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\begin{document}

\title{Title}



\author[1]{Rahul Satija}%
\affil[1]{New York Genome Center}%


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  \date{May 26, 2024}


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We thank the reviewers for their careful reading and constructive
feedback on our manuscript. We appreciated that each reviewer offered
support and enthusiasm for our work, but raised substantial concerns. We
have therefore added substantial new experimental, analytical, and
functional datasets to our manuscript. These new data are in agreement
with our ~original conclusions, but we believe fully address the
reviewer concerns and significantly strengthen the manuscript. We
provide a point-by-point response to each reviewer concern below, but
first summarize our new data and analyses, emphasizing points that were
raised by multiple reviewers.

\textbf{Technical limitations of scRNA-seq data.~}Reviewers \#2 and \#3
both highlighted that the sequencing depth in our original Drop-seq data
could limit the interpretation of our dataset, particularly our finding
that mitotic progenitors do not show transcriptomic evidence of fate
specification. To address this, we first more than doubled the
sequencing depth of our Drop-seq dataset. In parallel, we used a version
of the SMART-Seq2 protocol to obtain deep sequencing data (4-7,000
genes/cell) from 400 mitotic progenitors. Both new datasets strongly
validate our original findings, producing nearly identical developmental
trajectories, and exhibiting no evidence of distinct subpopulations of
fate-restricted cells in the subventricular zone. As requested by
Reviewer \#2, we performed the SMART-Seq2 experiments with the FlashTag
system, providing an independent validation that our inferred maturation
trajectory is consistent with biological time. Finally, we added data
produced using the 10X genomics system for 7,000 postmitotic progenitors
with significantly increased sequencing depth (1,500-11,000 UMI/cell,
1,000-4,000 genes/cell), and observed identical branching patterns as in
our original Drop-seq dataset. Taken together, these new data
demonstrate that our original findings and interpretations were not
skewed by technical limitations, and could be fully reproduced from
complementary scRNA-seq techniques.

\textbf{Identifying the emergence of distinct subtypes.~}While our
initial manuscript focused on our unexpected finding that committed
progenitor subsets could not be identified in mitotic progenitors, all
reviewers requested further analysis of when distinct interneuron
subsets first emerge during development, and how heterogeneity in the
eminences could be connected to later timepoints. Leveraging a Dlx6a;
Cre~;Ai9 line that uniformly marks interneuron progenitors, We therefore
collected a set of 15,522 single cells across a developmental timecourse
(E13.5, E18, P10), obtaining 3,432 additional P56 cells from a public
resource hosted by the Allen Brain Atlas. We next applied our recently
developed `alignment' tool for scRNA-seq datasets (Butler et al., 2017),
to identify shared sources of variation that exist between developmental
and adult stages. This allowed us to link the emergence of interneuron
subtypes across embryonic, postnatal, and adult timepoints, to identify
the earliest emergence of non-overlapping populations that we
hypothesize are committed to Vip, Sst, Id2, and Pvalb interneuron fates,
and the initial transcriptomic markers that mark these groups across all
developmental stages. Remarkably, functional perturbation of one of the
strongest discriminating genes between early Pvalb and Sst progenitors,
the autism-associated transcription factor Mef2c, led to a complete yet
specific ablation of Pvalb interneurons, validating our computational
predictions. ~We believe that these aligned datasets will serve as a
valuable resource, in particular by revealing rich lists of genes that
likely play similarly important functional roles in the establishment
and maintenance of interneuron fates.

\textbf{Biological validation of key computational predictions.~}All
reviewers requested that complementary methods to scRNA-seq be used to
broaden support for our key conclusions, and Reviewer \#2 specifically
requested that specific computational predictions from the sequencing
data should be validated and functionally characterized. {[}NB from
Rahul : This paragraph is repetitive, but meant to highlight how new
experiments are in the revision in one place, most specificially for
Reviewer 2. We could add more here, but may want to highlight techniques
and data that are not more scRNA-seq, though we could add Lhx6
discussion here as well{]}. As discussed above, we generated data using
the tagging system from the Jabaudon lab (FlashTag system) as a
complementary technique to measure a cell's true biological birth date,
and validate our inferred maturation trajectory. We leveraged
trancriptomic markers of our three post-mitotic `branches' to perform
genetic fate mapping of MGE branch 3 cells based on specific expression
of Lhx8, and find that Lhx8+ progenitors do not give rise to cortical
interneurons as predicted, but instead of give rise to X. Finally, we
validate a functional role for a key transcription factor, Mef2c, that
we predict discriminates between early progenitors of MGE-derived
interneuron subtypes, by performing immunohistochemistry on a
conditional knockout mouse. These experiments demonstrate the high
quality of our computational predictions from Drop-seq data, and more
importantly, provide complementary support and validation for our key
conclusions.

\textbf{Conceptual insights that advance our understanding of
interneuron development.~}All reviewers expressed enthusiasm for our
dataset and analysis, but requested greater clarification of our new
insights and advances. In the revised manuscript, we have worked to
clearly articulate the following advances :~\textbf{First}, we discover
that mitotic progenitors exhibit tightly conserved maturation programs
across eminences, with downstream differences in fate seeded by only a
few genes that have largely been previously described. We see no
evidence for detailed fate restriction or non-overlapping subpopulations
for individual cells within an eminence, an important insight ~that
weighs heavily on outstanding questions in the field.~\textbf{Second},
we discover that upon cell-cycle exit, cells branch into one of three
distinct precursor states, representing fate-restricted populations of
interneurons and projection neurons. Surprisingly, the markers defining
the branches exhibit conservation across the MGE, CGE, LGE, although the
proportion of cells in each state does vary across eminences, along with
the exact expression programs activated along each
branch.~\textbf{Lastly}, we trace the emergence of interneuron
heterogeneity across a developmental time series, and identify subtle
heterogeneity in the postmitotic MGE that separates SST and
Pvalb-committed progenitors. We validate that Mef2c, a transcriptomic
marker that is enriched in PV -mapping progenitors at multiple time
points, is functionally required for the production of Pvalb but not Sst
interneurons. Similarly, we propose a list of early markers for early
progenitors of Sst, Vip, and Id2 interneurons which are conserved
throughout development, and may play similar functional roles.

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