CONTINUOUSLY ACTIVE TRANSCRIPTIONAL PROGRAMS ARE REQUIRED TO BUILD EXPANSIVE SEROTONERGIC ARCHITECTURES

By

LAUREN JANINE DONOVAN

Submitted in partial fulfillment of the requirements for the degree of Doctor of

Philosophy

Dissertation Advisor: Evan S. Deneris

Department of Neurosciences

CASE WESTERN RESERVE UNIVERSITY

January 2020 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

Lauren Janine Donovan candidate for the degree of Doctor of Philosophy*.

Committee Chair

Jerry Silver, Ph.D.

Committee Member

Evan Deneris, Ph.D.

Committee Member

Heather Broihier, Ph.D.

Committee Member

Ron Conlon, Ph.D.

Committee Member

Pola Philippidou, Ph.D.

Date of Defense

August 29th, 2019

*We also certify that written approval has been obtained for any proprietary material contained therein.

ii TABLE OF CONTENTS

List of Figures……………………………………………………………………….….vii Abstract………………………………………….………………………………..….…1 CHAPTER 1. INTRODUCTION………………………………………………...……..3 GENERAL INTRODUCTION TO SEROTONIN………………………………….….4

Serotonin: Discovery and function………………………….……………...4

Serotonin Biosynthesis…………………………..…………………………..6

Manipulation of the serotonin system in ……………………….6

Human mutations in 5-HT related ………………………………….9

SEROTONIN NEURON NEUROGENESIS……………..………………………….11

5-HT neuron specification……………..………………………………...…11

Development of 5-HT neurons……………..………………………………13

NEUROANATOMY……………..……………………………………………………..13

Cytoarchitecture ……………..………………………………………………13

Adult Ascending 5-HT axon projection system ………………………..14

5-HT axon innervation patterns in the forebrain……………………14

Serotonergic axon tracing studies……………..…………………….16

Varicosities in ascending 5-HT ……………………………….18

Adult Descending 5-HT axon projection system……………………….19

DEVELOPMENT OF SEROTONERGIC AXON PROJECTION SYSTEM……...20

Development of ascending projection system ………………………...20

Primary axon pathway formation…………………………………….21

Selective routing ………………………………………………………22

Terminal target arborization…………………………………………..24

iii Known molecules affecting 5-HT neuron ascending axon

development…………………………………………………………………..26

Wnt planar cell polarity pathway……………………………………..26

Slit-Robo signaling…………………………………………………….28

EphrinA5………………………………………………………………..28

Gap43…………………………………………………………….….....29

STOP (Map6) ………………………………………………………….30

Pcdhac2………………………………………………………………...31

5-HT……………………………………………………………………..34

Development of descending projection system ……………………….37

PET-1…………………………………………………………………………………...38

Gene/ expression……………………………………………………38

Protein structure………………………………………………………….…..39

Function………………………………………………………………………..39

LMX1B………………………………………………………………………………….40

Gene/protein structure………………………………………………………40

Lmx1b in peripheral tissues………………………………………………..41

Lmx1b in the CNS…………………………………………………………….42

Lmx1b in midbrain-hindbrain patterning…………………………….42

Lmx1b in midbrain Dopaminergic neurons………………………….43

Lmx1b in Noradrenergic neurons…………………………………….44

Lmx1b in Serotonergic neurons……………………………………...44

iv CHAPTER 2. PET-1 CONTROLS PATHWAY AND SLC22A3 TRANSPORTER GENES IN SEROTONIN NEURONS. Summary……………………………………………………………………………....60 Introduction…………………………………………………………………..……….61 Methods………………………………………………………………………………..65 Results and Discussion………………………………………………………….....68

CHAPTER 3. LMX1B IS REQUIRED AT MULTIPLE STAGES TO BUILD EXPANSIVE SEROTONERGIC AXON ARCHITECTURES Summary……………………………………………………………………………….90 Introduction……………………………………………………………………………91 Materials and Methods………………………………………………………………94 Results………………………………………………………………………………..104 Lmx1b controls formation of ascending 5-HT projection pathways…………….104 Lmx1b controls formation of descending 5-HT projection pathways…………...107

Delayed primary pathway formation and aborted selective pathway routing in

Lmx1bcKO mice…………………………………………………………………...... 108

Lmx1b acts temporally to control 5-HT axon selective pathways…………..…..110

Lmx1b switches function to control terminal arborization...…………………...... 112

Targeting of 5-HT synthesis does not impair formation of forebrain and spinal cord 5-HT arbors………………………………………………………………..…....113

Lmx1b controlled axon-related transcriptomes…………………………………...114

An ascending-specific axonal Lmx1Pet1 regulatory cascade………………...117

Lmx1b acts through Pet1 to temporally control postnatal stage-specific and forebrain arborization……………………………………………...120

Discussion…………………………………………………………………………...123

v CHAPTER 4. DISCUSSION AND FUTURE DIRECTIONS Serotonergic Heterogeneity………………………………………………………196

Lmx1b is continuously required for the formation of 5-HT axon

architectures………………………………………………………………………...198

Lmx1b in intrinsic 5-HT axon growth or guidance?...... 200

Short-range versus long-range 5-HT axon growth……………………….200

Lmx1b regulates axon growth and guidance genes …………………….202

Potential mechanisms of Lmx1bcKO axon failure to innervate…………206

From identity to axon development………………………………………….…..211

Transcription factor regulation of ascending vs descending 5-HT axon

architectures…………………………………………………………………………212

Lmx1bPet1 regulatory cascade functions in 5-HT arborization…………214

Possible role for Lmx1b in regeneration?...... 217

Investigation of mutations in the 5-HT GRN………………………….219

Conclusion…………………………………………………………………………...220 Bibliography………………………………………………………………………….228

vi LIST OF FIGURES CHAPTER 1 Figure 1. Enzymatic steps of 5-HT synthesis……………………………....47 Figure 2. regulatory network that defines serotonergic identity……………………………………………….…………..49 Figure 3. Cytoarchitecture of 5-HT neurons in the mouse ………….51 Figure 4. 5-HT axon innervation patterns in the forebrain………………...53 Figure 5. Single labeled 5-HT neurons reveal a great diversity of 5-HT axon innervation……………………………………………………………….55 Figure 6. Embryonic time series of 5-HT axon selective routing in the mouse…………………………………………………………………………..57 CHAPTER 2

Figure 1. Schematic of BH4 de novo synthesis, salvage, and regeneration pathways and its role in 5-HT synthesis………………………………….…77

Figure 2. Isolation of ePet-EYFP and Pet-1-/-;ePet-EYFP 5-HT neurons.……79

Figure 3. Microarray analyses………………………………………………..81

Figure 4. Slc22a3 expression………………………………………………...83

Figure 5. Slc22a3 and Htr1a expression in the adult dorsal raphe……....85

Figure 6. Pet-1 control of the 5-HT neuron-type gene battery…………….87

CHAPTER 3

Figure 1. Lmx1b is required for the formation of ascending 5-HT axon projection pathways…………………………………………………………..130

Figure 1—figure supplement 1. Surrogate marking of 5-HT cell bodies and axons and Lmx1b conditional targeting……………………………….132

Figure 1—figure supplement 2. Lmx1b deficiency disrupts 5-HT axon patterns in the forebrain……………………………………………………...135

Figure 2. Lmx1b is required for the formation of descending 5-HT axon projection pathways…………………………………………………...……...137

Figure 2—figure supplement 1. Conditional targeting of Lmx1b in the descending 5-HT projection pathway……………………………………….139

vii Figure 2—figure supplement 2. Progressive deficits of 5-HT axon fibers in Lmx1b deficient spinal cord white and gray matter…………………....141

Figure 3. Initial axon outgrowth is delayed and selective pathway routing fails in Lmx1b deficient 5-HT neurons………………………….....143

Figure 4. Lmx1b is temporally required for 5-HT projection pathway formation……………………………………………………………………....145

Figure 4—figure supplement 1. Efficiency of postnatal tamoxifen inducible targeting of Lmx1b………………………………………………...147

Figure 5. Lmx1b temporally controls postnatal 5-HT terminal arborization…………………………………………………………………....149

Figure 5—figure supplement 1. P3 targeted Lmx1bicKO mice display normal 5-HT axon routing but decreased 5-HT terminal arbors………...151

Figure 6. Specific targeting of 5-HT synthesis does not alter 5-HT arborization patterns…………………………………………………….…....153

Figure 7. Ascending and descending Lmx1b regulated transcriptomes…………………………………………………………...... ….155

Figure 7--supplement table 1. Lmx1b-regulated axon-related genes in rostral and caudal 5-HT neurons at E17.5……………………………..….169

Figure 8. Distinct transcription factor requirements in the formation of ascending and descending 5-HT projection pathways………………..….158

Figure 8—figure supplement 1. Pet1cKO and Lmx1bcKO mice exhibit distinct axon defects in thalamus…………………………………………...160

Figure 8—figure supplement 2. DKO and Pet1-/- analyses………………162

Figure 9. An ascending specific Lmx1bPet1 cascade controls stage specific 5-HT gene expression and postnatal terminal arborization….…164 Figure 9—figure supplement 1. Lmx1bPet1 cascade acts postnatally to control 5-HT terminal arborization…………………...…………………..167

CHAPTER 4

Figure 1. Lmx1b regulates both growth and guidance genes in 5-HT neurons………………………………………………………………………...222

Figure 2. The glial sling is a transient barrier in the embryonic mouse brain...... 224

viii Figure 3. Gap43 is significantly upregulated in maturing 5-HT neurons into postnatal stages………………………………………………………………226

ix Continuously Active Transcriptional Programs are Required to Build

Expansive Serotonergic Axon Architectures

Abstract by

LAUREN JANINE DONOVAN

Neurons express unique , extend axons either short or long distances, and distribute axon processes in specific patterns in order to functionally integrate into the complex network that is the brain. Neuronal diversity is ultimately shaped by gene expression diversity. Serotonin (5-HT) neuron identity is defined by expression of a known set of transcription factors that activate genes to enable 5-HT neurons to produce, transport and metabolize serotonin itself. Here, we have extended the role for Pet1, an ETS transcription factor, in control over global 5-HT identity through its regulation of genes encoding all rate limiting responsible for 5-HT synthesis (Tph2 and

Gch1), as well as all transporters of 5-HT that enable reuptake (Slc6a4 and

Slc322a3).

While acquisition of 5-HT identity is now well defined, nothing is known of the intrinsic transcriptional mechanisms that govern 5-HT axon development. The

5-HT axonal system is one of the most expansive axon systems of the CNS, produced by relatively few 5-HT neurons. It is unknown how serotonin neurons have the tremendous capacity for producing such an expansive axon system. 5-

1 HT neurons undergo multiple stages of axon growth including primary pathway formation, long-range routing, and terminal arborization. Here, we found that

Lmx1b is required for the formation of the long-range ascending and descending

5-HT axon architectures to the brain and spinal cord respectively. Stage-specific loss of Lmx1b revealed the requirement for this continuously expressed transcription factor at each 5-HT axon growth stage, as opposed to distinct transcription factors regulating each growth stage. We found that Lmx1b regulates another continuously expressed transcription factor, Pet1, during 5-HT neuron maturation. Pet1 was found to be critical for ascending, but dispensable for descending 5-HT axon development, highlighting differential transcription factor requirements for these two independent 5-HT projection systems. We identified that a Lmx1b→Pet1 regulatory cascade functions in a temporal manner to control the late-stage 5-HT axon arborization, in part through regulating

Pcdhac2 expression, a downstream gene known to be involved in 5-HT axon arborization. This work provides the first demonstration of a continuously expressed transcription factor that is essential at many distinct morphological stages of axon development.

2

CHAPTER 1. INTRODUCTION

3 GENERAL INTRODUCTION TO SEROTONIN

Serotonin: Discovery and function

5-Hydroxytryptamine (5-HT), colloquially known as serotonin, was initially discovered in the digestive tract in 1935 by an Italian scientist named Dr. Vittorio

Erspamer. His research of enterochromaffin cells, which line the gastrointestinal tract, led him to the discovery of this previously unknown amine he initially termed “enteramine”. He found that this substance was produced by enterochromaffin cells to regulate smooth muscle contraction to induce gut motility in (Erspamer and Vialli, 1937). Years later, the same substance was discovered in the blood serum of cattle by an American research group at the Cleveland Clinic (Rapport et al., 1948a). This group termed the substance

“serotonin” for its vasoconstrictive action of blood vessels (Rapport et al., 1948b).

The structure of each enteramine and serotonin was later discovered to be the same, 5-Hydroxytryptamine (5-HT), by these two independent groups (Rapport,

1949; Erspamer and Asero, 1952). It wasn’t until 1953 that Dr. Betty Twarog published the first report of 5-HT presence in mammalian brain tissues of dogs, rabbits, and rats (Twarog and Page, 1953). This groundbreaking discovery led to the decades long, and continuing fascination, of understanding the impact of serotonin on brain function.

5-HT functions both as a hormone in the blood and a in the brain. In fact, 95% of serotonin is produced in the periphery, mainly by enterochromaffin cells of the gut (Gershon and Tack, 2007). In addition, a small number of other peripheral cell types can also produce 5-HT (Lv et al., 2017;

4 Weitzman et al., 1985; Morishima T., 1970). Importantly, platelets have the ability to take up 5-HT and re-release it to regulate blood clotting, vasoconstriction, and hemostasis in the circulatory system (Watts et al., 2005; Schlienger and Meier,

2003). The remaining 5% of serotonin is produced by a small number of neurons in the brain and functions as a crucial neuromodulator to control a vast number of physiological and behavioral processes. While commonly thought of as the

“happy chemical” because serotonin contributes to mood regulation, serotonin also modulates behaviors such as maternal care, aggression, appetite, memory, reward, and sleep (Lerch-Haner et al., 2008; Lam et al., 2010; Muller and

Jacobs, 2010; Fernandez et al., 2017; Li et al., 2016). Less commonly known, 5-

HT has important functions in the modulation of many basic physiological processes such as thermoregulation, breathing, pain control, heart rate/rhythm, reproductive function, walking, and urination (Cohen et al., 2015; Correia et al.,

2017; Liu et al., 2014; Lottem et al., 2018, Berger et al., 2009).

As a neuromodulator, serotonin functions within the CNS by either increasing or decreasing neuronal excitability, which depends on the type of 5-

HT that is engaged on the receiving neuron (Barnes et al., 1999;

Hannon and Hoyer, 2008). There are 14 different 5-HT receptors, all of which are

G protein coupled receptors with the exception of the 5-HT3 receptor, which is a ligand-gated cation channel (Hannon and Hoyer, 2008). These receptors are widely expressed by a multitude of cell types throughout the CNS and can receive 5-HT signaling through volume transmission extrasynaptically as well as through direct synaptic transmission (Fuxe et al., 2010).

5 Serotonin Biosynthesis Serotonin synthesis is a multistep process in which the essential amino acid L-Tryptophan (TRP) is converted into 5-Hydroxytryptamine (Figure 1). In the brain, serotonin neurons produce the monoaminergic monooxygenase named tryptophan hydroxylase 2 (Tph2) along with its obligatory 6R-L-erythro-

5,6,7,8-tetrahydrobiopterin (BH4) that work together to enzymatically convert L-

Tryptophan to 5-Hydroxytryptophan (Kapatos, 2013; Watschinger et al., 2010;

Werner et al., 2011). The regeneration of BH4 and Tph2 production is rate limiting to this enzymatic step (Fitzpatrick, 1999) (Figure 1). Following the production of 5-Hydroxytryptophan, aromatic amino acid decarboxylase (Aadc) removes the carboxyl group thereby finishing the final conversion to serotonin. In peripheral cell types, serotonin biosynthesis is predominately conducted by an isoform of Tph called Tph1, rather than the central expressing isoform of Tph2

(Walther and Bader, 2003; Walther et al., 2003). In support of this, Tph1 germline knock out mice have little effect on brainstem 5-HT levels while gut and blood 5-

HT levels are almost completely depleted (Cote et al., 2003; Izikki et al., 2007;

Savelieva et al., 2008). In contrast, Tph2 germline knock out mice result in a 93-

97% loss of 5-HT in the forebrain with no effect on peripheral 5-HT levels (Alenia et al., 2009; Savelieva et al., 2008).

Manipulation of the serotonin system in humans

From prescribed drugs to illegal drugs, the serotonin system in the brain has been hijacked by humans and heavily reported about due to its strong psychoactive effects that alter perception. In fact, many serotonergic

6 psychedelics structurally resemble 5-HT itself, including N,N-Dimethyltyprtamine

(DMT), Lysergic acid diethylamide (LSD), and psilocybin, and act as agonists of

5-HT2A receptors, thought to be the main contributor of hallucinogenic effects

(Nichols, 2016). In support of this, blockade of 5-HT2A receptors using antagonists ketanserin and pirenperone were successful in blocking the stimulus effects of LSD and other tryptamine hallucinogens in rodents (Nichols, 2016).

These psychedelics can also modulate activity of 5-HT1A and 5-HT2C receptors and contribute to the intoxication process. Clinical use of LSD in terminal cancer patients at the spring Grove State Hospital in Maryland in the 1960s showed that modulation of the 5-HT system by this drug improved mood as well as reduced anxiety and fear of death in two-thirds of these patients. Multiple reports of the effects of LSD caught the attention of the media and sparked recreational use which led to a restriction of this class of drugs in the late 60s and 70s (Hoffman,

1970; Nichols, 2016). Today, there has been an effort to reintroduce LSD as well as psilocybin into research to better understand the mechanisms that underly the positive effects of psychedelic drugs and potentially use them to treat patients with advanced-stage cancer, depression, and other psychiatric disorders (Grob et al., 2011; Geyer, 2015).

The psychoactive effects/altered consciousness caused by various drugs that modulate the 5-HT system launched interest to investigate the serotonin system in psychiatric disorders in the 1950s. The same researchers who experimented with a class of agonists of the 5-HT system, called ergot alkaloids, which includes LSD, also proposed the 5-HT deficiency hypothesis, that stated

7 low levels of brain 5-HT may be causative of mental health disorders (Woolley and Shaw, 1952; Shaw and Woolley, 1953). They further stated that raising 5-HT levels in patients could potentially help treat patients with psychiatric disorders

(Woolley and Shaw, 1954). Supportive of these concepts, it was found that clinically depressed patients have decreased serotonin in the brain as well as decreased 5-HT metabolites in cerebral spinal fluid (Belmaker and Agam, 2008).

Additionally, many drugs that target different components of 5-HT neurotransmission, to raise 5-HT signaling, have had success in alleviating depressive and anxiety type behaviors in many, but not all patients. One example of a synthesized antidepressant is fluoxetine (also known as Prozac), which functions as a selective serotonin reuptake inhibitor (SSRI) and promotes longevity of 5-HT signaling in the extracellular space by blocking 5-HT reuptake by the serotonin transporter, SERT (Cipriani et al., 2018). Many types of SSRI drugs are on the market and all have been effective to some level in treating depression, including fluoxetine, however, it does take several weeks for these beneficial therapeutic effects to happen and it is still unclear why (Charnay and

Leger, 2010; Cipriani et al., 2018). In addition, blocking 5-HT degradation through inhibiting monoamine oxidase (MAO) using a drug called iproniazid also effectively raised 5-HT levels in the brain by 2- to 3-fold in mice (Udenfriend et al., 1957b). Iproniazid was later found to effectively treat depressed patients by improving mood and attitude, as well as heighted an interest in both self as well as environment in a clinical study (Loomer et al., 1957). Iproniazid is still used today as a psychiatric medication. Drugs that target the 5-HT system are the

8 most widely prescribed still today to treat various neuropsychiatric disorders including depression.

Human mutations in 5-HT related genes

Many neuropsychiatric disorders including depression, anxiety, autism, schizophrenia, attention deficit hyperactivity disorder (ADHD), and obsessive- compulsive disorder (OCD) have been repeatedly linked to dysfunction of serotonin signaling (Deneris and Wyler, 2012). 5-HT neuron dysfunction can be caused by mutations in genes required to produce 5-HT, transport 5-HT, or metabolize 5-HT, resulting in most cases, in reduced serotonergic signaling

(Caspi et al., 2003; Albert et al., 2011; Waider et al., 2011, Liu et al., 2015).

Several mutations in genes that are known to be important in serotonin signaling have been identified in humans (Deneris and Wyler, 2012; Doan et al., 2019).

Many of these gene variants have been linked to dysfunctional behaviors thought to be related to defective serotonergic signaling (Deneris and Wyler, 2012; Doan et al., 2019; Prasad et al., 2009; Muller et al., 2016; Lopez et al., 2010). One example is SERT (gene name: Slc6a4), a 5-HT transporter that is necessary for movement of 5-HT across the 5-HT neuron membrane, and thus 5-HT neurotransmission (Blakely et al., 1991). Several variants of human SERT mutations that induce a gain of function and hyperactivity of SERT have been linked to patients with autism spectrum disorders (Prasad et al., 2009; Muller et al., 2016). In addition, many human variants of LMX1B, a gene found to be necessary for the differentiation of 5-HT neurons in mice (Zhao et al., 2006),

9 cause a disease called Nail patella syndrome (NPS) (Sweeney, 2003). In one small study, patients with NPS were found to have higher incidence of major depression disorder (MDD) and ADHD (Lopez et al., 2010). However, due to the pleiotropic nature of LMX1B, these patients have many compounding effects resulting from LMX1B mutation including developmental problems of the eye, kidney, knee caps, and fingernails (Sweeney, 2003). These peripheral developmental defects obscure whether the psychiatric disorders observed in

NPS patients are due to 5-HT system dysfunction. Rare human mutations have also been identified in genes responsible for BH4 production such as GCH1,

PTS, SPR, PCBD1, and QPDR genes (Thony and Blau, 2006). In humans containing these mutations, serotonin synthesis is disrupted, and behaviors include motor control disorders due to monoamine deficiencies (Thony and Blau,

2006). Recently this year, human mutations have also been identified in the gene FEV (Doan et al., 2019), which has been found to be essential for serotonin neuron differentiation in mice (Hendricks et al., 2003). Interestingly, FEV is solely expressed in serotonin neurons of the human brain (Maurer et al., 2004; Iyo et al., 2005). Two brothers were identified as having the same biallelic mutations in

FEV that caused a stop-gain function (Doan et al., 2019). One brother was clinically diagnosed with autism and presented with low IQ and aggressive behaviors while the other brother could not be categorized in the ASD spectrum, however displayed “pervasive developmental disorder-not otherwise specified and intellectual disability” (Doan et al., 2019). This is extremely interesting as investigations of genetic knock-out mice for Pet1 (the mouse ortholog of human

10 FEV) revealed that these mice display aggressive and anxiety-like behaviors, thought to be the result of the 80% deficiency of 5-HT (Hendricks et al., 2003).

In addition to mutations in genes that impact 5-HT signaling, it has been postulated that disruptions in genes that impact the wiring of 5-HT neurons into brain circuitry during development could likely cause behavioral disorders, although there is no direct evidence for this at this time in mouse or human studies (Kiyasova and Gaspar, 2011).

SEROTONIN NEURON NEUROGENESIS

5-HT neuron specification

Over the past two decades, molecular profiling of distinct neuron cell types has enabled great progress in describing neuron-type specific gene batteries.

These specific gene batteries are defined as the co-expression of particular genes that enable a neuron to function in a specific manner, thereby determining its neuronal identity (Hobert, 2010, Fishell, 2013, Deneris and Hobert, 2014).

Neuronal phenotype is comprised of both generic neuronal features as well as specific features, such as specific neurotransmitter expression. Neuronal specification is dependent on both the morphogen gradient in surrounding developing tissue as well as specific intrinsic expression of transcription factors that enable expression of downstream neuron-type specific gene batteries

(Goridis & Rohrer, 2002; Cordes, 2005; Scott & Deneris, 2005; Flames & Hobert,

2011).

11 What makes a serotonin neuron, a serotonin neuron? Dissected individually, no single gene expressed can define this neuronal type. However, it is the combinatorial expression of a set of transcription factors that collectively enable 5-HT neurons to express a serotonergic-type gene battery that allow these neurons to use 5-HT as a neurotransmitter. This serotonergic gene battery is comprised of the following genes: tryptophan hydroxylase 2 (Tph2), aromatic amino acid decarboxylase (Aadc, also named Ddc), vesicular monoamine transporter (Slc18a2, also known as Vmat2), serotonin transporter (Slc6a4, also known as Sert), 5-HT1a autoreceptor (Htr1a), 5-HT1b (Htr1b), and monoamine oxidase A and B (Maoa and Maob). These genes enable the synthesis, transport, autosensing capability, inactivation of 5-HT signaling by reuptake, and of 5-HT (Deneris and Wyler, 2012). The expression of a well-defined set of eight transcription factors is required to activate this battery of serotonergic specific genes in the mouse (Figure 2). Initially, Ascl1 and Nkx2.2 expression is induced, likely in response to early Shh signaling in surrounding developing tissue (Briscoe et al., 1999; Pattyn et al., 2004; Craven et al., 2004). As progenitors begin to exit the cell cycle to become postmitotic precursors, Ascl1 and Nkx2.2 expression is diminished and Foxa2 expression is induced (Jacob et al., 2007). Next, Gata2, Gata3, and Insm1 expression is initiated to finalize the postmitotic precursor stage (Craven et al., 2004; Jacob et al., 2009; van

Doorninck et al., 1999). Lastly, the Lim homeodomain, Lmx1b, and the ETS factor, Pet-1, expression is induced in parallel just before detection of 5-HT and

Tph2 (Lillesaar et al., 2007; Hendricks et al., 1999; Pfaar et al., 2002) (Figure 2).

12 Development of 5-HT neurons

There are two main groups of 5-HT neurons, rostral and caudal, named for their relative position within the hindbrain and their specific projection patterns to either the forebrain or spinal cord respectively. In mice, rostral and caudal 5-

HT neurons are born in the ventral rhombencephalon between E9.5 and E10.5, however caudal 5-HT neurons do not express 5-HT until 1-2 days later (Deneris and Wyler, 2012; Lidov and Molliver, 1982). Morphogenic gradients of sonic hedgehog (Shh) and fibroblast growth factors 4 and 8 within the ventral hindbrain regionally direct the pattern of 5-HT neuron specification resulting in bilateral clusters of 5-HT neurons on each side of the midline at early embryonic stages

(Deneris and Wyler, 2012). Once 5-HT neurons are born, they enter a maturation phase that extends to the 4th postnatal week in the mouse (Lidov & Molliver,

1982; Maddaloni et al., 2017). This maturation phase consists of a series of complex steps in which 5-HT neurons migrate to their final raphe patterns, extend dendrites, form their highly collateralized axon processes, and undergo synaptogenesis throughout the CNS.

NEUROANATOMY

Cytoarchitecture

Despite its critical role in so many different behavioral and physiological processes, serotonin is produced by a remarkably small number of neurons in the brain. There are only 26,000 5-HT neurons of the 75 million neurons in the mouse brain and 400,000 5-HT neurons of the 86 billion neurons in the human

13 brain (Ishimura et al., 1988; Baker et al., 1990; Hornung, 2003). These 5-HT neurons are located in discrete clusters within the midbrain, pons, and medulla named the raphe B nuclei (B1-9) (Dahlstrom & Fuxe, 1964) (Figure 3). The rostral nuclei, named B4-9 nuclei, contain the largest proportion of serotonin neurons, about 85% of total serotonin neurons, and project their long axon processes primarily in an ascending trajectory to forebrain targets (Lidov &

Molliver, 1982; Hornung, 2003; Jensen et al., 2008). These rostral nuclei are comprised of the dorsal raphe nuclei (DRN, B4, B6, and B7 nuclei), median raphe nuclei (MRN, B8 and B5 nuclei), and the B9 nuclei (Deneris and Wyler,

2012) (Figure 3). In contrast to rostral nuclei, the caudal nuclei, named B1-3 nuclei, have a primarily descending axon projection system to the brainstem and spinal cord, as well as axon projections to the cerebellum. While these caudal neurons are fewer in number compared to rostral neurons, they are still able to innervate the entire rostro-caudal extent of the spinal cord and modulate many physiological processes.

Adult Ascending 5-HT axon projection system

5-HT axon innervation patterns in the forebrain

Rostral 5-HT neurons send out their axons to innervate nearly every region of the forebrain (Figure 4A). It is important to note that while 5-HT axons innervate the entire forebrain, they pattern in precise densities in different forebrain targets. One example of extremely high 5-HT axon arbor density is at the paraventricular nucleus of the thalamus (PVT). 5-HT arbors at the PVT are so

14 dense and closely associated with cells at this target region, that one can make out small holes that are the cell bodies of the PVT (Figure 4B). This is a unique area of the forebrain innervated by 5-HT neurons because of this extremely high density. Functionally, this dense innervation by 5-HT axons is thought to mediate stress signals as the PVT is interconnected with the accumbens nucleus, hypothalamus, basal amygdala, and subgenual cortex (Hsu and Price, 2009). In many other thalamic regions, 5-HT arbors are much more loosely tiled and resemble more of a web-like appearance, crossing over each other in different directions (Figure 4C). This web-like structure of 5-HT axons is most common throughout the forebrain, but at varying densities in different target regions. For example, the cortex and dorsal hypothalamus have higher axon densities compared to lateral thalamic subnuclei regions. Another example of high 5-HT axon density is within the molecular layer of the , also known as the lacunosum moleculare (LSM). Interestingly, this density of 5-HT axons in the

LSM is directly adjacent to the apical dendrites of CA1 pyramidal neurons (Lidov and Molliver, 1982). Indeed, it has been recently reported that 5-HT signaling of

5-HT1B receptors expressed on CA1 pyramidal apical dendrites of the hippocampus plays an important role in spatial memory consolidation (Zhou et al., 2019).

Uniquely, serotonin neurons have the ability to innervate areas of the forebrain where other axon-types are not known to innervate, such as the ependymal zone, the lining of the ventricular system of the brain and central canal of the spinal cord (Lorez and Richards, 1982; Azmitia and Whitaker-

15 Azmitia, 1991). Not only do 5-HT neurons have the ability to innervate the ependyma, but they do so in extremely dense patterns (Figure 4D), perhaps to supply CSF with 5-HT to aid in volume transmission, or perhaps to provide specific serotonergic input to neural stem cells (NSCs) within the ventricular walls, which may be important for their function (Tong et al., 2014). Indeed,

NSCs within the lateral ventricle express 5-HT receptors 2C and 5A and can be trans-synaptically traced to DRN 5-HT neurons in adult mice (Tong et al., 2014).

Activation of these receptors using a 5HT2C agonist was found to be sufficient to increase V-SVZ proliferation, and conversely application of an antagonist decreased proliferation in mice (Tong et al., 2014). These results demonstrate an important function for 5-HT signaling in the regulation of NSC neurogenesis in the adult ependyma.

Serotonergic axon tracing studies

Serotonin neurons collectively innervate the entire forebrain leaving no specific cytoarchitectural structure devoid of their axons, besides some areas of the cerebellum, based on my observations in the mouse. How do these small numbers of neurons have such widespread reach throughout the forebrain? Do all 5-HT neurons elaborate similar densities of arbors at any given target region?

Do individual 5-HT neurons innervate only one general target region, or multiple?

3D reconstructions of axonal processes of single biotin-labeled rat DRN neurons revealed that a single 5-HT neuron can produce an axon that bifurcates to innervate two very different target regions (Gagnon and Parent, 2014). A single

16 DRN 5-HT neuron was shown to elaborate extensive arbors in all six layers of the frontal cortex as well as send a collateral axon to the hippocampus and arborize abundantly in CA1, CA2, CA3, and dentate gyrus (Gagnon and Parent,

2014) (Figure 5A). In contrast, other 5-HT neurons have a single non-bifurcating axon that only travels a relatively short distance to a single target region (Figure

5B). When total axonal length was measured for each DRN neuron labeled in this study, it was found that the axon length could vary between 1cm all the way up to 18.7cm (Gagnon and Parent, 2014). These results not only highlight the immense ability of a single 5-HT neuron to profusely arborize, but also highlights the diversity of individual 5-HT neuron projections to different terminal target regions.

There has been great interest to understand specific projection patterns of the different 5-HT subnuclei. Several independent studies have performed anterograde labeling of these B subnuclei to better characterize their specific axonal connections and possibly function in the brain. Recent studies employing stereotaxic viral injections of a conditional AAV-GFP vector to specific raphe nuclei (B5-B9) have found that while 5-HT neuron location within the different raphe subnuclei are correlated with some projection targets, they are not exclusively related to their terminal arbor patterns in the forebrain (Muzerelle et al., 2016). This study revealed that innervation to several brain target regions are highly correlated to specific 5-HT neuron subnuclei, while others, including mPFC, hippocampus, and thalamic regions have a mixed innervation originating from multiple 5-HT neuron subnuclei. While overlap in projections between

17 subnuclei provide evidence for a mixed 5-HT neuron population, this work provides the best description of 5-HT neuron subnuclei axon projections to specific brain regions if a particular circuit is under investigation.

Varicosities in ascending 5-HT axons

In addition to the differential axon densities at target regions, there are varying morphologies of 5-HT axons in different target regions. 5-HT axons can either have numerous axon varicosities that are present throughout development and maintained in adulthood or have few to no varicosities. Most 5-HT axon processes, however, are highly varicose. These varicosities are bulbus structures that appear like beads on a string along the entire length of the axon and contain vesicles loaded with neurotransmitter (Brown and Molliver, 2000; Gagnon and

Parent, 2014). Vesicles can contain 5-HT alone or in combination with glutamate or other neuropeptides (Gagnon and Parent, 2014). It has been demonstrated that the presence of glutamate can increase 5-HT accumulation into vesicles if in the presence of VMAT2 and vesicular glutamate transporter type 3 (VGLUT3)

(Amilhon et al., 2010). In addition, the overall number of varicosities in 5-HT axons differ depending on the target region, and is thought to be indicative of input strength, as the number of axon varicosities have been shown to correlate with the concentration of neurotransmitter in extracellular space (Gagnon and

Parent, 2014). Interestingly, only a percentage of 5-HT axon varicosities have post-synaptic densities as analyzed by electron microscopy and these precise percentages depend on the terminal target region (Seguela et al., 1989; Parent

18 et al., 2010; Descarries et al., 2010). This observation of the presence of non- synaptic vesicle containing varicosities is part of the morphological evidence that supports the concept of volume transmission that occurs extra-synaptically

(Descarries and Mechawar, 2008).

Adult Descending 5-HT axon projection system

While the descending projecting 5-HT neurons of the B1-B3 nuclei primarily innervate the entire brainstem and spinal cord to modulate numerous important physiological functions, nothing is known of the differential topographic projection patterns originating from each specific caudal subnuclei. Anatomical tracing studies have not been conducted in these caudal B nuclei presumably because these 5-HT neurons are spread out along the base of the hindbrain and are hard to access by standard stereotaxic viral injections without compromising basic brainstem function needed to keep the animal alive. However, the adult patterns of descending 5-HT-labeled axons throughout the spinal cord have been described in rodents (Newton et al., 1986; Rajaofetra et al., 1989). 5-HT neuron axon patterns throughout the spinal cord is roughly similar from rostral to caudal levels. 5-HT axons of passage can be found in white matter tracts called funiculi and 5-HT axon terminal arbors are found in all layers of the gray matter throughout the entire spinal cord. The highest densities of 5-HT axons in the white matter can be found in the lateral funiculi, while other axons of passage concentrate along the periphery of the white matter that can be clearly seen in coronal sections of the cord. 5-HT axons within the lateral funiculi are highly

19 varicose whereas 5-HT axons in other white matter are typically smooth

(Rajaofetra et al., 1989). The highest densities of 5-HT axons in the gray matter are found in dorsal horn layer I and II, around the central canal, and throughout the ventral horn, where motor neurons reside, compared to other laminae.

While there have been an extensive number of studies focusing on describing 5-HT neuron innervation patterns and the morphology of these axons, many outstanding questions remain. How do these 5-HT neurons have the capacity to profusely arborize? How is a 5-HT neuron instructed to innervate one specific target region versus 2 or 3 target regions in the forebrain? How do individual 5-HT neurons pattern the forebrain and spinal cord in such a complementary way to each other to produce the overall specific 5-HT axon densities? While answers to these questions are still largely unknown, some insights into molecules that impact 5-HT neuron axon development are reviewed in the next developmental section of this chapter “Known molecules affecting 5-

HT neuron axon development”.

DEVELOPMENT OF THE SEROTONERGIC AXON PROJECTION SYSTEM

Development of ascending projection system

Lidov and Molliver were the first to provide the detailed description of how ascending 5-HT axons develop in the forebrain (Lidov and Molliver, 1982). They described the progression of 5-HT immunolabeled axon growth in the rat forebrain. They defined 3 distinct stages of 5-HT axon development: i. Primary

20 axon pathway formation; ii. Selective routing through pre-existing axon tracts; and iii. Terminal target arborization.

The following detailed timeline description of 5-HT axon innervation is based on my observations in the mouse. The order of consecutive axon developmental steps in the mouse were entirely consistent with Lidov and

Molliver’s description in the rat, however the timing is slightly shifted in the mouse. Lidov and Molliver, 1982 should be consulted for reference of the precise embryonic timeline of 5-HT axon development in the rat.

Primary axon pathway formation

Initial axon elongation from 5-HT neurons occurs simultaneously with the onset of 5-HT synthesis from E10.5-12.5, while they are still migrating to their final raphe positions (Lidov and Molliver, 1982; Wallace and Lauder, 1983;

Hawthorne et al., 2010). All newly formed ascending 5-HT axons orient rostrally towards the forebrain and travel bilaterally through the medial forebrain bundle

(MFB). Primary axon pathway formation is defined as the extension of 5-HT axons through this first distinct pathway of the MFB (Lidov and Molliver, 1982)

(Figure 6A). The boundaries of the MFB have already been defined by other ascending and descending fibers that connect the limbic forebrain, midbrain, and hindbrain (Lidov and Molliver, 1982). 5-HT axons fasciculate onto themselves in smaller tight bundles as well as fasciculate onto other axons within the MFB as they emanate from the various rostral raphe nuclei and travel through this pre- existing MFB tract. The MFB continues down to the basal forebrain headed

21 rostrally towards the septum. The intrinsic molecular determinants of 5-HT axon initiation are unknown, however some evidence for molecules that impact orientation of nascent 5-HT axons is described later in this Chapter in the “Wnt planar cell polarity pathway” section.

