003). Rab5 proteins aid in the formation of transport vesicles and regulate molecular cargo degradation and recycling (Gonzalez-Gaitan and Jülicher, 2014). Rab5 is required for endosome integrity in the presynaptic terminal in Drosophila neuromuscular synapses (Wucherpfennig et al., 2003). Impaired Rab5 function affects both exo- and endocytosis rates and decreases the ability of neurotransmitter release, while overexpression of Rab5 increases the release efficacy of neurotransmitter (Wucherpfennig et al., 2003). This is particularly interesting because Ca²⁺ is an important regulator of neurotransmitter release (Wucherpfennig et al., 2003). Recent work has shown that Ca²⁺ channels regulate bulk endocytosis, a form of endocytosis of synaptic vesicles at nerve terminals, in addition to coupling exo- and endocytosis (Yao et al., 2017, 2009). Further, Rab5-dependent endocytosis requires Ca²⁺ signaling to increase the rate of membrane capacitance, which determines how quickly the membrane potential can respond to a change in current and is linearly related to changes in membrane surface area (Sedej et al., 2005). The converse was also shown in which low Ca²⁺ concentrations decreased membrane capacitance. Thus, an increase in surface area reflects increased exocytosis and decreased surface area reflects an increase in endocytosis (Sedej et al., 2005). Additionally, discovery of a feedback loop showed BMP and Scrib, a scaffold protein associated with cellular proliferation, promotes Rab5 endosome-dependent BMP/Dpp signaling during morphogenesis in Drosophila (Gui et al., 2016). Given that Ca²⁺ affects Rab5-related endocytosis and Rab5 affects Dpp signaling, it can be inferred that Ca²⁺ concentrations may serve as a potential modulator of morphogen transport through coupled exo- and endocytosis. However, this inference remains to be tested directly.

Another possible mechanism through which physiological signals such as Ca²⁺ affect transport of morphogens is through Ca²⁺ binding domains of transport proteins. Evidence for this lies in the presence of Ca²⁺ binding EF-domains in proteins involved in the formation of BMP and Dpp gradients in the Drosophila wing disc (Vuilleumier et al., 2010). For example, Drosophila Pentagone (Pent) directly interacts with Dally to provide long range distribution of the Dpp ligand. Structurally Pent has a similar domain composition to that of human SMOC protein, and both Pent and SMOC proteins contain Ca²⁺ binding EF-domains. Xenopus SMOC-1 (XSMOC-1) protein acts as a BMP antagonist in Xenopus embryos even in the presence of constitutively active BMP receptor (Thomas et al., 2009). Further analysis, suggests that SMOC-1 antagonizes BMP signaling downstream of receptor binding through activation of MAPK signaling. Later studies demonstrated the ability of Drosophila-specific Pent to similarly inhibit BMP signaling in Xenopus downstream of the BMP receptor, after injection of synthetic pent mRNA into Xenopus embryos (Thomas et al., 2017).

Following this, Thomas et al. utilized the SMOC deletion mutant constructs XSMOC-1ΔEC (lacking the extracellular Ca²⁺ binding domain) and XSMOC-1EC (containing the extracellular Ca²⁺ binding domain only) to demonstrate that normal XSMOC-1 and XSMOC-1ΔEC, but not XSMOC-1EC convert the fate of naïve Xenopus ectoderm explants to anterior neural tissue. Thus, embryonic cell fate decisions to become neural tissue via SMOC, and BMP inhibition does not require the extracellular Ca²⁺ binding domain (Thomas et al., 2017). However, the potentiation of BMP signaling by the extracellular Ca²⁺ binding domain was shown in further studies by observing the affinities of XSMOC-1EC and BMP2 for each other and other heparan sulfate proteoglycans (HSPGs). Experiments utilizing in vitro diffusion assays on agarose gels embedded with or without HSPGs demonstrated that the binding of BMPs to HSPGs restricts their range of effect and that BMP diffusion can be enhanced by the binding of SMOC to HSPGs to expand the range of effect. Thomas et al. propose that the SMOC extracellular Ca²⁺ binding domain expands the range of BMP signaling through competitive binding to HSPGs (Thomas et al., 2017). These studies demonstrate the importance of the SMOC extracellular Ca²⁺ binding domain in the context of development. Further investigations are needed into the role of physiological signaling within the context of the extracellular environment.

