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Barhl2 Determines the Early Patterning of the Diencephalon by Regulating Shh

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Abstract

The diencephalon is the primary relay network transmitting sensory information to the anterior forebrain. During development, distinct progenitor domains in the diencephalon give rise to the pretectum (p1), the thalamus and epithalamus (p2), and the prethalamus (p3), respectively. Shh plays a significant role in establishing the progenitor domains. However, the upstream events influencing the expression of Shh are largely unknown. Here, we show that Barhl2 homeobox gene is expressed in the p1 and p2 progenitor domains and the in zona limitans intrathalamica (ZLI) and regulates the acquisition of identity of progenitor cells in the developing diencephalon. Targeted deletion of Barhl2 results in the ablation of Shh expression in the dorsal portion of ZLI and causes thalamic p2 progenitors to take the fate of p1 progenitors and form pretectal neurons. Moreover, loss of Barhl2 leads to the absence of thalamocortical axon projections, the loss of habenular afferents and efferents, and a gross diminution of the pineal gland. Thus, by acting upstream of Shh signaling pathway, Barhl2 plays a crucial role in patterning the progenitor domains and establishing the positional identities of progenitor cells in the diencephalon.

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Acknowledgments

We thank Drs. R. Libby, A. Kiernan, P. White, and the members of Gan laboratory for their insightful discussions and technical assistance. This research was supported by The National Institute of Health grant 1R21EY023104, Zhejiang Province Science Grant 2012C13023-1, and the Research to Prevent Blindness challenge grant to the Department of Ophthalmology at the University of Rochester.

Authors’ Contributions

Q.D. and L.G. designed and conceived the experiments. Q.D., R.B., D.Z., G.L, and L.G. performed the experiments. Q.D., R.B., and L.G wrote the manuscript.

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Author notes

  1. Revathi Balasubramanian

    Present address: Department of Ophthalmology, Columbia University, New York, NY, 10032, USA

Authors and Affiliations

  1. Department of Ophthalmology, University of Rochester, Rochester, NY, 14642, USA

    Qian Ding, Revathi Balasubramanian & Lin Gan

  2. Department of Neurobiology and Anatomy, University of Rochester, Rochester, NY, 14642, USA

    Revathi Balasubramanian & Lin Gan

  3. Institute of Life Sciences, Hangzhou Normal University, Zhejiang, 310036, China

    Dongwang Zheng, Guoqing Liang & Lin Gan

  4. Flaum Eye Institute, University of Rochester, Rochester, NY, 14642, USA

    Lin Gan

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  1. Qian Ding
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  2. Revathi Balasubramanian
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Corresponding author

Correspondence to Lin Gan.

Additional information

Qian Ding and Revathi Balasubramanian contributed equally to this work.

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Fig. S1

Loss of thalamic nuclei in Barhl2 mutants. PROX1 is expressed in Barhl2 expressing thalamic nuclei at E17.5. Loss of Barhl2 leads to the loss of anterior thalamic nuclei (arrow) (a). Scale bar equals 200 μm (DOC 202 kb)

Fig. S2

Loss of characteristic expression of caudal thalamic neuron markers. A distinct compartmentalization of PAX7 and DBX1 expression in the developing diencephalon is seen at E10.5 and E11.5 (a). This boundary distinction is lost in the Barhl2-null mutant where PAX7 is expressed in the presumptive p2 progenitor domain and the expression of DBX1 is fragmented. At E12.5, Lhx2 and LHX9 are expressed in caudal thalamic p2 progenitors. In Barhl2-null mutants, the expression of both LHX9 (b) and Lhx2 (c) is severely attenuated and is restricted to a thin portion of the mantle zone. Scale bars equal 200 μm (DOC 1010 kb)

Fig. S3

Mis-expression of ASCL1 and SHH in Barhl2-null mutants. DBX1 is expressed in a graded fashion along the p2 progenitor domain, while ASCL1 is expressed in the p1 progenitor domain at E11 (a). Their expression is largely boundary restricted. In the absence of Barhl2, ASCL1 expression extends into the presumptive p2 domain, the graded expression pattern of DBX1 and boundary restriction is lost. There exists a small population of p2 progenitors that express DBX1 and ASCL1. NKX2.2 is expressed in the rostral thalamic p2 and p3 progenitor domain, flanking the SHH expression in ZLI at E11 (c). In Barhl2-null mutants, expression of SHH is caudalized, NKX2.2 expression in the rostral thalamus p2 domain is affected while that in the p3 domain is unaffected. Scale bar equals 200 μm (DOC 915 kb)

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Ding, Q., Balasubramanian, R., Zheng, D. et al. Barhl2 Determines the Early Patterning of the Diencephalon by Regulating Shh . Mol Neurobiol 54, 4414–4420 (2017). https://doi.org/10.1007/s12035-016-0001-5

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  • DOI: https://doi.org/10.1007/s12035-016-0001-5

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