Selective routing

From the MFB, 5-HT axons enter several major pre-existing fiber tracts including: fasciculus retroflexus, mammillotegmental tract, stria medullaris, external capsule, fornix, supracallosal stria, and cingulum bundle (Lidov and

Molliver, 1982). The fasciculus retroflexus (fr) is the first selective pathway in which a choice few 5-HT axons start to exit the MFB in a dorsal trajectory around

E13.5 (Figure 6B). The fasciculus retroflexus tract is located medially just beyond the bend of the mesencephalic flexure of the embryonic forebrain and axons within this tract grow towards the habenula (Lidov and Molliver, 1982)

(Figure 6B). It is unknown whether these axons that enter the fasciculus retroflexus are collaterals or single axons turning away from the MFB. The main bundle of 5-HT axons within the MFB continues ventrally towards the base of the forebrain and then levels out by E14.5 to travel rostrally straight along the basal forebrain towards the septum. It is important to note that not all 5-HT neurons initiate axon elongation simultaneously, as 5-HT neurons are born and begin to produce axons over a span of 2-3 days (Hawthorne et al., 2010). The overall result is a progressive increased number of axons that fill the MFB as well as all selective routes as development proceeds (Lidov and Molliver, 1982).

22 By E14.5, 5-HT axons within the MFB are found at the level of the hypothalamus and appear to join the mamillotegmental tract and proceed rostrally. Sparse 5-HT axons individually leave the MFB to turn dorsally towards the ventral and medial thalamus as well as stray ventrally to innervate regions of dorsal hypothalamus. These 5-HT axons do not appear to arborize yet at this early E15-16 stage in the thalamus or hypothalamus. In coronal sections, a small number of 5-HT axons can be seen crossing the midline at the base of the forebrain. Around E15-16, a group of 5-HT axons turn laterally away from the medial MFB tract into the lateral telencephalon to innervate the lateral cortex

(Figure 6C, MFBL). The medial group of 5-HT axons continue straight through the MFB (Figure 6C). As soon as axons within the MFB pass the hypothalamic region, before they reach the septum, a prominent group of axons enter the stria medullaris (sm) tract which feeds dorsally into the most anterior thalamus and arches back towards the habenula (Figure 6C).

Between E16-E17, 5-HT axons reach the septal area where they all leave the MFB and either: i. fill lateral olfactory tracts (ot) to reach the olfactory bulb; ii. enter external capsule lateral to striatum to innervate the amygdala and other cortical areas; iii. turn dorsally, then posteriorly into fornix-fimbria pathway, located beneath the corpus callosum, to reach the hippocampus; iv. turn dorsally, then posteriorly into the marginal zone (mz) of the medial cortex; or v. turn dorsally, then posteriorly into the supracallosal stria (SCS) and cingulum bundles

(cg), directly adjacent to the dorsal aspect of the corpus callosum, to reach all cortical areas from prefrontal cortex to caudal occipital cortex (Figure 6D). Some

23 of these axons in the SCS and cingulum grow posteriorly along the entire extent of the corpus callosum and then turn ventrally into the hippocampus. All major 5-

HT pathways have been established by E17, however all tracts become more prominent as development proceeds. As discussed in Chapter 3, 5-HT axons will continue to fully fill SCS, cingulum, and the marginal zone of the cortex up to approximately postnatal day 3 (P3) in the mouse.

At E18, 5-HT axons can be found loosely scattered throughout the thalamus and dorsal hypothalamic regions, presumably the early beginning of arborization, while all other areas are still un-arborized at this time. 5-HT axons in the SCS and cingulum can be found as far reaching as the most posterior region of the developing corpus callosum at E18. Also, at this stage, 5-HT axons of passage in the cortex can be clearly seen in coronal sections in a bilaminar distribution within the marginal zone (located at the outer pial cortical surface) and the intermediate zone (located at the base of the cortical plate). This bilaminar distribution can be seen in both medial and lateral cortex. These 5-HT axons will later turn into intermediate cortical layers to arborize.

Terminal target arborization

5-HT axon arborization ensues during the following first 4-5 postnatal weeks of age in the mouse (Lidov and Molliver, 1982; Maddaloni, et al., 2017).

Some arborization begins as early as E18 in the mouse thalamus as described in the previous section. Arborization consists of axon branching, growth/extension, and tiling (the spacing of arbors relative to each other) to fill terminal target

24 regions with the appropriate densities of axon processes. Maddaloni et al., 2017 provide an informative postnatal timeline describing the increasing 5-HT arbor densities in many forebrain regions of the mouse. Generally speaking, the more proximal target regions to 5-HT cell bodies begin to arborize first. For example, areas of the thalamus, including the geniculate nuclei and midline thalamic nuclei are some of the first areas in which 5-HT axons arborize, while occipital cortical lobes, which are the most distal regions that 5-HT axons reach in the forebrain, arborize later. Interestingly, not every target region reached by 5-HT axons experience 5-HT axon arborization immediately upon initial target innervation. In many target regions of the brain, 5-HT axons delay their arborization and seem to parallel the onset of arborization with the maturation of the target region (Lidov and Molliver, 1982). For example, 5-HT axon innervation of the striatum, suprachiasmatic nucleus, ventromedial hypothalamus, and middle layers of the parietal and occipital cortex occurs long after these 5-HT axons have reached these structures (Lidov and Molliver, 1982). It is also noted that the first appearing 5-HT axon arbors coincide with identifiable mature target regions by

Nissl stain. For example, in the hippocampus, the first 5-HT axons to arborize do so in two distinct layers, above and below the maturing pyramidal cell layer that is recently prominent by Nissl stain at P3 in the rat (Lidov and Molliver, 1982).

This is also the first area of the hippocampus where 5-HT axon arbors are observed in the mouse. In addition, dense 5-HT innervation in the basolateral amygdala begins just as the cytoarchitecture begins to become apparent in the rat (Lidov and Molliver, 1982). Due to these observations, it is speculated that

25 extrinsic cues from maturing tissue at terminal target regions may be necessary to signal 5-HT axons to arborize (Lidov and Molliver, 1982; Kiyasova and

Gaspar, 2011).

Known molecules affecting 5-HT neuron ascending axon development

What are the intrinsic or extrinsic cues that endow 5-HT neurons to grow such long distances, guide their axons to appropriate target regions, and instruct axons to profusely arborize? The majority of data that give some insight to these questions come from several independent studies, most of which investigated different germline knock out mice, which identified specific molecules that impact

5-HT axon development to the forebrain. These molecules include guidance receptors, cadherins, growth cone molecules, and 5-HT itself.

Wnt planar cell polarity pathway

The Wnt planar cell polarity (PCP) pathway is an evolutionarily conserved pathway that plays important roles in a variety of cellular processes during development including migration, organization of cell position within a tissue, proliferation, and polarity of many cell types in the body, including neurons in the brain. This pathway involves a family of ligands called Wnt , which can bind to a family of receptors named ’s. Activation of Frizzled receptors results in downstream activation of Dishevelled proteins leading to intracellular signaling cascades (Devenport, 2014). In addition, the Vangl family of receptors

26 as well as Celsr proteins are important players in PCP pathway in many different tissue types (Devenport, 2014).

5-HT neurons express several proteins involved in the PCP pathway including several Wnt’s and Frizzled’s, as well as Vangl2 and Celsr3

(Fenstermaker et al., 2010). The Wnt pathway has been shown to control both the organization and polarity of 5-HT neurons (Kiyasova and Gaspar, 2011).

Studies using genetic knock out mouse models have identified specific components of this pathway as important for 5-HT neuron axon guidance. For example, in each Frizzled3-/-, Vang2-/-, and Celsr3-/- mice, rostral 5-HT neurons project their axons in incorrect directions during initial axon outgrowth at E12.5

(Fenstermaker et al., 2010). In all of these mutants, many rostral 5-HT neurons send inappropriate projections laterally and towards the spinal cord instead of their normal anterior course towards the forebrain. In Frizzled3-/- and Vang2-/- mice, caudal 5-HT neurons are also affected and project their axons in random directions. Due to the known rostro-caudal gradient of Wnt5a in the midline of the developing hindbrain, these researchers used an “open-book” embryonic hindbrain explant culture where they placed Wnt5a-coated beads along the midline. These coated beads were sufficient to misguide wild-type 5-HT axons towards the Wnt5a ligand, thereby supporting the role for Wnt signaling in anterior-posterior 5-HT axon guidance and orientation (Fenstermaker et al.,

2010).

27 Slit-Robo signaling

Slit and Robo proteins are expressed by many neuronal types in the brain, including 5-HT neurons. The Slit family of proteins are secreted and bind the family of Robo receptors. This signaling cascade is best known for regulating axon guidance mainly through repulsion mechanisms (Blockus and Chedotal,

2016). For example, Slit-Robo signaling is well known for its role in mediating the axonal guidance of corpus callosal axons at the midline during development (Shu et al., 2003). Slit1 and Slit2 have also been shown to play important roles in preventing improper midline crossing of 5-HT axons as they reach the basal telencephalon during development. Slit2-/- and Slit1::Slit2-/- double knock out mice exhibited abnormal ventral displacement and midline crossing, respectively, within the MFB at E14.5 (Bagri et al., 2002). However, the integrity of the pre- existing MFB was at least partially disturbed by displaced dopaminergic axons, and therefore it is unclear if 5-HT neurons were intrinsically impacted by loss of

Slit signaling.

EphrinA5

Different subsets of 5-HT neurons differentially express a variety of Eph receptors and their ephrin ligands that are known to act as short-range axon guidance molecules in other cell types (Wylie et al., 2010; O’Leary and

Wilkinson, 1999; Klein and Kania, 2014). For example, Eph-ephrin signaling is well known to play a role in establishing topographic maps in somatosensory cortex and influence auditory nuclei connectivity (Prakash et al., 2002; Miko et

28 al., 2007). Teng et al. 2017 demonstrate the importance of EphrinA5 signaling in

5-HT neuron projections to several target regions of the forebrain. They show that the receptor EphA5 is selectively expressed in dorsal raphe (DR) versus median raphe (MR) 5-HT neurons. Further, they confirm that application of ephrinA5 ligand can induce growth cone collapse of DRN neurons in culture.

Their analysis of ephrin A5 (Efna5) knockout mice showed that DR neurons inappropriately innervate the olfactory bulb, ventromedial hypothalamus, and suprachiasmatic nucleus target regions, along with MR neuron innervation, leading to an overall increased density of 5-HT fibers (Teng et al., 2017). These distinct brain structures highly express the ligand ephrinA in vivo, leading to the conclusion that the high expression of EphA5 in DR neurons normally induces a repulsion to these target regions. These results demonstrate that Eph-ephrin signaling provides a mechanism for region-specific targeting of distinct 5-HT neuron subnuclei.

Gap43

Gap43, or growth-associated protein 43, is a membrane associated protein that is widely expressed throughout the brain, typically concentrated in the axon growth cone during development to direct axon growth (Strittmatter et al., 1995; Donovan et al., 2002). In retinal ganglion cells (RGC), GAP-43 functions to control decisions for midline crossing at the optic chiasm, but not for extension of neurites, as Gap43-/- mice exhibit normal RGC axon growth up to the optic chiasm (Strittmatter et al., 1995). Gap43-/- mice also display aberrant

29 development of whisker barrels in cortical fields thought to be caused by the failure of thalamocortical axons (TCAs) to extend to specifically cortical layer IV

(Maier et al., 1999). Gap43 expression in the brain was later found to be critical for 5-HT axon innervation to the cortex and hippocampus in both Gap43+/- and

Gap43-/- mice, although heterozygous animals had a much milder phenotype

(Donovan (not me) et al., 2002). This result, however, is confounded by the fact that Gap43+/- and Gap43-/- mice have major defects of the corpus callosum (CC) and hippocampal commissure (HC) (Shen et al., 2002). The corpus callosum completely fails to join at the midline and the hippocampal commissure is never formed in Gap43-/- mice (Shen et al., 2002). Growing 5-HT axons innervate both the cortex and hippocampus by major routes called the cingulum bundles and supracallosal stria (SCS) directly adjacent to the corpus callosum at the midline, as well as through routes via the HC. The fact that these major brain structures are not formed in Gap43-/- mice and only partially formed in Gap43+/- mice, may be the major contributing factor of the 5-HT axon innervation failure to cortex and hippocampus.

STOP (Map6)

Microtubule-associated protein (Map6), otherwise known as ‘stable tubules only polypeptide’ (STOP), as its name implies, functions to stabilize in several neuronal types. The STOP protein is required for not only the stabilization and maintenance of microtubules in neurons, but also for the formation of new neurites during development (Guillaud et al., 1998). Adult

30 STOP-/- mice exhibited decreased numbers of SERT-labeled fibers in several regions of the forebrain including cingulate cortex, hippocampus and basal ganglia, but not other regions analyzed (Fournet et al., 2010). However, it is unclear whether this decrease in detection of SERT-labeled fibers is due to decreased 5-HT axon innervation or actually decreased SERT levels in 5-HT neurons that project to these areas. These mice also displayed increased 5-HT levels as well as increased 5-HT1A and SERT levels in the brainstem, speculated to be caused by an accumulation of these proteins, and not an overabundance of 5-HT neurons themselves (Fournet et al., 2010). Therefore, it may be that SERT was not appropriately transported to distal axon tips. Analysis of these mice was limited to adult mice, and only SERT levels were analyzed, therefore it is also unknown whether these results were due to a failure of innervation or a failure 5-HT axon extension during development.

Pcdhac2

Protocadherin (Pcdh) genes are defined as either non-clustered, meaning their genes are distributed differentially in the genome, or clustered. There are three distinct gene clusters of Pcdhs that are named either alpha, beta, or gamma. These clusters are differentially regulated by specific promoter choice of regulatory factors followed by differential pre-mRNA splicing to produce about 70

Pcdh genes in (Chen et al., 2012). All protocadherins are membrane associated with a variable intracellular signaling domain, a transmembrane domain, and a variable extracellular adhesion domain. Unlike classical cadherins

31 whose intracellular signaling domain directly attaches to cytoskeletal elements through , protocadherin intracellular domains do not directly attach, however can influence cytoskeletal dynamics through other signaling mechanisms (Chen and Maniatis, 2013). Clustered Pcdh genes are primarily expressed by neurons during development in various isoform combinations depending on the neuron type (Chen and Maniatis, 2013). Pcdh genes have been shown to play roles in self-avoidance and self-recognition to aid in synaptic connectivity in the developing brain. Pcdh proteins are found in any neuronal compartment including axons, dendrites, soma, as well as in developing growth cones and synapses (Chen and Maniatis, 2013). Pcdh proteins can form either homo- or hetero- dimers in cis and mainly have trans-homophilic interactions to allow for self-recognition and ultimately repulsion allowing for tiling to occur

(Chen and Maniatis, 2013).

The Protocadherin alpha (Pcdha) gene cluster is highly expressed in raphe 5-HT neurons at both embryonic as well as adult stages (Katori et al.,

2009). Genetic deletion of the Pcdha constant region (CR) (PcdhaΔCR/ΔCR), which encodes the cytoplasmic tail common to all Pcdha proteins, resulted in abnormal serotonergic projection patterns throughout the forebrain of mice, specifically due to altered arborization (Katori et al., 2009). PcdhaΔCR/ΔCR mice displayed a sparser axon pattern in many regions of the forebrain including the cortex, as well as very dense aggregations of 5-HT fibers in many regions including the hippocampus molecular layer, thought to be the result of innapproprate 5-HT axon tiling in the arborization stage (Katori et al., 2009). Deeper analysis of the

32 role of the Pcdha cluster was followed up in a second paper, Katori et al., 2017, where they specifically identified Protocadherin alpha c2 (Pcdhac2) as the predominately expressing isoform of the Pcdha gene cluster in 5-HT neurons.

PcdhaΔac2/Δac2 mice also displayed a very similar 5-HT axon arborization defect in the forebrain compared to PcdhaΔCR/ΔCR mice, indicating that this isoform is the predominate Pcdha gene that controls 5-HT axon arborization (Katori et al.,

2017). In contrast, PcdhaΔac2/Δac2 showed no abnormal 5-HT axon patterns in the spinal cord.

The Pcdhac2 gene was further investigated using a 5-HT neuron conditional approach by another group Chen et al., 2017. This group found, by

TRAP-seq methods, that Pcdhac2 is the predominately expressed alpha isoform in serotonin neurons. Conditional deletion of Pcdhac2 in 5-HT neurons using a

Slc6a4-Cre driver resulted in abnormal clumping of 5-HT axons in the molecular layer of the hippocampus and other areas after postnatal day (P)5, similar to that found in Katori et al. in PcdhaΔac2/Δac2 mice (Chen et al., 2017; Katori et al., 2017).

This study confirmed an intrinsic role for Pcdhac2 in the arborization of 5-HT neurons. Further, this group injected conditional Cre-dependent Brainbow- reporter aneno-associated viruses (AAVs) into the DRN or MRN to label 5-HT neurons and trace individual axons with different fluorescent labels. This technique allowed them to conclude that a majority of fiber clumps occurred between differentially labeled axons originating from different serotonergic neurons, supporting the idea that 5-HT neurons require Pcdhac2 for homophilic repulsion during arborization. In addition, these Pcdhac2 conditional knock out

33 mice exhibit depressive-related behavioral phenotypes including increased immobility in forced swim test and enhanced contextual fear memory (Chen et al., 2017). These results together indicated that Pcdhac2 functions cell autonomously and postnatally within 5-HT neurons to appropriately tile arbors throughout the forebrain, and that depressive-related behaviors result from this altered wiring in absence of Pcdhac2 (Chen et al., 2017).

5-HT

It is well established that 5-HT itself is an important signaling molecule for the development of brain circuitry (Gaspar et al., 2003). In fact, during the developmental stages E15-P10, the 5-HT transporter (SERT) is widely expressed throughout the brain in many non-serotonergic cell types (Gaspar et al., 2003). These transient SERT-expressing cells have the ability to take up 5-

HT across the membrane and re-release 5-HT as a signaling molecule (Cases et al., 1998; Upton et al., 1999). One hypothesis for why this occurs during development is to regulate the amount of available 5-HT in the extracellular space in order to establish proper innervation patterns of multiple types of neurons of the brain (Gaspar et al., 2003). Indeed, SERT-knockout mice display defective thalamocortical axonal arbor patterns as well as abnormal retinal projections to the lateral geniculate nucleus, due to increased 5-HT in the extracellular space, supporting the concept that uptake of 5-HT by surrounding cells can regulate developing axonal projections (Upton et al., 1999; Salichon et al., 2001; Persico et al., 2003).

34 It wasn’t until 2013 that a group in Italy sought to identify whether 5-HT signaling was necessary for normal serotonergic axon development itself. This group generated a mouse line in which enhanced green fluorescent protein

(eGFP) was knocked into the genetic locus of Tph2, the responsible for synthesis of 5-HT, thereby creating a knock out mouse for Tph2 while simultaneously labeling 5-HT neurons and their axons with eGFP (Migliarini et al., 2013). Indeed, they found that near-complete loss of 5-HT caused 5-HT neuron circuitry formation defects to several brain target regions including a reduction of innervation to the paraventricular nucleus of the thalamus and suprachiasmatic nucleus, and an increase in innervation to parts of the nucleus accumbens and hippocampus (Migliarini et al., 2013). These axonal patterning alterations were the result of specifically 5-HT terminal arborization defects, as all selective 5-HT axonal pathways were reported unperturbed (Migliarini et al.,

2013).

While all of the molecules discussed above impact 5-HT axon development in some way, none of the genes investigated in the various germline knock out mice are solely expressed by 5-HT neurons (except 5-HT itself). Therefore, it is unclear whether these molecules are acting intrinsically in

5-HT neurons or extrinsically in other tissues to impact 5-HT axon development.

In addition, the majority of these studies used the endogenous markers 5-HT or

Sert to label 5-HT axons. It is unclear if knock out of any of the molecules described above impacted this endogenous expression, which would obscure

35 interpretations of 5-HT axon development, as this was not investigated in any detail. A better approach would be to conditionally label 5-HT axons with a fluorescent marker to easily and unambiguously label 5-HT axons in various knock out mice. In the case of Gap43-/- mice, the reports of major brain structure abnormalities may account for the majority of 5-HT axon failure to innervate the cortex and hippocampus, making it necessary to further pursue conditional knock out mice to implicate Gap43 in 5-HT axon outgrowth in vivo. In addition, all studies focused on ascending 5-HT axon innervation to the brain, while descending 5-HT axon innervation was left unreported. The only study that directly linked an intrinsic function in 5-HT axon development was the conditional knock-out for Pcdhac2, revealing its role in 5-HT axon arborization (Chen et al.,

2017). However, the exact mechanisms of Pcdhac2 action has not yet been elucidated. Interestingly, Pcdhac2 impacts ascending 5-HT axon development at the arborization stage in specific forebrain target regions and does not impact any descending 5-HT axon growth as Pcdhac2-KO mice show no axon phenotype in the spinal cord (Katori et al., 2017). This may indicate that different guidance programs are involved in rostral versus caudal 5-HT axon development. Still, there is a major gap in understanding the transcriptional control over 5-HT axon growth and guidance, as it is likely that transcription factors could play important roles in organizing the expression of growth and guidance molecules that are responsible for carrying out this prolonged 5-HT axon growth that enables complex integration into brain circuitry (Kiyasova et al.,

2011).

36 Development of descending projection system

Compared to the numerous studies concentrating on ascending 5-HT axon development, few studies have focused on the descending 5-HT axon projection system. Studies describing the developmental progression of 5-HT descending axons have been performed in rats and mice (Rajaofetra et al., 1989;

Ballion et al., 2002). Similar to ascending projections, descending projections in the mouse follow analogous stages of axon growth compared to the rat, with the timing of these stages slightly altered (Ballion et al., 2002). Descending projection development is much more straightforward than forebrain ascending projection development as there are far fewer pathways by which descending axons travel through to reach terminal target regions of the spinal cord.

In the mouse, 5-HT caudal neurons project their axons down the ventral and lateral funiculi of the white matter tracts starting at E12.5 at the rostral-most cervical levels (Ballion et al., 2002). Similar to ascending projections, descending

5-HT axons continually fill white matter tracts in a progressively denser fashion as 5-HT neurons are born over a span of several days and therefore begin to send axons over this span of time. These 5-HT axon projections proceed caudally down the white matter tracts in a rostro-caudal progression with the first

5-HT axons beginning to reach thoracic levels by E14.5 in the mouse. These 5-

HT axons then reach lumbar levels by E16.5 (Ballion et al., 2002). By E16-17, 5-

HT axons begin to invade ventral and intermediate gray matter, with more 5-HT axons invading gray matter at cervical levels compared to lumber levels, most likely because some 5-HT axons of passage have yet to reach the lumbar levels

37 at these early stages. Dorsal gray matter does not begin to be invaded by 5-HT axons until postnatal stage (P0) (Ballion et al., 2002). By P3, there are already dense 5-HT innervations throughout the gray matter, again with slightly more 5-

HT axon arbors in cervical gray compared to lower sacral levels. By P10, densities of 5-HT axons in the mouse ventral horn is described as similar to the

P10 rat spinal cord, and already similar to the adult mouse pattern with higher densities of 5-HT arbors in dorsal horn layer I and II and ventral horn (Rajaofetra et al., 1989; Ballion et al., 2002). The full adult 5-HT axon pattern occurs by approximately in the rat (Rajaofetra et al., 1989).

PET-1

Gene/protein expression

Pet-1, or Phenochromocytoma ETS (E26 transformation-specific) domain transcription factor-1, was initially discovered in adrenal chromaffin-derived PC12 cell lines and its expression has been confirmed in many different tissue types in vivo such as enterochromaffin cells, pancreatic islets, adrenal cortex, hematopoietic stem cells, uterine buds of the kidney, as well as the brain

(Fyodorov et al., 1998; Ohta et al., 2011; Pelosi et al., 2014; Wang et al., 2010;

Wang et al., 2013). Interestingly, Pet-1 expression in the brain is completely restricted to serotonergic neurons (Hendricks et al., 1999). This extremely specific expression pattern allowed for the generation of both a Cre recombinase transgenic mouse line that is driven by the Pet-1 enhancer region (ePet) to specifically access 5-HT neurons as well as an ePet-enhanced yellow fluorescent

38 protein (ePet-EYFP) transgenic mouse line (Scott et al., 2005). Both of these lines are used in the following studies presented in Chapter 2 and 3.

Protein structure

Pet1 is an ETS domain transcription factor of the ERG (ETS-related gene) subfamily, which is highly conserved across different species (Cooper et al.,

2015; Wei et al., 2010; Hollenhorst et al., 2011). Pet1 contains an ETS domain, as its name implies, as well as an alanine rich c-terminal domain thought to mediate repressor activity (Fyodorov et al., 1998; Wang et al., 2013; Maurer et al., 2003). However, Pet1 is also a known transcriptional activator for many genes (Wyler et al., 2016).

Function

ETS transcription factors are generally found to play important roles in multiple aspects of cellular development such as migration, proliferation, differentiation, and apoptosis. Pet1 is no exception as it is critical for the development and function of hematopoietic stem cells, pancreatic islets, as well as 5-HT neurons (Fyodorov et al., 1998; Ohta et al., 2011; Pelosi et al., 2014;

Wang et al., 2010; Wang et al., 2013). In 5-HT neurons, Pet1 is continuously expressed throughout life and found to be critical for the differentiation, migration to distinct nuclei positions, expression of presynaptic receptors, and maintenance of 5-HT identity (Liu et al., 2010; Wyler et al., 2016). In Pet-1 null animals, around

70-80% of 5-HT neurons fail to fully differentiate due to dysregulation of genes

39 Tph2 and Ddc, which are necessary for 5-HT synthesis (Hendricks et al., 2003;

Liu et al., 2010). Adult conditional knock out of Pet1 also results in a failure of

~50% of 5-HT neurons to produce Tph2 and synthesize 5-HT, thereby determining its role in maintaining 5-HT neuron identity into adulthood (Liu et al.,

2010). More recent studies provided RNA-sequencing datasets generated from flow sorted 5-HT neurons of either Pet1-/- or control mice (Wyler et al., 2016). It was found that Pet1 controls the expression of a host of genes involved in neuron maturation. Interestingly, Pet1 conditional targeting experiments performed on early postnatal pups revealed that Pet1 switches transcriptional targets from early genes that play roles in 5-HT synthesis, to later expressed genes involved in maturation of 5-HT neurons (Wyler et al., 2016). Additionally, chromatin immunoprecipitation followed by RNA sequencing (ChIP-seq) of 5-HT neurons collected from a transgenic mouse line expressing the mycPet1 transgene revealed that many of these genes are direct transcriptional targets of

Pet1 (Wyler et al., 2016).

LMX1B

Gene/protein structure

Lmx1b is a LIM- homeodomain (HD) transcription factor within the LIM- gene family in the “Lmx” group (Hobert and Westphal, 2000). As the name implies, Lmx1b contains two protein interacting zinc-fingers called LIM domains as well as a homeodomain that allows for direct DNA binding (Hobert and Westphal, 2000). The LIM domain has the ability to bind many co-factors,

40 including bHLH factors and other transcriptional regulators as homo- or hetero- dimers, endowing them to control a host of developmental events (Johnson et al.,

1997). Lmx1b has a paralog called Lmx1a, that shares 64% homology of their amino acid configuration, including 100% homology of their DNA binding homeodomain (Hobert and Westphal, 2000).

Lmx1b in peripheral tissues

Lmx1b is a pleiotropic gene expressed in the dorsal mesenchyme of developing limb buds, bone, eye, kidney, and brain tissues (Kania et al., 2000;

Chen et al., 1998; Zhao et al., 2006). In the periphery, the chicken ortholog of mouse Lmx1b, Lmx1, is essential for dorsal-ventral patterning of limbs of chick embryos (Vogel et al., 1995; Riddle et al., 1995; Chen et al., 1998a). In the developing mammalian limb, Lmx1b is expressed by limb mesenchymal cells and controls the expression of EphrinA which guides lateral motor column (LMC) neuron axons, that express EphA receptors, to a dorsal trajectory within the limb

(Kania et al., 2000; Kania and Jessell, 2003). Lmx1b was also found to be essential for proper kidney development as homozygous Lmx1b mutant mice present with irregular glomerular development (Chen et al., 1998). Interestingly, both limb patterning defects as well as kidney abnormalities occurs in human patients with a disease called Nail Patella Syndrome (NPS) (Dreyer et al., 1998).

Further, NPS patients have been shown to have heterozygous mutations in either the LIM domains or homeodomains of LMX1B (Dai et al., 2009; Bongers et al., 2008). Further investigation of specific human mutations in mice is necessary

41 to directly link these mutations in LMX1B to defects observed in NPS patients and further understand the pathology.

Lmx1b in the CNS

Lmx1b is expressed in multiple cell types in the CNS including dopaminergic (DA), noradrenergic (NE), and serotonergic (5-HT) neurons of the brain, as well as specific interneuron subtypes within the spinal cord (Filippi et al.,

2007; Ding et al., 2003; Smidt et al., 2000; Matise and Joyner, 1997). In each neural type, Lmx1b plays crucial developmental roles as described through the investigation of either Lmx1b null mice (Lmx1b-/-) or conditional knock out mice.

In Lmx1b-/- mice, research has been limited to embryonic timepoints due to the perinatal lethality of the null allele, thought to be caused primarily by kidney failure (Chen et al, 1998a). Therefore, to further study Lmx1b in the context of postnatal maturation and adult maintenance, conditional knock out mice have been generated (Zhao et al., 2006; Song et al., 2011).

Lmx1b in midbrain-hindbrain patterning

Lmx1b expression can be detected at E9.5 specifically within the isthmic organizer (Guo et al., 2007). During embryonic brain development, the isthmic organizer is essential to define regionalization of the CNS. The isthmic organizer functions to define midbrain-hindbrain boundaries (MHB) by secretion of FGF8 and WNT1. Perturbation of either FGF8 or WNT1 signaling results in a failure of development of the tectum and cerebellum (Chi et al., 2003). Addition of FGF8

42 signaling by FGF8-coated beads is sufficient to induce tectum formation as well as cerebellar formation in the chick embryo (Martinez et al., 1999). Lmx1b-/- mice revealed both FGF8 and Wnt1 dependence on Lmx1b expression at this critical developmental timepoint of isthmic organizer activity (Guo et al., 2007). As a result of disrupted isthmic organizer activity, increased cell death was observed which led to severe defects in tectum and cerebellar size observed in Lmx1b-/- mice, thus highlighting Lmx1b’s critical role in mid-hindbrain patterning (Guo et al., 2007).

Lmx1b in midbrain Dopaminergic neurons

In midbrain dopaminergic (mDA) neurons, Lmx1b along with its paralog

Lmx1a, function cooperatively in mDA neuron differentiation and proliferation

(Yan et al., 2011). In Lmx1b-/- mice, DA neurons largely fail to become specified, however this is actually due to defects in midbrain-hindbrain organization, rather than intrinsic loss of Lmx1b itself (Smidt et al., 2000; Guo et al., 2007). This is supported by the fact that conditional knock out of Lmx1b is not sufficient to impact specification or maintenance of mDA neurons (Yan et al., 2011). In contrast, Lmx1a mutant embryos have a 46% reduction in number of mDA neurons (Yan et al., 2011; Ono et al., 2007). The residual DA neurons found in either Lmx1b or Lmx1a individual conditional knock out mice are presumably the result of compensation for one another. Only double Lmx1a/b mutant mice exhibit a very severe loss of mDA neuron number (Yan et al., 2011). Loss of both

Lmx1a/b results in a loss of tyrosine hydroxylase (TH), and transcription factors

43 Nurr1 and Pitx3 (Yan et al., 2011). In addition, Lmx1a/b conditional double knock out mice show a mild axon phenotype with deficiencies in mDA axon innervation to the dorsal striatum, but do not show defects in other terminal target regions

(Chabrat et al., 2017).

Lmx1b in Noradrenergic neurons

Much less is known about Lmx1b function in noradrenergic (NA) neurons.

Studies in zebrafish have identified Lmx1b.1 expression in the noradrenergic population in both the locus coeruleus and the medulla oblongata (Filippi et al.,

2007). Its ortholog lmx1b.2 in zebrafish is not found to be expressed in any catecholaminergic group (Filippi et al., 2007). Zebrafish embryos treated with lmx1b.1/2 morpholinos do display a reduction in the number of noradrenergic neurons in the area postrema but not the locus coeruleus (Filippi et al., 2007). In

Lmx1b-/- mice, it is noted that the locus coeruleus noradrenergic center is disrupted, as β-dopamine hydroxylase expression is lost (Ding et al., 2003).

Although Lmx1b is expressed in NA neurons in the mouse, no conditional knock- out mice have been studied so far to probe a possible further role for Lmx1b in

NA neuron specification and maintenance.

Lmx1b in Serotonergic neurons

Unlike DA neurons, 5-HT neurons do not express Lmx1a (Wyler et al.,

2016). Serotonergic fate is entirely dependent on Lmx1b expression as no 5-HT neurons are able to differentiate in Lmx1b-/- mice (Ding et al., 2003). Lmx1b expression alone however is not able to induce serotonergic fate ectopically. It is

44 the combination of Lmx1b, Pet-1, and another transcription factor called Nkx2.2 expression that is sufficient to induce ectopic 5-HT neuron specification in the ventral spinal cord of the chick embryo (Cheng et al., 2003). Lmx1b controls the specification of 5-HT neurons through regulating the expression of downstream genes necessary for the production and transport of 5-HT including Tph2, Sert, and Vmat2 (Cheng et al., 2003). Further, it was found that Lmx1b is not only necessary to initially induce these genes, but also to maintain their expression during maturation and into adulthood (Zhao et al., 2006; Song et al., 2011).

Behavior studies performed on conditional knock out ePet-Cre transgenic mice, that targets Lmx1b expression at E12.5, display phenotypes that correlate with low 5-HT levels in the brain including hyperactivity, enhanced contextual fear memory, increased pain sensitivity, respiratory irregularities, and disturbed sleep regulation (Dai et al., 2008; Hodges et al., 2009; Zhang et al., 2018; Zhao et al.,

2007a; Zhao et al., 2007b).

While Lmx1b has been shown to be critical to establish serotonergic identity as evidenced by the regulation of the few genes mentioned above, nothing else is known of its likely regulation of additional genes. Also, because

Lmx1b is continually expressed in 5-HT neurons into adulthood, it is intriguing to investigate whether Lmx1b plays additional roles in 5-HT neuron maturation or maintenance. Or perhaps Lmx1b’s role is solely to establish 5-HT identity in development and maintain that identity throughout life.

45 In the following two Chapters, I investigate the intrinsic roles of two key transcription factors, Pet1 and Lmx1b, in 5-HT neuron maturation. In Chapter 2, we sought to investigate whether Pet1 was required for the expression of key identity genes in all 5-HT neurons by examining the expression of Htr1a, the serotonin autoreceptor, as well as Oct3, the low-affinity high capacity transporter of 5-HT, in mice that lack Pet1. Thereby, determining whether all serotonergic neurons require Pet1 for their full neurochemical identity. In Chapter 3, we further probe Lmx1b’s role beyond regulation of the few 5-HT identity genes it has been shown to regulate (Zhao et al., 2006). Due to the continual expression of Lmx1b throughout the maturation phase in 5-HT neurons, we aimed to understand whether this terminal selector-type transcription factor was responsible for further maturation features of 5-HT neurons, such as the development of their expansive axonal systems.

46 Figure 1

Adapted from Wyler and Donovan et al., 2015

47 Figure 1. Enzymatic steps of 5-HT synthesis. Tph2, BH4, and Aadc are all necessary for the synthesis of serotonin (5-HT). Schematic depicts genes involved in BH4 de novo synthesis. Tph2, Tryptophan hydroxylase 2; Aadc

(Ddc), aromatic L-amino acid decarboxylase; Gfrp (Gchfr), GTP cyclohydrolase I feedback regulator; Gtpch (Gch1), GTP cyclohydrolase; Ptps (Pts), 6-pyruvoyl- tetrahydropterin synthase; SR (Spr), ; Akr1c3, aldo-keto- reductase family 1 member 3; Akr1b1, aldo-keto-reductase family 1 B1; CR

(Cbr1), carbonyl reductase; Dhfr ; Dhpr (Qdpr), dihydropteridine reductase; Pcd (Pcbd1, Pcbd2), pterin-4 alpha-carbinolamine dehydratase. Dashed lines indicate non-enzymatic steps.

48 Figure 2

Adapted from Deneris and Wyler, 2012

49 Figure 2. Transcription factor regulatory network that defines serotonergic identity. Schematic shows the transcription factors that are required to define a

5-HT progenitor as well as the 5-HT post mitotic precursor in the mouse. The three transcription factors, Gata3, Lmx1b, and Pet1 are all induced in parallel and are all required for the expression of at least some of the downstream genes necessary for 5-HT synthesis (Tph2, Ddc, Gch1, Qdpr), reuptake (Slc6a4,

Slc22a3), vesicular transport (Slc18a2), and metabolism (Maoa, Maob). Ovals/ rounded boxes indicate transcription factors and rectangles indicate terminal effector genes. Arrows indicate loss of gene expression in some or all 5-HT neurons in absence of a particular transcription factor.

50 Figure 3

Figure adapted from Deneris and Wyler, 2012

51 Figure 3. Cytoarchitecture of 5-HT neurons in the mouse brain. 5-HT neurons are localized in specific B-subnuclei (B1-B9) in the mid and hindbrain.

They are categorized in rostral and caudal groups. Rostral: DRN, dorsal raphe nucleus (B4, B6, B7); MRN, median raphe nucleus (B5, B8); supraleminscal nucleus (B9). Caudal: raphe pallidus (RPa, B1); raphe obscurus (ROb, B2); raphe magnus (RMg, B3).

52 Figure 4

A.

