Transcription factor Lhx2 is necessary and sufficient PNAS PLUS to suppress astrogliogenesis and promote neurogenesis in the developing hippocampus

Lakshmi Subramaniana,1, Anindita Sarkara,1, Ashwin S. Shettya, Bhavana Muralidharana, Hari Padmanabhana, Michael Piperb, Edwin S. Monukic, Ingolf Bachd, Richard M. Gronostajskie,f, Linda J. Richardsb, and Shubha Tolea,2 aDepartment of Biological Sciences, Tata Institute of Fundamental Research, Mumbai 400005, India; bQueensland Brain Institute and School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia; cDepartment of Pathology and Laboratory Medicine, School of Medicine, University of California, Irvine, CA 92697; dPrograms in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605; eDepartment of Biochemistry, State University of New York, Buffalo, NY 14203; and fDevelopmental Genomics Group, New York State Center of Excellence in Bioinformatics and Life Sciences, Buffalo, NY 14203 AUTHOR SUMMARY

During the development of the verte- memory. We used two approaches to brate CNS, progenitor cells generate disrupt Lhx2 function in progenitors at neurons, the primary players in circuits, the peak of hippocampal neurogenesis, and glia, the support cells. A character- embryonic gestation day (E) 15. Condi- istic feature of this process, common to tional KO mice were used, in which the all vertebrate species, is that the pro- gene encoding Lhx2 protein was dis- duction of neurons precedes that of glial rupted by the introduction of an enzyme cells (1). The transition from neuro- called “Cre recombinase,” which can cut genesis to gliogenesis determines the and paste DNA (2). The second ap- number of neurons vs. support cells that proach used a “dominant-negative” con- NEUROSCIENCE are produced in a given structure. This struct that encodes a protein capable process is also likely to be one of the of binding Lhx2 and prevents it from critical steps shaped by evolution to bring accessing essential cofactors, thus ren- about the diversity of nervous systems dering it inactive. Each agent was in- and subsystems seen across verte- troduced directly into the embryonic brate species. brain at E15 using in utero surgical A common progenitor for neurons and techniques, which allow the perturbation glia means that intrinsic mechanisms of a small population of cells in an oth- within the cell or extrinsic cues from erwise normal background of unaltered surrounding cells or more distant regions cells (3). Briefly, the uterus is exposed must act to initiate a switch from neu- and DNA is introduced into the brain of rogenesis to gliogenesis. The molecular the embryo using a fine-glass capillary. mechanisms that control this switch are Electroporation is carried out by not well understood, but a cellular sig- placing electrodes, such that the nega- naling pathway known to play a funda- tively charged DNA moves toward the mental role in this process is triggered by positive electrode placed on the side the membrane receptor protein Notch. of the target tissue. The uterus is During the early neurogenic period, Fig. P1. Molecular control of cell fate in the replaced in the abdomen, the opening Notch signaling maintains proliferating developing hippocampus. (A) Progenitor cells (blue is sutured, and the mouse is allowed circles) first give rise to neurons (green triangles) and to recover (3). The manipulated embryos progenitors in an undifferentiated state then glia (yellow stars). This change in cell fate is so that the precursor pool may be ex- can be examined several days later, paralleled by decreasing levels of transcription factor – panded (1). At later stages, however, this Lhx2. Removing Lhx2 from early progenitors causes and we selected a stage 6 8 d after pathway has a distinct role in instructing them to produce glia prematurely. Supplementing electroporation, by which time the pro- gliogenesis (1). Although Notch signal- Lhx2 levels in late progenitors is sufficient to prolong genitors that incorporated the DNA ing is active from the early stages of de- their neuron production phase. Therefore, Lhx2 is fi velopment in the progenitor cells, glia necessary and suf cient to suppress glia production (specifically, astroglia or astrocytes) are andenhanceneuronproductioninhippocampal Author contributions: L.S., A.S., H.P., and S.T. designed progenitors. (B) At the molecular level, Lhx2 research; L.S., A.S., A.S.S., B.M., H.P., and M.P. performed not generated during the neurogenic functions as a “brake” on glia-producing pathways research; E.S.M., I.B., R.M.G., and L.J.R. contributed new phase. This raises the question of how within the progenitors. A major target of these reagents/analytic tools; L.S., A.S., A.S.S., B.M., H.P., gliogenesis is suppressed until neuro- pathways is the GFAP gene, which is activated by the M.P., and S.T. analyzed data; and L.S., A.S., H.P., and genesis is complete. The molecular action of the Notch-Nfia pathway. Lhx2 overrides this S.T. wrote the paper. players that prevent the generation of activation, suppressing glia production until neuron The authors declare no conflict of interest. astrocytes during the neurogenic period production is complete. Freely available online through the PNAS open remain unknown. access option. In this study, we report a unique role This article is a PNAS Direct Submission. for a protein transcription factor belonging to the LIM-homeo- 1L.S. and A.S. contributed equally to this work. domain (Lhx) family, Lhx2: this protein acts as a gene switch in 2To whom correspondence should be addressed. E-mail: [email protected]. suppressing astrogliogenesis in the developing hippocampus, See full research article on page E265 of www.pnas.org. which is a cerebral cortical structure critical for learning and Cite this Author Summary as: PNAS 10.1073/pnas.1101109108.