The coupling between neuronal and non-neural tissues in defining positional information extends beyond similarities in signaling mechanisms. A recent study provided further insight into the role of the nervous system in morphogen-based pattern formation and regeneration in Planaria (Pietak et al., 2019). A remarkable feature of regeneration in Planaria is the reformation of key morphogen gradients such as Hh and Notum Regulating Factor (NRF) after fragmentation or wounding of the organism. Pietak et al. developed a quantitative model of regenerating Planaria to elucidate the mechanism of morphogen gradient activity that ensures robust body-plan regulation. The model predicts the fraction of heteromorphoses in regenerating Planaria fragments. Through an iterative combination of computational simulations and experiments, they found that vector transport of morphogens were required to explain regeneration of pattern formation. Morphogen vector-based transport is defined as the directional transport of morphogens by a vector field. The vector transport field coincided with the nerve polarity throughout regenerating planarian tissue. In their Markov chain model, the transport of morphogens such as Hh and NRF, is mediated by kinesin and dynein motor proteins along microtubules networks of axons (Pietak et al., 2019) (Figure 3B). They further demonstrated that the head-tail axis is controlled by the net polarity of neurons. In contrast, a purely diffusion-based model of patterning could not explain the scaling of steady state concentrations of morphogens of fragments of various sizes. Further, diffusion would be too slow to regenerate the pattern (requiring more than a week on a 1 cm scale organism), while Planaria can reform within 72 hours or less. Thus, the nervous system plays an instructive role in regulating long distance axial patterning repair during regeneration.

Bioelectric signals and resting membrane potentials across tissues are crucial for proper patterning in multiple organisms (Durant et al., 2019; Fukumoto et al., 2005). Changes in membrane potential in regenerating Planaria, induced by ionophore treatment, permanently impacted gene expression, patterning, and polarity (Durant et al., 2019). After ionophore washout from the treated tissue, the induced changes in resting membrane potential persisted. This mechanism parallels synaptic plasticity in the brain where action potentials, modulated by voltage-gated ion channels, propagate signals. Work done in Xenopus and chicks provides insight to this occurrence where serotonin (5-HT), an endogenous neurotransmitter, establishes left-right patterning in embryos through regulation of ion fluxes (Fukumoto et al., 2005). A follow-up study showed that extracellular 5-HT availability drives innervation through tissues via gap junctional communication modulation (Blackiston et al., 2015). Combining their findings, Levin and colleagues propose that extracellular 5-HT, a positively charged molecule, navigates through gap junctions to accumulate in hyperpolarized cells, mimicking the reuptake function of 5-HT transporters (SERTs). These 5-HT sequestering, hyperpolarized cells can depolarize in response to loss of chloride ions via glycine-gated chloride channel activation. Without normal hyperpolarization, 5-HT is exported to the extracellular space by SERTs, which can induce growth and hyperinnervation of tissue (Blackiston et al., 2015; Fukumoto et al., 2005; Levin, 2006).

The importance of ion channels in facilitating communication in bacterial biofilm communities shows the generality of physiological signaling for mediating cell-to-cell communication mechanisms (Prindle et al., 2015). For example, bacterial biofilm communities utilize synchronized oscillations of short-range connectivity among a few cells and community-wide signaling, to minimize competition for resources (Larkin et al., 2018; Liu et al., 2017).

In sum, further quantitative investigations are needed to explore the role of neurotransmission, gap junction communication, and the nervous system in morphogen-based transport systems. The establishment of morphogen gradients through neural signaling still needs to be integrated with known physiological regulators of patterning (Chan et al., 2014; Durant et al., 2019). This will be critical for considering how patterns form across large spatial domains where diffusion-based mechanisms become ineffective.

Physiological signals modulate morphogen-based positional information systems.