B. C.

D. TdTomato DAPI Merge

VL VL VL

53 Figure 4. 5-HT axon innervation patterns in the forebrain. (A) Sagittal section of the adult mouse forebrain. TdTomato-labeled 5-HT axons are shown by heatmap. Right, schematic depicting pathways of 5-HT axon innervation. Scale bar, 1000μm. OB, olfactory bulb; ctx, cortex; cg, cingulum; cc, corpus callosum; hipp, hippocampus; spt, septum; hyp, hypothalamus; thal, thalamus; sc, superior colliculus; MFB, medial forebrain bundle; DRN, dorsal raphe nucleus. Schematic

(right): ot, olfactory tract; cg/SCS, cingulum/supracallosal stria; fx, fornix; sm, stria medularis; fr, fasciculus retroflexus. (B) Confocal images of coronal brain sections show TdTomato+ 5-HT axons densely innervate the paraventricular nucleus of the thalamus (PVT). Scale bars, 50μm. 3V, third ventricle; PVT, paraventricular nucleus of the thalamus. (C) Confocal images of

TdTomato+ 5-HT axons show sparse innervation in lateral thalamic subregions, compared to other thalamic regions shown in (B). Scale bars, 20μm. (D) 5-HT axons densely innervate ventricular zones. VL, lateral ventrical. Scale bars,

50μm.

54 Figure 5

A.

B.

Adapted from Gagnon and Parent, 2014 https://doi.org/10.1371/journal.pone.0087709

55 Figure 5. Single labeled 5-HT neurons reveal a great diversity of 5-HT axon innervation. Sagittal view of 3D reconstructed axon projections of single biotin labeled 5-HT neurons of the rat DRN. The number of axon varicosities counted is displayed in parenthesis. Single 5-HT neurons can innervate multiple distal brain regions (A) or single, relatively shorter-range target regions (B). Scale bars,

1mm; inserts:10μm. DRN, dorsal raphe nucleus; Hip, hippocampus; LH, lateral hypothalamic area; LS, lateral septum; mfb, medial forebrain bundle; MRN, median raphe nucleus; PFC, prefrontal cortex; SN, substantia nigra; SUM, supramammillary nucleus; Th, thalamus; VP, ventral pallidum.

56 Figure 6

A. B. E12.5 E13.5

fr MFB MFB

C. D. E16 * E17

fr fr cg/SCS mz sm fx sm MFB MFB ot

MFBL MFBL

57 Figure 6. Embryonic time series of 5-HT axon selective routing in the mouse. (A) All 5-HT axons travel together in bilateral bundles through the MFB at E12.5. (B) By E13.5, the first selective route, fasciculus retroflexus (fr) can be seen. (C) At E16, more selective routes have formed including the stria medularis

(sm), and the lateral MFB route (MFBL). (D) All selective routes have been established by E17. MFB, medial forebrain bundle; fr, fasciculus retroflexus; sm, stria medularis; fx, fornix; cg/SCS, cingulum/supracallosal stria; ot, olfactory tract.

58

CHAPTER 2

PET-1 CONTROLS TETRAHYDROBIOPTERIN PATHWAY AND SLC22A3

TRANSPORTER GENES IN SEROTONIN NEURONS

By: Wyler SC*, Donovan LJ*, Yeager M, Deneris E.

*These authors contributed equally to this work

Reproduced with permission from ACS Chemical Neuroscience, 2015 July

15;6(7):1198-205 Copyright (2015). American Chemical Society http://pubs.acs.org/doi/abs/10.1021/cn500331z

59 Summary

Coordinated serotonin (5-HT) synthesis and reuptake depends on coexpression of TPH2, AADC (Ddc), and SERT (Slc6a4) in brain 5-HT neurons.

However, other gene products play critical roles in brain 5-HT synthesis and transport. For example, 5-HT synthesis depends on coexpression of genes encoding the enzymatic machinery necessary for the production and regeneration of tetrahydrobiopterin (BH4). In addition, the organic cation transporter 3 (OCT3, Slc22a3) functions as a low affinity, high capacity 5-HT reuptake protein in 5-HT neurons. The regulatory strategies controlling BH4 and

OCT3 gene expression in 5-HT neurons have not been investigated. Our previous studies showed that Pet-1 is a critical transcription factor in a regulatory program that controls coexpression of TPH2, AADC, and SERT in 5-HT neurons.

Here, we investigate rather a common regulatory program determines global 5-

HT synthesis and reuptake through coordinate transcriptional control. We show with comparative microarray profiling of flow sorted YFP+ Pet-1–/– and wild type 5-

HT neurons that Pet-1 regulates BH4 pathway genes, Gch1, Gchfr, and Qdpr.

Thus, Pet-1 coordinates expression of all rate-limiting enzymatic (Tph2, Gch1) and post-translational regulatory (Gchfr) steps that determine the level of mammalian brain 5-HT synthesis. Moreover, Pet-1 globally controls acquisition of

5-HT reuptake in dorsal raphe 5-HT neurons by coordinating expression of

Slc6a4 and Slc22a3. In situ hybridizations revealed that virtually all 5-HT neurons in the dorsal raphe depend on PET-1 for Slc22a3 expression; similar results were

60 obtained for Htr1a. Therefore, few if any 5-HT neurons in the dorsal raphe are resistant to loss of Pet-1 for their full neuron-type identity.

Introduction

Coexpression of unique gene combinations encoding numerous kinds of neuron-type and pan neuronal characteristics establishes the identity of different neurons (Hobert et al., 2010). However, the gene regulatory mechanisms controlling the acquisition of neuron-type identities are poorly understood. One key and obvious identity feature that distinguishes different neuron types is transmitter identity. Transmitter identity is commonly defined by the presence of a particular transmitter together with the coexpression of genes required for its synthesis, reuptake, and vesicular transport in specific neuron types (Deneris and Hobert, 2014). In the case of serotonin (5-HT) neurons, the gene products that typically define serotonergic transmitter identity are tryptophan hydroxylase 2

(TPH2), aromatic amino acid decarboxylase (AADC, gene symbol: Ddc), serotonin transporter (SERT, gene symbol: Slc6a4), vesicular monoaminergic transporter 2 (VMAT2, gene symbol: Slc18a2), and the 5-HT1A (gene symbol:

Htr1a) and 5-HT1B (gene symbol: Htr1b) autoreceptors.

A serotonergic gene regulatory network, comprising multiple interacting transcription factors, has been identified that coordinates expression of Tph2,

Aadc, Slc6a4, Slc18a2, Htr1a, and Htr1b in brain 5-HT neurons (Deneris and

Wyler, 2012). Transcription factors ASCL1, NKX2-2, and FOXA2 are required for specification of serotonergic progenitors in the ventral hindbrain. These factors subsequently activate a downstream transcription factor network comprising

61 GATA-2, INSM1, GATA3, LMX1B, Engrailed1/2, and PET-1, which acts in postmitotic serotonergic precursors to initiate 5-HT neuron-type differentiation

(Craven et al., 2004; Ding et al., 2003; Fox and Deneris, 2012; Hendricks et al.,

2003; Jacob et al., 2009; Pattyn et al., 2004). Germ line targeting of each of these factors results in aborted differentiation to varying extents depending on which factor is missing (Deneris and Wyler, 2012). For example, the Pet-1 ETS factor is required for coordinate expression of Tph2, Ddc, Slc6a4, Slc18a2,

Htr1a, and Htr1b in postmitotic serotonergic precursors as expression of each of these 5-HT identity genes is severely reduced in Pet-1–/– 5-HT neurons

(Hendricks, et al., 1999; Hendricks et al., 2003; Liu et al., 2010). In vivo chromatin immunoprecipitation and in vitro DNA binding assays have demonstrated that PET-1 coordinates expression of these serotonergic genes through direct binding to a common conserved ETS DNA binding site in their promoter regions (Jacobsen et al., 2011; Liu et al., 2010). Although PET-1 is expressed in what appears to be all brain 5-HT neurons, Tph2 continues to be expressed, albeit at reduced levels, in a subset of Pet-1–/– 5-HT neurons suggesting the presence of a PET-1 resistant subpopulation of 5-HT neurons

(Hendricks et al., 1999; Hendricks et al., 2003; Kiyasova et al., 2011).

In addition to the genes described above, other gene products play critical roles in 5-HT synthesis and transport and therefore are necessary for 5-HT to function as a transmitter. For example, in addition to TPH2 and AADC, 5-HT synthesis depends on coordinate expression of the enzymatic machinery catalyzing the production and regeneration of 6R-l-erythro-5,6,7,8-

62 tetrahydrobiopterin (BH4), an obligatory cofactor for Tph2 enzymatic activity as well as the enzymatic activity of other monoaminergic monooxygenases, nitric oxide synthases, and alkylglycerol monooxygenase (Kapatos, 2013; Tietz et al.,

1964; Watschinger et al., 2010; Werner et al., 2011). BH4 is synthesized de novo from the precursor guanosine triphosphate (GTP) in four or five enzymatic steps

(Figure 1) catalyzed by GTP cyclohydrolase I (GTPCH, gene symbol: Gch1), 6- pyruvoyltetrahydropterin synthase (PTPS, gene symbol: Pts), and sepiapterin reductase (SR, gene symbol: Spr). The enzymatic steps catalyzed by SR, however, can be alternatively catalyzed by aldo-keto-reductase family 1 member

3 (mouse orthologue: Akr1c18), aldo-keto-reductase family 1 B1 (mouse orthologue: Akr1b3), and carbonyl reductase (CR, gene symbol: Cbr1) in various combinations (Figure 1) (Kapatos, 2013). Although it is commonly accepted that

TPH2 is a rate-limiting step for the production of 5-HT, GTPCH activity is rate limiting for BH4 synthesis and therefore control of its expression level is a critical determinant of 5-HT synthesis. GTPCH enzymatic activity is also controlled post- translationally through negative feedback regulation by GTP cyclohydrolase I feedback regulatory protein (GFRP, gene symbol Gchfr) (Harada et al., 1993;

Yoneyama and Hatakeyama, 1998). The allosteric binding of BH4 to GTPCH stimulates the formation of a multimeric GFRP:GTPCH complex in which GTPCH activity is inhibited (Maita et al., 2004; Yoneyama and Hatakeyama, 1998). In contrast, l-phenylalanine can stimulate BH4 biosynthesis in a GFRP-dependent manner Kapatos et al. 1999). After its enzymatic conversion to 4α-hydroxy- tetrahydrobiopterin in a monooxygenase or synthase reaction, BH4 can be

63 regenerated in a two-step pathway catalyzed by pterin-4α-carbinolamine dehydratase (PCD, gene symbol Pcbd1 and Pcbd2) and dihydropteridine reductase (DHPR, gene symbol: Qdpr, quinoid dihydropteridine reductase)

(Werner et al., 2011). A large number of rare mutations, causing BH4 and monoamine deficiency, have been identified in human GCH1, PTS, SPR,

PCBD1, and QDPR genes and are responsible for several neurological motor control disorders such as dopa-responsive or Segawa disease (Thony and Blau, 2006). The 5-HT transporter, SERT (gene symbol Slc6a4), is responsible for high affinity reuptake of 5-HT (Blakely and Edwards, 2012;

Murphy and Lesch, 2008). However, other transporters are now recognized as playing important roles in clearing 5-HT from the synaptic cleft and extrasynaptic sites (Daws, 2009). For example, OCT3 (gene symbol: Slc22a3) has been shown to function as a low-affinity, high-capacity 5-HT transporter (Baganz et al; 2008).

Slc22a3 expression is abundant in brain 5-HT neurons, and importantly, it is a critical determinant of SSRI efficacy (Horton et al., 2013). However, the regulatory mechanisms that control expression of BH4 and Slc22a3 genes in 5-

HT neurons have not been investigated.

A possibility is that a regulatory network distinct from that controlling Tph2,

Ddc, Slc6a4, Slc18a2, Htr1a, and Htr1b controls these additional key serotonergic genes. This possibility seems plausible as BH4 enzymatic pathways are expressed in numerous neural and non-neural cell types of the brain and periphery and are required for other cellular functions in addition to 5-HT synthesis (Kapatos, 2013; Werner et al. 2011). Similarly, Slc22a3 is widely

64 expressed in many different neuron types and glia of the adult brain unlike

Tph2, Slc6a4, and Slc18a2 (Gasser et al., 2009; Vialou et al., 2008).

Alternatively, as for other neural expressed the complex expression patterns of

Slc22a3 and BH4 genes might be generated through separate cis regulatory control modules one of which positively responds to the same transcription factors that control other serotonergic genes such as Tph2 (Serrano-Saiz et al.,

2013; Wenick and Hobert, 2004).

To begin to understand the regulatory mechanisms that control Slc22a3 expression and BH4 production in mouse 5-HT neurons, we investigated whether their serotonergic expression is controlled by PET-1. We previously reported a microarray method for transcriptome studies of flow sorted yellow fluorescent protein (YFP)-expressing fetal 5-HT neurons obtained from the ePet-EYFP transgenic mouse line (Scott et al. 2005; Wylie et al., 2010). Here, we present a protocol for flow sorting of ePet-EYFP-marked Pet-1–/– 5-HT neurons from the fetal rostral hindbrain. We used this new protocol for comparative microarray

–/– analyses of Slc22a3 and BH4 gene expression in wild type and Pet-1 5-HT neurons. In addition, we investigated the relative PET-1 dependency of serotonergic genes in the adult dorsal raphe.

Methods

Animals

Pet-1–/– mice have been described previously (Hendricks et al., 2003). All in situ hybridizations were performed on age and sex matched adult mice 6–8 weeks of age unless otherwise stated. The National Institutes of Health guide

65 was followed for the care and use of laboratory animals. All experiments were approved by the Case Western Reserve University School of Medicine

Institutional Animal Care.

Perfusion and Sectioning

Mice were anesthetized with Avertin (0.5 g of tribromoethanol/39.5 mL of

H2O + 0.31 mL of tert-amyl alcohol) and transcardially perfused with saline followed by cold 4% paraformaldehyde (PFA). were extracted and post- fixed for 2 h and incubated overnight (O/N) in 30% sucrose-PBS solution at 4 °C.

Floating 25 μm coronal brain sections of the dorsal raphe serotonin system were taken, mounted on SuperFrost Plus slides (Fisher Scientific), and dried in a vacuum chamber for at least 1 h before use.

In Situ Hybridization (ISH)

Digoxigenin (Roche Diagnostics, Indianapolis, IN) labeled antisense RNA probes (∼600 bp) were synthesized using cDNA fragments of Slc22a3, Htr1a,

Gch1, Gchfr, or Tph2 that were PCR amplified with reverse primers containing bacteriophage T3 promoter sequences at their 5′ ends. A previously published in situ hybridization protocol was followed (Hendricks et al., 1999).

Quantitative Real-Time PCR (RT-qPCR)

RNA was isolated from E12.5 Pet-1+/– and Pet-1–/– animals from the rostral serotonin system using the PureLinkTM RNA Mini Kit (Ambion by Life

Technologies, Carlsbad, CA). Purified RNA was converted to cDNA by PCR using the Transcriptor FirstStrand cDNA Synthesis Kit (Roche Diagnostics,

66 Indianapolis, IN) and stored at −20 °C. qPCR was performed in triplicate using

Fast Start Universal Syber Green ROX Master solution (Roche Diagnostics,

Indianapolis, IN). Samples were normalized to β-.

Immunohistochemistry (IHC)

Fluorescent immunohistochemistry was performed using a polyclonal primary rabbit antibody against 5-HT (1:1000, ImmunoStar, Hudson, WI) or a primary chicken antibody against GFP (1:1000, Abcam, San Francisco, CA) O/N at 4 °C and an anti-rabbit or anti-chicken Alexa Fluor488 secondary antibody

(1:500, Invitrogen by Life Technologies, Carlsbad, CA) for 1 h. at room temperature in the dark. A standard IHC protocol was used (Fox and Deneris,

2012). Fluorescent images were taken using a SPOT RT color digital camera

(Diagnostic Instruments, Sterling Heights, MI) using an Olympus Optical BX51 microscope (Center Valley, PA).

FACS of YFP+ Cells

The rostral hindbrain domain from the mesencephalic flexure to the pontine flexure of either ePet-EYFP+/+ and ePet-EYFPPet-1–/– embryos was dissected and then treated with 0.25% trypsin-EDTA (Life Technologies) to dissociate cells as described previously. (Wylie et al., 2010). Cells were filtered through a 40 μm filter and sorted using a Becton Dickinson FACS Aria digital cell sorter with an argon laser (200 mW at 488 nm). Cells were sorted directly into

Trizol (Invitrogen) for RNA extraction. Approximately 7000 YFP+ cells were

67 isolated from +/+ or Pet-1–/– embryos. Each of the four biological replicates (4 +/+, 4

Pet-1–/–) consisted of between 20,000 and 30,000 cells from independent litters.

Microarray

Total RNA was isolated after the addition of 20 μg of glycogen (Invitrogen) using phenol chloroform extraction. RNA amplification and cDNA libraries were prepared using the AmbionWT Expression Kit (Life Technologies) according to the manufacturer’s protocol. An amount of 5.5 μg of single-stranded DNA was fragmented and labeled using the Affymetrix GeneChip WT Terminal Labeling

Kit. Probes were hybridized overnight at 45 °C to a GeneChip Mouse Gene 1.0

ST Array (Affymetrix). After hybridization, chips were washed in a Genechip

Fluidics Station (Affymetrix) and scanned at high resolution using an Affymetrix

High Density GeneChip Scanner 3000. The .CEL files from the eight chips were normalized using the Robust MultiChip Averaging (RMA) using Affymetrix

Expression Console Software version 1.1.

Results and Discussion

In previous histochemical studies, we found comparable numbers of Pet-

1–/– 5-HT neuron cells bodies and wild type 5-HT neuron cell bodies in the midbrain dorsal raphe Krueger and Deneris, 2008). Here, we crossed ePet-

EYFP+/+ and ePet-EYFPPet-1–/– mice to generate ePet-EYFPPet+/– offspring. These offspring were then interbred to generate ePet-EYFP+/+ and ePet-EYFPPet-1–/– littermate embryos. Anti-YFP immunostaining of fetal 5-HT neurons in the ePet-

EYFP+/+ and ePet-EYFPPet-1–/– brain revealed comparable levels of YFP

68 expression and similar numbers of wild type and mutant neurons (Figure 2A, B).

These findings suggested we should be able to sort sufficient numbers of Pet-1–

/– 5-HT neurons for microarray gene expression profiling. Embryonic (E) 12.5

YFP+ rostral hindbrain domains were dissected, dissociated, and purified by flow cytometry as previously described Wylie et al., 2010). Because PET-1 may regulate the rostral and caudal 5-HT system differently, we only used tissue from the rostral 5-HT system which gives rise to the dorsal and median raphe and B9 nuclei.

Both +/+ and Pet-1–/– 5-HT neurons were readily sorted (Figure 2C–F), and comparable numbers were obtained (Figure 2G). Quantitative real-time PCR

(RT-qPCR) revealed, as expected, a complete lack of Pet-1 expression in YFP+

RNA isolated from Pet-1–/– rostral hindbrain (Figure 2H). Moreover, Tph2 and

Slc6a4 RNA levels were also dramatically reduced in Pet-1–/– embryos compared to control levels as expected (Hendricks et al., 2003). Importantly, the expression of Lmx1b, a serotonergic transcription factor whose expression is independent of

Pet-1 at fetal stages, was unchanged Ding et al. 2003). Thus, flow sorted PET-1–

/– YFP+ 5-HT neurons can be used to determine the impact of Pet-1 loss of function on Slc22a3 and BH4 gene expression.

Having established a protocol for flow cytometry of Pet-1 mutant 5-HT neurons we set up crosses to generate sufficient numbers of embryos to perform four microarray biological replicates for both Pet-1–/– and +/+ 5-HT neurons. Based on our previous studies, we collected between 20, 000 and 30, 000 cells per replicate (Wylie et al., 2010). This yielded sufficient RNA to generate labeled

69 cDNA probes, which were then hybridized to the GeneChiP Mouse Gene 1.0 ST array.

As a validation of our microarray approach, we first examined Pet-1 probe intensities in arrays hybridized with +/+ and Pet-1–/– cDNA (Figure 3A). Analysis of probe intensities revealed background levels of Pet-1 expression. As a further test of our approach, we examined expression levels of several known Pet-1 downstream targets. Expression levels of Tph2, Ddc, Slc6a4, Slc18a2, and Htr1a were all significantly reduced in Pet-1–/– vs +/+ arrays (Figure 3A) to extents that correlate well with previous histochemical studies of these genes in Pet-1–/– mice.

We also note that expression of Lmx1b, a gene not regulated by PET-1 in fetal 5-

HT neurons, (Ding et al., 2003; Liu et al., 2010). was not altered in the present microarray analysis of its expression in mutant 5-HT neurons. Our array findings indicated very low expression of Hrt1a and Hrtr1b, which is consistent with our earlier findings that expression of these two autoreceptor genes is not strongly induced until after E14 (Liu et al., 2010) . Thus, the very low, near background levels, of these genes at E12.5 likely precluded detection of Htr1b’s Pet-1 dependency, although Htr1a did show significantly reduced expression in sorted

Pet-1–/– 5-HT neurons (Figure 3B). Importantly, our array findings for Tph2, Ddc,

Slc6a4, Slc18a2, Htr1a, and Lmx1b were perfectly consistent with our previously published studies of their Pet-1 dependency (Deneris 2011).

Having demonstrated the validity of our array approach for reproducible and accurate detection of gene expression changes in response to loss of Pet-1 we analyzed our data sets for expression of Slc22a3 and BH4 genes (Figure 3B).

70 Examination of the +/+ arrays revealed that, in comparison to Pet-1 and other 5-

HT genes, Slc22a3, Gchfr, Qdpr, and Pcbd1 are robustly expressed at E12.5.

Gchfr was the most abundantly expressed BH4 gene in our data set, which is consistent with previous studies showing strong expression of this feedback regulator gene in adult rat 5-HT neurons and as shown here it is expressed strongly in a serotonergic pattern in the adult mouse dorsal raphe (Figure 3C)

(Kapatos et al., 1999). In contrast, its expression is undetectable in other brain monoaminergic neuron types. This suggests that BH4 production in 5-HT neurons is especially sensitive to GFRP-dependent BH4 negative feedback and

GFRP-dependent l-Phe stimulation of BH4 biosynthesis. (Kapatos et al, 1999).

One possibility is that this provides for rapid and precise adjustments in 5-HT synthetic rates in response to changing behavioral and metabolic states. The

Pcbd1 paralogue, Pcbd2, has a relatively low expression level (Pcbd1 average probe intensity, 1897 ± 239; Pcbd2, 384 ± 13), suggesting Pcbd1 plays the major role in BH4 regeneration in 5-HT neurons. Although, Gchfr, Qdpr, and Pcbd1 were robustly expressed at E12.5, Gch1, Pts, and Spr expression was relatively low (Figure 3B). Yet, the level of Gch1, Pts, and Spr expression must be adequate to support evident abundant synthesis of 5-HT at this fetal stage. In situ hybridization (ISH) confirmed that, by adulthood, Gch1 is indeed strongly expressed in the mouse dorsal raphe (Figure 3D).

Besides SR, several other enzymes can catalyze the last three steps of the de novo BH4 synthesis pathway. Of these, enzymes encoded by Akr1b3 and

Cbr1 but not Akr1c18 are robustly expressed at E12.5, suggesting multiple BH4

71 synthesis pathways may be involved in 5-HT synthesis in immature 5-HT neurons

(Figures 3B and 1). Finally, the regeneration/salvage gene, Dhfr, is weakly expressed in immature 5-HT neurons (Figure 3B).

Interestingly, our array experiments revealed a severe loss of Slc22a3 expression in Pet-1–/– 5-HT neurons thus demonstrating that Pet-1 controls not simply high-affinity, low-capacity 5-HT transport (SERT) but also low-affinity, high-capacity transport (OCT3) (Figure 3B). Our findings further revealed substantially reduced expression of Gchfr, and Gch1 (Figure 3B). Therefore, Pet-

1 is a major transcriptional regulator of the rate limiting enzymatic and post- translational regulatory steps for BH4 synthesis. Given the substantial loss of expression of Gchfr and Gch1 expression in Pet-1–/– 5-HT neurons, it is somewhat surprising that the expression levels of the other BH4 synthetic genes,

Spr and Pts were unchanged. Perhaps the very low expression of Pts and Spr near background levels detected for these genes at E12.5 precluded detection of

Pet-1 dependency as was likely the case for Htr1b. Further studies of PET-1 dependency for these two genes at later stages of life will be required. The array results also indicated a role for PET-1 in regulation of the BH4 regeneration pathway as Qdpr levels were significantly reduced in Pet-1–/– 5HT neurons

(Figure 3B). However, loss of Pet-1 had no effect on Pcbd1, Pcbd2, Cbr1,

Akr1b3, Akr1c18, and Dhfr expression level. Given the strong and comparable levels of expression of Pcbd1, Cbr1, and Akr1b3 in the wild type and mutant arrays we conclude that Pet-1 is not necessary for their expression at least at

E12.5. One possibility is that LMX1B or GATA-3 is required for expression of

72 these other BH4 synthesis, regeneration, and salvage pathway genes.

Alternatively, all three transcription factors may play compensatory roles in the expression of these genes so that removal of a single regulatory partner has little or no effect on expression of these genes. A final possibility is that the more broadly expressed broadly functional BH4 genes such as Akr1b3 are controlled by another gene regulatory network.

Together these findings reveal an extended battery of serotonergic genes under the control of PET-1. Slc22a3 and the BH4 pathway genes are expressed in many cell types in brain and periphery, while Pet-1 expression in the brain is restricted to the 5-HT lineage. Therefore, it seems likely that the cis regulatory regions of the Slc22a3 and BH4 genes possess a PET-1 binding module that functions specifically to direct their expression to 5-HT neurons. Either LMX1B and/or GATA-3 or other unknown factors may also control expression of Gchfr,

Gch1, and Qdpr and may account for the residual expression of these BH4 pathway genes in Pet-1–/– 5-HT neurons.

Although we obtained highly concordant array results among our biological replicates for Slc22a3, Gch1, Gchfr, and Qdpr dependence on Pet-1, we selected Slc22a3 for RT-qPCR verification using dissected E12.5 neural tubes (Figure 4 4A). Consistent with the array results, we detected significantly reduced expression of Tph2, Slc6a4, and Slc22a3 but not Lmx1b in Pet-1–/– neural tubes (Figure 4A).

We next investigated adult Slc22a3 expression with ISH. Consistent with the Allen Brain Atlas ISH analysis of Slc22a3, we found a highly restricted

73 serotonergic raphe pattern of expression for this gene in the midbrain and pons

(Figure 4B, left panel, and data not shown), suggesting a highly selective serotonergic role for this gene in these brain regions.

Analysis of Slc22a3 expression in in adult Pet-1–/– mice revealed a striking nearly complete loss of its expression (Figure 4B, right panel). This finding was surprising given that expression of other serotonergic identity features such as

Tph2 and 5-HT itself are not absolutely dependent on Pet-1 for their full expression in about 20–30% of 5-HT neurons, (7, 13) which is shown here in

Figure 4C for comparison to the Slc22a3 dependency on Pet-1 (Hendricks et al.,

2003; Kiyasova et al., 2011). Thus, our Slc22a3 findings suggest that whether or not some 5-HT neurons are resistant to loss of Pet-1 depends on specific identity features expressed in these neurons. We note, however, that the residual expression of Tph2 in individual Pet-1–/– 5-HT neurons (Figure 4C, lower panels) appears to be substantially weaker than its expression level in individual wild type 5-HT neurons, suggesting Pet-1 controls Tph2 expression in all 5-HT neurons.

To further investigate Slc22a3 dependency on Pet-1, we systematically investigated its expression throughout the entire dorsal raphe nucleus of Pet-1–/– adults. ISH was performed on every other 25 μm section spanning the entire dorsal raphe. As shown in Figure 5A–H, we found a nearly complete elimination of Slc22a3 expression at all levels of the dorsal raphe in Pet-1–/– mice. Residual expression, if any, was confined to scattered cells in numbers far lower than the number of mutant neurons still expressing Tph2 or 5-HT in the Pet-1–/– brain

74 (Figure 4C). These findings indicate that in the case of the Slc22a3 identity feature, its expression is completely dependent on PET-1 as virtually no 5-HT neurons are resistant to loss of PET-1. Thus, few if any 5-HT neurons acquire their complete adult identity in the absence of Pet-1 and the number of PET-1 resistant 5-HT neurons that exist is far fewer than previously thought (Hendricks et al., 2003; Kiyasova et al., 2011).

To extend this analysis to an additional key identity gene, we investigated

Htr1a’s dependency on PET-1. ISH throughout the dorsal raphe indicated a similar severe loss of Htr1a expression in Pet-1–/– mice (Figure 5I–P). Few if any cells in the mutant dorsal raphe expressed Htr1a at levels comparable to levels of Htr1a in wild type dorsal raphe or levels of residual Tph2 expression in cells of the Pet-1–/– dorsal raphe (Figure 4C). Instead, a low uniform level of blue precipitate was present in cells of the mutant dorsal raphe, which might be background ISH signal, low residual expression in mutant 5-HT neurons, or normal expression in non-serotonergic cells. Thus, in the case of the Htr1a identity feature, far fewer 5-HT neurons are resistant to loss of PET-1 compared to the number of PET-1 resistant 5-HT neurons in the case of the Tph2 gene.

Further gene expression studies are likely to reveal other serotonergic identity features whose expression is dependent on PET-1 in all brain 5-HT neurons.

In summary, our findings provide additional insight into the regulatory strategies that enable 5-HT synthesis and reuptake in the brain. We show that

Pet-1 globally controls acquisition of the brain’s capacity for 5-HT synthesis by coordinating coexpression of Tph2, Ddc, and genes required for BH4 cofactor

75 synthesis, regeneration, and post-translational negative feedback (Figure 6).

Thus, PET-1 controls all of the known rate-limiting enzymatic (TPH2, GCH1) and post-translational regulatory (GFRP) steps that determine the level of mammalian brain 5-HT synthesis. In addition, we show that PET-1 globally controls acquisition of 5-HT reuptake in dorsal raphe 5-HT neurons by coordinating expression of high-affinity, low-capacity transport via SERT and low-affinity, high- capacity transport via OCT3 (Figure 6). We show that few, if any, 5-HT neurons in the dorsal raphe are resistant to loss of Pet-1 for expression of Slc22a3.

Similarly, PET-1 is indispensable for expression of Htr1a in what appears to be all or nearly all 5-HT neurons of the dorsal raphe that normally express this gene.

These findings indicate that while some 5-HT neurons are not absolutely dependent on PET-1 for expression of Tph2 virtually all dorsal raphe 5-HT neurons require Pet-1 for expression of other key identity genes. Therefore, few if any 5-HT neurons in the dorsal raphe are truly resistant to loss of PET-1 for their neurochemical differentiation.

Funding Information

This research was supported by NIH Grants P50 MH096972, RO1MH062723, and T32 NS067431.

76 Figure 1

77 Figure 1. Schematic of BH4 de novo synthesis, salvage, and regeneration

pathways and its role in 5-HT synthesis. TPH2, tryptophan hydroxylase 2;

AADC (Ddc), aromatic l-amino acid decarboxylase; GFRP (Gchfr), GTP cyclohydrolase I feedback regulator; GTPCH (Gch1), GTP cyclohydrolase; PTPS

(Pts), 6-pyruvoyl-tetrahydropterin synthase; SR (Spr), sepiapterin reductase;

AKR1C3, aldo-keto-reductase family 1 member 3; AKR1B1, aldo-keto-reductase family 1 B1; CR (Cbr1), carbonyl reductase; DHFR, dihydrofolate reductase;

DHPR (Qdpr), dihydropteridine reductase; PCD (Pcbd1, Pcbd2), pterin-4 alpha- carbinolamine dehydratase. Dashed lines indicate nonenzymatic steps.

78 Figure 2

79 Figure 2. Isolation of ePet-EYFP and Pet-1-/-; ePet-EYFP 5-HT neurons. (A, B).

Sagittal sections of E12.5 embryonic hindbrain. Dashed line indicates division

between rostral and caudal 5-HT neurons. (C, D). Flow cytometry data of sorted

YFP+ cells. X-Axis forward scatter area (FSC-A). Y-Axis 488 nm fluorescent intensity. (E, F). Sorted 5-HT neurons. (G). Number of YFP+ cells per embryo collected from either +/+ or Pet-1–/– animals (p = 1.00). (H). RT-qPCR validation of selected 5-HT genes in +/+ vs Pet-1–/– samples. Scale, 200 μm; zoom, 10 μm.

80 Figure 3

81 Figure 3. Microarray analyses. (A). Signal intensities of 5-HT neuron-type identity genes. (B). Signal intensities of BH4 pathway genes and Slc22a3. In situ hybridization of (C) Gchfr and (D) Gch1 probes at two rostro-caudal levels of the dorsal raphe of 3 week old mice. Scale, 200 μm. Genes were analyzed by a

Student’s t test followed by a Bonferroni correction. Corrected p-value: *p < 0.05,

**p < 0.01, ***p < 0.001.

82 Figure 4

83 Figure 4. Slc22a3 expression. (A). RT-qPCR analyses of 5-HT genes with RNA obtained from unsorted hindbrain dissections of E12.5 Pet-1+/– or Pet-1–/–

embryos. (B). Comparative in situ hybridization of Slc22a3 probe in +/+ and Pet-

1–/– mice. (C). 5-HT immunohistochemistry and Tph2 in situ hybridization in +/+

and Pet-1–/– adult mice. Scale, 200 μm. Genes were analyze by a Student’s t

test. p-value: *p < 0.05, **p < 0.01, ***p < 0.001.

84 Figure 5

85 Figure 5. Slc22a3 and Htr1a expression in the adult dorsal raphe. 25 μm coronal brain sections processed by in situ hybridization and developed for 15 h using an (A–H) Slc22a3 or (I–P) Htr1a probe on either wild type or Pet-1–/– tissue

sections. Alternate sections through the entire dorsal raphe were used for each

probe. “A” anterior; “P” posterior. Scale, 200 μm.

86 Figure 6

87 Figure 6. Pet-1 control of the 5-HT neuron-type gene battery. PET-1 coordinates expression of genes required for 5-HT synthesis (orange), vesicular transport and reuptake (purple), and autoreceptor function (blue). Filled red circles indicate direct control of genes as determined by chromatin

immunoprecipitation, reporter assays, and in vitro mobility shift assays. Other

genes in the schematic may also be direct targets of Pet-1 but have not yet been

investigated.

88

CHAPTER 3

LMX1B IS REQUIRED AT MULTIPLE STAGES TO BUILD EXPANSIVE

SEROTONERGIC AXON ARCHITECTURES

By: Donovan LJ, Spencer WC, Kitt MM, Eastman BA, Lobur KJ, Jiao K, Silver J,

Deneris ES.

DOI: https://doi.org/10.7554/eLife.48788.001

89 Summary

Formation of long-range axons occurs over multiple stages of morphological maturation. However, the intrinsic transcriptional mechanisms that temporally control different stages of axon projection development are unknown.

Here, we addressed this question by studying the formation of mouse serotonin

(5-HT) axons, the exemplar of long-range profusely arborized axon architectures.

We report that LIM homeodomain factor 1b (Lmx1b)-deficient 5-HT neurons fail to generate axonal projections to the forebrain and spinal cord. Stage-specific targeting demonstrates that Lmx1b is required at successive stages to control 5-

HT axon primary outgrowth, selective routing, and terminal arborization. We show a Lmx1b→Pet1 regulatory cascade is temporally required for 5-HT arborization and upregulation of the 5-HT axon arborization gene, Protocadherin- alphac2, during postnatal development of forebrain 5-HT axons. Our findings identify a temporal regulatory mechanism in which a single continuously expressed transcription factor functions at successive stages to orchestrate the progressive development of long-range axon architectures enabling expansive neuromodulation.

90 Introduction

Newly generated neurons dramatically transform their morphology to establish mature circuit connectivity. Some neurons, for example interneurons, connect to local circuits and thus need to extend their axons relatively short distances. In contrast, other neuron types, such as those giving rise to neuromodulatory systems, have the capacity to extend extremely long axons to innervate distant target fields. This is well exemplified in the case of serotonin (5-

HT) synthesizing neurons. 5-HT modulates the excitability of nearly all neural circuitry in the brain and spinal cord despite being produced in a relatively small population of neurons (Azevedo et al., 2009; Baker et al., 1991; Hornung, 2003).

Expansive serotonergic neuromodulation is achieved through the formation of long-range highly diffuse ascending and descending projection pathways that deliver 5-HT throughout the brain and spinal cord for synaptic or non-synaptic interactions with an array of receptors (Hannon and Hoyer, 2008; Steinbusch,

1981). Despite the decades-long knowledge of 5-HT’s importance as a global neuromodulator, it is not understood how these small numbers of neurons develop, over an extended period of morphological maturation, such elaborate axonal architectures.

5-HT neurons initiate axon outgrowth concomitant with their birth and onset of 5-HT synthesis (Hawthorne et al., 2010; Lidov and Molliver, 1982).

Classic neuroanatomical studies have defined three successive stages, collectively spanning several weeks, in the formation of 5-HT projection pathways: primary pathway formation during which 5-HT axon outgrowth is

91 initiated, selective pathway routing, and finally postnatal terminal arborization

(Lidov and Molliver, 1982). Following primary pathway outgrowth through the medial forebrain bundle (MFB), ascending 5-HT axons originating in the dorsal raphe (DRN), median raphe (MRN) and B9 groups of midbrain/pons 5-HT neurons are selectively routed along pre-existing fiber tracts and reach all forebrain targets at parturition in an unarborized state (Bang et al., 2012; Lidov and Molliver, 1982; Muzerelle et al., 2016). Descending 5-HT axons, originating in the medullary serotonergic clusters, raphe pallidus (RPa), raphe obscurus

(ROb), and raphe magnus (RMg), enter the spinal cord via the dorsolateral and ventral funiculi. These primary projections then route medially to invade nearly all lamina of the dorsal and ventral horns as well as the intermediate zone from cervical to sacral levels (Rajaofetra et al., 1989). After the major ascending and descending projection pathways are formed, a final, entirely postnatal, stage of 5-

HT projection pathway maturation ensues during which 5-HT axons originating in different anatomically defined sub-regions flourish profuse terminal arbors in complementary and topographically organized patterns (Muzerelle et al., 2016;

Ren et al., 2018). Terminal arborization develops at least through the first four postnatal weeks and leaves few, if any, regions of the brain and spinal cord devoid of serotonergic input (Gagnon and Parent, 2014; Hornung, 2003; Lidov and Molliver, 1982; Maddaloni et al., 2017; Steinbusch, 1981). What are the intrinsic mechanisms that govern successive stages in the formation of long- range profusely arborized 5-HT axon projection pathways and how are they temporally coordinated? One possibility is that each stage is governed by

92 different intrinsic regulatory factors. Alternatively, a single continuously expressed intrinsic regulator may act at successive stages to orchestrate progressive morphological maturation of 5-HT pathways.