www.pnas.org/cgi/doi/10.1073/pnas.1101109108 PNAS | July 5, 2011 | vol. 108 | no. 27 | 10937–10938 Downloaded by guest on September 27, 2021 normally give rise to neurons. DNA constructs were tagged with toward late gestation, which provides a mechanism for the re- a GFP marker so that electroporated cells could be identified. lease of the suppression, signaling the transition from neuro- These were examined by immunohistochemistry for neuronal- genesis to astrogliogenesis (Fig. P1A). and astrocyte-specific markers in sections of the Lhx2 has a fundamental role in the early development of electroporated brain. the brain, that of a “selector gene” for cerebral cortical fate. Using this procedure, we discovered that loss of Lhx2 causes In the absence of Lhx2, the cortical primordium (hippocampus a dramatic and premature increase in astrogliogenesis (base- and neocortex) is lost at the expense of alternative noncortical line levels of 26% increased to 86% in the Cre electroporation fates (2). This is an early role for Lhx2, the critical period case, and those of 35% increased to 80% in the dominant- which ends by E10.5, after which loss of Lhx2 does not cause negative construct electroporation case). Thus, loss of Lhx2 loss of the cortical primordium (2). However, even after this produces astrocytes prematurely from progenitors that would time, Lhx2 continues to be expressed in the progenitor cells of otherwise produce neurons. Even more striking was the finding fi the cerebral cortex, of which the hippocampus is one major that enhancement of Lhx2 levels by overexpression is suf cient component. Our study reveals a previously undescribed role for to suppress astrogliogenesis and prolong neurogenesis into Lhx2 as a regulator of the neuron-astrocyte cell fate switch the astrogliogenic phase. In WT mice that had reached E15, during hippocampal development. Notch signaling is also known overexpression of Lhx2 decreased the baseline level of 35% to have multiple roles in the telencephalic neuroepithelium, an astrogliogenesis to 10%. Overexpression at E17 decreased the 79% level of astrogliogenesis to 31%, similar to levels on E15. early role in maintaining proliferation of the progenitor pool, Therefore, Lhx2 is necessary and sufficient to enhance and and a later role in instructing astrogliogenesis (1). Interestingly, prolong neurogenesis by suppressing astrogliogenesis in the de- Notch and Lhx2 appear to have parallel early roles in main- “ ” veloping hippocampus (Fig. P1A). This raised the question of taining the proliferation and cortical identity of cortical pro- how Lhx2 suppresses pathways that lead to gliogenesis. genitors, respectively. In contrast, they play opposing roles in Activation of Notch signaling promotes astrogliogenesis via regulating the cell fate of the progeny arising from these pro- the transcription factor Nfia, which is necessary and sufficient for genitors, such that Notch promotes gliogenesis and Lhx2 sup- the commitment to the astrocytic cell fate (4). In the hippo- presses it. How the balance between these opposing players is campus, astrogliogenesis can be prematurely induced by over- controlled to generate the choreography of neurogenesis, fol- expressing constitutively active Notch or its target, Nfia. If, at the lowed by astrogliogenesis, remains an open question. same time as Notch or Nfia, we simultaneously overexpressed One final experiment revealed an unexpected spatial selec- Lhx2, we found that neurogenesis was restored. Lhx2 is a mem- tivity to this “glial-brake” function of Lhx2. When we inactivated ber of the transcription factor family of proteins, which means Lhx2 in the neocortex, neurons destined for different cortical that it can act as a master regulator, turning target genes on layers appeared to be produced normally despite loss of Lhx2 or off. We show that Lhx2 is able to repress expression of one function in the neocortical progenitors. This was surprising of the targets of Nfia, GFAP, which is one of the identifying because the two structures arise from a common primordium features of astrocytes. Removal of Lhx2 does not, however, and earlier studies examining astrogliogenic mechanisms had produce astrocytes if the Notch pathway is suppressed or if not uncovered region-specific functions of any molecular player. fi N a is not functional. This was shown in a hippocampal slice Our study not only identifies Lhx2 as a key regulator of the preparation that recapitulates many aspects of hippocampal neuron-astrocyte cell fate switch in the developing hippocampus development. We performed ex vivo electroporation in which but reveals an unexpected spatial selectivity within the cortex the brains of E15 embryos were dissected out, electroporated, for this critical function. This may open a new avenue of and sliced; these hippocampal explants were then maintained in exploration with respect to the diversity within the broad culture for 6 d. When a control construct was electroporated, category of astrocytes that is now beginning to be appreciated explants displayed robust axons after 6 d in vitro, indicative of (5). It is possible that the regional disparity we report in the normal neurogenesis. When Lhx2 function was interfered with regulation of astrocyte production also confers a unique identity by electroporation of a dominant-negative construct, no axons were detectable, consistent with the enhanced astrogliogenesis to hippocampal astrocytes and that other parallel mechanisms under these conditions. However, when a Notch inhibitor was operate elsewhere in the nervous system. used or when the explants were derived from an Nfia mutant embryo, removal of Lhx2 failed to produce the “no-axon” phe- 1. Miller FD, Gauthier AS (2007) Timing is everything: Making neurons versus glia in the developing cortex. Neuron 54:357–369. notype. This indicates that removing Lhx2 does not produce 2. Mangale VS, et al. (2008) Lhx2 selector activity specifies cortical identity and suppresses astrocytes unless Notch and Nfia are active. Therefore, astro- hippocampal organizer fate. Science 319:304–309. cytes produced as a result of loss of Lhx2 are generated by the 3. Saito T, Nakatsuji N (2001) Efficient gene transfer into the embryonic mouse brain Notch-Nfia pathway rather than through an independent path- using in vivo electroporation. Dev Biol 240:237–246. “ ” 4. Namihira M, et al. (2009) Committed neuronal precursors confer astrocytic potential on way. This suggests that Lhx2 acts as a brake on the Notch- residual neural precursor cells. Dev Cell 16:245–255. Nfia pathway, preventing premature gliogenesis until neuro- 5. Freeman MR (2010) Specification and morphogenesis of astrocytes. Science 330: genesis is complete (Fig. P1B). Lhx2 levels appear to decline 774–778.

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