Differences in size and number of embryonic type-II neuroblast lineages are associated with divergent timing of central complex development between beetle and fly
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eLife assessment
The study is a valuable contribution to the question of evolutionary shifts in neuronal proliferation patterns and the timing of developmental progressions. The authors present solid support for the presence of type-II NB lineages in the beetle Tribolium with the same molecular characteristics as the counterparts in the fly Drosophila, but differences in lineage size and number. While presenting a number of interesting observations, further evidence will be required to show that the observed differences are indeed responsible for the differences in developmental timing of the central complex in the two insect species.
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Abstract
Despite its conserved basic structure, the morphology of the insect brain and the timing of its development underwent evolutionary adaptions. However, little is known on the developmental processes that create this diversity. The central complex is a brain centre required for multimodal information processing and an excellent model to understand neural development and divergence. It is produced in large parts by type-II neuroblasts, which produce intermediate progenitors, another type of cycling precursor, to increase their neural progeny. These neural stem cells are believed to be conserved among insects, but their molecular characteristics and their role in brain development in other insect neurogenetics models, such as the beetle Tribolium castaneum have so far not been studied.Using CRISPR-Cas9 we created a fluorescent enhancer trap marking expression of Tribolium fez/earmuff , a key marker for type-II neuroblast derived intermediate progenitors. Using combinatorial labelling of further markers including Tc-pointed , Tc-deadpan , Tc-asense and Tc-prospero we characterized the type-II neuroblast lineages present in the Tribolium embryo and their sub-cell-types. Intriguingly, we found 9 type-II neuroblast lineages in the Tribolium embryo while Drosophila produces only 8 per brain hemisphere. In addition, these lineages are significantly larger at the embryonic stage than they are in Drosophila and contain more intermediate progenitors, enabling the relative earlier development of the central complex. Finally, we mapped these lineages to the domains of early expressed head pattering genes. Notably, Tc-otd is absent from all type-II neuroblasts and intermediate progenitors, whereas Tc-six3 marks an anterior subset of the type-II-lineages. The placodal marker Tc-six4 specifically marks the territory where anterior medial type-II neuroblasts differentiate.Homologous type-II neuroblasts show a conserved molecular signature between fly and beetle. Enhanced activity of the embryonic beetle neuroblasts-type-II and intermediate progenitors is associated with an earlier central complex development when compared to the fly. Our findings on the differentiation of beetle type-II neuroblasts and on specific marker genes open the possibility to decipher the cellular and molecular mechanisms acting at the stem cell level that contribute to evolutionary divergence in developmental timing and neural morphology.
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eLife assessment
The study is a valuable contribution to the question of evolutionary shifts in neuronal proliferation patterns and the timing of developmental progressions. The authors present solid support for the presence of type-II NB lineages in the beetle Tribolium with the same molecular characteristics as the counterparts in the fly Drosophila, but differences in lineage size and number. While presenting a number of interesting observations, further evidence will be required to show that the observed differences are indeed responsible for the differences in developmental timing of the central complex in the two insect species.
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Reviewer #1 (Public Review):
Summary:
Insects inhabit diverse environments and have neuroanatomical structures appropriate to each habitat. Although the molecular mechanism of insect neural development has been mainly studied in Drosophila, the beetle, Tribolium castaneum has been introduced as another model to understand the differences and similarities in the process of insect neural development. In this manuscript, the authors focused on the origin of the central complex. In Drosophila, type II neuroblasts have been known as the origin of the central complex. Then, the authors tried to identify those cells in the beetle brain. They established a Tribolium fez enhancer trap line to visualize putative type II neuroblasts and successfully identified 9 of those cells. In addition, they also examined expression patterns of several genes …
Reviewer #1 (Public Review):
Summary:
Insects inhabit diverse environments and have neuroanatomical structures appropriate to each habitat. Although the molecular mechanism of insect neural development has been mainly studied in Drosophila, the beetle, Tribolium castaneum has been introduced as another model to understand the differences and similarities in the process of insect neural development. In this manuscript, the authors focused on the origin of the central complex. In Drosophila, type II neuroblasts have been known as the origin of the central complex. Then, the authors tried to identify those cells in the beetle brain. They established a Tribolium fez enhancer trap line to visualize putative type II neuroblasts and successfully identified 9 of those cells. In addition, they also examined expression patterns of several genes that are known to be expressed in the type II neuroblasts or their lineage in Drosophila. They concluded that the putative type II neuroblasts they identified were type II neuroblasts because those cells showed characteristics of type II neuroblasts in terms of genetic codes, cell diameter, and cell lineage.