In contrast to the poor understanding of how 5-HT axonal pathways are formed, there is substantial knowledge of the gene regulatory networks (GRNs) that generate 5-HT neurons and control acquisition of 5-HT transmitter identity

(Deneris and Gaspar, 2018). The LIM homeodomain protein, Lmx1b, is a crucial factor in 5-HT GRNs as 5-HT neuron selective targeting of Lmx1b results in the failure to induce Tph2 expression for 5-HT synthesis and Slc6a4 expression for

5-HT reuptake (Zhao et al., 2006). This results in extremely low levels of 5-HT in the adult brain, which is associated with high neonatal mortality and several abnormal behavioral phenotypes including hyperactivity, delayed respiratory maturation, enhanced inflammatory pain sensitivity, deficient opioid analgesia, sleep regulation, and increased contextual fear memories (Dai et al., 2008;

Hodges et al., 2009; Zhang et al., 2018; Zhao et al., 2007a; Zhao et al., 2007b).

Lmx1b is a continuously expressed, terminal selector-type factor in 5-HT neurons (Hobert, 2008) raising the possibility that subsequent to its initial role in the induction of 5-HT synthesis and transport it may perform additional stage specific functions in the maturation of serotonergic connectivity. However, stage specific functions of continuously expressed terminal selectors, such as Lmx1b, in postmitotic neuronal morphological maturation are poorly understood (Deneris and Hobert, 2014; Hobert, 2016). Here, we report that lack of Lmx1b results in the failure to build long-range ascending and descending 5-HT axon projection

93 pathways. Using temporal conditional targeting approaches we dissect distinct stage-specific functions for Lmx1b. Our findings show that Lmx1b acts at successive stages to control primary pathway growth rate, selective pathway routing and terminal arborization of 5-HT axons. We identify an ascending- specific Lmx1b-controlled regulatory cascade that regulates selective pathway routing and then switches to control forebrain 5-HT axon arborization through stage specific expression of genes required for arborization. This study demonstrates that a single continuously expressed transcription factor, initially required for induction of 5-HT synthesis and reuptake, subsequently acts at successive stages to build the expansive axon pathway architectures enabling

CNS-wide serotonergic neuromodulation.

Materials and methods

Animals

All procedures were approved by the Institutional Animal Care and Use

Committees of Case Western Reserve University in accordance with the National

Institutes of Health Guide for the Care and Use of Laboratory Animals.

Experiments were performed on male and female mice using age-matched and sex-matched controls in triplicate unless otherwise noted. Adult mice between the ages 2.5 to 3.5 months were used unless otherwise noted. Early conditional knockout mice (designated Lmx1bcKO, Pet1cKO, and Tph2cKO) were generated by using the following genetic mouse lines: Pet1-Cre (original name ePet-Cre)

(Scott et al., 2005), Ai9 Rosa TdTomato (RosaTom; Jackson labs) and either

94 Lmx1bfl (Zhao et al., 2006), Pet1fl (Liu et al., 2010), or Tph2fl (Kim et al., 2014). All early conditional knockout mice were compared to non-floxed controls (+/+; Pet1-

Cre;Ai9). The following genetic lines were used to generate Lmx1bicKO or

Pet1icKO mice for postnatal stage conditional knockout: Tph2-CreERT2

(Jackson lab), Ai9 Rosa TdTomato (RosaTom; Jackson lab) and either Lmx1bfl

(Zhao et al., 2006) or Pet1fl (Liu et al., 2010). All postnatal stage conditional knockout mice were compared to non-floxed controls (iControls: +/+;Tph2-

CreER; Ai9). Pet1-/- mice (Hendricks et al., 2003) carrying the Pet1-YFP transgene (Scott et al., 2005) were used for embryo studies at E13.5. Tail or ear genomic DNA was used to determine genotypes of all animals. All mice were housed in ventilated cages on a 12 hr light/dark cycles with access to food and water with 2–5 mice per cage.

Embryos and postnatal pups

All embryo experiments at each time point were performed in triplicate unless otherwise noted and compared to littermate controls (Lmx1bfl/+ vs Lmx1bfl/fl;

Pet1fl/+ vs Pet1fl/fl; Pet1+/- vs Pet1-/-). Both male and female littermates were used for analysis. Embryonic day 0.5 (E0.5) was determined by presence of a vaginal plug. Postnatal day 0 (P0) was designated by date of birth.

Histology and immunohistochemistry

Adult mice were anesthetized with Avertin (44 mM tribromoethanol, 2.5% tert- amyl alcohol, 0.02 ml/g body weight) and perfused for 2–3 min with cold PBS followed by 20 min cold 4% paraformaldehyde (PFA) in PBS. Brains and/or spinal cords were removed, post-fixed in 4%PFA for 2 hr and placed in 30%

95 sucrose/PBS overnight (O/N) for cryoprotection. E12-E13 embryos were drop fixed in 4% PFA in PBS O/N followed by 30% sucrose incubation O/N. All embryos between age E15-E18 were transcardially perfused with 5 mL of 4%

PFA in PBS, incubated in 4% PFA in PBS O/N, and incubated in 30% sucrose

O/N. All tissue collected was frozen in Optimal Cutting Temperature (OCT) solution and sectioned on a cryostat at 25 mm. Tissue sections were mounted on

SuperFrost Plus slides (Thermo Fisher Scientific) and vacuum dried. Sections were then permeabilized in 0.3%Triton 100X-PBS (PBS-T) for 15 min followed by antigen retrieval in Sodium Citrate buffer for 5 min in the microwave at low power. Sections were blocked with 10% NGS in PBS-T for 1 hr followed by incubation in primary antibody using a rabbit anti-RFP antibody (1:200; p/n 600-

401-379, Rockland) at 4 ˚C O/N. Antigen retrieval step was not used for the following primary antibodies: rabbit anti-GFP (1:200; A6455, Invitrogen), rabbit anti-5-HT (1:200; Immunostar), rabbit anti-Tph2 (1:500; ABN60, Millipore), and rabbit anti-Lmx1b (1:200; Suleiman et al., 2007). For all co-stains with TdTomato a mouse anti-RFP (1:200; ab65856, Abcam) or a mouse anti-RFP (1:200; p/n

200-301-379, Rockland) primary antibody was used. Secondary antibodies were used at 1:500; goat anti-rabbit or mouse, Alexa Fluor 594 or 488 (Invitrogen).

In situ hybridization

In situ hybridization was performed using a standard protocol using a digoxigen-

11-UTP labeled antisense RNA probe to detect Pet1 and Pcdhac2 (Roche diagnostics) as described elsewhere (Wyler et al., 2016). Pcdhac2 probe (566 bp) was generated using the following primers:

96 F:5’AGCCACCTCTATCAGCTACCG 3’ and

R:5’AGAATTAACCCTCACTAAAGGGCTCATTTTGAGAGC-CAGCATCA 3’.

Pet1 probe (513 bp) was generated using primers:

F:5’CCAGTGACCAATCCCATCC TC3’ and

R:5’AGAATTAACCCTCACTAAAGGGTTAATGGGGCTGAAAGGGATA3’.

Cell counts

5-HT neurons were identified by Tph2 immunostaining. To calculate the Pet1-Cre efficiency, every 4th section was taken through the entire rostrocaudal extent of the DRN/MRN/B9 and medullary areas of control mice (n = 2 control mice) and

RFP+/TPH2+ and TPH2+/RFP+ ratios were calculated. Tph2-CreER efficiency in postnatal tamoxifen injected pups was calculated from sections taken through the entire rostrocaudal extent of the DRN/MRN/B9 and expressed as a ratio of

RFP+/TPH2+ and TPH2+/RFP+ cells (n = 4 iControl mice). A computer program that permits blinded manual cell counting was used (Fox and Deneris, 2012) to determine numbers of TdTomato+ cells between control vs Lmx1bcKO, control vs

Tph2cKO, and iControl vs Lmx1bicKO or Pet1icKO mice. Every 4th matched section was counted through the entire rostrocaudal extent of the DRN/MRN/B9 or medullary 5-HT system as described. To calculate the percentage of remaining Tph2+ neurons in Lmx1bcKO vs Tph2cKO mice, Tph2+ neurons were counted in every 4th section throughout the DRN/MRN/B9. Tph2+ cell numbers in cKO animals was normalized to the number of Tph2+ cells in control matched sections and expressed as a percentage for each genotype comparison.

97 Tamoxifen injections

P1 pups (Lmx1bfl/fl;Tph2-CreER;Ai9 and iControls; n = 4 mice/genotype) were injected

1X with 100 mg of tamoxifen (10 mg/mL stock in corn oil). P3 or P5 pups (P3:

Lmx1bfl/fl;Tph2-CreER;Ai9, Pet1fl/fl;Tph2-CreER;Ai9, and iControls; n = 3 mice/genotype; P5:

Pet1fl/fl;Tph2-CreER;Ai9 and iControls, n = 2 mice/genotype) were injected 1X with 200 mg tamoxifen. All tamoxifen injections were performed subcutaneously at the back of the neck using a 30-gauge needle on a 1 mL syringe. Pups were allowed to sit for a few minutes before returning to mother. Injected mice were taken at either 31 or 49 days of age for analysis as indicated.

Viral injections

Adult animals (Lmx1bfl/fl;Pet1-Cre;Ai9 and Lmx1b+/+;Pet1-Cre;Ai9; n = 2 mice/genotype) were anesthetized with Isoflurane and stereotaxically injected unilaterally at two sites with 1.5 ml and 1 ml rAAv2/Ef1a-DIO-hchR2 (H134R)-EYFP (UNC GTC

Vector Core Lot# AV4378K) at X = 0.6 mm, Y = 4.2 mm, Z = 3.2 mm and X = 0.6 mm, Y = 4.2 mm, Z = 4.2 mm relative to Bregma respectively. Animals were treated with a local anesthetic (bupivacaine HCL; Hospira) administered subcutaneously prior to surgery and with analgesic (carprofen 5 mg/Kg; Pfizer) for 3 days following. Holes were drilled through the skull to expose brain, following which a Hamilton syringe was slowly lowered to desired coordinates.

Infusion rate was set to 0.1 ml/min with 10 min after each injection to allow diffusion of virus. Animals were returned to group housing following recovery and sacrificed 10 weeks after surgery.

98 5-HT neuron dissociation and flow cytometry

For RNAseq analyses rostral and caudal E17.5 YFP+ 5-HT neurons were collected from Lmx1bfl/fl;Pet1-EYFP or Pet1fl/fl;Pet1-EYFP (controls) and Lmx1bfl/fl;Pet1-

EYFP;Pet1-Cre or Pet1fl/fl;Pet1-EYFP;Pet1-Cre (Lmx1bcKO or Pet1cKO) mice using flow cytometry. To dissociate embryonic 5-HT neurons, hindbrains were initially dissected to separate rostral 5-HT neurons from caudal 5-HT neurons in cold

PBS. Tissue was transferred to 1.5 mL ependorf tubes and centrifuged at 1500 rpm for 1 min at 4 ˚C. PBS was removed and 500 mL 1X TrypLE Express (Gibco) was added to the tissue and incubated at 37 ˚C for 15 min followed by addition of

L-15 media (Gibco). Samples were centrifuged for 1 min at 1500 rpm and washed 3X with PBS. Tissue was then resuspended in 500 mL

L15/0.1%BSA/DNase solution and slowly triturated 30X with fire-polished

Pasteur pipettes of decreasing bore size until fully sus-pended. Samples were then filtered, and flow sorted on a FACS Aria I or Sony iCyt cell sorter.

Postnatal 5-HT neuron dissociation was performed in either P2

Lmx1bcKO or 4 week old Lmx1bicKO or Pet1icKO mice. Brains were sliced at

400 mm on a vibratome (Pelco easiSlicer) in continuously bubbling (95%O2;

5%CO2) aCSF solution (3.5 mM KCl, 126 mM NaCl, 20 mM NaHCO3, 20 mM

Dextrose, 1.25 mM NaH2PO4, 2 mM CaCl2, 2 mM MgCl2, 50 mm AP-V (Tocris),

20 mm DNQX (Sigma), and 100 nM TTX (Abcam)). Sections containing rostral 5-

HT neurons were incubated in 1 mg/mL Protease from Streptomyces griseus

(Sigma; P8811) in bubbling aCSF solution for 30min (P2 mice) or 45min (4-week- old mice) at room temperature. Slices were then incubated for 15min in bubbling

99 aCSF alone at RT. TdTomato+ neurons were microdissected from slices in cold aCSF/10%FBS solution. Samples were slowly triturated 30-100X with fire- polished Pasteur pipettes of decreasing bore size until fully suspended. Samples were then filtered, and flow sorted on a FACS ARIA-SORP sorter.

RNA sequencing

Total RNA was isolated from flow-sorted neurons using Trizol LS (Invitrogen) and the RNA Clean and Concentrator-5 kit (Zymo Research). RNA concentration and quality was determined using Quanti-fluor RNA system (Promega) and Fragment analyzer (Advanced Analytics). Samples were converted to cDNA, depleted of rRNA transcripts, and amplified using the TRIO RNA-seq kit for mouse (Nugen

Inc). Single-end sequencing was performed on a Nextseq 550 (Illumina) for 76 cycles. Read quality was assessed using FASTQC

(https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and adapters were trimmed using Trimmomatic (Bolger et al., 2014). Filtered and trimmed reads were aligned to the mouse genome (mm10, UCSC) using Tophat2 (v2.1.1) (Kim et al., 2013).

RNA sequencing analysis

Gene expression quantification and differential expression were analyzed using

Cufflinks v2.2.2 (Trapnell et al., 2010). In all differential expression comparisons, a gene was called differentially expressed if fold-change was ≥1.5 and false discovery rate (FDR) was ≤ 5%. Hierarchical clustering of differentially expressed genes in rostral Lmx1bcKO and caudal Lmx1bcKO samples was performed in R

(v. 3.5) using row-scaled values with Euclidean distance and complete linkage.

100 The heatmap was plotted with the gplots library. analysis was performed using Webgestalt (http://www.webgestalt.org), requiring five genes per category and FDR ≤ 5%. Venn diagrams were gener-ated using Biovenn

(http://www.biovenn.nl) (Hulsen et al., 2008; Wang et al., 2017). Differential exon expression analysis was performed to test for differences of the unique Pcdhac2 first exon with DEXSeq (Anders et al., 2012). RNA-seq reads were mapped to the Mus musculus genome (Ensembl, v. 96) and reads falling within annotated exons were counted using featureCounts (Rsu-bread). DEXSeq default settings were used and significant exon usage differences were determined at FDR ≤ 5%. qPCR

RNA was isolated from YFP+ flow sorted cells in Trizol LS (Invitrogen) using chloroform extraction followed by the RNA Clean and Concentrator-5 kit (Zymo

Research). cDNA was then synthesized using equal input RNA with the

Transcriptor First Strand cDNA Synthesis Kit (Roche). cDNA was then amplified

(8 PCR cycles) using PerfeCTa PreAmp SuperMix (QuantoBio) followed by ExoI digest to remove excess primers. RT-qPCR was performed using PerfeCTa

FastMix II ROX mastermix (QuantaBio) with TaqMan probes (Thermofisher

Scientific).

Chromatin immunoprecipitation analysis

ChIP-seq data for mycPet1 was obtained from Wyler et al. (2016). The data was re-analyzed using the ENCODE Transcription Factor ChIP-seq analysis pipeline

(https://github.com/ENCODE-DCC/chip-seq-pipeline). Reads were mapped to

101 the Mus musculus genome (UCSC mm10) using the Bur-roughs-Wheeler aligner

(BWA) and peaks were called with MACS2 with FDR ≤ 1% (Zhang et al., 2008).

Axon quantification

Spinal cord: Coronal sections of spinal cords were taken at 25 mm. Two sections were taken blindly from each cervical and lumbar spinal segment from each genotype (n = 3, control; n = 3, Lmx1bcKO mice) and (n = 3, control; n = 3,

Pet1cKO). Whole gray area or whole white matter area was selected independently, and pixels were quantified using Zeiss 2.3 Image Analysis module. Pixel values were normalized to selected area (mm2) for each section.

Two-way ANOVA with Welch’s correction analysis was performed. p values for each comparison are detailed in Fig. Legends.

Forebrain: Coronal sections of hippocampus, cortex, and axon tracts (SCS and cingulum) were taken and TdTomato+ pixels were quantified using Zeiss 2.3

Image Analysis module in P3 targeted iControls (n = 3 non-littermate mice) and

P3 Lmx1bicKO mice (n = 3 non-littermate mice). Pixel values were normalized to selected area (mm2). Unpaired t-test with Welch’s correction statistical analysis was performed. p values for each comparison are detailed in Fig. Legends.

Cell volume analysis

Thick tissue sections were sliced at 150 mm and cleared using CUBIC clearing technique to increase the optical transparency of sections (Susaki et al., 2015).

CUBIC reagent was prepared using urea (25 wt% final concentration), Quadrol

(25 wt% final concentration), Triton X-100 (15 wt% final con-centration) and dH2O. Tissue sections were incubated in CUBIC reagent at 4˚C overnight. To

102 avoid the distortion of tissue by compression of the glass cover slip, Blu-Tack reusable adhesive was used to create a reservoir with a 0.3–0.5 mm depth. The transferred tissue sections were then sealed in CUBIC reagent using a glass cover slip for z-stack confocal imaging of the endogenous TdTomato fluorescence of cell bodies. The 3D neuron images were reconstructed and measured using the Sur-faces or Filament Tracer tool in Imaris 7.4.2 (Bitplane,

Zurich, Switzerland). Unpaired t-test with Welch’s correction statistical analysis was performed. Sample size and p values for each comparison are detailed in

Fig. Legends.

Image acquisition and processing

Immuno-stained slides were imaged on an LSM800 confocal microscope (Carl

Zeiss). All embryonic forebrain images were captured using a BZ-X700 fluorescence microscope (Keyence) or Olympus Optical BX51 microscope.

Global brightness and contrast were edited across whole images equally between genotypes. Whole sagittal sections (Figure 1A,B) were acquired using confocal 10X objective and both control and Lmx1bcKO sections were processed in ImageJ and subjected to equal background subtraction followed by Lookup

Table-Fire to enhance visualization of TdTomato+ axon intensities in the forebrain. Axon tracing in Figure 5D and Figure 9K was performed using the

Filament Tracer module in Imaris 7.4.2 (Bitplane, Zurich, Switzerland).

Accession codes

All data generated in this study are deposited in NCBI GEO under accession code GSE130514.

103 Results

Lmx1b controls formation of ascending 5-HT projection pathways

Conditional targeting of Lmx1b with the Pet1-Cre transgene results in loss of endogenous 5-HT neuron markers, Sert, Tph2, and 5-HT at E12.5 (Zhao et al.,

2006). Therefore, we generated control (Lmx1b+/+;Pet1-Cre;Ai9) and Lmx1bcKO

(Lmx1bfl/fl;Pet1-Cre;Ai9) mice in which the Ai9 reporter allele was used as a surrogate marker to specifically label Pet1+ cell bodies and their axons with red fluorescent protein, TdTomato. In control adult mice, 82% of Tph2+ neurons in the DRN and

MRN express TdTomato. Conversely, approximately 88% of TdTomato+ cells co- label with Tph2; the remaining TdTomato+ cells likely express Tph2, but at a level insufficient for detection with standard IHC (Deneris and Gaspar, 2018; Okaty et al., 2015) (Figure 1—figure supplement 1A,B).

TdTomato+ cells were not found outside of the raphe nuclei (Figure 1— figure supplement 1B). TdTomato+ axons in control mice were widely distributed throughout the adult brain and colocalized with 5-HT (Figure 1—figure supplement 1C,D). The pattern of TdTomato+ axons in the control forebrain corresponded closely with the pattern of 5-HT axon distribution previously determined in the rat with an anti-5-HT antibody (Lidov and Molliver, 1982;

Steinbusch, 1981). Similarly, TdTomato+ axon distribution in control brains was highly concordant with 5-HT axon projection patterns determined more recently using a mouse line in which GFP was knocked into the Tph2 coding region, thus validating our surrogate marking of 5-HT axons using a soluble reporter

(Migliarini et al., 2013).

104 Abundant numbers of TdTomato+ cell bodies were detected in each of the raphe nuclei of Lmx1bcKO animals (Figure 1—figure supplement 1E). The vast majority of Lmx1bcKO TdTomato+ cells did not co-express Lmx1b or Tph2

(Figure 1—figure supplement 1E,F). Further, RT-qPCR analyses verified severe deficits of Lmx1b and Tph2 mRNAs in flow sorted YFP-labeled Pet1+ neurons in

Lmx1bcKO mice, confirming appropriate Lmx1b targeting (Figure 1—figure supplement 1G). Counts of TdTomato+ cell bodies in Lmx1bcKO mice indicated that equivalent numbers were present compared to controls at 3 months and that their numbers remained stable for at least 13 months (Figure 1—figure supplement 1H,I). Lmx1bcKO TdTomato+ cell bodies were located within the normal cytoarchitectural boundaries of the raphe nuclei with a mildly altered distribution and smaller size (Figure 1—figure supplement 1I,J). Together, these data indicate specific fluorescent labeling of 5-HT neurons with Ai9 and efficient

Lmx1b knock-down in Lmx1bcKO animals.

Analysis of TdTomato+ axons in adult Lmx1bcKO mice revealed a dramatically different pattern compared to that in control mice (Figure 1A,B).

Although TdTomato+ axons were present in normal density and with proper ascending trajectory within the MFB of Lmx1bcKO mice, TdTomato+ axons were nearly completely missing throughout the forebrain of Lmx1bcKO animals distal to the thala-mus (Figure 1B). Indeed, the vast majority of TdTomato+ axons failed to reach various distal fiber tracts including the fimbria-fornix, supracallosal stria, cingulum bundle, and olfactory tract (Figure 1B). Consequently, few if any

TdTomato+ axons were present in the olfactory bulb, cortex, amygdala,

105 hippocampus, striatum or many regions of the hypothalamus (Figure 1B–D;

Figure 1—figure supplement 2A,B). It is likely that the few TdTomato+ 5-HT axons that did reach the distal forebrain were the result of a small number of 5-

HT neurons in which Ai9 expression was activated, but Lmx1b targeting failed

(Figure 1—figure supplement 1F).

Although Lmx1bcKO TdTomato+ axons were present in the thalamus, the distribution of their terminal arbors was quite different from that in controls

(Figure 1B,E). We found aberrant clumping of mutant TdTomato+ terminal arbors in several intralaminar thalamic nuclei (Figure 1E; Figure 1—figure supplement

2C). Many regions were completely devoid of arbors unlike the relatively homo- geneously tiled distribution in the control thalamus (Figure 1E, arrowheads). In addition, the conspicuously dense 5-HT arborization that demarcates the paraventricular nucleus of the thalamus (PVT) was completely absent in

Lmx1bcKO mice (Figure 1E; Figure 1—figure supplement 2D).

To verify the deficit of TdTomato+ axons in the forebrain of Lmx1bcKO mice, we performed a second method of axon labeling by stereotaxic injection of an AAV2 virus expressing a Cre-dependent membrane bound channelrhodopsin with a YFP tag (rAAV2/Ef1a-DIO-hchR2-EYFP) into the midbrain of adult control and Lmx1bcKO mice. In Lmx1bcKO mice, we found an absence of YFP+ axons in all of the distal areas in which TdTomato+ axons were absent. Moreover, YFP+ axons were colocalized in all forebrain areas with TdTomato+ axons (Figure 1— figure supplement 1K,L).

106 Lmx1b controls formation of descending 5-HT projection pathways

As Lmx1b is strongly expressed in all medullary raphe 5-HT neurons, we next investigated descending 5-HT axon development. In control animals, 80% of

Tph2+ medullary cell bodies expressed TdTomato and 92% of TdTomato+ cell bodies were co-labeled with Tph2 (Figure 2—figure supple-ment 1A). TdTomato+ axon patterns in control E15.5 embryos corresponded closely with the pattern of developing 5-HT-labeled axons in the ventral and lateral funiculi of the spinal cord, thus indicating specific labeling of descending 5-HT axons with TdTomato

(Figure 2—figure supplement 1B). In addition, the pattern of control TdTomato+ axon distribution throughout the embryonic and adult spinal cord corresponded to descending 5-HT axons patterns in gray and white matter described in the rat and mouse with an anti-5-HT antibody (Ballion et al., 2002; Rajaofetra et al.,

1989). We confirmed that the number of TdTomato-labeled Pet1+ neurons in the medullary raphe nuclei did not differ between Lmx1bcKO and control mice at 3 months of age (Figure 2—figure supplement 1C). Further, Lmx1bcKO TdTomato+ cells did not express Tph2, confirming targeting of Lmx1b (Figure 2—figure supplement 1D).

We found a severe lack of TdTomato+ axons in the white matter funiculi, through which 5-HT axon projections normally extend caudally through the spinal cord (Figure 2A; Figure 2—figure supplement 2A). In addition, a severe reduction of Lmx1bcKO TdTomato+ axons was found in gray matter at all levels of the spinal cord (Figure 2A). The normally dense innervation to the dorsal and ventral horns of control spinal cords was dramatically decreased in cervical, thoracic and

107 lumbar levels in Lmx1bcKO mice (Figure 2A; Figure 2—figure supplement 2B,C).

At cervical levels there was a 67% deficit of TdTomato+ axons in white matter while at lumbar levels there was a 92% deficit (Figure 2B). Quantitation of

TdTomato+ axons in gray matter revealed a progressive cervical to lumbar deficit, 73% and 94%, respectively in Lmx1bcKO cords compared to control cords (Figure 2C). Collectively, our results indicate that Lmx1b deficiency severely disrupts long-range 5-HT axon architecture in the forebrain and spinal cord and that the deficit becomes increasingly profound with increasing distance from 5-HT cell bodies.

Delayed primary pathway formation and aborted selective pathway routing in Lmx1bcKO mice

We next followed the development of Lmx1bcKO TdTomato+ axons to distinguish among several possible explanations for the dramatic defects in ascending and descending 5-HT axon pathways: i, disrupted primary pathway formation; ii, failure to selectively route axons through pre-existing tracts; iii, abnormal axon trajectories with subsequent failure to extend, iv, normal pathway development followed by dieback. We first investigated TdTomato+ axons at

E13.5 when primary pathway formation is underway and Lmx1b conditional targeting has occurred. At this stage, TdTomato+ axon out-growth and trajectory in Lmx1bcKO mice were normal as they coursed through the MFB over the mesencephalic flexure (Figure 3A). At E16.5, however, Lmx1bcKO TdTomato+ axons exhibited a clear failure to extend as far rostrally as control axons,

108 suggesting either aborted or delayed axon outgrowth along the more rostral portions of the MFB during late stage primary pathway formation (Figure 3B). At

E18.5, the density of control TdTomato+ axons was maximal throughout the MFB and TdTomato+ axons were present as far rostral as the septum and diagonal band, where 5-HT axons turn dorsally to navigate through the cingulum, supracallosal stria, and fimbria-fornix (Figure 3C). Lmx1bcKO TdTomato+ axons now appeared at normal density throughout the MFB, thus supporting delayed axon outgrowth at E16.5 (data not shown). However, at this stage, most

Lmx1bcKO TdTomato+ axons failed to turn dorsally and remained stalled within the MFB. A smaller number of Lmx1bcKO TdTomato+ axons did turn dorsally away from the MFB to pass through the septum and diagonal band (Figure 3C).

Virtually all Lmx1bcKO axons failed to extend and selectively route into the cingulum and fimbria-fornix, thus failing to reach the cortex and hippocampus.

Lmx1bcKO TdTomato+ axons still did not exhibit ectopic trajectories. Further examination in one-month old Lmx1bcKO animals revealed a similar deficit of

TdTomato+ axons in distal forebrain tracts (data not shown). These findings suggest that conditional targeting of Lmx1b at E12.5 results in delayed primary pathway formation followed by a profound failure of ascending axons to selectively route into pre-existing fiber tracts.

Analysis of descending TdTomato+ fibers at E15.5 revealed similar initial axon outgrowth through the caudal medulla to the upper cervical spinal cord from

Lmx1bcKO and control medullary 5-HT cell bodies (Figure 3D). Although

Lmx1bcKO TdTomato+ axons appropriately entered the lateral and ventral

109 funiculi of the cervical spinal cord, greatly reduced densities were evident beginning at mid-cervical levels (Figure 3D). At lumbar levels, Lmx1bcKO

TdTomato+ axons were nearly undetectable in the lateral and ventral funiculi

(Figure 3D). These results indicate that TdTomato+ axons were not able to extend into the funiculi, which caused a severe and progressive cervical to lumbar deficit of 5-HT innervation in the adult Lmx1bcKO spinal cord (Figures 2 and 3D).

Lmx1b acts temporally to control 5-HT axon selective pathways

As formation of 5-HT axon projection pathways occurs over several weeks of embryonic to early postnatal neural maturation we next sought to determine whether Lmx1b is temporally required for 5-HT axon pathway formation. To address this question, we developed a tamoxifen inducible targeting strategy

(Figure 4A) to knock-down Lmx1b at different early postnatal timepoints. Thus, we generated Lmx1bicKO (Lmx1bfl/fl; Tph2-CreER; Ai9) and iControl (Lmx1b+/+; Tph2-

CreER; Ai9) mice. Subcutaneous delivery of tamoxifen into iControl pups resulted in approximately 94% of Tph2+ neurons co-labeled with TdTomato in the

DRN/MRN/B9 (Figure 4—figure supplement 1A,B). Conversely, 94% of

TdTomato+ cells were co-labeled with Tph2 (Figure 4—figure supplement 1B). As with the Pet1-Cre driver, the Tph2-CreER activated TdTomato+ cells that are

Tph2- evidently express Tph2 at a low level (Deneris and Gaspar, 2018; Okaty et al., 2015). RT-qPCR analysis of flow sorted TdTomato+ cells verified that Lmx1b mRNA was significantly reduced in Lmx1bicKO mice (Figure 4B). Equivalent

110 numbers of TdTomato-labeled cell bodies were generated in Lmx1bicKO versus iControl mice after injection of tamoxifen (Figure 4—figure supplement 1C,D). In contrast to Lmx1bcKO TdTomato+ cells, cell body size and distribution of tamoxifen treated Lmx1bicKO TdTomato+ cells were not different from that of iControl TdTomato+ cells (Figure 4—figure supplement 1C,E).

We analyzed TdTomato+ axons in P1 targeted Lmx1bicKO mice four weeks after tamoxifen delivery, when 5-HT axon pathways have fully matured

(Lidov and Molliver, 1982; Maddaloni et al., 2017). We found a dramatic deficit in

TdTomato+ arbors throughout the hippocampus in Lmx1bicKO mice compared to controls (Figure 4C). In addition, TdTomato+ arbors were severely reduced in all layers of the cortex with the most medial layers almost completely devoid of arbors in Lmx1bicKO mice (Figure 4D). These results clearly demonstrate that

Lmx1b is temporally required for postnatal development of 5-HT terminal axons.

However, we noticed that in addition to reduced 5-HT arbors in the hippocampus and cortex, TdTomato labeling within the supracallosal stria and cingulum, major routes to the hippocampus and cortex, were consistently less intense in P1 targeted Lmx1bicKO mice compared to controls (Figure 4C, asterisk). Indeed, further detailed examination revealed that these major 5-HT routes had not yet fully formed (Figure 4E). Quantification of TdTomato+ axons within the cingulum and supracallosal stria tracts confirmed a significant decrease in P1 targeted

Lmx1bicKO mice compared to controls (Figure 4F). These results indicate that i, long-range 5-HT axon routing is continuing to develop in the early neonatal

111 period and ii, Lmx1b is required at this stage for completion of 5-HT axon selective routing.

Lmx1b switches function to control terminal arborization

We next targeted Lmx1bicKO mice at P3 to further investigate temporal requirements for Lmx1b (Figure 5A). Importantly, in contrast to findings obtained in P1 targeted mice, we found that the density of TdTomato-labeled fibers in the supracallosal stria was now similar in P3 targeted Lmx1bicKO and iControl mice

(Figure 5—figure supplement 1A). Furthermore, TdTomato-labeled fibers in the cingulum, one of the longest of 5-HT forebrain tracts, was now comparable in P3 targeted Lmx1bicKO and iControl mice (Figure 5B). Quantification of the cingulum and supracallosal stria confirmed no significant difference between P3 targeted Lmx1bicKO and iControl mice (Figure 5—figure supplement 1B). These findings indicate that 5-HT axon routing was complete in P3 targeted Lmx1bicKO mice.

Despite fully developed 5-HT selective pathway routes in the forebrain,

TdTomato+ arbors were significantly decreased throughout the hippocampus

(Figure 5C; Figure 5—figure supplement 1B). A majority of remaining arbors detected within the hippocampus of P3 targeted Lmx1bicKO mice were found in the molecular layer (SLM), which are the first 5-HT arbors to appear in the hippocampus during rat development (Lidov and Molliver, 1982). TdTomato+ arbors were also severely decreased in all cortical layers of the P3 targeted

Lmx1bicKO brain including in layer I and VI where axons of passage also exist

112 (Figure 5D) (Lidov and Molliver, 1982). Quantification of the cortex confirmed a significant decrease of TdTomato+ axon arbors in P3 targeted Lmx1bicKO compared to iControls (Figure 5—figure supplement 1B). It is important to note that many 5-HT terminal arbors have already been generated in the forebrain at

P3 (Lidov and Molliver, 1982; Maddaloni et al., 2017). Thus, Lmx1b targeting at

P3 results in the failure of new arbors to form while leaving previously generated arbors intact.

Interestingly, arborization deficits were also found within the thalamus of

P3 targeted Lmx1bicKO mice. Similar to what we observed in Lmx1bicKO mice, the normally dense arborization within the PVT was absent in P3 targeted

Lmx1bicKO mice, rendering the PVT indiscernible (Figure 5E). Together, these results reveal a successive stage of continuous Lmx1b function during which it switches to control terminal arborization of forebrain 5-HT axons.

Targeting of 5-HT synthesis does not impair formation of forebrain and spinal cord 5-HT arbors

Lack of 5-HT itself has been reported to affect development of forebrain 5-

HT terminal arbor patterns (Migliarini et al., 2013). Therefore, we next investigated whether the arborization defects present in Lmx1b deficient mice resulted from reduced Tph2 expression and consequently reduced 5-HT levels.

To specifically knock down Tph2 to a level comparable to that in Lmx1bcKO mice, we generated Tph2cKO (Tph2fl/fl;Pet1-Cre;Ai9) and control (Tph2+/+;Pet1-Cre;Ai9) mice. The numbers of TdTomato+ cells in each raphe nuclei did not differ in

113 Tph2cKO and control mice (Figure 6A). In the DRN/MRN/B9 nuclei, we found a

74% reduction of Tph2+ neurons in Tph2cKO compared to the 79% reduction of

Tph2+ neurons in Lmx1bcKO mice (Figure 6B,C). Additionally, we confirmed loss of 5-HT itself in Tph2cKO mice (Figure 6D).

We analyzed TdTomato+ terminal arbors throughout the forebrain in adult

Tph2cKO mice. We did not find differences in Tph2cKO versus control

TdTomato-labeled arbor patterns anywhere in the forebrain including the hippocampus, cortex, and the PVT (Figure 6E). Further, despite loss of Tph2 in medullary neurons of Tph2cKO mice (Figure 6F), analysis of the spinal cord from cervical to lumbar levels revealed no differences in axon densities compared to control mice (Figure 6G). Collectively, these results indicate that the loss of Tph2 and thus reduced levels of 5-HT in Lmx1bcKO or Lmx1bicKO mice did not contribute to either the routing or arborization defects. Evidently, Tph2 and 5-HT levels need to be reduced to a greater extent than we achieved in either our

Lmx1bcKO or our Tph2cKO mice to generate the 5-HT arborization defects observed in mice engineered to completely eliminate Tph2 function and brain 5-

HT (Migliarini et al., 2013).

Lmx1b controlled axon-related transcriptomes

Genome-wide analysis of Lmx1b controlled serotonergic transcriptomes has not been performed and consequently only a few serotonergic genes are known Lmx1b targets (Zhao et al., 2006). We performed RNA-sequencing (RNA- seq) on flow sorted rostral hindbrain 5-HT neurons, which give rise to the

114 ascending system, and caudal hindbrain 5-HT neurons, which give rise to the descending system, obtained from Lmx1bcKO and control E17.5 embryos

(Figure 7A). Differential expression analysis revealed that the known Lmx1b target genes, Tph2, Slc18a2, Slc6a4, SCG II, Ctr, were significantly decreased in our E17.5 rostral and caudal Lmx1bcKO datasets. Moreover, we found significantly decreased expression of other 5-HT pathway genes, Ddc, Slc22a3,

Htr1a, Gch1, Gchfr, in both rostral and caudal Lmx1bcKO sorted 5-HT neurons that were previously unknown Lmx1b tar-gets (Figure 7B,C).

In addition to these changes, expression of many other genes was altered in the rostral and caudal sorted neurons (Figure 7D,E). In rostral Lmx1bcKO neurons, expression of 784 genes were significantly decreased, while 118 genes showed significantly increased expression (Figure 7F,G). In caudal Lmx1bcKO neurons, expression of 1529 genes were significantly decreased and 772 were significantly increased (Figure 7F,G). Although there were many genes commonly regulated by Lmx1b, many more were uniquely regulated by Lmx1b in rostral versus caudal 5-HT neurons (Figure 7F–H).