Ca²⁺ signaling also plays a key role in mediating cellular responses to morphogens. Several studies in various model systems have shown the interplay between Hh signaling and Ca²⁺ dynamics (Belgacem and Borodinsky, 2011; Klatt Shaw et al., 2018). For example, Shaw et al. showed that intracellular Ca²⁺ mobilization regulates the level of Sonic hedgehog (Shh)-dependent expression domains of nkx2.2b, isl1, nkx6.1, and pax3 genes in the developing nervous system of zebrafish embryos during the 18-somite stage. Furthermore, they showed that reduced expression of ryanodine receptor (RyR), an intracellular Ca²⁺ release channel, shifted the allocation of Shh-dependent cell fates in the somitic muscle and neural tubes in a manner that resembled the effects of reduced Shh signaling (Klatt Shaw et al., 2018). These findings were discovered by utilizing loss-of-function mutations, antisense morpholino knockdowns, and pharmacological treatments to perturb RyR activity.

In another study, Belgacem et al. demonstrated that Shh acutely increases Ca²⁺ through the activation of the transducer Smoothened (Smo), which then recruits heterotrimeric GTP-binding protein-dependent pathways. They demonstrated that Shh increases Ca²⁺ spike activity of developing spinal neurons, and propose that Ca²⁺ spike frequency encodes Shh concentration and is required for proper neuronal differentiation (Belgacem and Borodinsky, 2011) (Figure 4A). In addition to mediating cellular differentiation and proliferation, Ca²⁺ signals also influence cellular migration in response to morphogen gradients. A study by Li and colleagues demonstrated that coordinated cell migration during chicken feather elongation is accompanied by dynamic changes of bioelectric currents and Ca²⁺ signaling. Specifically, Shh-responsive cells contained synchronized Ca²⁺ oscillations in which Shh plays a key role in mediating interactions between the epithelium and mesenchyme during feather morphogenesis (Li et al., 2018; Ting-Berreth and Chuong, 1996). Li et al. showed that voltage-gated Ca²⁺ channels and Connexin43-based gap junctions regulate Ca²⁺ dynamics during feather elongation. Their study reveals a novel mechanism whereby Shh signaling and β-catenin signaling activates Connexin-43 expression transcriptionally to establish the gap junctional network (Li et al., 2018) (Figure 4B). The establishment of gap junctional networks in growing tissues thus alters the spatial range of Ca²⁺ signaling. Further, a review of the role of gap junctions in regulation of pattern formation details that left-right asymmetry in C. elegans neurons involves Ca²⁺ signaling and communication through gap junctions (Chuang et al., 2007; Mathews and Levin, 2017). These studies further demonstrate the role of second messenger systems in mediating morphogen-induced responses of cells and the role of physiological signaling in morphogen-based pattern formation. Quantitative experiments investigating the interplay between second messengers and gap junctions during pattern formation are greatly needed.

Figure 4. Physiological roles of Ca²⁺ in mediating morphogen response.

A) A gradient of the morphogen Sonic Hedgehog (Shh) directs the patterning of neuronal differentiation in the developing Xenopus spinal cord. Shh increases Ca²⁺ spike activity in developing spinal neurons. A current model suggests that Ca²⁺ spikes convert noisy Hh signaling into binary outputs to specify cell fates. This was demonstrated by a positive relationship between activation of Hedgehog signaling through activation of the Shh transducer Smoothened (Smo) and stimulating Ca²⁺ spike activity. Loss of the Ca²⁺ spikes resulted in decreased GABAergic neuronal cell fates (Belgacem and Borodinsky, 2011; Brodskiy and Zartman, 2018). This suggests Ca²⁺ spike frequency encodes Shh concentration and is required for proper neuronal differentiation. Figure is adapted with permission from Brodskiy and Zartman, 2018. B) Tissue-wide, long-range Ca²⁺ oscillations have been observed in mesenchymal cells. Synergistic actions of Shh and Wnt signaling allowed synchronized Ca²⁺ oscillations to coordinate cell movements during chicken feather elongation (adapted with permission from Li et al., 2018).

Feedback from physiological signaling ensures robustness of morphogen-based signaling.

Important aspects of morphogen gradients as a source of positional information are robustness in the presence of genetic or environmental noise and proper scaling of morphological patterns with respect to size (Werner et al., 2015). Robustness is a ubiquitous feature of biological systems that ensures specific functions of the system are maintained despite external and internal perturbations (Kitano, 2004). Robustness of positional information can influence development significantly. Several recent studies have begun to elucidate the mechanisms governing pattern robustness. For instance, in the context of Planarian regeneration, it was shown that pure reaction-diffusion mechanism of morphogens fails to provide scale-free morphogen gradients (Pietak et al., 2019). The authors hypothesized that in addition to the classical mechanism involving diffusion, directional transport of substances through the nervous system are necessary to achieve scale-free morphogen patterning and body axis polarity determination. Overall, this study supports the idea of scale invariance in developing systems in which morphogen gradients are scaled properly despite external and internal perturbations. However, despite extensive work into the scaling mechanisms that regulate scaling of patterns at the tissue, organ, and organism level, biochemical mechanisms underlying patterning robustness remain to be discovered (Umulis and Othmer, 2013).

