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Lhx3/4 initiates a cardiopharyngeal-specific transcriptional program in response to widespread FGF signaling [1]

['C. J. Pickett', 'Department Of Biology', 'Swarthmore College', 'Swarthmore', 'Pennsylvania', 'United States Of America', 'Hannah N. Gruner', 'Bradley Davidson']

Date: 2024-02

Individual signaling pathways, such as fibroblast growth factors (FGFs), can regulate a plethora of inductive events. According to current paradigms, signal-dependent transcription factors (TFs), such as FGF/MapK-activated Ets family factors, partner with lineage-determining factors to achieve regulatory specificity. However, many aspects of this model have not been rigorously investigated. One key question relates to whether lineage-determining factors dictate lineage-specific responses to inductive signals or facilitate these responses in collaboration with other inputs. We utilize the chordate model Ciona robusta to investigate mechanisms generating lineage-specific induction. Previous studies in C. robusta have shown that cardiopharyngeal progenitor cells are specified through the combined activity of FGF-activated Ets1/2.b and an inferred ATTA-binding transcriptional cofactor. Here, we show that the homeobox TF Lhx3/4 serves as the lineage-determining TF that dictates cardiopharyngeal-specific transcription in response to pleiotropic FGF signaling. Targeted knockdown of Lhx3/4 leads to loss of cardiopharyngeal gene expression. Strikingly, ectopic expression of Lhx3/4 in a neuroectodermal lineage subject to FGF-dependent specification leads to ectopic cardiopharyngeal gene expression in this lineage. Furthermore, ectopic Lhx3/4 expression disrupts neural plate morphogenesis, generating aberrant cell behaviors associated with execution of incompatible morphogenetic programs. Based on these findings, we propose that combinatorial regulation by signal-dependent and lineage-determinant factors represents a generalizable, previously uncategorized regulatory subcircuit we term “cofactor-dependent induction.” Integration of this subcircuit into theoretical models will facilitate accurate predictions regarding the impact of gene regulatory network rewiring on evolutionary diversification and disease ontogeny.

Here, we show that the homeobox TF Lhx3/4 serves as the Ets1/2.b partner responsible for coactivation of the transcriptional program driving C. robusta CPP specification. Because Lhx3/4 also participates in CPF lineage specification ( Fig 1A ) [ 34 ], it effectively regulates CPP gene expression through a multistep coherent feed-forward loop. Strikingly, we find that misexpression of Lhx3/4 in the anterior neural plate (ANP) lineage is sufficient to suppress the native FGF/MapK-dependent ANP program and initiate ectopic activation of the CPP specification program [ 18 , 48 , 52 ]. However, Lhx3/4 misexpression also initiates ectopic activation of a neuroectodermal motor ganglion (MG) program. The resulting execution of incompatible morphogenetic programs appears to drive highly aberrant cell behaviors. Based on these findings, we propose that lineage-determining cofactors play an instructive role, dictating rather than merely facilitating lineage-specific transcriptional responses to pleiotropic signals. We also argue that integration of subcircuits delineating the contribution of lineage-determining factors is essential for accurate modeling of developmental GRNs and the impact of GRN rewiring on evolutionary diversification and disease ontogeny.

C. robusta’s cellular simplicity has facilitated characterization of a wide array of FGF-dependent inductive events [ 46 ]. Indeed, FGF plays a predominant role in C. robusta embryonic patterning. During early cleavage stages, FGF secreted from the central endoderm progenitors specifies notochord and neural plate progenitor lineages, along with a range of “mesenchymal” lineages that give rise to postlarval mesodermal tissues. As mentioned above, FGF also specifies the CPP lineage during this early embryonic period. In later stages, FGF subspecifies a number of distinct lineages within the neural plate, CPPs, ectoderm, and endoderm [ 36 , 47 – 49 ]. As with CPP induction, many of these FGF-dependent specification events have been shown to alter transcription through the MapK/Ets pathway [ 47 , 48 ]. According to current models, shared reliance on FGF is translated into differential specification through deployment of distinct, lineage-determining Ets cofactors, some of which have been identified. For example, Gata.a and Zic-r.b serve as the Ets cofactors for neuroectodermal and primary notochord specification, respectively ( Fig 1G ) [ 18 , 50 , 51 ]. However, many of these presumptive partner factors, including the ATTA-binding factor driving Ets-dependent expression of CPP genes, have not been characterized.

