FGF and MafB regulated cadherin expression drives lamina formation in the auditory hindbrain
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eLife Assessment
The authors conducted a valuable study that investigates a molecular pathway mediating the transformation of a cell aggregate into a sheet known as the nucleus laminaris, a crucial site for auditory processing. While the study offers a comprehensive view of the sequence of developmental events and suggests possible roles for FGF signaling, the transcription factor Mafb, and the cell surface adhesive molecule Cadherin-23 in this process, the current data were considered incomplete.
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Abstract
Abstract
The avian auditory brainstem contains specialized nuclei critical for sound localization, including the nucleus laminaris (nL), which forms as a single-cell-thick lamina essential for computing interaural time differences. Despite its functional importance, the molecular mechanisms guiding nL lamina formation have remained poorly understood. Here, we identify a signalling cascade involving FGF8, MafB, and cadherin-22 that orchestrates this morphogenetic process.
We show that FGF8 is selectively expressed in the developing auditory hindbrain and correlates spatiotemporally with lamina formation in the nL. Disruption of FGF signalling—either via misexpression of FGF8 or dominant-negative FGFR1—perturbs the formation of the nL and alters cadherin-22 expression. In vitro culture experiments further reveal that nL lamination is sensitive to FGF8 dosage, with an optimal concentration required for both FGF8 and MafB expression and correct structural organization.
We demonstrate that FGF8 induces MafB, which in turn regulates cadherin-22 expression. Functional disruption of cadherins impairs lamina formation and leads to reduced FGF8 expression, indicating a feedback loop between adhesion and signalling. Cadherin protein expression appears enriched in the dendrites of nL neurons and computational models—both static and dynamic—show that bipolar, dendrite-localized, adhesion can drive laminar architecture as the maximum adhesion configuration.
These findings establish a novel molecular and biophysical mechanism for neuronal lamination in the vertebrate hindbrain, showing how local FGF signalling, transcriptional regulation, and dendritic adhesion converge to shape neural circuitry essential for sound localization.
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eLife Assessment
The authors conducted a valuable study that investigates a molecular pathway mediating the transformation of a cell aggregate into a sheet known as the nucleus laminaris, a crucial site for auditory processing. While the study offers a comprehensive view of the sequence of developmental events and suggests possible roles for FGF signaling, the transcription factor Mafb, and the cell surface adhesive molecule Cadherin-23 in this process, the current data were considered incomplete.
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Reviewer #1 (Public review):
Summary:
In this study, the authors sought to define a molecular pathway that mediates the transformation of an aggregate of cells into a sheet known as the nucleus laminaris, a key site for auditory processing. The data offer a comprehensive view of the sequence of developmental events and suggest possible roles for FGF signaling, the transcription factor Mafb, and the cell surface adhesive molecule Cadherin-23 in this process.
Strengths:
The description of nL development is thorough and well-done, with extensive quantification of the overall structure of the nucleus and also of neuron number. Additionally, the study implicates several molecules in nL development, starting with a clear description of when and where FGF8, Mafb, and several cadherins are expressed, including antibody stains suggesting that …
Reviewer #1 (Public review):
Summary:
In this study, the authors sought to define a molecular pathway that mediates the transformation of an aggregate of cells into a sheet known as the nucleus laminaris, a key site for auditory processing. The data offer a comprehensive view of the sequence of developmental events and suggest possible roles for FGF signaling, the transcription factor Mafb, and the cell surface adhesive molecule Cadherin-23 in this process.
Strengths:
The description of nL development is thorough and well-done, with extensive quantification of the overall structure of the nucleus and also of neuron number. Additionally, the study implicates several molecules in nL development, starting with a clear description of when and where FGF8, Mafb, and several cadherins are expressed, including antibody stains suggesting that one cadherin, cdh2, is localized to the neuronal dendrites. A series of perturbation experiments supports the idea that these three molecules play a role in nL formation. The computational model is an interesting addition that helps to conceptualize how cadherin-mediated adhesion might influence nL morphogenesis.
Weaknesses:
A number of weaknesses limit the impact of this work.