Regulation of downstream responses by physiological signals also suggest that physiological signals alter the robustness of morphogenetic processes. A study by Creton and colleagues on the Drosophila embryo showed that Ca²⁺ gradients are generated along the dorsal-ventral axis of the Drosophila embryo (Créton et al., 2000). These concentration gradients are formed during embryonic stage 5 with higher Ca²⁺ levels on the dorsal side. They also showed that manipulation of the Ca²⁺ gradient affects the specification of amnioserosa, located dorsally in Drosophila. This study underscores the importance of physiological signaling pathways that utilize Ca²⁺ in contributing to the specification of the dorsal embryonic region. Furthermore, this study also shows that Ca²⁺ gradients affect robustness of morphogenetic processes. An outstanding question is whether this mechanism of specification of positional information is influenced by Ca²⁺ gradients in other model systems.

The role of physiological signaling impacting robustness during organ development has been inferred from morphogenesis of sepals, which are floral organs that aid in protection for the flower in bud (Hong et al., 2016). To investigate how plants maintain robustness of morphogenesis, Hong et al. screened for thale cress Arabidopsis mutants with disrupted sepal uniformity (reduced robustness) within an individual plant, and isolated several variable organ size and shape (vos) mutants. Interestingly, vos1 mutants have increased variability in sepal size and shape due to reduced local spatial variability in the cell growth of vos1 sepals (Hong et al., 2016). Genetic analysis and map-based cloning determined that the vos1 phenotype is caused by recessive mutations in the mitochondrial protease gene FtsH4. Mitochondrial dysfunction results in perturbations in reactive oxygen species (ROS) (Pulliam et al., 2012). Hong et al. compared ROS levels between wild-type and mutant sepals and demonstrated that FtsH4 mutants have higher levels of both hydrogen peroxide (H₂O₂) and superoxide (O₂⁻). Further, the O₂⁻ gradient patterning paralleled the pattern of cellular maturation from the tip to the base of wild-type sepals (Hong et al., 2016). Decreasing ROS levels promotes sepal growth as overexpression of ROS-quenching enzymes resulted in larger sepal sizes. Abnormal accumulation of ROS interferes with the averaging at the cellular scale, which is necessary for robustness at the tissue scale.

Increasingly, evidence is accumulating for significant crosstalk of Ca²⁺ and ROS between endoplasmic reticulum, an established site of Ca²⁺ storage, and mitochondria, a generation site of ROS (Görlach et al., 2015). In particular, Ca²⁺ diminished ROS from ROS generation sites within the mitochondria under normal conditions and enhanced ROS generation when generation sites were inhibited by pharmacological agents (Brookes et al., 2004). Further, quantitative in vivo microscopy in Drosophila and zebrafish embryos identified ROS as crucial signals that regulate cell polarity after wounding (Hunter et al., 2018). Hunter et al. utilized fluorescent imaging after wounding Drosophila embryos and demonstrated Ca²⁺-dependent mitochondrial ROS production correlates with the site of actomyosin cable assembly. This suggests that wound-induced ROS production promotes healing in Drosophila and zebrafish embryos. Taken together, these studies demonstrate that physiological signaling, in the form of ROS and Ca²⁺, act to regulate robustness of morphogenesis. Further experiments are needed to elucidate how ROS and Ca²⁺ affect robustness of positional information in the context of development, regeneration and disease.

Open questions and future directions.