Strengths:
The authors established a useful enhancer trap line to visualize type II neuroblasts in Tribolium embryos. Using this tool, they have identified that there are 9 type II neuroblasts in the brain hemisphere during embryonic development. Since the enhancer trap line also visualized the lineage of those cells, the authors found that the lineage size of the type II neuroblasts in the beetle is larger than that in the fly. They also showed that several genetic markers are also expressed in the type II neuroblasts and their lineages as observed in Drosophila.
Weaknesses:
I recommend the authors reconstruct the manuscript because several parts of the present version are not logical. For example, the author should first examine the expression of dpn, a well-known marker of neuroblast. Without examining the expression of at least one neuroblast marker, no one can say confidently that it is a neuroblast. The purpose of this study is to understand what makes neuroanatomical differences between insects which is appropriate to their habitats. To obtain clues to the question, I think, functional analyses are necessary as well as descriptive analyses.
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Reviewer #2 (Public Review):
The authors address the question of differences in the development of the central complex (Cx), a brain structure mainly controlling spatial orientation and locomotion in insects, which can be traced back to the neuroblast lineages that produce the Cx structure. The lineages are called type-II neuroblast (NB) lineages and are assumed to be conserved in insects. While Tribolium castaneum produces a functional larval Cx that only consists of one part of the adult Cx structure, the fan-shaped body, in Drosophila melanogaster a non-functional neuropile primordium is formed by neurons produced by the embryonic type-II NBs which then enter a dormant state and continue development in late larval and pupal stages.
The authors present a meticulous study demonstrating that type-II neuroblast (NB) lineages are indeed …
Reviewer #2 (Public Review):
The authors address the question of differences in the development of the central complex (Cx), a brain structure mainly controlling spatial orientation and locomotion in insects, which can be traced back to the neuroblast lineages that produce the Cx structure. The lineages are called type-II neuroblast (NB) lineages and are assumed to be conserved in insects. While Tribolium castaneum produces a functional larval Cx that only consists of one part of the adult Cx structure, the fan-shaped body, in Drosophila melanogaster a non-functional neuropile primordium is formed by neurons produced by the embryonic type-II NBs which then enter a dormant state and continue development in late larval and pupal stages.
The authors present a meticulous study demonstrating that type-II neuroblast (NB) lineages are indeed present in the developing brain of Tribolium castaneum. In contrast to type-I NB lineages, type-II NBs produce additional intermediate progenitors. The authors generate a fluorescent enhancer trap line called fez/earmuff which prominently labels the mushroom bodies but also the intermediate progenitors (INPs) of the type-II NB lineages. This is convincingly demonstrated by high-resolution images that show cellular staining next to large pointed labelled cells, a marker for type-II NBs in Drosophila melanogaster. Using these and other markers (e.g. deadpan, asense), the authors show that the cell type composition and embryonic development of the type-II NB lineages are similar to their counterparts in Drosophila melanogaster. Furthermore, the expression of the Drosophila type-II NB lineage markers six3 and six4 in subsets of the Tribolium type-II NB lineages (anterior 1-4 and 1-6 type-II NB lineages) and the expression of the Cx marker skh in the distal part of most of the lineages provide further evidence that the identified NB lineages are equivalent to the Drosophila lineages that establish the central complex. However, in contrast to Drosophila, there are 9 instead of 8 embryonic type-II NB lineages per brain hemisphere and the lineages contain more progenitor cells compared to the Drosophila lineages. The authors argue that the higher number of dividing progenitor cells supports the earlier development of a functional Cx in Tribolium.