Ciona robusta (also referred to as Ciona intestinalis Type A [ 27 , 28 ]) provides an excellent platform for investigating developmental GRN structure and function. C. robusta is a tunicate, a clade of invertebrate chordates that are the closest sister group to the vertebrates [ 29 ]. However, tunicate genomes were not subjected to 2 rounds of duplications that occurred within the vertebrate lineage. The resulting lack of paralogs for many key signaling and TFs leads to greatly reduced GRN complexity. For example, there is a single gene encoding the FGF receptor (FGFR) in C. robusta versus at least 4 distinct FGFR genes in most vertebrate genomes. Additionally, the C. robusta genome is extremely compact. Relatively short intergenic regions permit rapid identification of C. robusta regulatory elements, and the ability to easily generate large numbers of transgenic C. robusta embryos has facilitated in-depth characterization of these elements [ 30 ]. These advantages have been productively exploited to generate a high-resolution map of the C. robusta cardiopharyngeal GRN [ 31 , 32 ].

Signals play an integral role in coordinating and refining the deployment of nearly all territorial GRNs [ 15 ]. Remarkably, this rich array of developmental functions largely involves only 10 signaling pathway families [ 15 – 17 ]. Although these families have diversified to generate numerous paralogous signaling components, each family still regulates transcription through a limited set of signal-dependent TFs, each of which binds a similar, family-specific binding site motif. This paradigm is well illustrated by the receptor tyrosine kinases (RTKs), a signaling pathway family that encompasses many key developmental signals, including epidermal growth factors (EGFs), vascular/endothelial growth factors (VEGFs), and fibroblast growth factors (FGFs). All of these signals mediate changes in transcription through a shared set of transduction cascades that includes the Map kinase (MapK) pathway. MapKs, regardless of the upstream signal that drives their activity, often regulate transcription through phosphorylation of Ets family TFs [ 18 , 19 ] ( Fig 1B ). All Ets factors bind a highly stereotyped motif (GGAW) [ 20 , 21 ]. The resulting tendency for diverse, widely deployed signaling pathway families to regulate transcription through a single, shared motif represents a conundrum. How does the genome encode such a vast array of temporally and spatially distinct transcriptional responses to functionally indistinguishable inputs? Lineage-specific transcriptional responses to pleiotropic signaling are thought to be driven by differential deployment of cofactors, referred to as lineage-determining TFs [ 22 , 23 ]. However, despite the central role of Ets family factors and other signal-dependent TFs in developmental patterning, the transcriptional partners that mediate discrete transcriptional outputs remain poorly characterized [ 24 – 26 ]. More broadly, the mechanisms by which these partners determine signal-dependent transcriptional outputs have not been characterized and relevant subcircuits have not been incorporated into theoretical models [ 8 ]. We aim to address these fundamental gaps in inductive specificity through in-depth analysis of the C. robusta cardiopharyngeal GRN.

Developmental GRNs consist of semi-independent, hierarchical modules or circuits. Distinct populations of progenitor or precursor cells are programed by distinct territorial gene networks. Each territorial GRN can be divided into regulatory modules, or subcircuits, that execute discrete, cascading functions as first elucidated in reference to echinoderm skeletogenic mesoderm specification [ 10 ]. Network motifs, such as feedback or feedforward loops, have also been considered to act as regulatory modules [ 11 ]. It has been proposed that there may be a limited set of recurring subcircuits or motifs that are deployed for related functions across both developmental and physiological GRNs [ 8 , 11 – 14 ]. Because theories regarding network motifs were derived from studies of bacterial signaling, extrinsic inputs are well integrated. In contrast, initial theories regarding developmental subcircuits were largely derived from knockdown of lineage-specific TFs as opposed to highly pleiotropic signal-dependent TFs. Thus extrinsic inputs have not been fully integrated into current theoretical overviews of developmental subcircuits.

