InteractiveFly: GeneBrief

LIM homeobox transcription factor 1 alpha: Biological Overview | References

The Drosophila ovary serves as a model for pioneering studies of stem cell niches, with defined cell types and signaling pathways supporting both germline and somatic stem cells. The establishment of the niche units begins during larval stages with the formation of terminal filament-cap structures; however, the genetics underlying their development remains largely unknown. This study shows that the transcription factor Lmx1a is required for ovary morphogenesis. Lmx1a is expressed in early ovarian somatic lineages and becomes progressively restricted to terminal filaments and cap cells. Lmx1a is required for the formation of terminal filaments, during the larval-pupal transition. Finally, the data demonstrate that Lmx1a functions genetically downstream of Bric-a-Brac, and is crucial for the expression of key components of several conserved pathways essential to ovarian stem cell niche development. Importantly, expression of chicken Lmx1b is sufficient to rescue the null Lmx1a phenotype, indicating functional conservation across the animal kingdom. These results significantly expand understanding of the mechanisms controlling stem cell niche development in the fly ovary (Allbee, 2018).

Drosophila ovaries are composed of about 15-20 functional units called ovarioles that produce eggs. In each ovariole, somatic and germline stem cells are maintained by a group of specialized cells that form the niche. These niches are composed of terminal filaments (TFs) and cap cells, which provide signals required for the maintenance and function of the stem cell populations in adult ovaries, including BMPs and Hh. Although the origin of the somatic lineage in the female gonad has been established, the genetic factors underlying the morphogenetic processes required for the specification and establishment of TF-cap structures are largely unknown. Ovarian somatic cells originate from three mesoderm cell clusters on each side of the embryo. During the mid-larval third instar (ML3), TF cells start to specify, expressing the TF marker Engrailed (En). By late third larval instar (LL3), and continuing in white pre-pupae (WPP), TFs form well-organized stacks and promote the proliferation and migration of muscle precursors that will develop into the muscular sheath and delimit individual ovarioles. This process ensures that a single TF stack and a defined population of germline and somatic cells are incorporated in each unit (Allbee, 2018).

The number of ovarioles and the developmental timing of the niche have been shown to be controlled by several signaling pathways, including Insulin, Hippo, Notch, Activin and Ecdysone pathways. In addition, the two transcription factors Bab1 and Bab2, encoded by the bric-á-brac (bab) locus, are expressed in TFs and are essential for the formation of these structures. Other BTB transcription factors, Pipsqueak, Trithorax-like and Batman (Lola like), also control TFs and ovariole numbers. Finally, loss of the Engrailed transcription factor causes abnormal TF cell stacking. Although these signaling pathways and transcriptional regulators are essential for development of stem cell niches, the early genetic events coordinating TF-cap cell specification, formation and function remain largely unknown (Allbee, 2018).

This study describes the expression and function of the LIM-homeodomain transcription factor Lmx1a in developing Drosophila ovaries. LIM-homeodomain proteins have essential roles during tissue patterning and cell differentiation in metazoans, from nematodes to vertebrates. Mammalian Lmx1a and Lmx1b are pleiotropic regulators of cell differentiation and tissue development in many organs, as shown by the defects and syndromes caused by loss-of-function mutations in these genes (Doucet-Beaupré, 2015). For instance, haploinsufficiency of Lmx1b causes nail-patella syndrome (NPS), a disorder affecting dorsal limb structures, kidneys, anterior eye components and the nervous system. Studies in genetic mouse models have confirmed a regulatory function of Lmx1b in the development of these organs and the behavior of associated cell types, including serotonergic neurons, podocytes and several eye tissues. Similarly, studies of the mouse Lmx1a factor have revealed diverse developmental functions, including brain patterning and cell fate decision, dopaminergic neuron differentiation and insulin expression. Finally, previous work has implicated Lmx1a and Lmx1b in a variety of other pathologies, including non-NPS renal disease, Parkinson's disease, and various forms of cancer, including ovarian epithelial carcinoma, highlighting the complexity of Lmx1a/b biology, as well as the urgent need to understand the function of these factors better (Allbee, 2018 and references therein).