Over-representation analysis of Gene Ontology (GO) terms from rostral and caudal Lmx1b-regulated 5-HT neuron genes revealed a pattern of strong enrichment for genes involved in axon development and morphogenesis (Figure

7I). In fact, the top enriched GO term, axon development, represents over 150 genes with a Benjamini-Hochberg (BH) adjusted p-value of 0. Many other axon- related terms included axon part, cell leading edge, regulation of supramolecular fiber organization, neuron projection fasciculation, tissue migration, cell-substrate

115 adhesion, negative chemotaxis, morphogenesis of a branching structure, cell leading edge, extracellular matrix binding, and semaphorin receptor binding were enriched at FDR ≤ 0.05. Grouping the genes from all of these axon-related categories together produced a dataset of 422 genes regulated by Lmx1b in rostral and/or caudal 5-HT neurons that have known roles in axon development or function (Supplementary file 1). Sixty-six of these downstream genes were significantly altered in both the ascending and descending 5-HT subsystems; 71 genes were uniquely altered in the ascending subsystem while 285 were uniquely altered in the descending subsystem. This analysis suggests that

Lmx1b coordinates distinct axonal regulatory programs and transcriptomes to build the two divergent axonal subsystems.

Previous studies have implicated several effector genes in the growth and patterning of 5-HT axons (Chen et al., 2017; Donovan et al., 2002; Fournet et al.,

2010; Katori et al., 2017; Lee et al., 2005). In most cases, however, is it not known whether these genes perform cell intrinsic functions in 5-HT neurons.

Examination of our rostral and caudal RNA-seq datasets revealed significantly decreased expression of most but not all of these genes (Figure 7J,K). We validated the expression changes of these genes with RT-qPCR from flow-sorted

E17.5 rostral 5-HT neurons (Figure 7O). Together, these expression studies suggest Lmx1b controls ascending and descending 5-HT projection pathways in part through Gap43, Pcdhac2, Ndn, Ret, Ntrk2, Map6 (STOP), and Celsr3 but that potentially hundreds of other functionally diverse Lmx1b-controlled genes

116 are likely involved in the formation of 5-HT projection pathways (Figure 7J,K,O;

Supplementary file 1).

An ascending-specific axonal Lmx1→Pet1 regulatory cascade

The regulatory interactions among transcription factors in the 5-HT GRN are poorly understood but are likely to be important for the development of 5-HT neurons. We reasoned that further study of these interactions might illuminate the regulatory mechanisms through which Lmx1b temporally controls formation of ascending and descending 5-HT axon pathways. Our whole genome expression analyses revealed complex effects of Lmx1b deficiency on the expression of other regulatory factors in the 5-HT GRN (Figure 7L–O).

Decreased Pet1 expression was the most consistent and persistent change among the regulatory factors in the rostral and caudal 5-HT GRNs after conditional targeting of Lmx1b, which raised the possibility that an Lmx1b→Pet1 regulatory cascade acts in the formation of 5-HT axons.

To investigate this idea, we generated Pet1cKO (Pet1fl/fl;Pet1-Cre;Ai9) and control (Pet1+/+;Pet1-Cre; Ai9) mice (Liu et al., 2010). We imagined three alternative experimental outcomes: i, Pet1cKO fully phenocopies the axon defects found in

Lmx1bcKO supporting an exclusive Lmx1b→Pet1 regulatory cascade in 5-HT axon formation, ii, 5-HT axon projection pathways are intact in Pet1cKO mice and therefore the Lmx1b→Pet1 regulatory path is not operational in 5-HT axon development or iii, Pet1cKO incompletely phenocopies Lmx1bcKO axon defects

117 suggesting that the Lmx1b→Pet1 cascade operates in parallel with other Lmx1b orchestrated regulatory programs.

We first examined TdTomato+ axon patterns in adult Pet1cKO versus control spinal cords. We confirmed Pet1 transcript loss in medullary 5-HT neurons by in situ hybridization (ISH) (Figure 8—figure supplement 1A).

TdTomato+ axon patterns from cervical to lumbar levels of the spinal cord were similar in Pet1cKO and control mice (Figure 8A). Quantitation of total TdTomato+ axons in gray and white matter revealed no significant difference between

Pet1cKO and control mice (Figure 8B). These findings indicate that Pet1, and consequently the hypothetical Lmx1b→Pet1 cascade, is not required for the formation of descending 5-HT axon pathways (Figure 8—figure supplement 1E).

We next investigated the Pet1cKO forebrain and found in striking contrast to the spinal cord that few if any TdTomato+ axons were present in the olfactory bulb, cortex, amygdala, hippocampus, striatum and many regions of the hypothalamus in Pet1cKO forebrains (Figure 8C–E). Furthermore, the pattern of

TdTomato+ axons in the thalamus of Pet1cKO mice was severely disrupted with notable patches of clumped fibers and other areas with few, if any, fibers (Figure

8—figure supplement 1B). Similar to Lmx1bcKO mice, TdTomato+ axons were present in normal density and with proper ascending trajectory within the MFB of

Pet1cKO mice (Figure 8—figure supplement 1C). These findings clearly demonstrate a requirement for Pet1 in forebrain 5-HT pathway formation and thus distinct transcription factor requirements in the generation of ascending and descending 5-HT axon projection pathways.

118 Although Pet1 conditional targeting largely phenocopies the forebrain 5-

HT axon defects found in Lmx1bcKO mice, we noticed distinctly different patterns of TdTomato+ arbors in the Pet1cKO vs. Lmx1bcKO thalamus (Figure 8—figure supplement 1B). The Pet1cKO thalamus lacked TdTomato+ axons in lateral regions while in other thalamic regions we found abnormal clumping of Pet1cKO

TdTomato+ axons (Figure 8—figure supplement 1B). Further, in contrast to the failure of region (Figure 8—figure supplement 1B). This suggests Lmx1b may act independently of Pet1 in this specific highly discrete target field. Interestingly, early Pet1 deficiency did not result in reduced cell body size thus demonstrating that reduced cell body size did not contribute to the axon defects in Lmx1bcKO mice (Figure 8—figure supplement 1D). The robust similarities and yet distinct differences in TdTomato+ axon patterns in Pet1cKO compared to Lmx1bcKO forebrain support a model in which Lmx1b→Pet1 is the main regulatory program but that additional minor Lmx1b- or Pet1-orchestrated regulatory pathways operate in building ascending 5-HT axonal architectures (Figure 8—figure supplement 1F).

We investigated whether Lmx1b and Pet1 compensate for one another in the formation of 5-HT axons, by examining double-targeted mice (DKO: Lmx1bfl/fl;

Pet1fl/fl;Pet1-Cre;Ai9) and controls (Lmx1b+/+; Pet1+/+;Pet1-Cre;Ai9). Analysis of DKO spinal cords revealed deficits in TdTomato+ axons patterns to similar levels as

Lmx1bcKO spinal cord, further confirming Pet1 does not play a role in descending 5-HT axon development (Figure 8—figure supplement 2A). In the forebrain, we did not find a more extreme deficit in TdTomato+ axons in DKO

119 mice compared to Lmx1bcKO mice. The DKO thalamus mirrored the Lmx1bcKO thalamus in the specific clumping of arbors and inability to properly pattern arbors in the PVT (Figure 8—figure supplement 2B).

To investigate whether the initial 1–2 days of Pet1 expression, not targeted by Pet1-Cre in Pet1cKO mice is required for primary ascending pathway formation, we analyzed Pet1-/-;Pet1-YFP mice in which the Pet1-YFP transgene labels control and mutant Pet1+ cell bodies and their axons with YFP (Hawthorne et al., 2010). We found comparable initial 5-HT primary axon outgrowth in Pet1-/- and control 5-HT neuron cell bodies at E13.5 (Figure 8—figure supplement 2C).

Lmx1b acts through Pet1 to temporally control postnatal stage-specific gene expression and forebrain arborization

Stage-specific regulatory functions suggest potential stage-specific control of gene expression to fulfill successive steps in the morphological maturation of axon projection pathways. Thus, we next sought to determine whether the

Lmx1b→Pet1 cascade temporally regulates expression of axon-related genes that are required at specific stages of 5-HT pathway formation. We found that

Lmx1b continues to control Pet1 postnatally as Pet1 transcript levels were decreased in P3 targeted Lmx1bicKO mice (Figure 9A,B). Next, we used RNA- sequencing to find common Lmx1b and Pet1 targets. Comparing rostral Lmx1b and Pet1 regulated genes, we found 82 regulated genes present in both datasets

(Figure 9C). By intersecting rostral Lmx1b and Pet1 regulated genes with the set of Lmx1b-regulated axon-related genes (Supplementary file 1), we found 15 co- regulated genes (Figure 9C). Interestingly, one of these coregulated genes is

120 Pcdhac2. Extensive studies of Pcdhac2 have demonstrated its key intrinsic role in the formation and patterning 5-HT axon arbors (Chen et al., 2017; Katori et al.,

2009; Katori et al., 2017). In particular, Pcdhac2 deficient mice exhibit a lack of forebrain 5-HT arbors and severe clumping of remaining arbors, thus raising a functional link to Lmx1b and Pet1.

Since Pcdhac2 is in the Pcdh alpha gene cluster and shares 3 (out of 4) exons with all other Pcdh alpha genes, we performed differential exon expression analysis with the Lmx1b and Pet1 RNA-seq datasets. By testing the only unique exon for Pcdhac2, we found that Pcdhac2 was significantly regulated by Lmx1b and Pet1 at E17.5 (Figure 9D). In contrast, Pcdhac2 expression was not significantly regulated by Pet1 at earlier stages (Wyler et al., 2016). Furthermore, our time-series RNA-seq analyses (Wyler et al., 2016) showed a significant and dramatic upregulation of Pcdhac2 only at the onset of the arborization stage

(Figure 9E). Thus, Pcdhac2 expression is precisely controlled at the stage in which it is required for morphological maturation of 5-HT neurons.

To determine whether Lmx1b was required to control the postnatal upregulation of Pcdhac2, we treated Lmx1bicKO mice with tamoxifen at postnatal day three and analyzed Pcdhac2 expression by ISH at P14, when arborization is profusely developing. Indeed, we found substantially reduced expression of

Pcdhac2 in P3 Lmx1bicKO mice (Figure 9F). To further probe whether the

Lmx1b→Pet1 regulatory cascade is required for upregulation of Pcdhac2 during arborization, we generated Pet1icKO (Pet1fl/fl; Tph2-CreER; Ai9) mice. We first verified that Pet1 was effectively targeted in P3 Pet1icKO mice by RT-qPCR of flow

121 sorted TdTomato+ cells as well as ISH for Pet1 (Figure 9—figure supplement

1A,B). We confirmed that similar numbers of TdTomato+ neurons were activated in P3 Pet1icKO mice (Figure 9—figure supplement 1C,D) and that long-range routes were filled compared to controls (Figure 9—figure supplement 1E). We found that Pet1 did not regulate Lmx1b at this stage (Figure 9—figure supplement 1A). Interestingly, we found that Pet1 was also required for the dramatic postnatal upregulation of Pcdhac2 expression (Figure 9G). These findings show that the Lmx1b→Pet1 regulatory cascade acts during the arborization stage to temporally control Pcdhac2 upregulation.

To determine if Pcdhac2 is a direct target of Lmx1b→Pet1, we analyzed our previously published ChIP-seq datasets (Wyler et al., 2016) for mycPet1 binding sites within the Pcdhac2 locus. Notably, we identified only two significant mycPet1 ChIP-seq peaks throughout the entire 250 kb Pcdha locus (Figure 9H).

One peak was located at the 5’ end of the unique Pcdhac2 first exon. The second peak was located precisely within the third-intronic DNase I hypersensitivity site, HS7, which marks a well characterized transcriptional enhancer known to regulate midbrain expression of the Pcdha gene isoform

(Kehayova et al., 2011; Ribich et al., 2006) (Figure 9H). Although the mycPet1 peak at the TSS of Pcdhac2 does not contain a match to the known Pet1 binding motif, the second peak within HS7 does contain a significant match to the Pet1 motif (Wei et al., 2010) (Figure 9I).

To determine whether Lmx1b→Pet1 cascade acts postnatally to control terminal arborization we analyzed the forebrains of P3 targeted Pet1icKO mice.

122 Analyses performed at P49 revealed a severe deficit of 5-HT arbors in the hippocampus and cortex of P3 targeted Pet1icKO mice, comparable to the deficit found in P3 targeted Lmx1bicKO mice (Figures 5C, 5D; Figures 9J, 9K). The timing of 5-HT terminal arborization varies widely in different regions of the early postnatal forebrain (Lidov and Molliver, 1982; Maddaloni et al., 2017). Thus, we next sought to determine whether the Lmx1b→Pet1 cascade continues to control arborization in forebrain regions that are late to develop their mature axon patterns. To investigate this, we administered a single dose of tamoxifen to

Pet1icKO pups at P5. We focused our subsequent analyses on the striatum, which is one of last regions of the forebrain to develop mature 5-HT arborization patterns (Lidov and Molliver, 1982). Interestingly, we found a notable deficit in the

5-HT arbors throughout the striatum (Figure 9L; Figure 9—figure supplement 1F).

Together, our findings demonstrate that the Lmx1b→Pet1 cascade operates continually over an extended postnatal period to control stage-specific gene expression and to generate both early and late 5-HT terminal arbors patterns in different forebrain regions.

Discussion

The process of long-range axon pathway formation occurs in temporally defined stages over an extended period during which successive morphological events occur (Fame et al., 2011; Lidov and Molliver, 1982; Shirasaki and Pfaff,

2002). Many regulatory factors have been implicated in the intrinsic control

(Santiago and Bashaw, 2014) of axon outgrowth, target selection, and terminal

123 arborization (Arlotta et al., 2005; Chen et al., 2005; Galazo et al., 2016; Livet et al., 2002; Srivatsa et al., 2015). What is not understood are the intrinsic programs that operate temporally to progressively control the prolonged, multistage process of long-range axon projection pathway formation (Paolino et al., 2018).

Our findings uncover a temporal regulatory strategy through which a continuously expressed transcription factor, Lmx1b, operates at successive stages to control progressive steps in the postmitotic morphological maturation of long-range highly diffuse axonal projection pathways. Thus, Lmx1b through its continuous expression not only controls the capacity for 5-HT synthesis and reuptake (Zhao et al., 2006), but also the formation of long range profusely arborized projection pathways that enable delivery of the transmitter throughout the CNS.

Our findings suggest Lmx1b acts to successively control 5-HT axon primary outgrowth, selective routing, and terminal arborization. We uncovered a delay in primary 5-HT axon outgrowth between E16.5 to E18.5 in Lmx1bcKO mice. However, initiation of primary axon outgrowth toward the fore-brain or spinal cord was not disrupted in either Lmx1bcKO, Pet1cKO, or Pet1-/- mice. As

5-HT axonogenesis occurs concomitant with the onset of 5-HT synthesis in newborn 5-HT neurons (Hawthorne et al., 2010), perhaps upstream regulatory programs operating at the progenitor stage control initial axon outgrowth from newborn 5-HT neurons (Briscoe et al., 1999; Jacob et al., 2007; Pattyn et al.,

2004).

The profound defect in the subsequent selective 5-HT axon routing through the cingulum, SCS, and fornix suggests a failure of intrinsic growth

124 extension beyond selective routing choice points. In support of this, we never observed ectopic 5-HT axon trajectories. Yet, we leave open the possibility that axon guidance defects contribute to the failure of 5-HT axons to selectively extend through proper routes. 5-HT axons normally turn dorsally through the septal area at E18.5, which might constitute a critical choice point in building expansive axon 5-HT architectures. In contrast, the vast majority of mutant 5-HT axons fail to make the dorsal turn and then fail to continue into selective tracts. In support of a possible guidance defect, our RNA-seq studies indicated that Lmx1b controls expression of a large number of genes encoding guidance receptors, guidance receptor ligands, and adhesion molecules.

Transcription factors function within GRNs as elucidated in the specification and differentiation of various types of neurons (Deneris and Gaspar,

2018; Guillemot, 2007; Shirasaki and Pfaff, 2002). How transcription factors temporally interact within networks to progressively generate precise connectivity patterns is poorly understood (Paolino et al., 2018). A focus of our experiments was to determine whether Lmx1b functions in the context of the 5-HT GRN to control progressive development of 5-HT axonal pathways. We uncovered a temporal requirement for Lmx1b in maintaining Pet1 expression during the early postnatal stage of 5-HT pathway formation. This finding together with postnatal temporal targeting of Pet1 revealed the Lmx1b→Pet1 regulatory cascade acts stage specifically to control selective pathway routing and arborization of forebrain 5-HT axons. Despite the requirement for Pet1 in the acquisition of medullary 5-HT neuron-type identity (Hendricks et al., 2003; Kiyasova et al.,

125 2011), it was surprising to find that Pet1 was not required for routing and arborization of descending 5-HT axons, thus revealing distinct transcription factor requirements in the generation of ascending and descending 5-HT axon pathways. Perhaps, Lmx1b operates with other continuously expressed serotonergic transcription factors in a regulatory cascade to control descending

5-HT axonal pathways. Gata3 is an interesting candidate as it functions more prominently in the medullary 5-HT neurons that generate the descending subsystem than 5-HT neurons that give rise to the ascending subsystem (Pattyn et al., 2004; van Doorninck et al., 1999).

The hundreds of axon related genes controlled by Lmx1b in the ascending and descending sub-systems suggests that mis-expression of scores of functionally diverse effector genes accounts for the axon pathway defects we have reported. One gene of note is Gap43. Telencephalic commissures of

Gap43 null mice fail to form, including the hippocampal commissure and the corpus callosum (Shen et al., 2002). This may largely explain the 5-HT neuron axonal projection deficits reported in the Gap43 null forebrain (Donovan et al.,

2002). Although Gap43 is expressed in 5-HT neurons, it is not yet known whether it plays an intrinsic role in 5-HT axon development (Bendotti et al.,

1991). Our findings highlight the potential importance of an intrinsic

Lmx1b→Pet1→Gap43→5-HT axon regulatory path given that Gap43 expression is reduced in both Lmx1bcKO and Pet1cKO mice. It will be interesting to investigate this putative path in 5-HT conditionally targeted Gap43 mice to

126 determine whether it accounts for the selective routing defects present in

Lmx1bcKO and Pet1cKO mice.

The stage specific regulatory roles we have uncovered suggests Lmx1b may temporally control certain genes to fulfill stage-specific events in the morphological maturation of 5-HT neurons. In support of this idea, our findings reveal that Pcdhac2, a key intrinsic effector of 5-HT terminal arbor growth and patterning (Chen et al., 2017; Katori et al., 2009; Katori et al., 2017), is dynamically regulated by the ascending Lmx1b→Pet1 cascade. Tamoxifen- inducible targeting of Lmx1b and Pet1 revealed a temporal requirement for

Lmx1b and Pet1 in the postnatal upregulation of Pcdhac2. Notably, our ChIP-seq datasets revealed Pet1 occupancy at the Pcdhac2-specific promoter and HS7 suggesting Lmx1b→Pet1 directly controls upregulation of Pcdhac2 during the postnatal arborization stage. Perhaps Pet1 occupancy at HS7 facilitates cohesin- mediated looping from HS7 to the Pcdhac2 promoter thus accounting for Pet1 occupancy at this promoter in the absence of a high affinity Pet1 binding motif

(Guo et al., 2012).

Deficient expression of a single stage-specifically expressed target gene such as Pcdhac2 likely does not account for the severe and complex multi-stage defects in long-range 5-HT axon pathway formation reported here. However, our studies do serve to illustrate the concept that Lmx1b and Pet1 are critical terminal selectors of neuronal morphology and likely do so by dynamically regulating downstream genes that are required at specific stages for the progressive morphological maturation of 5-HT neurons. Perhaps subsets of

127 Lmx1b controlled genes act in different 5-HT neuron subtypes and at distinct stages to control the specific routes of 5-HT axons to diverse forebrain and spinal cord targets.

We speculate that our findings illustrate a possible general mechanism through which continuously expressed terminal selectors build long-range diffuse axon pathways. In addition to 5-HT neurons, noradrenergic, dopaminergic, and histaminergic neurons also generate expansive and highly arborized axonal architectures (Bjo¨rklund and Dunnett, 2007; Haas et al., 2008; Moore and

Bloom, 1979). Lmx1b plays a critical role in dopaminergic (DA) neuron development. However, in contrast to 5-HT neurons, Lmx1b is co-expressed in postmitotic DA neurons with its paralog, Lmx1a, up to about 2 months of age

(Laguna et al., 2015). These two LIM HD factors play compensatory roles in mesDA neuron specification and differentiation (Laguna et al., 2015; Yan et al.,

2011). Simultaneous DA targeting of Lmx1a and Lmx1b beginning at E14 resulted in diminished DA axon outgrowth to the dorsal striatum while DA axon projections to the ventral striatum and other DA axon targets throughout the forebrain remained intact (Chabrat et al., 2017). These findings suggest that temporal control of axon routing and arborization of the DA mesostriatal, mesolimbic and mesocortical projection pathways may be controlled by different continuously acting terminal selector transcription factors expressed in DA neurons.

In addition to their tremendous intrinsic capacity for long-range axonal growth and arborization during development, 5-HT neurons are noted for their

128 rare ability to sprout and regenerate axons after injury (Hawthorne et al., 2011;

Jin et al., 2016). Given Lmx1b’s continuous expression into adulthood and crucial function in the formation of 5-HT projection pathways an intriguing line of investigation will be to determine whether Lmx1b transcriptionally powers 5-HT neuron’s intrinsic potential for axon regrowth following injury in adult brain or spinal cord, which may suggest ways to harness that power for development of new repair strategies.

129 130 Figure 1. Lmx1b is required for the formation of ascending 5-HT axon projection pathways. (A, B) Ascending 5-HT axonal projection system immunolabeled using an anti-RFP antibody to TdTomato in whole sagittal forebrain sections of 3 month old mice displayed by heatmap. Lmx1bcKO

TdTomato+ axons were nearly absent in numerous brain regions (B) compared to controls (A) (n = 6, controls; n = 7, Lmx1bcKO adult mice). Right, schematics depicting 5-HT axon trajectories in Lmx1bcKO vs. control brains. Scale bars,

1000 µm. OB, olfactory bulb; ctx, cortex; cg, cingulum; cc, corpus callosum; hipp, hippocampus; spt, septum; hyp, hypothalamus; thal, thalamus; sc, superior colliculus; MFB, medial forebrain bundle; DRN, dorsal raphe nucleus. Schematic

(right): ot, olfactory tract; cg/SCS, cingulum/supracallosal stria; fx, fornix; sm, stria medularis; fr, fasciculus retroflexus. (C) Confocal images of

TdTomato+axons in sagittal sections. Lmx1bcKO axons failed to fill cingulum bundles or innervate the hippocampus. Scale bars, 200 µm. cg, cingulum; cc, corpus callosum; LSM, lacunosum moleculare; DG, dentate gyrus; CA1 of hippocampus. (D) Coronal sections of cortex show near complete lack of Lmx1bcKO TdTomato+ axons Scale bars, 50 µm. (E) Coronal view of altered patterns of TdTomato+ axons in Lmx1bcKO thalamus. Arrowheads indicate areas devoid of axons in Lmx1bcKO thalamus. Numbers correspond to areas of axon clumping in Lmx1bcKO thalamus. See Figure 1—figure supplement 2 for high magnification images. Scale bars, 100 µm. PVT, paraventricular nucleus of the thalamus; 3V, third ventricle.

131 132 Figure 1—figure supplement 1. Surrogate marking of 5-HT cell bodies and axons and Lmx1b conditional targeting. (A) Pet1-Cre efficiency: 82% Tph2+ cells expressed TdTomato+ (RFP+/Tph2+) and 88% of TdTomato+ cells expressed Tph2 (Tph2+/RFP+) (n = 2 control mice). Data are represented as mean ± SEM. (B) TdTomato+ cells co-labeled with serotonergic marker Tph2 in the DRN. Immunofluorescence of Tph2 (green) and TdTomato (red). Scale bars,

100 µm. (C) TdTomato+ axons were found throughout the adult brain.

Lacunosum moleculare layer (LSM) of hippocampus shown here. Scale bars, 10

µm. (D) Co-immunolabeled axons at embryonic day (E) 14 with anti-5-HT and anti-RFP antibodies. Scale bars, 20 µm. (E) A majority of TdTomato+ Lmx1bcKO mutant cells did not co-localize with Tph2 (n = 6, control; n = 7, Lmx1bcKO; 2.5–

3.5 month old mice). Scale bars, 100 µm. (F) A majority of

TdTomato+ Lmx1bcKO mutant cells did not co-localize with Lmx1b protein (E15.5 shown). A small number of cells were TdTomato+ and Lmx1b+ (bottom row, arrow). Scale bars, 5 µm. (G) RT-qPCR of Lmx1b and Tph2 in flow sorted YFP+ cells from control and Lmx1bcKO E17.5 embryos (n = 4, control; n = 4, Lmx1bcKO embryos). Unpaired t-test with Welch's correction, *p<0.05,

**p<0.001, and ***p<0.0001. Data are represented as mean ± SEM. (H) Counts of TdTomato+ cells in Lmx1bcKO mice did not differ from controls. Every 4th section counted throughout DRN/MRN/B9 raphe nuclei (n = 3 mice/genotype).

Data are represented as mean ± SEM. (I) Lmx1bcKO cell bodies survive at least up to 13 months. Counts of TdTomato+ neurons in 6 matched sections for each animal (right) (n=1 animal per time point; 1mo, 3mo, 7mo, 13mo). Scale bars,

133 100µm. (I) Lmx1bcKO cell bodies survive at least up to 13 months. Counts of

TdTomato+ neurons in 6 matched sections for each animal (right) (n=1 animal per time point; 1mo, 3mo, 7mo, 13mo). Scale bars, 100µm. (J) Volume of Lmx1bcKO cell bodies were significantly smaller than controls (n = 587 control cells; n = 442 Lmx1bcKO cells). Unpaired t-test with Welch's correction, *p<0.05,

**p<0.001, and ***p<0.0001. Data are represented as mean ± SEM. (K, L)

Expression of a YFP tagged channel by AAV2 viral injection to

DRN/MRN of Lmx1bcKO and control mice. No YFP expression was found in Lmx1bcKO hippocampus (K) while YFP-labeled axons co-localized with

TdTomato in the Lmx1bcKO thalamus (L). Scale bars, 200 µm (K), 50 µm (L).

134 135 Figure 1—figure supplement 2. Lmx1b deficiency disrupts 5-HT axon patterns in the forebrain. (A) Sagittal sections of olfactory bulb show near complete lack of Lmx1bcKO TdTomato+ axons. Scale bars, 50 µm. gl, glomerular layer; gr, granule layer. (B) TdTomato+ axons severely reduced throughout Lmx1bcKO amygdala compared to controls (BLA, shown right). Scale bars, 100 µm. BLA, basolateral amygdala; st, stria terminalis; opt, optic tract. (C)

High magnification showing areas of clumping (correlate to numbers in Figure

1E) found in Lmx1bcKO thalamus compared to controls. Scale bars, 20 µm. (D)

Lack of TdTomato+ axons in the PVT of Lmx1bcKO mice. Scale bars, 50 µm.

3V, third ventricle; PVT, paraventricular nucleus of the thalamus.

136 137 Figure 2. Lmx1b is required for the formation of descending 5-HT axon projection pathways. (A) Coronal sections taken at cervical (C4), thoracic (T6), and lumbar (L3) levels of the spinal cord (diagram, left). Immunolabeling for

TdTomato shows Lmx1bcKO axons were severely reduced at every level of the cord in both gray and white matter compared to controls. Scale bars, 200 µm. m, midline; med, medulla; cc, central canal; dh, dorsal horn; vh, ventral horn; lat fun, lateral funiculi. (B, C) Quantification of total TdTomato+ axons (pixels/µm2) in white (B) and gray (C) matter at cervical and lumbar levels (n = 3, control; n = 3 Lmx1bcKO mice). Two-way ANOVA with Welch’s correction, *p<0.05,

**p<0.001, and ***p<0.0001. Data are represented as mean ± SEM.

138 139 Figure 2—figure supplement 1. Conditional targeting of Lmx1b in the descending 5-HT projection pathway. (A) Pet1-Cre efficiency: 80% of Tph2+ cells expressed TdTomato+ (RFP+/Tph2+); 92% of TdTomato+ cells expressed

Tph2 (Tph2+/RFP+) in medullary nuclei (n = 2 control mice). Data are represented as mean ± SEM. (B) TdTomato+ axon patterns in funiculi of the developing spinal cord aligned with 5- HT labeled axon patterns at E15.5 in controls (coronal view).

Scale bars, 100 µm. (C) Comparable numbers of TdTomato+ medullary cells in Lmx1bcKO and control mice (n = 2 mice/genotype). (D) Few Lmx1bcKO

TdTomato+ cell bodies expressed Tph2, a downstream target of Lmx1b. Scale bars, 100 µm.

140 141 Figure 2—figure supplement 2. Progressive deficits of 5-HT axon fibers in

Lmx1b deficient spinal cord white and gray matter. (A-C) Coronal sections immunolabeled using an anti-RFP antibody to TdTomato. Lmx1bcKO

TdTomato+ axon deficits in spinal cord white matter funiculi (A), gray matter dorsal (B), and ventral (C) horns. Red box in diagram outlines area imaged

(right). (n = 3, control; n = 3 Lmx1bcKO mice). Scale bars, 50 µm.

142 143 Figure 3. Initial axon outgrowth is delayed and selective pathway routing fails in Lmx1b deficient 5-HT neurons. (A–C) Immunolabeled

TdTomato+ ascending axons in sagittal slices at different embryonic stages.

Diagrams (left) show area of image (red box) presented for each time point.

Arrows indicate direction of growing axons. E13.5 Lmx1bcKO axons exhibited similar ascending trajectories and densities as controls (A). E16.5 Lmx1bcKO axons did not extend as far (asterisk) and were less abundant (under bracket) compared to control axons (B). E18.5 Lmx1bcKO axons failed to fill multiple axon tracts (cg, fx; asterisks) compared to controls (C). Scale bars, 50 µm (A), 200 µm

(B,C). DRN, dorsal raphe nucleus; MFB, medial forebrain bundle; mes, mesencephalic flexure; cg, cingulum bundle; fx, fornix; ac, anterior commissure; spt, septum; db, diagonal band; cc, corpus callosum. (D) Diagram

(left) depicting coronal section of an embryonic spinal cord.

TdTomato+ descending axons at E15.5 in control vs Lmx1bcKO embryos. Lmx1bcKO axons exit caudal medulla similar to controls but were severely reduced in funiculi at lower levels of the cord (mid-cervical and lumbar).

Boxed region of lateral funiculi enlarged to the right of each image. Scale bars,

100 µm (low magnification), 50 µm (high magnification-medulla), 20 µm (high magnification- cervical/lumbar insets). Lat fun, lateral funiculi; cc, central canal; m, midline; D, dorsal; V, ventral.

144 145 Figure 4. Lmx1b is temporally required for 5-HT projection pathway formation. (A) Schematic of tamoxifen-inducible approach to target Lmx1b at postnatal day (P)1. (B) RT-qPCR of flow sorted TdTomato+ neurons from postnatal targeted mice (n = 3, iControl; n = 4, Lmx1bicKO mice). Unpaired t-test with Welch's correction, *p<0.05. Data are represented as mean ± SEM. (C)

Coronal sections of P1 targeted Lmx1bicKO hippocampus compared to iControls analyzed at P31. *, incomplete formation of SCS and cingulum in P1 targeted Lmx1bicKO brain. Scale bars, 200 µm. (D) Coronal sections of P1 targeted Lmx1bicKO cortex compared to iControls analyzed at P31. Scale bars,

200 µm. (E) Coronal sections at level of corpus callosum showing incomplete formation of major 5-HT axon routes, SCS and cingulum, in P1 targeted Lmx1bicKO forebrain compared to iControls (above dotted line). Scale bars, 100 µm. cg, cingulum; SCS, supracallosal stria; cc, corpus callosum; m, midline. (F) Quantification of axons within SCS and cingulum tracts (n = 3, iControl; n = 3, Lmx1bicKO mice). Unpaired t-test with Welch's correction, p=0.0112. Data are represented as mean ± SEM.

146 147 Figure 4—figure supplement 1. Efficiency of postnatal tamoxifen inducible targeting of Lmx1b. (A) TdTomato+ cells co- localized with serotonergic marker

Tph2 in the DRN of iControl mice. Scale bars, 100 µm. (B) Tph2-CreER efficiency: 94% of TdTomato+ cells expressed Tph2 (Tph2+/RFP+) and 94% of

Tph2+ cells expressed TdTomato+ (RFP+/Tph2+) in DRN/MRN/B9 nuclei (n = 4, iControl mice). Data are represented as mean ± SEM. (C) DRN of P1 targeted Lmx1bicKO and iControl mice. Scale bars, 100 µm. (D) Cell counts confirmed comparable numbers of TdTomato+ cells in Lmx1bicKO and iControl mice. (E) Normal cell body volume in postnatal targeted Lmx1bicKO mice

(n = 464, control cells; n = 651, P3 Lmx1bicKO cells; p=0.9353). Unpaired t-test with Welch's correction. Data are represented as mean ± SEM.

148 149 Figure 5. Lmx1b temporally controls postnatal 5-HT terminal arborization.

(A) Schematic of tamoxifen inducible targeting of Lmx1b at postnatal day (P)3.

(B) Sagittal view of cingulum shows fully formed long-range axon routes in P3 targeted Lmx1bicKO mice compared to iControls. Scale bars, 200 µm. (C)

Coronal sections of hippocampus in P3 targeted Lmx1bicKO mice compared to iControls. Dashed boxed region: higher magnification image at right highlighting reduced TdTomato+ axons in Lmx1bicKO SLM. Scale bars, 200 µm (low magnification), 50 µm (high magnification). SLM, stratum lacunosum moleculare;

DG, dentate gyrus. (D) Coronal sections of cortex of P3 targeted Lmx1bicKO mice compared to iControls. Imaris tracing; right panels. Scale bars, 100 µm. (E)

Decreased TdTomato+ arbors detected in P3 targeted Lmx1bicKO PVT compared to iControls (arrows). Scale bars, 50 µm.

150 Figure 5--Figure Supplement 1

151 Figure 5—figure supplement 1. P3 targeted Lmx1bicKO mice display normal 5-HT axon routing but decreased 5-HT terminal arbors. (A) Coronal view of SCS and cingulum demonstrating complete formation of long-range axon routes in P3 targeted Lmx1bicKO mice compared to iControls. Scale bars, 100

µm. (B) Quantification of axons within SCS and cingulum tracts, cortex, and hippocampus of postnatal targeted P3 iControls and P3 Lmx1bicKO mice (n = 3, iControl; n = 3, Lmx1bicKO mice). Unpaired t-test with Welch's correction; axon tracts, p=0.1474; cortex, p=0.04; hippocampus, p=0.0277. Data are represented as mean ± SEM.

152 153 Figure 6. Specific targeting of 5-HT synthesis does not alter 5-HT arborization patterns. (A) Counts of TdTomato+ cells in each raphe nucleus of Tph2cKO mice did not differ from controls (n = 2 mice/genotype). Data are represented as mean ± SEM. (B) Comparable Tph2 knock-down in Tph2cKO and Lmx1bcKO mice. Scale bars, 100 µm. (C) Cell counts of residual

Tph2+ neurons in Tph2cKO and Lmx1bcKO mice expressed as a percentage

(n = 2 mice/genotype). Data are represented as mean ± SEM. (D)

Immunolabeling shows 5-HT was severely reduced in Tph2cKO mice. Scale bars, 100 µm. (E) Coronal forebrain sections showing no deficits of

TdTomato+ axon densities in Tph2cKO hippocampus, PVT, and cortex (n = 3 mice/genotype). LSM, lacunosum moleculare; DG, dentate gyrus; CA1 of hippocampus. Scale bars, 100 µm (PVT, cortex); 200 µm (hippocampus). (F) Co- immunolabeling for Tph2 and TdTomato in medullary neurons. Tph2 expression was severely reduced in medullary neurons of Tph2cKO mice. Scale bars, 50

µm. (G) No deficits of TdTomato+ axons were present throughout the Tph2cKO spinal cord (n = 3 mice/genotype). Scale bars, 200 µm.

154 Figure 7

A

Microdissec1ion/ '.� RNA-seq of Control Cell dissociation ·�t ii and Lmx1bcKO rostral � - Caudal FAGS "' and caudal cells D rostral E caudal

. significant f signi1icant . GO term enrichment axon developmento . : · ' . 20 categorv ··: . u . . 0" t-·- 1 • cc ..:..· 'lc,_20� �-,�,-�,-�.,��" 0 "' log2(foldchange) log2(foldchange) 15 0 U 2.0 0 oneurotransmitter transport H Q 2.S eaxonpart 0 3.0 oregulation of syn�pse s1ructure or ac1ivi1y Ql.S _ - 0 positive regulationofneuron d1fferent1at1on 0 �0- • I::, 10 Ovesiclelocalization 0 adi d •cell le ng e ge 0> regulation ofsupramolecular fiber organization i d Rostral control i lopmenial growlh 0 r r a i a i l ssue � Qneu on p ojection f sc cul t on ���� :::::�:: : � b di s n •central nervoussystem neuron fferent1at1on 5 a ra Lmx1bcKO omono n:iine1 nsport a i i di Rostral a i a • d nb n ng i Oneg t ve chemot x1s ves cle membrane id ri a a i i nucleos e-1 phosph 1aser!Qulator ct v ly •regulatio� oriontransmembrane ar a d ra o Caudal control ext cell� =i a a i �::�� ;�1�;t;��11�:�? br nch ng structure t n$p rt � � . :� a o d sem phonnreceptorbin 1ng Caudal Lmx1bcKO 0

50 Number of100 genes 150 J K L M Lmx1bcKO rostral E17.S Lmx1bcKO caudal E17.S Lmx1bcKO rostral E17.5 Lmx1bcKO caudal E17.5 2.0 1.5 1.00 2.0 1.5

1.0l------·······-- ··+----+ --·I 1.5 I i 'i1.o

0.5

0.0 '=' ro 0., t:.. � �o� 0 'ti l)j 0,0' �'l,, 4"'J o" �'l- 01,'y � f �� cf Y. -V..�0� �c .11.1 N Lmx1bcKO rostral P2

.2 i i)10 ---·------··------·· · --··---

3 1 • i o.o 2 Iii i .... CT . ,1 •. Iii ,_ 11 ii 111111

155 Figure 7. Ascending and descending Lmx1b regulated transcriptomes.

(A) Schematic for dissection of E17.5 brain to isolate rostral and caudal 5-HT neurons genetically labeled by Pet1-EYFP. After dissection, EYFP+ neurons were flow-sorted separately and prepared for RNA-sequencing (n = 3, controls; n = 2 Lmx1bcKO embryos). (B) Relative expression level of 5-HT pathway genes in rostral control versus Lmx1bcKO 5-HT neurons. Control gene expression levels were normalized to one. * indicates FDR ≤ 0.05. Data are represented as mean ± SEM. (C) Relative expression level of 5-HT pathway genes in caudal control versus Lmx1bcKO 5-HT neurons. Control gene expression levels were normalized to one. * indicates FDR ≤ 0.05. Data are represented as mean ± SEM. (D) Volcano plot for rostral control versus Lmx1bcKO differential expression. Significantly altered genes are in red with ≥log2(1.5X) and

FDR ≤ 0.05. (E) Volcano plot for caudal control versus Lmx1bcKO differential expression. Significantly altered genes are in red with ≥log2(1.5X) and

FDR ≤ 0.05. (F) Venn diagram of genes upregulated in rostral and caudal Lmx1bcKO 5-HT neurons. (G) Venn diagram of genes downregulated in rostral and caudal Lmx1bcKO 5-HT neurons. (H) Heatmap of differentially- expressed genes in rostral and caudal Lmx1bcKO 5-HT neurons. (I) GO term enrichment of Lmx1b regulated genes. BP = biological process, CC = cellular component, MF = molecular function. GO terms were enriched with FDR ≤ 0.05.