One problem is how the data is interpreted. The logic is often circular in that the same molecules are used both as markers of nL and also as players in its development. An independent measure of nL formation is needed. Along the same lines, while the experiments implicate each molecule, the data do not actually demonstrate that FGF directly modulates Mafb, which in turn modulates cadherin expression, especially as overexpression of cdh2 has no effect on FGF8 expression or lamina organization, and no manipulations of cdh22 are presented.
The other type of problem relates to how the experiments were performed and analyzed. Important details about the experiments, as well as key controls, are missing throughout. Sample sizes are rarely presented, and there is no evidence that either dominant negative construct actually acts as proposed. Some results are not well quantified, which further undermines the strength of the conclusions. For instance, the changes in mafb and cdh22 expression (Figure 7) are subtle and were not quantified for any of the conditions. Likewise, the claim that FGF8 has a dose-dependent effect on lamina size and neuron number needs to be supported by statistics.
There are also some questions about the quality of the data. Much of the histology is of poor quality and does not always show the same piece of brain in the same orientation from experiment to experiment, which makes it challenging to interpret the results. In particular, the quality of the in situ hybridization varies, with much more background in some cases than others, which makes it hard to know what signal is real.
Finally, there are some misstatements and problems with citations that weaken the scholarly nature of the paper. FGF signaling has been studied extensively in the hindbrain and even in auditory nucleus development (Abraira et al., 2007), but this literature is not discussed at all.
Due to these weaknesses, the authors have achieved their aims only in part. The data are suggestive, but the results do not yet fully support their conclusions.
Few labs study how populations of neurons assemble into spatially organized structures. This work has the potential to be very interesting to other developmental neuroscientists studying brain morphogenesis.
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Reviewer #2 (Public review):
Summary:
The overall goal of this study by Smith et al. was to understand the mechanisms through which groups of cells form specific nuclei during development. These cell groupings may have importance for the development of nervous system connections. Smith et al. have taken advantage of the ordered structure of the nucleus laminaris of the chick, which plays an important role in sound source localization. They used a candidate gene approach to both mark cells in nL and to test for signaling pathways that regulate nucleogenesis. They found that MafB, FGF8, and cadherins were expressed in the auditory hindbrain at the critical ages. They used in ovo electroporation to test gene function effects on nL lamina formation. They found that both increasing and decreasing FGF signaling (through introduction of mouse …
Reviewer #2 (Public review):
Summary:
The overall goal of this study by Smith et al. was to understand the mechanisms through which groups of cells form specific nuclei during development. These cell groupings may have importance for the development of nervous system connections. Smith et al. have taken advantage of the ordered structure of the nucleus laminaris of the chick, which plays an important role in sound source localization. They used a candidate gene approach to both mark cells in nL and to test for signaling pathways that regulate nucleogenesis. They found that MafB, FGF8, and cadherins were expressed in the auditory hindbrain at the critical ages. They used in ovo electroporation to test gene function effects on nL lamina formation. They found that both increasing and decreasing FGF signaling (through introduction of mouse FGF8 and expression of a dominant negative FGF receptor, respectively) reduced lamina formation in the nL. An optimal concentration of FGF needed for this process was obtained using cultured hindbrain slices. Misexpression of cadherins also perturbed the normal lamina formation. The authors showed that FGF regulates MafB expression, which in turn regulates cadherin expression, suggesting a pathway that shapes lamina development. They constructed computational models of adhesion on the development of nL cells and found that laminar formation is favored by nL cells modeled as bipolar adhesive units. Overall, the study has demonstrated the importance of these adhesion pathways for the formation of the nucleus laminaris, and the findings likely have significance for the development of other nuclei as well.
Strengths:
The experiments have used in situ hybridization, immunofluorescence, electroporation, and brainstem slice cultures to test their hypotheses, which were based on well-selected candidate molecules. The modeling adds to the rigor of the studies, particularly in light of the observation that cadherin expression is localized to nL dendrites.
Weaknesses:
(1) Some references should be considered more carefully for accuracy, and additional references may be needed (introduction and results).
(2) Information on animal numbers and statistical tests should be added.
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