Two LIM-homeodomain proteins of the LMX subgroup are encoded in the Drosophila genome: Lmx1a/CG32105 and Lmx1b/CG4328. Lmx1a is expressed in the LL3 eye imaginal disc and its overexpression in this tissue causes eye defects. However, the molecular, cell biological and developmental functions of this transcription factor have yet to be described. This study shows that Lmx1a is expressed in somatic lineages in the developing ovary. Its expression becomes restricted to TFs and cap cells by LL3/P0 (freshly pupariated) and is maintained in the adult stem cell niche. Analyzing a CRISPR-generated Lmx1a knockout allele as well as cell type-specific and stage-specific knockdown of Lmx1a, this study has determined that Lmx1a is required for ovary development specifically in the TF-cap cell niche at the time at which it forms. Transcriptional profiling of developing ovaries was performed, and Lmx1a was placed downstream of Bab1/2 in the specification of TF cells. Without Lmx1a, several components of signaling pathways crucial to the forming niche are not properly expressed, including Hh, the transcription factors Sox100B, Engrailed and Invected, and confirmed that these genes are required in the Lmx1a lineage. Strikingly, expression of a chicken ortholog of Lmx1a in forming TF-cap cells is sufficient to rescue the Lmx1a null phenotype. It is anticipated that these results will further elucidate the genetic and cell biological mechanisms underlying the establishment of the Drosophila ovary stem cell niche and provide insight into the role of LIM-HD factors in tissue development and patterning, homeostasis and disease (Allbee, 2018).

Despite longstanding interest in the Drosophila ovary as a model for the adult stem cell niche, the genetic mechanisms underlying the initial establishment and development of the niche remain largely unexplored. To date, only two transcription factor loci, bric-á-brac and engrailed/invected, have been identified as required for the development or proper stacking of TFs, and their function remains largely unknown. This study has characterized the function of Lmx1a, a highly conserved LIM homeobox transcription factor, in the developing Drosophila ovary. By generating an Lmx1a knockout line as well as taking advantage of a novel collection of Gal4 drivers that produce spatially restricted developing ovary expression patterns, Lmx1a was shown to be required in TF-cap structures at the time at which they form and initiate ovarian stem cell niche establishment and ovariole morphogenesis. It remains unclear whether Lmx1a plays a role in TFs, cap cells, or both. The data also suggest that Lmx1a expression is maintained in the adult niche. Further studies will be required to determine whether it is essential for the function of the adult niche, for example by controlling the expression of self-renewal factors (Allbee, 2018).

Using RNAseq, a novel list of genes enriched in LL3/P0 ovaries was generated, and from this list several pathways have been identified that are potentially affected by the loss of Lmx1a. Within this list are several transcription factors and components of signaling pathways necessary for ovarian niche development, function and maintenance, including Hh, FGF, Notch, Engrailed and Sox100B. Indeed, RNAi-mediated knockdown of several of these transcripts within the Lmx1a-Gal4 lineage results in ovary developmental defects and reduced fertility. It is therefore proposed that Lmx1a controls many aspects of the development of this tissue, including proliferation, migration or cell shape changes, by regulating a series of developmental factors and signal pathways. It is important to note that this study does not allow distinguishing between a direct transcriptional regulation of these genes by Lmx1a and an indirect effect of an impaired development of the TF-cap structure in Lmx1a mutants. The data analyzed, in young adult females, the consequence of manipulating Hh, Sox100B, Engrailed and Invected in the Lmx1a lineage. Investigating the function of these genes, specifically during the LL3-P0 transition or later during metamorphosis or adulthood, will allow testing of whether their genetic requirement and expression are compatible with a direct regulation by Lmx1a and to understand better the sequence of events required for normal ovarian morphogenesis (Allbee, 2018).

Interestingly, Engrailed has been previously suggested to be required for terminal filament (TF) stacking based on clonal analysis of TF cells homozygous mutant for both Engrailed and Invected (Bolívar, 2006). The current work suggests that the stacking defects caused by these mutations lead to a mild developmental phenotype in young adults. However, consistent with other work revealing a crucial role for Engrailed in the adult niche, this study also shows that persistent knockdown of Engrailed/Invected well into adulthood leads to an eventual loss of ovaries. It is speculated that the progressiveness of this defect is the consequence of a continuous impairment of adult niche function that worsens a mild initial developmental phenotype (Allbee, 2018).