While the manuscript clearly shows that type-II NB lineages similar to Drosophila exist in Tribolium, it does not considerably advance our understanding of the heterochronic development of the Cx in these insects. First of all, the contribution of these lineages to a functional larval Cx is not clear. For example, how do the described type-II NB lineages relate to the DM1-4 lineages that produce the columnar neurons of the Cx? What is the evidence that the embryonically produced type-II NB lineage neurons contribute to a functional larval Cx? The formation of functional circuits could rely on larval neurons (like in Drosophila) which would make a comparison of embryonic lineages less informative with respect to understanding the underlying variations of the developmental processes. Furthermore, the higher number of progenitors (and consequently neurons) in Tribolium could simply reflect the demand for a higher number of cells required to build the fan-shaped body compared to Drosophila. In addition, the larger lineages in Tribolium, including the higher number of INPs could be due to a greater number of NBs within the individual clusters, rather than a higher rate of proliferation of individual neuroblasts, as suggested. What is the evidence that there is only one NB per cluster? The presented schemes (Fig. 7/12) and description of the marker gene expression and classification of progenitor cells are inconsistent but indicate that NBs and immature INPs cannot be consistently distinguished.
The main difference between Tribolium and Drosophila Cx development with regard to the larval functionality might be that Drosophila type-II NB lineage-derived neurons undergo quiescence at the end of embryogenesis so that the development of the Cx is halted, while a developmental arrest does not occur in Tribolium. However, this needs to be confirmed (as the authors rightly observe).
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Reviewer #3 (Public Review):
Summary:
In this paper, Rethemeier et al capitalize on their previous observation that the beetle central complex develops heterochronically compared to the fly and try to identify the developmental origin of this difference. For this reason, they use a fez enhancer trap line that they generated to study the neuronal stem cells (INPs) that give rise to the central complex. Using this line and staining against Drosophila type-II neuroblast markers, they elegantly dissect the number of developmental progression of the beetle type II neuroblasts. They show that the NBs, INPs, and GMCs have a conserved marker progression by comparing to Drosophila marker genes, although the expression of some of the lineage markers (otd, six3, and six4) is slightly different. Finally, they show that the beetle type II neuroblast …
Reviewer #3 (Public Review):
Summary:
In this paper, Rethemeier et al capitalize on their previous observation that the beetle central complex develops heterochronically compared to the fly and try to identify the developmental origin of this difference. For this reason, they use a fez enhancer trap line that they generated to study the neuronal stem cells (INPs) that give rise to the central complex. Using this line and staining against Drosophila type-II neuroblast markers, they elegantly dissect the number of developmental progression of the beetle type II neuroblasts. They show that the NBs, INPs, and GMCs have a conserved marker progression by comparing to Drosophila marker genes, although the expression of some of the lineage markers (otd, six3, and six4) is slightly different. Finally, they show that the beetle type II neuroblast lineages are likely longer than the equivalent ones in Drosophila and argue that this might be the underlying reason for the observed heterochrony.
Strengths:
- A very interesting study system that compares a conserved structure that, however, develops in a heterochronic manner.
- Identification of a conserved molecular signature of type-II neuroblasts between beetles and flies. At the same time, identification of transcription factors expression differences in the neuroblasts, as well as identification of an extra neuroblast.
- Nice detailed experiments to describe the expression of conserved and divergent marker genes, including some lineaging looking into the co-expression of progenitor (fez) and neuronal (skh) markers.
Weaknesses:
- Comparing between different species is difficult as one doesn't know what the equivalent developmental stages are. How do the authors know when to compare the sizes of the lineages between Drosophila and Tribolium? Moreover, the fact that the authors recover more INPs and GMCs could also mean that the progenitors divide more slowly and, therefore, there is an accumulation of progenitors who have not undergone their programmed number of divisions.
- The main conclusion that the earlier central complex development in beetles is due to the enhanced activity of the neuroblasts is very handwavy and is not the only possible conclusion from their data.
- The argument for conserved patterns of gene expression between Tribolium and Drosophila type-II NBs, INPs, and GMCs is a bit circular, as the authors use Drosophila markers to identify the Tribolium cells.
An appraisal of whether the authors achieved their aims, and whether the results support their conclusions: Based on the above, I believe that the authors, despite advancing significantly, fall short of identifying the reasons for the divergent timing of central complex development between beetle and fly.
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