Developmental gene regulatory networks (GRNs) program spatiotemporal gene expression, ultimately determining embryonic cell fate decisions and morphogenesis [ 1 – 4 ]. The cardiogenic GRN in the invertebrate chordate Ciona robusta provides a useful overview of one such network ( Fig 1 ). During early stages of development, network circuits composed of genes encoding transcription or signaling factors dictate changes in the regulatory state or signaling status of each cell lineage ( Fig 1A–1C ). As development proceeds, these early circuits feed into effector circuits driving transient changes in cell behavior or more stable changes in cell identity associated with differentiation ( Fig 1D–1F ). Some portions of developmental GRNs encode cell autonomous programs in which inherited regulatory states dictate cascading shifts in gene expression ( Fig 1A ). However, substantial portions of developmental GRNs encode non-cell-autonomous programs, in which signals regulate the cell fate of neighboring lineages through modifications of signal-dependent transcription factor (TF) activity ( Fig 1B ). Previous studies have elucidated many fundamental features of GRNs, including recurrent network motifs [ 5 ], the bistability of cell fate [ 6 ], modular subcircuits [ 7 , 8 ], along with linkages between regulatory and effector circuits [ 3 , 9 ]. However, critical aspects of developmental GRN structure and function remain poorly described. One major gap involves integration of microenvironmental, extrinsic cues, including paracrine signals and matrix factors. How are subcircuits structured to permit robust responses to inherently noisy extrinsic cues? How do GRNs process a limited set of signal-dependent inputs to generate a vast array of differential transcriptional outputs? Addressing these gaps is required to productively investigate GRN rewiring associated with evolution and disease progression.

Results

Knockdown of Lhx3/4 does not impact Ets1/2.b expression in the CPPs Based on our knockdown results, we hypothesized that Lhx3/4 has 2 critical roles in CPP specification (Fig 3E). During early cleavage stages, Lhx3/4 serves as a cofactor for cell-autonomous specification of the CPF lineage [34]. Subsequently, during gastrulation, Lhx3/4 serves as the Ets1/2.b cofactor mediating signal-dependent specification of the CPP lineage. Although our results demonstrate that Lhx3/4 is required for Hand.r expression and CPP migration, it is not clear whether this is due to the hypothesized role of Lhx3/4 in CPP specification or reflects an earlier, previously characterized role of Lhx3/4 in CPF lineage specification (Fig 3E). In particular, if Mesp>nls::Cas9::nls-dependent knockdown of Lhx3/4 disrupted Mesp expression, this may lead to loss of Ets1/2.b expression, indirectly blocking signal-dependent specification of CPPs. Although it is unlikely that the Mesp>nls::Cas9::nls would knockdown Lhx3/4 early enough to disrupt founder lineage specification, we addressed this concern by examining Ets1/2.b expression. In control trials (Mesp>nls::Cas9::nls and U6>sgGFP), normal, bilateral Ets1/2.b expression was consistently observed in 93.1% of transgenic embryos (Fig 3F and 3H). Knockdown of Lhx3/4 generated a similar impact on Ets1/2.b expression (Fig 3G and 3H). These results provide further support for the hypothesis that Lhx3/4 serves as the Ets1/2.b cofactor during signal-dependent CPP specification.

Lhx3/4 knockdown disrupts heart formation According to our model, knockdown of Lhx3/4 and the subsequent loss of CPP specification should disrupt the formation of a beating heart, which is first observed after metamorphosis in 4-day old juveniles. To test this prediction, we reared Lhx3/4-knockdown and control animals to an early juvenile stage (Stage 6: Days 7 to 9; [65]). We first examined gross heart morphology using low-power imaging of living juveniles and then fixed them for higher-resolution confocal analysis. At this stage of juvenile development, the heart is composed of an outer pericardial sphere surrounding a single-cell layer myocardial tube that undergoes regular peristaltic contractions (Fig 3I and 3L and S1 Movie). In control trials, an average of 13% of animals exhibited mildly abnormal phenotypes, largely consisting of abnormally slow peristaltic contraction (Fig 3K). As predicted by our model, Lhx3/4 knockdown led to a significant increase in the percentage of juveniles displaying phenotypically abnormal hearts. In these experimental trials, 44% of Lhx3/4-knockdown animals exhibited a range of morphological defects including frequent gross abnormalities (Fig 3J, 3K and 3M). In some of these samples, the heart was composed of an empty pericardial cavity with no discernible myocardium (Fig 3J and S2 Movie). In other samples, heart size was reduced and the myocardium was severely disorganized (Fig 3M and S3 Movie). These results align with defects in CPP migration and unilateral loss of CPP gene expression observed in Lhx3/4-knockdown embryos (Figs 2E and 3B).