In the five decades since Lewis Wolpert proposed that positional information is a universal mechanism (Wolpert, 1969), there has been significant progress in elucidating the many patterning mechanisms that utilize morphogen-based signaling. Specifically, both computational and experimental approaches have uncovered mechanisms behind the specification and interpretation of positional information. However, integrative models incorporating physiological processes that directly mediate cellular processes into mechanisms of positional information remains a significant challenge. Several recent studies suggest that second messenger systems, such as Ca²⁺, play critical roles in morphogen-based signaling. First, physiological signaling controls morphogen secretion through regulation of exocytosis. Second, morphogen transport is dependent on physiological processes such as Ca²⁺-dependent exo- and endocytosis, interactions that occur extracellularly, that are dependent on the chemical milieu, and active cellular neurotransmission-based mechanisms. Third, physiological signals play an additional role in the interpretation of positional information by modulating and mediating morphogen-based downstream decision-making (Levin, 2006; Pezzulo and Levin, 2015). Perturbations to the physiological state can in turn impact the robustness of morphogenesis. Together, physiological signaling, by regulating the secretion, transport, and downstream responses, ensures that the morphogen-based specification of positional information is robust.

An emerging theme is the dynamic roles of key second messengers such as Ca²⁺ dynamics for downstream morphogen response. However, whether Ca²⁺ signaling is required for downstream activity is current debated in the field. A recent study by Li and colleagues demonstrated that transport of the hormone auxin is involved in patterning of plant morphogenesis by mechanical signals. They reported that transient changes of Ca²⁺ is required for downstream changes in PIN-FORMED 1 (PIN1) polarity, the plasma membrane-localized transporter of auxin (Li et al., 2019). While Ca²⁺ transients were required for downstream changes in PIN1 polarity, it is not sufficient. In contrast to the previous study, another study by Li and colleagues demonstrated that eliciting Ca²⁺ oscillations artificially through light activation of opto-CRAC channels in skin explants resulted in elongated feathers in chicken (Li et al., 2018). This suggests that Ca²⁺ oscillations were sufficient to induce a change in a key morphogenetic process. In particular, understanding redundancy between multiple physiological signals will be important, and signaling changes downstream of Ca²⁺ require further characterization. Optogenetic tools will help decipher the interplay between Ca²⁺ signaling and morphogen-based signaling.

Existing methods to measure Ca²⁺ activity using genetically encoded Ca²⁺ sensors allow one to study Ca²⁺ dynamics in short timescales. This approach is not sufficient to fully decode Ca²⁺ signaling in morphogenetic processes since the time scale of morphogen induced changes are slower than Ca²⁺ measurements obtained by Ca²⁺ sensors. Additional studies using transcriptional reporters of Ca²⁺ activity to report Ca²⁺ dependent interactions between calmodulin and its target peptide (Gao et al., 2015). This method could thus be used to study longer time-scale changes in physiological states that reflect the integration of physiological signaling dynamics. Furthermore, this methodology could be very helpful in studying morphogenetic processes controlled by gap junction mediated Ca²⁺ patterns. Since gap junctional communication is dynamic throughout development and is reported to be controlled by morphogens (Mathews and Levin, 2017; Pietak and Levin, 2018), using a transcriptional reporter would thus enable one to capture slow changes in Ca²⁺ activity in response to morphogenic signaling.

In conclusion, recent studies have demonstrated that physiological signals play key roles in the secretion, transport and downstream responses of morphogen signals. Despite tremendous progress, we still do not understand all the molecular players involved in the process. Application of novel sensors and other optogenetic tools is enabling quantitative studies into the roles of physiological signals, such as Ca²⁺ and other second messengers, during the specification of positional information or regulation of morphogenesis. Along with Ca²⁺, it will also be informative to look at how other signals, such as cyclic adenosine monophosphate (cAMP) and adenosine triphosphate (ATP), tune morphogen-based specification of positional information. In parallel, how other bioelectric signals, such as K⁺ channel activity, control morphogenetic processes requires further characterization. We are only beginning to understand how physiological signaling processes are interconnected with morphogen-based signaling. Moving beyond genetic relationships to systems dynamics is critically needed to gain a deeper understanding of morphogenetic processes in development and disease. It is also critical toward the goal of developing synthetic multicellular systems (Kamm et al., 2018; Narciso and Zartman, 2018).

Highlights:

• The molecular machinery of neural synapses and cell-mediated active morphogen transport shares striking similarities.

• At larger spatial scales, positional information is supplemented by directional information along nerves. Nerve polarity can provide an additional level of information for positional information.

• Physiological signaling contributes to robustness of tissue morphogenesis and organ growth through the regulation of level of cell-cell heterogeneity.