(J) Relative expression (FPKMs) of known 5-HT neuron axon-related genes in rostral Lmx1bcKO 5-HT neurons. Data are represented as mean ± SEM. (K)

Relative expression (FPKMs) of known 5-HT neuron axon-related genes in

156 caudal Lmx1bcKO 5-HT neurons. Data are represented as mean ± SEM. (L)

Relative expression (FPKMs) of 5-HT GRN transcription factors in rostral Lmx1bcKO 5-HT neurons. Data are represented as mean ± SEM. (M)

Relative expression (FPKMs) of 5-HT GRN transcription factors in caudal Lmx1bcKO 5-HT neurons. Data are represented as mean ± SEM. (N)

Relative expression (FPKMs) of 5-HT GRN transcription factors in flow sorted

TdTomato+ rostral Lmx1bcKO 5-HT neurons at postnatal day 2 (n = 3, controls; n = 4, Lmx1bcKO mice). Data are represented as mean ± SEM. (O) RT-qPCR verification of 5-HT GRN transcription factors and known axon-related genes from flow sorted rostral YFP+ Lmx1bcKO 5-HT neurons relative to control levels

(n = 4 mice/genotype). * indicates p-value≤0.05, # indicates p<0.1, t-test with

Welch’s correction. Data are represented as mean ± SEM.

157 Figure 8

A Cervical Lumbar B

White matter (funiculi)

ec u0

Gray matter Cervical Lumbar

dh dh s fun o s fun · • 0 lln l·:·llo NI' . cc � 01 � cc o 0. 0

O' 0 O' 0 � "J- � "J- vh vh cP �e\ cP �e\

C • cg cg cc cc . CA1 CA1 . LSM LSM

D • 11/111

IV IV gl >< gl Q) V V VI gr gr VI

158 Figure 8. Distinct transcription factor requirements in the formation of ascending and descending 5-HT projection pathways. (A) TdTomato+ axon innervation in Pet1cKO vs control spinal cords in 3 month old mice. Coronal semi-section views of cervical and lumbar levels. Scale bars, 200 µm. cc, central canal; dh, dorsal horn; vh, ventral horn; fun, funiculi. (B) Quantification of

TdTomato+ axons (pixels/µm2) in cervical and lumbar spinal cords (n = 3, controls; n = 3, Pet1cKO animals; Two-way ANOVA; white matter: cervical p=0.1372; lumbar p=0.6764; gray matter: cervical p=0.4440; lumbar p=0.1995).

Data are represented as mean ± SEM. (C) Decreased TdTomato+ arbors detected in Pet1cKO hippocampus compared to controls at 3 months of age.

Scale bars, 200 µm, sagittal view. (D) Decreased TdTomato+ arbors detected in Pet1cKO cortex compared to controls at 3 months of age. Scale bars, 100 µm, coronal view. (E) Decreased TdTomato+arbors detected in Pet1cKO olfactory bulb compared to controls at 3 months of age. Scale bars, 50 µm, sagittal view. cg, cingulum; cc, corpus callosum; LSM, lacunosum moleculare; DG, dentate gyrus; CA1 of hippocampus; gr, granule layer; gl, glomerular layer.

159 160 Figure 8—figure supplement 1. Pet1cKO and Lmx1bcKO mice exhibit distinct axon defects in thalamus. (A) Pet1 ISH shows Pet1 was targeted in medullary neurons of Pet1cKO mice. Scale bars, 200 µm. (B) Distinctly different clumping pattern in Pet1cKO thalamus (asterisks) compared to Lmx1bcKO thalamus.

Patterning in the PVT was normal in Pet1cKO mice (arrows). Scale bars, 200 µm. (C) No differences in early embryonic (E13.5) primary growth through the MFB in Pet1cKO embryos. Scale bars, 50 µm. (D) Cell volume analysis in Pet1cKO DRN revealed no significant difference in cell body size (n = 587 control cells; n = 549 Pet1cKO cells, p=0.4745). Compare to Lmx1bcKO cell volume, see Figure 1—figure supplement 1J.

Unpaired t-test with Welch's correction. Data are represented as mean ± SEM. (E)

Scheme illustrating evidence-supported model for control of descending 5-HT axon development (Model 2). (F) Scheme illustrating evidence-supported model for control of ascending 5-HT axon development (Model 3).

161 162 Figure 8—figure supplement 2. DKO and Pet1-/- analyses. (A) Similar 5-HT axons defects in spinal cord of DKO and Lmx1bcKO mice (n = 3 mice/genotype).

Scale bars, 200 µm. cc, central canal; dh, dorsal horn; vh, ventral horn; fun, funiculi. (B) DKO PVT was unarborized similar to Lmx1bcKO PVT (n = 3 mice/genotype). Scale bars, 100 µm. PVT, paraventricular nucleus of the thalamus; 3V, third ventricle. (C) Immunolabeled YFP+ axons in Pet1-/- embryos at E13.5 showed no deficit in primary ascending axon growth through the MFB

(n = 2 mice/genotype). Scale bars, 50 µm. MFB, medial forebrain bundle; mes, mesencephalic flexure.

163 Figure 9

A B C Pe/1 4wk post P3 P3 iControl P3 Lmx1bicKO

P3 iControl P3 LmxJbicKO

D Pcdhac2 unique exon E F P3 iControl P3 Lmx1bicKO

fo.75 20 0 "' ;iio.5o

10 ,1!� 0.25 □ G P3 iControl P3 Pel1icKO E11J5 E155 PN Pcdhac2

H 30 kb

10-6.00]

mycPet1 ChlP .....l_ Lo._. d. - ____ ... .I. ... •.J ·- L.�---� .1._ ... _l�------• HS7 DNase I site 3rd intron ...... Pcdhac2 ...... - .. - ...... --.. I I .... I ...... 2200bp ...... [0-5.50] [0-6.00] mycPet1 ChlP _____._....__.._._..__ _ _ _ �_ Pet1 motif match mycPet1 peaks - ...... Pet1 motifs '!!!!1!!17-- exon 1 --..HS q-val = 4. 75e-04 J

K

164 Figure 9. An ascending specific Lmx1b→Pet1 cascade controls stage specific 5-HT gene expression and postnatal terminal arborization.

(A) RT-qPCR analysis of Pet1 expression in flow sorted TdTomato+ neurons 4 weeks post P3 tamoxifen treatment (n = 3, iControl; n = 4, P3 Lmx1bicKO mice). Unpaired t-test with Welch's correction, *p<0.05. Data are represented as mean ± SEM. (B) Pet1 in situ hybridization in P3 targeted Lmx1bicKO mice.

Scale bars, 100 µm. (C) Venn diagram showing overlap of rostral Lmx1b and

Pet1 regulated genes and the axon-related gene dataset. Lmx1b rostral: genes controlled by Lmx1b in rostral 5-HT neurons; Pet1 rostral: genes controlled by

Pet1 in rostral 5-HT neurons; Axon genes: Lmx1b regulated rostral and caudal axon-related genes. (D) Relative expression of the unique Pcdhac2 exon in rostral Lmx1bcKO and Pet1cKO 5-HT neurons. * indicates FDR ≤ 0.05. Data are represented as mean ± SEM. (E) Developmental expression profile of Pcdhac2 in 5-HT neurons from E11.5 to early postnatal (PN). * indicates

FDR ≤ 0.05. Data are represented as mean ± SEM. (F) Pcdhac2 in situ hybridization at postnatal day 14 in P3 targeted Lmx1bicKO mice. Representative image from n = 3 mice/genotype. Scale bars, 100 µm. (G) Pcdhac2 in situ hybridization at postnatal day 14 in P3 targeted Pet1icKO mice. ISH experiments done in parallel, iControl section is the same section in (F) and (G) to compare icKOs at the same tissue level. Representative image from n = 3 Pet1icKO mice.

Scale bars, 100 µm. (H) Visualization of the Pcdhac2 gene locus showing mycPet1 ChIP peaks at the TSS and 3rd intron (shaded) and matches to the known Pet1 motif. Zoomed regions of the significant mycPet1 binding sites are

165 shown at the bottom. (I) The mycPet1 binding region located within the

3rd Pcdhac2 intron DNAse I hypersensitivity site (HS7) contains a significant match to the known Pet1 position-weight matrix. TOMTOM q-value = 4.75×10−04.

(J) Decreased TdTomato+ arbors detected in P3 targeted Pet1icKO hippocampus compared to iControl mice. Scale bars, 200 µm. (K) Decreased

TdTomato+ arbors detected in all layers of P3 targeted Pet1icKO cortex (Imaris tracing; right panels) compared to iControl mice. Scale bars, 50 µm. (L)

Decreased TdTomato+ arbors in striatum of P5 targeted Pet1icKO mice. Scale bars, 20 µm. See also Figure 9—figure supplement 1F.

166 167 Figure 9—figure supplement 1. Lmx1b→Pet1 cascade acts postnatally to control 5-HT terminal arborization. (A) RT-qPCR of flow sorted

TdTomato+ cells in P3 targeted Pet1icKO mice at 4 weeks of age. Pet1 was not detected by qPCR in flow sorted cells. Lmx1b was not significantly regulated by Pet1 (p=0.1791) (n = 3, iControl; n = 3, Pet1icKO mice). Unpaired t-test with

Welch's correction, *p<0.05. Data are represented as mean ± SEM. (B) Pet1

ISH confirmed Pet1 targeting in the DRN. Scale bars, 100 µm. (C, D) Similar numbers of TdTomato+ cells were counted in P3 Pet1icKO and P3 iControl mice.

Scale bars, 100 µm. (E) Long-range cingulum route was fully formed in P3 targeted Pet1icKO mice. Scale bars, 100 µm. (F) Analysis of TdTomato+ arbors in P5 targeted Pet1icKO mice revealed continuing requirement for Pet1 late- stage arborization in the striatum. Boxed regions presented in Figure 8L. Scale bars, 50 µm. CP, caudate putamen; Acb, nucleus accumbens; ac, anterior commissure; VL, lateral ventricle.

168 Figure 7--supplement table 1. Lmx1b-regulated axon-related genes in rostral and caudal 5-HT neurons at E17.5. Axon genes that are also regulated by Pet1 in rostral 5-HT neurons at E17.5 are in bold. Rostral Caudal (log2fold (log2 fold Gene_name Gene_description GO_terms change) change) RIKEN cDNA 2900011O08 gene regulation of [Source:MGI supramolecular 2900011O08Rik Symbol;Acc:MGI:1914504] fiber organization -1.2925 AP2 associated kinase 1 [Source:MGI axon part, cell Aak1 Symbol;Acc:MGI:1098687] leading edge -1.34758 abl-interactor 1 [Source:MGI axon part, cell Abi1 Symbol;Acc:MGI:104913] leading edge -2.08274 regulation of supramolecular abl-interactor 2 [Source:MGI fiber organization, Abi2 Symbol;Acc:MGI:106913] cell leading edge -1.26797 actin-binding LIM protein 1 [Source:MGI axon development, - Ablim1 Symbol;Acc:MGI:1194500] cell leading edge 0.986226 actin binding LIM protein family, member 3 [Source:MGI Ablim3 Symbol;Acc:MGI:2442582] cell leading edge -1.4282 axon development, acetylcholinesterase [Source:MGI extracellular matrix Ache Symbol;Acc:MGI:87876] binding -2.97281 actin, alpha 2, smooth muscle, aorta [Source:MGI tissue migration, Acta2 Symbol;Acc:MGI:87909] cell leading edge 4.47255 actin, beta [Source:MGI axon development, - Actb Symbol;Acc:MGI:87904] axon part -1.03241 0.884202 actin, gamma, cytoplasmic 1 [Source:MGI Actg1 Symbol;Acc:MGI:87906] axon part -1.43707 a disintegrin and metallopeptidase axon development, domain 17 [Source:MGI tissue migration, Adam17 Symbol;Acc:MGI:1096335] cell leading edge 1.88974 tissue migration, a disintegrin and metallopeptidase cell-substrate domain 9 (meltrin gamma) adhesion, [Source:MGI extracellular matrix Adam9 Symbol;Acc:MGI:105376] binding 2.6695 adenylate cyclase activating polypeptide 1 [Source:MGI Adcyap1 Symbol;Acc:MGI:105094] axon part -1.93698 regulation of adducin 2 (beta) [Source:MGI supramolecular Add2 Symbol;Acc:MGI:87919] fiber organization -1.4117 regulation of adducin 3 (gamma) [Source:MGI supramolecular Add3 Symbol;Acc:MGI:1351615] fiber organization -1.16861 allograft inflammatory factor 1-like Aif1l [Source:MGI cell leading edge 3.95673

169 Symbol;Acc:MGI:1919598] adherens junction associated protein 1 [Source:MGI cell-substrate Ajap1 Symbol;Acc:MGI:2685419] adhesion -1.9163 activated leukocyte cell adhesion molecule [Source:MGI Alcam Symbol;Acc:MGI:1313266] axon development -0.765743 adhesion molecule with Ig like axon development, domain 1 [Source:MGI neuron projection Amigo1 Symbol;Acc:MGI:2653612] fasciculation -2.04702 angiomotin [Source:MGI tissue migration, Amot Symbol;Acc:MGI:108440] cell leading edge 2.51561 regulation of adaptor-related protein complex 1 supramolecular associated regulatory protein fiber organization, [Source:MGI cell-substrate Ap1ar Symbol;Acc:MGI:2384822] adhesion -0.612637 -1.42124 adaptor protein complex AP-1, sigma 1 [Source:MGI Ap1s1 Symbol;Acc:MGI:1098244] axon part -2.79248 adaptor-related protein complex 3, beta 1 subunit [Source:MGI Ap3b1 Symbol;Acc:MGI:1333879] axon part -1.06225 adaptor-related protein complex 3, mu 2 subunit [Source:MGI Ap3m2 Symbol;Acc:MGI:1929214] axon part -1.12645 adaptor-related protein complex 3, sigma 2 subunit [Source:MGI Ap3s2 Symbol;Acc:MGI:1337060] axon part -1.67152 axon development, regulation of supramolecular apolipoprotein E [Source:MGI fiber organization, Apoe Symbol;Acc:MGI:88057] tissue migration 5.45882 axon development, regulation of supramolecular amyloid beta (A4) precursor protein fiber organization, [Source:MGI axon part, cell App Symbol;Acc:MGI:88059] leading edge -0.667049 regulation of supramolecular ADP-ribosylation factor 6 fiber organization, [Source:MGI tissue migration, Arf6 Symbol;Acc:MGI:99435] cell leading edge -0.919736 Rho GTPase activating protein 31 [Source:MGI Arhgap31 Symbol;Acc:MGI:1333857] cell leading edge 2.69056 actin related protein 2/3 complex, regulation of subunit 1A [Source:MGI supramolecular Arpc1a Symbol;Acc:MGI:1928896] fiber organization -1.15811 actin related protein 2/3 complex, regulation of subunit 1B [Source:MGI supramolecular Arpc1b Symbol;Acc:MGI:1343142] fiber organization 6.51629 Arpc5 actin related protein 2/3 complex, regulation of -1.67729

170 subunit 5 [Source:MGI supramolecular Symbol;Acc:MGI:1915021] fiber organization, axon part, cell leading edge actin related protein 2/3 complex, regulation of subunit 5-like [Source:MGI supramolecular Arpc5l Symbol;Acc:MGI:1921442] fiber organization -1.59761 arylsulfatase B [Source:MGI Arsb Symbol;Acc:MGI:88075] tissue migration 1.82901 ArfGAP with SH3 domain, regulation of repeat and PH domain 3 supramolecular [Source:MGI fiber organization, Asap3 Symbol;Acc:MGI:2684986] cell leading edge 2.39956 atlastin GTPase 1 [Source:MGI Atl1 Symbol;Acc:MGI:1921241] axon development -0.731784 -1.34143 ATPase, Na+/K+ transporting, alpha 3 polypeptide [Source:MGI Atp1a3 Symbol;Acc:MGI:88107] axon part -2.17889 ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1 [Source:MGI Atp5a1 Symbol;Acc:MGI:88115] tissue migration -0.694647 ATPase, H+ transporting, lysosomal V0 subunit D1 [Source:MGI Atp6v0d1 Symbol;Acc:MGI:1201778] axon part -1.62577 ATPase, H+ transporting, lysosomal V1 subunit B2 [Source:MGI Atp6v1b2 Symbol;Acc:MGI:109618] cell leading edge -1.15726 ATPase, aminophospholipid transporter-like, class I, type 8A, member 2 [Source:MGI Atp8a2 Symbol;Acc:MGI:1354710] axon development -0.733312 AXL receptor tyrosine kinase [Source:MGI cell-substrate Axl Symbol;Acc:MGI:1347244] adhesion 3.94595 B cell leukemia/lymphoma 11B [Source:MGI Bcl11b Symbol;Acc:MGI:1929913] axon development -1.81451 BCL2-like 11 (apoptosis facilitator) [Source:MGI cell-substrate Bcl2l11 Symbol;Acc:MGI:1197519] adhesion -1.505 brain derived neurotrophic factor [Source:MGI axon development, Bdnf Symbol;Acc:MGI:88145] axon part -3.41876 BMP-binding endothelial regulator [Source:MGI Bmper Symbol;Acc:MGI:1920480] tissue migration 1.7843 bone morphogenetic protein receptor, type 1B [Source:MGI Bmpr1b Symbol;Acc:MGI:107191] axon development 4.08211 biregional cell adhesion molecule- related/down-regulated by oncogenes (Cdon) binding protein [Source:MGI axon development, Boc Symbol;Acc:MGI:2151153] axon part 2.43231

171 BR serine/threonine kinase 2 [Source:MGI axon development, Brsk2 Symbol;Acc:MGI:1923020] axon part -0.653647 -1.30374 beta- repeat containing protein [Source:MGI morphogenesis of a Btrc Symbol;Acc:MGI:1338871] branching structure -0.76619 -1.12134 calcium channel, voltage-dependent, P/Q type, alpha 1A subunit [Source:MGI Cacna1a Symbol;Acc:MGI:109482] axon development -0.67991 -2.73529 calcium channel, voltage-dependent, N type, alpha 1B subunit [Source:MGI Cacna1b Symbol;Acc:MGI:88296] axon part -1.48743 calmodulin 1 [Source:MGI Calm1 Symbol;Acc:MGI:88251] axon part -1.16808 regulation of calmodulin regulated - supramolecular associated protein family, member 3 fiber organization, [Source:MGI cell-substrate Camsap3 Symbol;Acc:MGI:1916947] adhesion -1.51755 capping protein (actin filament) regulation of muscle Z-line, alpha 2 [Source:MGI supramolecular Capza2 Symbol;Acc:MGI:106222] fiber organization 1.11768 calcium/calmodulin-dependent serine protein kinase (MAGUK family) [Source:MGI cell-substrate Cask Symbol;Acc:MGI:1309489] adhesion -0.587634 1, caveolae protein regulation of [Source:MGI supramolecular Cav1 Symbol;Acc:MGI:102709] fiber organization 4.46864 cholecystokinin A receptor [Source:MGI axon development, Cckar Symbol;Acc:MGI:99478] axon part -2.32076 morphogenesis of a branching CD44 antigen [Source:MGI structure, cell Cd44 Symbol;Acc:MGI:88338] leading edge 5.56888 CD47 antigen (Rh-related antigen, integrin-associated signal regulation of transducer) [Source:MGI supramolecular Cd47 Symbol;Acc:MGI:96617] fiber organization -0.847659 -1.62198 tissue migration, CD63 antigen [Source:MGI cell-substrate Cd63 Symbol;Acc:MGI:99529] adhesion 1.34976 tissue migration, cadherin 13 [Source:MGI cell-substrate Cdh13 Symbol;Acc:MGI:99551] adhesion -0.980126 cadherin 2 [Source:MGI axon development, Cdh2 Symbol;Acc:MGI:88355] cell leading edge -1.77896 cadherin 4 [Source:MGI Cdh4 Symbol;Acc:MGI:99218] axon development -2.59285 cyclin-dependent kinase 5 axon development, [Source:MGI cell-substrate Cdk5 Symbol;Acc:MGI:101765] adhesion, axon -1.42201

172 part, cell leading edge cyclin-dependent kinase 5, axon development, regulatory subunit 1 (p35) neuron projection [Source:MGI fasciculation, axon Cdk5r1 Symbol;Acc:MGI:101764] part -1.31658 cyclin-dependent kinase 5, regulatory subunit 2 (p39) [Source:MGI Cdk5r2 Symbol;Acc:MGI:1330828] axon part -3.12076 CDK5 regulatory subunit associated regulation of protein 2 [Source:MGI supramolecular Cdk5rap2 Symbol;Acc:MGI:2384875] fiber organization 1.59211 cyclin-dependent kinase-like 3 [Source:MGI Cdkl3 Symbol;Acc:MGI:2388268] axon development -1.06921 cadherin, EGF LAG seven-pass G- type receptor 1 [Source:MGI morphogenesis of a Celsr1 Symbol;Acc:MGI:1100883] branching structure 2.56377 cadherin, EGF LAG seven-pass G- axon development, type receptor 3 [Source:MGI neuron projection Celsr3 Symbol;Acc:MGI:1858236] fasciculation -1.63288 1 [Source:MGI Chn1 Symbol;Acc:MGI:1915674] axon development -0.83102 Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 1 morphogenesis of [Source:MGI a branching Cited1 Symbol;Acc:MGI:108023] structure -1.4645 associated protein 2 regulation of [Source:MGI supramolecular Ckap2 Symbol;Acc:MGI:1931797] fiber organization 3.29679 regulation of supramolecular fiber organization, CLIP associating protein 1 tissue migration, [Source:MGI cell-substrate Clasp1 Symbol;Acc:MGI:1923957] adhesion -1.04159 axon development, regulation of supramolecular fiber organization, tissue migration, cell-substrate CLIP associating protein 2 adhesion, axon [Source:MGI part, cell leading Clasp2 Symbol;Acc:MGI:1923749] edge -1.10211 chloride channel, voltage-sensitive 3 [Source:MGI Clcn3 Symbol;Acc:MGI:103555] axon part -0.827676 -1.05774 chloride intracellular channel 4 (mitochondrial) [Source:MGI morphogenesis of a Clic4 Symbol;Acc:MGI:1352754] branching structure 1.6356 CAP-GLY domain containing linker regulation of Clip1 protein 1 [Source:MGI supramolecular -0.757095

173 Symbol;Acc:MGI:1928401] fiber organization, cell leading edge axon development, 1 (brain) neuron projection [Source:MGI fasciculation, axon Cnr1 Symbol;Acc:MGI:104615] part -1.31172 axon development, neuron projection fasciculation, cell- contactin 2 [Source:MGI substrate adhesion, Cntn2 Symbol;Acc:MGI:104518] axon part -1.37312 cordon-bleu WH2 repeat axon development, [Source:MGI axon part, cell Cobl Symbol;Acc:MGI:105056] leading edge -1.66649 collagen, type XXV, alpha 1 [Source:MGI Col25a1 Symbol;Acc:MGI:1924268] axon development -1.2187 -2.93916 collagen, type IV, alpha 1 [Source:MGI morphogenesis of a Col4a1 Symbol;Acc:MGI:88454] branching structure 3.15123 tissue migration, coronin, actin binding protein 1C cell-substrate [Source:MGI adhesion, cell - Coro1c Symbol;Acc:MGI:1345964] leading edge -0.656429 0.924617 cytoplasmic polyadenylation element binding protein 1 [Source:MGI Cpeb1 Symbol;Acc:MGI:108442] axon part -3.17369 cytoplasmic polyadenylation element binding protein 4 [Source:MGI Cpeb4 Symbol;Acc:MGI:1914829] axon part -1.56273 complexin 2 [Source:MGI Cplx2 Symbol;Acc:MGI:104726] axon part -0.806513 -1.11007 v-crk avian sarcoma virus CT10 oncogene homolog-like [Source:MGI cell-substrate Crkl Symbol;Acc:MGI:104686] adhesion -1.59903 collapsin response mediator protein 1 [Source:MGI axon development, Crmp1 Symbol;Acc:MGI:107793] axon part -1.43007 axon development, cartilage acidic protein 1 neuron projection [Source:MGI fasciculation, axon Crtac1 Symbol;Acc:MGI:1920082] part -2.26935 casein kinase 1, epsilon [Source:MGI Csnk1e Symbol;Acc:MGI:1351660] axon part -1.50132 chondroitin sulfate proteoglycan 4 [Source:MGI Cspg4 Symbol;Acc:MGI:2153093] cell leading edge 2.59491 (cadherin associated protein), delta 2 [Source:MGI morphogenesis of a Ctnnd2 Symbol;Acc:MGI:1195966] branching structure -1.05421 cathepsin D [Source:MGI morphogenesis of a Ctsd Symbol;Acc:MGI:88562] branching structure 2.17818 chemokine (C-X3-C motif) ligand 1 regulation of Cx3cl1 [Source:MGI supramolecular -2.72845

174 Symbol;Acc:MGI:1097153] fiber organization coxsackie virus and adenovirus receptor [Source:MGI Cxadr Symbol;Acc:MGI:1201679] axon part -1.22955 axon development, morphogenesis of a branching chemokine (C-X-C motif) receptor 4 structure, axon [Source:MGI part, cell leading Cxcr4 Symbol;Acc:MGI:109563] edge -2.76769 axon development, regulation of supramolecular cytoplasmic FMR1 interacting fiber organization, protein 1 [Source:MGI axon part, cell Cyfip1 Symbol;Acc:MGI:1338801] leading edge 1.64498 cytoplasmic FMR1 interacting protein 2 [Source:MGI Cyfip2 Symbol;Acc:MGI:1924134] axon development -1.41702 disabled 2, mitogen-responsive phosphoprotein [Source:MGI cell-substrate Dab2 Symbol;Acc:MGI:109175] adhesion 6.33366 disabled 2 interacting protein [Source:MGI Dab2ip Symbol;Acc:MGI:1916851] tissue migration -1.1787 axon development, regulation of supramolecular fiber organization, cell-substrate adhesion, axon drebrin 1 [Source:MGI part, cell leading Dbn1 Symbol;Acc:MGI:1931838] edge -1.5702 regulation of supramolecular drebrin-like [Source:MGI fiber organization, Dbnl Symbol;Acc:MGI:700006] cell leading edge -1.479 dachsous cadherin related 1 [Source:MGI morphogenesis of a Dchs1 Symbol;Acc:MGI:2685011] branching structure -1.87541 doublecortin-like kinase 1 [Source:MGI Dclk1 Symbol;Acc:MGI:1330861] axon development -1.16246 discs large MAGUK scaffold protein 2 [Source:MGI Dlg2 Symbol;Acc:MGI:1344351] axon part -1.05242 discs large MAGUK scaffold protein 4 [Source:MGI Dlg4 Symbol;Acc:MGI:1277959] axon part -1.66863 , muscular dystrophy cell-substrate [Source:MGI adhesion, cell Dmd Symbol;Acc:MGI:94909] leading edge 1.1451 1 [Source:MGI Dnm1 Symbol;Acc:MGI:107384] axon part -1.38927 Dpysl2 dihydropyrimidinase-like 2 axon development, -0.807003 -1.14169

175 [Source:MGI axon part Symbol;Acc:MGI:1349763] dihydropyrimidinase-like 5 [Source:MGI Dpysl5 Symbol;Acc:MGI:1929772] axon development -2.02166 dorsal inhibitory axon guidance protein [Source:MGI Draxin Symbol;Acc:MGI:1917683] axon development 1.33403 dual specificity phosphatase 3 (vaccinia virus phosphatase VH1- related) [Source:MGI cell-substrate Dusp3 Symbol;Acc:MGI:1919599] adhesion -1.01818 EGF-like repeats and discoidin I-like domains 3 [Source:MGI cell-substrate Edil3 Symbol;Acc:MGI:1329025] adhesion -2.16612 eukaryotic translation 1 alpha 1 [Source:MGI Eef1a1 Symbol;Acc:MGI:1096881] cell leading edge 1.04097 axon development, cell-substrate ephrin A5 [Source:MGI adhesion, negative Efna5 Symbol;Acc:MGI:107444] chemotaxis -0.842871 -2.05945 ELAV like RNA binding protein 4 [Source:MGI Elavl4 Symbol;Acc:MGI:107427] axon part -0.702067 -1.66109 ER complex subunit 10 [Source:MGI Emc10 Symbol;Acc:MGI:1916933] tissue migration -1.66665 epithelial membrane protein 2 tissue migration, [Source:MGI cell-substrate Emp2 Symbol;Acc:MGI:1098726] adhesion 3.7826 enolase 2, gamma neuronal [Source:MGI Eno2 Symbol;Acc:MGI:95394] axon part -3.09225 ectonucleotide /phosphodiesterase tissue migration, 2 [Source:MGI cell-substrate Enpp2 Symbol;Acc:MGI:1321390] adhesion 1.5434 axon development, neuron projection Eph receptor A4 [Source:MGI fasciculation, axon Epha4 Symbol;Acc:MGI:98277] part 1.18643 axon development, Eph receptor B2 [Source:MGI neuron projection Ephb2 Symbol;Acc:MGI:99611] fasciculation -1.33054 ELKS/RAB6-interacting/CAST family member 2 [Source:MGI Erc2 Symbol;Acc:MGI:1098749] axon part -0.890619 ezrin [Source:MGI Ezr Symbol;Acc:MGI:98931] cell leading edge 4.12702 F-box and WD-40 domain protein 7 [Source:MGI Fbxw7 Symbol;Acc:MGI:1354695] tissue migration -0.902157 feminization 1 homolog b (C. morphogenesis of a Fem1b elegans) [Source:MGI branching structure -0.714161

176 Symbol;Acc:MGI:1335087] FYVE, RhoGEF and PH domain containing 5 [Source:MGI Fgd5 Symbol;Acc:MGI:2443369] cell leading edge -0.980325 -2.65118 regulation of fibroblast growth factor 13 supramolecular [Source:MGI fiber organization, Fgf13 Symbol;Acc:MGI:109178] axon part -0.735893 -1.73578 fibroblast growth factor 18 [Source:MGI Fgf18 Symbol;Acc:MGI:1277980] tissue migration -2.24721 FK506 binding protein 1a [Source:MGI Fkbp1a Symbol;Acc:MGI:95541] axon part -0.834117 -2.02553 regulation of FK506 binding protein 4 supramolecular [Source:MGI fiber organization, Fkbp4 Symbol;Acc:MGI:95543] axon part -1.53352 regulation of supramolecular fiber organization, , alpha [Source:MGI cell-substrate Flna Symbol;Acc:MGI:95556] adhesion, axon part 1.37815 fibronectin rich axon development, transmembrane protein 3 negative [Source:MGI chemotaxis, axon Flrt3 Symbol;Acc:MGI:1918686] part -1.93475 axon development, fibronectin 1 [Source:MGI cell-substrate Fn1 Symbol;Acc:MGI:95566] adhesion 2.12294 3.25558 forkhead box P1 [Source:MGI axon development, Foxp1 Symbol;Acc:MGI:1914004] tissue migration -1.09187 -1.3937 fascin actin-bundling protein 1 [Source:MGI axon part, cell Fscn1 Symbol;Acc:MGI:1352745] leading edge -2.18864 gamma-aminobutyric acid (GABA) B receptor, 1 [Source:MGI axon part, cell Gabbr1 Symbol;Acc:MGI:1860139] leading edge -1.08896 gamma-aminobutyric acid (GABA) A receptor, subunit alpha 3 [Source:MGI Gabra3 Symbol;Acc:MGI:95615] cell leading edge -1.69127 gamma-aminobutyric acid (GABA) A receptor, subunit alpha 5 [Source:MGI Gabra5 Symbol;Acc:MGI:95617] cell leading edge -1.66817 gamma-aminobutyric acid (GABA) A receptor, subunit beta 3 [Source:MGI Gabrb3 Symbol;Acc:MGI:95621] axon part -1.52886 gamma-aminobutyric acid (GABA) A receptor, subunit epsilon [Source:MGI Gabre Symbol;Acc:MGI:1330235] cell leading edge -5.67539 gamma-aminobutyric acid (GABA) A Gabrg2 receptor, subunit gamma 2 cell leading edge -1.49442

177 [Source:MGI Symbol;Acc:MGI:95623] glutamate decarboxylase 1 [Source:MGI Gad1 Symbol;Acc:MGI:95632] axon part -1.54061 growth associated protein 43 axon [Source:MGI development, Gap43 Symbol;Acc:MGI:95639] axon part -0.708076 -2.06642 growth arrest specific 6 [Source:MGI cell-substrate Gas6 Symbol;Acc:MGI:95660] adhesion 1.31985 GATA binding protein 2 [Source:MGI Gata2 Symbol;Acc:MGI:95662] tissue migration -2.74766 GATA binding protein 3 [Source:MGI axon development, Gata3 Symbol;Acc:MGI:95663] tissue migration -0.664617 -3.45285 gastrulation brain homeobox 2 axon development, [Source:MGI morphogenesis of a Gbx2 Symbol;Acc:MGI:95668] branching structure 3.97612 GIT ArfGAP 1 [Source:MGI Git1 Symbol;Acc:MGI:1927140] axon part -2.18834 glutamate-ammonia ligase (glutamine synthetase) [Source:MGI tissue migration, Glul Symbol;Acc:MGI:95739] axon part -0.72056 1.7699 golgi autoantigen, golgin subfamily a, 4 [Source:MGI Golga4 Symbol;Acc:MGI:1859646] axon development 1.3961 glypican 1 [Source:MGI extracellular matrix Gpc1 Symbol;Acc:MGI:1194891] binding -1.46627 glypican 3 [Source:MGI morphogenesis of a Gpc3 Symbol;Acc:MGI:104903] branching structure -1.68811 glucose phosphate isomerase 1 [Source:MGI Gpi1 Symbol;Acc:MGI:95797] tissue migration -0.745484 glycoprotein m6a [Source:MGI Gpm6a Symbol;Acc:MGI:107671] axon part -1.33119 glycoprotein m6b [Source:MGI cell-substrate Gpm6b Symbol;Acc:MGI:107672] adhesion 1.39957 , ionotropic, AMPA4 (alpha 4) [Source:MGI Gria4 Symbol;Acc:MGI:95811] axon part -1.26559 -1.35345 glutamate receptor, ionotropic, kainate 5 (gamma 2) [Source:MGI Grik5 Symbol;Acc:MGI:95818] axon part 0.6188 glutamate receptor, ionotropic, axon development, NMDA1 (zeta 1) [Source:MGI axon part, cell Grin1 Symbol;Acc:MGI:95819] leading edge -2.14365 glutamate receptor, metabotropic 7 [Source:MGI Grm7 Symbol;Acc:MGI:1351344] axon part -1.4093 glycogen synthase kinase 3 beta axon development, [Source:MGI cell-substrate Gsk3b Symbol;Acc:MGI:1861437] adhesion, axon part -0.829383 -1.27975 hypoxia inducible factor 1, alpha subunit [Source:MGI tissue migration, Hif1a Symbol;Acc:MGI:106918] axon part -0.62537

178 heme oxygenase 1 [Source:MGI Hmox1 Symbol;Acc:MGI:96163] tissue migration 3.30135 hydroxysteroid (17-beta) dehydrogenase 12 [Source:MGI cell-substrate Hsd17b12 Symbol;Acc:MGI:1926967] adhesion 1.3184 regulation of heat shock protein 1A [Source:MGI supramolecular Hspa1a Symbol;Acc:MGI:96244] fiber organization 3.94728 regulation of 5-hydroxytryptamine (serotonin) supramolecular receptor 1A [Source:MGI fiber organization, Htr1a Symbol;Acc:MGI:96273] axon part -2.14415 -3.44427 insulin-like growth factor 1 tissue migration, [Source:MGI morphogenesis of a Igf1 Symbol;Acc:MGI:96432] branching structure 5.59491 insulin-like growth factor 2 [Source:MGI Igf2 Symbol;Acc:MGI:96434] tissue migration 3.82525 inositol polyphosphate-5- phosphatase F [Source:MGI - Inpp5f Symbol;Acc:MGI:2141867] axon development 0.926979 immunoglobulin superfamily containing leucine-rich repeat 2 [Source:MGI Islr2 Symbol;Acc:MGI:2444277] axon development 1.4986 -2.69087 cell-substrate adhesion, integrin alpha 6 [Source:MGI extracellular matrix Itga6 Symbol;Acc:MGI:96605] binding 3.42505 tissue migration, cell-substrate adhesion, negative chemotaxis, cell leading edge, integrin alpha V [Source:MGI extracellular matrix Itgav Symbol;Acc:MGI:96608] binding 2.87909 axon development, tissue migration, cell-substrate adhesion, cell integrin beta 1 (fibronectin receptor leading edge, beta) [Source:MGI extracellular matrix Itgb1 Symbol;Acc:MGI:96610] binding -1.27078 1.55091 regulation of junction-mediating and regulatory supramolecular protein [Source:MGI fiber organization, Jmy Symbol;Acc:MGI:1913096] cell leading edge -0.813646 jun proto-oncogene [Source:MGI axon development, Jun Symbol;Acc:MGI:96646] tissue migration 1.09278 tissue migration, junction [Source:MGI cell-substrate Jup Symbol;Acc:MGI:96650] adhesion -1.30925 KN motif and ankyrin repeat regulation of domains 1 [Source:MGI supramolecular Kank1 Symbol;Acc:MGI:2147707] fiber organization, 2.80551