Ectopic expression of Lhx3/4 disrupts anterior neural plate morphogenesis Specification programs often activate morphogenetic modules that dictate the cell behavior of newly specified lineages [9,69]. Thus, cofactor-dependent induction of lineage-specific specification programs in response to a shared signal will also lead to differential execution of downstream morphogenetic modules. This logical corollary to the cofactor-dependent induction model predicts that ectopic, overlapping expression of 2 cofactors would result in execution of 2 likely incompatible, morphogenetic modules. We tested this prediction by examining the impact of ectopic Lhx3/4 expression (Dmrt>Lhx3/4) on morphogenesis of Dmrt>LacZ-labeled ANP cells. Our model predicts that ectopic expression of Lhx3/4 will activate incompatible ANP and CPP morphogenetic modules and disrupt normal cell behavior (Fig 5A). As seen in control embryos, by the mid-tailbud stage, posterior rows of ANP cells that express both Dmrt and Zic-r.b invaginate to form the anterior sensory vesicle (ASV) of the central nervous system, which lies beneath the epidermis (arrowhead, Fig 5B) while anterior rows, in which Zic-r.b expression is repressed, form the palps (arrow, Fig 5B). Transgenically labeled ANP cells in all control mid-tailbud stage embryos displayed these behaviors (Fig 5B and 5D). In contrast, ectopic expression of Lhx3/4 (Dmrt>Lhx3/4) appeared to block invagination (Fig 5C and 5D). Transgenically labeled cells were consistently observed to lie superficial to the epidermis, forming a cluster that protruded dorsally (Fig 5C, arrowhead). Additionally, ANP lineage cells failed to spread anteriorly to form the palp primordia. These results align with our model, indicating that overlapping cofactor expression leads to execution of incompatible morphogenetic programs in response to a shared signal. In particular, disruption of ANP morphogenesis could reflect Lhx3/4-dependent activation of a CPP migration module (Fig 1C). Alternatively, overlapping activation of ANP and CPP morphogenetic modules might disrupt ANP morphogenesis through misregulation of cell proliferation. To begin testing these hypotheses, we examined the impact of Lhx3/4 on ANP morphogenesis in more detail. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Ectopic Lhx3/4 expression causes abnormal, migratory cell behavior in the neural plate but does not alter cell numbers. (A) Predicted activation of incompatible morphogenetic modules due to ectopic expression of the CPP lineage-determining factor Lhx3/4. (B) Representative control embryo in which Dmrt>LacZ-positive ANP cells are observed in the palps (arrow) and invaginated cells of the developing ASV. Note that ASV cells are at this stage covered by a layer of unstained epidermis (arrowhead). (C) Representative early tailbud embryo electroporated with Dmrt>LacZ and Dmrt>Lhx3/4. Note that Dmrt>LacZ-positive cells protrude from the anterior dorsal trunk and are not covered by epidermis (arrowhead). Staining is also absent from the anterior tip of the embryo (arrow) where the palps normally develop. (D) Graph of average percent of early tailbud embryos displaying neural plate defects across 2 trials; controls (n = 200, 0%) and Dmrt>Lhx3/4 samples (n = 200, 69%). Underlying data can be found in S1 Appendix. (E, F) Effect of Lhx3/4 on neural plate morphogenesis. Stages indicated above each image or illustration column. (E) Still images taken from representative movies of control (i-iii; S4 Movie) and experimental Dmrt>Lhx3/4 embryos (iv-vi; S6 Movie). See text for details. (F) Schematics illustrating phenotypes shown in the previous panel. D = dorsal view, L = lateral view. (G-K) Effect of Lhx3/4 on cell division. Representative neurulae (G, I) and larvae (H, J) expressing transgenes as indicated on the left. Transgenic GFP expression domain shown in magenta and DAPI-labeled nuclei in cyan. (K) Quantification of GFP-positive (GFP+) cells from experiments represented in panels G, I (left plot) and panels H, J (right plot). Two trials, control neurulae n = 9 (avg. 27.6 GFP+ cells), Dmrt>Lhx3/4 neurulae, n = 9 (avg. 26.8 GFP+ cells), control larvae n = 5 (avg. 170.8 GFP+ cells), Dmrt>Lhx3/4 larvae n = 6 (avg. 178.5 GFP+ cells). Underlying data can be found in S1 Appendix. ANP, anterior neural plate; ASV, anterior sensory vesicle; CPP, cardiopharyngeal progenitor. https://doi.org/10.1371/journal.pbio.3002169.g005

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002169

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