This study investigated where Lmx1a functions in relation to the transcription factors Bab1 and Bab2. In support of earlier work, this study found that both bab1 and bab2 are required for ovary morphogenesis. In addition, a strong reduction os shown in Lmx1a mRNA expression in the ovaries of LL3/P0 bab1 homozygous mutant animals, placing Bab1 genetically and/or temporally upstream of Lmx1a. It is worth noting that, although reduced, the expression of Lmx1a is not eliminated in Bab1 mutants. It is speculated that Bab2 activity may be maintaining residual Lmx1a expression in this background.Heterozygosity for Bab1 was found to be sufficient to enhance the ovary developmental defects caused by RNAi-mediated knockdown of the Lmx1a-dependent genes tested here, consistent with a model in which the bric-ì-brac locus lies genetically upstream of Lmx1a (Allbee, 2018).

Finally, this study found that the Lmx1a null homozygous phenotype can be significantly rescued by the expression of chicken Lmx1b, in terms of TF-cap formation, sheath formation and fertility. This reveals striking conservation between the Lmx1a/b factors across the animal kingdom. Even more striking is the conserved relationship between Lmx1a/b and Engrailed. For example, Lmx1b is required for Engrailed expression during both the establishment of the isthmic organizer in the developing mouse midbrain, and the specification and differentiation of dopaminergic neurons. Additionally, simultaneous lentiviral-mediated expression of Lmx1b and Engrailed has been shown to aid in the differentiation of dopaminergic neurons in vitro. Altogether, these observations point to a likely conserved Lmx1a/b-Engrailed transcriptional module involved in tissue patterning and cell differentiation, across different developing tissues, cell types and species. It is therefore anticipated that studies on the function of Lmx1a in the Drosophila ovary will lead to a better understanding of the developmental function of Lmx1a in animals ranging from invertebrates to mammals. This study also indicates that Drosophila represents a valuable model in which to investigate the mechanisms underlying complex diseases caused by a dysfunction of Lmx1a/b, such as nail-patella syndrome, ovarian carcinoma and Parkinson's disease (Allbee, 2018).

Drosophila eye developmental defect caused by elevated Lmx1a activity is reliant on chip expression

The LIM-homeodomain (LIM-HD) family member Lmx1a has been successfully used to induce dopaminergic neurons from other cell types, thus showing significant implications in replacement therapies of Parkinson's disease, but the underlying mechanism remains elusive. This study used Drosophila eye as a model system to investigate how forced expression of CG4328 and dLmx1a (CG32105), the fly homologs of human Lmx1a, alters cell identify. Ectopic expression of dLmx1a suppresses the formation of Drosophila eye tissue; the LIM and HD were found to be two essential domains. dLmx1a requires and physically binds to Chip, a well-known cofactor of LIM-HD proteins. Chip connects two dLmx1a proteins to form a functional tetrameric complex. In addition, evidence is provided showing that dLmx1a expression results in the suppression of two retina determination gene eyes absent (eya) and string (stg). Taken together, these findings identified Chip as a novel partner of dLmx1a to alter cell differentiation in Drosophila eye through repressing eya and stg expression, and provide an animal model for further understanding the molecular mechanism whereby Lmx1a determines cell fate (Wang, 2015).

The LIM-HD proteins play fundamental roles in a wide variety of biological processes, such as cell fate determination and organ development. The LIM-HD family members are characterized by two cysteine-rich LIM domains that associate with other proteins, and a unique DNA-binding homeodomain (HD) that interacts with specific DNA sequences. The functions of LIM-HD proteins are modulated by various cofactors including NLI/LDB/CLIM/Chip, MRGI, SLB, and RLIM through their interactions with the LIM domain. NL1/LDB/CLIM/Chip is the best-known cofactor of LIM-HD and harbors two distinct functional domains: the LIM-interaction domain (LID) that mediates the interaction with LIM-HD proteins, and the dimerization domain (DD) that mediates homodimerization (Wang, 2015).