179 tissue migration, cell-substrate adhesion, cell leading edge regulation of KN motif and ankyrin repeat supramolecular domains 2 [Source:MGI fiber organization, Kank2 Symbol;Acc:MGI:2384568] tissue migration 2.72834 voltage-gated channel, shaker-related, subfamily, member 6 [Source:MGI Kcna6 Symbol;Acc:MGI:96663] axon part -1.81428 potassium voltage gated channel, Shab-related subfamily, member 1 [Source:MGI Kcnb1 Symbol;Acc:MGI:96666] cell leading edge -1.57546 potassium voltage gated channel, Shaw-related subfamily, member 1 [Source:MGI axon part, cell Kcnc1 Symbol;Acc:MGI:96667] leading edge -1.01559 potassium voltage gated channel, Shaw-related subfamily, member 2 [Source:MGI axon part, cell Kcnc2 Symbol;Acc:MGI:96668] leading edge -2.31674 Kv channel interacting protein 3, calsenilin [Source:MGI Kcnip3 Symbol;Acc:MGI:1929258] axon part 3.44892 potassium voltage-gated channel, subfamily Q, member 2 [Source:MGI Kcnq2 Symbol;Acc:MGI:1309503] axon part -1.48528 family member 1B [Source:MGI - Kif1b Symbol;Acc:MGI:108426] axon part -0.626212 0.902574 kinesin family member 3A [Source:MGI axon development, Kif3a Symbol;Acc:MGI:107689] axon part -1.29656 -1.54262 kelch-like 2, Mayven [Source:MGI Klhl2 Symbol;Acc:MGI:1924363] cell leading edge -1.43319 B1 [Source:MGI cell-substrate Lamb1 Symbol;Acc:MGI:96743] adhesion 4.4541 LIM domain binding 2 [Source:MGI Ldb2 Symbol;Acc:MGI:894670] cell leading edge 1.0571 lymphoid enhancer binding factor 1 [Source:MGI morphogenesis of a Lef1 Symbol;Acc:MGI:96770] branching structure 1.97895 leucine-rich repeat LGI family, member 1 [Source:MGI Lgi1 Symbol;Acc:MGI:1861691] axon development -1.42152 legumain [Source:MGI Lgmn Symbol;Acc:MGI:1330838] tissue migration 3.43697 regulation of LIM domain and actin binding 1 supramolecular [Source:MGI fiber organization, Lima1 Symbol;Acc:MGI:1920992] cell leading edge 2.40127 Lmtk2 lemur tyrosine kinase 2 [Source:MGI axon development, -0.628425 -2.07108

180 Symbol;Acc:MGI:3036247] axon part receptor 1 regulation of [Source:MGI supramolecular Lpar1 Symbol;Acc:MGI:108429] fiber organization -1.64849 leucine-rich repeats and immunoglobulin-like domains 2 [Source:MGI axon development, Lrig2 Symbol;Acc:MGI:2443718] axon part -1.33965 low density lipoprotein receptor- related protein 4 [Source:MGI Lrp4 Symbol;Acc:MGI:2442252] axon development 2.77733 leucine rich repeat containing 4C [Source:MGI Lrrc4c Symbol;Acc:MGI:2442636] axon development -0.705496 melanoma antigen, family L, 2 regulation of [Source:MGI supramolecular Magel2 Symbol;Acc:MGI:1351648] fiber organization -2.89638 axon development, regulation of -associated protein 1 A supramolecular [Source:MGI fiber organization, Map1a Symbol;Acc:MGI:1306776] axon part -0.666585 axon development, regulation of supramolecular microtubule-associated protein 2 fiber organization, [Source:MGI axon part, cell Map2 Symbol;Acc:MGI:97175] leading edge 0.816556 mitogen-activated protein kinase tissue migration, kinase kinase kinase 4 [Source:MGI cell-substrate Map4k4 Symbol;Acc:MGI:1349394] adhesion -0.602763 microtubule-associated protein 6 [Source:MGI axon development, Map6 Symbol;Acc:MGI:1201690] axon part -1.75568 microtubule-associated protein, RP/EB family, member 1 regulation of [Source:MGI supramolecular Mapre1 Symbol;Acc:MGI:891995] fiber organization -0.686779 -1.24738 microtubule-associated protein, RP/EB family, member 2 [Source:MGI Mapre2 Symbol;Acc:MGI:106271] tissue migration -2.00285 axon development, regulation of supramolecular microtubule-associated protein tau fiber organization, [Source:MGI axon part, cell Mapt Symbol;Acc:MGI:97180] leading edge -1.67853 mutated in colorectal cancers [Source:MGI Mcc Symbol;Acc:MGI:96930] tissue migration -0.795106 regulation of methyl CpG binding protein 2 supramolecular [Source:MGI fiber organization, Mecp2 Symbol;Acc:MGI:99918] tissue migration, -0.614402 -1.18061

181 morphogenesis of a branching structure regulation of myocyte enhancer factor 2C supramolecular [Source:MGI fiber organization, Mef2c Symbol;Acc:MGI:99458] tissue migration 2.4051 multiple endocrine neoplasia 1 [Source:MGI cell-substrate Men1 Symbol;Acc:MGI:1316736] adhesion 1.20735 meteorin, glial cell differentiation regulator [Source:MGI Metrn Symbol;Acc:MGI:1917333] axon development 3.42335 melanoma inhibitory activity 3 [Source:MGI Mia3 Symbol;Acc:MGI:2443183] tissue migration -0.828284 cell-substrate matrix metallopeptidase 14 adhesion, (membrane-inserted) [Source:MGI morphogenesis of a Mmp14 Symbol;Acc:MGI:101900] branching structure 3.89168 matrix metallopeptidase 9 [Source:MGI Mmp9 Symbol;Acc:MGI:97011] tissue migration 3.51627 membrane protein, palmitoylated 2 (MAGUK p55 subfamily member 2) [Source:MGI Mpp2 Symbol;Acc:MGI:1858257] cell leading edge -2.72701 5-methyltetrahydrofolate- homocysteine methyltransferase [Source:MGI Mtr Symbol;Acc:MGI:894292] axon development 2.46132 metastasis suppressor 1 regulation of [Source:MGI supramolecular Mtss1 Symbol;Acc:MGI:2384818] fiber organization -1.42134 regulation of supramolecular , heavy polypeptide 9, non- fiber organization, muscle [Source:MGI tissue migration, Myh9 Symbol;Acc:MGI:107717] cell leading edge 3.78983 myosin X [Source:MGI Myo10 Symbol;Acc:MGI:107716] cell leading edge 2.42607 nanos C2HC-type 1 [Source:MGI Nanos1 Symbol;Acc:MGI:2669254] tissue migration -1.69318 neuron navigator 1 [Source:MGI Nav1 Symbol;Acc:MGI:2183683] axon part -1.52079 regulation of neuron navigator 3 [Source:MGI supramolecular Nav3 Symbol;Acc:MGI:2183703] fiber organization -1.54438 axon development, neural cell adhesion molecule 1 neuron projection [Source:MGI fasciculation, axon Ncam1 Symbol;Acc:MGI:97281] part -1.25731 axon development, necdin [Source:MGI neuron projection - Ndn Symbol;Acc:MGI:97290] fasciculation -0.632285 0.884451

182 neuron-derived neurotrophic factor [Source:MGI cell-substrate Ndnf Symbol;Acc:MGI:1915419] adhesion -2.11619 N- downstream regulated gene 2 [Source:MGI Ndrg2 Symbol;Acc:MGI:1352498] axon part 2.50966 neurofascin [Source:MGI axon development, Nfasc Symbol;Acc:MGI:104753] axon part -1.47021 nuclear factor, erythroid derived 2, like 2 [Source:MGI Nfe2l2 Symbol;Acc:MGI:108420] tissue migration 5.005 cell-substrate adhesion, nidogen 1 [Source:MGI extracellular matrix Nid1 Symbol;Acc:MGI:97342] binding 2.71017 NK6 homeobox 1 [Source:MGI Nkx6-1 Symbol;Acc:MGI:1206039] axon development -2.17344 NME/NM23 nucleoside diphosphate kinase 1 [Source:MGI Nme1 Symbol;Acc:MGI:97355] cell leading edge -1.34415 axon development, tissue migration, cell-substrate adhesion, morphogenesis of a branching notch 1 [Source:MGI structure, cell Notch1 Symbol;Acc:MGI:97363] leading edge 1.45405 neuronal pentraxin 1 [Source:MGI Nptx1 Symbol;Acc:MGI:107811] axon development -1.97758 neuropeptide Y [Source:MGI Npy Symbol;Acc:MGI:97374] axon part 2.61346 Y2 [Source:MGI cell-substrate Npy2r Symbol;Acc:MGI:108418] adhesion -2.49744 -2.89667 subfamily 2, group F, member 2 [Source:MGI Nr2f2 Symbol;Acc:MGI:1352452] tissue migration -0.733782 -2.56305 nuclear receptor binding protein 1 [Source:MGI Nrbp1 Symbol;Acc:MGI:2183436] cell leading edge -0.609058 neuregulin 3 [Source:MGI negative Nrg3 Symbol;Acc:MGI:1097165] chemotaxis -1.53498 axon development, neuropilin 2 [Source:MGI negative Nrp2 Symbol;Acc:MGI:1100492] chemotaxis -0.982098 neurensin 1 [Source:MGI Nrsn1 Symbol;Acc:MGI:894662] axon part -2.14189 netrin 1 [Source:MGI Ntn1 Symbol;Acc:MGI:105088] axon development 2.69235 netrin G1 [Source:MGI Ntng1 Symbol;Acc:MGI:1934028] axon development -2.32904 neurotrophic tyrosine kinase, receptor, type 2 [Source:MGI axon development, Ntrk2 Symbol;Acc:MGI:97384] axon part -0.637499

183 numb-like [Source:MGI Numbl Symbol;Acc:MGI:894702] axon development -2.03233 olfactomedin 1 [Source:MGI axon development, Olfm1 Symbol;Acc:MGI:1860437] axon part -1.1814 , kappa 1 [Source:MGI Oprk1 Symbol;Acc:MGI:97439] axon part -2.89531 ORAI calcium release-activated calcium modulator 2 [Source:MGI Orai2 Symbol;Acc:MGI:2443195] axon part -1.48563 protein kinase C and casein kinase substrate in neurons 2 [Source:MGI Pacsin2 Symbol;Acc:MGI:1345153] cell leading edge 1.74718 axon development, p21 (RAC1) activated kinase 3 regulation of [Source:MGI supramolecular Pak3 Symbol;Acc:MGI:1339656] fiber organization -1.01958 paired box 2 [Source:MGI morphogenesis of a Pax2 Symbol;Acc:MGI:97486] branching structure 2.31743 paired box 8 [Source:MGI morphogenesis of a Pax8 Symbol;Acc:MGI:97492] branching structure 3.35012 PAX interacting (with transcription- activation domain) protein 1 [Source:MGI Paxip1 Symbol;Acc:MGI:1890430] tissue migration -1.19749 protocadherin alpha subfamily C, 2 [Source:MGI Pcdhac2 Symbol;Acc:MGI:1891443] cell adhesion -1.82227 -3.6608 proprotein convertase subtilisin/kexin type 5 [Source:MGI cell-substrate Pcsk5 Symbol;Acc:MGI:97515] adhesion -2.27439 -3.43747 platelet derived growth factor receptor, alpha polypeptide [Source:MGI morphogenesis of a Pdgfra Symbol;Acc:MGI:97530] branching structure 2.68538 cell-substrate podoplanin [Source:MGI adhesion, cell Pdpn Symbol;Acc:MGI:103098] leading edge 3.65016 regulation of pyridoxal (pyridoxine, B6) supramolecular phosphatase [Source:MGI fiber organization, Pdxp Symbol;Acc:MGI:1919282] cell leading edge -2.02707 preproenkephalin [Source:MGI Penk Symbol;Acc:MGI:104629] axon part -1.31513 -2.58404 morphogenesis of a [Source:MGI branching Pgr Symbol;Acc:MGI:97567] structure, axon part -1.92335 -2.48984 phosphatidylinositol binding clathrin assembly protein [Source:MGI Picalm Symbol;Acc:MGI:2385902] axon development -0.647028 phosphatidylinositol transfer protein, alpha [Source:MGI Pitpna Symbol;Acc:MGI:99887] axon development -0.599331 Pkd2 polycystic kidney disease 2 morphogenesis of a 1.62692

184 [Source:MGI branching Symbol;Acc:MGI:1099818] structure, cell leading edge regulation of supramolecular pleckstrin [Source:MGI fiber organization, Plek Symbol;Acc:MGI:1860485] cell leading edge 3.88851 pleckstrin homology domain containing, family G (with RhoGef regulation of domain) member 2 [Source:MGI supramolecular Plekhg2 Symbol;Acc:MGI:2141874] fiber organization 2.37471 axon development, plexin A1 [Source:MGI morphogenesis of a Plxna1 Symbol;Acc:MGI:107685] branching structure -1.40299 axon development, plexin A3 [Source:MGI negative Plxna3 Symbol;Acc:MGI:107683] chemotaxis -1.32445 axon development, plexin A4 [Source:MGI negative - Plxna4 Symbol;Acc:MGI:2179061] chemotaxis 0.925581 plexin B2 [Source:MGI Plxnb2 Symbol;Acc:MGI:2154239] axon development 0.952712 plexin C1 [Source:MGI Plxnc1 Symbol;Acc:MGI:1890127] axon development -1.25938 peripheral protein 22 [Source:MGI Pmp22 Symbol;Acc:MGI:97631] axon development 4.45394 peroxisome proliferator activator tissue migration, receptor delta [Source:MGI cell-substrate Ppard Symbol;Acc:MGI:101884] adhesion 2.3985 protein phosphatase 3, regulatory subunit B, alpha isoform (calcineurin B, type I) [Source:MGI morphogenesis of a Ppp3r1 Symbol;Acc:MGI:107172] branching structure -0.654313 -1.34912 regulation of phosphatidylinositol-3,4,5- supramolecular trisphosphate-dependent Rac fiber organization, exchange factor 1 [Source:MGI cell-substrate Prex1 Symbol;Acc:MGI:3040696] adhesion, axon part 2.66111 protein kinase C, beta [Source:MGI - Prkcb Symbol;Acc:MGI:97596] axon part 0.776683 regulation of supramolecular fiber organization, protein kinase C, epsilon tissue migration, [Source:MGI cell-substrate Prkce Symbol;Acc:MGI:97599] adhesion -1.1059 regulation of protein kinase C, theta [Source:MGI supramolecular Prkcq Symbol;Acc:MGI:97601] fiber organization -1.29833 cell-substrate adhesion, axon protein kinase C, zeta [Source:MGI part, cell leading Prkcz Symbol;Acc:MGI:97602] edge -1.7815 Prrt2 proline-rich transmembrane protein 2 axon part -1.27291

185 [Source:MGI Symbol;Acc:MGI:1916267] pleckstrin and Sec7 domain containing [Source:MGI Psd Symbol;Acc:MGI:1920978] cell leading edge -2.21259 pleckstrin and Sec7 domain containing 3 [Source:MGI Psd3 Symbol;Acc:MGI:1918215] cell leading edge -1.05814 polypyrimidine tract binding protein 2 [Source:MGI Ptbp2 Symbol;Acc:MGI:1860489] axon part -1.03372 prostaglandin E receptor 4 (subtype regulation of EP4) [Source:MGI supramolecular Ptger4 Symbol;Acc:MGI:104311] fiber organization -3.81965 pleiotrophin [Source:MGI cell-substrate Ptn Symbol;Acc:MGI:97804] adhesion 2.46984 protein tyrosine phosphatase, non- receptor type 13 [Source:MGI Ptpn13 Symbol;Acc:MGI:103293] cell leading edge 1.72154 protein tyrosine phosphatase, receptor type, F [Source:MGI axon development, Ptprf Symbol;Acc:MGI:102695] axon part -0.63967 protein tyrosine phosphatase, axon development, receptor type, M [Source:MGI tissue migration, Ptprm Symbol;Acc:MGI:102694] cell leading edge -0.774414 -1.06726 protein tyrosine phosphatase, receptor type, N polypeptide 2 [Source:MGI Ptprn2 Symbol;Acc:MGI:107418] axon part -1.36907 protein tyrosine phosphatase, receptor type, O [Source:MGI Ptpro Symbol;Acc:MGI:1097152] axon development -1.49485 protein tyrosine phosphatase, receptor type, R [Source:MGI Ptprr Symbol;Acc:MGI:109559] tissue migration -1.5954 protein tyrosine phosphatase, receptor type, S [Source:MGI Ptprs Symbol;Acc:MGI:97815] axon development -1.47617 axon development, neuron projection protein tyrosine phosphatase, fasciculation, cell- receptor type Z, polypeptide 1 substrate adhesion, [Source:MGI axon part, cell Ptprz1 Symbol;Acc:MGI:97816] leading edge 1.60443 RAB10, member RAS oncogene family [Source:MGI - Rab10 Symbol;Acc:MGI:105066] axon development -0.866468 0.802575 RAB3A, member RAS oncogene family [Source:MGI axon development, Rab3a Symbol;Acc:MGI:97843] axon part -2.01307 Rac family small GTPase 2 cell-substrate [Source:MGI adhesion, cell Rac2 Symbol;Acc:MGI:97846] leading edge 4.68948 Rac family small GTPase 3 regulation of [Source:MGI supramolecular -1.57276

186 Symbol;Acc:MGI:2180784] fiber organization, cell-substrate adhesion, axon part, cell leading edge GTPase activating protein 1 [Source:MGI Rangap1 Symbol;Acc:MGI:103071] axon part -1.11683 -1.85507 Ras association (RalGDS/AF-6) and pleckstrin homology domains 1 [Source:MGI axon development, Raph1 Symbol;Acc:MGI:1924550] cell leading edge -0.734261 -1.08447 RAS protein-specific guanine nucleotide-releasing factor 1 [Source:MGI Rasgrf1 Symbol;Acc:MGI:99694] axon part -2.65424 regulation of supramolecular radixin [Source:MGI fiber organization, Rdx Symbol;Acc:MGI:97887] cell leading edge 2.20706 [Source:MGI Reln Symbol;Acc:MGI:103022] axon development -2.58788 ret proto-oncogene [Source:MGI Ret Symbol;Acc:MGI:97902] axon development -0.92342 -2.58715 regulation of supramolecular regulator of cell cycle [Source:MGI fiber organization, Rgcc Symbol;Acc:MGI:1913464] tissue migration 4.87577 repulsive guidance molecule family member A [Source:MGI Rgma Symbol;Acc:MGI:2679262] axon development 0.74546 regulator of G-protein signalling 10 [Source:MGI Rgs10 Symbol;Acc:MGI:1915115] axon part 1.07879 regulation of supramolecular fiber organization, tissue migration, cell-substrate ras homolog family member A adhesion, negative [Source:MGI chemotaxis, cell Rhoa Symbol;Acc:MGI:1096342] leading edge -0.796751 ras homolog family member B [Source:MGI Rhob Symbol;Acc:MGI:107949] tissue migration -0.628854 -0.97183 ras homolog family member J [Source:MGI Rhoj Symbol;Acc:MGI:1931551] tissue migration 3.26961 Ras-like without CAAX 2 [Source:MGI semaphorin Rit2 Symbol;Acc:MGI:108054] receptor binding -2.25335 regulation of supramolecular ribosomal protein S3 [Source:MGI fiber organization, Rps3 Symbol;Acc:MGI:1350917] cell leading edge -1.66868

187 ras responsive element binding tissue migration, protein 1 [Source:MGI cell-substrate Rreb1 Symbol;Acc:MGI:2443664] adhesion 4.05549 axon development, tissue migration, neuron projection fasciculation, [Source:MGI morphogenesis of a - Rtn4 Symbol;Acc:MGI:1915835] branching structure -0.752739 0.782122 reticulon 4 receptor-like 1 [Source:MGI Rtn4rl1 Symbol;Acc:MGI:2661375] axon development 2.02618 RUN and FYVE domain containing 3 axon development, [Source:MGI axon part, cell - Rufy3 Symbol;Acc:MGI:106484] leading edge -0.854967 0.981065 receptor-like tyrosine kinase axon development, [Source:MGI negative Ryk Symbol;Acc:MGI:101766] chemotaxis -0.846894 regulation of supramolecular S100 calcium binding protein A10 fiber organization, (calpactin) [Source:MGI cell-substrate S100a10 Symbol;Acc:MGI:1339468] adhesion -1.73933 -4.08309 S100 calcium binding protein A11 [Source:MGI S100a11 Symbol;Acc:MGI:1338798] cell leading edge -0.753519 , beta polypeptide, neural [Source:MGI S100b Symbol;Acc:MGI:98217] cell leading edge 3.11031 sphingosine-1-phosphate receptor 1 regulation of [Source:MGI supramolecular S1pr1 Symbol;Acc:MGI:1096355] fiber organization 3.77763 scavenger receptor class B, member 1 [Source:MGI Scarb1 Symbol;Acc:MGI:893578] tissue migration 0.908871 sodium channel, voltage-gated, type VIII, alpha [Source:MGI Scn8a Symbol;Acc:MGI:103169] axon part -0.73226 axon development, negative chemotaxis, sema domain, immunoglobulin morphogenesis of a domain (Ig), short basic domain, branching secreted, (semaphorin) 3C structure, [Source:MGI semaphorin Sema3c Symbol;Acc:MGI:107557] receptor binding -1.56475 axon development, sema domain, immunoglobulin tissue migration, domain (Ig), transmembrane domain negative (TM) and short cytoplasmic domain, chemotaxis, (semaphorin) 4A [Source:MGI semaphorin Sema4a Symbol;Acc:MGI:107560] receptor binding -1.68114 sema domain, immunoglobulin axon development, domain (Ig), transmembrane domain negative Sema4d (TM) and short cytoplasmic domain, chemotaxis, -0.753722

188 (semaphorin) 4D [Source:MGI semaphorin Symbol;Acc:MGI:109244] receptor binding axon development, sema domain, immunoglobulin negative domain (Ig), TM domain, and short chemotaxis, cytoplasmic domain [Source:MGI semaphorin Sema4f Symbol;Acc:MGI:1340055] receptor binding -1.79924 sema domain, immunoglobulin axon development, domain (Ig), transmembrane domain negative (TM) and short cytoplasmic domain, chemotaxis, (semaphorin) 4G [Source:MGI semaphorin Sema4g Symbol;Acc:MGI:1347047] receptor binding -1.82732 sema domain, seven thrombospondin repeats (type 1 and axon development, type 1-like), transmembrane domain negative (TM) and short cytoplasmic domain, chemotaxis, (semaphorin) 5B [Source:MGI semaphorin Sema5b Symbol;Acc:MGI:107555] receptor binding 1.99165 cell-substrate secreted frizzled-related protein 1 adhesion, [Source:MGI morphogenesis of a Sfrp1 Symbol;Acc:MGI:892014] branching structure 2.45135 secreted frizzled-related protein 2 [Source:MGI morphogenesis of a Sfrp2 Symbol;Acc:MGI:108078] branching structure 2.18826 serum/glucocorticoid regulated regulation of kinase 1 [Source:MGI supramolecular Sgk1 Symbol;Acc:MGI:1340062] fiber organization -0.873794 SH3 and PX domains 2B regulation of [Source:MGI supramolecular Sh3pxd2b Symbol;Acc:MGI:2442062] fiber organization -1.46685 SH3 domain containing ring finger 1 [Source:MGI Sh3rf1 Symbol;Acc:MGI:1913066] cell leading edge -2.23312 SH3 and multiple ankyrin repeat domains 2 [Source:MGI Shank2 Symbol;Acc:MGI:2671987] axon part 1.02854 SH3 and multiple ankyrin repeat regulation of domains 3 [Source:MGI supramolecular Shank3 Symbol;Acc:MGI:1930016] fiber organization -2.17284 shisa family member 7 [Source:MGI Shisa7 Symbol;Acc:MGI:3605641] cell leading edge -1.8682 short stature homeobox 2 axon development, [Source:MGI morphogenesis of a Shox2 Symbol;Acc:MGI:1201673] branching structure -1.72665 signal-regulatory protein alpha [Source:MGI cell-substrate Sirpa Symbol;Acc:MGI:108563] adhesion 1.6794 sirtuin 1 [Source:MGI tissue migration, Sirt1 Symbol;Acc:MGI:2135607] axon part -1.9342 solute carrier family 12, member 2 [Source:MGI morphogenesis of a Slc12a2 Symbol;Acc:MGI:101924] branching structure 2.106 solute carrier family 12, member 5 Slc12a5 [Source:MGI cell leading edge -1.52563

189 Symbol;Acc:MGI:1862037] solute carrier family 17 (sodium- dependent inorganic phosphate cotransporter), member 6 [Source:MGI Slc17a6 Symbol;Acc:MGI:2156052] axon part 1.37734 solute carrier family 17 (sodium- dependent inorganic phosphate cotransporter), member 8 [Source:MGI Slc17a8 Symbol;Acc:MGI:3039629] axon part -3.06482 solute carrier family 18 (vesicular monoamine), member 2 [Source:MGI Slc18a2 Symbol;Acc:MGI:106677] axon part -2.40196 -3.97069 solute carrier family 9 (sodium/hydrogen exchanger), member 3 regulator 1 [Source:MGI Slc9a3r1 Symbol;Acc:MGI:1349482] cell leading edge 2.59146 solute carrier family 9 (sodium/hydrogen exchanger), member 6 [Source:MGI axon development, Slc9a6 Symbol;Acc:MGI:2443511] axon part -1.32038 axon development, regulation of supramolecular fiber organization, tissue migration, negative chemotaxis, morphogenesis of a branching structure, slit guidance ligand 2 [Source:MGI extracellular matrix Slit2 Symbol;Acc:MGI:1315205] binding -1.40234 SLIT and NTRK-like family, member 1 [Source:MGI Slitrk1 Symbol;Acc:MGI:2679446] axon development -2.5382 SLIT and NTRK-like family, member 3 [Source:MGI Slitrk3 Symbol;Acc:MGI:2679447] axon development -1.32093 SLIT and NTRK-like family, member 4 [Source:MGI Slitrk4 Symbol;Acc:MGI:2442509] axon development -2.0305 SLIT and NTRK-like family, member 5 [Source:MGI Slitrk5 Symbol;Acc:MGI:2679448] axon development -0.838027 -1.61212 axon development, , frizzled class receptor morphogenesis of a [Source:MGI branching Smo Symbol;Acc:MGI:108075] structure, axon part 3.19804 cell-substrate SPARC related modular calcium adhesion, binding 1 [Source:MGI extracellular matrix Smoc1 Symbol;Acc:MGI:1929878] binding 2.84505

190 synaptosomal-associated protein 91 [Source:MGI axon development, Snap91 Symbol;Acc:MGI:109132] axon part -1.27 , beta [Source:MGI Sncb Symbol;Acc:MGI:1889011] axon part -2.27091 synuclein, gamma [Source:MGI Sncg Symbol;Acc:MGI:1298397] axon part -2.29134 sorting nexin 5 [Source:MGI Snx5 Symbol;Acc:MGI:1916428] cell leading edge 2.55875 sorting nexin 9 [Source:MGI Snx9 Symbol;Acc:MGI:1913866] cell leading edge 2.25576 SRY (sex determining region Y)-box tissue migration, 9 [Source:MGI morphogenesis of a Sox9 Symbol;Acc:MGI:98371] branching structure 1.89538 trans-acting transcription factor 1 [Source:MGI Sp1 Symbol;Acc:MGI:98372] tissue migration -0.861824 secreted acidic cysteine rich tissue migration, glycoprotein [Source:MGI extracellular matrix Sparc Symbol;Acc:MGI:98373] binding 4.73567 SPARC-like 1 [Source:MGI extracellular matrix Sparcl1 Symbol;Acc:MGI:108110] binding 2.96139 spermatogenesis associated 13 [Source:MGI Spata13 Symbol;Acc:MGI:104838] cell leading edge 2.86461 sparc/osteonectin, cwcv and kazal- cell-substrate like domains proteoglycan 2 adhesion, [Source:MGI extracellular matrix Spock2 Symbol;Acc:MGI:1891351] binding -1.58152 sparc/osteonectin, cwcv and kazal- like domains proteoglycan 3 [Source:MGI extracellular matrix Spock3 Symbol;Acc:MGI:1920152] binding -0.783579 -1.76355 morphogenesis of a sprouty RTK signaling antagonist 2 branching [Source:MGI structure, cell Spry2 Symbol;Acc:MGI:1345138] leading edge 2.30277 regulation of supramolecular spectrin beta, non-erythrocytic 1 fiber organization, [Source:MGI axon part, cell Sptbn1 Symbol;Acc:MGI:98388] leading edge -0.627532 SRC kinase signaling inhibitor 1 cell-substrate [Source:MGI adhesion, cell Srcin1 Symbol;Acc:MGI:1933179] leading edge -0.999121 -1.3632 axon development, slingshot protein phosphatase 2 regulation of [Source:MGI supramolecular Ssh2 Symbol;Acc:MGI:2679255] fiber organization -0.671567 StAR-related lipid transfer (START) domain containing 13 [Source:MGI Stard13 Symbol;Acc:MGI:2385331] tissue migration -1.82446 -1.89672 serine/threonine kinase 25 (yeast) [Source:MGI Stk25 Symbol;Acc:MGI:1891699] axon development -1.13159

191 regulation of supramolecular fiber organization, -like 2 [Source:MGI axon part, cell Stmn2 Symbol;Acc:MGI:98241] leading edge -0.640204 -1.04456 stathmin-like 3 [Source:MGI Stmn3 Symbol;Acc:MGI:1277137] axon part -2.26573 stathmin-like 4 [Source:MGI Stmn4 Symbol;Acc:MGI:1931224] axon part -0.593573 -2.01138 [Source:MGI axon part, cell Stx3 Symbol;Acc:MGI:103077] leading edge -1.10137 syntaxin binding protein 1 [Source:MGI Stxbp1 Symbol;Acc:MGI:107363] axon development -1.86731 syntaxin binding protein 5 (tomosyn) [Source:MGI Stxbp5 Symbol;Acc:MGI:1926058] axon development -0.703511 -1.01002 sulfatase 1 [Source:MGI morphogenesis of a Sulf1 Symbol;Acc:MGI:2138563] branching structure 1.88848 [Source:MGI Syp Symbol;Acc:MGI:98467] axon part -0.776693 -2.5161 synaptotagmin XI [Source:MGI Syt11 Symbol;Acc:MGI:1859547] axon part -0.806483 synaptotagmin VII [Source:MGI Syt7 Symbol;Acc:MGI:1859545] axon part -1.54504 regulation of supramolecular tachykinin 1 [Source:MGI fiber organization, Tac1 Symbol;Acc:MGI:98474] tissue migration 1.67824 -4.59322 3 [Source:MGI Tacr3 Symbol;Acc:MGI:892968] cell leading edge -1.41873 TBC1 domain family, member 24 [Source:MGI Tbc1d24 Symbol;Acc:MGI:2443456] axon part -0.665784 regulation of transforming growth factor, beta supramolecular receptor I [Source:MGI fiber organization, Tgfbr1 Symbol;Acc:MGI:98728] tissue migration -1.12567 1.61954 thioesterase superfamily member 4 [Source:MGI Them4 Symbol;Acc:MGI:1923028] cell leading edge -1.6154 timeless circadian 1 [Source:MGI morphogenesis of a Timeless Symbol;Acc:MGI:1321393] branching structure 4.0778 1 [Source:MGI Tln1 Symbol;Acc:MGI:1099832] cell leading edge 2.78874 regulation of toll-like receptor 2 [Source:MGI supramolecular Tlr2 Symbol;Acc:MGI:1346060] fiber organization 3.66994 transmembrane protein with EGF- like and two follistatin-like domains 2 regulation of [Source:MGI supramolecular Tmeff2 Symbol;Acc:MGI:1861735] fiber organization -1.34819 -1.18733 transmembrane protein 8B cell-substrate Tmem8b [Source:MGI adhesion -2.37205

192 Symbol;Acc:MGI:2441680] regulation of supramolecular 2 [Source:MGI fiber organization, - Tmod2 Symbol;Acc:MGI:1355335] axon part -0.627287 0.957407 regulation of thymosin, beta 10 [Source:MGI supramolecular Tmsb10 Symbol;Acc:MGI:109146] fiber organization -1.68187 axon development, morphogenesis of tenascin C [Source:MGI a branching Tnc Symbol;Acc:MGI:101922] structure 1.2064 2.77012 tumor necrosis factor receptor axon superfamily, member 21 development, [Source:MGI neuron projection - Tnfrsf21 Symbol;Acc:MGI:2151075] fasciculation -0.612938 0.874129 3, gamma [Source:MGI Tpm3 Symbol;Acc:MGI:1890149] axon part -0.881865 TPX2, microtubule-associated [Source:MGI Tpx2 Symbol;Acc:MGI:1919369] axon part 1.99256 trafficking protein, kinesin binding 2 [Source:MGI axon development, Trak2 Symbol;Acc:MGI:1918077] axon part -0.792756 tripartite motif-containing 46 [Source:MGI axon development, Trim46 Symbol;Acc:MGI:2673000] axon part -2.4763 transient receptor potential cation channel, subfamily V, member 2 axon development, [Source:MGI axon part, cell Trpv2 Symbol;Acc:MGI:1341836] leading edge -1.91614 teashirt zinc finger family member 3 [Source:MGI Tshz3 Symbol;Acc:MGI:2442819] axon part -1.58469 tweety family member 1 [Source:MGI cell-substrate Ttyh1 Symbol;Acc:MGI:1889007] adhesion 2.56986 , beta 3 class III [Source:MGI Tubb3 Symbol;Acc:MGI:107813] axon development -1.99367 regulation of tubulin, beta 4A class IVA supramolecular [Source:MGI fiber organization, Tubb4a Symbol;Acc:MGI:107848] axon part -1.01426 regulation of twinfilin actin binding protein 1 supramolecular [Source:MGI fiber organization, Twf1 Symbol;Acc:MGI:1100520] cell leading edge 1.24913 ubiquitin carboxy-terminal L1 [Source:MGI axon development, Uchl1 Symbol;Acc:MGI:103149] axon part -2.34401 unc-51 like kinase 1 [Source:MGI Ulk1 Symbol;Acc:MGI:1270126] axon development -1.84794 unc-13 homolog A [Source:MGI Unc13a Symbol;Acc:MGI:3051532] axon part -1.49314

193 unc-5 netrin receptor A [Source:MGI axon development, Unc5a Symbol;Acc:MGI:894682] cell leading edge -1.59302 unc-5 netrin receptor D [Source:MGI Unc5d Symbol;Acc:MGI:2389364] axon development -0.654463 -2.15262 uronyl-2-sulfotransferase [Source:MGI Ust Symbol;Acc:MGI:2442406] axon development -1.63322 [Source:MGI axon development, Vim Symbol;Acc:MGI:98932] cell leading edge 3.49899 vitrin [Source:MGI cell-substrate Vit Symbol;Acc:MGI:1921449] adhesion 3.071 cell-substrate adhesion, vitronectin [Source:MGI extracellular matrix Vtn Symbol;Acc:MGI:98940] binding 5.45535 von Willebrand factor C domain containing 2 [Source:MGI cell-substrate Vwc2 Symbol;Acc:MGI:2442987] adhesion -2.90498 Wiskott-Aldrich syndrome regulation of [Source:MGI supramolecular Was Symbol;Acc:MGI:105059] fiber organization 4.97577 regulation of WAS protein family, member 1 supramolecular [Source:MGI fiber organization, Wasf1 Symbol;Acc:MGI:1890563] cell leading edge -1.41666 regulation of WAS protein family, member 2 supramolecular [Source:MGI fiber organization, Wasf2 Symbol;Acc:MGI:1098641] cell leading edge 2.06921 axon development, tissue migration, neuron projection fasciculation, negative wingless-type MMTV integration site chemotaxis, family, member 5A [Source:MGI morphogenesis of a Wnt5a Symbol;Acc:MGI:98958] branching structure -1.48537 xylosyltransferase 1 [Source:MGI Xylt1 Symbol;Acc:MGI:2451073] axon development 1.77683 tyrosine 3- monooxygenase/tryptophan 5- monooxygenase activation protein, zeta polypeptide [Source:MGI Ywhaz Symbol;Acc:MGI:109484] cell leading edge -0.6969 -1.76361 zinc finger, FYVE domain containing 27 [Source:MGI axon development, Zfyve27 Symbol;Acc:MGI:1919602] axon part -2.34971

194

CHAPTER 4. DISCUSSION AND FUTURE DIRECTIONS

195 The cumulation of the work reported in this dissertation elucidate i. the transcriptional control over the global identity of dorsal raphe 5-HT neurons

(Chapter 2); and ii. the transcriptional programs that control the successive morphological stages required to build the highly diffuse serotonergic axon projection pathways (Chapter 3).