The LIM-HD family proteins are evolutionally conserved. Based on the features of the homeodomain, 12 mammalian LIM-HD proteins are divided into 6 subgroups. Lmx1a is the founding member of Lmx subgroup and expressed in multiple tissues. To date, Lmx1a has been implicated in various biological processes, including controlling the formation of CNS roof plate, specification of dopaminergic neurons, morphogenesis of inner ear and oncogenesis. More interestingly, Lmx1a has been successfully used to induce neurons from non-neuronal cell types. Lmx1a, in conjunction with Mash1 (also known as Ascl1) and Nurr1 (also known as Nr4a2), is able to generate functional dopaminergic neurons directly from mouse and human fibroblasts. Lmx1a in conjunction with Neurturin also has been shown to successfully convert human umbilical cord mesenchymal stem cells to dopamine neurons. However, the precise molecular mechanism whereby elevated Lmx1a activity changes cell identity remains elusive (Wang, 2015).

This study demonstrates that ectopically expressed dLmx1a suppresses eyes absent (eya) and string (stg), thus restricts Drosophila eye development in a Chip-dependent manner. These findings provide new insight into the understanding of Lmx1a in cell fate determination (Wang, 2015).

Previous studies have indicated that Lmx1a is essential for proper development of roof plate and cortical hem structures, as well as the development of floor and midbrain dopaminergic neurons. And recently Lmx1a has been successfully used to induce dopaminergic neurons from fibroblast cells or human umbilical cord mesenchymal stem cells. However, the mechanism whereby forced expression of Lmx1a alters cell identity remains elusive. This study showed that the fly homology dLmx1a requires its LIM and HD domains to determine cell fate in Drosophila eye. Biochemical and genetic analysis indicated that the functional tetrameric complex consisting of two dLMX1a molecules bridged by a Chip dimer is required for ectopic expressed dLmx1a to change cell differentiation in Drosophila eye. Furthermore, genes eya and stg function downstream of dLmx1a to modulate the cell fate determination caused by dLmx1a misexpression (Wang, 2015).

Many intriguing questions still remain. First, Chip is a cofactor of multiple LIM-HD proteins. Thus, the functional significance of stoichiometry between dLmx1a and Chip as well as the contribution of the dynamic association of Chip between dLmx1a and other LIM-HD proteins remain to be determined. Second, dLmx1a, LIM1 and Awh all suppress eya and stgexpression. It remains to investigate whether these LIM-HD/Chip complexes assemble on all LIM-HD targets or they are promoter specific. Third, it is still unknown whether dLmx1a can also alter cell fate determination in other Drosophila tissues. Further investigation will help to better understand the mechanism of mammalian Lmx1a in human diseases and induced formation of neurons (Wang, 2015).

Search PubMed for articles about Drosophila Lmx1a

Allbee, A. W., Rincon-Limas, D. E. and Biteau, B. (2018). Lmx1a is required for the development of the ovarian stem cell niche in Drosophila. Development 145(8). PubMed ID: 29615466

Bolivar, J., Pearson, J., Lopez-Onieva, L. and Gonzalez-Reyes, A. (2006). Genetic dissection of a stem cell niche: the case of the Drosophila ovary. Dev Dyn 235(11): 2969-2979. PubMed ID: 17013875

Doucet-Beaupre, H., Ang, S. L. and Levesque, M. (2015). Cell fate determination, neuronal maintenance and disease state: The emerging role of transcription factors Lmx1a and Lmx1b. FEBS Lett 589(24 Pt A): 3727-3738. PubMed ID: 26526610

Wang, P., Chen, Y., Li, C., Zhao, R., Wang, F., Lin, X., Cao, L., Li, S., Hu, L., Gao, Y., Li, Y. and Wu, S. (2015). Drosophila eye developmental defect caused by elevated Lmx1a activity is reliant on chip expression. Biochem Biophys Res Commun. PubMed ID: 26718403

date revised: 10 September 2018

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