Serotonergic Heterogeneity

It had been previously reported that Pet1 regulates essential genes involved in 5-HT synthesis and transport including Tph2, Aadc, Vmat2, and Sert in serotonin neurons (Hendricks et al., 2003). In Chapter 2, we investigated whether Pet1 regulates additional genes required for 5-HT synthesis and transport. Indeed, we found that Pet1 controls the expression of additional 5-HT identity genes including the genes required for the regeneration of tetrahydrobiopterin (BH4) as well as the 5-HT transporter called organic cation transporter 3 (Oct3, Slc22a3). This indicated that Pet1 globally controls 5-HT synthesis and 5-HT reuptake genes. However, a previous publication (Kiyasova et al., 2011) questioned whether all serotonin neurons depend on Pet1 for their neurochemical identity. This statement was based on the fact that about 30% of

5-HT neurons in Pet1 knock out (Pet1-/-) mice still express the gene Tph2, necessary for 5-HT synthesis, and still produce 5-HT (Hendricks et al., 2003;

Kiyasovia et al., 2011). Kiyasova et al. 2011 state that these cells are Pet1- independent for 5-HT identity, and therefore may be a distinct subtype of 5-HT neuron. As discussed in Chapter 2, we found that other 5-HT genes in the 5-HT

196 gene battery, Htr1a as well as Slc22a3, are completely dependent on Pet1 for their expression, as in situ hybridization (ISH) confirmed the complete loss of detection of these genes in Pet1-/- DRN neurons. Therefore, we conclude that all

5-HT neurons are dependent on Pet1 for the expression of certain 5-HT battery genes crucial for normal 5-HT neurotransmission.

It is unclear why there are a percentage of 5-HT neurons that can still produce Tph2 and 5-HT in absence of Pet1. Perhaps Pet1 functions in parallel with other transcription factors to turn on some 5-HT identity genes, including

Tph2, in subpopulations of 5-HT neurons. Maybe these Tph2+Pet1-/- cells do have lowered Tph2 expression compared to Pet1+ cells, however Tph2 is still detectable by immunohistochemistry and in situ techniques. Studies using

SABER- methods (Kishi et al., 2019) to detect individual Tph2 transcript in

Tph2+Pet1-/- cells compared to control cells would be necessary to conclude that

Pet1 does regulate Tph2 to some degree. Indeed, Okaty et al., 2015 performed single cell RNA-sequencing and showed that there are varying levels of Tph2 transcript in individual 5-HT neurons. So, it could be possible that the compensatory action of other transcription factors that have to ability to individually regulate Tph2 expression, could be the source of the residual Tph2 detected in the 30% of 5-HT neurons in Pet1-/- mice. However, given the findings that Pet1 is required for Oct3 and Htr1a expression in all 5-HT neurons, even if

Tph2 is expressed and 5-HT is produced in some Pet1-/- neurons, 5-HT neurotransmission must still be altered.

197 Lmx1b is continuously required for the formation of 5-HT axon architectures

The formation of long-range axon pathways occurs in successive stages over an extended period of time (Fame et al., 2011; Lidov and Molliver, 1982;

Shirasaki and Pfaff, 2002). How these temporally distinct stages of morphological maturation are transcriptionally regulated in neurons is not understood. The findings presented in Chapter 3 provide answers to the temporal regulatory strategy of serotonergic long-range axon formation. A single continuously expressed transcription factor, Lmx1b, is essential at multiple stages of morphological maturation of 5-HT axon architectures. This is the first transcription factor identified in 5-HT neurons to control axon development.

Investigation of early Lmx1b conditional knock out (Lmx1bcKO) mice at adult stages confirmed a critical intrinsic role for Lmx1b in the formation of 5-HT axons, as mutant 5-HT axons failed to grow into a majority of target regions distal to 5-

HT cell bodies in both the forebrain as well as the spinal cord. Embryonic analysis of Lmx1bcKO mice showed delayed primary 5-HT axon outgrowth in the medial forebrain bundle (MFB) as well as in spinal cord funiculi, major primary axon routes of ascending and descending projecting 5-HT neurons respectively.

At the end of embryonic development, we found a clear inability of these axons to selectively route into multiple pathways beyond the MFB in the Lmx1bcKO forebrain, indicating that either growth or guidance of these axons is perturbed in

Lmx1bcKO mice. All of these Lmx1bcKO axons reach the end of the MFB tract, however only some of these axons make the dorsal turn during development into

198 the septum before stalling. These stalled Lmx1bcKO axons remain in this septal area into mid-adulthood and do not appear to degenerate, demonstrating that

Lmx1b absence does not impact the stability of these axons. However, higher magnification analysis would be necessary to detect possible early subtle changes of these axons in adult animals that may reveal early degenerative features. Perhaps prolonged absence of Lmx1b may cause destabilization of 5-

HT neurons and later cause 5-HT axon degeneration. Further analysis of axon processes in older adult Lmx1bcKO mice would be necessary to start to see possible changes in 5-HT axon morphology.

Stage-specific conditional targeting approaches of Lmx1b at early postnatal periods (Lmx1bicKO) confirmed a continuing requirement for Lmx1b in

5-HT neuron arborization, as we found a significant decrease in TdTomato+ axon arbors in multiple late arborizing terminal target regions. Interestingly, those arbors that formed prior to Lmx1b targeting, for example arbors within the thalamus and hypothalamus, were left unaffected, indicating that Lmx1b is not required for the maintenance of 5-HT axon arbors in early adulthood. Taken together, results obtained from Lmx1bcKO and Lmx1bicKO mice demonstrated that Lmx1b is continuously required during the morphological maturation of 5-HT neurons to complete the formation of their axon architectures by controlling i. primary axon extension, ii. selective axon routing, and iii. terminal arborization.

This is an impressive feat for a single transcription factor, as serotonergic axon architectures are one of the most expansive axon systems present in the brain. It was previously unknown whether single transcription factors play continual roles

199 over complex multistage developmental processes such as axon formation, or if different transcription factors are required for each individual stage. It is feasible to imagine different transcription factors could be necessary to turn on specific gene sets required for individual axon developmental stages. For instance, the process of long-range axon routing through the developing forebrain compared to recognition of specific target regions and formation of specific arbor patterns likely require distinct gene expression profiles that encode molecules necessary to complete these two very different functions. However, in the case of 5-HT axon development, we found that Lmx1b continually controls 5-HT axon formation throughout each of these different stages. This model of a continuously expressed transcription factor orchestrating many stages of axon development may be representative of how other neurons such as dopaminergic, noradrenergic, and histaminergic neurons, that also produce widespread innervation to the forebrain, control their axon formation.

Lmx1b in intrinsic 5-HT axon growth or guidance?

Short-range versus long-range 5-HT axon growth

The Lmx1bcKO 5-HT axon phenotype supports the idea that Lmx1b provides a crucial motor to power 5-HT axons to expansively grow throughout the forebrain, given that deficiency of Lmx1b results in failure of 5-HT axonal growth in almost all regions of the forebrain, especially distal regions. There are a few short-range targets including the dorsal hypothalamus that appear to have many terminals in Lmx1bcKO animals. However, this region also contains many axons

200 of passage within the MFB, which is located in this area, making it difficult to fully conclude that normal innervation occurs in this region. In addition, many brainstem target regions appear to have normal innervation, however these axons may have reached these very short-range targets before full Lmx1b knock- down, as the Pet1-Cre transgene targets Lmx1b starting at E12.5. The thalamus also contains many axons, albeit in altered densities compared to controls, however again it is not clear whether these areas were innervated prior to full

Lmx1b knock-down. If it is true that Lmx1b is not required for 5-HT axon growth to some of the short-range targets, it may be key for specifically the growth of long-range 5-HT axons. Further analysis of developing 5-HT axons in even earlier Lmx1b-targeted or Lmx1b-/- animals would be necessary to conclude whether Lmx1b is critical for specifically 5-HT axon innervation to far-reaching targets versus more proximal targets.

Some neurons extend very short axons and others have extremely long axons with profuse arbors. What is so special about 5-HT neurons that enable them to grow so profusely compared to an interneuron, for example? Perhaps this transcription factor, Lmx1b, controls an unknown specialized intrinsic growth program that enables these neurons to grow to such an enormous extent. Maybe

5-HT neurons express more growth promoting molecules in their growth cones and fewer repulsive markers than other neurons, enabling them to grow long distances. Possibly Lmx1b differentially regulates growth and guidance molecules at specific times to enable the 5-HT axon to extend until it reaches a target region to innervate. If this is true, then how does Lmx1b differentially

201 upregulate genes over a span of time? Maybe other transiently expressed transcription factors differentially open chromatin over this maturation period to allow Lmx1b to regulate different targets over time. Or perhaps these transiently expressed transcription factors complex with Lmx1b and guide Lmx1b to certain promoter targets to regulate downstream genes. Yet another possibility is that the subpopulation of 5-HT neurons that has the ability to grow to very distal regions of the brain, could have a particular transcription factor code compared to the relatively short-range targeting 5-HT neurons. In any case, Lmx1b seems to be a crucial common transcription factor in almost all 5-HT neurons that extend to the forebrain, except perhaps in some of the 5-HT neurons with very short- range targets discussed above. There are, however, some short-range targets such as the ventromedial hypothalamus that appear specifically avoided by

Lmx1bcKO axons as well. This opens the question as to whether Lmx1b could also play roles in regulating guidance mechanisms in addition to long-range growth.

Lmx1b regulates axon growth and guidance genes

While it is likely that Lmx1b controls the growth of 5-HT axons, we cannot rule out a guidance defect contributing to the Lmx1bcKO phenotype. In RNA- sequencing datasets generated from flow sorted rostral and caudal Lmx1bcKO 5-

HT neurons at E17.5, Lmx1b was found to regulate genes involved in not only axon growth (GO term: axon extension) but also axon guidance. Of the 422 axon-related genes that Lmx1b regulates in 5-HT neurons, 38 genes are

202 associated with the GO term axon guidance while 25 genes are associated with the GO term axon extension, and another 21 genes are associated with both of these GO terms (Figure 1). Lmx1b regulates genes that encode guidance ligands including several Sema isoforms, Wnt5a, Slit2, Ntn1, Ncam1, Cdh4,

Cntn2, and Efna5 (protein name: ephrin A5). Lmx1b also regulates several known guidance receptors including Nrp2, Plxnc1, Epha4, EphB2, and Celsr3. I did further investigate Nrp2 in the possible role of long range 5-HT axon guidance in a conditional knock out mouse where Nrp2 was knocked out specifically in 5-HT neurons at E12.5 and analyzed for TdTomato-labeled axons.

However, I did not see any substantial overall patterning defects in either the forebrain or lumbar spinal cord of adult Nrp2cKO mice. It is unknown whether there were any transient developmental phenotypes in these animals as embryonic time points were not evaluated. It is possible that the homolog Nrp1 may compensate for loss of Nrp2, as it is also known in other cell types to be important in axon guidance (Piper et al., 2009). Further experiments investigating

5-HT neuron conditional knock out mice of receptors or growth/guidance molecules known to impact axon development found in the list of Lmx1b regulated genes will very likely uncover new downstream targets that impact intrinsic 5-HT axon growth or guidance.

It is known that 5-HT neurons are heterogeneous in gene expression based on single cell RNA sequencing studies (Okaty et al., 2015). Also, the morphological evidence that individual groups of 5-HT neurons project to very different target regions of the forebrain is evidence for possible differential

203 expression of growth and guidance genes between 5-HT neurons (Muzerelle et al., 2016). The RNA sequencing datasets generated in Chapter 3 are likely representative of a mixed population of 5-HT neurons that differentially express subsets of genes within the 422 Lmx1b axon-related regulated gene list. It could be that Lmx1b differentially regulates specific downstream gene sets in specific subpopulations of 5-HT neurons by way of combinatorial control based on co- expression with different regulatory factors. Studies using SABER-FISH methods could begin to elucidate the heterogeneity of expression of some of these growth and guidance molecules found to be dysregulated in Lmx1bcKO 5-HT neurons.

Using this RNA detection method, it may be determined that specific subnuclei of

5-HT neurons express specific genes that correspond with their ability to reach particular terminal target regions. Establishing the location of 5-HT neurons that express specific genes that impact growth and/or guidance of 5-HT axons combined with the knowledge of subnuclei-specific target regions provided by 5-

HT neuron tracing studies could open opportunities to investigate differential growth and guidance mechanisms of different 5-HT neuron subnuclei. One specific example to investigate could be the paraventricular thalamic nucleus

(PVT). Tracing studies identified the majority of innervation at the PVT comes from 5-HT neurons located in the B8 nucleus, or median raphe nucleus (MRN)

(Muzerelle et al., 2014). Perhaps these neurons express distinct guidance molecules that promote the extremely dense 5-HT axon patterns that form at the

PVT. If specific molecules expressed by the B8 neurons are identified, RNAi experiments could be conducted for individual genes using stereotaxic injections

204 to target this B8 subregion in early postnatal control pups. This experiment is feasible because, as we found in Chapter 3, 5-HT axon arborization at the PVT occurs sometime after P3, as P3 tamoxifen-targeted Lmx1bicKO animals completely lack the dense 5-HT axon innervation to this region (see Chapter 3,

Figure 5E).

Why do 5-HT neurons produce so many growth and guidance ligands as well as receptors? It is interesting that Lmx1b plays roles in regulating both guidance ligands and receptors that are implicated in both attraction and repulsion. One theory is that 5-HT neurons produce growth and guidance ligands to help other developing axons navigate or to instruct target regions to mature following terminal target innervation. Another theory is that perhaps 5-HT neurons produce attractive guidance molecules to regulate local corresponding receptors expressed by neighboring 5-HT growth cones to promote small subgroups of 5-HT axons within the MFB to grow in a particular direction in order to send a subset of their axons to particular regions of the forebrain. Repulsive cues could also serve as local guidance cues to prevent all axons turning in the same direction, as 5-HT axons create many routes that emanate at different times and locations from the MFB. Lmx1b may temporally direct the up- or down- regulation of specific growth or guidance molecules over the 5-HT axon maturation period to instruct these axons to selectively route. While the

Lmx1bcKO phenotype does not show any obvious ectopic trajectories, many selective routes that emanate from the MFB fail to form. It is also unclear whether some of the earlier selective routes such as the fasciculus retroflexus, and stria

205 medularis are able to fully form. Most likely the ability of groups of 5-HT axons to turn away from the MFB and enter other pre-existing axon tracts is directed by a combination of extrinsic and intrinsic factors. Investigation of multiple timepoints in development would be necessary to understand whether Lmx1b does temporally regulate different growth or guidance genes, and whether expression of those genes correlate with selective routing choices made over the embryonic timeline.

Potential mechanisms of Lmx1bcKO axon failure to innervate

An indication that 5-HT axon guidance may be disrupted is the curious location of the stalled mutant axons in developing Lmx1bcKO animals. There are two groups of these stalled Lmx1bcKO axons. All Lmx1bcKO axons fully fill the

MFB and do one of two things, either i. stop within the MFB, thereby failing to dorsally turn into the septum, or ii. make the dorsal turn into the septal area where they then stall and fail to fill subsequent selective routes.

This first group of Lmx1bcKO axons, that stall just before the septal area within the MFB and fail to make the dorsal turn, could have a possible guidance defect because these axons fail to grow in a particular direction at this critical choice point of the forebrain. 5-HT axons need to first turn dorsally out of the

MFB and into the septum in order to then choose between multiple subsequent routes to reach many areas of the forebrain. TdTomato-labeled Lmx1bcKO axons reach this choice point region of the forebrain around E17-E18. Further experiments performed at this time point to detect actin filaments of developing

206 growth cones by immunohistochemistry could identify whether these 5-HT axons have active or collapsed growth cones. If many healthy and active Lmx1bcKO growth cones are found, this could indicate that intrinsic growth is specifically stunted. Indeed, Lmx1b was found to positively regulate many genes involved in axon growth including Gap43, Ndn, Ntn1, Map6, Ret, and Gfra1. However, if the

Lmx1bcKO growth cones appeared collapsed in this region, this could indicate that a negative repulsive cue causes the failure of axon extension. Collapsed

Lmx1bcKO growth cones could be the result of upregulated receptors that are typically repulsive. Indeed, an Ephrin receptor, EphA4, implicated in repulsive axon guidance, is found to be upregulated in the Lmx1bcKO RNA-sequencing datasets. This upregulated expression of EphrinA4 needs to be further validated by secondary methods like immunohistochemistry to confirm its accumulation in

Lmx1bcKO growth cones. Additionally, staining for Ephrin ligands in the relevant region of this septal area could begin to help understand which ephrin ligands may be involved and start to provide evidence of Eph-ephrin signaling within 5-

HT neurons at this choice point during development.

In vitro experiments could also be conducted on 5-HT raphe explants at early embryonic stages to test for intrinsic growth or guidance defects of developing Lmx1bcKO versus control 5-HT axons. These 5-HT raphe explants have been shown to grow for many days in culture in specific media and on certain substrates (Kiyasova et al., 2011). Functional growth assays could be performed on these explants by plating them on growth-promoting laminin and observing whether Lmx1bcKO axons have the intrinsic ability to grow.

207 Additionally, guidance stripe assays can be conducted to test specific sensitivities of Lmx1bcKO axon growth direction compared to control axons. For instance, Ephrin ligands can be plated within the substrate in a stripe pattern, with neighboring stripes containing growth promoting substrates. This may be a way to test whether possible upregulation of EphrinA4 causes enhanced repulsion of Lmx1bcKO axons to begin to validate this signaling mechanism in 5-

HT axon guidance. In addition, RNAi knock down experiments in wild-type 5-HT explant cultures could further validate individual genes necessary for either growth or guidance of 5-HT axons.

The second group of stalled Lmx1bcKO axons are first able to make the dorsal turn out of the MFB and into the septal area but then subsequently fail to grow into selective routes. One possibility for this group of Lmx1bcKO axons, that may have developmentally preceded the first group of Lmx1bcKO axons that stalled within the MFB, is that their growth capacity was fully reached, and they stall because of an inability to grow any farther. A second theory is that these

Lmx1bcKO axons, which curiously all stop within the mid-septal area, experience a transient developmental obstruction in the embryonic forebrain. The subcallosal glial sling (also called glial wedge), discovered in the laboratory of Dr. Jerry

Silver, is a transient structure within the embryonic brain that is primarily made up of a dense population of glial cells that starts to form between E15-E16 in the mouse. By E16-E17, this midline glial sling structure is fully formed and is located under the corpus callosum and extends rostrally up to the olfactory bulb, along the corticoseptal boundary (Hankin and Silver, 1988; Hankin et al., 1988) (Figure

208 2). This structure serves as a transient barrier between the cortex and septum during development and provides support and guidance to decussating axons of the corpus callosum, which begins to form at E17 in the mouse (Hankin and

Silver, 1988). Glial cells within this structure serve as guidepost cells for callosal axons that is mediated, in part, by secretion of Slit2 (Shu et al, 2003). The glial sling then undergoes cell death at E18 and completes its full degeneration postnatally, resulting in a cavity called the cavum septi, which is a fluid filled gaping hole under the corpus callosum (Hankin et al., 1988). Many developing 5-

HT axons normally begin to pass this septal area between E15-E16 during development in the mouse. However, Lmx1bcKO axons experience a delayed primary pathway growth in the MFB between E15 and E18, as described in

Chapter 3. Therefore, it may be possible that Lmx1bcKO axons that make the dorsal turn around E17-E18, encounter the glial sling that either physically, or molecularly prevents Lmx1bcKO axons from crossing, resulting in a failure to fill subsequent routes of the cingulum, supracallosal stria, and fornix. Slit2 is produced by cells within the sling and acts as a repulsive cue for developing callosal axons (Shu et al, 2003). This signaling mechanism may also mediate the

Lmx1bcKO failure to grow past this region, as mutant axons appear to have stopped just at glial sling edge (see Chapter 3: Figure 3C). Normally, 5-HT axons induce Robo2 expression in late embryonic development (Wyler et al.,

2016), however Pet1-/- RNA-seq datasets showed a significant increase of Robo2 expression at early stage E15.5. While Robo2 upregulation was not found to reach significance in E17.5 Lmx1bcKO RNA-seq datasets, it was found to be

209 upregulated in P2 Lmx1bcKO RNA-seq datasets, making this signaling mechanism an interesting target to pursue further. Perhaps early upregulation of

Robo2 by these specific 5-HT axons that encounter the barrier, experience enhanced repulsion by Slit2 expressing cells within the barrier. This then poses the question, how do wild-type 5-HT axons penetrate the barrier? The timing of

5-HT axon arrival to the barrier precedes the full formation of the barrier by about a day. Therefore, the 5-HT axons that make it across early may serve as a bridge for later developing 5-HT axons to fasciculate onto and grow through the barrier to reach subsequent routes. Or perhaps 5-HT axons need to digest extracellular matrix within the barrier by releasing proteases to cross. To test whether

Lmx1bcKO axons are blocked by this barrier, one could irradiate pregnant mice at E14.5, which has been shown to prevent this glial sling formation (Schneider and Silver, 1990). This could be performed in Lmx1bcKO vs control animals and observe whether Lmx1bcKO axons can grow past this region of the forebrain and continue into subsequent routes. It would also be interesting to closely look at the early postnatal stages that follow E18 to see if any Lmx1bcKO axons are able to pass this septal area after the barrier is normally fully degenerated. Yet another experiment could be to perform in utero electroporation of shRNA expression vectors to target receptors like Robo2 and observe whether Lmx1bcKO axons have to ability to cross the barrier.

210 From identity to axon development

As a terminal selector-type transcription factor Lmx1b controls the acquisition of 5-HT identity in early fetal development and continues to control expression of genes required to maintain 5-HT identity into adulthood in mice

(Zhao et al., 2006; Song et al., 2011). Here, we found that Lmx1b is also required for 5-HT axon development. This concept of a single continuously expressing transcription factor that controls identity as well as multiple axon maturation steps could extend to other neuronal cell types, such as dopaminergic (DA) neurons. In fact, Lmx1b is also an important factor in mDA differentiation and functions cooperatively with its paralog, Lmx1a, which is co-expressed in mDA neurons but not serotonergic neurons (Yan et al., 2011; Blaess and Ang, 2015). Single conditional knock out of Lmx1b is not sufficient, however, to show DA specification defects, only double knock out mutants for Lmx1a/b display failure to induce DA neuron specification (Yan et al., 2011). In addition, both Lmx1b and

Lmx1a dual knockout is necessary to observe some DA axon projection defects to specific striatal areas, leaving other DA terminal target region innervation unaffected (Chabrat et al., 2017). While Lmx1b does not seem to have the same impressive control over DA axon development or specification as it does in 5-HT neurons, another candidate terminal selector transcription factor in DA neurons that could parallel Lmx1b’s control in 5-HT neurons, is Nurr1 (gene name:

Nr4a2). It is already established in DA neurons that the continuously expressed regulatory factor, Nurr1, is responsible for the neurochemical identity of DA neurons (Blaess and Ang, 2015). Nurr1 knockout disrupts DA differentiation

211 through loss of key DA identity genes TH, DAT, VMAT2, and AADC (Blaess and

Ang, 2015). Further, adult ablation of Nurr1 was reported to result in dystrophic axons and dendrites of midbrain DA neurons in mice (Kadkhodaei et al., 2013).

However, these results were concluded from immunolabeling with the DA endogenous marker, tyrosine hydroxylase (TH), which Nurr1 controls the expression of, making it difficult to analyze the true morphology of DA axons

(Kadkhodaei et al., 2013). Still, Nurr1 would be a good candidate to test whether it is required for successive stages of DA axon development. Conditional targeting of Nurr1 at different critical axon developmental time windows with better axon-labeling methods using fluorescent markers would solidify whether it plays similar roles in DA neurons as Lmx1b does in 5-HT neurons. Further, RNA seq studies at relevant stages of development would help elaborate the extent to which Nurr1 controls gene expression that could influence these developmental steps.

Transcription factor regulation of ascending vs descending 5-HT axon architectures

Through RNA-sequencing analysis of conditional Lmx1b-targeted flow sorted 5-HT neurons at either E17.5 or postnatal day 2 (P2), we found that

Lmx1b regulates several other transcription factors that operate within the 5-HT

GRN in either rostral or caudal 5-HT neurons including Pet1, Gata2, Gata3, En1, and En2. The most consistently and differentially regulated gene in this group was Pet1. We found Pet1 was significantly downregulated in both Lmx1bcKO

212 rostral and caudal 5-HT neurons at E17.5 and P2. Given the severe axon deficits at all Lmx1bcKO spinal levels, we investigated whether the Lmx1b→Pet1 regulatory cascade functioned in descending 5-HT axon development.

Interestingly, analysis of TdTomato+ axons in Pet1cKO spinal cords revealed no quantifiable 5-HT axon innervation defect from cervical to lumbar levels, indicating that Pet1 plays no major role in either routing or arborization of descending 5-HT axon innervation. We did, however, identify Pet1’s critical role in rostral 5-HT neuron axon development, supporting the operation of a

Lmx1b→Pet1 regulatory cascade in the development of ascending 5-HT axonal architectures. Taken together, these results revealed a differential transcription factor requirement for ascending and descending 5-HT axon innervation, where the Lmx1b→Pet1 regulatory cascade operates in early embryonic development for ascending 5-HT axon development while descending 5-HT axon development is Pet1 independent. This raised the question of additional transcription factors that may operate under Lmx1b regulation in descending 5-HT axon development.

We speculate that possibly other continuously expressed transcription factors found to be regulated by Lmx1b in caudal 5-HT neurons, such as Gata3, may play a role in descending 5-HT axon innervation. Further analysis of labeled 5-HT axons in conditional knock-out mice of Gata3 will be necessary to investigate this possibility.

213 Lmx1b→Pet1 regulatory cascade functions in 5-HT arborization

Due to the arborization defects seen in postnatal targeted Lmx1bicKO mice, we further investigated whether the same Lmx1b→Pet1 regulatory cascade that functions in early 5-HT axon formation was continually required during 5-HT axon arborization. Initial analysis of RNA-sequencing datasets generated from

Lmx1bcKO and Pet1cKO flow sorted 5-HT neurons revealed a commonly regulated axon-related gene called Pcdhac2. This gene has been extensively investigated in the context of 5-HT axon development and is known to intrinsically regulate the growth and patterning of 5-HT arbors during the arborization stage (Chen et al., 2017; Katori et al., 2009; Katori et al., 2017). In addition, previously generated RNA-seq datasets comparing the RNA gene expression profile in wild-type 5-HT neurons at E11.5, E15.5, and early postnatal stages identified Pcdhac2 as a gene whose expression profile is significantly upregulated at the later postnatal stages compared to early embryonic stages

(Wyler et al., 2016). We further found that each Lmx1b and Pet1 individually postnatally regulate Pcdhac2 expression supporting the idea that Lmx1b→Pet1 cascade may induce Pcdhac2 expression in a temporal manner to control 5-HT arborization. Interestingly, re-analysis of a previously generated ChIP-seq study using mycPet1 transgenic mice, revealed direct Pet1 binding of two relevant regulatory regions that have been shown to drive expression of the specific

Pcdhac2 isoform (Wyler et al., 2016; Kehayova et al., 2011; Ribich et al., 2006).

Investigation of postnatal, tamoxifen-induced, Pet1 conditional knock out

(Pet1icKO) mice supported a temporal requirement for the Lmx1b→Pet1

214 regulatory cascade as Pet1icKO phenotype very closely paralleled the

Lmx1bicKO phenotype in the inability of mutant axons to fully arborize in the cortex and hippocampus. Together these results suggested that the

Lmx1b→Pet1 regulatory cascade is required at the arborization stage of 5-HT axon development, likely in part through the upregulation of Pcdhac2. The regulation of Pcdhac2 by the Lmx1b→Pet1 regulatory cascade serves as an example of how this cascade can temporally operate to regulate downstream genes necessary for a stage-specific function such as arborization. Ideally, further ChIP experiments to pull down Lmx1b would confirm whether Lmx1b, with

Pet1, co-transcriptionally regulates downstream genes such as Pcdhac2, however no ChIP-grade antibody exists for Lmx1b at the moment.

While the role for Pcdhac2 in 5-HT axon arborization has been well studied and validated, further rescue experiments inducing Pcdhac2 expression in Lmx1bicKO and Pet1icKO mice would be necessary to help describe the level of involvement of Pcdhac2 in the disrupted arborization of each of these conditional knock-out mice. Stereotaxic injections of lentiviral expression vectors, containing the Pcdhac2 gene along with simultaneous targeting of Lmx1b or Pet1 by tamoxifen IP injection in early postnatal icKO mice would i. allow for conditional knock out of these transcription factors in the relevant arborization time period in which Pcdhac2 acts; and ii. restore expression of Pcdhac2 in 5-HT neurons. It is possible that the outcomes of these experiments would result in partial rescue, or possibly full rescue of arbor growth and patterning in specific terminal fields over others that may depend more heavily on Pcdhac2

215 expression. Another way to test that the Lmx1b→Pet1→Pcdhac2 cascade functions in 5-HT axon arborization would be to create a new mouse in which the two Pcdhac2 regulatory sites that Pet1 binds are mutated to prevent direct transcriptional regulation by Pet1. Further analysis of any 5-HT arborization disruption would confirm whether Pcdhac2 plays a role at this arborization stage that is dependent on Pet1 binding.

Most likely, Pcdhac2 is not the sole downstream gene of the Lmx1b→Pet1 cascade that impacts 5-HT axon arborization. We identified other commonly regulated genes of Lmx1b and Pet1 in the cKO RNA-seq datasets, including

Gap43. Gap43 is highly expressed in 5-HT neurons in early development, and its expression continues to significantly rise into postnatal stages (Figure 3). Gap43 is known to be an elongation factor and can drive arborization of astrocytes when constitutively active (Hung et al., 2016). It may be possible that a combination of

Gap43 and Pcdhac2 could mediate 5-HT axon arborization, which may be why our icKO mice have fewer arbors in some regions including the molecular layer of the hippocampus compared to the phenotype observed at this region in Chen et al., 2017. However, down regulation of Gap43 in later postnatal targeted

Lmx1bicKO and Pet1icKO mice would first need to be verified. In addition, there are many unannotated genes regulated by each Lmx1b and Pet1 that follow the same upregulated expression trajectory in 5-HT neurons in the postnatal period.

Possibly these undefined genes could be contributing factors of the arborization defects observed in Lmx1bicKO and Pet1icKO mice. RNAi studies using AAV viral expression vectors injected stereotaxically to newborn pups could be

216 performed systematically to test whether these unannotated genes impact 5-HT axon growth in vivo at later arborization stages, thereby identifying novel genes.

Alternatively, in vitro DRN explant studies using RNAi techniques could also reveal other genes involved in 5-HT axon growth, however this type of experiment would test early 5-HT axon growth, and not arborization. Further,

RNA-sequencing at later postnatal time periods in icKO mice could help identify additional genes that may be involved specifically in the postnatal maturation period, when arborization occurs.

Possible role for Lmx1b in regeneration?

During development, 5-HT neurons have a remarkable ability to grow profuse arbors throughout the forebrain and spinal cord. Here, we have uncovered that this developmental 5-HT axonal growth is dependent on Lmx1b expression. In the adult, 5-HT neurons also have a striking ability to regenerate their axons after injury (Hawthorne et al., 2011; Jin et al., 2016). Indeed, it has been historically challenging to prove regeneration of axons after injury because it is unclear whether axons that do regrow into injury sites have been severed by the injury or are actually spared axons that have the ability to grow into the injury site. While there has been evidence in the brain and spinal cord of 5-HT axon sprouting into injury sites, it was not fully proven that severed 5-HT axons were contributing to regrowth until Jin et al., 2016 used live two-photon imaging showing actively re-growing severed 5-HT axons. In addition, they found that these re-growing axons did not depend on the same specific pathways left by

217 degenerated 5-HT axons after injury (Jin et al., 2016). Therefore, 5-HT neurons have an intrinsic ability to activate regrowth of injured axons and re-find correct targets, seemingly without additional aid of their own pre-existing axon tracts.

How do they do this? It may be possible that Lmx1b, which is continuously expressed throughout adulthood, could transcriptionally activate a regrowth program after injury that may parallel the developmental growth program it activates to initially generate profuse 5-HT axons and innervate target regions.

Interestingly, it has been found that 5-HT re-growth capacity takes several weeks to substantially grow into the lesion site after initial retraction following injury in several independent studies using different lesion strategies (Hawthorne et al.,

2011; Jin et al., 2016; Alilain et al., 2011; Mamounas et al., 2000; Camand et al.,

2004). This observation is interesting given that this weeks-long timing corresponds to the approximate time it could take for transcriptional activation of downstream genes and possible impact on regrowth in injured axons.

Cortical stab injury experiments could be performed in adult-targeted

Lmx1bicKO and iControl mice to test whether Lmx1bicKO axons have a reduced capacity to regrow through the lesion site. If Lmx1b does appear to control regeneration of 5-HT axons after injury, it would be interesting to do single cell

RNA-sequencing of microdissected 5-HT neurons from the specific DRN subnuclei that innervate the region where the lesion was performed. Comparative analysis of RNA-sequencing datasets generated from control or Lmx1bicKO mice that underwent stab injury would uncover genes differentially regulated by Lmx1b in the context of regeneration. Additionally, one could also perform single cell

218 ATAC-sequencing from each sham and stab injured control and Lmx1bicKO mice. This method would unveil the change in chromatin states in 5-HT neurons during regeneration compared to their resting state, as well as changes in chromatin state in absence of Lmx1b. Perhaps Lmx1b or other transcription factors are necessary to change the chromatin state of 5-HT neurons to induce a regeneration program. Further, motif analysis for Lmx1b binding sites could be performed to identify possible Lmx1b-regulated genes that could impact regeneration. Alternatively to stab injury, PCA injections could be performed in

Lmx1bicKO and iControl mice to induce general 5-HT axon degeneration to test whether Lmx1b functions in 5-HT axon regeneration in many terminal target regions. Gap43 is an interesting possible downstream target as it is highly expressed in regenerating axon growth cones and is expressed in 5-HT neurons

(Skene and Willard, 1981a, b). Possibly Lmx1b continues to control genes such as Gap43 in a state-dependent manner to initiate axon regrowth after injury.

Investigation of human mutations in the 5-HT GRN

Now that it is known that both Lmx1b and Pet1 are essential for proper 5-

HT axon innervation to the forebrain, an interesting line of investigation would be to use CRISPR technology to introduce the known human mutations of Lmx1b and Pet1 into mice. True, there are over 100 variants of human Lmx1b mutations that cause a varying degree of the disease NPS, making it more difficult to choose which mutations to investigate further (Sweeney et al., 2003; Dai et al.,

2009; Bongers et al., 2008). Perhaps mutations within the homeodomain would

219 be the first to test as Lmx1b is a known transcription factor and mutations in the

DNA binding site may unveil more changes in transcription that would have a larger impact on neuronal and behavioral phenotype. Also selecting mutations based on severity of the human phenotype could serve as another criteria. These studies would be the first to implicate human variants of genes within the 5-HT gene regulatory network (GRN) that may impact 5-HT neuron development in multiple ways, including 5-HT axonogenesis as well as 5-HT identity. While there is likely some impact on the level of 5-HT production by 5-HT neurons in any of these human variants, additional altered circuitry could further exacerbate a mental phenotype.

Conclusion I have uncovered that Lmx1b, a transcription factor known to be essential for initial acquisition and maintenance of 5-HT neuron-type identity, functions continuously over 5-HT neuron maturation to control 5-HT axon formation to the far reaches of the brain and spinal cord. Through regulation of Pet1, Lmx1b intrinsically enables the relatively few 5-HT neurons to expansively grow their axons throughout the forebrain, and ultimately enables the widespread 5-HT neurotransmission that modulates many behaviors. I have uncovered a potential general principle in which a continuously expressed transcription factor controls many distinct stages of long-range axon pathway formation. This finding was somewhat unexpected, as it may have been that distinct transcription factors control the different stages of 5-HT axon growth. It will be important to further understand how Lmx1b controls stage specific gene expression to regulate

220 primary growth, selective routing, and terminal arborization. The RNA- sequencing datasets generated in Chapter 3 reveal for the first time, many axon- related genes under Lmx1b regulatory control that are now implicated in the intrinsic growth and guidance of 5-HT axons. These datasets serve as an important resource for future investigation of the molecular mechanisms that drive 5-HT long-range axon formation. It will be important to try and elucidate the intrinsic growth program that Lmx1b controls to power long-range 5-HT axon growth. Perhaps this will reveal why some neurons are only able to grow their axons short distances while others have the capacity to grow very long distances, like 5-HT neurons. Finally, it will be important to determine whether

Lmx1b also controls a 5-HT re-growth program after injury, as 5-HT neurons have this unique capability that other neurons do not. Overall, further identification of molecular mechanisms behind 5-HT axon growth could lead to a better understanding of underlying 5-HT neuron dysfunction in psychiatric disorders.

221 Figure 1

222 Figure 1. Lmx1b regulates both growth and guidance genes in 5-HT neurons. Venn diagram represents Lmx1b-regulated axon-related genes obtained from RNA-sequencing datasets of flow-sorted Lmx1bcKO vs control 5-

HT neurons at E17.5. All Lmx1b-regulated genes that are categorized as axon- related genes by gene ontology is shown in the green circle (L_Ax); all annotated axon guidance genes are shown in the blue circle; all annotated genes involved in axon extension are shown in the yellow circle. Lmx1b regulates 38 genes associated with the gene ontology term axon guidance, 25 genes associated with the gene ontology term axon extension, and 21 genes that fall under both categories.

223 Figure 2

Adapted from Hankin et al., 1988 https://doi.org/10.1002/cne.902720204

224 Figure 2. The glial sling is a transient barrier in the embryonic mouse brain.

Hematoxylin-stained sagittal section of the embryonic mouse brain at E17 shows a high density of cells that form the glial sling (Ba) that has been shown as a guidance structure for developing corpus callosum axons. SL, sling region

(arrowheads 1-2); Ba, rostral barrier region (arrowheads 2-3); CC, corpus callosum; OB, olfactory bulb; CG, cingulate cortex; HC, hippocampal commissure.

225 Figure 3

*

Gap43

*

226 Figure 3. Gap43 is significantly upregulated in maturing 5-HT neurons into postnatal stages. RNA-sequencing from flow sorted 5-HT neurons taken at either E11.5, E15.5, or early postnatal stages (P0-P3). Graph depicts the gene

Gap43 is highly expressed during the maturation period, and significantly upregulated at E15.5 compared to E11.5, as well as significantly upregulated in postnatal periods compared to E15.5. RNA-sequencing datasets were generated in Wyler et al., 2016 and reanalyzed for Gap43 expression changes.

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